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Klopfenstein, N.B.; Chun, Y.W.; Kim, M.-S.; Ahuja, M.A., eds. Dillon, M.C.; Carman, 

R.C.; Eskew, L.G., tech. eds. 1997. Micropropagation, genetic engineering, and 

molecular biology of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain Forest and 

Range Experiment Station. 326 p. 

Abstract: 

Thirty-four Populus biotechnology chapters, written by 85 authors, are comprised 

in 5 sections: 1) in vitro culture (micropropagation, somatic embryogenesis, 

protoplasts, somaclonal variation, and germplasm preservation); 2) transformation and foreign gene expression; 3} molecular biology (molecular/genetic 

characterization); 4) biotic and abiotic resistance (disease, insect, and pollution); and 5) biotechnological applications (wood properties, flowering, phytoremediation, breeding, commercialization, economics, and bioethics). 

Keywords: plant transformation; gene expression; tree biotechnology; molecular 

genetics; bioethics; plant tissue culture 

Editors' note: The views expressed in each chapter are those of the author(s) 

and not necessarily those of the USDA Forest Service. Trade names are used 

!or the information and convenience of the reader and do not imply endorsement or preferential treatment by the USDA Forest Service. 

Publisher: 

Rocky Mountain Forest and Range Experiment Station 

Fort Collins, Colorado 

September 1997 

You may order additional copies of this publication by sending your mailing information in label form through one of the following media. Please send the publication title 

and number. 

Telephone (970) 498-1719 

DG message R.Schneider:S28A 

FAX (970) 498-1660 

E-mail /s;::r.schneider/ou1 ;::S28a@ mhs-fswa.attmail.com 

Mailing Address Publications Distribution 

Rocky Mountain Forest and Range 

Experiment Station 

3825 E. Mulberry Street 

Fort Collins, CO 80524 

Cover art by Joyce Patterson; concept inspired by Claude Monet's Peupliers au bord 

de I'Epte, vue du marais, 1891. 
Micropropagation, 

Genetic Engineering, and 

Molecular Biology of Populus 

Ned B. Klopfenstein, Young Woo Chun, Mee-Sook Kim, 

and M. Raj Ahuja, Editors 

Madelyn C. Dillon, Richard C. Carman, and Lane G. Eskew, 

Technical Editors 

Contents 

Preface 

N.B. KLOPFENSTEIN, Y.W. CHUN, M.-S. KIM, and M.R. AHUJA ....... . 

Introduction- Sentinels of the Prairie Speak (by Populus Species) 

H.S. McNABB, Jr ............................................. . 

Section I. In Vitro Culture 

Chapter 1 - Poplar Shoot Cultures: Their Generation and 

Use in Biotechnology 

iv 

1 

B.H. McCOWN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 

Chapter 2 - Somatic Embryogenesis From Poplar Leaf Tissue 

Y.G. PARK and S.H. SON. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 

Chapter 3 - Plant Regeneration Through Organogenesis in Poplar 

H. KANG and Y.W. CHUN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 

Chapter 4 - Protoplast Isolation and Culture 

J. RUSSELL KIKKERT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 

Chapter 5 - Somaclonal Variation in Populus: An Evaluation 

D.J. FRY, G.C. DOUGLAS, and N. Th. SAIEED. . . . . . . . . . . . . . . . . . . . . . 33 

Chapter 6 - Germplasm Preservation of Populus Through 

In Vitro Culture Systems 

S.H. SON, Y.G. PARK, Y.W. CHUN, and R.B. HALL . . . . . . . . . . . . . . . . . . 44 

Section II. Transformation and Foreign Gene Expression 

Chapter 7 - Agrobacterium-mediated Transformation of Populus Species 

M.-S. KIM, N.B. KLOPFENSTEIN, and Y.W. CHUN. . . . . . . . . . . . . . . . . . . 51 

Chapter 8 - Direct Gene Transfer in Poplar 

P.J. CHAREST, Y. DEVANTIER, C. JONES, J.C. SELLMER, 

B. H. McCOWN, and D.O. ELLIS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 

Chapter 9 - Gene Expression Studies 

L. JOUANIN and G. PILATE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 
ii 

Chapter 1 0 - Differential Expression of aroA Gene in Transgenic Poplar: 

Influence of Promoter and Ozone Stress 

D.F. KARNOSKY, G.K. PODILA, D. SHIN, and 

D.E. RIEMENSCHNEIDER ..................................... . 

Chapter 11 -Growth and Development Alteration in Transgenic Populus: 

Status and Potential Applications · 

B. SUNDBERG, H. TUOMINEN, 0. NILSSON, T. MORITZ, 

C.H.A. LITTLE, G. SANDBERG, and 0. OLSSON .................. . 

Chapter 12 - Transgene Expression in the Field-Grown Poplar 

G. PILATE, D. D. ELLIS, and S. HAWKINS ......................... . 

Chapter 13 - Transgenes and Genetic Instability 

M.A. AHUJA ................................................ . 

Section Ill. Molecular Biology 

Chapter 14 - Applications of Molecular Marker Technologies in 

Populus Breeding 

M.T. CERVERA, M. VILLAR, P. FAIVRE-RAMPANT, M.-C. GOUE, 

70 

74 

84 

90 

M. VAN MONTAGU, and W. BOERJAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 

Chapter 15 - Differentiation of Poplar Clones Using Random Amplified 

Polymorphic DNA Fingerprinting 

D.C. LIN, M. HUBBES, and L. ZSUFFA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 

Chapter 16 - Seasonal Vegetative Storage Proteins of Poplar 

G.D. COLEMAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 

Chapter 17 - Poplar Seed Storage Proteins 

T.L. BEARDMORE, S. WETZEL, and S.M. REGAN................... 131 

Chapter 18 - Molecular Genetic Analysis of Populus Chloroplast DNA 

E.W. NOH and J.S. LEE........................................ 143 

Chapter 19 - Molecular Marker Techniques to Study Variability of 

Populus Pathogens 

G.M. TABOR and H.S. McNABB, Jr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 

Section IV. Biotic and Abiotic Resistance 

Chapter 20 - In Vitro Screening and Selection for Disease Resistance 

M.E. OSTRY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 

Chapter 21 -Transformation of Hybrid Aspen for Resistance to 

Crown Gall Disease 

H. EBINUMA, E. MATSUNAGA, K. YAMADA, and M. YAMAKADO 161 

Chapter 22 - Designing Small Antimicrobial Peptides and Their Encoding Genes 

W.A. POWELL and C.A. MAYNARD............................... 165 

Chapter 23 - Potential of Proteinase Inhibitors for Enhanced Resistance 

to Populus Arthropod and Pathogen Pests 

S.A. HEUCHELIN, L JOUANIN, N.B. KLOPFENSTEIN, and 

H.S. McNABB, Jr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 

Chapter 24 - Expression of Transgenic Bacillus thuringiensis o-endotoxin 

in Poplar 

D.D. ELLIS and K.F. RAFFA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 
Chapter 25 - Genetic Engineering for Air-Pollutant Resistance in Hybrid Aspen 

S. ENDO, E. MATSUNAGA, K. YAMADA, and H. EBINUMA.. . . • . . . . . . . . 187 

Section V. Biotechnological Applications 

Chapter 26 - Genetic Modification of Lignin Biosynthesis in Quaking Aspen 

and Poplar 

W. BOERJAN, M. BAUCHER, B. CHABBERT, M. PETIT-CONIL, J.-C. LEPLE, 

G. PILATE, D. CORNU, B. MONTIES, D. INZE, J. VAN DOORSSELAERE, 

L. JOUANIN, and M. VAN MONTAGU; with C.-J. TSAI, G.K. PODILA, and 

V.L. CHIANG • . . . . • • • • • • • . . . . . . . . . . . . • . . . . . . . . . . . • . . . . . . . . . . . . 193 

Chapter 27 - Potential Use of Populus for Phytoremediation of 

Environmental Pollution in Riparian Zones 

M.E. DIX, N.B. KLOPFENSTEIN, J.W. ZHANG, S.W. WORKMAN, and 

M.-S. KIM • . . . . . . . . • . . . . . • . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . 206 

Chapter 28 - Poplar Genetically Engineered for Reproductive Sterility and 

Accelerated Flowering 

A. MEILAN and S.H. STRAUSS . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . 212 

Chapter 29 - Integration of Molecular and Classical Genetics: A Synergistic 

Approach to Tree Improvement 

R.J. DINUS and G.A. TUSKAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 

Chapter 30 - Application of Tissue Culture Systems for Commercial 

Plant Production 

K.A. LOUIS and L.E. ElLS. .. . .. . . . .. . . . . • . .. .. .. . .. . . . . . . . . . . . . . 236 

Chapter 31 - Economics of Producing Populus Biomass for Energy and 

Fiber Systems 

C.H. STRAUSS and S.C. GRADO. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 

Chapter 32 - Environmental Risk Assessment and Deployment Strategies 

for Genetically Engineered Insect-Resistant Populus 

K.F. RAFFA, K.W. KLEINER, D.O. ELLIS, and B.H. McCOWN . . . . . . . . . . . 249 

Chapter 33 - Biotechnology Risk Assessment: A View From Plant Pathology 

X.B. YANG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264 

Chapter 34 - Biotechnology, Biodiversity, and Bioethics 

M.A. AHUJA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 

Section VI. Appendices 

Authors and Affiliations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 

Reviewer Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 

Scientific and Common Names of Biological Organisms Associated With 

Populus Biotechnology 

M.C. DILLON, R.C. CARMAN, and M.-S. KIM. . . . . . . . . . . . . . . . . . . . . . . . 290 

Glossary of Populus Tissue Culture and Molecular Biology 

M.-S. KIM, M.C. DILLON, and R.C. CARMAN.. . . . .. .. . .. . . . .. .. . . .. . 295 

Index of Populus.................................................... 311 

General Index....................................................... 313 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. iii 
Preface 

Species of the genus Populus (poplars and aspens) are 

interspersed primarily in temperate and cold-region forests of the northern hemisphere. They are relatively shortlived, fast-growing trees that can grow on marginal soils 

and are widely adaptable. Populus systems are rapidly being developed for application in fiber, fuel, and environmental plantings. In addition, contributions of in vitro and 

molecular biological studies of Populus are apparent in 

virtually every area of forest biology and ecology. The future of Populus research appears bright and promises significant biotechnological advances toward our 

understanding of processes and interactions in forest ecosystems. 

The concepts for this book were generated from a survey that identified the need to synthesize diverse, yet related, information from the rapidly developing studies on 

Populus molecular biology and in vitro culture. The topics 

included were designed to complement the recently published book, Biology of Populus and its implications for management and conseroation (Stettler, R.F.; Bradshaw, H. D., Jr.; 

Heilman, P.E.; Hinckley, T.M., eds. 1996. Ottawa, Ontario, 

Canada: NRC Research Press. 539 p.). Although we attempted to direct chapters toward a "more general" audience, not all topics were amenable. Lead authors were 

invited and encouraged to collaborate with co-authors 

working in similar areas, and we were especially supportive of collaboration among diverse, international laboratories. We thank all authors for their stellar efforts and 

valuable contributions. We also appreciate the constructive advice from peer reviewers, who are listed in the appendix, on all chapters. 

1his book was designed to foster information exchange 

and to encourage research cooperation. U.S. Department of 

Agriculture (USDA) funding was obtained to distribute this 

publication at a minimal cost and to broaden information 

access. Unfortunately, hampered by USDA Forest Service 

budget cuts and downsizing, associated publication delays 

ensued. These delays were minimized by the efforts and 

iv 

perseverance of the Rocky Mountain Forest and Range Experiment Station publication staff. We appreciate their patience during the extended publication process. 

Administrative support is essential for the success of all 

projects, especially those of substantial proportion. USDA 

Forest Service administrative support for this significant 

endeavor was provided by Dr. Barbara C. Weber (Associate Deputy Chief for Research, USDA Forest Service), Dr. 

Calvin F. Bey (Director, Forest Management Research, 

USDA Forest Service, retired), Dr. J. Robert Bridges (Staff 

Research Entomologist, Forest Insect and Disease Research), Dr. Denver P. Burns (Director, Rocky Mountain 

Forest and Range Experiment Station [RMFRES]), Dr. Brian 

M. Kent (Assistant Director for Research, RMFRES), Dr. 

Michele M. Schoeneberger (Project Leader, RM-4551, 

RMFRES), and Mr. Lane G. Eskew (Supervisory Technical 

Publications Editor, RMFRES). We appreciate the administrative support from these people that helped this project 

proceed during difficult times. 

The diligent perseverance of several people provided 

the driving force behind this publication and was instrumental in improving overall quality. The proficient efforts 

of Richard C. Carman were especially outstanding. Richard maintained general organization in the multi-step process, conducted correspondence, performed preliminary 

technical editing, prepared the index, and sustained the 

voluminous records. Special recognition is extended to 

Madelyn C. Dillon, who conducted the primary technical 

editing and ensured continued progress with her devoted 

vigilance and persistence. Karen Mora helped to produce 

this publication's high-quality visual information and professional layout. Joyce Patterson designed and created the 

book's aesthetically pleasing cover art. Rudy M. King provided the biometrics review of all chapters. Loretta J. 

Ulibarri with the assistance of Derald L. Dunagan, and 

Sharon M. Powers (U.S. Geological Survey, Water Resources Division) completed the indexing process, a task 

of monumental proportions. We are grateful to all these 

people for their valuable contributions. 

We especially thank our friends and colleagues for their 

overall assistance, and hope that this publication will serve 

as a basis for future research with Populus and other tree 

genera. 

Ned B. Klopfenstein 

Young Woo Chun 

Mee-Sook Kim 

M. Raj Ahuja 

USDA Forest Service Gen. Tech. Rep. AM·GTA·297. 1997. 
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Introduction 

Sentinels of the Prairie Speak (by Populus Species) 

Harold S. McNabb, Jr. 

This chapter is dedicated to the late Dr. Marie-France 

Michel, Station d' Ameliora tion des Arbres Forestiers, 

INRA, Centre de Recherches d'Orleans, Ardon - 45160 

Olivet, France. Dr. Michel bridged the old and the new, 

first researching the crown gall disease of poplar in nurseries, then helping to identify strains of the causal agent, 

Agrobacterium tumefaciens, which were ideal for foreign 

gene transfer. 

As we approach the 21st century, our American Indian 

friends should be proud of us poplars. As the research 

writings in the following pages show, we are the model 

system for new scientific advances in woody plant biology. We continue to be the "guinea pig" of forest-tree breeding (Pauley 1949). This distinction means more than the 

mere fact tha t human s ca n easily ma nipulate ou r 

germp lasm to meet their needs. The following is our a ttempt to convey the mystique poplars have possessed in 

recent human history. 

Although we resemble our ancestors in the Salicaceae 

from approximately 58 million years ago, we are comprised 

of around 29 different species (Eckenwa lder 1996). Poplars were selected as the symbol of liberty during the 

French Revolution of 1789, later becoming the banner of 

the resulting new republic (Tucker 1989). During the 18th 

and 19th centuries, poplars contributed to the overa ll 

beauty, stability, and economy of rural life. Our wood was 

important to the everyd ay Jives of people; shoes in the lowland a reas of western Europe, fuel and building material 

for rural areas, and later, matches that fueled specialized 

industrial development. Poplars were also part of an established economic system that designated b oundaries 

between landowners and tenants. Mone t's famous popJar series from the 1890s illustrate the significance of popJars to p eople of that period. Our importance is conveyed 

to subsequent socie ties as p eople continue to enjoy the 

spirituality of these works of art (Tucker 1989). 

Monet's tall, slender images of poplars suggests an early 

selection of Populus x euramericana. A number of us were 

first recognized in 1775 as "hybrids spontanes" between 

the recently imported P. deltoides and the European na tive 

P. nigra (Muhle Larsen 1960). Then, we were called black 

Italian poplars (P. serotina Hartig). The hybrid name P. x 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

canadensis was introduced in 1795 by Mi:inch (Pauley 1949). 

Members of these early spontaneous hybrids are used by 

our human friends throughout the world and constitute a 

vast network of our poplar clan today. 

Many of our natural hybrids were identified and used 

during the 19th century. However, in the beginning of the 

20th century, artificial breeding was introduced at Kew 

Gardens when Professor Henry fertilized flowers of P. 

angulata with pollen from P. trichocarpa. The resulting progeny were subsequently named P. generosa (Muhle Larsen 

1960). Later, many breeding p rograms were initiated with 

our species. For example, the Oxford Paper Co. in the 

northeastern United States in 1924 with A. B. Stout and 

E. J. Schreiner, C. Syrach Larsen in Denmark in 1926, 

P. Guinier in France in 1926, and C. Heimburger and his 

colleagues in Canada in 1930. Probably the most intensive 

breeding efforts occu rred in Italy where the Institu te of 

Poplar Culture at Casale Monferrato was founded in 1948 

by Professor G. Piccarolo. A similar insti tute was established in 1949 by the l'Union Allumettiere, S.A. (U AL), a 

match producing group, at Geraardsbergen (Grammont), 

Belgium. C. Muhle Larsen came from Denmark to head 

this new Belgium institute . 

Vic Steenackers, recently retired head of the Belgian laboratory, remarked once while showing colleagues his famous female poplar parents, "My supervisors always 

wanted to know when I would begin biotechnology research?" My answer was, "What do you think I have been 

doing d uring my career? Isn't breeding a form of biotechnology?" Although the modern biotechnology tools enable the gene ticist to quickly a nd compreh ensive ly 

understand germplasm questions, early poplar selection, 

b reeding, and basic genetic studies p roduced valuable 

practical and sc ientific benefits and were the basis of 

today's work. Current use of the word biotechnology implies use of molecular tools, which was absent from early 

poplar selection, breeding, and basic genetic studies. 

We poplars believe that molecular tools will help alleviate many problems, such as disease susceptibility and insect injury, with establishmen t, maintenance, and use of 

our productive systems. Our interpreter, H. S. McNabb, 

Jr., and his colleagues believe that host resistance provides 

the fundamental basis of management stra tegies for these 
damaging agents. Breeding programs with our species 

have recognized this principle and have used pest resistance as a major selection criteria. Consequently, people 

look to genetic engineering and gene mapping as tools to 

develop resistance in our poplar descendants. Molecular 

tools should also reduce the time needed by breeders to 

produce individuals with the desired traits. 

In early genetic engineering discussions about foreign, 

pest-resistance genes, our interpreter expressed concern 

·about using genes that could promote higher applications 

of silvicultural pesticides (e.g., herbicide tolerance genes) 

(Fillatti et al. 1987) or genes that coded for constitutive internal production of pesticides (e.g., the Bt endotoxin genes) 

(Raffa 1989). Based on their interactions with insects and 

pathogens, resistant (R) genes can be classified as either 

subtle or radical (Klopfenstein et al. 1992; McNabb et al. 

1990). For example, subtleR genes (e.g., proteinase inhibitor genes) are generally nonspecific and may reduce growth 

and/ or reproduction of pests, making them more susceptible to parasites, predators, and adverse environments. 

Alternatively, radical R genes (e.g., Bt endotoxin genes) are 

typically pest-specific and pesticidal in their mode of action. Misuse of these radical I specific genes can place high 

selection pressure on pest populations; whereas, subtle I 

nonspecific genes are less likely to be rapidly overcome by 

shifts in pest populations. 

Attempts to reduce selection pressure has fostered interest in wound-inducible constructs such as the Proteinase Inhibitor II (PIN2) gene. Such constructs were 

used in genetic engineering research with three of our 

hybrid relatives (Chun et al. 1988; Heuchelin et al. 1997; 

Kang et al. 1997; Klopfenstein et al. 1989, 1991, 1997) 

and were tested in an early field planting of transgenic 

poplar in July 1989 (Klopfenstein et al. 1991; McNabb 

et al. 1991). However, questions remain: Do subtle reductions in pest populations significantly diminish the 

selection pressure on pests? Would subtle reduction be 

sufficient to manage these pest populations below economic loss levels? The age-old questions remain about 

the proper planting design for pest resistant poplars in 

woody crop systems. 

Although we poplars believe that pest problems are 

potentially the most serious, molecular techniques can also 

help in other areas. An intriguing topic concerns competition among ourselves. The phytochrome system has been 

shown to be associated with a "communication mechanism" among adjacent individuals (Smith 1995). Poplars 

"talk" to each other. In competition with each other, we 

compensate by favoring height growth instead of diameter growth. But, if this effect was mitigated, you humans 

might achieve greater biomass production from us. Genetic 

modification of the phytochrome recognition of infra-red 

reflectance could change this competitive growth response. 

The potential importance of this research area has 

2 

prompted the recent establishment of an issue team at Iowa 

State University led by R. B. Hall. People continue to unravel our secrets. 

This book addresses the multi-faceted biological studies that are inspired and challenged by molecular tools. 

Use of these tools wil.l rapidly convey greater understanding about us poplars, our relatives, and our role in the 

diverse natural ecosystems in which we reside. Concurrently, our natural ecosystems can be preserved when 

humans develop special needs and artificial systems, such 

as fiber farms, for us. However, appropriate precautions 

are needed to prevent pollution of our native germplasm 

via nonindigenous seed and pollen. As more molecular 

techniques are applied to poplars, the resulting information will become a vital part of our genetic and breeding 

programs around the world. This new biology does not 

exist in a vacuum. If people wish to optimize poplar 

potential for sustainable growth in these artificial systems, future research must be integrated into system 

development. This publication attempts to promote this 

integration. 

This book is divided into 5 general sections each containing chapters on related topics. The initial section discusses what has been achieved with in vitro cultures of 

our poplar relatives including micropropagation, somatic 

embryogenesis, protoplast culture, somaclonal variation, 

and germplasm preservation. The second section concerns 

methods of genetic engineering and evaluation of 

transgene expression. Poplar researchers have included 

topics on genetic marker technologies and molecular characterization of Populus in the third section. Methods to 

improve our resistance to insects, pathogens, and air pollution are addressed in the fourth section. We find the fifth 

section most interesting. These chapters include the biotechnological applications of modification of our wood 

properties, alteration of our flowering processes, integration of molecular techniques into breeding programs, the 

commercialization of propagule production, bioethics, and 

economic considerations. We speak for all members of our 

Populus genus when we say that this new treatment of 

poplar biology is a welcome event. Humans have honored 

us as woody plant leaders in the basic biological knowledge explosion. 

This introductory chapter closes by asking the following questions: With these new techniques, will humans be 

able to determine our gender before our reproductive structures develop? At the turn of the 20th century, when our 

ancestors along the Platte River were cut, the loggers always harvested the female trees because they believed that 

the wood was stronger. Is this true? And, if so, why? As 

humans unravel more and more of our secrets, will they 

have the same reverence for the natural systems as the 

American Indian people possessed? Will we remain the 

"Sentinels of the Prairie?" 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Literature Cited 

Chun, Y.W.; Klopfenstein, N.B.; McNabb, H.S., Jr.; Hall, R.B. 

1988. Transformation of Populus species by an 

Agrobacterium binary vector systems. J. Korean For. Soc. 

77: 199-207. 

Eckenwalder, J.E. 1996. Systematics and evolution of 

Populus. In: Stettler, R.F.; Bradshaw, H.D., Jr.; Heilman, 

P.E.; Hinckley, T.M., eds. Biology of Populus and its implications for management and conservation. Ottawa, 

Ontario, Canada: NRC Research Press: 7-32. Chapter 1. 

Fillatti, J.J.; Sellmer, J.; McCown, B.; Haissig, B.; Comai, L. 

1987. Agrobacterium mediated transformation and regeneration of Populus. Mol. & Gen. Genet. 206: 192-199. 

Heuchelin, S.A.; McNabb, H.S., Jr.; Klopfenstein, N.B. 1997. 

Agrobacterium-mediated transformation of Populus x 

euramericana 'Ogy' using the chimeric CaMV 35S-pin2 

gene fusion. Canadian Journal of Forest Research. in 

press. 

Kang, H.; Hart, E.R.; Hall, R.B.; Heuchelin, S.A.; McNabb, 

H.S., Jr.; Mize, C.W. 1997. Transgenic Populus: In vitro 

screening for resistance to cottonwood leaf beetle (Coleoptera: Chrysomelidae). Canadian Journal of Forest 

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Klopfenstein, N.B.; Allen, K.K.; Avila, F.J.; Heuchelin, S.A.; 

Martinez, J.; Carman, R.C.; Hall, R.B.; Hart, E.R.; 

McNabb, H.S., Jr. 1997. Proteinase inhibitor II gene in 

transgenic poplar: Chemical_ and biological assays. Biomass and Bioenergy. in press. 

Klopfenstein, N.B.; McNabb, H.S., Jr.; Hart, E.R.; Hall, R.B.; 

Hanna, R.D.; Heuchelin, S.A.; Allen, K.K.; Shi, N.-Q.; 

Thornburg, R.W. 1992. Molecular approaches toward 

understanding and improving defense systems of 

Populus. In: Hall, R.B.; Hanna, R.D.; Nyong'o, R.N., eds. 

Proceedings of the International Energy Agency 1991 

joint meeting of the task V activity groups on exchange 

of genetic material, pest I disease management, and joint 

trials of Alnus, Populus, and Salix; 1991 August 22-27; 

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Ames, IA, U.S.A. Ames, IA, U.S.A.: Iowa State University Printing Service: 79-91. 

Klopfenstein, N.B.; McNabb, H.S., Jr.; Thornburg, R.W.; 

Hall, R.B.; Hart, E.R. 1989. Insertion and expression of 

foreign genes in aspen hybrids. In: Abstract booklet, 

Aspen symposium '89. 1989 July 25-27; Duluth, MN, 

U.S.A. Abstract No. 8. 

Klopfenstein, N.B.; Shi, N.-Q.; Kernan, A.; McNabb, H.S., 

Jr.; Hall, R.B.; Hart, E.R.; Thornburg, R.W. 1991. A 

transgenic Populus hybrid expresses a wound-inducible 

potato proteinase inhibitor II-CAT gene fusion. Can. J. 

Forest Res. 21: 1321-1328. 

McNabb, H.S., Jr.; Klopfenstein, N.B.; Hanna, R.D.; Hall, 

R.B.; Hart, E.R.; Heuchelin, S.A.; Allen, K.K.; Thornburg, 

R.W. 1991. Afield trial of transgenic hybrid poplar trees: 

Establishment and growth through the second season. 

In: MacKenzie, D.R.; Henry, S.C., eds. Biological monitoring of genetically engineered plants and mcrobes. 

Bethesda, MD, U.S.A.: Agricultural Research Institute: 

155-159. 

McNabb, H.S., Jr.; Thornburg, R.W.; Heuchelin, S.A.; 

Klopfenstein, N.B. 1990. Insect and disease resistance 

genes. In: Proc. XIX world congress, International Union 

of Forestry Research Organizations, Vol. 3, Division 2, 

Forest plants and forest protection. Montreal, Canada: 

148. Abstract. 

Muhle Larsen, C. 1960. L' Amelioration du peuplier par voie 

genetique. Bull. de Ia Societe Royale Forestiere de 

Belgique. March-Apri11960. 48. 

Pauley, ScottS. 1949. Forest-Tree Genetics Research: Populus 

L. Economic Botany. 3: 299-330. 

Raffa, K.F. 1989. Genetic engineering of trees to enhance 

resistance to insects. BioScience. 39: 524-534. 

Smith, H. 1995. Physiological and ecological function 

within the phytochrome family. Annual Review of 

Plant Physiology and Molecular Biology. 46: 289-

315. 

Tucker, Paul Hayes. 1989. Monet in the '90s - The series 

paintings. Museum of Fine Arts, Boston. New Haven, 

CT, U.S.A.: Yale University Press: 114-151. 

3 
Section I In Vitro Culture 
Chapter 1 

Poplar Shoot Cultures: Their Generation and 

Use in Biotechnology1 

Brent H. McCown 

Introduction 

Although most biotechnologists would welcome an opportunity to transform a plant, such as poplar, without resorting to in vitro techniques (microculture), currently 

microculture is central to any genetic engineering protocol commonly used for plants. Functional microculture 

methods are more essential to tree biotechnology than to 

annual crop biotechnology. Unlike annual crops where 

seed-based technologies can be used for various aspects 

of biotechnology, many tree selections are don ally propagated. Thus, access to uniform and responsive tissues can 

most readily be achieved only through in vitro culture. Although the need for microculture is highest in tree biotechnology, optimizing such procedures for wood y 

perennials is among the most challenging of any in vitro 

work. 

Fortunately, a microculture tool that is relatively easy to 

perfect and is useful for various aspects of tree biotechnology is shoot culture (figure 1A). In this chapter, a shoot 

culture is defined as an in vitro culture that: 1) is derived 

from shoot explants that contain at least 1 preformed meristem (shoot-tip or node); new shoots are derived solely 

from preformed meristems (apical or nodal) and adventitious meristem generation is avoided; and 2) remains actively growing through most or all of the culture cycle; the 

culture can be maintained indefinitely by subculturing 

shoot explants. Since poplar has become the model species for tree biotechnology, detailed aspects of poplar microculture have been extensively reviewed (Ahuja 1987; 

Chun 1993; Douglas 1986; Ernst 1993). This chapter examines the various uses of shoot cultures to complement tree 

' Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 326 p. 

biotechnological efforts and summarizes major factors and 

challenges involved in establishing shoot cultures of trees, 

especially poplar. 

Use of Shoot Cultures in 

Tree Biotechnology 

The responsiveness, stability, and reproducibility of 

shoot cultures are particularly suitable for tree biotechnology. However, the complex physiological growth cycles 

of woody perennials is a major limiting factor. The phase 

change and mid-seasonal growth cycles are of interest. The 

vegetative life cycle is exemplified by strong juvenile and 

adult phases, the former is typified by rapid vegetative 

growth and the latter by flowering (Hackett 1987). As a 

woody plant progresses through the juvenile and into the 

adult phase, its tissue responsiveness to microculture manipulation usually decreases markedly (Bonga 1987; 

Francelot et al. 1987; Hackett 1987). Strong physiological 

changes accompany seasonal growth cycles (e.g., spring 

flush, bud set, and dormancy) and confound this progressive life cycle change. For microculture, the most responsive tissues are usually obtained from the spring flush 

growth. Shoot cultures of trees are a powerful research tool 

because, once established, they suspend these progressive 

changes in a more or less permanent condition equivalent 

to the juvenile phase and the spring flush of growth. Thus, 

a well-established shoot culture will provide a tissue source 

that typically is more responsive to various biotechnological manipulations than most other sources, excluding embryonic materials that are usually unsuitable for clonal 

crops. 

Besides providing responsive tissues, shoot cultures stop 

progression through life and seasonal cycles. Plant shoot 

growth is essentially suspended while plants are maintained as shoot cultures. For example, shoot cultures of a 

birch clone (Betula populifolia cv. 'Whitespire') and a poplar clone (Populus tremula cv. 'Erecta') have been maintained 

continuously for more than 15 years without noticeable 

5 
Section 1 In Vitro Culture 

6 

Figure 1 A. Steps in establishing a shoot culture of a Populus alba hybrid clone. Nodal 

and tip explants are taken from actively growing shoots, sterilized, and 

placed in culture. New shoot growth is rapidly subcultured until uniform and 

continuous growth is obtained. 

Figure 1 B. Microcuttings (center) are harvested from mature shoot cultures and rooted/ 

acclimated ex vitro to produce young plants for testing and evaluation. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
changes in their shoot growth characteristics (Deborah D. 

McCown, Knight Hollow Nursery, Middleton, WI, USA, 

personal communication). Plants derived from these cultures appear to begin their ex vitro growth at the same lifecycle stage (i.e., as a seedling-like plant). Thus, shoot 

cultures can supply tissues of relatively uniform and reproducible physiology throughout the year. 

A second aspect of stability and reproducibility is genetics. Because shoot culture depends on growth from preformed meristems (apical or nodal) of subcultured 

explants, any single cell mutations and other aberrations 

rarely develop into aberrant shoots. Mutated shoots should 

appear at a rate approximately equal to the rate that such 

aberrant shoots appear in source plants grown ex vitro. 

Some plant genotypes, especially those that are chimeric 

in character, may be inherently genetically unstable, 

whether grown as shoot cultures or in the field. However, 

a vast majority of plants are genetically stable when maintained properly (i.e., without adventitious shoot generation) as shoot cultures. For example, no aberrant shoots 

have been seen in the long-term shoot cultures of the poplar and birch clones mentioned previously. 

Because of their responsiveness, stability, and reproducibility, shoot cultures are an excellent tissue source for various in vitro manipulations. For poplar, shoot cultures 

provide useful source tissues to establish cell I callus I nodule cultures (McCown et al. 1988), protoplast cultures 

(Chun 1985; McCown 1985; Russell and McCown 1986; 

Smith and McCown 1983), and genetic engineering. Tissues from shoot cultures were proven adaptable to vector 

(Confalonieri et al. 1994; DeBlock 1990; Fillatti et al1987; 

Riemenschneider and Haissig 1991) and particle-bombardment transformation of Populus (McCown et al. 1991). 

Conducting routine assays can be hampered by complex secondary compounds (e.g., phenolics or tannins) 

often present in the tissues of many woody perennials. 

Protein isolations, DNA extractions, or common assays of 

gene activity (e.g., the reporter gene GUS) (Vainstein et al. 

1993) are frequently difficult with woody perennials. Culture-derived shoots usually contain few interfering compounds and often provide the best source tissue for such 

assays (Francis 1996). With at least some poplar genotypes, 

DNA for molecular analyses is more readily obtained from 

shoot cultures than greenhouse- or field-grown leaves 

(Francis 1996). However, unlike many other woody plants, 

there were no compounds interfering with the GUS reporter gene assays detected in poplar leaf tissues, even in 

tissues collected from field plants (Francis 1996). 

Shoot cultures also provide the basis for the most widely 

used method of micropropaga tion in commerce today. 

Shoot cultures provide uniform microcuttings that can be 

rooted and acclimated (figure 1B) to produce liners that 

are handled like seedlings in container and field plantings 

(Douglas 1986). Micropropagation is particularly useful 

to provide a multitude of uniform plants in a relatively 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Poplar Shoot Cultures: Their Generation and Use in Biotechnology 

short time for use in biotechnological tests. Transgene expression in poplar was tested under laboratory conditions 

(Confalonieri et al. 1994; Francis 1996), in growth chambers/ greenhouses (McCown et al. 1991; Riemenschneider 

and Haissig 1991; Robison et al. 1994), and in the field 

(Kleiner et al. 1995) using shoot cultures directly or plants 

derived from shoot cultures. 

Finally, shoot cultures provide a convenient way to store 

selections under evaluation. Cultures can be continuously 

subcultured and I or maintained under cold storage conditions (Chun 1993). We have routinely maintained more 

than 100 transformants as shoot cultures, which preserves 

these unique genotypes and provides a convenient "on 

demand" source of new plants for further testing or distribution. 

Generation of Shoot Cultures 

As previously described (McCown and McCown 1987), 

new isolations from most woody perennials pass through 

an in vitro stage called stabilization before reproducible 

and uniform shoot growth is achieved. Physiological processes of the stabilization phase are not well understood; 

however, rejuvenation apparently is involved (reviewed 

in McCown 1986; for poplar example, see Whitehead and 

Giles 1977). Stabilized shoot cultures are achieved most 

rapidly when the explant sources are juvenile materials 

such as collar shoots, adventitious shoots, or rejuvenated 

plants (Hartmann et al. 1990). However, gradual rejuvenation often can be achieved by repeated subculturing of 

new shoots (Mullins 1987). 

Once stabilized, the growth and multiplication rate of 

the shoot cultures can be optimized in the production phase 

(McCown and McCown 1987). Shoots can be multiplied 

by either cytokinin stimulation of axillary bud growth, 

often in conjunction with the loss of apical dominance 

(shoot tip removal), or by manually dividing a shoot explant into its component nodes during subculture. Both 

methods are useful with poplars (e.g., Chun 1993; Sellmer 

et al. 1989). 

With Populus, the optimal growth and multiplication regime usually depends on the specific genotype. Although 

many specific differences and variations are observed, 3 

general groups of poplars can be discerned from the literature. 

1. Many clones, particularly those in the Leuce (currently termed Populus) section can be successfully 

cultured as shoot cultures grown on standard MS 

medium (Murashige and Skoog 1962) supplemented 

with the cytokinin benzyladenine (Sellmer et al. 

1989). Auxins are usually not required. 

7 
Section I In Vitro Culture 

2. Some clones, especially those of P. tremula and P. 

tremuloides species/hybrids, often perform better as 

shoot cultures on media with a lower salt formulation, such as Woody Plant Media (WPM) (Lloyd and 

McCown 1980; McCown and Sellmer 1987) or its 

derivatives (Ahuja 1987; Sellmer et al. 1989}, supplemented with benzyladenine. Again, auxins are usually not required. 

3. Other clones, especially those belonging to the 

Aigeiros and Tacamahaca sections, do not perform 

particularly vigorously as shoot cultures on any medium so far defined. Some selections may not be amenable to long-term maintenance as shoot cultures 

(SeUmer et al. 1989). For others, acceptable shoot cultures may be generated using more complex cultural 

regimes. MS medium supplement~d with cytoki~ins 

and auxins may be helpful (Whttehead and Giles 

1977). For some selections, benzyladenine may be 

phytotoxic, but naturally-occurring cytokinins~ such 

as zeatin, may be stimulatory (Ernst 1993). Multistage 

protocols separating bud stimulation and elongation, 

each with a separate medium requirement, was useful in other work (Chun 1993; Ernst 1993; Whitehead 

and Giles 1977). DeBlock (1990) used a buffered medium supplemented with calcium gluconate to overcome shoot-tip necrosis (Sha et al. 1985) problems 

associated with some poplar clones. 

Conclusion 

Although not a comprehensive remedy fo~ the difficu~­

ties associated with working with trees for biotechnological research, shoot cultures are a major and often essential 

tool. Shoot cultures provide tissues to begin manipulations 

and offer an effective avenue for moving plant materials 

from culture to testing or production. For a program beginning work on a selection of poplar, establishing t~e 

genotype in shoot culture will sensitiz.e researchers to Its 

idiosyncrasies in the microculture environment. 

Acknowledgments 

This work was supported by the University of Wisconsin-Madison College of Agricultural and Life Sciences, 

HATCH, the UW-Graduate School, and the Plant Biotechnology Consortium. 

8 

Literature Cited 

Ahuja, M.R. 1987. In vitro propagation of poplar and aspen. In: Bonga, J.M.; Durzan, D.J., eds. Cell and tissue 

culture in forestry, Vol. 3. Case histories: Gymnosperms, 

angiosperms, and palms. Dordrecht, The Netherlands: 

Martinus Nijhoff Publishers: 207-223. 

Bonga, J.M. 1987. Clonal propagation of mature trees: 

· Problems and possible solutions. In: Bonga, J.M.; 

Durzan, D.J., eds. Cell and tissue culture in forestry, 

Vol. 1. General principles and biotechnology. 

Dordrecht, The Netherlands: Martinus Nijhoff Publishers: 249-271. 

Chun, Y.W. 1985. Isolation and culture of in vitro cultured 

Populus alba x P. grandidentata protoplasts. J. Korean For. 

Soc. 71:45-59. 

Chun, Y.W. 1993. Clonal propagation in non-aspen poplar 

hybrids. In: Ahuja, M.R., ed., Micropropagation of 

woody plants. Dordrecht, The Netherlands: Kluwer 

Academic Publishers: 209-222. 

Confalonieri, M.; Balestrazzi, A.; Bisoffi, S. 1994. Genetic 

transformation of Populus nigra by Agrobacterium 

tumefaciens. Plant Cell Rep. 13: 256-261. 

DeBlock, M. 1990. Factors influencing the tissue culture 

and the Agrobacterium tumefaciens-mediated transfo~ation of hybrid aspen and poplar clones. Plant Phystol. 

93: 1110-1116. 

Douglas, G.C. 1986. Poplar (Populus spp.). In: Bajaj, Y.P.S., 

ed. Biotechnology in agriculture and forestry, Trees II, 

Vol. 5. Berlin: Springer-Verlag: 300-323. 

Ernst, S.G. 1993. In vitro culture of pure species non-aspen 

poplars. In: Ahuja, M.R., ed. Micropropagation of 

woody plants. Dordrecht, The Netherlands: Kluwer 

Academic Publishers: 195-207. 

Fillatti, J.J.; Sellmer, J.; McCown, B.; Haissig, B.; Comai, L. 

1987. Agrobacterium mediated transformation and regeneration of Populus. Mol. Gen. Genet. 206: 192-199. 

Francelot, A.; Boulay, M.; Bekkaoui, F.; Fouret, Y.; 

Verschoore-Martouzet, B.; Walker, N. 1987. Rejuvenation. In: Bonga, J.M.; Durzan, D.J., eds. Cell and tissue 

culture in forestry, Vol. 1. General principles and biotechnology. Dordrecht, The Netherlands: Martinus 

Nijhoff Publishers: 232-248. 

Francis, K.E. 1996. Genetic transformation and transgene 

analysis of hybrid poplar NM6 (Populus.nigr~ x Popul.us 

maximowiczii). Madison, WI, U.S.A.: Umverstty of Wisconsin: 80 p. M.S. thesis. 

Hackett, W.P. 1987. Juvenility and maturity. In: Bonga, 

J .M.; Durzan, D.J., eds. Cell and tissue culture in forestry, Vol. 1. General principles and biotechnology. 

Dordrecht, The Netherlands: Martinus Nijhoff Publishers: 216-231. 

USDA Forest Service Gen. Tech. Rep. AM-GTA-297. 1997. 
Hartmann, H.T.; Kester, D.E.; Davies, F.T. 1990. Plant propagation: Principles and practices. Englewood Cliffs, NJ, 

U.S.A.: Prentice Hall. 647 p. 

Kleiner, K.W.; Ellis, D.O.; McCown, B.H.; Raffa, K.F. 1995. 

Field evaluation of transgenic poplar expressing a Bacillus thuringiensis cryiA(a) d-endotoxin gene against forest tent caterpillar (Lepidoptera: Lasiocampidae) and 

gypsy moth (Lepidoptera: Lymantriidae) following winter dormancy. Environ. Entom. 24: 129-135. 

Lloyd, G.; McCown, B. 1980. Commercially-feasible 

micropropagation of mountain laurel, Kalmia latifolia, 

by use of shoot-tip culture. Proc. Intern. Plant Prop. Soc. 

30:421-426. 

McCown, B.H. 1985. From gene manipulation to forest 

establishment: Shoot cultures of woody plants can be a 

central tool. TAPPI J. 68: 116-119. 

McCown, B.H. 1986. Adventitious rooting of tissue cultured plants. In: Jackson, M.B., ed. New root formation 

in plants and cuttings. Boston: Martinus Nijhoff Publishers: 289-302. 

McCown, B.H.; Zeldin, E.L.; Pinkalla, A.H.; Dedolph, R.R. 

1988. Nodule culture: A developmental pathway with 

high potential for regeneration, au tom a ted 

micropropagation and plant metabolite production 

form woody plants. In: Hanover, J.; Keathley, D.E., eds. 

Genetic manipulation of woody plants. New York: Plenum Press: 149-166. 

McCown, D.O.; McCown, B.H. 1987. North American hardwoods. In: Bonga, J.M.; Durzan, D.J., eds. Cell and tissue culture in forestry,. Vol. 3. Case histories: 

Gymnosperms, angiosperms, and palms. Dordrecht, 

The Netherlands: Martinus Nijhoff Publishers: 247-260. 

McCown, B.H.; Sellmer, J.C. 1987. General media and vessels suitable for woody plant microculture. In: Bonga, 

J.M.; Durzan, D.J., eds. Cell and tissue culture in forestry, Vol. 1. General principles and biotechnology. 

Dordrecht, The Netherlands: Martin us Nijhoff Publishers: 4-14. 

McCown, B.H.; McCabe, D.E.; Russell, D.R.; Robison, D.J.; 

Barton, K.A.; Raffa, K.R. 1991. Stable transformation of 

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Poplar Shoot Cultures: Their Generation and Use in Biotechnology 

Populus and incorporation of pest resistance by electrical discharge particle acceleration. Plant Cell Reports. 

9:590-594. 

Mullins, M.G. 1987. Propagation and genetic improvement 

of temperate fruits: the role of tissue culture. In: Somers, 

D.A., ed. Proc. VI Congress of Plant Tissue and Cell 

Culture. Minneapolis, MN, U.S.A.: Univ. of Minnesota: 

395-406. 

Murashige, T.; Skoog, F. 1962. A revised medium for rapid 

growth and bioassays with tobacco tissue cultures. 

Physiol. Plant. 15: 473-479. 

Riemenschneider, D.E.; Haissig, B.E. 1991. Producing herbicide tolerant Populus using genetic transformation 

mediated by Agrobacterium tumefaciens C58: A summary 

of recent research. In: Ahuja, M.R., ed. Biotechnology of 

woody plants. New York: Plenum Press: 247-263. 

Robison, D.J.; McCown, B.H.; Raffa, K.F. 1994. Responses 

of gypsy moth (Lepidoptera: Lymantriidae) and forest 

tent caterpillar (Lepidoptera: Lasiocampidae) to 

transgenic poplar, Populus spp., containing a Bacillus 

thuringiensis d-endotoxin gene. Environ. Entomol. 23: 

1030-1041. 

Russell, J .A.; McCown, B.H. 1986. Techniques for enhanced 

release of leaf protoplasts in Populus. Plant Cell Reports. 

5:284-287. 

Sellmer, J.C.; McCown, B.H.; Haissig, B.E. 1989. Shoot culture dynamics of six Populus clones. Tree Physiol. 5:219-

227. 

Sha, L.; McCown, B.H.; Peterson, L.A. 1985. The occurrence 

and cause of shoot-tip necrosis in shoot cultures. J. A mer. 

Soc. Hort. Sci. 110: 631-634. 

Smith, M.A.L.; McCown, B.H. 1983. A comparison of source 

tissue for protoplast isolation from three woody plant 

species. Plant Sci. Lett. 28: 149-156. 

Whitehead, H.C.M.; Giles, K.L. 1977. Rapid propagation 

of poplar by tissue culture methods. N.Z. J. For. Sci. 7: 

40-43. 

Vainstein, A.; Fischer, M.; Ziv, M. 1993. Application of reporter genes to carnation transformation. HortScience. 

28: 1122-1124. 

9 
Chapter2 

Somatic En1bryogenesis From Poplar Leaf Tissue1 

Young Goo Park and Sung Ho Son 

Introduction 

An efficient in vitro regeneration system with cell and 

tissue cultures is a prerequisite for biotechnological applications to plant improvement programs. Although 

whole plants have been regenerated by several techniques 

from various species and explants, somatic embryogenesis is one of the most powerful morphogenetic schemes. 

First reported using carrot (Steward et al. 1958}, somatic 

embryogenesis was later defined by Haccius (1978) as a 

nonsexual developmental process leading to the differentiation of zygotic embryo-like structures from somatic 

cells. Two advantages of somatic embryogenesis are that: 

1) genetically identical propagules are provided by avoiding genetic recombination that occurs during meiosis in 

sexual reproduction; and 2) it provides a fast, reliable, reproducible method for mass production. In contrast to organogenesis, somatic embryogenesis exhibits a bipolar 

structure with a closed vascular system and differentiated 

shoot and root meristems. 

Poplars, considered an economically important species 

in many countries, are usually propagated by cuttings 

and/ or root suckers. Thus, cloning techniques can propagate superior genotypes that show heterosis in the F1 progeny from artificial hybridization. For interspecific crosses, 

some species from the Leuce (currently termed Populus) 

section have superior traits such as a straight trunk and 

rapid growth Giang et al. 1980). Nevertheless, most hybrids from this section have been difficult to propagate 

asexually using standard techniques. Tissue culture systems, especially somatic embryogenesis are advantageous 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 326 p. 

10 

for application to mass cloning. Techniques developed for 

poplar may also be applied to related tree species such as 

willow (Salix spp.). 

Literature Review 

Studies on poplar tissue culture began in the early 1930s 

(Gautheret 1934). After 30 years, Mathes (1964}, Wmton 

(1968}, and Wolter (1968) have established plant regeneration methods based on callus culture. In their systems, 

shoots and roots were induced through organogenesis 

from different callus sites at different developmental 

stages. Since then, similar morphogenetic responses were 

reported by other authors (Chen and Huang 1980; Sellmer 

et al.1989; Whitehead and Giles 1977). Because of the potential applications associated with somatic embryogenesis, many studies have focused on crop, horticultural 

species, and woody perennials (Gingas and Lineberger 

1989; Tremblay 1989). 

Pioneering studies on somatic embryogenesis in poplar were reported by Michler and Bauer (1987, 1991), Park 

and Son (1988}, Cheema (1989). Somatic embryogenesis 

at relatively high frequency was obtained directly and/ 

or indirectly by leaf-tissue and cell culture of hybrid aspen (Populus alba x P. grandidentata cl. 'NC5339'). With this 

hybrid, short-term auxin treatment stimulated embryo 

maturation and eventually allowed plants to harden under greenhouse conditions (Michler and Bauer 1991). 

Cheema (1989) obtained callus and cell suspension cultures using semi-organized leaf culture from mature Himalayan poplars (P. ciliata). Similar to most other species, 

somatic embryogenesis was induced in P. ciliata by exposure to a high level of 2,4-dichlorophenoxyacetic acid (2,4-

0), followed by reduced auxin concentration. Callus 

retained its embryonic potential over a year; however, embryogenic suspension cultures lost this ability after 6 subcultures. In another study, organogenesis and somatic 

embryogenesis occurred simultaneously when punctured 

leaves of hybrid poplar (Populus nigra x P. maximowicziz) 

were cultured (Park and Son 1988). 
With Populus, embryogenic capacity has been associated with leaf-tissue explants. For most other species including woody plants, immature or mature zygotic 

embryos are commonly used as explants to initiate somatic embryogenesis (Tremblay 1989). For many woody 

plant species, immature embryos are available only once 

a year for a very short time. Using leaf tissue, especially 

that grown in vitro, for somatic embryogenesis provides 

Populus species with a substantial advantage. 

Poplar Somatic Embryogenesis 

In poplar, the developmental process, morphology, and 

physiology of somatic embryogenesis is similar to zygotic 

embryogenesis. Because reports on somatic embryogenesis of poplar are limited, summarizing a general method 

is difficult. However, based on our research and other reports, some critical steps for somatic embryogenesis in 

Populus spp. are described. The steps in somatic embryogenesis from leaf explants of poplar are: 1) explant preparation and adjustment to culture conditions; 2) induction 

of direct and/ or indirect embryogenesis; and 3) maturation and germination. 

Explant Preparation and Culture Conditions 

As donor plants, greenhouse- or field-grown plants can 

provide explants to initiate in vitro plantlets or callus. Typically, stem-node sections with or without buds are disinfested 

using 0.5 to 1 percent of sodium hypochlorite for 5 to 10 

min. Murashigeand Skoog (1962) (MS)culture medium with 

a low concentration of cytokinin (6-benzyladenine; BA), 

alone and in combination with high levels of auxin (2,4-D), 

is used to induce in vitro bud break and callus induction, 

respectively. To multiply the shoots for source material, apices are excised from shoots before subculturing on proliferation medium containing 0.88 J.lM BA. A 16-h photoperiod 

provided by cool-white, fluorescent light is used for shoot 

elongation and direct embryogenesis. Complete darkness is 

used to establish and maintain callus and embryogenic cell 

suspension cultures. Suspension cultures are routinely agitated at 100 to 120 rpm using a gyratory shaker, and subcultured at 2-week intervals. 

Direct Embryogenesis 

Isolated leaves (< 2 em in diameter) from in vitro cultured plantlets are cultured on MS medium supplemented 

with BA and 2,4-D. Leaves are further prepared by puncturing with a pin or slicing into leaf disks with a cork borer 

(1 em in diameter). Amendments such as coconut milk, 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Somatic Embryogenesis From Poplar Leaf Tissue 

malt extract, and glutamine may be used to stimulate somatic embryogenesis. In this method, direct and indirect 

embryogenesis and organogenesis can occur simultaneously. To maintain somatic embryos or embryogenic 

callus for further development, embryoids and I or embryogenic callus must be selected at an early stage. 

Indirect Embryogenesis 

Isolated embryogenic callus or embryogenic callus-derived cell suspension cultures can be used for indirect somatic embryogenesis. A simple, reliable method for indirect 

somatic embryogenesis is obtained by altering the 2,4-D 

level. A semi-solid agar medium can support further development of embryos harvested at an early developmental stage. Overall, the culture media for maintenance of 

embryogenic cell lines are similar to induction media except that plant growth regulator levels are lower. 

Maturation and Germination 

Harvested embryos require several washes in MS liquid medium without plant growth regulators. Somatic 

embryos originating directly or indirectly from poplar leaf 

cultures typically do not exhibit dormancy. Maturation of 

somatic embryos in poplar can be triggered by the changing plant growth regulators. In 2 cases, the maturation 

medium included BA, alone or combined with anaphthaleneacetic acid. Increased levels of osmoticum 

may effectively stimulate maturation. 

Limitations 

Although somatic embryogenesis was successfully 

demonstrated in Populus species, the induced somatic 

embryo is strikingly plastic in its development. Many of 

the induced embryos exhibited abnormal growth characteristics including: 1) bearing multiple secondary embryoids among the embryonic axes; 2) rapid growth of 

nonembryonic callus during embryogenesis; 3) abnormal 

cotyledons growth; and 4) growth failure of either cotyledon or root radical. This plasticity may result from the 

altered gene expression during development. Another 

problem associated with poplar embryogenesis is its low 

success rate and poor reproducibility in comparison with 

carrot. Because all stages from induction to maturation 

are very labor intensive, large-scale propagation through 

somatic embryogenesis is impractical without improving 

overall efficiency. A critical limitation is lack of knowledge on the somatic embryogenic processes of poplar. 

Continued intensive studies are needed. 

11 
Section I In Vitro Culture 

Conclusion and Prospects 

Somatic embryogenesis can be induced from leaf explants of poplar. Many factors such as basal media composition, undefined substances, amino acids, plant growth 

regulator dosage, and treatment methods influence somatic embryogenesis. Among these factors, 2,4-0 plays a 

paramount role as an auxin source. However, a phytohormone imbalance usually causes the formation of abnormal or incomplete structures. Because the role of auxins 

in embryoid formation is complex, purification and identifi~ation of various auxin receptors and understanding 

the1r molecular action will contribute to our knowledge 

of cellular differentiation mechanisms related to somatic 

embryogenesis (LoSchiavo 1995). Using leaf explants of 

poplar, the somatic embryogenic response was examined 

using inverted stereo microscopy (Michler and Bauer 1991; 

Park and Son 1988). When embryos were produced from 

cultured leaves, they appeared to arise directly or via 

callogenesis from wounded portions or from sliced leaf 

ends. 

In recent years, embryogenesis through cell suspension 

culture has provided opportunities for gene transformation by microparticle bombardment of intact cells or direct gene transfer using protoplasts. Liquid plating of 

protoplast allows stringent selection of transformed cells. 

Cell suspension culture-based somatic embryogenesis is 

also applicable to germplasm preservation and mutant selection. If successfully developed, these technologies can 

be employed in bioreactor systems for mass production. 

Atte~pts at. large-scale embryogenesis are being applied 

to vanous btoreactor systems such as air-lift, impeller, and 

hybrid types. 

Induction of somatic embryogenesis and organogenesis 

have usually occurred simultaneously, but embryoid formation efficiency was relatively low. Abnormalities in 

m?rphology and growth were evident. It is our hope that 

th1s s~mmary of somatic embryogenesis methods for poplar wtll be useful to future studies. Continued studies are 

needed to improve culture systems and for further understanding of somatic embryogenesis for application to poplar and other woody plants. 

Acknowledgments 

Part of this manuscript was reported in: Bajaj, Y.P.S., 1995. 

ed .. Biotechnology in agriculture and forestry 30. Berlin: 

Spnnger-Verlag Publication: 446-454. 

12 

Literature Cited 

Cheema, G.S. 1989. Somatic embryogenesis and plant regeneration from cell suspension and tissue culture of 

mature Himalayan poplar (Populus ciliata). Plant Cell 

Rep. 8: 124-127. 

Chen, D.; Huang, M. 1980. Culture of the apical tissue of 

Populus nigra cv. Blanc de Garonne and the variation of 

their isozymes. J. Nanjing Tech. Coli. For. Products. 3: 

104-107. 

Gautheret, R.J. 1934. Culture du tissu cambial. CR Acad. 

Sci. (Paris). 198: 2195-2196. 

Ging~s, V.M.; Lineberger, R.D. 1989. Asexual embryogenesis and plant regeneration in Quercus. Plant Cell Tiss. 

Org. Cult. 17: 191-203. 

Haccius, B. 1978. Question of unicellular origin on nonzygotic embryos in callus cultures. Phytomorphology. 

28: 74-81. 

Jiang, H.; Han, Y.; Zhao, H.; Gu, W.; Yang, C.; Fan, K.; Xing, 

X. 1980. Breeding of the superior hybrid clone 741 of 

poplar. Hebei Linye Keji. 1: 72-75. 

LoSchiavo, F. 1995. Early events in embryogenesis. In: 

Bajaj, Y.P.S., ed. Biotechnology in agriculture and forestry 30. Berlin: Springer-Verlag Publication: 20-29. 

Mathes, M.C. 1964. The culture of isolated triploid aspen 

tissue. For. Sci. 10: 35-38. 

Mi~hler, C.H.; Bauer, E.O. 1987. Somatic embryogenesis 

m plant cell culture of Populus. In vitro. Cell Oev. Bioi. 

23 {3), Partll: 46A. 

Michler, C.H.; Bauer, E.O. 1991. High frequency somatic 

e~bryogenesis from leaf tissue of Populus spp. Plant 

Set. 77: 111-118. 

Murashige, T.; Skoog, F. 1962. A revised medium for rapid 

growth and bio assays with tobacco tissue cultures. 

Physiol. Plant. 15: 473-479. 

Park, Y.G.; Son, S.H. 1988. In vitro organogenesis and somatic 

embryogenesis from punctured leaf of Populus nigra x P. 

maximowiczii. Plant Cell Tiss. Org. Cult. 15: 95-105. 

Sellmer, J.C.; McCown, B.H.; Haissig, B.E. 1989. Shoot culture 

dynamics of six Populus clones. Tree Physiol. 5: 219-227. 

Steward, F.C.; Mapes, M.O.; Mears, K. 1958. Growth and 

organized development of cultured cells II. Organization in cultures grown from freely suspended cells. 

Amer. J. Bot. 45: 705-708. 

Tremblay, F.~. 1989. Somatic embryogenesis and plantlet 

regeneration from embryos isolated from stored seeds 

of Picea glauca. Can. J. Bot. 68: 236-242. 

Whitehead, H.C.M.; Giles, K.L. 1977. Rapid propagation of poplar by tissue culture methods. N. Z. J. For. Sci. 7: 40-43. 

Winton, L.L. 1968. Plantlet formation from aspen tissue 

culture. Science. 160: 1234-1235. 

Wolter, K.E. 1968. Root and shoot initiation in aspen callus culture. Nature. 219: 503-510. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Chapter3 

Plant Regeneration Through Organogenesis in Poplar1 

Hoduck Kang and Young Woo Chun 

Introduction 

Among woody genera, Populus has been extensively 

studied as a model system for biotechnological research. 

This genus of the Salicaceae family, widely distributed 

across the Northern Temperate Zone (FAO 1980), is composed of 5 sections: Leuce (currently termed Populus), 

Aigerios, Tacamahaca, Turanga, and Leucoides (Dickman 

and Stuart 1983). Leuce is a large group that includes aspen and white poplars, which have great economic importance. Aigeiros, considered the "true poplars," include 

the cottonwoods and black poplars. Tacamahaca, containing the balsam poplars, is the largest poplar species group. 

The remaining 2 sections (Turanga and Leucoides) have 

minor economic importance. 

Populus species and hybrids are ideal for plywood and 

lumber production, and woody biomass production because of rapid growth, ease of establishment through stem 

or root cuttings, and relative ease of coppice regeneration 

(Ahuja 1987; Behrens and Melchior 1978; Hallet al. 1989; 

Herrmann and Seuthe 1982). Trees of this genus represent 

wide genetic diversity, exhibit widely ranging site requirements, and respond well to cultural input (Schreiner 1974). 

Many new, high-quality, rapidly growing poplar clones 

were developed in various breeding programs from diverse geographic regions (Hall et al. 1989). Vegetatively 

propagated Populus species and hybrid clones have been 

planted extensively in many countries (Zsuffa 1985). For 

the difficult-to-root poplar (section Leuce), tissue culture 

techniques for micro-propagation have become a primary 

alternative to more conventional propagation procedures. 

In vitro propagation techniques were applied to poplars to 

overcome limitations such as production cost and availabil1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 326 p. 

ity of specific genotypes for planting material (Ahuja 1987). 

Furthermore, as a model system for biotechnology programs, 

poplars have been intensively studied for in vitro micropropagation, genetic transformation, and gene expression. 

The objectives of this review are to summarize: 1) organogenic regeneration of poplar by adventitious bud induction 

from various explants; and 2) physiological malformation 

of in vitro plantlets, which is a fundamental problem in poplar micro-propagation. 

In Vitro Organogenesis 

Since 1980, poplars have been intensively studied for 

in vitro establishment (Chun et al. 1988). Such studies reveal that Populus tissue exhibits a high degree of developmental plasticity, similar to tobacco in the herbaceous 

species. Many in vitro cloning techniques were developed 

that provide an integral basis for biotechnological applications. Published reports on in vitro regeneration of poplars are summarized in table 1. Types of shoot regeneration 

with organs or tissues are ordinarily classified as: 1) adventitious shoot formation or 2) axillary shoot induction. 

Adventitious shoot formation is amorphous shoots originating from the various explants of leaf, internode, catkin, immature embryo, pollen, vascular or bark cambium, root, etc. 

Cultures for axillary shoot induction are composed of 

morphous shoots originating from an apical meristem or axillary bud. Apical meristems containing shoot primordia can 

produce multiple shoots. For more information on in vitro 

morphogenesis of Populus hybrids and species, refer to reviews by Ahuja (1987, 1993), Chun (1993), and Ernst (1993). 

Adventitious Shoot Organogenesis 

Through In Vitro Cultured Explants 

Adventitious poplar shoots can be induced from in vitro 

cultured leaf, internode, petiole, and root explants that 

13 
Section I /n Vitro Culture 

Table 1. Published reports on in vitro establishment with Populus species. 

Culture type Species Reference 

Leaf culture 

immature lamina discs P. ciliata Mehra and Cheema 1980 

leaf, internode, root P. alba x P. grandidentata Chun 1987 

leaf discs P. maximowiczii Michler and Bauer 1988 

x P. trichocarpa 

leaf, stem, root P. maximowiczii Kang 1991 

x P. trichocarpa 

Root culture 

bud, stem, leaf, root P. tremula Ahuja 1983 

bud, stem, leaf, root P. tremuloides Ahuja 1983 

root P. alba x P. grandidentata Son and Hall 1990 

leaf, stem, root P. x euramericana Kang 1991 

Anther culture 

anther P. glandulosa Kim et al. 1983 

anther P. deltoides Uddin et al. 1988 

Embryo culture 

immature embryo P. deltoides Kouider et al. 1984 

immature embryo P. deltoides Savka et al. 1987 

ovary, embryo P. alba Raquin et al. 1993 

ovary, embryo P. deltoides Raquin et al. 1993 

ovary, embryo P. trichocarpa Raquin et al. 1993 

ovary, embryo P. lasiocarpa Raquin et al. 1993 

ovary, embryo P. trichocarpa x P. deltoides Raquin et al. 1993 

immature ovary P. deltoides Kang and Hall 1996 

Callus culture 

callus P. tremuloides Winton 1968 

callus P. tremuloides Wolter 1968 

callus P. tremuloides Winton 1970 

callus P. tremuloides Wolter and Gordon 1975 

callus P. tremuloides Noh and Minocha 1986 

callus P. maximowiczii Ostry and Skilling 1988 

x P. trichocarpa 

callus P. nigra var. charkowiensis Ostry and Skilling 1988 

x P. nigra var. caudina 

callus P. nigra var. betulifolia Ostry and Skilling 1988 

x P. trichocarpa 

callus P. nigra var. charkowiensis Ostry and Skilling 1988 

x P. deltoides 

callus P. nigra var. betulifolia Ostry and Skilling 1988 

x P. nigra 

callus P. nigra x P. maximowiczii Park and Son 1988 

cell suspension P. ciliata Cheema 1989 

callus P. deltoides Coleman and Ernst 1990 

callus P. alba x P. grandidentata Michler and Bauer 1991 

normally never reproduce vegetatively in vivo (Ahuja 1993; 

Chun 1993; Ernst 1993). Most aspen, cottonwood, and their 

hybrids produce adventitious shoots in Murashige and 

Skoog (MS) (Murashige and Skoog 1962) media or Woody 

Plant Media (WPM) (Lloyd and McCown 1980) supplemented with 0.2 to 0.5 mg/1, 6-benzyladenine (BA) or 2.0 

mg/1 zeatin as a cytokinin source. Many Populus species 

were successfully regenerated through adventitious shoot 

14 

induction from cultured explants including node, internode, leaf, and root segment (Agrawal and Gupta 1991; 

Chun 1990; Coleman and Ernst 1990; Douglas 1984; Kim 

et al. 1994a, 1994b; Nadel et al. 1992; Rutledge and Douglas 1988). 

Adventitious regeneration in vitro may generate a much 

higher rate of shoot production than proliferation of axillary shoots. Commercial-scale regeneration systems were 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 
established from leaf and root cultures of European aspen (P. tremula), quaking aspen (P. tremuloides), and their 

hybrids (Ahuja 1984). Plant regeneration from stem internodes was accomplished in P. trichocarpa x P. tacamahaca 

(Douglas 1984). Park and Son (1988) demonstrated that 

an average of 178 shoots was directly produced from a 

punctured leaf of P. nigra x P. maximowiczii after 6-weeks 

culture. 

Morphogenetic responses of explants from in vitro 

cultured poplar plantlets depend on the explant source 

and the combination of exogenously applied plant 

growth regulators. Chun (1990) demonstrated that 

abaxial side culture of whole-leaf explants was best to 

induce adventitious shoot buds from a hybrid aspen 

(P. alba x P. grandidentata) plantlet. Kim et al. (1994a) 

showed that the form and concentration of nitrogen 

sources (ammonium and nitrate) and combination of 

BA with naphthaleneacetic acid (NAA) greatly affect 

adventitious shoot induction from leaf-explant cultures 

of P. davidiana. 

Kang and Hall (1996b) successfully established a 

nodal culture system to maximize shoot production of 

a cottonwood hybrid (P. x euramericana). They used 4 

explants: node, internode, node with the axillary bud 

excised, and reutilized stem. The shoot proliferation 

capacity of nodal explants and modified explants was 

compared, and the origin of multiple shoots was investigated. For these explants, most shoots were derived 

from the axillary meristems. Shoots also originated from 

the vascular cambium and qccasionally from the lenticels (figures 1-4 to 1-6). A conventional method of directly using nodal explants produced a mean (± 

standard error) of 5.7 ± 0.6 shoots by treatment with 

2.0 mg/1 zeatin. However, shoot production from internodes and modified nodes was better using 0.5 mg I I 

BA with 2.0 mg/1 zeatin, which produced averages of 

21.7 ± 1.1 and 29.0 ± 0.9 shoots, respectively (figures 1-1 

to 1-3). Shoot formation from lenticels was examined 

in thin sections using bright-field microscopy, and intact specimens were observed with a stereo microscope 

(figures 1-8 and 1-9). 

For adventitious shoot production from in vitro cultured leaves of hybrid aspen clones (P. alba x P. 

grandidentata cv. 'Crandon' and cv. 'Hansen'), thidiazuron (TDZ) and NAA test combinations were more 

effective than BA and NAA test combinations or TDZ 

test concentrations without NAA (Kim et al. 1994b). 

Among 40 combinations of TDZ and NAA tested, 0.05 

J.lM TDZ with 1.0 J.LM NAA produced the most adventitious shoots; 18 shoots per leaf explant for 'Crandon' 

and 15 shoots per leaf explant for 'Hansen.' Increasing 

TDZ concentrations (0.1, 0.2, 0.5, 1.0, and 2.0 J!M) had 

varying effects on the frequency of regeneration and 

vitrification of the 2 hybrid aspen clones (Kim et al. 

1994b). Significant clonal differences were observed for 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Plant Regeneration Through Organogenesis in Poplar 

the number of adventitious shoot regenerated. After 2 

to 3 weeks on TDZ/NAA regeneration media, adventitious shoots initiated on the proximal cut surface of the 

petiole and near punctured areas on the leaf surface. 

On BA/NAA regeneration media, most adventitious 

shoots formed at the proximal cut surface of the petiole, while only a few formed on the cultured leaf surface. 

Shoot proliferation occurred on cultured root segments 

of quaking aspen, hybrid aspen (P. alba x P. grandidentata), 

and hybrid cottonwood (P. x euramericana) (Ahuja 1983; 

Chun 1990; Kang 1991; Son and Hall 1990). Using 

'Crandon' and 'Hansen' clones of P. alba x P. grandidentata, 

Son and Hall (1990) tested root segments of various ages 

for shoot regeneration. They reported that root tip containing explants from 60-day-old roots produced the most 

shoots (an average of 111 shoots per root segment for 

'Crandon' and 98 for 'Hansen') on WPM supplemented 

with 4.8 mg I l or 3.0 mg /1 zeatin, respectively. Clonal 

propagation was used with root segments of hybrid cottonwood (P. x euramericana) clones (Kang 1991). Among 4 

clones ('Canada Blanc,' 'Eugenei,' '1-45/51,' and 'Wisconsin #5'), the most shoots (17.60 ± 8.96) were induced from 

'Canada Blanc' explants on WPM with either a low concentration (0.2 mg/1) of BA or a high concentration (5.0 

mg/1) of zeatin. 

Generally, morphogenetic responses of in vitro cultured 

explants depend on the species, explant source, and combination of exogenously applied plant growth regulators. 

Research efforts to develop more generalized organogenesis techniques for explant culture of poplar species and 

hybrids include: 1) applications of physical treatment, such 

as the leaf-surface puncturing technique of Park and Son 

(1988); 2) the effects of various plant growth regulator combinations, such as 2,4-dichlorophenoxyacetic acid (2,4-D) 

vs. NAA; and 3) optimization of culture conditions for 

explant source material. 

Adventitious Shoot Induction 

From Anther, Ovule, Embryo, 

and Catkin Culture 

Haploid plantlets were produced from anther cultures 

of P. glandulosa, P. deltoides, and P. maximowiczii (Kim et al. 

1983; Stoehr and Zsuffa 1990; Uddin et al. 1988). In a breeding program, embryo cultures can potentially rescue aborting embryos of interspecific hybrids. Several cultures were 

reported with ovules or embryos of Populus species 

(Kouider et al. 1984; Raquin et al. 1993; Savka et al. 1987). 

Recently, Kang and Hall (1996a) reported shoot formation 

15 
Ol 

<0 

<0 

-.J 

4 

Figure 1. Shoot proliferation from different explants of the 'Ogy' clone (Populus x euramericana). 1-1 ) Multiple shoots from the 

modified node with the axillary bud excised (2.0 mg/1 zeatin), reutilized stem (0.5 mg/1 BA), internode (0.5 mg/1 BA), a

1-2) Shoot formation from the internodal explants; 1-3) Shoot initiation from the vascular cambium of a stem internode

tion and elongation from lenticel. 1-7) Shoot induction with a high concentration of zeatin (5.0 mg/1) from a modified n

1-9) Longitudinal section of a partial lenticel with a shoot. l=lenticel (Kang and Hall 1996a). 
c.o 

c.o 

-...1 

Figure 2. Shoot formation from immature ovules of Populus deltoides. 2-1) Shoot initiation from immature embryo and expand

cates the multiple shoots from the cotyledon. 2-2 and 2-3) Shoot elongation of multiple shoots in half-strength mediu

2-4) Plants after transplanting to soil mix and 6-weeks growth in a greenhouse (Kang and Hall 1996b) 
Figure 3. Shoot vitrification from stem nodal cultures of Populus alba x P. grandidentata, 'Crandon' clone. 3-1) Normal shoot 

3-2) Vitrified shoots induced from an axillary bud. 3-3) Shoot elongation on the half-strength Murashige and Skoog 

stomate of nonvitrified leaf. 3-5) Abnormal stomate of vitrified leaf (Kang and Hall 1996c). 
from developing ovules cultured on vitamin-supplemented WPM containing BA and zeatin as cytokinins. 

Multiple shoots were obtained from immature ovules collected 20 days after pollination. Generally, zeatin was better than BA for inducing multiple shoots. The highest mean 

number (56.1 ± 7.4) of shoots was from immature ovules 

on WPM supplemented with 5.0 mg/1 zeatin (figures 2-1 

and 2-2). Roots formed after small clumps of shoots were 

transferred to half-strength WPM containing 0.02 mg I 1 

indole-3-butyric acid (IBA) (figure 2-3). A survival rate of 

97 percent was achieved by transferring plantlets to a 

greenhouse environment (figure 2-4). 

Development of shoots from excised flower buds is extremely rare in tree species. Bawa and Stettler (1972) cultured female catkin primordia of P. trichocarpa. Although 

unsuccessful in developing whole plants, they found a 

general tendency for callus formation with increasing culture age, occasionally followed by a reversal to vegetative 

growth. Chung et al. (1993) successfully established a 

flower-bud culture system to maximize shoot regeneration of P. deltoides and P. maximowiczii. They used small 

slices (5 to 6 mm thick) of catkin prepared from well-developed (3 to 4 em long) flower buds that were cultured 

on MS medium. An average of 3.8 shoots from P. deltoides 

and 4.1 shoots from P. maximowiczii were regenerated from 

catkin after culturing on MS medium containing 1.0 mg/1 

BA with 0.05 mg/1 NAA or 0.5 mg/1 BA with 0.05 mg/1 

N AA, respectively. More than 20 shoots can be regenerated from a single mature catkin of poplar such as P. 

deltoides, which is typically recalcitrant to shoot regeneration. Adventitious shoots were initiated from 3 locations 

on the explant; the outer and the cut surface of stalks, and 

the marginal area of small bracts. 

Callus Culture 

Calli, groups of undifferentiated cells, are induced from 

various explants, such as stem, leaf, root, embryo, pollen, 

or protoplast. Plants regenerate in 2 ways from calli manipulation: 1) organogenesis in which shoots form directly 

from calli; and 2) somatic embryogenesis from embryogenic development. Pioneering studies for in vitro shoot 

regeneration were conducted on callus cultures of triploid 

quaking aspen (Winton 1968; Wolter 1968) and quaking 

aspen (Winton 1970). Wolter (1968) suggested that shoots 

were induced on callus explants of P. tremuloides by supplementing the basal medium with 0.2 to 0.5 mg/1 BA, and 

that fresh shoots were rooted on the same medium supplemented with 0.04 mg/12,4-0 and 1.0 mg/1 kinetin. In vitro 

plantlets were regenerated through organogenesis in 

Populus species (Coleman and Ernst 1990; Noh and 

Minocha 1986; Park and Son 1988). Cytokinin treatments 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Plant Regeneration Through Organogenesis in Poplar 

of 0.2 to 1.0 mg/1 BA or 2.0 to 5.0 mg/1 zeatin were used 

for shoot regeneration. 

McCown et al. (1988) defined nodules from cell suspension culture as independent, spherical, dense cell clusters 

that form a cohesive unit and display a consistent internal 

cell/ tissue differentiation. They demonstrated that nodule culture has a high potential for organogenesis. Chung 

and Chun {1991) obtained fine nodules from cell suspension cultures of P. x euramericana and P. nigra x P. 

maximowiczii on MS medium with an optimal combination of BA and NAA. When these fine nodules were transferred to liquid regeneration medium with BA and NAA, 

an average of 27 shoots per nodule was induced after 8-

weeks culture. 

Overall, plant morphogenesis through in vitro culture 

depends on species, explant, type and concentration of 

plant growth regulators, culture medium composition, and 

culture environment. For further application, systems for 

successful plant regeneration need careful study to optimize these parameters. 

Physiological Changes of 

In Vitro Cultured Plantlets 

Vitrification is a physiological disorder affecting in 

vitro plantlets (Kevers et al. 1984). Although most vitrification studies have focused on herbaceous species, 

some woody species have been studied (Letouze and 

Daguin 1987; Marin et al. 1988; McLaughlin and 

Karnosky 1989). Numerous studies have reported on 

the abnormalities caused by factors such as medium 

type (Earle and Langhans 1975), gelling agents 

(Debergh 1983), organics (Zimmerman and Cobb 1989; 

Ziv et al. 1983), inorganics (Vieitez et al. 1987), and plant 

growth regula tors (Leshem et al. 1988; Paques and 

Boxus 1987). In comparison with normal leaves of in 

vitro plantlets, vitrified plantlets have malfunctional 

stomata (Brainerd et al. 1981; Marin et al. 1988; Miguens 

et al. 1993; Ziv et al. 1987); unorganized vacuolated, 

spongy mesophyll cells (Earle and Langhans 1975; 

Vieitez et al. 1987); large intercellular spaces (Brainerd 

et al. 1981); reduced epicuticular waxes (Sutter 1985); 

and low chlorophyll content (Phan and Letouze 1983; 

Ziv et al. 1983). Vitrified in vitro plantlets also poorly 

acclimatize after transfer to soil, a problem for commercial production (Debergh et al. 1981). Vitrified plantlets demonstrated an increased cellular volume (Paques 

and Boxus 1987; Vieitez et al. 1985; von Arnold and 

Ericksson 1984), and vascular bundles were less developed in vitrified leaves (Leshem 1983; Ziv et al. 1983). 

Several rescue methods using modifications of media 

19 
Section I In Vitro Culture 

0 

Figure 4. Morphological characteristics of leaf vitrification in the culture of Populus alba x P. grandidentata. 4-1) Normal leaf 

of 'Crandon' clone. 4-2) Adaxial orientation of normal 'Crandon' clone, P=palisade parenchyma. 4-3 and 4-4) 

Abnormal leaf of 'Crandon' clone; M=spongy mesophyll. 4-5) Normal leaf of 'Hansen' clone. 4-6) Abnormal leaf of 

'Hansen' clone; !=enlarged intercellular space. 4-7) Normal leaf of 'Hansen' clone. 4-8) Abnormal leaf of 'Hansen' 

clone (Kang and Hall 1996c}. ·. 

20 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
components have been reported (McLaughlin and 

Karnosky 1989; Sato et al. 1993). 

In liquid culture, vitrified plantlets produce more ethylene, suggesting that increased 1-aminocyclo-propane-1-

carboxylic acid (ACC) and ethylene production is a 

nonwound response to stress (Kevers et al. 1984). Subsequently, ACC and ethylene cause metabolic changes that 

activate peroxidases such as indole-3-acetic acid (IAA) and 

ACC oxidases. Increased peroxidase activities were detected in vitrified tissue of herbaceous and woody plantlets (Kervers et al. 1984). 

Kang and Hall (1996c) studied in vitro morphological 

abnormalities in hybrid aspen (P. alba x P. grandidentata cv. 

'Crandon' and cv. 'Hansen'). Morphological differences in 

stomata and other cell types were investigated through 

scanning electron microscopy. Phenotypic differences are 

shown in figures 3-1 to 3-3. In vitrified leaves of 'Crandon' 

and 'Hansen,' stomatal shape was circular and inflated 

with larger stomatal apertures (figures 3-4 and 3-5); however, stomatal densities were similar between normal and 

vitrified leaves. Vitrified leaves were thicker than those of 

normal leaves (figure 4). Mesophyll cells were arranged 

irregularly and palisade parenchyma was less developed 

in vitrified leaves (figures 4-1 to 4-4). Chloroplasts of vitrified leaves were arranged sparsely within the larger mesophyll cells, whereas they were highly condensed in 

normal leaves (figures 4-5 to 4-8). 

Although many reports relate to vitrification studies in 

herbaceous horticultural species, only a few papers have 

been published for woody species. The morphological 

study of Kang and Hall (1996c) is the only report on the 

physiological variations related to vitrification in Populus 

species. Studies on such physiological phenomena provide 

a basis for developing successful in vitro culture systems. 

Summary 

Organogenesis has become a basic technique for advancing studies such as genetic manipulation and can provide 

a primary plant regeneration system to obtain "true-totype" plantlets. Fundamental studies on in vitro physiology are prerequisites for developing and optimizing a 

successful tissue culture system. 

Biotechnology can support future industrial applications to produce synchronized plantlets for tree-breeding 

programs and secondary metabolites for pharmaceutical 

programs. Further applications of biotechnology require 

more studies on: 1) in vitro physiological phenomena; 2) 

development of generalized regeneration systems for 

poplar species and hybrids; 3) variation among plantlets 

from different explants; and 4) transplanting for greenhouse growth. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Plant Regeneration Through Organogenesis in Poplar 

Acknowledgments 

We thank Dr. Ned B. Klopfenstein (USDA Forest Service, Rocky Mountain Forest and Range Experimental Station, National Agroforestry Center) for reviewing an earlier 

draft of this manuscript. 

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For. Serv.: 12-25. 

23 
Chapter4 

Protoplast Isolation and Culture1 

Julie Russell Kikkert 

Introduction 

Protoplasts are cells without cell walls. In plants protoplasts are usually liberated by tissue digestion with cellulases and pectinases purified from wood-rotting fungi. To 

keep the plasma membrane from bursting during protoplast isolation, an isotonic or slightly hypertonic medium 

is used. Because of the lack of external constraints on the 

membrane, protoplasts are spherical (figure 1B). One use 

of plant protoplasts is to isolate single cells for physiological studies. In Populus (poplar), protoplasts have been used 

to study reactions of the photosynthetic pathway 

(Dalakishvili et al. 1989; Mgaloblishvili et al. 1988; Sanadze 

et al. 1986, 1990). 

Mter the demonstration of totipotency of tobacco protoplasts by Takebe et al. (1971), many laboratories began 

developing protocols to regenerate plants from herbaceous 

and woody plant protoplasts to use in genetic engineering. Protoplasts were considered essential for gene transfer because the cell wall is usually impermeable to DNA. 

Several protoplast-based genetic engineering techniques 

emerged, including protoplast fusion, DNA microinjection, 

electroporation, and polyethylene glycol (PEG)-mediated 

gene transfer. Today, other methods, such as Agrobacteriummediated gene transfer and biolistics, are available to transform cells within intact tissues. Still, protoplasts are 1 

method that has proven valuable for genetic engineering 

research. 

To obtain transgenic plants, the genetically altered protoplasts must regrow their cell walls, divide, and regener1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 326 p. 

24 

ate into whole plants. Figure 1 shows the steps involved 

and table 1lists reports of poplar protoplast isolation and 

culture. Woody plants were once considered difficult to 

regenerate from protoplasts, but breakthroughs occurred 

in the mid-1980s with Populus spp. protoplasts as one of 

the first succesful genera (McCown 1988; McCown and 

Russell1987; Russell and McCown 1986a,1986b). Critical 

to this success was the use of juvenile or juvenile-like source 

tissues, such as in vitro grown plants (shoot cultures) or 

embryogenic cell cultures (McCown 1988), and optimum 

protoplast isolation and culture conditions (Russell1993). 

Currently, whole plants have been regenerated from a wide 

range of poplar species and hybrids (table 1, section D). 

~chapter compares reports of plant regeneration from 

poplar protoplasts and highlights the important common 

success factors. For a more thorough description of general protoplast culture, refer to reviews by Kirby et al. 

(1989); Rashid (1988); and Russell (1993). 

Key Factors 

Numerous factors affect the isolation and culture of protop lasts. A variety of poplar genotypes have been successfully cultured by several laboratories. When the procedures 

are compared, the following common features emerge. 

Protoplast Isolation 

The source tissue is a common and important factor in 

successful poplar protoplast culture. In 10 of the 12 reports 

listed in table 1, section D, the protoplast source was leaves 

from shoot cultures. Further, most of the shoot cultures 

were from nonseedling trees, which allowed researchers 

to work with already proven clone varieties. In woody 

species, shoot cultures yielded higher numbers of protoplasts with better viability than did leaves from greenhouse- or field-grown plants (Smith and McCown 1982). 

In addition to being juvenile, shoot cultures are aseptic, 

adapted to tissue culture conditions, and maintain genepc 

and physiological tissue uniformity (McCown 1988). To 
Protoplast Isolation and Culture 

Figure 1. Stages in the culture of protoplasts through plant regeneration for Populus alba x P grandidentata. A) Leaves from 

shoot cultures are the source tissue. B) Newly isolated protoplasts plated in liquid medium with a polyester screen 

disc for support (openings in the screen are 150 11m). C) Cell divisions approximately 2 weeks after protoplast 

isolation. D) Protoplast-derived calli developing on the screen disc (ruler markings represent 1 mm). E) Shoot 

regeneration. F) Shoot multiplication. G) Harvest and rooting of micro cuttings that are ready for transfer to the 

greenhouse. For detailed procedures, see Russell and McCown (1988). 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 25 
Section I In Vitro Culture 

Table 1. Reports of protoplast isolation and culture in Populus species, which are categorized by the most advanced 

protoplast development for a given report. 

Species Source1 tissue Reference 

Protoplast isolation 

P. alba 

P. nigra var. charkowiensis 

x P. nigra var. caudina 

P. deltoides 

P. x euramericana 

P. glandulosa 

P. nigra x P. laurifolia 

P. tacamahaca x P. trichocarpa 

P. tremuloides 

P. trichocarpa x P. tacamahaca 

Protoplast division 

P. alba x P. glandu/osa 

P. alba x P. grandidentata 

P. ciliata 

P. davidiana 

P. glandulosa 

P. sieboldii 

P. tremula 

P. tremuloides 

Organogenesis from 

protoplast-derived calli 

P. alba 

Recovery of whole plants from 

protoplasts 

P. alba x P. glandulosa 

P. alba x P. grandidentata 

P. nigra var. charkowiensis 

x P. nigra var. caudina 

somatic hybrid with 

Hibiscus sabdariffa 

P. glandulosa 

P. koreana x P. nigra 

somatic hybrid with 

P. x euramericana 

P. nigra 

P. nigra x P. maximowiczii 

P. nigra x P. trichocarpa 

P. simonii 

P. tomentosa 

P. tremula 

P. tremula x P. alba 

C,CS 

SH 

L 

L 

L-SDL 

SH 

SH 

L 

SOL 

cs 

cs 

L 

SH 

SH 

c 

SH 

SH 

SH 

L 

L 

SH 

SH 

SH 

SH 

SH 

SH 

c 

SH 

SH 

cs 

SH 

SH 

SH 

Park and Son 1987 

Park et al. 1987 

Ito et al. 1986 

Sanadze et al. 1986 

Xin et al. 1991 

Saito, 1976, 1980a 

Park and Son 1986 

Jang et al. 1987 

Russell and McCown 1986a 

Butt 1985 

Verma and Wann 1983 

Douglas 1982 

Youn et al. 1985 

Kim et al. 1988 

Park and Han 1986 

Chun 1985 

Cheema 1988 

Park et al. 1988 

Park et al. 1988 

Saito et al. 1987 

Ahuja 1983a,b 

Ahuja 1983b 

Sasamoto et al. 1989 

Park and Son 1988 

Russell and McCown 1986b, 1988 

Ito et al. 1986; 

Oji-Paper 1989 

Park et al. 1990 

Park et al. 1992 

Lee et al. 1987 

Park and Son 1992 

Russell and McCown 1988 

Wang et al. 1995 

Wang et al. 1991 

Russell and McCown 1988 

Chupeau et al. 1993 

1 C=callus; CS=cell suspensions; L=leaf; SDL=seedlings; SH=shoot cultures 

ensure high yields of viable protoplasts, growth conditions 

of shoot cultures must be optimum. Lighting conditions 

(Sasamoto et al. 1989) and the growth medium (Russell and 

McCown I988) are especially important. 

26 

A second component of protoplast isolation is tissue 

digestion. Table 2 lists the enzymes and treatment times 

that have been used for poplars. Cellulase RIO and 

Macerozyme RIO were the most commonly used, often in 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Protoplast Isolation and Culture 

Table 2. Enzyme treatments used to liberate protoplasts from Populus shoot culture-derived leaves. Only reports in 

which whole plants were regenerated from protoplasts are listed. 

Enzyme concentration (percent w/v) 

Cellulase Cellulase Cellulase Macerase Macerozyme Hemi- Driselase PectoSpecies (Cooper) R1 0 RS 

P. alba 

(Sasamoto et al. 1989) 1.0 

P. alba x P. glandulosa 

(Park and Son 1988) 

P. nigra x P. maximowiczii 

(Park and Son 1992) 2.0 

P. alba x P. grandidentata 

P. nigra x P. trichocarpa 

P. tremula 

(Russell and McCown 1988) 0.5 

P. x euramericana 

(Park et al. 1992) 1.0 

P. g/andulosa 

(Park et al. 1990) 1.5 

P. koreana x P. nigra 

(Park et al. 1992) 2.0 

P. tomentosa 

(Wang et al. 1991) 2.0 

P. tremula x P. alba 

(Chupeau et al. 1993) 0.1 

combination with Hemicellulase, Driselase, or Pectolyase. 

However, the exact concentrations and time of treatment 

vary with the genotype and the researcher preferences. The 

digestion environment may contain many toxic compounds, such as enzyme impurities and components released after tissue wounding. Thus, digestive time is a 

critical factor for good protoplast viability (table 2). Because enzymes are relatively large molecules, they penetrate tissues slowly. To increase penetration, leaf tissue is 

usually sliced or chopped before enzyme treatment. However, cell exposure to the enzymes is still not uniform, 

which results in over digested cells near the edges of the 

cuts and under digested cells in the interior of the leaves. 

Russell and McCown (1986a) demonstrated that when 

leaves of poplar shoot cultures were processed in an Omnimixer, the epidermis was stripped away and the cells were 

more uniformly exposed to the enzymes. The results were 

higher protoplast yields and better viability. Leaves processed with an Omni-mixer required only a 4-h digestion, 

whereas other labs used a 10- to 16-h digestion (table 2). Park 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

R10 cellulase lyase Time 

0.25 1.5 h 

0.8 1.2 2.0 0.05 1.8 h 

0.1 4h 

0.4 1.2 2.0 0.05 10 h 

0.5 0.5 0.5 0.05 12 h 

0.4 1.2 2.0 0.05 10 h 

0.5 14 h 

0.02 0.05 16 h 

and Son (1988, 1992) digested poplar leaf tissue for only 1.8 h, 

even though the leaves were sliced with a scalpel. The shorter 

digestion may have been possible because the leaves were 

placed in fresh enzyme solution 4 times. Thus, toxic components were removed along with the old enzyme solution. 

Osmoticum, salts, growth regula tors, and buffers in the 

isolation medium are also important for long-term viability 

of poplar protoplasts {Park and Son 1992; Chupeau et al. 

1993 ). However, no general recommendations are made here. 

Protoplast Culture and Regeneration 

Key factors for poplar protoplast culture through sustained division and plant regeneration are listed on tables 

3 and 4. One of the most common factors is plating in 

liquid medium (10 of the 12 reports) (table 3). Agar or 

ctgarose plating generally produces few or no protoplastderived calli ( Chupeau et al. 1993; Park and Son 1988, 1992; 

Russell and McCown 1986b ). One reason for using liquid 

plating is to avoid buildup of toxic exudates around the 

27 
~ 

Section I In Vitro Culture 

'\;;jjjj/ 

VJiil 

Table 3. Conditions for the culture of Populus protoplasts that led to sustained cell division and eventual plant recovery. 

V/Jiiil 

V!iiil 

Culture Plating Basal NH/ Growth 

Species method density1 medium2 (ammonium nitrate) Osmotic urn regulators ~ 

P. alba Liquid 2.5x104 MS No 0.08 M sucrose 1.0 J.LM 2,4-D ...:# 

(Sasamoto et al. 1989) 0.6 M mannitol 0.1 J.1M BA ~ 

P. alba x P. glandulosa Agar/ 2.4x105 MS No 0.2 M mannitol 9.0 J.LM 2,4-D .) (Park and Son 1988) Gauze 0.4 M glucose 22.2 J.1M BA 

P. alba x P. grandidentata Liquid 5.0x104 WPM No 0.366 M sucrose 1.0 J.1M NAA ~ 

(Russell and McCown 1988) Floating 0.046 M mannitol 0.1 J.1M BA 

Disc 0.046 M sorbitol 'o:!Jfl 

0.046 M xylitol ~ 

0.046 M inositol 

P. glandulosa Liquid 1.0x105 KM8P Yes 0.6 M sucrose 5.0 J.1M 2,4-D -.a/ 

(Park et al. 1990) 0.5 J.1M BA ~ P. koreana x P. nigra Liquid un- KM8P Yes 0.6 M sucrose 0.4 J.1M 2,4-D 

somatic hybrid with available 0.5 J.LM BA ~ 

P. x euramericana 

(Park et al. 1992) ~ 

P. nigra Liquid 3.7x104 WPM No 0.366 M sucrose 1.0 J.1M NAA ~ 

(Lee et al. 1987) 0.046 M mannitol 0.1 J.lM BA 

0.046 M sorbitol viii~ 

0.046 M xylitol ~ 

0.046 M inositol 

P. nigra x P. maximowiczii Agar/ 2.4x105 MS No 0.2 M mannitol 9.0 J.LM 2,4-D \.:J!i) 

(Park and Son 1992) Gauze 0.4 M glucose 0.4 J.1M BA -..9 P. nigra x P. trichocarpa Liquid 0.5x104 WPM No 0.366 M sucrose 1.0 J.1M NAA 

(Russell and McCown 1988) Floating 0.046 M mannitol 0.1 J.LM BA ~ 

Disc 0.046 M sorbitol 

0.046 M xylitol ...;6) 

0.046 M inositol ...;6) 

P. simonii Liquid 3.0x106 KM8P Yes 0.4 M glucose 13.6 J.tM 2,4-D 

(Wang et al. 1995) 1.1 J.1M NAA -w) 

0.9 J.1M KT 'Q!ii) 

P. tomentosa Liquid 2.5x105 KM8P Yes glucose 2.2 J.lM 2,4-D 

(Wang et al. 1991) 2.5 J.1M NAA .v) 

2.2J.1M BA ~ P. tremu/a Liquid 1.0x105 WPM No 0.366 M sucrose 1.0 JlM NAA 

(Russell and McCown 1988) Floating 0.046 M mannitol 0.1 J.LM BA 'Qii) 

Disc 0.046 M sorbitol 

0.046 M xylitol ...a) 

0.046 M inositol ~ 

P. tremula x P. alba Liquid 5-8x104 Chupeau Yes 0.55 M glucose 14.0 J.tM 2,4-D 

(Chupeau et al. 1993) 0.05 J.1MTDZ \8) 

1 Protoplasts per mi. ~ 

2 Three basal media formulations have been used for protoplast culture of poplars in an equal number of cases. WPM is a lower ~ salt and lower chloride medium than MS. KM8P is a complex medium containing numerous vitamins, sugars and sugar alcohols, 

organic acids, casamino acids, and coconut water. WPM=Lioyd and McCown (1980); MS=Murashige and Skoog (1962); ~ 

KM8P=Kao and Michayluk (1975). ~ 

~ 

cells (Chupeau et al.1993; Russell and McCown 1986b). plating ~ethod, in which the protoplasts were cultured ~ 

In liquid culture, the medium can be refreshed periodi- in contact with a polyester screen at the surface of the ~ 

cally, thus reducing the concentration of toxic exudates. medium (figure 1). The protoplasts adhered to the screen, 

Russell and McCown (1986b, 1988) used a floating disc ~ which aided in medium replenishment and observation. 

-c) 

28 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. ~ 

\$I 

lou# 
Protoplast Isolation and Culture 

Table 4. Plant growth regulators used for shoot regeneration from protoplast-derived calli of Populus. 

Species 

P. alba 

(Sasamoto et al. 1989) 

P. alba x P. glandulosa 

(Park and Son 1988) 

P. alba x P. grandidentata 

(Russell and McCown 1988) 

P. glandu/osa 

(Park et al. 1990) 

P. koreans x P. nigra 

somatic hybrid with 

P. x euramericana 

(Park et al. 1992) 

P. nigra 

(Lee et al. 1987) 

P. nigra x P. maximowiczii 

(Park and Son 1992) 

P. nigra x P. trichocarpa 

(Russell and McCown 1988) 

P. simonii 

(Wang et al. 1995) 

P. tomentosa 

(Wang et al. 199·1) 

P. tremula 

(Russell and McCown 1988) 

P. tremula x P. alba 

(Chupeau et al. 1993) 

1 benzyladenine 

2 kinetin 

3 thidiazuron 

4 zeatin 

5 2,4-dichlorophenoxyacetic acid 6 a-naphthaleneacetic acid 

0.1 

0.1 

4.44 

2.2 

0.4 

Protoplasts will float only when sucrose is used as the 

osmoticum; however, sucrose is toxic (Chupeau et al. 1993) 

or not optimal for some genotypes (table 3). Sucrose, glucose, and mannitol were the most commonly used osmotica. 

Another apparent requirement for poplar protoplasts, 

though not listed on table 3, is culturing under dark conUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Concentration in Regeneration Medium (J.lM) 

I(T2 TDZ3 ZT4 2,4-05 

2.32 

1.0 

4.6 

0.1 

7.5 

5.0 

1.0 

6.8 

0.1 

2.28 0.54 

1.0 

0.01 

0.1 

ditions until colonies have formed. Poplar protoplasts produced anthocyanins and did not divide even when cultured in dim light (Chupeau et al. 1993; Russell and 

McCown 1986b). Plating density also affects protoplast 

development. With poplars, the optimal densities range 

from 0.5 x 104 to 3.0 x 106 protoplasts per ml of culture 

29 
Section I In Vitro Culture 

medium (table 3). Optimal plating densities vary with the 

genotype, culture medium (Russell and McCown 1988), 

and culture method. 

Protoplasts can be sensitive to the types and concentrations of nitrogen in the culture medium. The usual concentrations of ammonium nitrate in the basal formulations (4.94 

mM in WPM; 20.6 mM in MS; 7.4 mM in KM8P) are sometimes toxic and thus, are eliminated or reduced in the protoplast culture medium (Russell and McCown 1988; Russell 

1993). In poplars, 7 of the 10 reports of successful culture 

were without ammonium ions (NH4 ·)in the medium (table 

3). Similarly, the response to organic nitrogen sources is genotype dependent. When Russell and McCown (1988) added 

casein hydrolysate and coconut water to their WPM-based 

protoplast medium, the supplements were required for 1 

genotype, toxic to another, and neither required nor toxic to 

yet another. Organics in the medium lowered the optimal 

plating density. Wang et al. (1995) found that either ammonium nitrate or glutamine plus aspartic acid supported low 

rates of colony formation, but that a combination doubled 

the rates of colony formation. With P. tremula x P. alba, organic nitrogen produced no apparent benefits. Rather, optimal protoplast culture occurred when a low initial 

concentration of mineral nitrogen was successively increased 

over time (Chupeau et al. 1993). 

Plant growth regulators are another important component of the protoplast culture medium. However, the optimal type and concentration varies (table 3). The most 

commonly used auxin and cytokinin are 2,4-D {2,4-dichlorophenoxyacetic acid) and BA (benzyladenine), respectively. Chupeau et al. {1993) were the only group to test 

thidiazuron (TDZ) for poplar protoplast culture. TDZ is a 

substituted phenylurea with cytokinin-like activity, and 

in combination with 2,4-D, was required for sustained 

development of protoplast-derived cells of P. tremula x P. 

alba. TDZ also reduced the release of exudates from the 

cells. In another study, Sasamoto et al. {1989) found that 

the optimal growth regulator combination of 1 J.1.M 2,4-D 

with 0.1 J1M BA could overcome inhibitory conditions of 

osmoticum or ammonium ions to promote cell division 

and colony formation. 

Plant growth regulators are also important in the medium for organogenesis from the protoplast-derived calli. 

The requirements appear to be highly genotype dependent (table 4). Zeatin and BA were commonly used, with 

TDZ sometimes required. Standard micropropagation 

techniques were used to propagate and move regenerated 

plants to the greenhouse and field. 

Protoplast-Based Genetic Engineering in 

Poplars 

There are few reports of protoplast-based genetic engineering in poplars. This is because of the availability of 

other gene transfer techniques and because regeneration 

30 

systems have only recently been accomplished for many· 

genotypes. Somatic hybridization, which requires protoplasts, has been reported in 3 cases. Saito {1980b) fused 

protoplasts of P. x euramericana with those of either the 

same species or of Paulownia taiwaniana. Heterokaryons 

were identified by differential staining, but were not cultured. In a second report, leaf protoplasts of P. nigra var. 

charkowiensis x P. nigra var. caudina were fused with callusderived protoplasts of Hibiscus sabdariffa (Ito et al. ·1986; 

Oji-Paper 1989). Plants were regenerated that were poplarlike. Thirdly, protoplasts of P. koreana x P. nigra were fused 

with protoplasts of P. x euramericana (Park et al. 1992; Park 

and Son 1994). Two of the regenerated plants showed intermediate protein band patterns in SDS-PAGE analysis. 

Chupeau et al. (1994) recovered plants after electroporation 

of protoplasts with genes encoding resistance to 3 selective 

agents; paromomycin, chlorsulfuron, and phosphinothricin. 

Standard electroporation techniques developed for herbaceous crops were used. Transgenic plants that expressed the 

genes were obtained with relatively high frequency. The 

patterns of gene integration were single and clear. 

Conclusions 

Several poplar species and hybrids have been cultured 

from protoplasts to whole plants. Common requirements 

for protoplast culture are the use of shoot cultures as a 

source tissue, liquid plating medium, and culture in the 

dark. Other important factors for each genotype include 

the enzyme concentration and incubation time, the isolation medium, and the protoplast culture medium, including osmoticum, nitrogen, and plant growth regulators. 

Growth regulators for plant regeneration from protoplastderived calli are also genotype dependent. Though protoplast isolation and culture must be optimized for each new 

genotype, the parameter ranges for poplars are better defined than in the past. The ability to successfully isolate 

and culture poplar protoplasts allows for their use in physiological and genetic engineering studies. 

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Lloyd, G.; McCown, B. 1980. Commercially-feasible 

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McCown, B.H.; Russell, J.A. 1987. Protoplast culture of 

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Badridze, G.S.H. 1988. The effect of dithiothreitol on the 

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Park, Y.G.; Choi, M.S.; Kim, J.H. 1990. Plant regeneration 

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Park, Y.G.; Han, K.H. 1986. Isolation and culture of mesophyll 

protoplasts from in vitro cultured Populus alba x glandulosa. 

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Park, Y.G.; Kim, J.H.; Son, S.H. 1992. Induction of somatic 

hybrid by protoplast fusion between Populus koreana x 

Populus nigra var. italica and Populus euramericana cv. 

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279. 

Park, Y.G.; Shin, 0.1.; Woo, J.H.; Sui, I.W.; Son, S.H. 1987. 

Protoplast isolation from mesophyll of Populus alba. Korean Journal of Plant Tissue Culture. 14: 49-54. 

Park, Y.G.; Son, S.H. 1986. Factors affecting the isolation of 

mesophyll protoplasts from Populus euramericana cv. 1-

214. Journal of the Korean Forestry Society. 74: 29-36. 

Park, Y.G.; Son, S.H. 1987. Protoplast isolation from callus 

and suspension cultured cells of Populus alba. Korean 

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Park, Y.G.; Son, S.H. 1988. Culture and regeneration of 

Populus alba x P. glandulosa leaf protoplasts isolated from 

in vitro cultured explant. Journal of the Korean Forestry 

Society. 77: 208-215. 

31 
Section I In Vitro Culture 

Park, Y.G.; Son, S.H. 1992. In vitro shoot regeneration from 

leaf mesophyll protoplasts of hybrid poplar (Populus 

nigra x P. maximowiczii). Plant Cell Reports. 11:2-6. 

Park, Y.G.; Son, S.H. 1994. Somatic hybridization in Populus 

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470-480. 

Park, Y.G.; Son, S.H.; Han, K.H. 1988. Isolation and culture 

of mesophyll protoplasts from Populus species. Basic Life 

Science. 44: 483. 

Rashid, A. 1988. Protoplast isolation and cell regeneration. 

In: Rashid, A., ed. Cell physiology and genetics of higher 

plants, Vol. II. Boca Raton, FL, U.S.A.: CRC Press: 1-46. 

Russell, J.A. 1993. Advances in the protoplast culture of 

woody plants. In: Ahuja, M.R., ed. Micropropagation 

of woody plants. Dordrecht, The Netherlands: Kluwer 

Academic Publishers: 67-91. 

Russell, J.A.; McCown, B.H. 1986a. Techniques for enhanced release of leaf protoplasts in Populus. Plant Cell 

Reports. 5: 284-287. 

Russell, J.A.; McCown, B.H. 1986b. Culture and regeneration of Populus leaf protoplasts isolated from non-seedling tissue. Plant Science. 46: 133-142. 

Russell, J.A.; McCown, B.H. 1988. Recovery of plants from 

leaf protoplasts of hybrid-poplar and aspen clones. Plant 

Cell Reports. 7: 59-62. 

Saito, A. 1976. Isolation of protoplasts from mesophyll cells. 

of Paulownia fortunei Hemsl. and Populus 

euramericana cv. 1-45/51. Journal of the Japanese Forestry Society. 58: 301-305. 

Saito, A. 1980a. Isolation of protoplasts from mesophyll 

cells of Paulownia and Populus. Bulletin of the Forestry 

and Forest Products Research Institute, Japan. 309: 1-6. 

Saito, A. 1980b. Fusion of protoplasts isolated from somatic 

cells of tree species. Bulletin of the Forestry and Forest 

Products Research Institute, Japan. 309: 7-12. 

Saito, A.; Hosoi, Y.; Ishii, K.; Sato, T. 1987. Callus formation 

from protoplasts of mesophyll cells of Populus plantlets. 

Journal of the Japanese Forestry Society. 69: 472-477. 

Sanadze, G.A.; Mgaloblishvili, M.P.; Dalakishvili, K.G. 

32 

1986. Independence of carbon conversion pathways in 

the Benson-Calvin cycle. Fiziologiya Rastenii. Moscow. 

33: 856-863. 

Sanadze, G.A.; Mgaloblishvili, M.P.; Dalakishvili, K.G. 

1990. Effects of cerulenin organophosphorus compounds and synthetic analogs of natural carbohydrates 

on carbon dioxide assimilation and on the isoprene effect in isolated poplar protoplasts. Fiziologiya Rastenii. 

Moscow. 37:6-11. 

Sasamoto, H.; Hosoi, Y.; Ishii, K.; Sato, T.; Saito, A. 1989. 

Factors affecting the formation of callus from leaf protoplasts of Populus alba. Journal of the Japanese Forestry 

Society. 71: 449-455. 

Smith, M.A.L.; McCown, B.H. 1982. A comparison of source 

tissue for protoplast isolation from three woody plant 

species. Plant Science Letters. 28: 149-156. 

Takebe, I; Labib, G.; Melchers, G. 1971. Regeneration of 

whole plants from isolated mesophyll protoplasts of tobacco. Naturwissen. 58:318-320. 

Verma, D.C.; Warm, S.R. 1983. Isolation of high yields of viable protoplasts from quaking aspen seedlings and cultured loblolly pine cell suspensions. In: Potrykus, 1.; 

Harms, C.T.; Hinnen,A.; Hutter, R.; King, P.J.; Shillito, R.D., 

eds. 6th international protoplast symposium; Basel, Switzerland: Experientia Supplementum. 45: 10-11. 

Wang, S.P.; Xu, Z.H.; Wei, Z.M. 1991. Culture and regeneration of poplar mesophyll protoplasts. Science in 

China, Series B, Chemistry, Life Sciences & Earth Sciences. 34: 587-592. 

Wang, Y.; Huang, M.R.; Wei, Z.M.; Sun, Y.R.; Chen, D.M.; 

Xu, Z.H.; Zhang, L.M.; Xu, N. 1995. Regeneration of 

simon poplar (Populus simonii) from protoplast culture. 

Plant Cell Reports. 14:442-445. 

Xin, S.; Kangfeng, M; Houman, F. 1991. Technique for the 

isolation of mesophyll protoplasts from Populus deltoides 

Bartr. cv. Lux. Journal of Beijing Forestry University. 13: 

112-115. 

Youn, Y.; Lee, J.S.; Lee, S.K. 1985. Isolation and culture of 

protoplasts from suspension-cultured cells of Populus 

alba X P. glandulosa Fl. Research Report of the Institute 

of Forest Genetics. Korea. 21: 109-113. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
ChapterS 

Somaclonal Variation in Populus: An Evaluation1 

D. John Fry, Gerry C. Douglas, and N. Th. Saieed 

Introduction 

Substantial genetic and phenotypic variation occurs 

within plant cell, protoplast, anther, or tissue cultures. 1his 

variation also occurs among plantlets regenerated from 

these cultures, and it is assumed that this variation is genetically based (Michler 1993). Larkin and Scowcroft (1981) 

proposed that the variation occurring within cultures, or 

recoverable from cultures following plantlet regeneration, 

be termed somaclonal variation. They believed that the 

term described the variation among calliclones and 

protoclones from stem callus material and protoplast culture, respectively (Ahuja 1987a). However, some still consider protoclonal variation a separate category (Serres et 

al. 1991) and use the word gametoclonal to denote variation recoverable from haploid cultures. Ahuja (1988) proposed that somatoclonal variation encompass variability 

among genetically transformed plant materials. 

Demonstrating this capacity for variation is dependent 

upon developing culture protocols for the species in question, but some genera, species, or varieties are recalcitrant 

to micropropagation techniques and have yet to show genetic variation within culture. Most micropropagation protocols were developed for herbaceous plants, but successful 

culture and regeneration was achieved in diverse woody 

perennial species (Dhawan 1993; Thorpe et al. 1991). 

Dhawan (1993) lists 7 culture systems for Populus and 3 

for Salix; although, cell, protoplast, and anther culture systems exist for many species of the Salicaceae, including 

some poplars that are difficult to propagate (Ahuja 1987b; 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 326 p. 

Cheema 1989; Chen 1987; Chun 1993; Douglas 1989; 

Gebhardt 1989; Lu and Liu 1990; Rutledge and Douglas 

1988). The first clonally propagated forest trees were from 

juvenile aspen cultures (Winton 1970). Populus was the first 

genus used for in vitro production of haploid plants (Lu 

and Liu 1990). Ahuja (1987b) and Chun (1993) recorded 

plantlet regeneration from tissue cultures of at least 29 

Populus species, hybrids, or clones. 

One purpose of micropropagation was to obtain genetic and morphological stability in culture and fidelity of the regenerants to the parental phenotype. 

Therefore, initially, acknowledgment of somaclonal 

variation in the literature probably depended upon the 

researchers' paradigm; particularly whether they 

viewed variation as an experimental failure. Populus 

and Salix species are generally easy to stabilize in culture (Thorpe et al. 1991) and were used for early experimental tissue culture systems. However, the genetic 

stability of regenerants was frequently doubted and callus culture was usually replaced by organ culture for 

propagation work (Ahuja 1987a, 1987b, 1993). 

Somaclonal variation is recorded among regenerants of 

widely ranging species and is particularly well documented for herbaceous plants; reflecting their ability for 

adventitious shoot regeneration and micropropagation, 

and their pre-eminence among species of economic interest. Frequently, variation was a concern, but it is not universal among regenerants of successfully cultured species. 

Diverse semi-woody and woody perennials also exhibited 

somaclonal variation (table 1). Gynmosperms (especially 

the genus Pinus) are considered more genetically stable in 

culture than angiosperms (Diner and Karnosky 1987), but 

somaclonal variants were recovered in Pinus, Abies, and 

Picea (table 1). Among perennial angiospenris, somaclonal 

variants were recovered in woody monocots including 

Musa (Skirvin et al. 1994), oil palm, and bamboo; in dicot 

fruit crops, it was observed in semi-woody Rubus species, 

Olea, and various species of Citrus, Malus, Prunus, Pyrus, 

and Carica (Hammerschlag 1992; Manshardt 1992); in timber yielding hardwoods, excluding the Salicaceae, it was 

observed in Ulmus sp., Eucalyptus sp., Albizia lebbeck, and 

Santalum album, and in ornamentals such as Paulownia 

tomentosa (Marcotrigiano and Jagannathan 1988) (table 1). 

33 
Section I In Vitro Culture 

Table 1. Range of woody perennial species that showed 

somaclonal, protoclonal or gametoclonal variation. 

Genus/species 

Abies 

Picea 

Pinus 

Mus a 

Elaeis guineensis (oil palm) 

Bambusa 

Rubus 

Olea 

Citrus 

Malus 

Prunus 

Pyrus 

Carica 

Coffea arabica 

Santalum album (sandal wood) 

Eucalyptus 

Albizzia lebbeck 

Ulmus 

Paulownia tomentosa 

(foxglove tree) 

Reference 

Gajdosova and Vookova 1991 

Isabel et al. 1995 

Berlyn et al. 1986 

Hammerschlag 1992 

Benbadis 1992 

Huang and Hunag 1993 

Swartz et al. 1983 

Skirvin et al. 1994 

Hammerschlag 1992 

Gmitter et al. 1992 

Hammerschlag 1992 

Hammerschlag 1992 

Hammerschlag 1992 

Manshardt 1992 

Sandahl and Lauritius 1992 

Sita 1992 

Haines 1994b 

Varghese and Kaur 1991 

Lineberger et al. 1990 

Domir and Schreiber 1993 

Marcotrigiano and 

Jagannathan 1988 

Poplar Somaclonal Variation 

In the Salicaceae, somaclonal variation is well documented among Populus species, but apparently has not 

been recorded for Salix. This is probably due to the relative ease in culturing Populus, the general recalcitrance of 

Salix spp. in vitro, and the economic encouragement for 

working with Populus. Retrospectively, one of the earliest 

demonstrations of recoverable somatic variation in poplar was a semi-evergreen mutation on a branch of P. nigra 

cv. 'Italic' in Chile before 1900 (Pryor and Willing 1965). 

Somaclonal variation was also apparently observed in the 

1960s when concern was expressed that regenerants from 

poplar cultures might be abnormal or genetically unstable 

(Ahuja 1987b ), but no significant survey was initiated. 

Lester and Berbee (1977) first published the systematic 

demonstration of within-done variation (soma clonal variation) in P. nigra cv. 'ltalica' (P. nigra var. italica) and P. x 

euramericana. Since then, somaclonal variation has been 

repeatedly demonstrated within the genus (table 2), as 

have protoclonal (Serres et al. 1991) and gametoclonal 

variation (Chen et al. 1981; Chen 1987; Liu et al. 1992; Wu 

et al. 1981). Ignoring duplications at the clonal level, 

34 

somaclonal variation was demonstrated in cultures of at 

least 21 poplar species or hybrids (table 2; table 3). Furthermore, DNA analyses by Keirn et al. (1989) demonstrated natural intra specific polymorphism in overlapping 

populations of P. angustifolia, P. fremontii, and their F1 (first 

generation) hybrid progeny. Like the semi-evergreen 

Populus nigra cv. 'ltalica', this might reflect in vivo somatic 

mutation, particularly since the hybrid genome was apparently perpetuated by natural vegetative propagation. 

The use of callus culture (especially repeated subculturing) to rejuvenate hardwood tissues might be similar to 

soma clonal variation since it attempts to reverse the physiological change accompanied by dramatic alterations in 

cell DNA (Berlyn et al. 1986; Bonga and vonAderkas 1993; 

Franclet et al. 1987). Rejuvenation by repeated subculturing also was achieved with Salix babylonica {Franclet et al. 

1987); but the technique is not widely applied to the 

Salicaceae because juvenile tissues from these species are 

regenerated by other means. 

From the research perspective, somaclonal variation is 

considered highly undesirable since it may complicate 

studies on genetic transformation and transmission. Horticultural or silvicultural applications are of further concern becau~e variation raises doubts about the stability of 

any micropropagated perennial plant over a long cropping cycle or rotation (Berlyn et al. 1986; Hammerschlag 

1992; Skirvin et al. 1994). Alternatively, it has long been 

argued that somaclone production is useful to uncover 

variants that could combine new desirable traits with the 

favorable characteristics of the existing variety or vegetative clone (Larkin and Scowcroft 1981; Phillips et al. 1990). 

The potential application of biotechnology to selective tree 

improvement and the drawbacks and potential benefits 

of somaclonal variation was generally reviewed (Ahuja 

and Libby 1993; Haines 1994a, 1994b; Huang et al. 1993; 

Zimmerman 1988); in relation to selection and improved 

disease resistance (Michler 1993; Ostry 1994); and for individual groups or species including conifers (Berlyn et 

al. 1986; Dunstan 1988) and poplars (Nelson et al. 1984; 

Ostry and Michler 1993 ). 

Cultures or regenerants within the genus Populus exhibit 

the full range of genetic, physiological, anatomical, and 

morphological variation suggested by Parfitt and 

Arulsekar (1987). Lester and Berbee (1977) observed variation in root cell chromosome counts; Noh and Minocha 

(1990) detected isozyme variation in 4 of 5 enzymes tested; 

and Son et al. {1993) recorded correlations between growth 

rates and specific SDS-PAGE protein banding patterns. At 

the physiological level, differences were recorded and I or 

selected for disease resistance (Antonetti and Pinon 1993; 

Ostry and Skilling 1988; Prakash and Thielges 1989; Valentine et al. 1988); herbicide tolerance (Michler and Haissig 

1988); and gas exchange characteristics (Saieed et al. 1994a, 

1994b). In some cases, variability is only detectable at the 

physiological level because regenerants are morphologiUSDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 
(. 

\cl 

~ 

~ 

Somactonal Variation in Populus: An Evaluation 

~ Table 2. Taxonomic distribution of Populus species, hybrids, or clones that exhibited somaclonal (calliclone or 

protoclonal) variation for one or more features. 

Species/hybrid 

Populus alba 

P. alba; cl. '5972-S38' 

P. alba x P. grandidentata 

P. alba x P. grandidentata; cv. 'Favorit' 

P. alba x P. grandidentata; cv. 'Crandon' ('NC5339') 

P. alba x P. grandidentata; cv. 'Crandon' ('NC5339') 

P. alba x P. tremuloides; cl. '802-36-7' 

P. angustifolia 

P. angustifolia x P. fremontli 

P. deltoides cl. 'K417' 

P. x euramericana 

P. x euramericana cv. 'Ogy' 

P. x euramericana cv. 'Robusta' 

P. fremontii 

P. x interamericana cv. 'Barn' 

P. x interamericana cv. 'Rap' 

P. maximowicziix P. trichocarpa; cv. 'Androscoggin' 

P. maximowicziix P. trichocarpa; cl. 'NC11390' 

P. nigra var. italica 

P. nigra cl. 'R1 029' 

P. nigra x P. laurifolia; cl. 'NC5272' 

P. nigra var. betulifolia x P. nigra; cv. 'Volga' 

P. nigra var. betulifolia x P. trichocarpa 

P. nigra var. betulifolia x P. trichocarpa; cl. 'NE299' 

P. nigra var. betulifolia x P. trichocarpa; cl. 'NC5331' 

P. nigra var. charkowiensis x P. deltoides 

P. nigra var. charkowiensis x P. nigra var. caudina 

P. tremula 

P. tremula x P. alba; cl. '717 -1-84' 

P. tremula x P. alba; cl. '706-8' 

P. tremuloides 

P. tremuloides 

P. trichocarpa x P. deltoides; cv. 'Hunnegem' 

P. trichocarpa x P. balsamifera cl. 'TT32' 

P. hybrid?-protoclone 

P. hybrid?-shoot culture 

1

A = Aigeiros 

L = Leuce (currently termed Populus) 

T = Tacamahaca 2natural intra specific polymorphism 

? = unavailable 

cally similar to the parental lines (Michler and Haissig 1988; 

Ostry and Skilling 1988; Pr.akash and Thielges 1989). Differences also were noted in various leaf characteristics 

including albinism, variegation, color, and chlorophyll 

content (Ahuja 1983; Douglas 1989; Michler 1993; Noh and 

Minocha 1990; Saieed et al. 1994a, 1994b; Serres et al. 1991; 

Son et al. 1993); and marginal serration and pubescence 

(Michler and Haissig 1988; Serres et al. 1991}. Leaf 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Section1 Author(s) 

L lordan-Costache et al. 1991 

L Antonnetti and Pinion 1993 

L Son et al. 1993 

L lordan-Costache et al. 1991 

LxL Michler and Haissig 1988 

LxL Serres et al. 1991 

LxL Antonnetti and Pinion 1993 

T Keirn et al. 19892 

TxA Keirn et al. 19892 

A Prakash and Thielges 1989 

AxA Lester and Berbee 1977 

AxA Antonnetti and Pinion 1993 

AxA Antonnetti and Pinion 1993 

A Keirn et al. 19892 

AxT Antonnetti and Pinion 1993 

AxT Antonnetti and Pinion 1993 

TxT Ostry and Skilling 1988 

TxT Michler and Haissig 1988 

A Lester and Berbee 1977 

A lordan-Costache et al. 1991 

AxT Michler and Haissig 1988 

A Ostry and Skilling 1988 

AxT Ostry and Skilling 1988 

AxT Ostry et al. 1994 

AxT Michler and Haissig 1988 

AxA Ostry and Skilling 1988 

A Ostry and Skilling 1988 

L Ahuja 1983 

LxL Antonnetti and Pinion 1993 

LxL Antonnetti and Pinion 1993 

L Valentine et al. 1988 

L Noh and Minocha 1990 

TxA Jehan et al. 1994 

TxT Saieed et al. 1994a/b 

? McCown (In: Michler 1993) 

? Michler (In: Michler 1993) 

morphotype and thickness also vary (Antonetti and Pinon 1993; Lester and Berbee 1977; Saieed et al. 1994a, 1994b; 

Serres et al. 1991; Son et al. 1993) with the morphotype 

and its ratio of leaf length to width, showing apparent correlation with other characteristics (Lester and Berbee 1977; 

Saieed et al. 1994a, 1994b; Serres et al. 1991). In addition, 

gross morphological variation is recorded for root form, 

stem girth, stem height, degree of branching, and overall 

35 
Section I In Vitro Culture 

Table 3. Taxonomic distribution of Populus species, 

hybrids, or clones that exhibited gametoclonal or 

somatoclonal variation for one or more features. 

Species/hybrid Section1 Author(s) 

Gametoclonal variation 

P. x berolinensis TxA Lu et al. {In: Chen 1987) 

P. simonii x P. nigra TxA Wu et al. 1981 

P. xiaohei T Liu et al. 1992 

Populussp. ? Chen et al. 1981 

Somatoclonal variation 

P. tremula x P. alba Lxl Michel et al. 1988 

'A= Aigeiros 

L = Leuce (currently termed Populus) 

T = Tacamahaca 

? = unavailable 

growth rates (Ahuja 1983; Antonetti and Pinon 1993; Lester 

and Berbee 1977; Saieed et al. 1994a, 1994b; Serres et al. 

1991). 

Somaclonal Variation Basis· and 

Frequency 

Having demonstrated somaclonal variation, scientific 

and practical questions remain. Where does the variation 

come from, how is it evoked, and can it be avoided? Does 

soma clonal variation have practical use, and if so, how can 

it be evaluated and selected for? Once selected, will the 

variation remain stable throughout the life of a tree crop? 

Ensuring culture stability is a crucial first step to maintaining genetic and phenotypic fidelity of regenerants. 

This applies to mass-propagation protocols and to culture 

systems used for germ plasm preservation (Ahuja 1993; 

Chun 1993; Wang et al. 1993). In the latter case, it is reassuring that prolonged below- or above-zero tissue cold 

storage apparently does not induce somaclonal variation 

(Cyr et al. 1994; Hausman et al. 1994). However, culture 

systems that produce somaclonal variation or rejuvenation involve presumed genetic changes, and these are occasionally observed at a physical level. With somaclonal 

variation, separate studies on the same parental material 

indicate that specific culture conditions can induce genetic 

and somaclonal variation that was not produced by 

gamma radiation (Douglas 1986; Saieed 1994a). Suggested 

causes of variation include minor point mu~ations in 

nuclear or organelle DNA, activation of transposable ele36 

ments, polyploidy, aneuploidy, and epigenetic change 

(Michler 1993; Ostry and Michler 1993). Point mutations 

could have caused some albino variants that Son et al. 

(1993) correlated with the loss of 2 proteins, and other 

minor rearrangements may have caused the isozyme 

variation reported by Noh and Minocha (1990). Nevertheless, genetic analysis suggests that herbaceous 

somaclones principally change at the chromosome level 

rather than single genes (Michler 1993). Poplar somaclonal 

variation also has been correlated with changes at the chromosome level, but this is not universal. Native poplars 

are generally diploid (2n = 38), although some naturally 

occurring triploids and tetraploids exist (Ahuja 1993; Chen 

and Ahuja 1993; Sarkilahti et al. 1988). 

In contrast, poplar callus cultures often demonstrate 

variable ploidy levels (Son et al. 1993), and several poplar 

somaclones were fully or partially tetraploid or exhibited 

a leaf morphology characteristic of polyploid plants 

(Antonetti and Pinon 1993; Jehan et al. 1994; Lester and 

Berbee 1977; Saieed et al. 1994b; Serres et al. 1991). However, some desirable variants from the parental type were 

either diploid or similar to the parental leaf phenotype, 

suggesting that ploidy levels were unchanged (Antonetti 

and Pinon 1993; Lester and Berbee 1977; Saieed et al. 1994a, 

1994b). Again, this reinforces evidence from herbaceous 

culture systems where most beneficial mutations are single 

trait characteristics (Michler 1993). 

Somaclonal variation occurs inconsistently; a variability that presumably relates to tissue source and culture 

conditions. In most systems where somaclonal variation 

is undesirable and detectable by gross morphological 

changes, the variant poplars constitute less than 25 percent of the total regenerant population (Ahuja 1983; 

Antonetti and Pinon 1993; Noh and Minocha 1990; Son et 

al.1993). Nevertheless, after observing the distribution of 

inter- and intra-treatment variation within 166 regenerants 

from 18 differing culture regimes that were subsequently 

aggregated into 4 morphotype groups, Saieed et al. (1994a, 

1994b) concluded that each regenerant should be regarded 

as a separate somaclone. Further evidence for a significantly higher and subtler level of variation is provided 

by Noh and Minocha (1990) who showed a 25 to 34 percent variation for isozyme changes among tested 

regenerants. A considerably higher variation percentage 

among successful regenerants is expected when 

somaclonal selection is employed (Michler 1993) and variants are actively selected in culture (e.g., in systems to 

produce disease resistance or herbicide tolerance) (Michler 

and Haissig 1988; Ostry et al. 1990; Prakash and Thielges 

1989; Riemenschneider et al. 1988; Valentine et al. 1988). 

The frequency of variation also is high among haploid 

plants, because some anther culture regenerants originate 

somatically and because of late chromosomal adjustments; the percentage of haploid cells decreases with age 

(Lu and Liu 1990). 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 
Induction of Somaclonal 

Variation in Culture 

Variability in the apparent level of induction suggests 

certain predisposing conditions. These conditions include 

differences in the inherent genetic variability within the 

parental material, variation with the type of culture, agerelated changes amplified by the duration of culture or 

repeated subculturing, and dosage-related effects associated with the use of potentially mutagenic chemicals 

(Parfitt and Arulsekar 1987; Skirvin et al. 1994). 

Genotype significantly influences the ability to micropropagate poplars (Rutledge and Douglas 1988), and evidence from herbaceous systems and Musa (Hwang and 

Ko 1986) suggests that intra generic differences affect culture stability and the level of somaclonal induction. In a 

broad survey of poplar regenerants, Antonetti and Pinon 

(1993) examined 1,092 regenerants from 13 clones and identified 44 morphological and disease-resistant somaclones 

representing 8 parental lines. They suggested that 

somaclonal induction might occur more frequently in species from the Leuce (currently termed Populus) section than 

from Aigeiros or Aigeiros x Tacamahaca hybrids. Circumstantial support for this hypothesis is that Leuce calli can 

be grown on modified MS (Murashige and Skoog 1962) 

medium without potentially mutagenic growth regulators, 

and that only 1 of their 5 stable clones was from Leuce 

(table 4). Furthermore, although the calli were heteroploid 

or tetraploid and most somaclones were tetraploid, the 

more pronounced Leuce variants were diploid. This suggests that Leuce somaclones might be induced without 

undergoing major chromosomal change. 

In contrast, Ostry et al. (1994) suggested that the Aigeiros x 

Tacamahaca (d 'NE299') (P. nigra var. betufolia x P. trichocarpa) 

clone is particularly unstable in culture. In addition, in a limited study of herbicide tolerance in 4 different clones, 2 

Somaclonal Variation in Populus: An Evaluation 

Aigeiros x Tacamahaca clones produced the lowest numbers 

of tolerant microplants, although they yielded more 

microshoots than either the Leuce or Tacamahaca lines 

(Michler and Haissig 1988). Unfortunately, the separate studies cannot be fully compared since culture and regeneration 

conditions were not identical for all lines. 

Ignoring duplications, a survey of published reports on 

somaclonal variation at the species or hybrid level (table 

2) indicates the following: Leuce (6), Aigeiros (6), 

Tacamahaca (2), Aigeiros x Tacamahaca hybrids (4). Given 

that Tacamahaca is the largest section of the genus and 

that most cultivated poplars are in section Aigeiros, Leuce 

does seem to dominate. However, Leuce species may be 

over represented because they are prime candidates for 

micropropagation due to difficulties in stem cutting propagation (Ahuja 1987b; Ernst 1993; Zsuffa 1992). 

As in herbaceous systems, studies of poplar link 

somaclonal induction to aspects of culture history (Ahuja 

1983; Iordan-Costache et al. 1991; Jehan et al. 1994; Ostry 

et al. 1994). Lester and Berbee (1977) were concerned that 

their observed variation was associated with incomplete 

elimination of culture viruses. This was not observed in 

subsequent poplar studies, but it has been suggested that 

the explant source may be a contributing factor. Ostry et 

al. (1994) produced calli from several tissue sources and 

showed that only regenerants from hardwood cuttings 

were resistant to somaclone formation. The cultural stability of tissue derived from hardwood cuttings may correlate with the general clonal fidelity of poplars propagated 

by rooted stem cuttings. While studying somaclonal variation, Saieed et al. (1994a) demonstrated that propagation 

by stem cuttings ensured phenotypic stability among individual somaclones and the parental ('TT32') clone from 

which the original callus was derived. 

Evidence from herbaceous plants suggests that somaclones are more likely when protocols involve callus formation. Poplar micropropagation systems designed .to 

ensure donal stability generally involve little or no callus 

formation because it is assumed that callus or suspension 

Table 4. Taxonomic distribution of Populus species, hybrids, or clones that seemed stable in culture. 

Species/hybrid 

Populus ciliata 

P. x euramericana cv. 'Guariento' 

P. x euramericana cv. 'L Avanzo' 

P. x euramericana cv. 'Carpaccio' 

P. x interamericana cv. 'Beaupre' 

P. tremula x P. tremuloides; cl. '333-4' 

1 A = Aigeiros 

L = Leuce (currently termed Populus) 

T = Tacamahaca 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Section1 

T 

AxA 

AxA 

AxA 

AxT 

Lxl 

Author(s) 

Cheema 1989 

Antonnetti and Pinion 1993 

Antonnetti and Pinion 1993 

Antonnetti and Pinion 1993 

Antonnetti and Pinion 1993 

Antonnetti and Pinion 1993 

37 
Section I In Vitro Culture 

cultures have an inherent risk of somaclonal induction 

(Ahuja 1987a, 1987b; Ahuja et al. 1988; Jehan et al. 1994; 

Ostry et al. 1994; Park and Son 1988). Saieed et al. (1994a) 

compared 52 plants (stem cuttings from primary 

regenerants) from suspension culture with those from 

short- and long-term callus culture. They suggested that 

factors related to callus culture duration or repeated subculturing, rather than callus formation, contribute to instability. Degenerative changes (i.e., the ability of cells to 

proliferate or regenerate) were shown to increase with 

poplar callus age. For example, 2,4-D /BA (2,4-dichlorophenoxyacetic acid/benzyladenine) combinations induced 

regeneration or protoplast division in six-month old P. alba 

suspension cultures, but not in cultures over one-year old 

(Sasamoto et al. 1995). Other poplar studies were similarly 

explained or interpreted (Chen 1987; Jehan et al. 1994; Ostry 

and Skilling 1988), and studies on other diverse woody 

species offer further support (Ahuja 1987a; Domir and 

Schreiber 1993; Donovan et al. 1994; Gmitter et al. 1992; 

Hammerschlag 1992). 

Conversely, Cheema (1989) demonstrated that plants can 

be regenerated by either organogenesis or somatogenesis 

from long-term subculture systems with no immediately 

obvious somaclonal variation. Similarly, micropropagated 

apple shoots were repeatedly subcultured for up to 9 years 

without inducing discernible changes in the bark or leaf 

characteristics of regenerants (Webster and Jones 1992). 

This suggests that other factors, such as specific concen~ 

trations of. certain growth regulators, may have roles as 

mutagenic agents (Ahuja 1987a; Bayliss 1973}. 

Some Leuce cultures were maintained without regulators (Antonetti and Pinon 1993}, but controlling culture 

maintenance and regeneration usually required concentrations of potentially mutagenic regulators such as BA, 

NAA (a-naphthalEmeacetic acid), and 2,4-D (Cheema 1989; 

Douglas 1982) that were greater than endogenous amounts. 

Auxins in particular are known to enhance DNA methylation and their elimination fro~ culture systems might reduce the incidence of somaclonal variation (Phiilips et al. 

1990). In attempts to reverse tissue maturation changes, 

rejuvenation in shoot apices of diverse tree species was 

achieved by repeated subculturing in the presence of cytokinins (Thorpe et al. 1991). 

Some studies attempted to assess the long-term implications of using particular regulators or regulator combinations. Following a three-year study of regenerants from 

calli exposed to varying combinations of 2,4-D and BA, 

Saieed et al. (1994a) observed a simple negative relationship between final growth performance and the applied 

BA concentration range (0.25, 0.50, or 1.00 mg/1). Theresponse to the 2,4-D concentration range (0.1 to 0.6 mg/1) 

in callus culture was more complex, but superior lines were 

associated with either 0.1 or 0.4 mg/12,4-D. These results 

suggest that the chemical environment of final calli stages 

may have amplified or exerted a secondary screening af38 

feet on potential variation. Comparable screening of aneuploid cells by cytokinins was demonstrated in polymorphic, herbaceous callus cultures (Dolezel and Novak 1985; 

Lavania and Srivastava 1988). 

Poplar Somaclonal Stability 

Many soma clones are nonadaptive and should probably 

be screened out during post-regeneration steps. Some recorded variation would be beneficial if it becomes stably 

fixed in somaclones exhibiting otherwise normal characteristics (Michler 1993; Ostry and Michler 1993). Heritable 

somaclonal stability must be confirmed by genetic analysis following sexual reproduction. Nevertheless, in an 

asexually propagated genus such as Populus, the demonstration of stability following vegetative propagation may 

allow practical exploitation of beneficial traits (Ostry and 

Skilling 1987; Skirvin et al. 1994). 

Several authors report a variation in disease resistance 

or growth potential in somaclones that are superior or inferior to the parental line (Liu et al. 1992; Ostry and Skilling 

1988; Prakash and Thielges 1989; Riemenschneider et al. 

1988; Saieed et al. 1994a, 1994b; Son et al. 1993). These individual traits are often identified or selected in culture 

and are clearly sustained through the 51 (primary regeneration) phase. However, questions of long-term stability 

arise; whether the desirable 51 features will be continually 

expressed throughout the plant's life span under varying 

environmental conditions, and whether they persist into 

the 52 stage following further vegetative or sexual propagation. A degree of instability might be expected in haploid regenerants as the growing points change to approach 

the more normal diploid condition (Chen and Ahuja 1993). 

Wu et al. {1981) observed a mixture of stability and late 

developing changes in leaf characteristics of haploid P. 

simonii. Such random changes might produce further material for screening and selection purposes (Chen 1987; 

Ernst 1993; Lu and Liu 1990). 

In contrast, diploid or tetraploid plants are expected to 

exhibit greater levels of long-term stability. Some 

protoclones identified by Serres et al. (1991) expressed 

morphological variances in the greenhouse but not under 

field conditions, and vice versa. Variation in resistance to 

Melampsora rust identified by Antonetti and Pinon (1993) 

was not immediately detectable, but appeared in 2 

somaclones after a growing season in the field. Alternatively, the variant leaf morphology of many individual 

somaclones appeared stable over periods ranging from 4 

months to 3 years and persisted through a secondary 

propagation phase (Lester and Berbee 1977; Michler and 

Haissig 1988; Saieed et al. 1994a). These observations are 

reconcilable and the differences are probably due to norUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
l.ol 

mal adjustment to environmental change or internal maturation processes. For example, the leaf length to width ratio appears to show an age-related decline even in a stable 

poplar clone (Pieters 1974). 

Long-Term Field Stability 

Silvicultural applications of clonal forestry indicate that 

field trials are required for several years to fully evaluate 

the relative performances of nonsomaclonal poplar lines 

(Zsuffa 1992). Somaclones are a strong argument for longterm trials due to the need to prove field stability. Some 

studies have been promised (Iordan-Costache et al. 1991), 

and our own study of stability among poplar somaclones 

was extended to 8 years from regeneration. 

Saieed (1990) investigated 166 ('TT32') poplars from calli 

after 16-months culture with 6 different 2,4-D concentrations, then regenerated in 1985 with 3 different BAconcentrations. Regenerated plantlets from 15 of these 18 

treatment combinations (designated as lines A- 0) survived to potting. The variation in several morphological, 

physiological, and leaf gas exchange traits was evaluated 

Somaclonal Variation in Populus: An Evaluation 

among the primary regenerants in 1986, and in their 52 

vegetative propagules in 1987 and 1988. Qualitative differences among lines were detected by the end of 1986, 

most of which were statistically confirmed within the threeyear period. Significant correlations were detected between 

gas exchange parameters and morphological characters, 

with some lines (especially C, F, and G) performing better 

than the parental reference clone over a large range of parameters, while others (especially A, H, and M) performed 

worse. The relative ranking among the experimental lines 

remained consistent in successive years despite significant 

changes in individual parameters induced by annual 

growth changes (Saieed et al. 1994a). 

In 1989, the 105 surviving 52 plants (3 to5 per somaclone) 

were planted outdoors in a semi-randomized block at 

Kinsealy Research Centre, Dublin, and investigated for 

morphological growth characteristics between August 

1992 and January 1993. Some previously investigated parameters were reassessed, such as height, stem diameter, 

and characteristics of fully-expanded, primary sun leaves. 

Leaf thickness was investigated for the first time and displayed an inverse correlation with leaf length or area. The 

comparative performances of these lines are in table 5, 

where the accumulated data (except leaf thickness) are 

ranked in descending numerical order. The ranking among 

....., Table 5. Ranked tabulation in descending numerical order (except leaf thickness) of mean values1 for morphological 

measurements on field-grown plants of 15 poplar somaclonal lines A-02 and the ('TI32') parental reference line3• 

Stem characters Leaf characters 

Height Diameter FW ow o/oH2Q4 Length Width Area Thickness5 

F Ref Ref Ref Ref Ref Ref Ref Ref 

G G G G 0 F F F F 

Ref F B F K c G G c 

c c F c F G c c G 

B B A H A 0 E E E 

E E H A G E H 0 B 

0 0 0 E H B A B 0 

N N c B B M B H N 

A A M 0 E H 0 A H 

H H E M c A K M I 

K K N I M K M K M 

M J L L N L N L J 

L I I N L N L N L 

I 0 K K I 0 I I A 

J L J 0 J I J J 0 

0 M 0 J 0 J 0 D K 

1 Parameters measured August 1992 - January 1993 (3-5 plants per line). 

2 Lines regenerated from fifteen 2,4-0/BA regimes, designated as treatments A - 0. 3 Reference line produced by vegetative propagation from the parentai'TI32' clone. 4 %H20 = % leaf water content 5 Leaf thickness ranked in ascending numerical order. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 39 
Section I In Vitro Culture 

lines is fairly consistent across the range of parameters, 

with the reference and somaclonallines C, G, and F dominating. No significant differences were apparent in stem 

height or diameter among lines F, G, and the reference; 

these lines significantly outperformed line A and all lower 

ranking lines for these parameters (P > 0.999). This overall 

performance pattern is comparable to that obtained by 

Saieed {1990) and further demonstrates the long-term stability of the somaclones. Lineberger et al. (1990) used a 

comparable ranking technique as evidence of the fixation 

of somaclonal variation following a secondary propagation phase in elm somaclones. The original growth advantage of lines F and G over the reference appears to have 

eroded and it is unknown whether these somaclones will 

retain superior performance in respect to gas exchange or 

total biomass. No gas exchange measurements were conducted in 1992, but estimates of abaxial stomatal frequency 

were obtained for 5 lines. Frequencies for the reference and 

somaclonallines C and G were essentially comparable, and 

were twice as high as those of lines K and H. This corresponds with a previous demonstration of high photosynthetic performances in lines C and G, and poor gas 

exchange by lines K and H {Saieed et al. 1994a). These results provide continuing evidence of stability within the 

somaclonallines and reinforce the earlier belief that morphological and gas exchange measurements of newly regenerated plants are useful to predict final growth and 

yield. 

Acknowledgments 

Any new data presented in this paper were collected by 

Aisling Cusack. 

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41 
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42 

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International: 86-97. · 

43 
Chapter6 

Germ plasm Preservation of Populus Through In Vitro 

Culture Systems1 

Sung Ho Son, Young Goo Park, Young Woo Chun, and Richard B. Hall 

Introduction 

Demand for germplasm preservation of various plant 

species has recently increased due to air pollution, climate 

change, acid rain, natural destruction, illegal collection, and 

human impact to ecosystem biodiversity. Forested land in 

the world is rapidly declining at an annual rate of 9.9 million ha (Singh and Janz 1995). As a result, there has been a 

decrease in the genetic pool of forest tree species, including wild genotypes that could serve as future breeding 

sources. 

Seed storage is common for germplasm preservation of 

most plant species. For Populus species, seed longevity 

under natural conditions is from 2 weeks to 1 month, depending on the species and/ or clone, time of collection, 

and environmental conditions (Graham et al. 1964; Trappe 

1964). The longevity of seed storage can be greater than 2 

years by drying the seeds soon after collection, adjusting 

seed moisture content to 8 percent, using sealed containers in cold storage, or storing vacuum-packed seeds under freezing conditions (Trappe 1964). Even then, seed 

prppagation of selected tree genotypes may not guarantee 

the preservation of genetic traits such as fast growth and . 

Because of the vast land area requirement and the difficulty in controlling pests and/ or disease, in situ and ex 

situ conservation of vegetatively propagated plants is challenging. 

In this review we: 1) suggest an alternative approach 

for germplasm preservation through an in vitro culture 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: U.S. 

Department of Agriculture, Forest Service, Rocky Mountain Research Station. 326 p. 

44 

system; 2) discuss methods of germ plasm preservation; 3) 

discuss the ·currently available teclu,ology; and 4) suggest 

future strategies. 

Poplars as a Model System for 

Germplasm Preservation In Vitro 

The genus Populus includes about 30 species that are 

widely distributed throughout the North Temperate Zone 

(between the Arctic Circle and the Tropic of Cancer). This 

wide distribution represents considerable species' adaptability and demonstrates its potential over a wide geographic 

region. Among the woody plants, poplar is one of the most 

intensively studied species for breeding because of the enormous genetic variation that exists. Thousands of poplar 

clones are being tested and hundreds of these are commercially propagated throughout the world (Sa to 1959). Populus 

spp. are fast growers and have modest nutritional requirements, therefore, they are an economically important source 

of pulp for paper industries and are considered an energy 

crop. Also, cryopreservation of cells/tissues requires relatively little space because regeneration of Populus plantlets 

from single-cell/protoplast cultures is possible. Commercial 

production of selected individuals does not depend on seed 

reproduction because vegetative macro and micropropagation methods without adulteration or dilution of 

their genetic potential have been intensively studied (Ahuja 

1987; Park and Son 1988). Problems controlling open pollination under natural conditions makes conservation of taxonomically diverse and newly created clones by in situ or ex 

situ preservation programs for poplar germplasm difficult. 

Development of reliable micropropagation systems is 

essential for germ plasm preservation in vitro. Many reports 

of in vitro propagation technology of poplars have further 

developed this species as a model system for germplasm 

preservation (Aitken-Christie and Singh 1987; Ahuja 1987; 

Moon et al. 1987; Park and Son 1988; Son et al. 1991; Stoehr 
and Zsuffa 1990). In many clones with a high regeneration 

capacity in a microenvironment, plantlets from shoot-tip 

cultures were used in large-scale production and their productivity was found superior to conventionally propagated 

plants (Chen and Huang 1980; Lester and Berbee 1977; 

Mehra and Cheema 1980). Most tissues and organs can 

serve as initial explant materials for in vitro culture of Populus 

spp. Germplasm preservation through in vitro culture is advantageous because: 1) entire sets of genetic materials can 

be copied through regeneration schemes; 2) rapid proliferation is achievable; 3) minimal space is required; 4) diseasefree plants can be produced and maintained; and 5) risks 

related to environmental changes are avoided. 

Low-Temperature Storage 

Low temperature (about 4 °C) has been widely applied 

for short-term, minimal-growth storage of cultured plant 

cells, tissues, and organs. To prepare for nursery planting 

during the optimal growing season, low-temperature storage was used successfully for large-scale synchronization 

of transplanting propagules (Son et al. 1991 ). If suitable 

methods are developed, valuable genotypes can be selected 

at the in vitro level. 

For low-temperature storage in vitro, many techniques 

can be applied individually or in combination. For example, methods related to maintenance of plant materials 

under conditions of a mineral oil overlay (Caplin 1959), 

low-level illumination (Preil and Hoffmann 1985), complete darkness (Marino et al. 1985), reduced temperature 

(Bhojwani 1981 ), various osmotica or plant growth regulators (Henshaw et al. 1978), modified culture atmosphere 

(Moon and Kim 1987), and modified subculturing schedules (Withers 1985) have been described. 

Although nonfrozen storage systems for germplasm 

preservation have been reported, such studies focus on 

herbaceous and/ or crop species and may not apply directly to woody plants. During low-temperature poplar 

storage, the duration, previous subculturing, and various 

culture media affect the survival rate (Son et al. 1991 ). With 

hybrid aspen (Populus alba x P. grandidentata), the previous 

subculturing period and medium composition are critical 

variables for the success of germplasm preservation under low-temperature storage (Son et al. 1991). In our experiment, after 9-months of storage at low temperature, 

hybrid aspen shoots exhibited slow growth, yet continued to possess some healthy, green leaves and stems (figure 1b). After 20-months of cold storage, shoot cultures 

had nearly stopped incremental height growth and had 

thin stems and small, chlorotic leaves. According to one 

report, explant size can greatly influence survival during 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Germplasm Preservation of Populus Through In Vitro Culture SY.stems 

cold storage (Aitken-Christie and Singh 1987). In our experiment, no significant difference in cold-storage survival 

was observed among the five shoot sizes {1, 3, 5, 7, and 9 

em) tested; although 3 em shoots did exhibit a slightly 

higher survival rate. Under low-temperature storage, survival rates after 2-years and 5-years cold storage were 75 

percent and 25 percent, respectively. From 2,000 plantlets 

transplanted in the greenhouse and I or nursery, less than 

1 percent displayed phenotypic variation related to growth 

performance or pigment accumulation. 

Cryopreservation 

Cryopreserved plant cells, tissues, and organs stopped 

their metabolic functions when exposed to ultra-low temperature ( -196 °C); yet they retained viability. In this condition, plant cells, tissues, and organs are preserved for a 

long period of time. Cryopreservation systems can reduce 

the cost of conventional labor-intensive nursery practices 

and minimize genetic changes, such as point mutation 

and I or ploidy level change, which may occur during longterm subculture. However, developing a reliable 

cryopreservation system involves several complicated 

steps. In many examples of cryopreservation, only a few 

species were successfully preserved (Bajaj 1977; Kartha et 

al. 1979; Kartha et al. 1980; Uemura and Sakai 1980). In 

other examples, further investigations are needed to optimize treatments at general stages of pregrowth, 

cryoprotection, freezing, thawing, and recovery (Chen and 

Kartha 1987). Preconditioning, such as cold treatment during culture or applying osmotically active chemicals to the 

medium before cryopreservation, was beneficial for viabiiity (Fuchigami et al. 1981). Plant samples require sufficient 

desiccation before cooling in liquid nitrogen. Without such 

care, most samples produce intracellular ice crystals that 

eventually cause cell mortality. 

When poplar callus was used for ultra low-temperature 

storage, optimal results were obtained with the cells from 

the end of lag phase or exponential growth phase, which 

occurs approximately 1 week after subculture (data is not 

shown). This may relate to lower water content and smaller 

vacuole size of optimal source cells. Pretreatment with 

cryoprotective chemicals, dimethyl sulfoxide (DMSO), 

glycerol, polyvinylpyrrolidone (PVP), and dextran were 

useful separately or in combination. Although its mode of 

action is not clearly understood, DMSO may form multiple hydrogen bonds that prevent water from crystallizing at intracellular levels. Although cryoprotective 

chemicals were harmful to plant cells in our experiment, 

DMSO treatments produced 62 percent survival after longterm preservation; until now, calli were stored for 5 years 

and survival tests were conducted annually. 

45 
Section l .fn Vitro Culture 

Protocols 

Species used for low-tempera ture storage and 

cryopreservation were Populus alba x P. grandide11 tata and 

P. glandulosa (Suwon poplar), respectively. Protocols described here are based on preliminary experiments; supporting data are not included. 

Low-Temperature Storage 

a) Shoot cultures were established by bud cultures 

from greenhouse-grown stock plants. Shoot multip lica tion, elonga tion, and root induction med ia 

were Murashige and Skoog (MS) (Murashige and 

Skoog 1962) med ium containing 1.33 ~1M 6-

benzylad enine (BA), no plant growth regu lators, 

and 0.98 JIM indole-3-butyric acid (IBA), respectively. Cultures were mainta ined under a 16-h light 

regime (photosynthetic photon flux rate of 40 to 

60 1-1M m·

2 s·1 from cool-white, fluorescent tubes) 

at 26±1 °C. 

b) Shoots approximately 3 em long were used (figure 

1a). 

c) Shoots were maintained on shoot multiplication medium for 1 month before cold storage. 

d) Magenta GA-7 culture vessels (7.6 x 7.6 x 10.2 em, 

Magenta Corp., Chicago, IL) containing 50 ml medium and test tubes containing 15 ml of medium were 

used for cold storage. 

c) To prevent drying, culture vessels were double 

sealed with esco film (Banda Chemical Ind., Ltd., 

Kobe, japan) and stored within two transpa rent plastic bags. 

f) During cold storage, cultures were maintained at 4 °C 

without light. 

g) After 4-weeks subculture of cold-stored plants on 

fresh MS medium, each shoot was excised from its 

root system and transferred to a Poly terra ~ p lug 

styrofoam tray (M40045, Techniculture Co., Sa linas, 

CA) and eventually to pots containing artificial soil 

mix (vermiculite: perlite : peat at 1:1:1) (figure lf). 

Cryopreservation 

46 

a) Callus was induced from cambial tissues isola ted 

from i11 vitro-, greenhouse-, and field-grown d onor 

plants. 

Figure 1. Cold storage of in vitro shoot cultures of hybrid 

poplar (Populus alba x P grandidentata). a) Shoot 

cultures before storage. b) Shoot cultures after 9-

months storage at 4 oc. c) Multiplied shoot 

cultures on shoot proliferation medium after 5-

years storage. d) Shoot regeneration from the 

cold-stored materials. e) Regenerated plantlets 

derived from cold-stored shoots. f) Plants established in pots containing artificial soil mix. 

b) Call us proliferation was cond ucted on MS medium 

with 2.26 p M 2,4-dichlorophenoxyacetic acid (2,4-D). 

c) Cold hardening was performed at 10 oc for 1 week 

before placing the ca llus into cryogenic plastic vessels and adding DMSO (callus: DMSO at 1:5 weight/ 

volume) as a cryoprotectant solution. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
c 

d) Further cooling was initiated by incubating the callus on ice for 1 to 2 h. 

e) A freezing temperature ranging from -25 to -45 oc 

was obtained using a programmable freezer controller to drop the cooling temperature by 0.5 to 1 oc per 

minute, followed by liquid nitrogen immersion. 

f) For regrowth of stored callus, temperature was increased in reverse of the freezing procedures but 

quicker. Before culturing, calli were rinsed several 

times with MS liquid medium containing no plant 

growth regulators. 

g) After subculturing on MS basal medium supplemented with auxin at the appropriate levels, newly 

grown callus was collected for use as shoot induction material. 

h) For regeneration, selected callus was subdivided into 

small pieces (5 to 10 mm in diameter) and cultured 

on a shoot induction medium supplemented with 

optimal levels of zeatin (figure 2c). 

b 

Germ plasm Preservation of Populus Through In Vitro Culture Systems 

Limitations 

Plant cell and tissue culture techniques have developed 

rapidly and the knowledge associated with culturing systems has increased significantly in comparison to other 

plant science fields. Based on the theory of totipotency, any 

cells, tissues, and organs should be regenerable; however, 

successful establishment of reliable regeneration systems 

is limited, especially for woody plants. This restriction frequently confines the application of tissue culture methods 

and restricts plant biotechnological applications for many 

valuable species. 

A basic premise of i11 vitro conservation techniques for 

plant germplasm is that genetic materials must be preserved without genetic alteration at any level. Most tissue 

culture work is based on adventitious shoot regeneration 

systems. These systems a re typically associated with a prolonged undifferentiated phase before regeneration. However, such culture systems have generated phenotypic, 

genotypic, and I or biochemical variation (Bebeli and 

Kaltsikes 1990; D' Amato 1978; Whelan 1990). Although this 

variation frequently occurs at negligible levels,' the potential fo r genetic variation should be 

addressed. 

There is also potential for genetic 

alteration associated with low and 

ultra-low temperature storage methods. In a nonfrozen storage system, 

for example, phenotypic variation 

was reported for traits such as color 

accumulation in leaves (Son et al. 

1991 ), organ shape, seed yield, and 

photoperiodic response (Grout 1990). 

During cryopreservation at -196 oc, 

most cellular chemical reactions cannot occur because the energy levels 

are too low to allow sufficient molecular motion to complete enzymatic reactions. Nevertheless, certain 

chemical reactions that affect genetic 

alteration by damaging nucleic acids 

via ionizing radiation can accrue at 

unacceptable levels. Although this 

kind of chromosome damage is observed in plants and microorganisms 

under storage conditions, OJ A repair mechanisms to overcome damage from freezing or low temperature 

conditions are not fu lly understood. 

Figure 2. Cryopreservation of in vitro shoot cultures of poplar (Populus glandulosa). 

a) Cryopreserved callus recently subcultured to proliferation medium. b) 

Regrowth of selected callus. c) Shoot regeneration from callus. d) Plants 

established in pots containing artificial soil mix. 

Most storage systems have not provided 100 percent survival and are 

not adaptable for diverse species. Intensive studies are needed to develop 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 47 
Section I In Vitro Culture 

methodology and accumulate relevant knowledge for successful germplasm storage of target plant materials. 

Conclusions and Prospects 

Due to rapid environmental destruction and associated 

declines in biological diversity, genetic resource conservation has become critical. Recently, major approaches to 

genetic resource conservation such as ex situ and in situ 

preservation programs were established for forestry. Although these approaches have merit, a successful genetic 

conservation program will require modification and/ or 

a combination of existing methods with new technology 

to meet current needs. 

Advances in plant tissue culture systems are widely 

applicable throughout plant science. This technology is also 

a valuable tool for tree germplasm conservation through 

low- or ultra-low temperature storage. Recent progress in 

somatic embryogenesis may provide other opportunities 

for storage material. In particular, somatic embryos exhibit 

high genetic stability and are easily manipulated. Above 

all, relatively simple desiccation methods that reflect the 

physiology and development of zygotic embryos offer 

additional potential for extending the longevity of 

germplasm storage in vitro. · 

Because poplar has demonstrated totipotency at diverse 

cell levels, it is a model species for mass clonal propagation, 

genetic transformation, and other biological research of 

woody plants. For the conservation and preservation of tree 

germplasm, intensive studies are needed to refine associated methods and to provide reliable and reproducible results. 

Acknowledgments 

The authors are grateful to Dr. Gebhart, a senior researcher at Institute of Rapid Growth Tree Species, 

HannMuunden, Germany, for helpful advice and contributions to cryopreservation studies. 

Literature Cited 

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culture in forestry. Dordrecht, The Netherlands: 

Martinus Nijhoff Publishers: 207-223. Vol. 3. 

48 

Aitken-Christie,].; Singh, A.P. 1987. Cold storage of tissue 

cultures. In: Bonga, J.M.; Durzan, D.]., eds. Cell and tissue culture in forestry. Dordrecht, The Netherlands: 

Martinus Nijhoff Publishers: 285-304. Vol. 2. 

Bajaj, Y.P.S. 1977. Clonal multiplication and cryropreservation of cassava through tissue culture. Crop. Improv. 

4: 198-204. 

Bebeli, P.].; Kaltsikes, P.J. 1990. Somaclonal variation from 

cultured immature embryos of sister lines of rye differing in heterochromatic content. Genome. 33: 177-183. 

Bhojwani, S.S. 1981. A tissue culture method for propagation and low temperature storage of Trifolium repens 

genotypes. Physiol. Plant. 52: 187-190. 

Caplin, S.M. 1959. Mineral oil overlay for conservation of 

plant tissue cultures. Amer. ]. Bot. 46: 324-329. 

Chen, D.; Huang. M. 1980. Culture of the apical tissue of 

Populus nigra cv. Blanc de Garonne and the variation of their 

isozymes. ]. Nanjing Tech. Coli. For. Products. 3: 104-107. 

Chen, T.; Kartha, K.K. 1987. Cryopreservation of woody 

species. In: Bonga, J.M.; Durzan, D.]., eds. Cell and tissue culture in forestry. Dordecht, The Netherlands: 

Martinus Nijhoff Publishers: 305-319. Vol. 2. 

D' Amato, F. 1978. Chromosome number variation in cultured cells and regenerated plants. In: Frontiers of plant 

tissue culture. In: Thrope, T.A., ed. Int. Asso. Plant Tissue Culture, Univ. Calgary, Alberta, Canada: 287-295. 

Fuchigami, L.H.; Cheng, T.Y.; Soeldner, A. 1981. Abaxial 

transpiration and water loss in aseptically cultured 

plum.]. Amer. Soc. Hortic. Sci. 106: 519-522. 

Graham, S.A.; Harrison, R.P., Jr.; Westell, C.E., Jr. 1964. 

Aspen, phoenix tree of the Great Lakes region. Ann 

Arbor, Ml, U.S.A.: Univ. Press: 272. 

Grout, B.W.W. 1990. In vitro conservation of germplasm. 

In: Bhojwani, S.S., ed. Plant tissue culture: Application 

and limitations. Amsterdam: Elsevier: 394-411. 

Henshaw, G.G.; O'Hara,] .F.; Westcott, R.J. 1978. Tissue culture and germ plasm storage. In: Sala, F.; Parisi, B.; Cella, 

R.; Ci.fferi, R., eds. Plant cell cultures: Results and perspectives. Amsterdam: Elsevier, North-Holland Biomedical Press: 277-282. 

Kartha, K.K.; Leung, N.L.; Gamborg, O.L. 1979. Freezepreservation of pea meristems in liquid nitrogen and 

subsequent plant regeneration. Plant Sci. Lett. 15: 7-15. 

Kartha, K.K.; Leung, N.L.; Pahl K. 1980. Cryopreservation 

<:>f strawberry meristems and mass propagations of 

plantlets. ]. Amer. Soc. Hort. Sci. 105: 481-484. 

Lester, D.T.; Berbee, ].G. 1977. With-in clone variation 

among black poplar trees derived from callus culture. 

For. Sci. 61: 263-272. 

Marino, G.; Rosati., P.; Sagrati, F. 1985. Storage of in vitro 

cultures of Pnmus root stocks. Plant Cell Tiss. Org. Cult. 

5:73-78. 

Mehra, .P.H.; Cheema, G.S. 1980. Clonal multiplication in vitro 

of Htmalayan poplar (Populus ciliata). Phytomorphology. 

30: 336-343. 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 
Moon, H.K.; Kim, J.H. 1987. Cold storage of Populus spp. 

shoot tips in vitro. Kor. J. Tissue Culture. 14: 137-140. 

Murashige, T.; Skoog, F. 1962. A revised medium for rapid 

growth and bioassays with tobacco tissue cultures. 

Physiol. Plant. 15: 473-497. 

Park, Y.G.; Son, S.H. 1988. In vitro organogenesis and somatic 

embryogenesis from punctured leaf of Populus nigra x P. 

maximowiczii. Plant Cell Tiss. Org. Cult. 15: 95-105. 

Preil, W.; Hoffmann, M. 1985. In vitro storage in chrysanthemum breeding and propagation. In: Schafer-Menuhr, 

A., ed. In vitro technology: Propagation and long term 

storage. Dordrecht, The Netherlands: Martinus Nijhoff 

Publishers:161-165. 

Sato, Y. 1959. Production of poplar timber in Europe and 

its significance and application in the United States. U. 

S. Dep. Agric. Handbook 150: 124. 

Son, S.H.; Chun, Y.W.; Halt R.B. 1991. Cold storage of in 

vitro cultures of hybrid poplar shoots (Populus alba L. x 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Germplasm Preservation of Populus Through In Vitro Culture Systems 

P. grandidentata Michx.). Plant Cell Tiss. Org. Cult. 27: 

161-168. 

Singh, K.D.; Janz, K. 1995. Assessing the world's forest resources. Nature & Resources. 31: 32-40. 

Stoehr, M. U.; Zsuffa, L. 1990. Induction of haploids in 

Populus maximowiczii via embryogenic callus. Plant Cell 

Tiss. Org. Cult. 23: 49-58. 

Trappe, J.M. 1964. Populus trichocarpa seeds endure adversities. Tree Plant. Notes. No. 65: 31. 

Uemure, M.; Sakai, A. 1980. Survival of carnation (Dianthus 

caryophyllus L.) shoot apices frozen to the temperature of 

liquid nitrogen. Plant and Cell Physiol. 21:85-94. 

Whelan, E.D.P. 1990. Behavior of an alien telocentric chromosome in callus culture of wheat. Crop Sci. 30: 923-926. 

Withers, L.A. 1985. Long term storage in vitro cultures. In: 

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49 

I 

\iili:il 

Section II 

"" Transformation and Foreign Gene Expression 
Chapter7 

Agrobacterium-mediated Transformation of Populus 

Species1 

Mee-Sook Kim, Ned B. Klopfenstein, and Young Woo Chun 

Introduction 

Although molecular biology of woody plants is a relatively young field, it offers considerable potential for breeding and selecting improved trees for multiple purposes. 

Conventional breeding programs have produced improved growth rates, adaptability, and pest resistance; 

however, tree improvement processes are time consuming because of the long generation and rotation cycles of 

trees (Dinus and Tuskan this volume; Leple et al. 1992). 

Genetic engineering of trees helps to compensate for conventional breeding disadvantages by incorporating known 

genes into specific genetic backgrounds. Since the first 

successful plant transformation was reported in 1983 

(Herrera-Estrella et al. 1983; Murai et al. 1983), several 

nonsexual gene transfer methods were developed for important agronomic crops and forest tree species. These 

methods include biolistics (microprojectile bombardment), 

electroporation, and Agrobacterium-mediated transformation. Biolistics and electroporation are discussed by Charest 

et al. (this volume). This chapter focuses on Agrobacteriummediated gene transfer methods, which are widely-used 

for plant transformation of broad-leaved, woody plants 

because of their versatility and efficient application 

(Brasileiro et al. 1991; Chun 1994; Han et al. 1996; Leple et 

al. 1992). 

· Agrobacterium spp. are soil bacteria tJ:tat naturally infect 

many dicotyledonous and gymnospermous plants predis-

, Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

posed by wounding (Perani et al. 1986). Infection by A. 

tumefaciens causes crown gall disease (figure 1), whereas 

A. rhizogenes causes hairy root disease. In addition to its 

chromosomal DNA, Agrobacterium contains 2 other genetic 

components that are required for plant cell transformation; T-DNA (transferred DNA) and the virulence (vir) region, which are both located on the TI (tumor-inducing) or 

Ri (root-inducing) plasmid (Zambryoski et al. 1989). The 

T-DNA portion of the A. tumefaciens TI plasmid or the A. 

rhizogenes Ri plasmid is transferred to the nucleus of a host 

plant where it integrates into the nuclear DNA genetically 

transforming the recipient plant. A region of the 1i plasmid outside the T-DNA, referred to as the wirulence region, carries the vir genes. Expression of vir genes occurs 

during plant cell infection and is a prerequisite for the subsequent transfer of the T-DNA. Agrobacterium chromosomal regions are involved in attachment of Agrobacterium 

to plant cells. The T-DNA of A. tumefaciens contains auxin 

{iaaH, iaaM) and cytokinin (IPT) synthesis genes 

(Zambryoski et al. 1989). These genes are referred to as 

oncogenes and are responsible for tumor induction. In A. 

rhizogenes, T-DNA contains multiple rol genes that induce 

root formation (Zambryoski et al. 1989). The T-DNA also 

encodes several genes responsible for the synthesis of compounds called opines, which are metabolic substrates for 

the bacteria (Nester et al. 1984). Efficient transfer ofT-DNA 

is facilitated by 24-base pair direct repeats at the T-DNA 

borders. Genes within the T-DNA can be replaced with 

genes of interest without affecting transfer efficiency (Han 

et al. 1996; Jouanin et al. 1993). 

Members of the genus Populus have a small genome size, 

short rotation cycle, fast growth rate, and the capacity for 

vegetative propagation. In addition, Populus spp. demonstrate developmental plasticity to tissue culture manipulations. These traits and susceptibility to 

Agrobacterium-mediated transformation and techniques to 

regenerate transgenic trees make Populus a suitable mod~ I 

system for genetic engineerin? of deciduou~ trees.~ th1s 

chapter, we describe the ma1n Agrobacterzum-med1ated 

transformation procedures developed for Populus andreview the results obtained using several Populus species. 

51 
Section II Transformation and Foreign Gene Expression 

Figure 1. Crown gall produced by Agrobacterium 

tumefaciens strain A281 infection of hybrid 

poplar (Populus alba x P. grandidentata) stem 

after approximately 1 0 weeks. 

Gene Transfer to Populus Species 

Populus has been known as a natural host for Agrobacteriu111 

for many years. DeCleene and De Ley (1976) cite early literature tha t suggests the susceptibility of 3 Populus species to 

infection by A. tu111ejaciens. The presence of T-ON A sequences 

in gall and root tissue confirmed Populus as a host for A. 

tu111ejaciens and A. rhizogenes (Parsons e t al. 1986; Pythoud et 

al. 1987). These early pathogenicity studies of Agrobacteriu111 

provided the basis for its use as a tool to transfer foreign 

genes into the poplar genome. 

The process for prod ucing transgenic pop la r plants includes 5 main components (figure 2): 1) initiation: starting 

explants (host species/genotype/tissue type) a re selected 

52 

for infection and transformation; 2) infection: wounded starting explants are co-cultiva ted with an Agrobacteriz1111 strain 

containing co-integrate or binary vectors; 3) selection: after 

removal of residual Agrobacterium, transformed cells are selected for subsequent regeneration into transgenic plants (figure 3); 4) regeneration: transformed cells are regenerated 

during or after the selection period (figures3 and 4); and 5) confirmation: the presence or function of transgenes in the genome 

of transgenic plants is confirmed using molecular techniques 

such as polymerase chain reaction, Southern hybridization, 

northern hyb ridization, western blotting, enzyme-linked 

immunosorbent assay (ELISA), or enzyme activity assays. 

Transgenes 

Several silviculturally usefu l genes have been isolated and 

used for Agrobacterium-mediated transformation of Populus. 

A table listing genes used in Populus transformation (Chun 

1994) was updated for this chapter (table 1). These genes 

include the: 1) mutant aroA gene, which encodes glyphosate 

tolerance via a 5-enolpyruylshikimate-3-phosphate synthase 

(EPSP) that is less sensitive to the herbicide glyphosate 

(Donahue et al. 1994; Fillatti et al. 1987); 2) bar gene encoding the enzyme phosphinotricin acetytransferase (PAT) that 

inactivates the herbicide phos.phinotricin (glufosinate) (De 

Block 1990; Devillard 1992); 3) mutant crs1-1 gene from a 

chlorsulfuron-herbicide-resistant line of Arabidopsis thaliana 

(Brasileiro et al. 1992); 4) OCI (oryzastatin), a cysteih proteinase inhibitor, and PIN2 (proteinase inhibitor II), a trypsin / 

chymotryp sin inhibitor gene for pest resistance (Heuchelin 

et al. 1997 this volume; Klopfenstein et al.1991, 1993, 1997; 

Leple et al. 1995); and 5) insecticidal protein genes from Bacillus thuringiensis (Bt) (Howe et al. 1994). Other studies have 

focused on transgene regulation (Chun and Klopfenstein 

1995; Confa lonieri et al. 1994; Kajita et al. 1994; Klopfenstein 

et al. 1991; Leple e t al. 1995; ilsson et al. 1992) and developmental influences (Ah uja and Fladung 1996; Charest et al. 

1992; Ebinuma e t al. 1992; Nilsson e t a l. 1996a, 1996b; 

Schwartzenberg et al. 1994; Sundberg et al. this volume; 

Tuominen et al. 1995; Weigel and Tilsson 1995). 

Transgene Copy Number 

Few stud ies have reported the copy number of inserted 

transgenes by Agrobacteriu111-mediated transformation on 

Populus species. Transgenic microshoots of hybrid aspen (P. 

alba x P tremula) contained from 1 to 3 copies of the inserted 

foreign bar genes (De Block 1990); whereas, in vitro plants (P 

tre111 ula x P alba) regenerated from transformed roots contained 1 copy of the bar gene (Devillard 1992). Only a single 

copy of the chloramphenicol acetyltransferase (CAT) gene 

was inserted into the genome of transgenic hybrid poplar 

(P alba x P. grandidentata) (Klopfenstein et al. 1991). In addition, 1 to 4 copies of crs1 -1 gene had been inserted per hyUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Agrobacterium-mediated Transformation of Populus Species 

••• ... Wounding 

INITIATION 

.:!: Dark 

~ Conditions 

Field Test 

t 

CONFIRMATION 

(e.g .. Southern blot. PCR. 

northern hybridization . 

western blot. ELISA. 

'I= =I' 

Preculture (CIM or SIM) 

Greenhouse 

Growth 

and/or enzyme activity assay) 

'I = I' INFECTION 

/ Co-cultivation with 

A. tumefaciens or 

A. rhizogenes .:!: Secondary selection and In vitro propagation 

I ' regeneration to avoid 

chimeric transformants 

SELECTION 

REGENERATION 

Decontamination 

_:!: preselective culture 

'I.Je *' I' 

Selection of transformed 

cells 

:!: Additional selection for root 

formation in selective media 

Figure 2. The primary steps for Agrobacterium-mediated transformation of Populus species. CIM=callus inducing medium; 

SIM=shoot inducing medium. 

brid aspen (P. tremula x P alba) genome (Brasilciro ct a l. 1992). 

Also, Howe ct al. (1991) showed that the number of inserted 

0 A copies ranged from 1 to 10 after the maize transposable 

element Ac (Activator) was transferred into hybrid poplar (P 

alba x P grmzdidentata). However, it is unknown if all inserted 

gene copies were expressed (Chun 1994; Leple et al. 1992). 

Agrobacterium-mediated 

Transformation 

Host Species/Genotype/Tissue Type 

A prerequisite for any genetic transformation work using Agrobacterium is the ability of the bacterium to infect 

the plant of interest. The effect of 2 Agrobacterium 

tumefaciens strains, A281 and A348, on infection of P. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

triclzocarpa x P. del/aides (Parsons et al. 1986) was studied 

and add itional information was gathe red on the effect of 

popla r genotypes (Charest et al. 1992). Prev ious studies 

showed significan t differences among the geno types 

w ith in species and the clones with in gen otype 

(Confa lonie ri et al. 1994; De Block 1990; Riemenschneider 

1990). A differential response of Leuce (currently termed 

Populus) section cultivars to infection by A. tumefaciens 

was described by Nesme et a!. (1987), and susceptibility 

of aspen cu ltivars to A. tumefaciens was correlated to cytokinin sensitivi ty by Beneddra eta!. (1996). In addition, intra- and inter-specific hybrid poplars coming from Aigeiros 

or Tacamahaca sections differed in suscep tibility to A. 

lumefaciens C58 strain (Riemenschneider 1990). 

It is criti cal to select approp ria te starting materia ls 

(or explants) fo r Agrobacterium-mediated transformation . Po tentially, explant materia l can be derived from 

seed ling, leaf, in te rn ode, petio le, root, call us, or other 

cells, tissues, and organs. In vitro cultured leaves and 

internodes (stems) have been used most often to trans53 
Section II Transformation and Foreign Gene Expression 

Figure 3. Regeneration of a transformed shoot on selective medium. After co-cultivation of hybrid poplar 

(Populus alba x P grandidentata) leaf pieces 

with Agrobacterium tumefaciens containing 

NOS-NPT/1 and PIN2-CAT genes, transformants 

were selected on Murashige and Skoog (MS) 

{1962} regeneration medium supplemented with 

40 1-1g/ml kanamycin. 

form many Populus species. Greenwood stem internode sections of P. tremuloides are the most susceptible 

to tumor fo rmation and leaf disks are the least susceptible (Kubisiak et a l. 1993). Leple e t a l. (1992) showed 

that inte rnode explants of P. tremula x P. alba produced 

more transformed calli than leaf explants. 

A suspension culture transformation system for inserting genes into poplar might offer severa l advantages including: 1) the ability to screen large numbers 

of potentially transformed cells; 2) effective inhibition 

of residual Agrobacterium following co-cultivation; and 

3) hig h transformation frequencies d ue to rapi dl y dividing s uspension cultures that may be amenable to 

stable integration of foreign D A (Howe e t al. 1994). 

However, it is freque ntly unknown w hi ch cell type 

within an explant is the most transformable or the most 

capable of regenerating into a ferti le plant. The small 

amount of availab le d a ta indicates that the most regenerable cells do no t necessarily correspond with the most 

transformable cells (De Block 1993). 

54 

Figure 4. Secondary selection of transformants occurred 

on Murashige and Skoog (MS) rooting medium 

containing 20 1-1g/ml kanamycin. Rooted plantlets 

of transgenic hybrid poplar (Populus alba x P 

grandidentata) were propagated in vitro 

(Klopfenstein et a l. 1991 ). 

Agrobacterium Strain 

To assure high infectivity levels for effective transformation, the most suitable Agrobacterium strain should be 

determined for each host species I genotype I tissue. Generally, tree species respond better to the nopaline strains 

than octopine strains of A. tumefaciens (Ahuja 1987). Most 

transgenic poplars have been produced using nopaline 

strains of Agrobacterium (Han e t a l. 1996). The p lasmid 

rather than the chromosomal background was the most 

critical determinant for infection (Kubisiak et al. 1993). 

However, influence of plasmid type on infection levels has 

varied with host species/genotype/tissue type (Kubisiak 

et al. 1993). 

Two designed vector systems are used in Agrobacteriummediated transformation: 1) co-integrate: T-D A includes 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Agrobacterium-mediated Transformation of Populus Species 

Table 1. Transformation research using Agrobacterium-mediated transformation systems with Populus species. 

Bacterial 

Species Transgenes 1 spp.3 Reference 

P. trichocarpa x P. deltoides T-DNA2 A.t. Parsons et al. 1986 

P. trichocarpa x P. deltoides T-DNA A.r. Pythoud et al. 1987 P. trichocarpa x P. deltoides bar, NPT/1 A.t. De Block 1990 P. trichocarpa x P. deltoides GUS, NPT/1 A.t. Wang et al. 1994 

P. alba x P. grandidentata aroA, NPT/1 A.t. Fillatti et al. 1987 P. alba x P. grandidentata CAT, NPT/1 A.t. Klopfenstein et al. 1991 P. alba x P. grandidentata aroA, NPT/1 A.t. Donahue et al. 1994 P. alba x P. grandidentata Ac, Bt, HPT, NPT/1 A.t. Howe et al. 1994 P. alba x P. grandidentata PIN2, NPT/1 A.t. Klopfenstein et al. 1997 

P. alba x P. glandulosa T-DNA A.r. Chung et al. 1989 

P. davidiana T-DNA A.r. Lee et al. 1989 

P. tomentosa CAT, NPT/1 A.t. Wang et al. 1990 

P. alba x P. tremula bar, NPT/1 A.t. De Block 1990 P. tremula x P. alba GUS, NPT/1, T-DNA A.t. Brasileiro et al. 1991 P. tremula x P. alba crs1-1, NPT/1 A.t. Brasileiro et al. 1992 P. tremula x P. alba bar, NPT/1 A.r. Devillard 1992 P. tremula x P. alba GUS, NPT/1 A.t. Leple et al. 1992 P. tremula x P. alba IPT, NPT/1 A.t. Schwartzenberg et al. 1994 P. deltoides x P. nigra T-DNA A.t.IA.r. Charest et al. 1992 P. deltoides x P. nigra PIN2, NPT/1 A.t. Heuchelin et al. 1997 P. nigra x P. maximowiczii T-DNA A.t.IA.r. Charest et al. 1992 

P. sieboldii x P. grandidentata iaaM, GUS, MPT/1 A.t. Ebinuma et al. 1992 

P. sieboldii x P. grandidentata prxA 1, GUS, NPT/1 A.t. Kajita et al. 1994 

P. sieboldii x P. grandidentata GR, NPT/1 At. Endo et al., this volume 

P. tremula x P. tremu/oides luxF2, HPT, NPT/1 A.t. Nilsson et al. 1992 

P. tremu/a x P. tremu/oides OC/, NPT/1 A.t. Leple et al. 1995 

P. tremula x P. tremu/oides OCI, NPT/1 A.t. Leple et al. 1995 

P. tremula x P. tremu/oides iaaH, iaaM, HPT, NPT/1 A.t. Tuominen et al. 1995 

P. tremula x P. tremu/oides LFY, NPT/1 A.t. Weigel and Nilsson 1995 

P. tremula x P. tremu/oides Ac, ro/C, NPT/1 A.t. Ahuja and Fladung 1996 

P. tremu/a x P. tremuloides GUS, HPT A.t. Nilsson et al. 1996a 

P. tremula x P. tremuloides ro/C, NPT/1 A.t. Nilsson et al. 1996b 

P. tremula x P. tremu/oides phyA, phyB, NPT/1 A.t. Sundberg et al., this volume 

P. tremuloides T-DNA A.t. Kubisiak et al. 1994 

P. tremuloides GUS, NPT/1 A.t. Tsai et al. 1994 

P. nigra GUS, HPT, NPT/1, T-DNA A.t. Confalonieri et al. 1994 

P. nigra GUS, NPT/1, T-DNA A.t. Confalonieri et al. 1995 

P. tremula Ac, roiC, NPT/1 A.t. Ahuja and Fladung 1996 

P. deltoides T-DNA A.t. Riemenschneider 1990 

P. deltoides GUS, NPT/1 A.t. Dinus et al. 1995 

1 Ac (Activato,~transposable element from maize; aroA=bacterial5-enolpyruvylshikimate-3-phosphate synthase chimeric gene; 

bar=phosphinotricin acetyltransferase gene; Bt=endotoxin gene from Bacillus thuringiensis; CAT=chloramphenicol 

acetyltransferase gene; crs 1-1=mutant acetolactate synthase gene; GR=glutathione reductase gene; GUS=~-glucuronidase gene; 

HPT=hygromycin phosphotransferase gene; iaaH=agrobacterial indoleacetamide hydrolase gene; iaaM=agrobacterial tryptophan 

monooxygenase gene; /PT=agrobacterial isopentenyltransferase gene; LFY=flower-meristem-identity gene; luxF2=1uciferase 

gene; NPT//=neomycin phosphotransferase gene; OC/=cystein proteinase inhibitor g~ne; phyA, phyB=phytochrome ge~es; 

P/N2=wound-inducible potato proteinase inhibitor II gene; prxA 1=peroxidase gene; and ro/C=one of the genes responsible for 

hairy root disease, caused by the Agrobacterium rhizogenes 

2 Transferred DNA 

3 A.t.=Agrobacterium tumefaciens; A.r.=Agrobacterium rhizogenes 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 55 
Section II Transformation and Foreign Gene Expression 

gene(s) of interest with a selectable marker gene instead 

of oncogenes on the Ti-plasinid; and 2) binary: T-DNA is 

located on a separate vector plasmid instead of the Ti-p lasmid. T-DNA also includes the gene(s) of interest and selectable marker gene (Walkerpeach and Velten 1994}. No 

recombination event is necessary for the binary vector system, unlike the co-integrate vector system. Overall, A. 

tumefaciens strains C58, A281, EHA101, and LBA4404 were 

commonly used with binary vectors for transformation of 

many poplars and seem to generate suitable transformation efficiencies (Brasileiro et al. 1991, 1992; Confalonieri 

et al. 1994, 1995; De Block 1990; Ebinuma et al. this volume; Howe et al. 1994; Kajita et al. 1994; Klopfenstein et 

al. 1991, 1993, 1997; Leple et al. 1992, 1995; Nilsson et al. 

1992; Schwartzenberg et al. 1994; Sundberg et al. this volume; Tuominen et al. 1995). 

Transformation Procedures 

Several factors should be considered to improve transformation efficiency such as the Agrobacterium inoculum 

titer, vir inducer, selectable marker system, and in vitro tissue culture manipulation techniques. Optimal results were 

obtained by dipping initial host explants into a bacterial 

suspension (5 to 6 x 108 cells/ ml) for 20 min to 4 h, then cocultivating them for 24 to 72 h on a liquid or semisolid 

regeneration medium that contained plant growth regulators such as benzyladenine (BA}, 2,4-dichlorophenoxyacetic acid (2,4-D}, naphthaleneacetic acid (NAA}, or 

thidiazuron (TDZ) (Confaloniei et al. 1994; Wang et al. 

1994). 

Acetosyringone (AS) and hydroxy-acetosyringone (OHAS) elicited the expression of Agrobacterium vir region genes 

(Stachel et al. 1985). AS and OH-AS occur specifically in 

exudates of wounded and metabolically active plant cells 

and perhaps allow Agrobacterium to recognize susceptible 

cells (Stachel et al. 1985). Transformation efficiency could 

be increased during co-cultivation by using a vir region 

inducer such as AS (10 to 200 ~M) (Confalonieri et al. 1995; 

Howe et al. 1994; Kubisiak et al. 1993; Nilsson et al. 1992; 

Weigel and Nilsson 1995). 

A practical selectable marker system is essential to obtain high efficiency transformations while avoiding 

nontransformed plants that escape selectioh (Leple et al. 

1992). Selectable marker genes used for Populus transformation have encoded traits such as hygromycin resistance 

(hygromycin phosphotransferase; HPT), neomycin resistance (neomycin phosphotransferase II; NPTII), 

phosphinotricin (glufosinate) resistance (phosphinotricin 

acetyltransferase; bar), and chlorsulfuron resistance (mutant acetolactate synthase; crs1-1). Because the NPTII gene 

has been frequently employed in several woody plants 

including Populus species to select transformants (table 1), 

kanamycin is one of the most commonly used antibiotics 

56 

for a transformation selection system. Even modest kanamycin concentrations (10 mg/1) can inhibit regeneration 

of untransformed hybrid poplar (P. alba x P. grandidentata) 

(Chun et al. 1988). Culture on nonselective medium (without selective antibiotics) for 2 days to 2 weeks before transfer to a selective medium (with selective antibiotics) has 

been used to obtain higher transformation frequencies 

(Charest et al. 1992; Dinus et al. 1995; Tuominen et al. 1995; 

Wang et al. 1994). 

The transfer of explants to light conditions after decontamination using cefotaxime (250 to 500 mg/1) and/or 

carbenicillin (250 to 500 mg/1), a preculture (shoot-inducing or callus-inducing medium induding BA, 2,4-D, NAA, 

or TDZ) period before Agrobacterium-mediated infection, 

or a prolonged infection period can enhance transformation frequencies dramatically (Confalonieri et al. 1994, 

1995; De Block 1993; Leple et al. 1992; Schwartzenberg et 

al. 1994; Tsai et al. 1994). Several studies demonstrate that 

the Agrobacterium plasmid, explant type, in vitro techniques, 

and use of a vir region inducing compound can substantially influence stable transformation frequency 

(Confalonieri et al. 1994, 1995; De Block 1990; Kubisiak et 

al.1993). 

Reporter genes used to detect transgene expression have 

included CAT, rl-glucuronidase (GUS), and luciferase 

(luxF2) genes (table 1). To date, GUS has been used most 

often and has been effective as a reporter gene in poplar 

(Jouanin and Pilate this volume; Pilate et al. this volume). 

Use of luxF2 as a reporter allows in vivo monitoring of gene 

expression by nondestructive imaging (Nilsson et al. 1992; 

Schneider et al. 1990). Inhibitors present in poplar leaf extracts can interfere with CAT-activity assays reducing the 

advantage of CAT as a reporter gene in poplar (Klopfenstein 

et al. 1991). 

Limitations and Prospects 

Although transformation technology has reached a relatively advanced level, many variables exist that can interfere with the generation of stable transformed plants that 

express transgenes in a predictable manner (Ahuja this 

volume; De Block 1993). Recently, there have been several 

papers about the quantitative and qualitative instability 

of transgenes in primary transformed plants and subsequent generations (reviewed by De Block 1993; Ahuja this 

volume). Agrobacterium-mediated transformation is believed to result in random integration of transgenes into 

the genome causing high variation in quantitative and 

qualitative expression levels of transgenes in primary 

transformants and I or subsequent generations. However, 

an Agrobacterium-mediated system is a desirable method 

USDA Forest Service Gen. Tech: Rep. RM-GTR-297. 1997. 
to transform Populus because it is relatively inexpensive, 

easy to use, can produce an acceptable transformation rate, 

and transfers a limited copy number of transgenes. 

Acknowledgments 

This paper was supported in part by the USDA Forest 

Service, funds from contract #DOE OR22072-17 with the 

Consortium for Plant Biotechnology Research, Inc., and 

the Biotechnology Graduate Research Associateship program of the Center for Biotechnology, University of Nebraska-Lincoln. Use of trade names in this paper does not 

constitute endorsement by the USDA Forest Service. 

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59 
ChapterS 

Direct Gene Transfer in Poplar1 

Pierre J. Charest, Yvonne Devantier, Cathy Jones, Jim C. Sell mer, 

Brent H. McCown, and David. D. Ellis 

Introduction 

Agrobacterium-mediated genetic transformation and regeneration of transgenic Populus has been achieved with selected 

genotypes (Kim et al. this volume). Direct gene transfer 

using electroporation and microprojectile-mediated DNA 

delivery has also been accomplished (Chupeau et al. 1~94; 

Devantier et al. 1993; McCown et al. 1991), with successful 

regeneration of transgenic Populus from a few genotypes 

(Chupeau et al. 1994; McCown et al. 1991). Microprojectile 

DNA delivery has been also used to study transient expression of chimeric genes (Devantier et al. 1993). 

Several differences exist between Agrobacterium-mediated DNA delivery and direct gene transfer. Using 

Agrobacterium, only a fragment of the transformation vector within the T-DNA border of the TI plasmid is transferred to the host cell and, in most cases, only 1 copy or a 

few copies of this fragment are integrated into the host 

genome. With direct DNA delivery, the whole transformation vector is introduced into the host cell and often 

several copies of the delivered DNA are integrated into 

the host genome. Although Agrobacterium-mediated DNA 

delivery is a more precise transformation method, direct 

DNA delivery has two advantages; it is genotype independent, and it bypasses the long life cycle of tree species 

by delivering DNA into a variety of plant tissues for gene 

expression studies (Charest et al. 1993). For instance, somatic and zygotic embryos, embryonal masses, pollen, 

germinated seedlings, flower organs, differentiating 

wood, needles, and vegetative buds of coniferous trees 

were used for transient gene expression using micro1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

60 

projectile-mediated DNA delivery (reviewed by Aronen 

et al. 1994, 1995; Charest et al. 1993; Ellis 1995). Electroporation is less flexible because, as with protoplast culture, it is often genotype dependent and has a more 

restricted range of tissues that respond to the procedure 

(Adbudl-Baki et al. 1990; Dekeyser et al. 1990; D'Halluin 

et al. 1992; Laursen et al. 1994; Luong et al. 1995; Songstad 

et al. 1993; Xu and Li 1994; Yang et al. 1993). Both microprojectile-mediated DNA delivery and electroporation 

could be useful alternatives to Agrobacterium-mediated 

transformation for recalcitrant genotypes or transient gene 

expression studies. Regeneration of transgenic poplar trees 

following Agrobacterium-mediated transformation, which 

required several months to obtain the transgenic plant 

material, was used for gene expression studies (Leple et 

al. 1992; Miranda Brasileiro et al. 1992; Strohm et al. 1995). 

Similar results to evaluate the relative strength of promoters could have been obtained with microprojectile-mediated DNA delivery within a few days. 

The most limiting factors to enhance foreign· DNA integration and/or the recovery of stable transformants for 

direct DNA delivery are the host cell biology and defining 

cellular factors that interact with the introduced DNA. 

Experimental protoplast systems can be used for genetic 

transformation but assessing cellular changes that interact with the integration of the introduced DNA is difficult. 

Problems associated with protoplast systems include the 

enzymatic cell treatment, gene transfer environment, and 

need to transfer cells through a sequential series of different media. This difficulty is illustrated by the high mortality (50 percent) of poplar protoplasts following 

electroporation (Sellmer 1991). In contrast, microprojectile 

bombardment offers more potential for examining cellular factors involved in DNA incorporation because DNA 

can be delivered into any plant cell or tissue. One drawback of this approach is the heterogeneity of the cell population used for bombardment; changes in 1 tissue may alter 

a response by another tissue within an explant. For both 

methods, regulation of the transiently expressed genes may 

be less stringent than with transgenic plants because multiple copies of the gene can occur transiently, thus gene 

expression is not necessarily controlled through chro~o-
some structure (Katagari and Chua 1992; Quail et al. 1987; 

Vernet et al. 1982). 

Electroporation 

DNA transfer using electroporation is due to increased 

cell wall and membrane permeability caused by a short electrical pulse applied to the target plant tissue, whether protoplasts or whole tissue explants (Van Wert and Saunders 1992). 

This method has been used to study transient gene expression and produce transgenic tissues and plants (Chupeau et 

al. 1993; Russell and McCown 1986; Wang et al. 1991). A prerequisite is the availability of tissue cultures amenable to 

electroporation. For poplar, protoplast culture and tree regeneration were achieved (Chupeau et al. 1993; Russell and 

McCown 1986; Wang et al. 1991) that provided a basis for 

gene transfer via electroporation. Transgenic P. tremula x P. 

alba trees were produced following electroporation of protoplasts with a decay-wave electroporation device (Chupeau 

et al. 1994). Transformation selection was achieved using 

phosphinotricin with the phosphinotricin acetyl transferase 

(PAT) gene, and chlorsulfuron with the acetolactate synthase 

(ALS) gene. The transformation frequency was from 1.3 to 

7.5 x 10-6, and 56 independent transgenic trees were regenerated. An interesting finding of this study was that the introduced genes were integrated in a relatively simple and 

straightforward pattern. 

Microprojectile-mediated DNA 

Delivery 

In poplar, the propulsion of microprojectiles coated with 

DNA was accomplished using 3 different mechanisms: electric discharge, gunpowder, and helium gas (Devan tier 1992; 

Devantier et al. 1993; McCown et al. 1991; Sellmer 1991). 

These methods were optimized primarily using cell suspensions and were not directly compared. Transgenic trees were 

obtained using the electric discharge method (McCown et 

al. 1991), while only transgenic. calli were obtained with the 

gunpowder method (Devantier 1992; Devantier et al. 1993). 

Electric Discharge Propulsion 

Optimization with Cell Suspension 

With the electric discharge method, optimization for 

transient expression was performed using a cell suspension of P. alba x P. grandidentata cl. 'NC5339' (cv. 'Crandon') 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Direct Gene Transfer in Poplar 

from leaf callus. The experiment included an evaluation 

~f the effects of particle size, discharge voltage, and particle load (Sellmer 1991). Although no strong correlation 

between transient expression and stable expressi.on of 

transgenes has been reported, maximizing transient expre~sion in the cell suspension provides a uniform, reproduCible system to evaluate biological parameters that affect 

transgene expression. 

The first step toward developing a reproducible method 

for gene transfer is characterization of the target cells. Cell 

density (initial volume of cells/ volume of tissue culture 

medium) on the target plate did not affect transient gene 

expression 2 days after bombardment for cell concentrations of 5 percent, 10 percent, and 15 percent; however, 50 

~ercent yielded a significant increase in transient expressiOn. Interestingly, no differences in f3-glucuronidase (GUS) 

gene expression were observed among the 4 concentrations 1 week after bombardment. Cell culture age significantly affected transient expression levels of the GUS gene. 

Based on weekly subculturing (5 ml packed cells into 50 

ml fresh medium), 3-day-old cell suspensions had significantly higher GUS gene expression compared to cells harves.ted at other times in the culture cycle. Based on dry 

weight, the cell suspension culture was in a lag phase 

through day 4, underwent a rapid growth phase during 

the next 4 days, and were in a stationary phase at day 9. 

Maximum transient gene expression corresponded to the 

mid-lag phase. During this 3-day period, growth was not 

noticeable, but a profound change in cellular morphology 

and the initiation of active cell division occurred. Cytologically, 32 percent of the cells on day 3 contained a division plate, in contrast with only 13 percent on day 1 and 9 

percent on day 5. Furthermore, cells from 3-day-old cultures had almost twice as much 3H-thymidine incorporation compared with cells from other stages. Cell size was 

also the smallest (less than 100 11m) from 3-day-old cultures. Because of this smaller size, more cells per volume 

were used causing a bias in transient GUS expression. 

When adjusted for cell number, cells from 1-day-old and 

3-day-old cultures exhibited similar expression levels, but 

both produced higher GUS expression than those at other 

stages. However, this difference was diminished in GUS 

expression assays 21 days after bombardment, and no difference was detectable 56 days after bombardment. This 

long-term pattern of gene expression was independent of 

transformation vector size (Sellmer 1991 ). 

Later experiments focused on factors that could promote 

cell competence for transient gene expression and stable 

transgene incorporation. These factors were associated 

with diverse approaches including preculturing cells with 

growth regulators, cell synchronization, and DNA breakage experiments. To test the effect of cell division (rate and 

synchronization) on transient GUS gene expression, cultures were grown on various hormone regimes. Experiments were inspired by previous cell-age experiments and 

61 
Section II Transformation and Foreign Gene Expression 

other reports that preculturing of tissue culture materials 

can enhance gene transfer and transient gene expression 

by promoting axillary and adventitious meristem development before bombardment (Ellis et al. 1991; McCown et 

al. 1991; Serres et al. 1992). In 1 set of experiments, 2,4-

dichlorophenoxyacetic acid (2,4-D) was required for cell 

growth. Higher cytokinin levels or the inclusion of 

thidiazuron (TDZ) did not significantly change the growth 

rate of the cultures. Although growth rates were unchanged, 

incorporation of 3H-thymidine was the highest with the 

highest concentration of cytokinin tested (1.0 ~-tM benzyladenine). However, transient GUS gene expression did not 

change (Sellmer 1991). These results suggest that tissue cultures responded toward differentiation and meristematic 

development, but transient gene expression was unaffected. 

Continuing this rationale of enhancing cell competence 

and GUS gene expression, experiments were conducted 

to synchronize poplar cell suspension to potentially increase transient gene expression and stable incorporation 

of genes. Some success was achieved with aphidicolin, a 

tetracyclic diterpenoid that reversibly blocks cell division 

before DNA strand synthesis. Aphidicolin was used to halt 

cell division and was effective at blocking DNA synthesis 

based on 3

H-thymidine incorporation in early exponential 

(3-day-old) and stationary (8-day-old) cultures. Effects of the 

block apparently remained for 12 to 24 h after release from 

the aphidicolin because little 3H-thymidine incorporation 

was observed. In both cell ages, a sharp peak of 3

H-thymidine incorporation was observed after 24 h. In the cells from 

3-day-old cultures, this peak dropped back to the baseline 

level suggesting synchronization of DNA synthesis. The 

highest level of transient GUS gene expression was observed 

at the 3-day-old stage although the ~evel was not significantly 

different from cell suspensions at other times following 

aphidicolin release. Differences were not apparent in GUS 

gene expression, nor in the level of stably transformed calli 

recovered from the different time points (Sellmer 1991). 

In a complementary study, ionizing radiation (gamma 

rays) was tested alone and in combination with aphidicolin 

to evaluate if DNA repair in conjunction with synchronized 

division could increase stable transformation. Radiation 

levels up to 3,000 rads did not affect cell growth, although 

no difference on the transient or long-term expression of 

GUS was observed, even at higher radiation levels. Also, 

no difference in GUS gene expression was evident when 

this treatment was combined with the aphidicolin treatment (Sellmer 1991). 

Gene Delivery into Protoplast-Derived Cells and 

Nodules 

Electric discharge propulsion was used to deliver 

microprojectiles in cells from protoplasts and nodule cultu.res of P. alba x P. grandidentata cl. 'NC5339' and P. nigra x P. 

trichocarpa cl. 'NC5331,' and to obtain transgenic trees con62 

taining a chimeric Bacillus thuringiensis 8-endotoxin gene 

conferring resistance to some lepidopteran pests (McCown 

et al. 1991). Transient GUS gene expression was obtained 

with 2 cell types (cell microcalli and nodules) and in stem 

sections. Bombardment intensity, particle load, and target 

tissue type were studied. Transgenic nodules resistant to 

kanamycin were obtained with both genotypes, but only 

nodules of P. alba x P. grandidentata regenerated transgenic 

trees. Transgenic tree tissues were tested for feeding toxicity 

against forest tent caterpillar and gypsy moth larvae. Reduced survival was observed for the forest tent caterpillar 

only, but both insect species manifested reduced growth. 

Gene Delivery into Plantlet Tissues 

As with poplar cell suspensions, the developmental and 

physiological stage of the poplar explant influences transient 

expression following particle bombardment. With poplar 

hybrid clones 'NC5339' (P. alba x P. grandidentata cv. 

'Crandon') and 'NM6' (P. nigra x P. maximowiczii), explant 

pretreatment on shoot inducing media significantly increased 

transient GUS gene expression, driven by an enhanced 

CaMV 35S promoter, in nodules, petioles, internodes, and 

leaf explants (Sellmer 1991). Pretreatment of 0.5 to 1 em petiole segments in a liquid Woody Plant Medium (WPM) (Lloyd 

and McCowm 1980) supplemented with 0.1 ~-tM each 

benzyladenine (BA), naphthaleneacetic acid (NAA), and 

thidiazuron (TDZ) progressively increased the number of 

cells histochemically staining for GUS activity for up to 7 

days (Wraith et al. 1994). Although proliferation at the cut 

end of the petiole explant had initiated, cells ·were not yet 

sloughing off. Interestingly, pretreatment on callus induction media did not markedly increase transient gene expression in petiole segments, despite the similar morphological 

appearance of the explants (Wraith et al. 1994). 

To increase the number of GUS-expressing cells, pretreatment must either increase the number of cells impacted or 

alter cell competency for transiently expressing inserted 

DNA. Explant pretreatment with a bud-induction medium 

may induce differentiation of cells capable of forming 

shoots or meristematic cells. These pretreated cells may 

be similar to the cells of the late-lag-phase cell suspensions. 

For stable integration of delivered genes, a callus induction phase before culture on a bud-induction medium may 

improve the recovery of transgenic tissues after bombardment. Although this phase is not always required when 

no selection is applied, it is necessary when kanamycin 

selection is performed after bombardment of the hybrid 

poplar l~ne 'NM6' (Wraith et al. 1994). There was no strong 

correlation between level of transient gene expression and 

number of stably transformed tissues recovered. Eight 

weeks after particle bombardment, the low number of 

GUS-expressing cells was constant regardless of gene construct, level of transient gene expression, and prebombardment treatments. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Gunpowder and Helium Propulsions 

Optimization With Cell Suspension 

Similar to work with the electric discharge method, optimization of gene delivery conditions with the gunpowder and the helium propulsion methods (Biolistic 

PDS-1000, Biolistic PDS-1000/He) was done with a cell 

suspension of P. nigra x P. maximowiczii cl. 'NM6' 

(Devantier 1992; Devantier et al. 1993). Evaluations were 

conducted on the effects of propulsion mechanism type, 

DNA precipitation method, DNA amount, bombardment number, microprojectile type and size, use of 

screens on top of the target tissues, target sample position in the bombardment chamber, and transient gene 

expression assay time. All these factors influenced gene 

expression levels. In addition, the helium propulsion 

device, using gold particles on which DNA was precipitated with CaC12 (calcium chloride), produced a 

higher level of transient gene expression. The optimum 

cell suspension age was between 7 and 9 days after subculturing, and the maximum transient gene expression 

was 1 day after bombardment. No study of growth rate 

or cell division was conduct~d. Following cell suspension bombardment, subcellular localization of 

microprojectiles revealed that most GUS-expressing 

cells contained microprojectiles in their cytoplasm suggesting that a mechanism may be available to transport 

the DNA to the nucleus. 

In this study, microprojectile-mediated DNA delivery in a cell suspension of 4 different poplar genotypes 

(P. nigra x P. maximowiczii cl. 'NM1' and cl. 'NM6,' P. 

deltoides x P. nigra cl. 'DN106,' and P. tremula x P. alba cl. 

'7171-84') was used to evaluate the strength of different chimeric promoters (Devantier et al. 1993). A genotype effect was observed, with 'NM1' yielding the 

highest level of transient gene expression by a vector 

containing the GUS gene linked to a double 35S promoter with an alfalfa mosaic virus translational enhancer. This effect was perhaps associated with different 

morphologies of the cell suspensions for each genotype. 

Lower expression levels were obtained with chimeric 

promoters containing only the single 35S promoter with 

the alfalfa mosaic virus promoter, the double 35S promoter, and the single 35S promoter (in order of decreasing strength). Similar results were obtained with the 

single and double 35S promoter in transgenic poplar 

(Leple et al. 1992). 

Stable integration of the introduced genes was obtained 

following selection on 500 mg/1 kanamycin. Expression 

of the introduced neomycin phosphotransferase II (NPTII) 

gene was detected using a radioactive assay. However, 

because· the cell suspension was over 5 years old, no 

transgenic plants were regenerated (Devantier 1992; 

Devantier et al. 1993). 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Direct Gene Transfer in Poplar 

Gene Delivery Into Plantlet Tissues 

As with the electric discharge method, explants were used 

to attempt recovery of transgenic poplar trees (Devantier 

1992; Devantier et al. 1993). Three genotypes (P. nigra x P. 

maximowiczii cl.'NM1' and cl. 'NM6,' and P. tremula x P. alba 

cl. '7141-84') were tested to optimize transient gene expression. The Biolistic Helium device using gold particles was 

superior, and 6-day-old explants gave higher transient gene 

expression. Different hormone treatments, a callusing phase, 

bombardment time after excision, different chimeric promoter strengths, and the timing of kanamycin selection were 

evaluated for stable genetic transformation; however, no 

transgenic tissues were obtained Uones and Charest, unpublished). Furthermore, selection with hygromycin and methotrexate was attempted without success. A current approach 

is to obtain cell suspensions from in vitro grown plantlets 

and use these cells to produce transgenic calli from which 

trees will be regenerated. 

Conclusions 

Direct gene transfer by electroporation.or microprojectile-mediated DNA delivery was achieved in poplar, and transgenic trees were obtained. These methods can 

also be used for fast experiments on transient gene expression. However, only a few genotypes were useful for regenerating transgenic trees, despite several attempts to 

extend these methods to a wider range of genotypes. Several biol.ogical parameters need exploring, and a better 

understanding of the mechanisms of DNA integration into 

plant cell genomes is required to improve currently used 

protocols. Once DNA delivery parameters are established, 

careful consideration must be given to the: 1) target cell 

response to the delivery process; 2) cell competence for 

DNA uptake, transient gene expression, and stable expression of the delivered DNA; and 3) interactions between 

the delivered DNA and the target cell machinery. 

Literature Cited 

Abd ul-Baki, A.A.; Saunders, J .A.; Matthews, B.F.; Pittarelli, 

G.W. 1990. DNA uptake during electroporation of germinating pollen grains. Plant Sci. 70: 181-190. 

Aronen, T.; Haggman, H.; Hohtola, A. 1994. Transient -

glucuronidase expression in Scots pine tissues derived 

from mature trees. Can. J. For. Res. 24: 2006-2011. 

Aronen, T.; Hohtola, A.; Laukkanen, H.; Haggman, H. 1995. 

Seasonal changes in the transient expression of a 35S 

63 
Section II Transformation and Foreign Gene Expression 

CaMV-GUS gene construct introduced into Scots pine 

buds. Tree Physiol. 15: 65-70. 

Charest, P.J.; Calero, N.; Lachance, D.; Mitsumune, M.; Yoo, 

B. 1993. The use of microprojectile DNA delivery to bypass the long life cycle of tree species in gene expression studies. Curr. Topics Bot. Res. 1: 151-163. 

Chupeau, M.C.; Lemoine, M.; Chupeau, Y. 1993. Requirement of thidiazuron for healthy protoplast development 

to efficient tree regeneration of a hybrid poplar (Populus 

tremula x P. alba). J. Plant Physiol. 141: 601-609. 

Chupeau, M.C.; Pautot, V.; Chupeau, Y. 1994. Recovery of 

transgenic trees after electroporation of poplar protoplasts. Trans. Res. 3: 13-19. 

Dekeyser, R.A.; Claes, B.; De Rycke, R.M.U.; Habets, M.E.; 

van Montagu, M.C.; Caplan, A.B. 1990. Transient gene 

expression in intact and organized rice tissues. Plant 

Cell. 2: 591-602. 

Devantier, Y. 1992. Microprojectile-mediated DNA delivery to the Salicaceae family. Waterloo, Ontario, Canada: 

University of Waterloo. 159 p. M.S. thesis. 

Devantier, Y.A.; Moffatt, B.; Jones, C.; Charest, P.J. 1993. 

Microprojectile-mediated DNA delivery to the 

Salicaceae family. Can. J. Bot. 71: 1458-1466. 

D'Halluin, K.; Bonne, E.; Bossut, M.; De Beuckeleer, M.; 

Leemans, J. 1992. Transgenic maize plants by tissue 

electroporation. Plant Cell. 4: 1495-1505. 

Ellis, D.O. 1995. Transformation of gymnosperms. In: Jain, 

S.; Gupta, P.; Newton, R., eds. Somatic embryogenesis 

in woody plants Vol. 1. Dordrecht, The Netherlands: 

Kluwer Academic Publishers: 227-251. 

Ellis, D.O.; McCabe, D.; Russell, D.; Martinell, B.; McCown, 

B.H. 1991. Expression of inducible angiosperm promoters in a gymnosperm, Picea glauca (white spruce). Plant 

Mol. Bioi. 17: 19-27. 

Katagari, F.; Chua, N.H. 1992. Plant transcription factors: 

present knowledge and future challenges. Trends 

Genet. 8: 22-27. 

Laursen, C.M.; Krzyzek, R.A.; Flick, C.E.; Anderson, P.C.; 

Spencer, T.M. 1994. Production of fertile transgenic 

maize by electroporation of suspension culture cells. 

Plant Mol. Bioi. 24: 51-61. 

Leple, J.C.; Brasileiro, A.C.M.; Michel, M.J.; Delmotte, F.; 

}ouanin, L. 1992. Transgenic poplars: expression of chimeric genes using four different constructs. Plant Cell 

Rep. 11: 137-141. 

Lloyd, G.; McCown, B. 1980. Commercialli-feasible 

micropropagation of mountain laurel, Kalmia latifolia, 

by use of shoot-tip culture. Proc. Inter. Plant Prop. Soc. 

30: 421-426. 

Luong, H. T.; Shewry, P.R.; Lazzeri, P.A. 1995. Transient gene 

expression in cassava somatic embryos by tissue 

electroporation. Plant Sci. 107: 105-115. 

McCown, B.H.; McCabe, D.E.; Russell, D.R.; Robison, D.J.; 

Barton, K.A.; Raffa, K.F. 1991. Stable transformation of 

Populus and incorporation of pest resistance by electric 

64 

discharge particle acceleration. Plant Cell Rep. 9: 590-594. 

Miranda Brasileiro, A. C.; Toumeur, C.; Leple, J.C.; Combes, 

V.; Jouanin, L. 1992. Expression of the mutant Arabidopsis 

thaliana acetolactate synthase gene confers chlorsulfuron 

resistance to transgenic poplar plants. Transg-enic Res. 

1: 133-141. 

Quail, P.N.; Christensen, A.H.; Jones, A.M.; Lissemore, 

L.; Parks, B.M.; Sharrock , R.A. 1987. The phytochrome molecule and the regulation of its gene. In: 

Integration and control of metabolic processes: pure 

and applied aspects. Cambridge, UK: Cambridge U. 

Press: 41-54. 

Russell, J.A.; McCown, B.H. 1986. Culture and regeneration of Populus leaf protoplasts isolated from non-seedling tissue. Plant Sci. 46: 133-142. 

Sellmer, J.C. 1991. Examination and manipulation of Populus 

cell competence for direct gene transfer. Madison, WI, 

U.S.A.: University of Wisconsin. Ph.D. dissertation. 

Serres, R.; Stang, E.; McCabe, D.; Russell, D.; Mahr, D.; 

McCown, B.H. 1992. Gene transfer using electric discharge 

particle bombardment and recovery of transformed cranberry plants. J. Amer. Soc. Hort. Sci. 117: 174-180. 

Songstad, D.O.; Halaka, F.G.; DeBoer, D.L.; Armstrong, 

C.L.; Hinchee, M.A.W.; Ford-Santino, C. G.; Brown, S.M.; 

Fromm, M.E.; Horsch, R.B. 1993. Transient expression 

of GUS and anthocyanin constructs in intact maize immature embryos following electroporation. Plant Cell 

Tiss. Org. Cult. 33: 195-201. 

Strohm, M.; Jouanin, L.; Kunert, K.J.; Pruvost, C.; Polle, A.; 

Foyer, C.H.; Rennenberg, H. 1995. Regulation of glutathione synthesis in leaves of transgenic poplar 

(Populus tremula x P. alba) overexpressing glutathione 

synthetase. Plant J. 7: 141-145. 

Van Wert, S.L.; Saunders, J.A. 1992. Electrofusion and 

electroporation of plants. Plant Physiol. 99: 365-367. 

Vemet, T.; Fleck, J .; Durr, A.; Fritsch, C. H.; Pinck, M.; Hirth, 

L. 1982. Expression of the gene coding for the small subunit of ribulosebisphosphate carboxylase during differentiation of tobacco plant protoplasts. Eur. J. Biochem. 

126: 489-494. 

Wang, S.P.; Xu, X.H.; Wei, Z.M. 1911. Culture and regeneration of poplar mesophyll protoplasts. Sci. China. B. 

Chern. Life Earth Sci. 34: 587-592. 

Wraith, S.; Ellis, D.; Raffa, K.; Kleiner, K.; Stanosz, G.; 

McCown, B. 1994. Biorational strategies for deploying 

pest-resistant poplars in bioenergy plantations. In: Proceeding of the joint meeting of the Poplar Councils of the 

U.S. and Canada; 1994 July 26-28; St. Paul, MN, U.S.A. 

Xu, X.; Li, B. 1994. Fertile transgenic Indica rice plants obtained by electroporation of the seed embryo cells. Plant 

Cell Rep. 13: 237-242. 

Yang, J.; Ge, K.; Wang, Y.; Wang, B.; Tan, C.C. 1993. Highly 

efficient transfer and stable integration of foreign DNA 

into partially digested rice cells using a pulsed electrophoretic drive. Transgenic Res. 2: 245-251. 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 
Chapter9 

Gene Expression Studies1 

Lise Jouanin and Gilles Pilate 

Introduction 

In this chapter, we discuss published research on the 

isolation and expression of poplar genes, including 

complementary eDNA (eDNA) and genomic DNA of 

several characterized genes. The over and under expression of introduced genes in homologous or heterologous host plants is discussed and published results are 

presented. Gene expression studies in poplar are now 

possible because procedures to transform and regenerate different genotypes are commonly available (reviewed in Jouanin et al. 1993 and this volume). Genes 

of plant origin are the focus of this chapter. Sequences of nonplant origin are discussed elsewhere 

in this book. 

Poplar Gene Isolation 

The list of genes from woody plants that have been 

isolated, sequenced, and published is relatively small. 

Continued effort is directed toward understanding the 

metabolic aspects of trees. Poplar is particular! y amenable to gene isolation because of its small genome size 

(1.12 picograms DNA/diploid genome), which promotes the production of genomic libraries representative of the complete genome. 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, LG., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

Among trees, most of the available gene sequences 

are from poplar (mainly eDNA sequences). This information is summarized in table 1. Research efforts have 

focused on genes: 1) involved in the wounding response 

(Bradshaw et al. 1989; Davis et al. 1991; Parsons et al. 

1989); 2) involved in lignification (Bugos et al. 1991; 

Dumas et al. 1992; Kawai et al. 1993; Osakabe et al.1994, 

1995a, b, c; Subramanian et al. 1993; Tsai et al. 1995; 

van Doorsselaere et al. 1995a); and 3) encoding storage 

proteins (Clausen and Apel1991; Coleman et al. 1992). 

In most studies, gene sequences from annual dicotyledonous plants were the basis of probes or polymerase 

chain reaction primers used to isolate pop!ar cDNAs 

(potato PAL eDNA: Subramanian et al. 1993; tobacco 

CAD eDNA: van Doorsselaere et al. 1995a; aspen OMT 

primers: Tsai et al. 1995). 

Where possible, the poplar eDNA sequences that were 

obtained were compared with those of the corresponding genes in annual dicot plants (WIN: Davis et al. 1991; 

OMT: Dumas et al. 1992; PAL: Osakabe et al. 1995a). A 

high degree of homology was observed between poplar eDNA and annual dicot plant eDNA; frequently 

more than 80 percent at the amino acid level. However, 

the highest homology was found with sequences originating from other deciduous trees; for example, comparison of CAD and OMT sequences between poplar and 

eucalyptus (CAD 80.9 percent, van Doorsselaere et al. 

1995a; OMT 84 and 80 percent, Hayakawa et al. 1996). 

When present, intron sequences were found in the expected locations, as demonstrated by genes encoding 

chitinases (Davis et al. 1991) and anionic peroxidases 

(Osakabe et al. 1995b). Overall, poplar genes showed a 

closer relationship to dicotyledonous angiosperms than 

to gymnosperms or monocotyledons as shown in table 2 

(PAL: Osakabe et al. 1995a; CAD: van Doorsselaere et al. 

1995a; OMT: Hayakawa et al. 1996). 

Most of the studied genes are members of small 

multigene families (WIN: Davis et al. 1991; OMT: 

Hayakawa et al. 1996; PRX: Osakabe et al. 1995b; PAL: 

Osakabe et al. 1995c, CHS: Lurin and Jouanin 1995). In 

some cases, they are clustered in the genome (WIN: Davis 

et al. 1991; PRX: Osakabe et al. 1995b; PAL: Osakabe et al. 

1995c). 

65 
Section II Transformation and Foreign Gene Expression 

Table 1. eDNA and genomic sequences cloned in poplar. 

Name Enzyme Origin Poplar type References 

Ptom1 0-methyl transferase (OMT) eDNA P. tremuloides Bugos et al. 1991 

PTOMT 0-methyl transferase (OMT) genomic P. tremuloides Tsai et al. 1995 

pPCL4 0-methyl transferase (OMT) eDNA P. deltoides x P. trichocarpa Dumas et al. 1992 

HOMT1 & HOMT2 0-methyl transferase (OMT) genomic P. sieboldii x P. grandidentata Hayakawa et al. 1996 

PAL Phenylalanine ammonia-lyase (PAL) eDNA P. deltoides x P. trichocarpa Subramaniam et al. 1993 

cDNA1 Phenylalanine ammonia-lyase (PAL) eDNA P. sieboldii x P. grandidentata Osakabe et al. 1995a 

PALG1 & G2A Phenylalanine ammonia-lyase (PAL) genomic P. sieboldii x P. grandidentata Osakabe et al. 1995a 

PALG28 & PALG4 Phenylalanine ammonia-lyase (PAL) genomic P. sieboldii x P. grandidentata Osakabe et al. 1995c 

POPCAD Cinnamyl alcohol dehydrogenase eDNA P. deltoides x P. trichocarpa van Doorsselaere et al. 

(CAD) 1995a 

WIN3 Wound-inducible (similar to sporamin eDNA P. trichocarpa x P. deltoides Hollick and Gordon 1993 

gWIN3 and Kunitz proteinase inhibitor) genomic P. trichocarpa x P. deltoides Bradshaw et al. 1989 

WIN6& 8 Chitinase eDNA P. trichocarpa x P. deltoides Parsons et al. 1989 

gWIN6,8&X Chitinases genomic P. trichocarpa x P. deltoides Davis et al. 1991 

pPOP1 Malic enzyme eDNA P. deltoides x P. trichocarpa van Doorsselaere et al. 

1991 

samPdxPt S-adenosyi-L-Methionine synthetase eDNA P. deltoides x P. trichocarpa van Doorsselaere et al. 

1993 

DHDPS Dihydrodipicolinate synthase eDNA P. deltoides x P. trichocarpa Vauterin and Jacobs 1994 

BSP Bark storage protein eDNA P. deltoides Coleman et al. 1992 

bspA Bark storage protein genomic P. deltoides Coleman and Chen 1993 

WS1 Xylem ray cell storage protein eDNA P. nigra x P. deltoides Clausen and Apel 1991 

pA2, pA3 Anionic peroxidases eDNA P. sieboldii x P. grandidentata Osakabe et al. 1994 

PRXA1 Anionic peroxidase genomic P. sieboldii x P. grandidentata Kawai et al. 1993 

PRXA3A 

PRXA4A Anionic peroxidases genomic P. sieboldii x P. grandidentata Osakabe et al. 1995b 

Table 2. Identity comparison at the amino acid level (%) among the PAL, OMT, and CAD genes of poplar and other plants. 

Dicot Monocot Conifer 

Gene Plant (%) Plant 

PALG2 bean (85.9) 

parsley (84.7) 

PAL G2 bean (81.5) 

parsley (82.8) 

HOMT1 tobacco OMT1 (77) maize 

OMT2 (56) 

eucalyptus (84) 

HOMT2 tobacco OMT1 (76) maize 

OMT2 (54) 

eucalyptus (80) 

CAD tobacco (79.8) 

alfalfa (84) 

Gene Expression at the RNA Level 

Isolated cDNAs have been used as probes to monitor 

gene expression in different tissues and under various 

environmental conditions. The most detailed studies have 

focused on the systemic accumulation of specific messen66 

(%) Plant (%) References 

pine (66.1) Osakabe et al. 1995a 

pine (64.2) Osakabe et al. 1995a 

(28) Hayakawa et al. 1996 

(26) Hayakawa et al. 1996 

spruce (69.2) van Doorsselaere et al. 

1995a 

ger RNA (mRNAs) in response to wounding (Parsons et 

al. 1989; Bradshaw et al. 1989), seasonal changes and tissue-specific expression of mRNAs encoding bark and xylem ray cell storage proteins (Clausen and Apel 1991; 

Coleman .et al. 1992), and genes involved in the phenylpropanmd pathway (PAL: Subramanian et al. 1993; 

Osaka beet al. 1995a, c; OMT: Bugos et al. 1991, Hayakawa 

et al. 1996; anionic peroxidases: Osaka beet al. 199Sb; CHS: 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

~. 
Lurin and Jouanin 1995). In these studies, mRNA abundance was measured either by northern hybridization or 

by reverse transcriptase-polymerase chain reaction. From 

these studies, we can infer that gene regulation often occurs at the transcriptional level. 

Poplar Heterologous Promoters 

When establishing a genetic transformation procedure 

for a specific poplar hybrid,· vectors frequently contain a 

neomycin phosphotransferase gene (NPTII), which is a 

selectable marker that confers resistance to kanamycin, and 

a ~-glucuronidase gene (GUS), which is used as a reporter 

of gene expression Qouanin et al. 1993). Binary vectors such 

as pBI121 Oefferson et al. 1987) or its derivatives, in which 

the NPTII and the GUS genes are placed under control of 

the Agrobacterium tumefaciens nopaline synthase (NOS) 

promoter and either the 35S or 705 (double enhancer sequen~es) CaMV promoters, function efficiently in poplar 

(Leple et al. 1992). Thus, vectors used for dicot transformation are frequently suitable for poplar use. 

Reports that illustrate the function of heterologous plant 

promoters in poplar include promoters from the potato 

proteina.se inhibitor II (PIN2) gene (Klopfenstein et al. 

1991 ), the Arabidopsis thaliana acetolacta te synthase (AtALS) 

gene (Brasileiro et al. 1992), and the Eucalyptus cinnamyl 

alcohol dehydrogenase (EuCAD) gene (Feuillet et al. 1995). 

Expression of an associated reporter gene (CAT or GUS) 

indicates that regulation is conserved. This is represented 

by the wound induction of the PIN2-CAT construct 

(Klopfenstein et al. 1991), and the tissue-specificity of the 

EuCAD-GU5 construct in relation to lignification {Feuillet 

et al. 1995). These studies illustrate the basis of considerable interest in poplar, an easily transformable woody 

plant, in which sequences originating from other plant 

species have conserved patterns of expression. 

Poplar Promoters in 

Heterologous Plants 

In some cases, the characterization of poplar genomic 

sequences has involved promoter isolation. These promoters have been linked to a reporter gene such as GUS to 

study their expression pattern in transgenic plants. Two 

wound-inducible poplar promoters, WIN3 (Hollick and 

Gordon 1993) and WIN6 (Clarke et al. 1994), are also induced upon wounding in tobacco. This demonstrates conservation of expression mechanisms between an annual 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Gene Expression Studies 

dicot and a woody angiosperm. It may be feasible to study 

woody plant gene promoters in a herbaceous plant model 

such as tobacco. However, reintroduction of sequences into 

the same species is required to study promoter function in 

relation to unique structural, metabolic, and developmental features. Currently, there are no reports of poplar promoters reintroduced into poplar. This research field is 

expected to develop rapidly. 

Over and Under Expression of 

Poplar Genes in Plants 

Of major interest, at the fundamental and applied levels, is the manipulation of gene expression to alter levels 

of specific proteins and shift metabolic processes. Such 

work in poplar is just emerging. As of this writing, the 

only report of over expression of a gene concerns a peroxidase gene in poplar callus (Kajita et al. 1994). The only 

report of under expression of a gene is that of a caffeic 

acid/5-hydroxyferulic acid 0-methyltransferase (OMT) 

gene in poplar stems (van Doorseelaere et al. 1995b). 

A chimeric gene containing the CaMV 355 promoter and 

the 5' end of the poplar OMT gene in antisense orientation 

was introduced into tobacco by Dwivedi et al. {1994). In 

some transgenic tobacco plants, the level of OMT activity 

was decreased and modification of the lignin composition 

was observed. This demonstrates that homology between 

the tobacco and poplar OMT genes is sufficient for a functional antisense strategy. Another study reported under 

expression of OMT in transgenic poplars expressing a poplar antisense RNA (van Doorsselaere et al. 1995b ). OMT 

activity was reduced up to 95 percent and critical modification of the lignin composition was observed. Reducing 

the activity of enzymes involved in lignin monomer biosynthesis is potentially important for the paper pulp industry and can be effectively tested in woody plants. 

Detailed information on this subject is included in the chapter by Boerjan et al. in this volume. 

Summary 

Poplar is considered a model plant for gene expression 

studies in woody angiosperm species. The advantages of 

the poplar model system are the availability of transformation procedures and its small genome size, which allows for relatively easy gene isolation. High levels of 

homology and conservation of the regulatory systems have 

been observed between poplar (a dicot) genes and other 

67 
Section II Transformation and Foreign Gene Expression 

dicots. Poplar promoters and genes potentially can be studied in other model plant species such as tobacco. However, for specific metabolic pathways, such as lignification, 

more representative information will result if gene expression is tested in poplar. The ability to transform many poplar genotypes affords diverse opportunities to study 

expression of genes from other woody species, including 

conifers. In the future, the development of such studies 

could provide critical information on gene expression in 

woody plants. 

Literature Cited 

Bradshaw, H.D.; Hollick, J .J .B.; Parsons, T.J .; Clarke, H.R.G.; 

Gordon, M.M. 1989. Systematically wound-responsive 

genes in poplar trees encode proteins similar to sweet 

potato sporamins and legume Kunitz trypsin inhibitors. 

Plant Mol. Bioi. 14: 51-59. 

Brasileiro, A.C.M.; Toumeur, C.; Leple, J.C.; Combes, V.; 

Jouanin, L. 1992. Expression of the mutant Arabidopsis 

thaliana acetolactate synthase gene confers chlorsulfuron 

resistance to transgenic poplar plants. Transgenic Res. 

1: 133-141. 

Bugos, R; Chiang, V.L.C.; Campell, W.H. 1991. eDNA cloning, 

sequence analysis and seasonal expression of ligninbispecific caffeic I 5-hydroxyferulic acid 0-methyltransferase of aspen. Plant Mol. Bioi. 17: 1203-1215. 

Clarke, H.R.G.; Davis, J.M.; Wilbert, S.M.; Bradshaw, H. D.; 

Gordon, M.P. 1994. Wound-induced and developmental activation of a poplar tree chitinase gene promoter 

in transgenic tobacco. Plant Mol. Bioi. 25: 799-815. 

Clausen, S.; Apel, K. 1991. Seasonal changes in the concentration of the major storage protein and its mRNA 

in xylem ray cells of poplar trees. Plant Mol. Bioi. 17: 

669-678. Erratum 1992. Plant Mol. Bioi. 20: 365. 

Coleman, G.D.; Chen, T.H.H.; Fuchigami, L.H. 1992. 

Complementary DNA cloning of poplar bark storage 

protein and control of its expression by photoperiod. 

Plant Physiol. 98: 687-693. 

Coleman, G.D.; Chen, T.H.H. 1993. Sequence of a poplar 

bark storage protein gene. Plant Physiol. 102: 1347-1348. 

Davis, J.M.; Clarke, H.R.G.; Bradshaw, H. D.; Gordon, M.P. 

1991. Populus chitinase genes: structure, organization, 

and similarity of translated sequences to herbaceous 

plant chitinases. Plant Mol. Bioi. 17: 631-639. 

Dumas, B.; van Doorsselaere, J.; Gielen, J.; Legrand, M.; 

Fritig, B.; van Montagu, M.; Inze, D. 1992. Nucleotide 

sequence of a complementary DNA encoding 0-

methyltransferase from poplar. Plant Physiol. 98:796-797. 

Dwivedi, U.N.; Campbell, W.H.; Yu, J.; Datla, R.S.S.; Bugos, 

R.C.; Chiang, V.L.; Podila, G.K. 1994. Modification of 

lignin biosynthesis in transgenic Nicotiana through ex68 

pression of an antisense 0-methyltransferase gene from 

Populus. Plant Mol. Bioi. 24: 61-71. 

Feuillet, C.; Lauvergeat, V.; Deswarte, C.; Pilate, G.; Bourlet, 

A.; Grima-Pettenati, J. 1995. Tissue- and specific expression of a cinnamyl alcohol dehydrogenase promoter in 

transgenic poplar plants. Plant Mol. Bioi. 27: 651-667. 

Hayakawa, T.; Nanto, K.; Kawai, S.; Katayama, Y.; 

Morohoshi, N. 1996. Molecular cloning and tissue-specific expression of two genes that encode caffeic acid 0-

methyltransferases from Populus kitakamiensis. Plant Sci. 

113: 157-165. 

Hollick, J.B.; Gordon, M.P. 1993. A poplar tree proteinase 

inhibitor-like promoter is responsive to wounding in 

transgenic tobacco. Plant Mol. Bioi. 22: 561-572. 

Jefferson, R.A.; Kavanagh, T.A.; Bevan, M.W. 1987. GUS fusions: f3-glucuronidase as a sensitive and versatile gene 

fusion marker in higher plants. EMBO J. 6:3901-3907. 

Jouanin, L.; Brasileiro, A.C.M.; Leple, J.C.; Pilate, G.; Cornu, 

D. 1993 .. Genetic transformation: A short review of methods and their applications, results and perspectives for 

forest trees. Ann. Sci. For. 50: 325-336. 

Kajita, S.; Osakabe, K.; Katayama, Y.; Kawai, S.; Matsumoto, 

Y.; Hata, K.; Morohoshi, N. 1994. Agrobacterium-mediated transformation of poplar using a disarmed binary 

vector and the over expression of a specific member of 

a family of poplar peroxidase in transgenic poplar cell. 

Plant Sci. 103: 231-239. 

Kawai, S.; Matsumoto, Y.; Yamada, K.; Katayama, Y.; 

Morohoshi, N. 1993. Nucleotide sequence for the genomic DNA encoding an anionic peroxidase gene from 

a hybrid poplar, Populus kitakamiensis. Biosci. Biotech. 

Biochem. 57: 131-133. 

Klopfenstein, N.B.; Shi, N.Q.; Kernan, A.; McNabb, H.S., 

Jr.; Hall, R.B.; Hart, E.R.; Thornburg, R.W. 1991. 

Transgenic Populus hybrid expresses a wound-inducible potato proteinase inhibitor II-CAT fusion. Can. J. 

For. Res. 21: 1321-1328. 

Leple, J.C.; Brasileiro, A.C.M.; Michel, M.F.; Delmotte, F.; 

Jouanin, L. 1992. Transgenic poplars: expression of chimeric genes using four different constructs. Plant Cell 

Rep. 11: 137-141. 

Lurin, C.; Jouanin, L. 1995. RFLP of RT-PCR products: 

Application to the expression of chs multigene family 

in poplar. Mol. Breeding. 1:411-417. 

Osakabe, K.; Koyama, H.; Kawai, S.; Katayama, Y.; 

Morohoshi, N. 1994. Molecular cloning and the nucleotide sequences of two eDNA that encode anionic peroxidases of Populus kitakamiensis. Plant Sci. 103: 167-175. 

Osakabe, K.; Ohtsubo, Y.; Kawai, S.; Katayama, Y.; 

Morohoshi, N. 1995a. Structure and tissue-specific expression of genes for phenylalanine ammonia-lyase 

from a hybrid aspen, Populus kitakamiensis. Plant Sci. 105: 

217-226. 

Osakabe, K.; Koyama, H.; Kawai, S.; Katayama, Y.; 

Morohoshi, N. 1995b. Molecular cloning of two 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
I 

~ 

tandemly arranged peroxidase genes from Populus 

kitakamiensis and their differential regulation in the stem. 

Plant Mol. Bioi. 28: 677-689. 

Osakabe, K.; Osakabe, K.; Kawai, S.; Katayama, Y.; 

Morohoshi, N. 1995c. Characterization of the structure 

and determination of mRNA levels of the phenylalanine ammonia-lyase gene family from Populus 

kitakamiensis. Plant Mol. Bioi. 28: 1133-1141. 

Parsons, T.J.; Bradshaw, H.D.; Gordon, M.P. 1989. Systemic 

accumulation of specific mRNAs in response to wounding in poplar trees. Proc. Natl. Acad. Sci. USA. 86: 7895-

7899. 

Subramaniam, R.; Reinold, S.; Molitor, E.K.; Douglas, C.J. 

1993. Structure, inheritance, and expression of hybrid 

poplar (Populus trichocarpa x Populus deltoides) phenylalanine ammonia-lyase genes. Plant Physiol. 102: 71-

83. 

Tsai, C-J.; Polida, G.K.; Chiang, V.L. 1995. Nucleotide sequence of a Populus tremuloides gene encoding bispecific 

caffeic acid/ 5-hydroxyferulic acid 0-methyltransferase. 

Plant Physiol. 107: 1459. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Gene Expression Studies 

van Doorsselaere, J.; Villarroel, R.; van Montagu, M.; Inze, 

D. 1991. Nucleotide sequence of a eDNA encoding malic 

enzyme from poplar. Plant Physiol. 96: 1385-1386. 

van Doorsselaere, J.; Gielen, J.; van Montagu, M.; Inze, D. 

1993. A cON A encoding 5-adenosyl-L-methionine synthetase from poplar. Plant Physiol. 102: 1365-1366. 

van Doorsselaere, J .; Baucher, M.; Feuillet, C.; Boudet, A.; 

van Montagu, M.; Inze, D. 1995a. Isolation of cinnamyl 

alcohol dehydrogenase cDNAs from two important economic species: alfalfa and poplar. Demonstration of a 

high homology of the gene within angiosperms. Plant 

Physiol. Biochem. 33: 105-109. 

van Doorsselaere, J.; Baucher, M.; Chognot, E.; Chabbert, 

B.; Tollier, M.T.; Leple, J.C.; Pilate, G.; Cornu, D.; Monties, 

B.; van Montagu, M.; lnze, D.; Boerjan, W.; Jouanin, L. 

1995b. A novel lignin in poplar trees with a reduced caffeic acid I 5 - hydroxyferulic acid 0-methyltransferase 

activity. Plant J. 8: 855-864. 

Vauterin, M; Jacobs, M. 1994. Isolation of a poplar and an 

Arabidopsis thaliana dihydrodipicolate synthase eDNA 

clone. Plant Mol. Bioi. 25: 545-550. 

69 
Chapter 10 

Differential Expression of aroA Gene in Transgenic Poplar: 

Influence of Promoter and Ozone Stress1 

David F. Karnosky, Gopi K. Podila, Dongill Shin, and Don E. Riemenschneider 

Introduction 

Fillatti et al. (1986, 1987a) first demonstrated Agrobacterium-mediated transformation and regeneration of whole 

plants with hybrid poplar clone 'NC5339' (Populus alba x 

P. grandidentata cv. 'Crandon'). They inserted a mutant bacterial aroA gene with a mannopine synthetase (MAS) promoter using a binary vector plasmid (pPMG 85/587) of 

Agrobacterium tumefaciens. Riemenschneider et al. (1988) 

conducted glyphosate spray trials that suggested that 5 of 

6 transgenic hybrid poplars had improved glyphosate tolerance compared with non transformed controls. However, 

the maximum glyphosate concentration tolerated (0.28 kg/ 

ha) was still much less than that used in commercial applications. Riemenschneider et al. (1988) attributed the low 

glyphosate tolerance to arr ineffective MAS promoter and 

the lack of transit peptide coding sequences that would 

target the modified EPSP synthase to the chloroplasts 

where native EPSP synthase is localized (Mousdale and 

Coggins 1985). 

To determine if herbicide tolerance could be increased 

in hybrid poplar by a more efficient promoter and to target aroA gene product activity to the chloroplasts, 

Riemenschneider and Haissig (1991) cocultivated hybrid 

poplar 'NC5339' with a second binary vector (pCGN 1107). 

The second binary vector (pCGN 1107) contained the mutant aroA gene, which was fused to the cauliflower mosaic 

virus promoter (CaMV 35S), and RUBISCO small subunit 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

70 

transit peptide coding sequences from Pisum sativum and 

Glycine max (Fillatti et al. 1987b ). Transformed plants from 

this experiment were tested for their growth, photosynthetic physiology, and herbicide tolerance (Donahue et al. 

1994). As expected, glyphosate tolerance was markedly 

higher for transgenic plants containing the 35S promoter 

and the RUBISCO transit peptide sequences. 

In this study, we sought a molecular explanation of the 

Donahue et al. (1994) report that suggested that the 35S promoter and the RUBISCO transit peptide sequences had conveyed increased glyphosate tolerance. We examined: 1) 

transcription and translation of the aroA gene by transformed 

hybrid poplar 'NC5339' using both MAS and 35S promoters; and 2) intracellular translocation of modified EPSP synthase (via cell fractionization). In addition, we examined the 

stability of transgene expression under stress by assessing 

the effects of acute oxidative stress (200 ppb ozone [03

] for 4 

h per day for 1 to 5 days) on aroA gene expression. 

Methods 

We used Agrobacterium tumefaciens strain C58-based binary 

vectors. One strain contained the engineered plasmid pPMG 

85/587 with the aroA gene under control of the mannopine 

synthase (MAS) promoter. A second strain contained plasmid pCGN 1107 with the aroA gene under control of the 35S 

(CaMV 35S) promoter and also ligated to RUBISCO small 

subunit transit peptide leader sequences. The leader sequences should target intracellular translocation of EPSP 

synthase from the cytosol to chloroplasts. T-DNA vectors 

were separately inserted into hybrid poplar 'NC5339' using 

the tobacco feeder layer, leaf disc transformation method 

(Fillatti et al. 1986, 1987a, 1987b). 

Shoots were regenerated via organogenesis from cocultivated leaf discs on selective media (Riemenschneider and 

Haissig 1991 ). Shoot cuttings from stabilized shoot cultures 

were collected from 3 different transgenic hybrid poplar 

clones and 1 untransformed control and rooted ex vitro 
DiHerential Expression of aroA Gene in Transgenic Poplar: Influence of Promoter and Ozone Stress 

(Riemenschneider and Haissig 1991). Clone PT-1 and PT7 contained the aroA gene controlled by the 35S promoter 

fused to the RUBISCO leader sequence for chloroplast targeting. Clone 12182 contained the aroA gene controlled by 

the MAS promoter. 

RNA was isolated (Bugos et al. 1995) and analyzed via 

northern blots using an aroA gene probe (Shin et a!. 1994). 

Protein extraction and western blot analyses of aroA protein (EPSP synthase) were performed (Shin et al. 1994). 

Cell fractionization and isolation of chloroplasts were performed (Shah et al. 1986). Protein quantification was performed (Brad ford 1976). Scanning densitome tr y o f 

northern and western blot autoradiograms was done using a mirror scanner and scananalysis softwa re. 

Ozone (03 ) fumiga tions were conducted in a continuously stirred tank reactor (CSTR) in a greenhouse at a concentration of 200 ppb for 4 h per day from 10 AM to 2 PM 

for 1, 3, or 5 d ays. Ozone was generated and monitored 

(Karnosky et a!. 1996). Four-month-old plants (40 to 60 em 

in height) were mainta ined under standard greenhouse 

condi tions before and d uring fumigations. Immediately 

after the 1-, 3-, or 5-day exposures, all leaves were removed 

and stored in -80 oc after being frozen in liquid nitrogen. 

Results and Discussion 

The 35S promoter was more .efficient than the MAS promoter for transcription of the aroA gene (figu re la) and 

translation of the aroA gene to EPSP synthase (figure 1b). 

Densitometer readings (da ta not shown) suggest that about 

5 times more messenger R 'A (mRNA) and more protein 

was produced in transgenic hybrid poplar plants with the 

35S p romoter than with the MAS promoter. These results 

are consistent with those of Donahue et al. (1994) and explain why the 35S promoter conveyed a higher level of 

glyphosate tolerance than the MAS promoter in previous 

studies (Donahue et al. 1994). 

EPSP synthase was found in chloroplasts of poplars 

transformed with construct pCGN 1107 containing the 35S 

promoter and transit peptide coding sequences (figure 1c). 

In contrast, EPSP synthase produced by poplars transformed with construct pPMG 85 / 587 was not found in 

chloroplasts (figure 1c). We conclude that heterologous 

RUBISCO small subunit leader sequences were e ffective 

in targeting the translocation of the modified EPSP synthase from the cytosol to chlorop lasts. Size differences between the chloroplastic and cytosolic fractions (figure 1c) 

suggest that the cytosolic EPSP synthase possessed an attached leader sequence, whereas the chloroplastic EPSP 

synthase is smaller, ind icating effective post-translocation 

excision of the leader sequences. 

For genetic engineering to be successful in the long term, 

the transgenes must be stably expressed, even in the presence of commonly occurring p lant stresses. To examine 

the effects of acute oxidative stress on aroA gene expression in transgenic hybrid poplar plants, we exposed plan ts 

lu 0 3 fur 4 h per day fur either 1, 3, ur 5 d ays in a cuntrolled environment chambe r. As is shown in figu re 2a, 

0 3 stress resulted in a d own regulation of the aroA gene 

under control of the MAS promoter. However, transcription of the aroA gene under the influence of the 35S pro2 3 4 5 6 2 3 4 

La--------------_, b ____________ _. 

1- -4c ! ~ 

Figure 1. Transcriptional and translational expression of the aroA gene in transgenic hybrid poplar and subcellular localization of the EPSP synthase protein. a) Northern blot analysis was performed using 1 0 J.lg each of total RNA from 

untransformed control (lane 1), clone 12182 (lane 2), clone PT-1 (lane 3), and clone PT-7 (l ~ne 4). Blots were . 

probed with Jzp labeled aroA gene probe. Arrow points to aroA RNA. b) Western blot analysis was performed us1ng 

20 J.l9 each of total cellular proteins from untransformed control an~ 1) •. clone 1~2 (lane 2), clone PT-1 (i an~ 3), 

and clone PT-7 (lane 4). Blots were probed with EPSP synthase antibodies. Detection of EPSP synthase ~r m 

was done using HAP-conjugated, secondary antibodies and ECL detecti ~ method (Amersham Corp ... Arhngton 

Heights, IL). c) Western blot analysis of EPSP synthase was pe~ormed usmg 20 J.lg each from cytosohc and 

chloroplastic protein fractions. Lanes 1 through 4 are chloroplastlc EPSP synthase from control, clone 12182, 

clone PT-1 and clone PT-7, respectively. Lanes 5 through 8 are cytoplasmic EPSP synthase from control, clone 

12182, cl e PT-1, and clone PT-7. Arrow points to mature chloroplastic form of EPSP synthase and arrowhead 

points to cytosolic precursor form of EPSP synthase. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 7 1 
Section 11 Transformation and Foreign Gene Expression 

"' CJ 

> 

CJ 

6 

5 

4 

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z 

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E: 

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10 

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n n II n ·n 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 

a) Poplar Clones 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 

b) Poplar Clones 

Figure 2. Influence of acute ozone exposure (200 ppb for 

4 h per day for 1, 3, or 5 days) on the transcriptional a nd transla tional level expression of the 

aroA gene in trans genic hybrid poplars. a) 

Densitome te r scan of aroA RNA northern blot 

from untreated and 0 3 treated untransformed 

and transformed plants. Lanes 1, 5, 9, and 13 

a re untransformed control plants treated with 0 3 

for 0, 1, 3, or 5 days. Lanes 2, 6, 10, and 14 a re 

transforme d clone 12182 treated with 0 3 for 0, 1, 

3, or 5 days. La nes 3, 7, 11, and 15 are transformed clone PT-1 treated with 0 3 for 0, 1, 3, and 

5 days. Lanes 4, 8, 12, and 16 a re transformed 

clone PT-7 treate d with 0 3 for 0, 1, 3, a nd 5 

72 

days. b) Densitometer scan of EPSP synthase 

weste rn blot from untreated a nd 0 3 treated 

untransformed and tra nsformed plants. Lanes 

are as described for figure 2a. 

mote r was not detectably d ifferent from control conditions 

afte r 1, 3, or 5 days of 0 3 (figure 2a). Concentration of 

modified EPSP synthase in transgenic popla rs containing 

the 355 promoter was affected by 0 3 exposure with both 

promoters (figure 2b). We do not know if this was due to 

reduced EPSP synthase biosynthesis or a n increased 

protein d egra d ation ra te. However, if degrada tion was 

a major factor, we woul d expec t to see an increase in 

degrad a tion products resulting in new bands in fi gure 

2b. Since this d id not occur, we conclude that the main 

in flu ence of 0 3 w as in decreasing the transla ti on of 

mR A into protein. 

Summary 

The he rbicide glyphosate [N-(phosphonomethyl) glycine] kills plants prima rily by inhibiting the enzyme EPSP 

synthase (5-enolpyruvylshik.ima te-3-phosphate synthase). 

A mutant nroA gene encod ing a modified EPSP synthase 

was used to convey increased glyphosa te tolerance to hybrid poplar cl. 'NC5339' and othe r plants. While earlier 

studies showed that tolerance of transgenic ' NC5339' to 

glyphosa te was increased, transgenic 'NC5339' was not 

immune to recommended commercia l application rates. 

In this study, we showed tha t: 1) the 355 promoter was 

more efficient than the MAS promoter in eliciting both transcriptional and translational expression of the nroA gene; 

2) the RUBISCO transit pep tide coding sequees successfully mediated translocation of the aroA gene p roduct to 

chloroplasts, and 3) acute oxidative stress (200 ppb ozone 

for 4 h pe r day for 1, 3, or 5 days) decreased the effectiveness of the MAS promoter more significantly than that of 

the 355 promoter in regula ting nroA gene expression. These 

results suggest that promote r selection plays an important 

role in transgene expression, and tha t we can design organelle-specific transgene expression. Our results with 

oxid c. tive stress suggest tha t additiona l resea rch is needed 

to d ete rmine the e ffectiveness of transgene expression 

unde r stress, and that stress tole rance of promote rs should 

be conside red in developing long-te rm biogenic products 

in forest trees. 

Acknowledgments 

This research was partially supported by the State of 

Michigan Resea rch Excellence Fund and by the Departmen t of Energy und er Inte ragency Agreement DE-A105-

800R20763. . 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

-
Differential Expression of aroA Gene in Transgenic Poplar: Influence of Promoter and Ozone Stress 

Literature Cited 

Bradford, M.M. 1976. A rapid and sensitive method for 

the quantification of microgram quantities of protein 

utilizing the principle of protein-dye binding. Anal. 

Biochem. 72: 248-254. 

Bugos, R.C.; Chiang, V.L.; Zhang, X.-H.; Campbell, E.R.; 

Podila, G.K.; Campbell, W.H. 1995. RNA isolation from 

plant tissues recalcitrant to extraction in guanidine. 

BioTechniques. 19: 734-737. 

Donahue, R.A.; Davis, T.D.; Michler, C.H.; 

Riemenschneider, D.E.; Carter, D.R.; Marquardt, P.E.; 

Sankhla, D.; Haissig, B.E.; Isebrands, J.G. 1994. Growth, 

photosynthesis, and herbicide tolerance of genetically 

modified hybrid poplar. Can. J. For. Res. 24:2377-2383. 

Fillatti,J.J.; McCown, B.H.; Sellmer, J.; Haissig, B. 1986. The 

introduction and expression of a gene conferring tolerance to the herbicide glyphosate in Populus NC-5339. Proc. 

TAPPI research and development conference. Raleigh, 

NC, U.S.A. Atlanta, GA, U.S.A.: TAPPI Press: 83-85. 

Fillatti, J.J.; Sellmer, J.; McCown, B.; Haissig, B.; Comai, L. 

1987a. Agrobacterium-mediated transformation andregeneration of Populus. Mol. Gen. Genet. 206: 192-199. 

Fillatti, J.J.; Kiser, J.; Rose, R.; Comai, L. 1987b. Efficient 

transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Bio/Technology. 5: 726-730. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Karnosky, D.F.; Gagnon, Z.E.; Dickson, R.E.; Coleman, 

M.D.; Lee, E.H.; Isebrands, J.G. 1996. Changes in 

growth, leaf abscission, and biomass associated with 

seasonal tropospheric ozone exposures of Populus 

tremuloides clones and seedlings. Can. J. For. Res. 26: 

23-37. 

Mousdale, D.M.; Coggins, J.R. 1985. Subcellular localization of the common shikimate-pathway enzymes in 

Pisum sativum L. Planta. 163: 241-249. 

Riemenschneider, D.E.; Haissig, B.E.; Sellmer, J.; Fillatti, 

J.J. 1988. Expression of a herbicide tolerance gene in 

young plants of a transgenic hybrid poplar clone. In: 

Ahuja, M.R., ed. Somatic cell genetics of woody plants. 

Dordrecht, The Netherlands: Kluwer Academic Publishers: 73-80. 

Riemenschneider, D.E.; Haissig, B.E. 1991. Producing 

herbicide-tolerant Populus using genetic transformation mediated by Agrobacterium tumefaciens C58: A 

summary of recent research. In: Ahuja, M.R., ed. Biotechnology of woody plants. New York: Plenum 

Press: 247-263. 

Shah, D.M.; Horsch, R.B.; Klee, H.J.; Kishore, G.M.; Winter, J.A.; Turner, N.E.; Hironaka, C.M.; Sanders, P.R.; Gasser, C.S.; Aykent, S.; Siegel, N.R.; Rogers, S.G.; Fraley, 

R.T. 1986. Engineering herbicide tolerance in,transgenic 

plants. Science. 233: 478-481. 

Shin, D.-I.; Podila, G.K.; Huang, Y.; Kamosky, D.F. 1994. 

Transgenic larch expressing genes for herbicide and insect resistance. Can. J. For. Res. 24: 2059-2067. 

73 
Chapter 11 

Growth and Development Alteration in Transgenic Populus: 

Status and Potential Applications1 

Bjorn Sundberg, Hannele Tuominen, Ove Nilsson, Thomas Moritz, 

c. H. Anthony Little, Goran Sandberg, and Olof Olsson 

Introduction 

With the development of gene-transfer techniques applicable to forest tree species, genetic engineering is becoming 

an alternative to traditional tree breeding. To date, routine 

transformation methods for several hardwood species, particularly Populus and Eucalyptus, have been established and 

promising advances have occurred in the development ~f 

transformation protocols for conifers (Olarest et al. 1996; Ellis 

et al. 1996; Jouanin et al. 1993; Walters et al. 1995). Rapid 

progress in transformation technology makes it possible to 

develop genetic engineering tools that modify economically 

tractable parameters related to growth and yield in tree species. Such work will also increase our understanding of genetic and physiological regulation of growth and 

development in woody species. 

Several hybrid aspen lines with phenotypes modified 

by genetic engineering were produced and characterized 

in our laboratory. Their existence shows, unequivocally, 

that tree growth and development are alterable by genetic 

engineering. Some modified phenotypes are described in 

this chapter. We also briefly discuss how genetic engineering might be used to generate trees with properties, such 

as modified wood structure, which are desirable to the 

forest industry. 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

74 

Using Transgenic Populus to 

Study Growth and Development 

in Woody Species 

The best model system for understanding the genetic 

and physiological control of tree growth and wood formation is a perennial species containing a vascular cambium, which is the meristem that produces secondary 

xylem and phloem. Presently, Populus is the preferred treemodel system because it has several useful fea.tures. 

Populus has a small genome, approximately 5 x lOS base 

pairs (bp ), which encourages molecular mapping, library 

screening, and rescue cloning. Saturated genetic maps are 

already constructed for several Populus spp. (Bradshaw et 

al. 1994; Cervera et al. 1996). Moreover, Populus can beefficiently transformed and regenerated, and it grows rapidly. Thus, isolated genes inserted into proper vectors can 

easily be introduced into Populus, readily producing 

transgenic plants. 

The major disadvantage of the Populus model system is 

that controlled crosses are time consuming and difficult to 

perform. Consequently, expression of introduced genes in 

the F2 generation remains undetermined. To date, all physiological and phenotypic investigations involving 

transgenic Populus have been restricted to the primary 

transformant. However, as research on Populus increases, 

novel schemes will be developed to induce early flowering by physiological, biochemical, or genetic manipulation. 

As discussed below, the potential for genetic manipulation has been shown by the expression of the Arabidopsis 

LEAFY (LFY) gene in a hybrid aspen. 

We used a hybrid aspen, Populus tremula x P. tremuloides, 

as a model system for genetic engineering experiments. 

Expression vectors containing the gene(s) and promoter(s) 

of interest were introduced by electroporation into the C58 

Agrobacterium tumefaciens strain GV3101 (pMP90RK). To 

move the transfer DNA (T-DNA) from these vectors to the 
Growth and Development Alteration in Transgenic Populus: Status and Potential Applications 

Populus genome, sterile, internodal stem segments were 

cocultivated with the Agrobacterium for 2 days on solid 

Murashige and Skoog (MS) medium (Murashige and 

Skoog 1962). Cocultivated explants were transferred to new 

media containing plant growth substances to initiate shoot 

formation and antibiotics to select for transformants and 

eliminate remaining Agrobacterium cells (Nilsson et a!. . 

1992). Using this protocol, transformation frequency was 

typically about 20 percent, regeneration success was higher 

than 95 percent, and rooted plants 7 to 10 em tall were 

obtained after about 4 months in sterile culture. Transgenic 

plantlets were either placed in pots and cultured in a greenhouse or placed in a controlled environment chamber (figure 1). For the first 7 to 10 days of culture, plants are covered 

with a plastic bag for acclimation to decreased humidity. 

Gene expression, and therefore phenotype, is significantly affected by environmental conditions such as light 

quality and quantity, nutrient availability, and temperature. To compare phenotypes from different transformation events, plants must be cultured under controlled and 

reproducible environmenta l conditions, especia lly when 

performing experiments at different times of the year. For 

critical evaluation of transgenic phenotypes, we used controlled environment chambers and potted the plants in 

mineral wool. This potting medium was used for a stable 

and defined nutrient supply through daily surplus watering with a n optimal nutrient solution (Ingestad 1970). 

These culture conditions induced rapid growth; about 1 

leaf primordia was produced every 2 days in wild-type 

plants, and a plant 1 m tall was produced after about 6 

weeks in the growth chamber. 

The Populus model system enables the function of genes 

isolated from any organism to be evaluated in a tree species by transgene expression. Alternatively, the corresponding endogenous Populus gene can be cloned and used for 

transformation in either sense (overproduction of the gene 

product) or antisense (suppression of the gene expression) 

orientation. 

Transgenic Populus With Altered 

Growth and Development 

Transgenic Hybrid Aspen Expressing the 

Arabidopsis LEAFY Gene 

Knowledge of gene function in a distantly related model system, such as Arabidopsis, can be directly applied to a tree species. 

This was recently shown by expression of the Arabidopsis gene 

LEAFY (LFY) in hybrid aspen to promote precocious flowering 

(Weigel and Nilsson 1995). Arabidopsis plants with lossof-function mutations in LFYhave shoots where typically 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Figure 1. Regeneration and culture of hybrid aspen plants. 

A) Internodal stem segments. B) Shoots regenerated on stem segments. C) Shoot proliferation 

on cytokinin-rich medium. D) Initial culture of 

potted plants under plastic bags. E) Plants 

cultured under controlled environment conditions 

in a climate chamber. F) Plants cultured in a 

greenhouse. 

flowers grow (Huala and Sussex 1992; Schultz and Haughn 

1991; Weigel eta!. 1992), which shows that LFYis necessary 

for normal flower initiation in that species. Furthermore, LFY 

is the first gene expressed in the flower primordium, and 

LFY transcription is detected in the predicted positions of 

the flower primordia before any other signs of primordium 

formation are evident (Weigel eta!. 1992). 

75 
Section II Transformation and Foreign Gene Expression 

Figure 2. Typical phenotypes of hybrid aspen transformants. A) and B) Plants expressing the Arabidopsis LFY gene. Note 

flowers formed by the axillary (A) (Weigel and Nilsson 1995) and apical (B) meristems. C) and E) Plants expressing the Agrobacterium tumefaciens T-DNA IAA-biosynthesis genes. Note the slower growth, smaller leaves and, 

after decapitation, inhibited axillary bud outgrowth in transformants (C-left, E-right) compared with wild-type 

76 

plants (C-right, E-left). D) Plants expressing the oat phyA 

gene. Note the short internodes. F) and G) Plants expressing the Agrobacterium rhizogenes T-DNA ro/C gene. Note 

the bushy phenotype (F) and stem fasciation (G) (Nilsson 

et al. 1996b). 

These findings suggest that LFY is the "main switch" 

for the flower formation program, and that it might be 

necessary and su fficient to ind uce flowering. This function of LFY was shown by fusing the LFY coding region 

to the strong cauliflower mosaic virus (CaMV) 355 promoter and tran sforming the 355-LFY construct into 

Arabidopsis (Weigel and Nilsson 1995). The transgenic 355-

LFYplants flowered ea rlier than their wild-type counterparts because the meristem in the leaf axil, which norma lly 

produces a secondary shoot, produced a single flower. 

Eventually, the apical meristem of the prima ry shoot 

formed a termina l flower. However, because Arabidopsis 

natu rally flowers very early, a fter about 3 weeks under 

inductive long-d ay conditions, the induced difference in 

flowering time was not very dramatic. 

A more stringent test of the flower-ind ucing capability 

of LFY and for the conserva tion of LFY function between 

species was the introd uction of the same 355-LFY construct into a hybrid aspen whose pa rental species take 8 

to 20 years to flower under natural conditions (Schreiner 

1974}. Remar kably, the 355-LFY hybrid aspen 

transformants flowered after only a few months in tissue 

culture (Weigel and 1 ilsson 1995) (figure 2A, 28). 

Transformants with high LFYexpression prod uced a solita ry flower in the axil of a few leaves before the apical 

meristem of the shoot was consumed in the formation of 

an aberra nt te rmina l fl ower. Transformants with low 

LFY expression could be rooted and s ubsequently were 

transferred to the greenhouse. Some of these p lants also 

formed a single fl ower with normal appea rance in the 

lea f axils, bu t it remains unclear whethe r these fl owers 

are fe rtile . 

Th us, th e phenotype of hybrid aspen p lan ts that 

s trong ly express 355-LFY is similar to ana logous 

Arabidopsis transformants. This shows that the fu nction 

of the LEAFY protein is highly conserved between unrelated d icot species and raises the possibility that the 

Arn~idopsis LFY can be used to ind uce early flowering in 

a w1de ra nge of tree species. This range of tree speci'es 

may include conifers as Douglas-fir contains a gene that 

shows high homology with Arnbidopsis LFY (S. Strauss, 

personal communication). 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
I 

.., 

Growth and Development Alteration in Transgenic Populus: Status and Potential Applications 

Transgenic Hybrid Aspen Expressing phyA 

an6 phyB Genes From Oat and Arabidopsis 

In temperate-zone forest tree species, adaptation to a 

climate is based primarily on the proper timing of growth 

and dormancy during the year. Photoperiod and its interaction with temperature are the main environmental factors that control the periodicity of growth and frost 

hardiness (Heide 1974; Junttila 1989). Phytochrome is generally believed to be the pigment system responsible for 

the perception of light (i.e., dawn and dusk) in the photoperiodic regulation of plant growth and development 

(Sharrock 1992). However, the molecular mechanism of 

phytochrome action and the components of its signaling 

pathway are largely unknown. Most studies have focused 

on 2 of the 5 identified types of phytochrome genes in 

plants, phytochrome A (phyA) and phytochrome B (phyB). 

In collaboration with Drs. Junttila, Nilsen, and Olsen at 

the University of Troms0, Norway, the phyA and phyB genes 

from oat and Arabidopsis, respectively, were expressed in 

our hybrid aspen under the control of the CaMV 355 promoter. The most striking morphological alterations were 

shorter internodes in phyA transformants (figure 2D). Out 

of 22 phyA-expressing lines, as verified by RNA blot analysis, 6 lines with varying degree of phenotypic alteration 

were selected and characterized with respect to growth 

pattern and photoperiodic control of growth cessation. 

Preliminary data showed that lines with weak expression 

of phyA had internodes with normal elongation and that 

increased phyA expression was associated with reduced 

internode length. Morover, the lines with highest phyA 

expression lost their photoperiodic control almost completely. The normal, critical day length for wild-type plants 

is 15 h, but even exposure to a photoperiod of only 8 h did 

not induce bud set in these lines. 

Transgenic Hybrid Aspen Expressing the 

Agrobacterium tumefaciens T-DNA IAABiosynthesis Genes 

Plant hormones are important molecular regulators of 

growth and development. They are major signal:rs in environmentally induced growth responses and are mvo~v~d 

in integrating patterns of growth and ~evel?pment ~Ithm 

the plant. The hormone indole-3-acehc aod (IAA) IS t_he 

most abundant endogenous auxin, and it plays a maJOr 

role in the control of basic developmental processes such 

as cell division, expansion, and differentiation. IAA influences many aspects of plant anatomy and morp~ology; 

for example, leaf enlargement, inter~ode elo~gation, abscission, shoot and root induction, apical domma~ce, a~d 

tropisms (Davies 1995). IAA also maintai~s the.I~tegnty 

of the vascular cambium, promotes cambial activity, and 

affects xylem cell type and anatomical structure (Little and 

Pharis 1995). 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

To investigate the effect of IAA on wood formation using 

the transgenic approach, we transformed a hybrid aspen with 

the 1M-biosynthesis genes iaaM and iaaH, originating from 

the soil bacterium Agrobacterium tumefaciens T-DNA (Guadin 

et al. 1994). Resulting transformants showed that both tree 

phenotype and wood properties can be altered using genetic 

engineering to modify the hormone balance (Tuominen et 

al. 1995). The most severe phenotypic change was in a line 

expressing the iaaM and iaaH genes under control of the 

mannopine synthase (MAS) 1' and 2' promoters, respectively 

(figure 2C, 2E). This line exhibited reduced stem growth, 

radially and longitudinally, had an altered leaf and root morphology, and maintained apical dominance over axillary 

buds after removal of the apical shoot. Wood formed in 

transgenic poplars expressing the fAA-biosynthesis genes 

was characterized in considerable detail. Not surprisingly, 

the xylem in these plants was altered with respect to composition, pattern, and size of the rays, fibers, and vessels 

(figure 3). 

Hybrid aspen transformants expressing the fAA-biosynthesis genes were also characterized for free and conjugated IAA. Elevated concentrations of free and 

conjugated IAA were found in the basal leaves and root 

tips but not in the apical part of the shoot. Apparently 

these promoters increased the level of gene expression basipetally (Langridge et al. 1989). However, an elevated IAA 

concentration was not observed in the extraxylary tissues 

of the stem base, although RNA blot analysis showed that 

the iaaM and iaaH genes were both expressed in these tissues (unpublished data). This observation highlights the 

problem of separating the primary and secondary effects 

of ectopic gene expression on metabolic events and phenotypic expression. In this example, the ectopic expression of the IAA-biosynthesis genes resulted in a general 

growth inhibition. This growth reduction likely decreased 

the supply of endogenous IAA, which is produced predominantly in apical shoots and transported basipetally 

down the stem (Rinne et al. 1993; Sundberg and Little 

1987). This is supported by more detailed IAA measurements in specific extraxylary tissues (see Uggla et al. 1996) 

that showed that transgenic plants had a lower concentration of IAA in the cambial cell division zone, which is 

the major pathway of pol~u transport (unpubli~hed data_). 

However, expression of the introduced IAA-biOsynthesis 

genes seemed to increase IAA concentration in cambial 

derivatives, which resulted in an unchanged total concentration of IAA in the extraxylary tissues. Another 

possibility is that the transform~nts com~ens.ated for 

the induced increase in IAA biosynthesis either by 

down-regulating endogenous IAA biosynthesis thro~gh 

feedback inhibition or by enhancing IAA degradation. 

The important observation concerning the transformants described are that tree phenotype, and wood 

properties in particular, can be altered using the 

transgenic approach. 

77 
Section II Transformation and Foreign Gene Expression 

Figure 3. Transverse section of the xylem in hybrid aspen 

wild-type plants (left) and transformants expressing the Agrobacterium tumefaciens T-DNA IAAbiosynthesis genes (right). Note the distortion of 

radial files, the occurrence of intermediate-sized 

xylem elements, and the larger fibers and ray 

cells in transformants. V=vessel; F=fiber; R=ray. 

Transgenic Hybrid Aspen Expressing the 

Agrobacterium rhizogenes T-DNA ro/C Gene 

The most compelling example of an altered phenotype 

in a hyb rid aspen due to the expression of a single-gene 

insert resulted from transformation with the Agrobncterir1111 

rhizogenes rotC gene, which was fused to the CaMY 355 

promoter (Nilsson et al. 1996b) (figure 2F, 2G). The rotC 

gene is responsible for hairy root disease, which is caused 

by the plant pathogen Agrobacterium rhizogenes (Zambryski 

et al. 1989). Compared with wild-type hybrid aspen plants, 

rotC-expressing plants elongated slower, produced more 

leaves, generated dwarfed leaves, and displayed an altered 

phyllotaxis. In addition, the apex branched at irregula r 

interva ls to produce multiple leading shoots, which resu lted in a bushy phenotype. Following a period of environmentally induced dormancy, outgrowth of axillary buds 

in the second growing season always produced short 

shoots in the rotC trans formants, whereas wild-type p lants 

formed long shoots. Another conspicuous feature of the 

rotC-expressing hybrid aspen plants was the formation of 

fasciated stems, which occurs when the apical meristem 

loses its radial symmetry and expands in 1 plane to form a 

ridge-like meristem (Gorter 1965). The flat stem produced 

by such meristems occasionally grew in a spiral (figure 

2G). Overall, expression of the 355-rotC construct in a hybrid aspen resulted in a phenotype with drastically altered 

appearance. 

Endogenous levels of IAA, gibberellins (GAs), and cytokinins were measured in fasciated and nonfasciated 

shoots of rotC-expressing hybrid aspen plants. Like IAA, 

GAs and cytokinins are plant hormones involved in controlling cell division and elongation, which are important 

78 

for shoot and leaf expansion (Davies 1995). A marked reduction in the ratio of IAA to the cytokinin zeatin riboside 

([9R]Z) was found in the transformants, which resulted 

from a small decrease in free lAA and a large increase in 

the cytokinin. Moreover, GA1 was decreased . Because the 

biochemical activity of the rotC protein is unknown 

(Costantino et al. 1994), any observed changes in hormone 

levels as a primary effect of its presence is only speculation. Nevertheless, the large alterations in the auxin / cytokinin ratio and the GA1 level, together or separa tely, could 

explain several observed changes in phenotype. 

Improvement of Trees by Gene 

Technology 

Examples presented show that growth and development 

in woody species can be altered by genetic engineering. 

This has considerable potential for accelerating tree improvement and offers the possibility of directly altering 

specific parts of the genome either by manipulating the 

expression pattern of ndog~nous genes or by inserting 

genes from other organisms. Several levels of control can 

be genetically modified to obtain phenotypic alterations. 

These include the: 1) perception of external stimuli, as in 

the phyA transformants; 2) internal balance of signal substances, as in the plants expressing the IAA-biosynthesis 

genes; and 3) expression of specific cellular proteins determining developmental fa tes, as in the LFY transformants. The gene pool available for tree improvement 

by genetic engineering is not limited to the species of interest because traits outside the genetic range of the species can be incorporated. The potential of this approach to 

alter plan t development is best exemplified by the severely 

altered phenotype of the rotC-expressing hybrid aspen 

plants. 

Potential Applications 

LEAFY and Early Flowering 

If the resulting flowers are fertile, ectopic expression of 

the Arnbidopsis gene LFY in a tree species, which reduces 

the flowering time from 8 to 20 years to a few months, will 

have a major impact on tree breeding programs. Reducing flowering time would encourage construction of homozygous inbred transgenic lines and characterization of 

molecular markers over several generations and wou ld 

acce lerate existing tree-improvement programs. Traditional breeding techniques have increased the yield of 

many agricultura l crop species that have a short generation time. One example is the development of cereals from 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Growth and Development Alteration in Transgenic Populus: Status and Potential Applications 

their original long, slender, low-yielding grass species to 

today's ·short, sturdy varieties with a dramatically increased seed production. However, such breeding programs have operated for thousands of plant generations, 

whereas programs involving tree species are much less 

developed due to their long time to flowering. 

Recently, techniques for the construction of saturated 

genetic maps were improved tremendously. These techniques also detect small structural differences between 

DNA of individuals. This technology is used to link socalled "quantitative trait loci" (QTLs) with specific DNA 

markers (e.g., Bradshaw and Stettler 1995; Grattapaglia et 

al. 1995; Groover et al. 1994). Once a QTL has been tagged, 

growing full-sibling progeny to maturity to select a particular trait is unnecessary. Instead, the trait of interest can 

be sought at the seedling stage based on its association 

with a molecular marker. Using this approach, the limiting factor for the breeding program will again be time to 

flowering. Coupling early screening for QTLs with the 

application of LFY to shorten the generation time would 

accelerate traditional tree-improvement programs. Incorporating an inducible promoter system would be an additional refinement because flowering could be induced 

when wanted; for example, by applying a harmless chemical (e.g., Wilde et al. 1992). 

Plant Hormones and Tree Growth 

The morphological and anatomical changes in hybrid 

aspen plants expressing either the IAA-biosynthesis genes 

or the roiC gene were associated with an altered hormone 

balance. This is not surprising as hormones are involved 

in the regulation of many aspects of plant growth and development. Using genetic engineering to modify the endogenous hormone balance has obvious potential for obtaining a desired phenotype. However, the dramatic 

alteration of growth and development observed in our 

hybrid aspen transformants emphasizes the need to express factors involved in developmental control in the appropriate cells at the proper time. Depending on the target cell competence, this is particularly true with plant 

hormones that have distinct functions in different tissues. 

Controlling modification with such factors requires promoters capable of targeting gene expression both spatially 

and temporally. Incorporation of an inducible promoter 

system would be most beneficial. The molecular tools for 

manipulating hormone homeostasis in specific tissues of 

intact plants are available because the levels of IAA, cytokinin, ethylene, and GA have been altered by genetic engineering (e.g., Hedden et al. 1995; Klee and Romano 1994). 

Moreover, the number of tissue-specific promoters available for experimentation is increasing rapidly. Exploiting 

these tools is valuable to evaluate the role of hormones in 

wood formation and for future gene engineering of tree 

growth. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Modification of Wood Properties 

An obvious target for tree breeding is to improve the 

chemical and physical properties of wood itself (Whetten 

and Sederoff 1991). To date, efforts toward genetic engineering of wood have focused on the lignification process; van Doorsselaere et al. {1995) showed that lignin composition in Populus can be modified. However, our 

discussion is restricted to wood structure, which is a primary determinant of wood density and the most important factor for yield and quality of fibrous and solid wood 

products (Zobel and Jett 1995). The role of plant hormones 

in regulating wood formation was investigated for many 

years. Auxin, cytokinins, ethylene, and GAs are all involved to varying extents in regulating xylem cell production and structure and in determining the proportion 

of each xylem cell type formed in time and space (Aloni 

1991; Little and Pharis 1995). IAA, whose transport is basipetally polar from crown to root in the cambial region, is 

key to this regulation because it maintains the structure of 

the vascular cambium and induces cambial cell mitotic activity and xylem cell development (Little and Pharis 1995). 

Therefore, it seems likely that IAA is involved in controlling 

the integration of crown structure and stem form and in serving as a link between external stimuli such as photoperiod, 

gravity, and wood formation: Underlying mechanisms, by 

which auxin or any other plant hormone acts in regulating 

wood formation, remain to be determined; however, wood 

structure was altered in hybrid aspen plants expressing the 

IAA-biosynthesis genes. Although the potential for genetically engineering wood structure is evident, other possible 

tools besides plant hormones are available for modifying 

wood density. These prospects are briefly discussed. 

Wood density is determined by a complex of factors related to the: 1) structure of individual xylem cells (i.e., their 

secondary wall thickness and radial diameter); 2) proportion and distribution of the various types of xylem cells 

(i.e., fibers, vessels, and parenchyma in hardwoods, and 

tracheids and parenchyma in conifers); and 3) proportion 

and distribution of different types of wood (e.g., reactionwood, earlywood, and latewood). Xylem cell structure is 

determined as cambial derivatives differentiate. During 

differentiation, each derivative passes through several 

successive developmental phases, including primary wall 

expansion, secondary wall formation, and programmed 

death for the supportive fibers and the water-conducting 

vessels and tracheids (Larson 1994) (figure 4). Xylem cell 

size and wall thickness reflect the rate and duration of the 

expansion and secondary wall deposition phases (Dodd 

and Fox 1990; Wodzicki 1971 ). These phases probably are 

independently regulated, because wide- and narrow-lumen tracheids with thick and thin walls have been induced 

experimentally (Larson 1969). This variability supports the 

idea that xylem cell structure can be manipulated in specific ways by genetic engineering. 

79 
Section II Transformation and Foreign Gene Expression 

Figure 4. Transverse section of the cambial region in a 

current-year s hoot of wild-type hybrid aspen plants 

showing xylem cells in different developmental 

phases. CZ=meristematic cambial cell zone; 

REX=radially enlarging xylem cells undergoing 

primary-wall expansion; MX=maturing xylem 

cells undergoing secondary-wall formation. 

Na tural variability of traits related to wood density is 

high . Many wood properties have high heritability (Zobel 

and Jett 1995), but w ood formation is also very plastic, 

which suggests the importance of physiological factors in 

controlling wood density (figure 5). The p lasticity of wood 

formation shows that environmental cues, such as photoperiod, temperature, gravity, mineral availability, and 

water status, affect wood structure and sometimes result 

in different wood types such as latewood or reaction wood 

Figure 5. Transverse section of a current-year shoot of wildtype hybrid aspen plants in which the IAA balance 

was disturbed by applying the auxin-transport 

inhibitor N-1-naphthylphthalamic acid. Note the 

marked change in the type of wood formed after 

the treatment began (arrows), which exemplifies 

the plasticity of wood formation and highlights the 

major role of IAA in controlling wood properties. 

80 

(Creber and Chaloner 1990; Denne and Dodd 1981; Timell 

1986). Moreover, wood structure varies spatially in the 

stem; for example, wood with juvenile characteristics is 

formed within the crown while more la tewood to earlywood is produced at the base of the stem rather than at the 

top of the tree. Relative amounts of juvenilewood and latewood significantly influence wood density (Zobel and 

Buijtenen 1989). Variability of naturally formed wood provides additional evidence that wood formation processes 

are amenable to manipulation by genetic engineering. In 

fact, all processes that are directly or indirectly under genetic control can in theory be manipulated by this approach. Obvious targe ts are factors associated w ith 

controlling xylem cell production (see Savidge 1985). 

Development of New Tools for the 

Genetic Engineering of Wood 

Currently, the main factor limiting genetic engineering 

for wood improvement is insufficient knowledge about 

the regulation of wood formation. Be tter understanding is 

required of the mechanisms that operate at the molecular 

and cellular levels, and of the mechanisms that integrate 

environmental cues responsible for the seasonal and spatial changes in wood production at the whole-plant level. 

Further, characterization of key internal factors, which 

serve as lin ks between external stimuli and wood formation and control the cascade of cellular events that determine the properties of individual xylem cells, is essential. 

Many aspects of xylem cell formation a re best studied 

using simple experimental systems that arc easily cultiva ted and manipulated s uch as cell or tissue cultures of 

herbaceous model species (Fukada et a!. 1994). For example, cultured Zinnia mesophyll cells were useful for investigating gene expression and molecular function of key 

factors involved in trachcary element differentiation (e.g., 

Fukada 1994; Stacey eta!. 1995). Several genes induced 

during the differentiation process were characterized, and 

their spatia l expression pattern was visualized in intact 

Zinnia seed lings by in situ loca liza tion (Demura and 

Fukada 1994; Ye and Varner 1994). In another approach to 

isolate genes involved in xylem cell differentiation, chemical mutagenesis was used at the John Innes Institute (Norwich, England) to characterize several Arnbidopsis mutants 

with aberrant xy lem development (M. Bevan and K. 

Mctzlaff, personal communication). Molecular cloning of 

genes corresponding to known mutations in Arnbidopsis is 

promoted by dense genetic and physical maps that cover 

known markers. Because Arnbidopsis has become the most 

important model system for studying basic plant biology, 

it will be the major source of genes coding for conserved 

factors involved in plant growth and development regulation (Gibson and Sommerville 1993). As previously discussed, the usefulness of this source for the genetic 

engineering of wood y species was recently shown with 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
I 

I 

I 

Growth and Development Alteration in Transgenic Populus: Status and Potential Applications 

the Arabidopsis LFY gene that induced early flowering in 

our hybrid aspen model system. Knowledge about xylem 

cell formation in herbaceous model systems should also 

be applicable in woody species. 

Although experimental systems that are easily manipulated are attractive, using tree species to identify biochemical events and specific genes related to xylem cell 

development also has advantages. For example, due to the 

size of trees, specific cambial region tissues and cell types 

can be identified and isolated by microdissecting techniques (Hampp et al. 1990). This approach enables monitoring of the cellular status of enzymes, nutrients, and 

hormones with sensitive microanalytical techniques at 

varying stages of the differentiation of cambial derivatives 

(Catesson et al. 1994; Uggla et al. 1996). Similarly, polymerase chain reaction (PCR)-based techniques can be used 

to isolate genes that are uniquely expressed in time and 

space within the cambial region (Hertzberg and Olsson 

1995). In addition, using tree species allows for the characterization of cambial derivatives differentiating into different wood types. For example, the transition from 

earlywood to latewood and from norrnalwood to 

reactionwood can be induced experimentally by photoperiod and tilting, respectively. Thus, changes in cellular 

events and gene expression related to these wood types 

can be characterized. Furthermore, using Populus as a 

model system will enable isolation of genes and characterizatioh of their function in transgenic plants. 

A combination of biochemical, molecular, and genetic 

approaches will be required to <:>btain the tools for improving wood properties by genetic engineering. Unraveling 

metabolic pathways, characterizing their enzymes, and 

cloning the corresponding genes will provide the· means 

for manipulating known end products. Using this approach, lignin biosynthesis, carbon metabolism, and hormone balance was manipulated (Klee and Romano 1994; 

Stitt 1995; Whetten and Sederoff 1995). However, genes 

determining developmental fates, such as LFY, will not be 

isolated by this approach. For this purpose, screening for 

mutants with aberrant development is attractive. Additional advantages of the mutant approach include: 1) the 

effect of gene inactivation can be determined at both the 

whole-plant and cellular levels; and 2) very weakly expressed genes can be detected. Isolation of differentially 

expressed genes during defined phases of xylem cell formation, as with the Zinnia system, or during the production of specific wood types may result in unexpected and 

exciting discoveries that may lead to the characterization 

of novel cell- or tissue-specific promoters. 

Undoubtedly, an increasing number of genes, enzymes, 

and other factors involved in xylem cell formation will be 

characterized in herbaceous and woody model systems. 

In addition, control of the activities of these factors will be 

better understood (Catesson 1994; Northcote 1995; Savidge 

1995). This knowledge will give us tools to unravel the 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

control of wood density and, ultimately, to use genetic 

engineering for wood improvement. Although the stern's 

main functions of storage, transport, and support must be 

maintained, the variability of wood structure found in 

nature shows that considerable modification of the wood 

formation process is possible. 

Acknowledgments 

The authors thank Drs. Lars-Goran Sundblad and Sharon 

Regan for their critical comments on the manuscript. 

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83 
Chapter 12 

Transgene _Expression in Field-Grown Poplar1 

Gilles Pilate, David D. Ellis, and Simon Hawkins 

Introduction 

Genetic engineering is a powerful tool used to modify 

plants that are of economic interest. This is accomplished by 

introducing genes that confer desirable traits such as pathogen resistance, herbicide tolerance, and modification of key 

metabolites. The introduction of foreign genes (transgenes) 

or the transformation of plants is routine for many annual 

crop species. Many examples are undergoing field trials and 

commercialization (Dale 1995). Similar transgene research 

on forest trees is less advanced, primarily because of difficulties manipulating woody plants, which are generally less 

responsive in tissue culture than herbaceous material. Also, 

trees are not well analyzed or understood at the genetic level. 

Poplar is a model species for pioneering such biotechnological procedures because it is relatively easy to manipulate in. 

vitro and has a small genome. Since 1987, when the first 

transgenic poplar was obtained (Fillatti et al. 1987), several 

techniques were developed, which are now routinely used, 

to transform different Populus species or hybrids. Availability of such techniques has allowed the introduction of many 

genes aimed at conferring or modifying particular traits. For 

example, genetic transformation was used to improve herbicide resistance (Brasileiro et al. 1992; Donahue et al. 1994; 

Fillatti et al. 1988), insect tolerance (Leple et al. 1995; 

McCown et al~ 1991), and lignin metabolism (Baucher et 

al. 1996; Van Doorsselaere et al. 1995). Most of the above 

studies were conducted on plants grown under controlled 

greenhouse conditions. The promising results obtained 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

84 

from these studies need careful field evaluation before 

commercialization. 

Trees are grown for multiple years before harvest; they 

are large, complex plants, which presents sampling problems; and they undergo a phase change Quvenile to mature). All of these factors could significantly influence the 

expression of foreign genes. Furthermore, care must be 

taken to.prevent foreign genes from spreading into native, 

non transformed populations. The situation is further complicated because, depending on the nature of the crop, different growing practices are used for different species. 

Thus, designing a field trial that represents specialized 

commercial conditions is difficult. Initial commercial exploitation of transformed trees may be restricted to hightechnology, agroforestry plantations with intensive, 

short-rotation times. Poplar is a model system since clonal 

material is already grown commercially under such conditions allowing establishment of representative field trials. Additionally, the desirable traits cited above involve 

modifying plants grown under field conditions. 

Field trials must include an evaluation of the level and 

stability of transgene expression. Limited information is 

available about transgene stability in long-lived species 

such as trees. This information is critical because transgene 

expression in annual plants is less stable than originally 

believed (Finnegan and McElroy 1994; Meyer et al. 1994). 

Similar problems may occur in perennial woody species 

such as trees. 

Field Studies of Transgene 

Expression 

Seasonal and Developmental Influences 

Our laboratories at INRA, Orleans, France and the University of Wisconsin have established field trials of 

transgenic trees, including poplar and spruce hybrids, to 

~alyze the stability and variation of the transgene expression. In the first field trial, the poplar hybrid done 'INRA 

717 1B4' (P. tremula x P. alba) was transformed by 
·~ 

I . 

Agrobacterium tumefaciens with the gene uidA coding for 

the protein (3-glucuronidase or GUS. After supplying tissue with an appropriate enzymatic substrate, expression 

of the GUS reporter gene is detectable by blue coloration 

or other quantitative assays. The uidA gene under control 

of the cauliflower mosaic virus 35S promoter, was stably 

integrated into the host genome. Twelve plants representing 4 independently transformed lines were regenerated, 

characterized at the molecular level, and introduced into 

the field in 1991 (Pilate et al. 1994). Leaf and stem samples 

from these plants were harvested at regular intervals (1992-

1995}, and trans gene expression levels (GUS activity) were 

determined by GUS fluorometry and histochemistry 

Qefferson 1987). After 4 years in the field, all transformed 

lines continued to express the GUS gene in all living cells 

and no gene silencing was observed (Pilate et al. in prep.). 

Significant differences in transgene expression levels were 

observed among different transformants, but not among 

different individuals from the same line (Pilate et al. in 

prep.). GUS activity apparently was not correlated with 

the copy number of the introduced GUS gene. Similar 

variation was observed among 27 trans formants (35S-GUS) 

grown under greenhouse conditions (Pilate et al. in prep.); 

observing expression of these lines in the field is of further 

interest. 

Field trials were initiated in 1993 with the poplar hybrid 'NC5339' (P. alba x P. grandidentata cv. 'Crandon') that 

was transformed by particle bombardment with a similar 

GUS construct that was under the control of an enhanced 

35S promoter. Analyses per(ormed in 1994 and 1995 

showed significant differences in expression levels among 

different transformed lines (Ellis et al. 1996 ). Although gene 

silencing was observed in vitro, it did not occur after plants 

were transferred to field conditions. Similarly, silenced 

plants in vitro did not initiate transgene expression after 

transfer to the field (Ellis et al. 1996). This suggests a degree of stability of transgene expression in the field. 

In poplar clones 'NC5339' and 'NM6' (P. nigra x P. 

maxmiowiczii}, and in several transformed lines of spruce 

(Picea glauca}, the highest level of GUS activity and the least 

variation in GUS expression occurred with in vitro plants 

(Ellis et al. 1996). As plants were transferred to greenhouse, 

cold frame, and field conditions, the levels of GUS expression decreased while variation in expression among the 

different lines increased (Ellis et al. 1996 ). These differences 

in gene expression among different environments are possibly due to differences in relative growth rates, plant structure, and leaf anatomy. 

Leaves of field-grown poplar are more robust and morphologically different than leaves from in vitro cultures. 

Leaves from greenhouse-grown plants are morphologically 

intermediate between tissue culture- and field-leaves. 

These observations help design in vitro selection strategies 

for trans formants for subsequent field growth. Despite this 

variation, transgenic lines showing the highest expression 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Transgene Expression in Field-Grown Poplar 

in vitro also showed the highest expression level in the field 

(Ellis et al. 1996). With in vitro- or greenhouse-grown plants, 

minimal variation in GUS activity occurred among different individuals of the same transformant line. However, the 

variation increased substantially after these transformants 

were transferred to the field, despite using the leaf 

plastochron index to reduce variation due to differences in 

developmental stage (Ellis et al. 1996). One problem is that 

leaves in the field are highly variable in development, morphology, and physiology, which may reflect the variation in 

GUS expression. Relatively little variation in GUS expression of mature leaves from different individuals of the same 

transformant line indicates that developmental differences 

may be important in gene expression. 

While the above work indicates that the developmental 

stage of leaves may influence expression levels, studies 

on poplar clone 'INRA 717 184' help explain the effect of 

the leaf spatial position within the tree on transgene expression. Leaf samples from branches at 4 different tree 

heights showed no significant differences in GUS expression. However, significant differences were observed between leaf samples harvested either at the apex or close to 

the trunk of the same branch (Pilate et al. in prep.). In that 

study, the highest expression was observed in leaves closest to the trunk. Significant differences in the· transgene 

expression levels also were observed among different organs harvested from similar tree positions (Pilate et al. in 

prep.). Seasonal events also influence GUS gene expression in field-grown poplar trees. In poplar clone 'INRA 

717 1 84,' expression was significantly higher in summer 

than autumn (Pilate et al. in prep.). 

In the leaves of clone 'NC5339,' GUS expression was 

initially very high immediately after bud break, but within 

2 weeks expression was 5 to 8 times lower than that originally observed (Ellis et al. 1996). After this drop, GUS expression stabilized, although considerable variation still 

occurred among leaves of similar developmental stages. 

Overall, younger leaves that were still expanding showed 

higher and more variable levels of GUS expression 

throughout the year in comparison with older leaves (Ellis 

et al. 1996). This contrasts with field expression patterns 

of GUS observed with another hybrid poplar 'NM6,' where 

the older fully expanded leaves exhibited higher GUS expression (Francis et al. 1995). In older leaves of 'NC5339,' 

no significant differences were observed in GUS expression between the 2 years and GUS levels were significantly 

different in younger leaves on only a few dates. Although 

these studies suggest a general stability of transgene expression in field-grown trees, external factors (environment) and internal factors (organ, developmental stages, 

metabolism, source-sink relationships, etc.) affect such 

expression. In addition, these observations indicate the 

sampling difficulties associated with tree studies and illustrate the need to collect comparable material for subsequent analyses of transgene expression. 

85 
Section II Transformation and Foreign Gene Expression 

Targeted Gene Expression 

Until now, most tree species tested in the field were transformed with a reporter gene (usually uidA coding for GUS) 

under control of the cauliflower mosaic virus 355 promoter 

or derivatives. The 35S promoter reportedly induces transcription of the transgene coding sequence in all cells providing constitutive expression. However, recent results 

show that expression controlled by the 355 promoter is 

not always constitutive. At the University of Wisconsin, 

poplar and spruce lines transformed with the uidA gene 

under control of the 355 promoter exhibited tissue-specific (vascular) expression (Ellis et al. 1996). Similarly, at 

INRA, Orleans, of 27 poplar lines transformed with the 

355-GUS construct, 5 lines showed some type of tissue 

specificity in plants grown under greenhouse conditions 

(Pilate et al. in prep.). It should be emphasized that the 

expression pattern in all transformants appeared stable. 

Transformants expressing GUS in a tissue-specific manner continue to do so, as do transformants expressing GUS 

in a constitutive manner. We have never observed a switch 

from one pattern of expression to another. Although the 

use of a 355 promoter does not guarantee constitutive expression of an introduced gene, careful screening will probably result in the selection of transformed lines exhibiting 

the desired pattern of gene expression. 

While constitutive promoters can be used for controlling the expression of certain genes (e.g., bacterial endotoxins from Bacillus thuringiensis for insect resistance), 

transformation with other genes may involve other strategies. In the'se situations, effective transgene expression 

may require careful spacial and temporal regulation. For 

example, when sterility was induced in tobacco by using 

an RNase gene fused to a tapetum-specific promoter 

(Mariani et al. 1990), the use of a constitutive promoter 

was impossible. 

Little is known about the stability of tissue-specific or 

inducible promoters in trees, especially under field conditions. However, we recently began studies of such expression patterns at INRA, Orleans. Because it constitutes the 

timber, xylem is an interesting tree target tissue and is an 

ideal target for introducing genes aimed at modifying 

wood quality or quantity. A recent study showed that the 

Eucalyptus CAD (cinnamyl alcohol dehydrogenase) promoter (EuCAD) directs the expression of a reporter gene 

in mainly young xylem tissue (Feuillet et al. 1995). As an 

example of a tissue specific promoter, we used this promoter with the uidA gene to study the stability of targeted 

transgene expression in field-grown poplar trees. Anumber of transformants were analyzed under in vitro and 

greenhouse conditions. While differences in GUS expression levels were observed among different transformed 

lines, all transformants examined exhibited tissue-specific 

(vascular) expression. The pattern and stability of such 

expression will be analyzed under field conditions and will 

86 

allow an evaluation of seasonal affects on expression controlled by a tissue specific promoter. 

Similar field trials are underway at the University of 

Wisconsin where the uidA coding sequence has been functionally fused to the potato wound inducible PIN2 promoter and introduced into 'NC5339.' Initial results indicate 

that the PIN2 promoter is induced by wounding under 

field conditions and is active in a developmentally regulated manner (Ellis et al. 1996). While the GUS system is 

undoubtedly the most useful reporter gene for characterizing promoter expression patterns, such expression patterns should be independently confirmed. We intend to 

study the expression controlled by the EuCAD promoter 

under field conditions using another reporter gene (Green 

Fluorescent Protein-GFP; Haseloff and Amos 1995). 

Commercial Application and 

Environmental Impact 

Field trials with trees transformed by reporter gene-promoter combinations are useful for analyzing the expression pattern, level, and stability of any particular promoter. 

The potential of specific promoters to control the expression of genes of economic interest can be thoroughly evaluated by such studies. If a plant is transformed with a coding 

sequence for a gene of economic interest that is fused to a 

well-characterized promoter, we expect that the transgene 

expression should be controlled similarly to the previously 

sttidied reporter gene. However, interactions between the 

introduced gene or gene product (messenger RNA, protein) and the plant could affect the expression of the gene 

in an unexpected manner. Preliminary field trials using 

trees transformed with the actual gene of economic interest should be conducted on a scale and under conditions 

comparable to that of a commercial operation before commercial application. Such trials would help determine 

whether results from young plants grown under controlled 

conditions are consistent with those from field-grown trees. 

Since the range of possible transformations is almost 

infinite, the environmental impact of each transformation 

must be thoroughly evaluated. For example, a transformation conferring insect resistance could strongly impact 

the ecosystem and result in: 1) an adaptive advantage for 

the trees should they become weeds; 2) the possible evolution of resistant insect biotypes; and 3) altered competition among different insect species. Conversely, 

modifications of lignin metabolism may have less environmental impact, although such modifications could impact the transformed tree's disease and I or stress resistance. 

To assess field performance and environmental impact 

of trees transformed for insect resistance, Leple et al. (1995) 

separately introduced 2 genes into a poplar clone (P. tremula 

x P. tremuloides cl. '353-38') and investigated the affect on 

the clone's resistance to a poplar leaf beetle, Chrysomela 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
tremulae (Coleoptera: Chrysomelidae). A gene encoding a 

cysteine protease inhibitor (OCI) was used first. Then, a 

gene encoding an endotoxin from B. thuringiensis (Bt) 

(cry IliA) was used. Both of these gene products were shown 

to be effective against Coleoptera. Following molecular 

characterization, 3 transformants were selected that overexpressed the OCI gene. Greenhouse assessments of this 

approach showed that target insect mortality was increased 

by approximately 40 percent (Leple et al. 1995). Following 

this initial success, field trials were established under growing conditions comparable to commercial plantations, with 

10 individuals from each of 2 transformed lines (Leple et 

al. 1995). The performance of these transformed trees will 

be evaluated over an 8-year period for growth, resistance 

to the target insect, and effects on target and nontarget insect species. Such a study should provide valuable information on the long-term stability of expression with such 

constructs, and their environmental impact under field 

conditions. Over the last 3 growing seasons, field trials at 

Wisconsin with poplar hybrid 'NC5339' containing a 

cryiA(a) gene, have revealed no loss of Bt gene expression. 

This indicates that expression of an economically interesting coding sequence and reporter genes is apparently 

stable over multiple growing seasons (Kleiner et al. 1995). 

Lignin metabolism is another target for genetic transformation. When making paper pulp, lignin is separated 

from fibers by chemical processes that are economically 

and environmentally costly. In a European-funded research 

project (ECLAIR-OPLIGE (N° AGREE 0021 - C)/FAIRTIMBER (PL95424)), a poplar _CAD coding sequence was 

introduced into poplars in an antisense orientation to try 

to reduce the endogenous activity of CAD and modify lignin content (refer to chapter by Boerjan et al.). As part of 

the FAIR-TIMBER program, field trials are underway using transgenic poplar plants that show a reduced activity 

in CAD or 0-methyltransferase (OMT), another enzyme 

involved in lignin biosynthesis (refer to chapter by Boerjan 

et al.). The effects of these transformations, which were 

evaluated in plants grown under controlled conditions 

(Van Doorsselaere et al. 1995), on lignin quality and quantity will be investigated in field-grown plants after 4 and 8 

years. Transgene expression in such transformed plants 

can be followed by monitoring.enzymatic activity (CAD, 

OMT) and, in plants transformed with the antisense CAD 

sequence (ASCAD), by the phenotypic red coloration induced in the xylem. These genetically modified poplars 

were transformed with the antisense coding sequences 

(CAD, OMT) under the control of the 355 promoter (Van 

Doorsselaere et al. 1995). However, future commercialization of transgenic poplars will probably use an antisense 

sequence under the control of a tissue-specific promoter. 

As part of the FAIR TIMBER Program, poplar plants are 

being transformed with the ASCAD coding sequence under the control of the EuCAD promoter to investigate the 

above combination. The stability of transgene expression 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Transgene Expression in Field-Grown Poplar 

in these plants will be followed under in vitro, greenhouse, 

and field conditions. Such experiments should provide 

information about the interactions among a tissue specific 

promoter, an endogenous plant coding sequence in an 

antisense orientation, and in the field environment. 

Future Prospects 

Future research work should focus on the underlying 

mechanisms of transgene instability (Matzke and Matzke 

1995; Meyer et al. 1994) and ways to reduce such variation 

(Bhattacharyya et al. 1994; Dejong et al. 1994; Mlynarova 

et al. 1994) under controlled conditions. Research efforts 

should also focus on understanding the complex environmental effects, including temperature, light, abiotic stress, 

and biotic stress, on transgene expression in field-grown 

trees. Another important research area from a commercial 

viewpoint is the frequency of gene silencing and the reduction in expression levels that can occur after transfer 

from in vitro conditions to the field. Is this a genuine problem or can lines that are prone to expression instability be 

detected and eliminated before the field stage? 

Many commercial transformations may require targeted 

transgene expression using tissue-specific promoters. 

Thus, studies that characterize other tissue-specific promoters are an important area of future research. The rapid 

rate that new plant genes are being characterized will provide new coding sequences for desirable traits that can be 

combined with tissue-specific/inducible promoters to expand the range of plant improvements. Such advances will 

require continued trials to investigate these.promoter-coding sequence combinations in the field. 

Biotechnological research with transgenic trees is less 

advanced than with many other shorter-lived species. This 

situation is reflected in the strict regulations governing 

establishment of field trials and field introduction of genetically modified trees (GMTs). In Europe, the poplar root 

system, which is superficial, is contained by burying plastic sheeting in the soil surrounding the plantation and 

maintaining a plant-free zone. All flowers must be manually removed as soon as they appear. Although these labor-intensive precautions may seem detrimental to future 

commercialization of GMTs, strict regulations are not in 

force everywhere. In the United States, the regulations 

concerning the release of GMTs into the field are less strict 

than in Europe. In addition, Strauss et al.(1995) have presented a solution to the concerns about the spread of foreign genes by using transformation technology to produce 

sterile trees. Hopefully, risk assessment studies will be refined and enlarged to encompass more tree species and 

additional knowledge will be gained about the environmental impact of particular· introduced coding sequences. 

87 
Section II Transformation and Foreign Gene Expression 

This information should allow streamlining of regulatory 

procedures to facilitate transgenic tree commercialization 

(Dale 1995). 

Most field trials involving transgenic poplar have used 

clones that are not of immediate economic interest but are 

easy to transform. In contrast, poplar dones of commercial interest ('NM6') and potential commercial interest 

('NC5339') were used in field trials at Wisconsin, where 

transgene expression was stable (Ellis et aJ. 1996). Present 

efforts to transform poplar dones of economic interest will 

intensify, and field trials with such dones will likely be 

initiated shortly after transformation protocols are available. Another step toward the commercialization of 

transgenic poplar is to develop protocols and technology 

to introduce more than 1 transgene. The introduction of 

multiple genes may allow modification or introduction of 

completely different traits or affect a single trait contro11ed 

by several genes. For the most efficient expression of these 

transgenes, different combinations of coding sequences 

and promoters will probably be used. Field trials should 

prove useful and are essential for evaluating the complex 

interactions between the environment and introduced 

genes. 

Another important question concerns the future exploitation of these introduced genes and the plants containing 

them. Will such modified trees be propagated solely in vitro 

or be used as parents in crosses with dther non transformed 

or transformed elite plants? One prerequisite for evaluating the use of transformed plants is knowing what happens to the introduced gene(s) in such crossings; how it 

segregates ahd how stably it is expressed in the progeny. 

Pilate et aJ. (in prep.) are analyzing the results of a controlJed cross involving 1 of the field-grown, transgenic 

(355-GUS) poplar and will soon address this important 

point. 

Acknowledgments 

The work at INRA, Orleans was conducted within the 

framework of an EC-funded project. AIR2-CT94-1571 (Stability and Expression of Foreign Genes in Populus). 

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651-667. 

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387-405. 

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Dumanois Le Tan, V.; Delplanque, A.; Cornu, D.; 

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Populus and incorporation of pest resistance by electric 

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2088. 

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P.; Stiekema, W.J.; Nap, J.P. 1994. Reduced position effect 

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. Strauss, S.H.; Rottmann, W.H.; Brunner, A.M; Sheppard, 

L.A. 1995. Genetic engineering of reproductive sterility 

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Van Doorsselaere, J.; Baucher, M.; Chognot, E.; Chabbert, 

B.; Leple, J.C.; Pilate, G.; Cornu, D.; Monties, B.; Van 

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89 
Chapter 13 

Transgenes and Genetic lnstability1 

M. Raj Ahuja 

Introduction 

Genetic improvement of forest trees by traditional breeding and selection methods is a slow process. Because of 

long life cycles, these approaches may have limited application in forest trees. Even in annual plant species, traditional approaches involving hybridization and backcrosses 

may take several generations for transfer of desirable 

genes. Genetic engineering, on the other hand, offers prospects for genetic improvement at an accelerated pace in 

herbaceous and woody plants. 

A major problem regarding gene transfer is how to regulate integration and expression of foreign genes in plant 

and animal genomes. Although many promoters of viral, 

bacterial, and plant origins have been tested in herbaceous 

plants, littlE: is known about their regulatory control in forest trees. Another problem presented by gene transfer is 

stability of the integrated transgenes in forest tree genomes. 

Although originally transgenes were considered stably 

integrated and expressed, recent reports on herbaceous 

crop plants suggest that trans gene expression is not always 

stable in the transgenic plants. Gene silencing, partial or 

complete inactivation of recombinant genes or endogenous 

genes, has been observed in several herbaceous transgenic 

plants (Finnegan and McElroy 1994;Jorgensen 1992, 1995; 

Kooter and Mol 1993; Matzke and Matzke 1993, 1995; 

Meyer 1995; Paszkowski 1994). Furthermore, transgene 

expression levels vary among independent transformants, 

which may be determined by copy number and/ or integration transgene site (Hobbs et al. 1990, 1993; Jones et al. 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

90 

1985; Prols and Meyer 1992). This suggests that it is difficult to predict if a transgene would be stably integrated or 

expressed in plants, whether herbaceous annuals or woody 

perennials. Does a chimeric gene integrate either at a specific site or at any available slot in the genomic landscape? 

Are these locations assigned by specific endogenous genetic sequences, or does the alien gene choose any location in the genome? Although the answers to these 

questions are currently unknown, research in genetic engineering is progressing so rapidly that these questions 

may soon be resolved. 

Forest trees have long life cycles, with an extended vegetative phase ranging from 1 to several decades. Because 

trees are firmly anchored in 1location, they are exposed to 

changing environments over long periods that may influence their physiology and morphogenetic processes. For 

long-term survival, trees must adapt to the new challenges 

posed by the changing environment. Under such conditions, genes conferring low fitness in trees, for example 

some transgenes, may be silenced or eliminated. Therefore, genetic transformation in woody plants must be investigated to understand genetic instability on a short- and 

long-term basis. Questions on the genetic instability of 

transgenes in long-lived forest trees (Ahuja 1988a, 1988b, 

1988c) include: Will the foreign genes be stably integrated 

and expressed immediately in a specific tissue or in all 

tissues? Will foreign genes be expressed immediately but 

remain inactive for a long time, then be re-expressed? Will 

foreign genes undergo rearrangement or be lost during the 

long vegetative phase of forest trees? Will foreign genes 

cause genetic changes in the host genome via a position 

effect involving 1 or multiple copies of a transgene in the 

host genome? 

In this review, I will discuss published experimental data 

and present theoretical arguments on the stability and instability of transgenes in woody plants using published 

reports on transgene inactivation in annual herbaceous 

plants. Recent investigations are also presented on 

transgene stability and expression in Populus and other 

woody plants. Because the techniques and methods for 

genetic transformation by Agrobacterium-mediated gene 

transfer or DNA delivery through a microprojectile-bombardment system have been adequately described in ear-
lier reviews (Binns and Thomashow 1988; Charest and 

Miche l 1991; Chupeau e t al. 1994; H ooykaa s and 

Schilperoort 1992; Jouanin et al. 1993; Kung and Wu 1993; 

van Wordragen and Don 1992) and in other chapters in 

this volume, they are not discussed here. 

Genetic Transformation 

The following conditions must be present before gene 

transfer is possible in plants: 1) an efficient in vitro regeneration system; 2) a vector system for the transport of the 

chimeric gene(s); and 3) an efficient gene transfer system. 

Once the transgenic plants are regenerated, there is a risk 

from genetic instability (internal), and a risk to humans, 

animals, and the ecosystem (external). I will address the 

genetic risk, whether caused by gene interaction or external factors. 

Regeneration System 

Many excellent in vitro regeneration systems were developed in multiple plant species, including woody plants. 

Some of the reasons that Populus is a mode l system in forest biote.chnology and genetic engineering include: 

• fast growth, 

• short-rotation cycles, 

• growth on marginal sites, 

• vegetative phase normally lasts 7 to 10 years, 

• in vitro technology offers prospects for early maturation that may reduce the vegetative phase to3 to 4years, 

• unisexual flowers, 

• in vitro regeneration systems are developed for many 

species, 

• rejuvenation from mature trees is achievable by tissue culture, 

• relatively small genome (1 / 10th the size of a pine 

genome), 

• molecular genome maps are available, and 

• genetic transforma tion and regenera tion of 

transgenic plants are achievable in certain aspens and 

poplars. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Transgenes and Genetic Instability 

Excellent regeneration systems are available in aspens 

(Ahuja 1983, 1986, 1987a, 1993) and poplars (Chun 1993; 

Ernst 1993), which lend them to genetic transformation 

and regeneration of transgenic plants (Chupeau e t al. 1994; 

Confalonieri et al. 1994; Devillard 1992; Donahue et al. 

1994; Fillatti et al. 1987; Fladung et al. 1996; Howe et al. 

1994; Klopfenstein et al. 1991, 1993; 1 ilsson et al. 1992; 

Tsai et a l. 1994). 

Chimeric Gene and Vector System 

Several chimeric genes have been transferred to Populus 

by the Agrobacterium-mediated gene transfer system, particle acceleration Dt A delivery system (McCown et al. 

1991), and electroporation of plasmids into popla r protoplasts (Chupeau et al. 1994). In most studies, at least 2 chimeric genes were transferred simultaneously, 1 as a 

selectable marker and the other as a reporter gene. The 

selectable marker genes normally used are antibiotic-resistance genes that confer kanamycin or hygromycin resistance to the transformed tissue. Kanamycin resistance 

is conferred by a bacterial neomycin phosphotransferase 

(NPTII) gene and 2 promoters, NOS (nopa line synthase) 

and OCS (octapine synthase), which are used to control its 

expression. Alternatively, severa l alien genes have been 

used as reporter genes. These may include those tha t are 

biochemically detectable, and others that have a characteristic phenotypic expression. One biochemically detectable gene, the GUS (!}-glucuronidase) gene from E. coli 

bacteria, has been extensively used in many plant genetic 

transformation studies. In most studies, GUS expression 

is driven by a 35S promoter from the cauliflower mosaic 

virus (35S-GUS). GUS expression can be histochemica lly 

detected in transgenic plant tissues. Several different chimeric gene constructs were used to confer pest and herbicide tolerance in poplars. A proteinase inhibitor II (PIN2) 

gene from potato conferring pest tolerance under the control of a 35S or NOS promoter (35S-PIN2 or NOS-PIN2) 

was expressed in a transgenic hybrid poplar clone (P. alba 

x P. grandidentata cv. 'Hansen' ) (Klopfenstein e t al. 1991, 

1993). Another bacterial mutant gene, aroA coding for 5-

enolpyruvyl-shikimate 3-phosphate (EPSP), driven by a 

35S promoter, conferred herbicide tolerance in another 

hybrid poplar clone (P. alba x P. grandidentata cl.' C5339') 

(Donahue eta!. 1994). In an earlier study using a chimeric 

gene fusion carrying the aroA gene with a MAS (mannopine 

synthase) promoter (pMAS-aroA) (Fillatti et al. 1987), herbicide tolerance was not well expressed in hybrid popla r 

leaves (Riemenschneider and Haissig 1991 ). As with N PTII 

or GUS genes, the morphological effects of PIN2 or aroA 

expression were undetected, except for the action of the 

introduced gene product. 

In a recent investigation, we used the rotC gene from A. 

rhizogenes, under the expressive control of 35S and rbcS 

(from potato) promoters (355-ro/C and rbcS-ro/C), for ge91 
Section II Transformation and Foreign Gene Expression 

netic transformation of European aspen (P. tremula) and 

hybrid aspen (P tremula x P. tremuloides) clones (Ahuja and 

Fladung 1996; Fladung et al. 1996). Expression of the ro/C 

gene is de tected at the phenotypic level by reduced leaf 

size and red uced chlorophyll content that produces a palegreen colored lea f in comparison to the dark green leaves 

of untransformed aspens. Since expression of ro/C is detected at the morphological level, it offers prospects fo r 

continuously monitoring expression during growth and 

development of transgenic plants. Consequently, ro/C can 

be used to address questions regarding genetic stability of 

transgenes in woody plants at the phenotypic and molecular levels. Transgenic plants carrying other chimeric genes 

are also suited for similar analysis. In cases such as aroA 

and PIN2 genes, those lacking phenotypic expression must 

be analyzed at specific interva ls by biochemica l methods 

and then exposed to pests or herbicide for tolerance monitoring. 

It could be argued that chimeric genes, driven by promoters from viruses, bacteria, or plants, then introduced 

in herbaceous or tree species, are subject to rejection by 

the p lant genome as foreign entities. Because of specific 

integration sites or sequence homology in the genome, 

integrated transgenes are no t a mainstream part of the 

genetically and evolutiona rily adapted plant species in 

space and time. Such transgenes may be subject to gene 

interaction, inactivation, and loss depending on the endogenous and exogenous environments. Genetic va riation 

occurs in callus cultures and tissue culture-derived plants. 

Transgenic p lants apparently can inherit genetic instability from the 'tissue culture process and from foreign gene 

transfer; both sources are examined. 

Tissue Culture Related Genetic 

Instability 

Tissue culture-induced genetic va ria tion has been observed in many plant species (Ahuja 1987b; Bajaj 1990; 

Kaepplc r and Phillips 1993; Larkin and Scowcroft 1981; 

Phillips ct al. 1994; Skirvin 1978). In pa rticular, callus cu ltures are prone to genetic changes and exhibit diffe rent 

kinds of genetic abe rrations. Va rious terms have been used 

to describe plant va riants regenerated from tissue culture. 

Plants regene rated from stem callus arc called "calliclones" 

(Skirvin 1978), and those from leaf p rotoplasts are referred 

to as "protocloncs" (Shepa rd e t a l. 1980). Larkin and 

Scowcroft (1981) proposed the general term "somaclonal 

variation" for plant variants regenerated by any type of 

somatic cell culture. In contrast, variation detected in haploid cell cultures are ca lled "gamctoclonal variation" 

(Evans et al. 1984). 

92 

The va lue of somaclonal variation in horticultural and 

agricultural crops has been adequately described (Bajaj 

1990; Evans and Sharp 1983; Evans et al. 1984; Larkin and 

Scowcroft 1981; Larkin et a l. 1984). I will focus on the possible role of the media components, in particular hormones, 

in the induction of genetic variation du ring cell culture. I 

will also argue that using cultured cells for genetic engineering adds a risk of genetic instability in transgenic 

plants. Somaclonal variation apparently occurs at a higher 

frequency in cultured cells than in sp ontaneous muta tions 

in uncultured plant cells. Most commonly observed genetic variations include changes in chromosome number 

and structure (D' Amato 1978), gene mutations, and epigenetic changes involving DNA methylation {Brown 1989; 

Kaepp ler and Phillips 1993; Muller e t al. 1990). In addition, repeat-induced point mutation (RIP) may also contribute to the genetic instability of cultured plants cells 

(Phillips et al. 1994). Some variation induced in tissue culture is not heritable and may be an epigene tic modification. All types of soma clonal variation are not useful; most 

genetic variation induced in tissue culture is undesirable. 

Factors involved in somaclonal variation in plants include: genotype, donor plant age, explant sou rce (stem 

segments, meristems, leaf discs, root segments), medium 

composition, length of time tissues are kept in cultu re, and 

culture conditions. Exposing cells in the culture medium 

to relatively high levels of exogenous phytohormones, such 

as 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), 6-benzyladenine (BA), or other chemica ls, which are seve ra l magnitudes hi gher than the 

physiological endogenous concentrations, may strongly influ ence the ind uction of somaclonal variation. In particula r, 2,4-D and othe r auxins may be involved in increased 

DNA methylation and may induce RIP in plant tissue cultures (Phill ips et al. 1994). Therefore, changes in the genome a rc expected w hen cells are removed from their 

norma l surroundings and .p laced in an a rtificial tissue culture environment (McClintock 1984). Cul tured cells a re 

under constant stress in vitro. As a result, the genome 

adapts to this new environment and becomes more e rrorprone. This adap tation may depend on the genotype, but 

many genotypes exhibit genetic instability when placed 

in the tissue culture environment. 

Are Transgenic Plants 

Genetically Stable? 

In a 1987 symposium ti tled "Genetic Manipulation in 

Woody Plants" in East Lansing, Michigan (Hanover and 

Keathley 1988), severa l reports were presented on the status of biotechnology in woody plants including one on 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
gene transfer in forest trees (Ahuja 1988a). At that time, 

genetic engineering in annual plants was newly established 

but was still in its infancy in forest tree species. The few 

existing studies mainly involved testing the efficacy of 

Agrobacterium strains for inducing galls on trees (Ahuja 

1988a; Chapham and Ekberg 1986; Sederoff et al. 1986). 

Around that time, the first report was published on 

Agrobacterium-mediated transfer of the aroA gene from 

Salmonella bacteria to confer herbicide tolerance in the hybrid poplar clone 'NC5339' (Fillatti et al. 1987). Variation 

occurred in the type of shoots produced in vitro following 

genetic transformation (Fillatti et al. 1987). Also, aroAgene 

expression was less than expected when transgenic poplars were sprayed with the herbicide glyphosate 

(Riemenschneider and Haissig 1991; Riemenschneider et 

al. 1988). These variations may be attributed to genetic or 

epigenetic changes in the poplar genome, perhaps due to 

transgene position effect, bacterial hormonal genes, or less 

than optimal transgene expression. 

Based on transgenic poplars and earlier experience with 

the genetic tumor systems of Nicotiana and Lycopersicon, 

which also involved gene transfer from 1 species to another by conventional backcrossings (Ahuja 1962, 1965, 

1968; Doering and Ahuja 1967), it was proposed that foreign genes may be unstable or poorly expressed under 

greenhouse or field conditions. Unexpected changes or 

expression loss of a foreign gene can occur during the extended vegetative phase in long-lived forest trees. Variability occurring in transgenic plants, possibly associated 

with an in vitro transformation and a gene transfer event, 

which may be independent of somaclonal variation, was 

termed "somatoclonal variation" (Ahuja 1988a, 1988b, 

1988c). Somatoclonal variation may be epigenetic or heritable and due to any of the following factors (Ahuja 1988c), 

which occur singly or in combination in the plant genome 

after genetic transformation: 

• loss of expression of a foreign gene, 

• rearrangements in the copy number of a foreign gene, 

• loss of a foreign gene, 

• copy number of a foreign gene inserted in the host 

genome, 

• integration site of a foreign gene in the genome, 

• position effect, and 

• rearrangements caused by a foreign gene in the host 

genome. 

The first published report on transgene inactivation by 

another non linked transgene was based on a double transformation mediated by Agrobacterium in transgenic tobacco 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Transgenes and Genetic Instability 

(Matzke et al. 1989). Subsequently, many reports on gene 

silencing have been published involving the inactivation 

of 1 transgene by another, and widely ranging transgene 

inactivations involving homologous endogenes or 

ectogenes (de Lange et al. 1995; Finnegan and McElroy 

1994; Flavell1994; Flavell et al. 1995; Jorgensen 1992, 1995; 

Kooter and Mol1993; Matzke and Matzke 1990,1993, 1995; 

Meins and Kunz 1995). Levels of transgene expression 

seem unpredictable in the transgenic plants and appear to 

vary among independent transformants. 

In a recent informal survey of more than 30 biotechnology firms working on the commercialization of transgenic · 

crops, nearly all companies reported observations of 

transgene inactivation (Finnegan and McElroy 1994). Unfortunately, much of this information may or may not be 

published. 

We used Populus as a model system to investigate the 

stability and expression of transgenes at the morphological, physiological, and molecular levels (Ahuja and 

Fladung 1996; Faldunget al. 1996). Using anAgrobacterium 

binary vector system, 4 clones of European aspen (P. 

tremula) and hybrid aspen (P tremula x P tremuloides) were 

genetically transformed with different chimeric genes, inCluding the role gene from A. rhizogenes. A dominant, pleiotropic gene, role exhibits pronounced phenotypic and 

physiologic affects on transgenic plants. Since role expression is detectable at the morphological level, it can be used 

as a selectable marker for monitoring phenotypic response, 

molecular expression, and genetic stability in transgenic 

plants. Two types of promoters, 355 from the cauliflower 

mosaic virus and rbcS from potato, were used to control 

role gene expression. A second gene for kanamycin resistance (NPTII) under control of a NOS promoter was also 

included in the gene construct. By using an improved leafdisc transformation method, putative transformants were 

regenerated on a kanamycin-containing medium. 

During the past 2 years, more than 1,000 transgenic aspens have been regenerated and grown in the greenhouse 

to investigate their morphologic and genetic stability. 

Transgenic aspens carrying the 355-role gene construct had 

much smaller leaves when compared to untransformed 

controls. In contrast, rbc5-role transgenics showed only 

slightly smaller leaves when compared to the controls. 

However, transgenic aspens carrying either 355-rolC or 

rbc5-role transgene exhibited pale-green leaf color in the 

1-year-old plants when compared to the dark-green leaf 

color of the untransformed controls. In the second year of 

growth, the leaf size remained characteristic of the 355-

role or rbc5-role transgenic aspens, and the young leaves 

were pale-green; however, the leaves turned dark green 

with maturation. Several leaf abnormalities, including 1 

leaf showing half pale-green and half dark-green sectors, 

chimeras, and revertants to normal state, were observed 

in the transgenic aspens. In addition, a couple of transgenic 

aspens that tested positive for the rolC gene by PCR (poly93 
Section II Transformation and Foreign Gene Expression 

merase chain reaction) were negative for the rolC phenotype. Southern blot analysis, using a nonradioactive hybridization method (Fladung and Ahuja 1995), revealed that 

most of the independent transformants carried a single integrated copy of the rolC. However, some transgenic aspens 

carried 2 or more transgene copies (Fladung et al. 1996). 

We will continue molecular analysis of the transgenic 

aspens showing growth abnormalities including chimeras. In addition, we are investigating the mechanism of 

transgene inactivation in aspens. To learn how the 35SrolC or rbcS-rolC transgenic aspens perform under field 

conditions, it is important to determine if the rolC-controlled phenotypic traits are expressed under environmental conditions in the greenhouse and field. 

Expression regulation of another chimeric gene, uidA 

(GUS) under control of different promoters, was investigated in poplar and spruce (Ellis et al. 1996; Olsson et al. 

1995). In the rolC-uidA transgenic hybrid aspen (P. tremula 

x P. tremuloides), GUS activity was tissue-specific and 

mainly localized in the vascular tissue before the winter 

dormancy. However, during the onset of dormancy, GUS 

expression shifted from phloem to include cortex and pith 

cells. After exposure to temperatures that induced final dormancy changes, GUS expression disappeared from all stem 

tissues. In contrast, GUS expression in the stem of 35S-uidA 

transgenic hybrid aspen was strong in all tissues, except the 

vascular cambium and xylem, and did not vary in intensity 

during growth and dormancy (Olsson et aL 1995). 

Variation in GUS expression was also monitored in the 

35S-uidA transgenic poplars and spru·ce (Ellis et al. 1996). 

In 1 hybrid' poplar clone (P. alba x P. grandidentata cl. 

'NC5339'), GUS expression was more variable during the 

growing season in younger leaves than in mature leaves. 

However, GUS activity was relatively higher in older than 

younger leaves in another hybrid poplar clone (P. nigra x 

P. maximowiczii cl. 'NM6'). In spruce (Picea glauca), GUS 

activity was detected in needles only during their elongation but persisted throughout the growing season in the 

stems. Based on GUS levels in transgenic poplars and 

spruce, Ellis et al. {1996) concluded that GUS expression 

was least variable during in vitro culture of regenerated 

transformants and most variable during field growth of 

transgenic plants. These studies on GUS activity suggest 

that recombinant gene expression is dependent on promoter type, plant genotype, and environmental conditions. 

Inactivation of Transgenes and 

Endogenous Genes 

All alien genes, whether introduced into plants by genetic engineering or introgressive hybridization, are sub94 

ject to genomic scrutiny. Acceptance or rejection of foreign 

genes may depend on their interaction with endogenous 

genes, and on their integration site within the host genome. 

In a well-adapted organism, new genetic introductions, 

and most new endogenous mutations, are not well tolerated; transgenes are not exceptions. They are alien, reconstituted, hybrid genes, chimeric for the promoter and 

reporter genes located between the left and right borders 

of the T-DNA in a plasmid vector. Transgenes are subject 

to inactivation by the homologous or nonhomologous 

endogenes and by environmental factors in the greenhouse, 

under field conditions, and in vitro. Similar parameters may 

apply to the modification of expression or inactivation of 

1 endogene by another nonlinked endogene, a transposon, 

or alleles at 1 locus. 

Gene silencing may be due to transinactivation (unidirectional) or cosuppression (bidirectional) in transgenic 

plants. Several hypotheses on gene silencing have been 

recently proposed (Bester et al. 1994; Finnigan and McElroy 

1994; Flavell 1994; Jorgensen 1992, 1995; Kooter and Mol 

1993; Matzke and Matzke 1993, 1995; Meins and Kunz 

1994). Although these modes of gene silencing are not 

mutually exclusive, no single hypothesis can explain the 

origins of transgene instability. Alternatively, several different mechanisms for gene silencing might occur depending on: 1) the hybrid chimeric gene construct; 2) the 

promoter type; 3) the plasmid/DNA delivery system; 4) 

whether it is a single or double genetic transformation; 5) 

the extent of homology between the transgene(s) and the 

endogene(s); 6) the plant species; 7) the genotype; and 8) 

the hormonal regimes in the in vitro regeneration system 

used. Therefore, transgene inactivation may occur at several different DNA/RNA levels. Possible mechanisms of 

gene silencing at the transcriptional and post-transcriptional levels are briefly discussed. 

Transcriptional Silencing 

Transcriptional gene inactivation occurs at DNA/RNA 

levels presumably by increased methylation, 

paramutation, or other mechanism(s) (Flavell et al. 1995; 

Matzke and Matzke 1995; Meins and Kunz 1995). By using a hybrid chimeric gene 355-uidA/NOS-NPTII in tobacco, Hobbs et al. {1990) observed intertransformant 

variation in uidA gene expression by monitoring GUS activity in the R1 and R2 generations. High and low levels of 

GUS expression were observed in transgenic tobacco 

leaves. Transformants in the high group had a single copy 

of the transgene, while those with low GUS levels had 

multiple T-DNA integrations into the tobacco genome. 

Plants with multiple transgene copies exhibited increased 

methylation of the integrated T-ON A (Hobbs et al. 1990). 

However, transgene copy number and expression w~re 

not correlated in other studies (Dean et al. 1988; Hoeven 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
'l..w!IV 

et al. 1994; Jones et al. 1987; Shirsat et al. 1989). Later experiments by Hobbs et al. (1993) showed that multipleTDNA insertions per se were not associated with low GUS 

activity. However, the configuration of a particular insert 

due toT-DNA rearrangement during transformation determined whether the GUS activity was low or high. 

Further, transinteractions between single and multiple 

inserts caused partial or total suppression of GUS activity. Correlation between T-DNA methylation and inactivation of T-DNA encoded genes was observed in 

several other studies (Meyer 1995); however, it is unclear whether methylation was the cause or the effect 

of transgene inactivation. 

Another well-studied instance pf transgene silencing 

involves the transfer of the maize A1 gene, which produces brick-red pigmentation in flowers, into a whiteflower mutant of Petunia hybrida. Transgenic petunias 

exhibited a flower color ranging from brick red to variegated to white (Linnet al. 1990; Meyer et al. 1987). Most 

transgenic plants carrying multiple copies of the A 1 gene 

were white, while plants carrying a single copy of the 

transgene had brick-red flowers (designated line RL-01-

17). To test transgene stability under different environmental conditions, 30,000 transgenic petunias carrying a 

single copy of the A1 gene (line RL-01-17) were planted 

under field conditions (Meyer et al. 1992). During the 

growing season, variegation in flower color was observed; 

the number of plants with white, red, variegated, and 

weakly colored flowers was variable. Plants with red 

flowers during the early grow~ng season often produced 

flowers with weakly colored petals later in the season. 

Reduction in A1 gene expression was correlated with 

increased methylation of the 355 promoter that controlled transgene expression (Meyer et al. 1992). 

Hypermethylation was restricted to the transgene and 

did not spread to adjacent areas of the hypomethylated 

endogenous DNA in the transgenic petunia (Meyer and 

Heidmann 1994). Such studies suggest that a transgene 

is inactivated by the plant genome when it is recognized 

as a foreign entity. 

Environmental factors modify or silence genes introduced into 1 species from another by hybridization. Such 

introduced genes are not chimeric genes, but are alien 

genes with some or no homology to the endogenous genes, 

and they cause genetic instability and developmental 

changes in host plants. One such gene, frosty spot (Frs), 

was introduced from Lycopersicon chilense into the genetic 

background of L. esculentum by repeated backcrossing 

(Martin 1966). Frs is a dominantly inherited gene that 

causes many phenotypic and developmental abnormalities in the hybrid derivatives such as: 1) dwarfed, weak 

plants with poorly developed xylem in the stem; 2) tumor-like outgrowths on the abaxiel surface of the leaves; 

and 3) abortive flower set in the greenhouse (Ahuja and 

Doering 1967; Doering and Ahuja 1967). The tumor-prone 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Transgenes and Genetic Instability 

genotype carrying the Frs gene behaved quite differently 

under field conditions; it grew, blossomed, and set fruit 

like normal tomato plants but failed to develop tumors. 

On a morphological basis, tumor-prone and normal genotypes were indistinguishable under field conditions. This 

epigenetic change was reversible when progeny from 

these plants were grown in the greenhouse ·(Doering and 

Ahuja 1967). 

Genetic tumors from different interspecific combinations in Nicotiana also exhibited a different response to 

environmental conditions (Ahuja and Hagen 1967). Tumors generally develop on specific genotypes after the 

flowering stage; however, they can be induced at any stage 

of plant development by stresses including wounding, 

hormonal treatment, or irradiation (Ahuja 1965; Smith 

1972). Some tumor-prone genotypes are more sensitive to 

environmental conditions than others. For example, tumor-prone hybrid derivatives carrying a Tu gene on a specific chromosome fragment from N. longiflora in the genetic 

background of N. debneyi-tabacum (Ahuja 1962, 1968), developed tumors under greenhouse and field conditions 

(Ahuja and Hagen 1967). In contrast, a non tumor mutant 

isolated by irradiation of seed from tumor-prone amphidiploid N. glauca-langsdorffii (Izard 1957), behaved differently under greenhouse, field, and irradiation 

environments. Although questions remain regarding the 

dominant nature of the nontumor mutant (Ahuja 1996; 

Smith and Stevenson 1961), it or the nontumor F1 progeny from crosses between nontumor and tumor-prone 

genotypes developed variable tumors when grown in 

the greenhouse (Ahuja 1996) or when exposed to irradiation (Durante et al. 1982; Smith and Stevenson 1961). 

However, because tumors did not develop on these 

genotypes under field conditions, environmental factors must play an important role in the activation or 

silencing of another gene class, the tumor genes in Nicotiana. 

Besides inactivation of the A 1 trans gene by methylation of its 355 promoter in the petunia line 17-R (Meyer 

and Heidmann 1994), other mechanisms, such as 

paramutation (Brink 1973), may operate in gene silencing. The transgenic line 17-R carries a single copy of the 

maize A1 gene. However, a reduction or inactivation of 

A 1 gene expression can occasionally be observed in flowers of the same line. A white derivative, line 17-W, was 

isolated from the homozygous line 17-R in which the brickred pigmentation was undetectable (Meyer et al. 1993). 

In line 17-W, the 355 promoter of the A1 was found 

hypermethylated, in contrast to its hypomethylated state 

in the transgenic petunia line 17-R, which usually remains 

transcriptionally active (PrOls and Meyer 1992). Following hybridization between the red and white petunia lines, 

the heterozygous petunias carrying ~7-R and 17-W alleles did not exhibit the expected A1-mediated, brick-red 

color. Instead, variable expression of the allelic transgenes 

95 
Section II Transformation and Foreign Gene Expression 

(17-R/17-W} was observed in the flower color. Meyer et 

al. (1993) proposed that differential methylation of ectopic 

homologous alleles may cause transinactivation. Presumably the 17-W allele induced a paramutation or a directed 

epigenetic change of the 17-R allele in hetero·zygous 

petunias. 

Inactivation of homologous and nonhomologous 

transgenes can occur in transgenic plants. Transinactivation 

was observed in transgenic tobacco plants that were sequentially transformed with two selectable markers, kanamycin and hygromycin resistance, encoded by T-DNA I 

and T-DNA II, respectively (Matzke and Matzke 1990; 

Matzke et al. 1989). Inactivation ofT-DNA I occurred in 

the presence of T-ON A II, probably by methylation of the 

promoter in T-DNA I (Matzke and Matzke 1991). Following segregation of the two T-DNAs, transgene (T-ON A I) 

expression was restored after several generations. This 

change was accompanied by partial or complete 

demethylation of the promoters in the transgenic tobacco. 

Epistatic interaction between the two transgenes occurred 

again, when both the T-ON As were brought together in a 

hybrid following sexual hybridization (Matzke et al. 1994). 

Post-Transcriptional Silencing 

Transgene interaction usually results in gene silencing 

at the post-transciptional level. Cosuppression, which is 

coordinate suppression of the transgene and the homologous endogene, was described previously (Flavell et al. 

1995; Jorgensen 1995; Meyer 1995). Usually, cosuppression 

results from 'a post-transcriptional process involving RNA 

turnover. A flower color gene in petunia, chalcone synthase (CHS}, is necessary for the biosynthesis of the anthocyanin pigment responsible for the red and purple flower 

color. When a chimeric CHS gene was introduced into a 

purple flowering line of Petunia hybrida, the resulting 

transgenic plants produced either white flowers or variegated flowers with white and pale sectors (Napoli et al. 

1990; van der Krol et al. 1990}. In the white flowers, both 

t~e CHS transgene and the endogenous CHS genes were 

seemingly silent and were characterized by a reduction in 

steady-state mRNA levels (van Blokland et al. 1994}. Posttranscriptional silencing was also observed with other chimeric genes. For example, the meiotically reversible 

silencing of rolB in Arabidoposis thaliana (Dehio and Schell 

1994}, J3-1,3-glucanase (GN1) gene from Nicotiana 

plumbaginifolia in tobacco (de Carvalho et al. 1995}, and 

NPTII gene in tobacco (Ingelbrecht et al. 1994). Another 

variation on the cosuppression of genes invokes an autoregulatory model involving a biochemical switch that determines the degradation of excessive RNA produced by 

the transgene and its homologous endogene (Meins and 

Kunz 1994, 1995). Whether the degradation of a specific RNA 

occurs in the nucleus or cytoplasm remains unresolved in 

most cases of post-transcriptional gene silencing. 

96 

Transgene Stability: Limitations 

and Prospects 

Although transferring recombinant genes into plants is 

possible, several problems exist with their integration and 

expression. Investigations on several plant model systems, 

including Populus, indicate that transgenes are less stable 

in the transgenic plants than originally thought. Transgene 

inactivation was observed in many plant species. Levels 

of transgene expression vary among independent 

transformants, and this variability may be conditioned by 

the transgene copy number and/ or integration sites. This 

suggests that it is difficult to predict if a transgene will be 

stably integrated or expressed in plants; whether annual 

plants or a forest trees. Several hypotheses explain gene 

silencing in transgenic plants (Flavell 1994; Jorgensen 

1995; Matzke and Matzke 1995; Meins and Kunz 1995). 

Although several of these are mutually exclusive, none 

can explain all instances of transgene inactivation. Perhaps different mechanisms of transgene inactiv.ation 

occur that are interdependent on the promoter type, 

reporter gene, chimeric gene number, or other sequences between the T-DNA borders introduced into 

the host genome. 

An overall picture emerging from transgenic research 

in plants is that recombinant genes may not be compatible or well integrated with an evolutionary stable plant 

genome, particularly of a forest tree. However, it is possible that some transgenes will be well expressed, but 

may still cause instability in transgenic plants. 

Transgene expression may depend on the type of chimeric genes such as those conferring herbicide tolerance, disease and pest resistance, or causing hormone 

or phenotypic alterations. Transgene stability may also 

depend on the T-DNA insert and its sequence homology at the site(s) of integration within the plant genome 

(Koncz et al. 1994). 

Transgene loss or instability acquires a new dimension 

and perspective for long-lived trees. For example, a 

transgene conferring herbicide tolerance must be active 

during the major part of the life cycle of an annual 

transgenic crop. Alternatively, expression of a herbicidetolerant transgene may be required only during the first 

or second year of tree growth, when herbicide is sprayed 

to kill competing weeds. After a year or two, expression of 

the herbicide-tolerant gene may be unnecessary, and the 

gene could become nonfunctional, inactive, heterochromatized, or perhaps discarded from the tree genome 

during the extended vegetative phase of a tree species. In 

contrast, disease- or pest-resistant transgenes must remain 

active for many years to protect against disease or pests 

during tree growth. Therefore, transgene expression may 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
depend on its functional use during the life cycle of a 

woody plant. On the other hand, a transgene may become 

inactive at any stage of ·tree development by 

transinactivation, silencing, or eventually undergoing 

mutation. It is the fitness and adaptability of a transgene 

in the tree genome that determines its long-term survival. 

Improving transgene stability, particularly under field 

conditions, is important for commercialization of 

transgenic crops or forest trees. Many biotechnology firms 

worldwide are conducting field trials on improved 

transgenic crops, such as cotton, maize, and potato, to determine their performance before large-scale release. In 

1994, the transgenic "Flavr Savr"TM tomato was released 

into the marketplace in the United States (Ahl Goy and 

Duesing 1995). Overall, transgene inactivation or gene silencing apparently can occur at any stage of transgenic 

plant development, clonal propagation, or in the progeny of the transgenic plants. To address this problem, 

transformation methods should be devised that preferentially insert a single copy of the chimeric gene, perhaps at 

specific sites in the genome. While such technology is currently unavailable, methods should be developed that select transformants carrying a single integrated· copy of a 

transgene that remains stable over time. Because a 

transgene promoter may be the target of increased methylation, selecting promoters that are less prone to methylation is worthwhile. Recent studies show that stability 

of transgene expression may be increased in transgenic 

plants by including nuclear scaffold attachment regions 

(SAR) or matrix attachment r~gions (MAR) to flank the 

transgene (Allen et al. 1993; Breyne et al. 1992; Mlynarova 

et al. 1994). Although Agrobacterium-mediated gene transfer has served us well (Chilton 1993), insertion ofT-DNA, 

carrying the hybrid chimeric genes and extra genetic sequences between the left and right borders or outside of 

them, may contribute to genetic or epigenetic instability 

in transgenic plants. The particle gun DNA delivery 

method is an alternative, but is not without problems. 

Therefore, other innovative avenues for gene transfer 

should also be explored to promote the stability of transferred genes in annual crops and long-lived woody plant.s. 

What factors determine whether 1 or several copies of 

the chimeric gene will be integrated at 1 or several sites in 

the genome? Are transgenes regulated in the plant genome 

by their own promoter, or do plant regulatory genes also 

control transgene expression? Does increased methylation 

of the transgene promoter cause transgene inactivation, 

or is it the result of gene instability? Does transinactivation 

involve a paramutation or an epimutation? What causes 

cosuppression of a transgene and the homologous endogenous gene? Is gene silencing due to post-transcriptional 

events involving reduction in steady-state mRNA levels? 

These are some questions relevant to transgene instability 

in annual crops and in woody plants that future research 

efforts may answer. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Transgenes and Genetic Instability 

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USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Section Ill Molecular Biology 
Chapter 14 

Applications of Molecular Marker Technologies in 

Popf)/us Breeding1 

Maria Teresa Cervera, Marc Villar, Patricia Faivre-Rampant, 

Marie-Christine Goue, Marc Van Montagu, and Wout Boerjan 

Introduction 

In the 17th and 18th centuries, farmers and tree-breeders 

in Europe selected individual trees for traits such as fast 

growth and . Traditional breeding work was based on the 

analysis of phenotypes and has provided a wealth of information on genetic material through multigeneration pedigrees. Recent developments in DNA marker technologies have 

created the possibility of generating new breeding strategies. In 

forest trees, these technologies are developed for conifer, Eucalyptus, and Populus breeding. • 

Application of molecular markers to forest tree breeding 

is dependent on the advantages and limitations of tree genetics. The Populus genus (poplars, cottonwoods, and aspens) 

is a good model system for the diverse disciplines of forest 

tree biology (reviewed by Stettler et al. 1996). Populus species have considerable genetic variation; are highly heterozygous; interspecific hybrids can be easily obtained through 

artificial breeding techniques (Stanton and Villar 1996); and 

controlled sexual propagation can be conducted in a greenhouse over approximately 3 months. Moreover, the ease of 

vegetative propagation permits replicated clonal trials to 

estimate genetic and environmental components of phenotypic variance for traits of interest; the nuclear genome is 

relatively small (2C = 1.1 pg); and the chromosome number 

(2n = 38) is the same for all Populus species (Bradshaw and 

Stettler 1993; Wang and Hall1995). 

In the first part of this chapter, we describe marker technologies common! y used in plant breeding, and provide ref1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTA-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

erences to their application in forest trees including poplar. 

The second part focuses on Populus spp. and is an overview 

of current applications of molecular markers in phylogenetic 

analysis, genetic mapping, and marker-assisted selection. 

Molecular Markers Used as 

Genetic Marker~ ·in Breeding 

For centuries, morphological traits provided the--basis 

for plant breeding and contributed to the development of 

theoretical population genetics. The first genome maps 

were produced by analysis of genetic segregation and linkage between morphological markers. However, because 

morphological markers can be strongly influenced by the 

environment and are limited in number, they are less valuable in current breeding programs. 

Techniques to analyze proteins (Smithies 1955) have allowed molecular marker identification based on protein 

polymorphism.s (Lewontin and Hubby 1966; May 1992). 

Through developments in molecular biology, different 

techniques have emerged to detect molecular markers 

based on DNA. Using restriction endonucleases (Linn and 

Arber 1968; Meselson and Yuan 1968) allows evaluation 

of DNA sequence variation through the analysis of Restriction Fragment Length Polymorphism (RFLP). In 1985, the 

Polymerase Chain Reaction (PCR) technique was developed based on the amplification of DNA fragments using 

a thermostable DNA polymerase (Saiki et al. 1985, 1988). 

Many variants of the PCR strategy have since been established to detect DNA polymorphism.s. The objective of the 

PCR variant strategies was to increase the number of markers analyzed per experiment and the likelihood of identifying markers that display a high Polymorphism Information 

Content (PIC). Since the plant genotype is analyzed directly 

by DNA marker technology, environmental or developmental alterations of the phenotype have no effect. 

101 
Section Ill Molecular Biology 

Molecular Markers ,. According to Weising et al. (1995), an ideal marker technology has markers: 1) with .a codominant inheritance 

(homo- and heterozygotic states can be discriminated); 2) 

that are evenly distributed throughout the genome; and 

3) with selectively neutral behavior. Moreover, ideal 

marker technology: 1) can reveal many polymorphic loci 

with multiple alleles; 2) is easy, fast, and inexpensive; and 

3} is highly reproducible. 

Marker technologies that are currently available combine some of these properties. A marker system should be 

selected on the basis of the genetic analysis to be performed. In the following discussion, we describe the principles, advantages, and limitations of marker technologies 

commonly used in poplar breeding (table 1). 

Isozyme and Allozyme Markers 

Allozymes are enzymes encoded by different alleles of the 

same gene; thus, they can differ in their amino add sequence, 

protein structure, and kinetic properties. Allozyme analysis 

is based on the correlation between differences in mobility 

in an electric field and differences among alleles encoding 

these allozymes (Murphy et al. 1990). 

Allozyme analysis comprises: 1) preparation of tissue 

extracts using buffers that allow protein extraction while 

maintaining enzymatic activity; 2) electrophoretic (starch 

or polyacrylamide gel) separation of proteins according 

to net charge and size; and 3) allozyme detection after electrophoresis using specific stains. 

Allozyme analysis is easy to perform, inexpensive, and 

technically accessible. Allozyme expression is typically 

codominant allowing discrimination between homozygous and heterozygous loci and multiple alleles. However, 

allozyme analysis has several drawbacks. The limited number of loci that can be analyzed may restrict attempts to 

locate markers associated with traits of interest, and the 

level of genetic polymorphism within coding sequences is 

relatively low. A new allele can be detected as a polymorphism only if nucleotide differences cause amino add substitutions that affect the electrophoretic mobility. 

Furthermore, allozymes may be active only at a specific 

physiological stage, tissue, or cell compartment. Other limiTable 1. Comparison of DNA marker systems (adapted from Mazur and Tingey 1995). 

RFLP1 RAPD2 SSR3 AFLPTM 4 

Assay principle Endonuclease Amplification Amplification Selective 

digestion and with random of SSRs amplification of 

hybridization primers DNA fragments 

Polymorphism Single-base Single-base Repeat l~ngth Single-base 

type insertions or insertions or insertions or 

deletions deletions deletions 

Polymorphism Medium Medium High Medium 

level 

Number of 1-2 5-20 1 40-100 

different loci 

assayed in a 

single reaction 

Abundance High Very high Medium Very high 

Dominance Codominant Dominant Codominant DominanVcodominant 

Amount of DNA 2-10 J.tg 10-25 ng 25-50 ng 250 ng 

required 

DNA sequence No No Yes No 

required 

Radioactive Yes/no No No Yes/no 

detection 

Cost Medium/high Low High Medium 

1 Restriction Fragment Length Polymorphism 2 Random Amplified Polymorphic DNA 3 Simple Sequence Repeats 4 Amplified Fragment Length Polymorphism 

,.... Registered trademark in the Benelux 

102 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
tations, such as post-transcriptional modifications or the 

presence of isozymes with identical mobility but encoded 

by different loci, can hamper interpretation of the zymogram (banding pattern) analysis. 

Linkage analysis of isozyme loci was performed on 

many forest trees such as Populus spp. (Liu and Furnier 

1993a}, Larix laricina (Cheliak and Pitel 1985}, Eucalyptus 

regnans (Moran and Bell 1983), Pinus spp. (Conkle 1981), 

Pinus radiata (Moran et al. 1983), Pinus rigida (Guries et al. 

1978), Pinus strobus (Eckert et al. 1981), and Pinus taeda 

(Adams and Joly 1980). Isozyme analysis was used to identify clones of the genus Populus (Cheliak and Dancik 1982; 

Hyun et al. 1987; Liu and Fumier 1993b; Lund et al. 1992; 

Raj?ra 1988, 1989a; Rajora and Zsuffa 1989}. 

Restriction Fragment Length Polymorphism (RFLP) 

RFLP analysis, a DNA-based marker technology used 

in plant breeding and plant genetics (Neale and Williams 

1991; Tanksley et al. 1989}, is based on polymorphism observed after DNA digestion with 1 or more restriction enzymes. Resulting fragments are electrophoretically 

separated in agarose or acrylamide gels, according to their 

molecular weight, transferred to a membrane and visualized after DNA hybridization with probes containing homologous sequences. RFLP technology detects base 

substitutions within cleavage sites, insertions, deletions, 

and sequence rearrangements. 

RFLPs are codominant markers capable of distinguishing 

multiple alleles. They have been used to generate genome 

maps, study the architecture of complex genetic traits and 

monitor their inheritance, monitor trait introgression through 

RFLP-backcross breeding, evaluate germplasm diversity, and 

analyze genome homologies among plant species. Depending on the method used to select the probes, RFLP markers 

can cover the entire genome. DNA clones used as probes to 

detect RFLPs from nuclear DNA can be obtained from 

complementary DNA (eDNA) or genomic libraries. 

Although RFLP analysis reveals high levels of polymorphism within a population, it is technically demanding, 

slow to accomplish, and requires relatively large amounts 

of DNA. Another limitation is the availability of libraries 

with useful probes, although libraries are available for several main crops and some forest trees such as Populus spp. 

(Bradshaw and Stettler 1993; Liu and Furnier 1993a), Eucalyptus nitens (Byrne et al. 1994), and loblolly pine (Devey 

et al. 1991). RFLP genome maps were generated with these 

probes (Bradshaw et al. 1994; Byrne et al. 1995; Devey et 

al. 1994; Liu and Fumier 1993a). Ahujaet al. (1994) reported 

that informative eDNA probes for RFLP analysis of loblolly 

pine were also useful for other conifers. Associations between RFLP markers and traits that display Mendelian 

inheritance were established for several forest tree species 

including loblolly pine (Groover et al. 1994) and Populus 

spp. (Bradshaw and Stettler 1995). In addition, RFLP markUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Applications of Molecular Marker Technologies in Populus Breeding 

ers were used to study inter- and intraspecific variation in 

Populus (Keirn et al. 1989; Liu and Fumier 1993b). 

RFLPs can also be obtained from chloroplast DNA 

(cpDNA) and mitochondrial DNA (mtDNA). Two different approaches are useful to study RFLPs in cytoplasmic 

DNA: 1) isolation of cp or mtDNA, digestion with restriction enzymes, electrophoretic separation, and visualization of RFLPs by ethidium bromide or silver staining; and 

2) isolation and digestion of total DNA, electrophoretic 

separation, and Southern blotting using total cp or mtDNA 

or specific cp or mtDNA sequences as a probe. Various 

studies were conducted on Populus based on cpDNARFLPs 

(Mejnartowicz 1991; Rajora and Dancik 1995a, 1995b) and 

mtDNA RFLPs (Barrett et al. 1993; Radetzky 1990). 

Polymerase Chain Reaction (PCR) Technologies 

Many variant methods have been developed since the 

invention of PCR technology in 1985 (Innis et al. 1990); 

some allow detection of DNA polymorphisms. 

Random Amplified Polymorphic DNA (RAPD) markers are generated by PCR amplification using arbitrary 

primers. The following research teams established this 

technique: 1) DuPont, which patented the technique with 

the name RAPD (Williams et al. 1990); 2) Caetano-Ann6les 

et al. (1991a}, who proposed the name OAF for DNA Amplification Fingerprinting; and 3) Welsh and McClelland 

(1990), who described AP-PCR (Arbitrarily Primed PCR). 

RAPD markers have been used for DNA fingerprinting, 

genetic mapping, and localization of genes of interest 

(Caetano-Ann6les et al. 1991b; Neale and Harry 1994; 

Newbury and Ford-Lloyd 1993; Tingey and DelTufo 1992; 

Williams et al. 1993). 

RAPD protocol is based on use of a single short primer 

(10 to 12 bases in length with at least 40 percent G+C content) in the PCR reaction. To obtain an amplification product, the distance between both regions complementary to 

the primer sequence (but in an inverted orientation) should 

not exceed 1,000 to 2,000 bp. Amplified DNA fragments 

are electrophoretically separated on an agarose gel and visualized by staining with ethidium bromide. Experiments 

have shown that the number of amplified DNA fragments 

observed per primer is independent of the genome complexity. Typically, between 5 and 20 loci can be analyzed 

per experiment. Thus, when large genomes, such as those 

of conifers, or small genomes, such as that of Arabidopsis, 

are analyzed, RAPD patterns show similar complexity. 

These results are due to primer competition and imply that 

not all amplifications derive from the perfect pairing between the primer and DNA template (Williams et al. 1990). 

RAPD technology detects single nucleotide changes, 

deletions, and insertions within the primer annealing site 

(Williams et al. 1990). A RAPD marker of interest can be 

cloned and sequenced for conversion to a Sequence Characterized Amplified Region (SCAR) (Paran and 

103 
Section Ill Molecular Biology 

Michelmore 1993). Each SCAR is a specific PCR-based 

marker that defines a single locus. Approximately 20 percent of the RAPDs can be converted to codominant SCARs. 

RAPD technology does not require probe libraries; instead, direct detection of bands in the gel is possible, which 

avoids hybridization and autoradiography. Moreover, less 

(approximately 1,000-fold) tissue is required for the assay, 

and a universal primer set can be used for genome analysis of any organism. Another important advantage is that 

RAPD analysis can be automated. Overall, RAPD assays 

are relatively simple, quick, and inexpensive. 

However, this marker technology does present several 

limitations. RAPD markers are dominant, so they usually 

cannot distinguish between homozygous and heterozygous 

loci. This results in a loss of genetic information mainly for 

highly heterozygous organisms such as forest trees. RAPDs 

are also difficult to reproduce. This variation can be attributed to tnismatched annealing of the random primer to the 

ON A (Muralidharan and Wakeland 1993; Penner et al. 1993 ). 

Since Carlson et al. (1991) originally demonstrated Mendelian inheritance of RAPD markers based on segregation 

data in conifers, numerous maps were generated by analyzing haploid DNA from megagametophytes of white spruce 

(Tulsieram et al. 1992), loblolly pine (Grattapaglia et al. 1991 ), 

longleaf pine (Nelson et al. 1994), and maritime pine 

(Plomion et al. 1995a, 1995b). Grattapaglia and Sederoff 

(1994) combined the pseudo-testcross strategy and RAPD 

technoldgy to generate genetic linkage maps of Eucalyptus 

grandis and E. urophylla, while Bradshaw et al. (1994) used 

RAPDs, RFLPs, and STSs to cons.truct a Populus genome map. 

Associations were established between RAPD markers and 

economically important traits of Eucalyptus grandis, E. 

urophilla (Grattapaglia et al. 1995), sugar pine (Devey et al. 

1995), loblolly pine (Wilcox et al. 1995), and Populus spp. 

(Villar et al. 1996). Fingerprint analysis based on RAPD markers was also used to reveal inter- or intraspecific variation, 

and to study taxonomic relationships within the genus 

Populus (Castiglione et al. 1993; Liu and Fumier 1993b). 

Simple Sequence Repeats (SSRs), or microsatellites, are 

tandem repeats of sequence units, which can be as short as 

4, 3, 2, or even 1 nucleotide. SSRs are characterized by high 

levels of genetic polymorphism due to variation in the number of repeats (Hamada et al. 1982; Tautz and Renz 1984). 

Database searches of published sequences reveal that 

SSRs are abundant and widely dispersed in the plant genome with an average frequency of 1 every 50 kb 

(Morgante and Olivieri 1993). They were detected in 34 

plant species. The AT dinucleotide repeat was the most 

abundant, whereas AC/TG repeats, common in animals, 

were observed only in 1 plant species. Oligonucleotides 

containing TG and GATA/GACA repeats were used as 

probes in RFLP assays and revealed polymorphisms in 

plants (Lonn et al. 1992; Weising et al. 1989). 

SSR-based markers are generated by PCR amplification 

of the SSR using specific primers (20 to 25 bases) comple104 

mentary to their flanking regions. The number of repeat 

units at a locus is highly variable and can be easily detected as polymorphisms when the amplified DNA fragments are electrophoretically separated in polyacrylamide 

or high-resolution agarose gels. Bands are directly visualized by ethidium bromide staining or by autoradiography 

when labeled primers are used in the PCR reaction. One 

locus can be analyzed per specific primer combination. 

SSR-based markers have been used for genome mapping, variety identification, and germplasm analysis 

(Akkaya et al. 1992; Bell and Ecker 1994; Lynn and Heun 

1993; Morgante et al. 1994; Thomas and Scott 1993; Wu 

and Tanksley 1993). SSR-based markers are codominant 

and detect many different allele sizes per locus. SSRs have 

the highest PIC of any marker system. However, the effort 

required to obtain the sequences flanking SSR is the major 

disadvantage of SSR-based technology. The first step is to 

generate a genomic library enriched in repeated sequences. 

Then, clones must be hybridized with oligonucleotides 

complementary to the target sequence repeat. Positive clones 

are sequenced to design specific primers flanking the SSR. 

Due to this limitation, only few data based on characterizing microsatellites in forest trees were reported (Condit and 

Hubbel11991; Dow et al. 1995. Steinkellner et al. 1995). 

AFLPTMI (Amplified Fragment Length Polymorphism) 

marker technology is a powerful DNA fingerprinting technique developed by Keygene N.V. (Zabeau and Vos 1993), 

which is based on selective PCR amplification of restriction fragments from a complete digestion of genomic DNA. 

The AFLP technique is as reliable as the RFLP technique 

but avoids Southern blotting by detecting restriction fragments with specific PCR amplification (Vos et al. 1995). 

The assay consists of the following 5 steps (Vos et al. 

1995): 1) digestion of total genomic DNA with 2 different 

restriction enzymes, a rare cutter enzyme (e.g., EcoRI) and 

a frequent cutter enzyme (e.g., Msel); 2) ligation of oligonucleotide adapters (e.g., EcoRI-adapter and Msel-adapter) 

to the restriction fragments to obtain the primary template for PCRs, these oligonucleotide adapters contain the 

core sequence and an enzyme-specific cohesive sequence; 

3) selective preamplification of the primary template using PCR primers that contain 3 characteristic regions in 

their sequences: a core sequence, an enzyme-specific sequence (both homologous to the sequence of the adapter), 

and a 3' selective extension (1 nucleotide); 4) selective amplification with labeled PCR primers, similar to those used 

in the preamplification but with a longer 3' selective extension (2 or more nucleotides); and 5) electrophoretic 

separation of labeled fragments on polyacrylamide gels 

followed by autoradiography. 

Selective amplification does not require previous information about the sequence because it is conferred by se1 ALFP is a registered trademark in the Benelux. 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 
'~ 

lective extension of the primers; thus, only those fragments 

homologous to the nucleotides flanking the enzyme-specific sequence are amplified. The number of amplified DNA 

fragments can be controlled by the cleavage frequency of 

the rare cutter enzyme and the number of 3' selective nucleotides. Typically, between 60 and 120 restriction fragments 

are detected per reaction. 

The AFLP technique detects polymorphisms such as the 

presence or absence of restriction enzyme sites, sequence 

polymorphisms adjacent to these sites, insertions, deletions, and rearrangements. AFLP technology can be applied to clonal identification, germplasm analysis, 

construction of high-density linkage maps, and localization of monogenic and polygenic traits when close associations are established between AFLP markers and traits 

of interest. This technique will contribute significantly to 

map-based cloning (Cervera et al. 1996a, 1996b; Thomas 

et al. 1995; van Eck et al. 1995). 

The most important advantage of the AFLP technique 

is the high number of polymorphic markers that can be 

analyzed per experiment. Fingerprints are produced using a limited set of primers. Another advantage is its high 

reproducibility. AFLP markers are codominant; using specialized software, it is possible to identify whether a locus is homo- or heterozygous although it is impossible to 

detect multiallelism. However, AFLP technology is technically demanding, and is more difficult to automate and 

more expensive than RAPD technology. 

In forestry research, close associations between AFLP 

markers an9 genes of interest were reported in Populus 

(Cervera etal. 1996a). Clonal identification based onAFLP 

fingerprints has been performed, and high density AFLP 

maps are in progress for 3 Populus species (Cervera et al. 

1996b). 

Sequence Tagged Site (STS) markers are those obtained 

by converting RFLP markers into PCR-based markers 

(Olson et al. 1989). This conversion requires the terminal 

sequence of RFLP probes to synthesize specific primers. 

Codominant STS-based polymorphism can be detected 

directly as length variation (Amplified Sequence Polymorphism) or may require restriction enzyme digestion of the 

amplified product to reveal the polymorphism. STS markers are called Expressed Sequence Tags (ESTs) when the 

sequenced clones are cDNAs (Olson et al. 1989). In forestry research, Bradshaw et al. (1994) generated a genetic 

linkage map of a hybrid poplar composed of RFLP, RAPD, 

and STS markers. 

Single Strand Conformation Polymorphism (SSCP), a 

method that enables detection of DNA polymorphisms such 

as point mutations (Hayashi 1991 ), is based on differences 

in electrophoretic migration of single stranded, denatured 

DNAs in a nondenaturing polyacrylamide gel. SSCPs are 

codominant markers capable of distinguishing multiple alleles. SSCP was used by Bodenes et al. (1996) to differentiate 

between Quercus petraea and Q. robur, 2 white oak species. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Applications of Molecular Marker Technologies in Populus Breeding 

Applications of Molecular 

Markers to Poplar Breeding 

Molecular breeding is the application of molecular (protein- or DNA-based) marker technologies to breeding programs. Short-term applications of molecular markers 

involve identification and discrimination of genotypes, 

germ plasm analysis, and taxonomic studies. Medium- and 

long-term applications involve: 1) the generation of genetic linkage maps; 2) early selection of individuals with 

specific characteristics within larger progenies; 3) efficient 

selection of parents for new breeding programs; 4) efficient trait introgression; 5) genetic mapping of simple or 

complex traits; 6) the study of the architecture of Quantitative Trait Loci (QTLs); and 7) comparative or syntenic 

mapping. A more exhaustive list on applications of 

marker-based technologies in plant breeding has been provided by Beckmann (1991). The use of molecular breeding 

technologies for forest trees could radically reduce the 

breeding time, a crucial advantage given the long generation times (Grattapaglia et al. 1994). 

Genetic Fingerprints to Identify Genotypes 

in Populus 

Marker technologies have been used to: 1) fingerprint 

genotypes and identify characteristic inter or intraspecific 

variation (varietal identification can be a tool to legally 

protect a breeder's rights); 2) perform paternity analysis; 

3) identify superior pollen donors; 4) monitor fidelity of 

controlled crosses by analyzing specific alleles present in 

the homozygous dominant state in 1 parent and null in 

the other; and 5) characterize genetic diversity. Molecular 

information on genetic diversity is useful to screen for redundancy and deficiencies in germplasm collections of 

forest trees, and to analyze management and use efficiency 

(Millar 1993 ). 

Isozyme analysis (Cheliak and Dancik 1982; Hyun et al. 

1987; Liu and Fumier 1993b; Lund et al. 1992; Rajora 1988, 

1989a; Rajora and Zsuffa 1989) and gas chromatography 

(Greenway et al. 1989) provided the first molecular clonal 

identification within the genus Populus. Cheliak and 

Dancik (1982), Hyun et al. (1987), and Lund et al. (1992) 

examined levels and distribution of isozyme variation in 

several P. tremuloides populations. Liu and Fumier (1993b) 

compared allozyme RFLP and RAPD markers for their 

ability to reveal inter and intraspecific variation in P. 

tremuloides and P. grandidentata. Their results indicated that 

RAPDs are more powerful for fingerprinting individuals. 

To estimate taxonomic relationships and discriminate 

among all tested commercial clones, Castiglione et al. 

(1993) used RAPD fingerprints to analyze 32 clones be105 
Section Ill Molecular Biology 

longing to 10 Populus species. Based on ribosomal DNA 

(rONA) RFLP analysis, Faivre-Rampant et al. (1992) obtained characteristic inter and intraspecific variability 

among 5 different species of Populus (P. deltoides, P. nigra, 

P. trichocarpa, P. maximowiczii, and P. alba). Interspecific variability was displayed by different major ribosomal unit 

types, while intraspecific variability was displayed by the 

length variation of the ribosomal unit types. 

Based on RFLP analysis of interspecific poplar crosses, 

a study on cpDNA transmission revealed a maternal mode 

of inheritance (Mejnartowicz 1991 ). Rajora and Dancik 

(1995a, 1995b) conducted research on inter and intraspecific cpDNA variation in Populus (P. deltoides, P. nigra, and 

P. maximowiczii) by RFLP analysis using 16 restriction enzymes and 6 heterologous probes. Similar studies were 

performed by Barrett et al. (1993) using mtDNA RFLPs to 

examine inter and intraspecific mitochondrial variation 

among P. deltoides, P. nigra, P. maximowiczii, and P. 

tremuloides. Using mtDNA RFLPs, Radetzky (1990) established that mtDNA is inherited maternally in Populus. Recently, AFLP fingerprints were used to reveal inter or 

intraspecific variation in Populus (M.T. Cervera unpublished results). 

Molecular Systematics in Poplar 

Development of biochemical and molecular marker technologies provides new tools for plant systematic studies 

(Machan et al. 1995; Olmstead and Palmer 1994; Strauss et 

al. 1992). The genus Populus comprises approximately 35 

species native to the northern hemisphere (figure 1). The 

classification in 5 sections, Aigeiros, Tacamahaca, 

Leucoides, Leuce (currently termed Populus), and 

Turanga, is based on morphology, geographical localization, and crossability. Although Populus is considered a 

model in forestry, data on systematics are lacking for this 

genus and published studies involve only a limited number of species. Taxonomic analysis is difficult due to wide 

intraspecific variability, natural crossability of the members within this genus, and convergent morphology shown 

by hybrids and species (Eckenwalder 1984a, 1984b; Hu et 

al. 1985; Keirn et al. 1989; Ronald 1982). Because the variation in poplar morphological traits is difficult to interpret, 

molecular systematics was developed to evaluate genetic 

diversity in the framework of breeding and gene conservation programs. 

The first approach involved 12 enzyme systems used to 

study P. deltoides, P. nigra, and P. maximowiczii (Rajora and 

Zsuffa 1990). The results indicated that P. nigra was more 

closely related to P. maximowiczii than to P. deltoides, although both P. nigra and P. deltoides are classified in the 

section Aigeiros. 

Other marker technologies have been used for molecular phylogeny studies, involving nuclear and cytoplasmic 

markers. Smith and Sytsma (1990) analyzed restriction site 

106 

---compatibility 

c=:=:=:J incompatibility 

Figure 1 . Species of the genus Populus, with compatibility 

and incompatibility relationships shown among 

the 5 genus sections (Willing and Pryor 1976). 

variations in cpDNA and rONA in 10 poplar species from 

the sections Aigeiros, Tacamahaca, and Leuce. Based on 

cpDNA data, a single-parsimonious Wagner tree revealed 

a close relationship between the European poplars P. alba 

and P. tremula. Another cluster was composed of the American species P. deltoides, P. fremontii (Aigeiros), and P. 

balsamifera (Tacamahaca). Unexpectedly, the results supported a closer relationship of P. nigra to P. alba and P. 

tremula, than to the other Aigeiros species. Cladistic analysis performed on rONA revealed 3 distinct groups. One of 

these groups included the Leuce species. The second group 

was composed of Tacamahaca species and Aigeiros species except P. nigra. The third group was only represented 

by P. nigra. These results suggest that cpDNA phylogeny 

is correlated with geographic distribution, while rONA 

phylogeny is concordant with current botanical classifications. A~ suggested by the authors, the analyzed P. nigra 

might have resulted from hybridization/ introgression 

events between P. alba and P. nigra. 

Experiments by Barrett et al. (1993), using mtDNA RFLP, 

showed a close relationship between P. deltoides and P. 

maximowiczii. In addition, they revealed close similarities between P. nigra and 1 species of the Leuce section, P. tremuloides, 

confirming the results of Smith and Sytsma (1990). 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Using RAPD markers, Castiglione et al. (1993) studied 10 

species belonging to the 3 main sections, Aigeiros, 

Tacamahaca, and Leuce, 19 hybrids of P. x euramericana (P. 

deltoides x P. nigra), and 1 P. deltoides x 1~ maxinzowiczii hybrid. 

One cluster of P. x euramericana clones was revealed. All hybrid clones were closer to the maternal species, P. deltoides, 

than to the paternal species. Surprisingly, phenetic analysis 

revealed no cluster between the species. Nevertheless, the 

authors pointed out the unusual position of P. trichocarpa, 

which was separated from the other Tacamahaca species. P. 

nigra was also separated from the Aigeiros species. More 

recently, Rajora and Dancik (1995b) and Castiglione ( unpublished data) confirmed dissimilarity between P. nigra and P. 

deltoides using cpDNA RFLP. 

Investigation of nuclear DNA variation was reported by 

Faivre-Rampant et al. (1995a). This study involved 18 species and 1 hybrid belonging to the sections Aigeiros, 

Tacamahaca, Leuce, and Leucoides. STS markers developed 

by Bradshaw et al. (1994) were used. Sakal and Michener's 

(1958) genetic distance analysis and cluster analysis based 

on this distance analysis were performed. The results are 

presented as a neighbor-joining tree in figure 2. Species of 

the Leuce section were clearly clustered, except P. davidiana. 

As expected, P. deltoides, P. fremontii, and P. wislizenii formed 

a group, confirming that the 2 latter species can be considered subspecies of P. deltoides. Tacamahaca and Leucoides 

species, with P. nigra, formed a single large cluster. Again, P. 

nigra was isolated from the other Aigeiros species. These results indicate that P. nigra is not related to P. deltoides, P. 

fremontii, anq P. wislizenii, and that species of the Tacamahaca 

and Leucoides sections share a dose relationship. 

In conclusion, molecular classification obtained by polymorphism analysis of nuclear markers roughly matches the 

botanical classification. Molecular systematic studies do not 

classify P. nigra in the Aigeiros section. These results coincide with many elements on evolutionary relationships obtained from morphological (Eckenwalder 1977), crossability 

(Rajora 1989b }, pathological (Pinon and Teissier du Cros 1976; 

Steenackers 1972}, and cytological studies (Faivre Rampant 

et al. 1995b). According to Smith and Sytsma (1990), P. alba 

could be an ancestor of P. nigra. Further work is needed to 

confirm this interesting hypothesis. Recently, Rajora and 

Dancik (1995b) proposed to assign P. nigra in a new section 

called Nigrae. Additional studies are required on cpDl'\A, 

mtDNA, and nuclear DNA with markers equally distributed throughout the genome. Studies are needed on interspecific variation of species that were not previously 

analyzed, and on intraspecific variation involving individuals from the entire natural range. 

Genetic Linkage Maps of Populus 

Two linkage maps were published and 5 others are currently under construction from different pedigrees and 

with various objectives. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Applications of Molecular Marker Technologies in Populus Breeding 

1) The genetic linkage map generated at the University 

of Minnesota (USA) is based on an F1 P. tremuloides x P. 

tremuloides family (Liu and Fumier 1993a). This partial map incorporates 54 RFLP and 3 allozyme markers, and comprises 14 linkage groups covering 

664 centiMorgan (eM) of the genome. The objective of 

~------------dav 

------ang 

tri 

..... ---lsc 

._---cil 

..... -----yun 

..... ----nig 

.._-------sim 

..-----del 

wil 

"---fre 

..... ----alb 

.._--tom 

...._---tra 

'-------t re 

Figure 2. Neighbor-joining tree for some species of the 

genus Populus. The analysis was done with 

PHYLIP package programs (Felsenstein 1993; 

http://bimcore.cc.emory.edu/phylip.doc). 

alb= P. alba 

tra = P. tremula 

tre = P. tremuloides 

tom = P. tomentosa 

dav = P. davidiana 

ang = P. angustifolia 

tri = P. trichocarpa 

lrf = P. laurifofia 

szc = P. szechuanica 

mxw = P. maximowiczi 

bal = P. balsamifera 

yun = P. yunnanensis 

sim = P. simonii 

lsc = P. fasiocarpa 

cil = P. ciliata 

nig = P. nigra 

del = P. deftoides 

wit = P. wisfizenii 

fre = P. fremontii 

107 
Section Ill Molecular Biology 

this map was to understand the mode of inheritance 

and linkage relationships of these genetic markers. 

2) The genetic linkage map constructed at the University 

of Washington/Washington State University (UW I 

WSU), USA is based on a 3 generation P. trichocarpa x P. 

deltoides pedigree. This pedigree consists of an F 2 family of approximately 350 trees. This family was obtained 

from a controlled cross between 2 full-sib individuals 

from an interspecific hybridization between a female 

P. trichocarpa and a male P. deltoides. The published map 

is composed of 343 RFLP, STS, and RAPD markers, 

and the total distance contained within the 19 largest 

linkage groups is 1,261 eM (Bradshaw et al. 1994). Average spacing between the markers is 6.7 eM 

(Bradshaw and Stettler 1995). Limitations of this map 

are its size, which is approximately half the estimated 

length of the Populus genome. The UW /WSU map has 

been extended to 512 markers (H.D. Bradshaw unpublished data) and is of sufficient scope and density to 

permit the mapping of QTLs. This map will be. used .to 

analyze the genetics of adaptive and commeraal traits 

such as leaf phenology, stem basal area, height, wood 

quality traits, and disease resistance. Genotype data, 

available markers, and other data information may be 

obtained on the World Wide Web at http: I I 

wv:rw.poplar1.cfr.washington.edu. 

3) Linkage maps generated at the INRA (France) are based 

on an interspecific F1 P. deltoides x P. trichocarpa family 

of 125 individuals (Goue et al. 1996). This map is under construction according to the pseudo-testcross 

strategy described by Carlson et al. (1991) and 

Grattapaglia and Sederoff (1994) for conifers and Eucalyptus. This mapping strategy results in 2 maps corresponding to each parent. Four types of markers, 

RFLPs, RAPDs, STS, and allozymes, are currently being mapped. Of 255 Operon primers that were 

screened, 119 were selected to amplify a total of 274 

RAPD markers. These markers were grouped to produce a maternal P. deltoides map with a total of 113 

markers in 25 linkage groups, and a paternal P. 

trichocarpa map with 121 markers in 28 linkage groups. 

RFLP markers will be mapped with probes provided 

by H. D. Bradshaw (UW /WSU). A mixed marker map 

composed of numerous RAPDs and fewer codominant 

markers, such as RFLPs and I or allozymes, has been 

successful (Bradshaw et al. 1994). RFLPprobes will also 

permit the comparison of the 2 maps, searching for 

syntenic groups. These INRA maps will allow investigation of the genetic architecture of resistance to diseases such as rusts (Melampsora spp.), bacterial canker 

(Xanthomonas populi), and leaf spot (Marssonina brunnea) 

(Goue et al. 1996). The family being mapped in this 

108 

project is connected to other intra and interspecific 

families by a 9 x 9 factorial mating design, involving 8 

P. trichocarpa parents and 10 P. deltoides parents. This 

mating design will play a key role in determining QTL 

stability across genetic backgrounds. 

4) The linkage maps constructed at the Flander$ Interuniversity Institute for Biotechnology (Vffi) (Gent, Belgium) are based on 2 interspecific F1 families, P. deltoides 

x P. nigra and P. deltoides x P. trichocarpa (Cervera et al. 

1996b ). The 2 families share a common P. deltoides female parent. Three maps are in progress combining 

the pseudo-testcross strategy with the AFLP technology. The P. deltoides x P. nigra hybrid family consists of 

126 individuals, while the P. deltoides x P. trichocarpa 

family consists of 110 individuals. Each map will incorporate about 300 AFLP markers, and other ~LP 

and STS markers used previously by Bradshaw et al. 

(1994). RFLP and STS markers will be used to identify 

syntenic linkage groups. These maps will allow the 

genetic mapping of resistance to pathogens such as 

Melampsora spp., Xanthomonas populi, poplar mosaic virus, and Marssonina brunnea, along with other economically important traits such as growth, leaf phenology, 

wood density, and frost tolerance. 

Genetic Analysis of Simply and Complex 

Inherited Traits 

Genetic Analysis of Simply Inherited Traits 

Sax (1923) proposed the association between a morphological marker and a quantitative trait as a method for indirect selection. The utility of molecular markers in plant 

breeding, and more specifically in forestry breeding (because of the long generation times), is based on finding 

associations between these markers and traits of interest. This 

linkage allows the screening of a progeny for the presence 

of a desirable trait by detection of isozymes, RFLP, RAPD, 

SSR, STS, or AFLP markers. These linkages have been determined for several simply inherited traits (reviewed in 

Michelmore 1995 for resistance to pests and diseases). 

To introduce disease resistance into susceptible varieties, crosses with varieties that carry the resistance gene 

must be performed. In traditional breeding, progeny 

screening involves pathogen inoculation. For nursery 

tests, screening efficiency depends on environmental conditions, pathogen infection pressure, and the presence of 

other pathogens. For laboratory tests, screening efficiency depends on inoculation efficiency. Furthermore, 

screening requires large nursery and greenhouse space 

and several annual replications. In contrast, the use of 

molecular markers allows rapid screening of progeny 

to identify resistant clones by analyzing the presence 

of markers associated with the resistance gene. In adUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
clition, this analysis does not depend on biotic or abiotic factors. 

Molecular rust genetics (Melampsora larici-populina) resistance was initiated mainly by searching for markers 

associated with qualitative resistance using Bulked Segregant Analysis (Michelmore et al. 1991). RAPD markers 

tightly linked to M. larici-populina resistance were identified in 3 families of a 2 x 2 factorial mating design (Villar 

et al. 1994, 1996), and such linkage is currently under investigation in 2; interspecific P. deltoides x P. trichocarpa 

families originating from 10 P. deltoides and 8 P. trichocarpa 

parents (M. Villar unpublished data). Cervera et al. (1996a) 

identified 3 AFLP markers tightly linked to M. 

larici-populina resistance in an interspecific P. deltoides x P. 

nigra family. Because of their close linkage, these markers 

are exceptional tools for gene introgression and can be 

useful in cloning the resistance genes by chromosome landing (Tanksley et al. 1995). 

For breeding purposes, qualitative resistance is not as 

desirable as other resistance forms. In Europe, the breakdown of qualitative resistance has regularly occurred on 

interspecific F1 hybrids selected for immunity. When 

breeding for durable resistance, quantitative resistance 

must be considered. Durable resistance (horizontal resistance or tolerance), which is assumed to be under polygenic control, is currently under dissection through the 

search for QTLs on genetic maps (Goue et al. 1996; Lefevre 

et al. 1994, 1995). This is one mapping effort objective at 

the University of Gent (Belgium) and INRA (France). The 

USA group is focusing on resistance against M. medusae, 

for which rrlarkers have been revealed on the UW /WSU 

linkage map (Newcombe et al. 1996). 

Genetic Analysis of Complex Inherited Traits 

Most of the heritable characters of economic importance, 

such as vegetative propagation, growth, or development, 

are complex inherited traits. These traits are named polygenic or quantitative because they result from a combined 

action of several genes (Tanksley et al. 1989). Little is known 

about the number, localization, magnitude of effect, and 

interaction of genetic loci controlling expression of these 

traits. Manipulation of polygenic traits, more difficult than 

that of simple inherited traits, has always been challenging for traditional breeders. 

The first premise for identifying markers for polygenic 

traits is to consider the inheritance of quantitative traits. 

Quantitative traits depend on genes subject to the same 

properties and laws of transmission as genes displayed 

by qualitative (or monogenic) traits (Falconer 1989). The 

identification of markers associated to quantitative or 

qualitative traits requires a similar approach when selecting a DNA marker technology. Classical QTL mapping 

strategy involves analysis of a segregating population derived by crossing 2 progenitors that are genetically disUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Applications of Molecular Marker Technologies in Populus Breeding 

tinct for the trait under investigation. Two searches must 

be·conducted; identification of molecular markers associated to the character, and construction of a relatively dense 

linkage map to analyze the number, chromosomal position, magnitude of effect, and mode of action of genes controlling the expression of the trait. Not all QTLs are 

detectable through associations with molecular markers; 

detection of a QTL depends on the magnitude of its effect 

on the quantitative trait, heritability of the trait, size of 

the segregating population, and recombination frequency 

(map distance) between the marker and the QTL. 

Different statistical methods can determine the linkages 

between marker loci and QTLs. One of the most accepted 

approaches is interval mapping (Lander and Botstein 1989). 

The single marker approach, based on associations between 

a molecular marker and a QTL, cannot discriminate between 

a small-effect QTL that is close to a marker and a large-effect 

QTL that is further away from the marker. The interval mapping approach solves this problem because it is based on the 

analysis of 2 linked markers flanking an interval that may 

contain a QTL (Lander and Botstein 1989). Different software packages have been developed for the analysis of QTLs 

such as MapMaker-QTL (Lander and Botstein 1989; Lander 

et al. 1987), QTLStat (Liu and Knapp 1992), QTL cartographer, QGene, and MAPQTL. 

QTL analysis is difficult because age, genetic background, and environmental factors can affect phenotypic 

expression. Moreover, QTL analysis requires scoring the 

trait and genetic analysis of several hundreds of individuals to obtain a high mapping resolution. The number of 

progeny that is analyzed can be reduced while keeping a 

similar statistical power of QTL detection. To accomplish 

this, a selective genotyping can be carried out by choosing only those individuals showing extreme phenotypic 

values among the entire sample population (Darvasi and 

Soller 1992); this might be successful in tagging QTLs of 

large effect (Wang and Paterson 1994). Or, a genotype replication strategy can be used to reduce the environmental 

variation. In this approach, clonal replicates are planted 

and analyzed to increase the precision of the phenotypic 

measurement, without increasing the number of molecular marker assays. This strategy is especially useful for 

traits of low heritability (Bradshaw and Foster 1992). 

When planted in contrasting environments, this strategy 

allows analysis of QTL by environment interactions. 

Results show that large proportions of the total variation 

for many complex inherited traits may be controlled by a 

limited number of major genes (3 to 7) (Beavis et al. 1991; 

Edwards et al. 1987, 1992; Grattapaglia et al. 1995). This is 

also true for Populus hybrids, where QTLs were mapped for 

stem growth, form, and spring leaf flush (Bradshaw and 

Stettler 1995). Phenotypic data were collected over a 2-year 

period from a nursery clonal trial in Puyallup (Washington) 

containing ramets of the parental trees (P. trichocarpa and P. 

deltoides), F1 progeny, and F2 progeny. For each trait mea109 
Section Ill Molecular Biology 

sured, 1 to 5 QTLs were responsible for a large proportion of 

the genetic variance. For example, after 2-years growth, 44.7 

percent of the genetic variance in stem volume was controlled 

by just 2 QTLs (LOD = 3.86). For spring leaf phenology, 5 

QTLs were identified that explained 84."7 percent of the genetic variance (multilocus LOD = 16.63). This QTL mapping 

information provides the genetic basis for understanding the 

processes governing heterosis in hybrid poplars. 

The major positive QTL allele for 2-year height growth 

was dominant and was derived from the P. trichocarpa parent. This result agrees with the observation that hybrid F1 

progeny trees from a P. trichocarpa by P. deltoides cross are 

generally as tall as the P. trichocarpa parent. Additionally, 

QTLs controlling stem basal area growth, and those controlling sylleptic branch and leaf area traits were clustered (i.e., 

sharing a similar chromosomal position) suggesting a pleiotropic effect of QTLs ultimately responsible for stem diameter growth. This result corroborates previous statistical 

correlations among these traits (Hinckley et al. 1989). The 

QTL mapping effort at the UW I WSU was strengthened by 

growing this pedigree and another F 2 family, having the same 

P. trichocarpa maternal grandparent, in 2 other contrasting 

environments. The Boardman site (Oregon) is drier, more 

continental, and irrigated, while the Clatskanie site (Oregon) 

is wetter, coastal, and not irrigated (Stettler et al. 1994). The 

aim is to analyze QTL stability across genetic backgrounds 

and environments, and to measure other traits such as wood 

quality. After 1-years growth, the data indicate that the magnitude of the QTL effect for stem basal area was different for 

each family and environment (Stettler et al. 1994). However, 

some QTLs are conserved across different environments 

because their mapped chromosomal locations coincided. 

Stettler et al. (1994) identified similar positions for QTLs controlling stem volume, stem height, and height/ diameter that 

were mapped in 2 families with a different male grandparent P. deltoides. For some QTLs, different alleles control the 

trait in different environments, thus a strong genotype by 

environment (G x E) interaction is evident. 

Conclusion 

In recent decades, rapid technological evolution in the 

molecular marker field has allowed development of diverse methodologies for the detection of genetic variability. Choosing the best-suited marker technique should be 

based on the advantages and limitations of each molecular marker methodology, and other factors such as breeder 

friendliness, cost, time-efficiency, and automation ease. 

The introductiop of molecular markers in poplar breeding is providing vast information about genome structure, 

organization, evolution, and diversity. For breeding programs, the development of high-density linkage maps will 

110 

increase the efficiency of QTL mapping and consistently 

facilitate the introgression of more complex traits. In addition, these high-density linkage maps will facilitate positional cloning strategies (Tanksley et al. 1995). 

Using molecular markers for gene introgression programs 

(backcross breeding) is one example of Marker-Assisted Selection (MAS) (Hillel et al. 1990; Hospital et al 1992). Trait 

introgression is appropriate for monogenic or oligogenic 

traits (Dudley 1993). In this way, breeding for resistance controlled by a small number of loci could be improved and 

accelerated through MAS in forest trees (Nance et al. 1992), 

fruit trees, and other plants (Michelmore 1995). 

For the future, a primary challenge is to demonstrate 

that genetic markers can complement long-term breeding 

programs focused on improving complex polygenic traits. 

Determining the full potential of MAS depends on answering: Which QTLs are stable across different developmental stages, genetic backgrounds, and environments? 

Recent results on Populus (Bradshaw et al. 1995) and Eucalyptus (Grattapaglia 1996) are encouraging. In both cases, 

most of the markers transfer well to other pedigrees, and 

linkage relationships and map distances are conserved. Furthermore, QTLs with higher LOD scores and larger effects 

are more stable across environments (Bradshaw et al. 1995; 

Grattapaglia 1996), and some QTLs are conserved across 

genetic backgrounds (Bradshaw et al. 1995). Although several aspects need intensive investigation, these results provide interesting prospects for marker-assisted breeding. 

Acknowledgments 

The authors would like to thank Carlos A. Malpica for 

critical comments on the manuscript, and Martine De Cock 

for help with manuscript preparation. This work was supported by a grant from the Flemish Government (BNO I BB I 

6/1994) and the Commission of the European CommunitiesAIR program (AIR1-CT92-0349), called Inter-disciplinary 

Research for Poplar Improvement (IRPI). M.T.C. is funded 

by an individual fellowship from the Human Capital Mobility program of the European Union (41AS8694). 

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Epplen, J.T. 1989. Polymorphic simple GATA/GACA repeats in plant genomes. Nucleic Acids Research. 17: 10128. 

Welsh, J.; McClelland, M. 1990. Fingerprinting genomes 

using PCR with arbitrary primers. Nucleic Acids Research. 18: 7213-7218. 

Wilcox, P.L.; O'Malley, D.M.; Amerson, H.V.A.; Kuhlman, 

E.G.; Sederoff, R.R. 1995. Application of genomic mapping technology confirms resistance in loblolly pine to 

fusiform rust disease involves genes of large effect. In: 

Proceedings of the plant genome meeting III; 1995 January 15-19; San Diego, CA, U.S.A. Abstract. 

Williams, J.G.K.; Hanafey, M.K.; Rafalski, J.A.; Tingey, S.V. 

1993. Genetic analysis using RAPD markers. Methods 

in Enzymology. 218: 704-740. 

Williams, J.G.K.; Kubelik, A.R.; Livak, K.J.; Rafalski, J.A.; 

Tingey, S.V. 1990. DNA polymorphisms amplified by 

arbitrary primers are useful as genetic markers. Nucleic 

Acids Research. 18: 6531-6535. 

Willing, R.R.; Pryor, L.D. 1976. Interspecific hybridisation 

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Zabeau, M.; Vos, P. 1993. Selective restriction fragment 

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Publication No. 0 534 858 AI. 

115 
Chapter 15 

Differentiation of Poplar Clones Using Random 

Amplified Polymorphic DNA Fingerprinting1 

Dolly .c. Lin, Martin Hubbes, and Louis Zsuffa 

Introduction 

For over a decade, interest in poplars for short rotation 

intensive culture plantations for energy, chemicals, and fiber has been developing (Zsuffa et al. 1984). Due to easy 

clonal propagation, species and hybrid materials were inadequately defined. Conclusive identification of Populus 

species and hybrids is urgently required to: 1) further genetic improvement and characterization; 2) determine the 

linkage of important economic traits such as fast growth, 

superior wood quality, and ; and 3) enable advanced genetic engineering and breeding. 

Recent progress in molecular biology has generated new 

analytical tools that are well suited for taxonomic and genetic investigations. Several approaches have been studied 

in poplar such as isozyme markers (Rajora 1988, 1989a, 1989b; 

Rajora and Zsuffa 1989, 1990, 1991; Rajora et al. 1991), restriction fragment length polymorphism (RFLP) markers 

(Bradshaw et al. 1994; Soltes-Rak 1993), STS (sequence tagged 

site)/RFLP (Bradshaw et al. 1994), ribosomal DNA (rONA) 

polymorphisms (D'Ovidio 1992; D'Ovidio et al. 1990, 1991; 

Faivre-Rampant et al. 1992a, 1992b), mitochondrial DNA 

(mtDNA) (Barrett et al. 1993), chloroplast DNA (cpDNA) 

(Rajora and Dancik 1995a, 1995b, 199Sc; Smith and Sytsma 

1990), and random amplified polymorphic DNA (RAPD) 

markers (Castiglione et al. 1993; Lin et al. 1994a, 1994b). 

Although isozymes have served as genetic markers for 

fingerprinting clones, the small number of available alleles 

and loci made it impractical to identify all the clones under 

investigation (Bournival and Karban 1987). In recent years, 

RFLP of rONA, mtDNA, and cpDNA were used intensively 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

116 

for inter and intraspecies differentiation in Populus. However, these processes are labor intensive and require a relatively large amount of tissue. Other limitations of RFLP 

include low resolution for small size differences between relatively large DNA fragments (Gillet al. 1990) and ambivalent 

differences in the migration of DNA fragments across an agarose gel caused by band shifting (Landers 1989). 

Amplification of DNA sequences using polymerase chain 

reaction (PCR) (Saiki et al. 1988) is currently used with many 

organisms for studying populations and systematics, tagging major genes, and constructing genetic maps (Innis et 

al. 1990). Advantages of PCR-based DNA markers over the 

RFLPs are their rapidity, simplicity, and requirement for only 

small amounts of DNA (Castiglione et al. 1993). 

RAPD banding patterns were sensitive to slight changes 

in reaction conditions, which caused reproducibility problems (Ellsworth et al. 1993; Muralidharan and Wakeland 

1993; Penner et al. 1993). However, DNA fingerprints can 

be reproduced with standardized conditions such as consistent use of the same thermal cycler, cycling conditions, 

and concentrations of the reagents (i.e., template DNA, 

buffer, dNTPs, and DNA polymerase). DNA fingerprints 

by RAPD markers were chosen in the present study for 

species and hybrid differentiation and individual clonal 

characterization of Populus. 

Materials and Methods 

Plant Material 

Fifty-five poplar clones representing 8 species and hybrids (table 1) were analyzed. Clones were selected according to frequency of cultivation in Europe and North 

America (Castiglione et al. 1993), and for their potential 

use in plantations and biomass production (Steenackers 

eta1.1990;Zsuffa 1990). Twodormantcuttingsofeachclone 

were collected and rooted in the greenhouse at the Faculty of Forestry, University of Toronto. 
Differentiation of Poplar Clones Using Random Amplified Polymorphic DNA Fingerprinting 

Table l. List of poplar species and hybrids for clonal 

characterization. 

Species and hybrids 

P. x balsamifera 

P. xjackii 

P. deltoides 

P. trichocarpa x P. deltoides 

P. deltoides x P. trichocarpa 

P. x euramericana 

P. nigra 

P. nigra x P. maximowiczii 

P. maximowiczii 

Total 

DNA Extraction 

Number 

of clones 

3 

3 

5 

3 

2 

25 

6 

5 

3 

55 

Young leaves were collected from rooted cuttings. After washing with distilled water and blotting dry, leaf tissue (0.5 g) was ground to powder in liquid nitrogen, then 

transferred to 10 ml of preheated 2X CTAB isolation buffer 

(100 mM Tris-HCI, pH 8.0, 1.4 M NaCI, 20 mM EDTA, 2 

percent hexadecyltrimethylammonium bromide 'CTAB,' 

0.2 percent 2-mercaptoethanol). After thoroughly stirring, 

the mixture was incubated at 60 oc for 30 min, extracted 

twice with chloroform-isoamyl alcohol (24:1), and precipitated with 2/3 volume of cold isopropanol (Doyle and 

Doyle 1987). DNA concentration was estimated by comparing serial dilutions of DNA with those in agarose gels 

stained with ethidium bromide. 

Primers 

Sequences of 17 primers used for the PCR reaction are 

in table 2. 

Amplification of DNA by PCR 

The amplification reaction was performed in 15 JJI of 

buffer containing 50 mM Tris-HCl (pH 8.3), 5 mM 

MgCI 2

, 200 11M each of dATP, dCTP, dGTP and dTTP, 

0.5 units of Taq DNA polymerase (Boehringer 

Mannheim), 15 pmol primer and 10 ng of DNA. The 

amplification reaction was performed in the PerkinElmer DNA Thermal Cycler (Model TC-1). After aninitial denaturation at 94 oc for 2 min, the reaction program 

continued for 40 cycles consisting of 1 min denaturing 

at 94 oc, 1 min annealing at 40 oc, and 2 min extension 

at 72 °C. This program was followed by an extension 

phase at 72 oc for 10 min. DNA from all clones was subjected to 3 independent amplifications, and only fragments that were observed in all assays were scored for 

species and clonal characterization. 

Electrophoresis in Agarose Gel 

Amplification products (15 ill) were analyzed by electrophoresis in 1.5 percent agarose gel (15 x 15 em) at 100 V 

until the tracking dye (tartrazine) migrated toward the end 

Table 2. Sequences and sources of primers used in the RAPD analyses. 

Name Sequence References 

Chl-1 5'-GAGGCCTACGCCCCATAGAA-3' Castiglione et al. 1993 

Chl-2 5'-AATGCGTTGAGGCGCAGCAG-3' Castiglione et al. 1993 

Chl-4 5'-TTCCCGTGTCTTCCGGCTTAC-3' Castiglione et al. 1993 

Chl-10 5'-TTCTTCTCCTACCAGTATCG-3' Castiglione et al. 1993 

Deca-2 5'-GCGATCCGGC-3' Castiglione et al. 1993 

Deca-4 5'-CGTTGGCCCG-3' Castiglione et al. 1993 

Deca-5 5'-CCAAGGGGGC-3' Castiglione et al. 1993 

Deca-7 5'-CCGCCCGGAT-3' Castiglione et al. 1993 

Deca-9 5'-TGGCCCCGGT-3' Castiglione et al. 1993 

Deca-10 5'-AGCCGGCCTT-3' Castiglione et al. 1993 

Deca-11 5'-ATCGGCTGGG-3' Castiglione et al. 1993 

Deca-12 5'-CTTGCCCACG-3' Castiglione et al. 1993 

Deca-13 5'-GTGGCAAGCC-3' Castiglione et al. 1993 

2114 5'-GACTGCCTCT-3' Operon Technologies1 

2115 5'-GAAACGGGTC-3' Operon Technologies 

2116 5'-GTGACCGAGT-3' Operon Technologies 

2117 5'-CAGAAGCGGA-3' Operon Technologies 

1 1000-T Atlantic Ave. Suite 108, Alameda, CA 94501-1147, USA 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 117 
Section Ill Molecular Biology 

of the gel. Gels were stained in 0.5 11g/ml ethidium bromide solution and photographed in UV-light. 

4). The molecular sizes of RAPD markers used for species and 

hybrid differentiation are in table 4. Individual clones could be 

characterized by primer Chl-1 (figure 3) and/ or Deca-7 (figure 

4). DTAC clones from different parental origin (i.e., P. deltoides x 

P. trichocarpa and P. trichocarpa x P. deltoides) could be distinguished 

by primer Chl-1 (figure 3). Results and Discussion Recently, several specific PCR primers were developed for 

DNA fingerprinting. These primers were determined from 

known DNA sequences, which include the M13 universal 

primer (Chong et al. 1995), simple sequence repeats (SSRs) 

(Gupta et al. 1994), and microsatellite repeats (Morgante and 

Olivieri 1993). DNA fingerprinting with these specific primThe clone and origin of poplar materials subjected to RAPD 

analyses are listed in table 3. Of 17 primers tested, only 4 (DecatO, Deca-2, Chl-1, and Deca-7) (figures 1 to 4) were needed to 

distinguish all 55 poplar clones into species and hybrids (table 

Table 3. Poplar (Populus spp.) clones and their origin for RAPD analyses (note figures 1 - 4). 

Clone no. 

Upper Gel 

TAC8 

TAC21 

TAC51 

JAC4 

JAC7 

JAC28 

035 

039 

0196 

0391 

OTAC7 

DTAC8 

DTAC26 

DTAC9 

OTAC10 

ON5 

ON14 

DN16 

ON17 

Lower Gel 

DN93 

145/51 

1214 

1455 

DN21 

ON30 

ON173 

DN177 

N75 

N84 

N91 

N100 

NM1 

NM2 

NM8 

M900 

M901 

M908 

Species and hybrid 

P. balsamifera 

P. balsamifera 

P. balsamifera 

P.xjackii 

P.xjackii 

P.xjackii 

P. deltoides (W-1-2) Larsson 

P. deltoides 

P. x deltoides cv. 'Northwest' 

P. deltoides (Mixoploid) (Brockville #C136) 

P. trichocarpa x P. deltoides cv. 'Unal' 

P. trichocarpa x P. deltoides cv. 'Beaupre' 

P. trichocarpa x P. deltoides cv. 'Boelare' 

P. deltoides x P. trichocarpa cv. 'Donk' 

P. deltoides x P. trichocarpa cv. 'Barn' 

P. x euramericana cv. 'Gel rica' 

P. x euramericana cv. 'Hartt' 

P. x euramericana cv. 'Batarde d'Hauterive' 

P. x euramericana cv. 'Robusta' 

P. x euramericana cv. 'Triplo'; cl. '137/61' 

P. x euramericana 

P. x euramericana 

P. x euramericana 

P. x euramericana cl. '178B' (LW42) 

P. x euramericana cv. 'Canada Blanc' 

P. x euramericana (cv. 'Oorskamp') 

(Koster 925) 

P. x euramericana (cv. 'Spijk') (Koster 2195) 

P. nigra (#113) 

P. nigra var. italica (#555/50) 

P. nigra cv. 'Purkersdorf' (#44/62(1 0)) 

P. nigra cv. 'Kunovice' (312/65(002/66) 

P. nigra x P. maximowiczii 

P. nigra x P. maximowiczii (cl. 'Max-1 ') 

P. nigra x P. maximowiczii ( #62-7) 

P. maximowiczii 

P. maximowiczii 

P. maximowiczii 

Origin 

Ontario 

Ontario 

Manitoba 

Ontario 

Ontario 

Manitoba 

Ontario 

Ontario 

Saskatchewan 

Ontario 

Geraardsbergen, Belgium 

Geraardsbergen, Belgium 

Geraardsbergen, Belgium 

Wageningen, Holland 

Wageningen, Holland 

W. Germany 

W. Germany 

France 

France 

Casale Monferrato, Italy 

Casale Monferrato, Italy 

Casale Monferrato, Italy 

Casale Monferrato, Italy 

Italy 

Spain 

Holland 

Holland 

Hungary 

W. Germany 

Austria 

Czechoslavakia 

Japan 

W. Germany 

Korea 

118 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Differentiation of Poplar Clones Using Random Amplified Polymorphic DNA Fingerprinting 

T J 0 TO DT _ 0=-=--=N- M 

u 1 2 3 4 56 7 8 910111213141516171819 

M EU ON N NM Ma 

L 1 2 3 4 5 6 7 8 9 101112131415161718 

ers was also performed in our study (Lin unpublished data) to generate 

highly reproducible Dt A-banding patterns. However, our results from 

these experiments (1993 to 1995) indica te that either random or specific 

primers can be used for Dt A fingerprinting without major problems. 

The application of RFLP of rONA, mtDNA, and cpDNA in differentiation of Populus is limited to the inte r and intraspecies level. Compared to RAPD markers, the recently developed technique of amplified 

restriction fragment polymorphism (AFLP) (Vas et a!. 1995) may provide an advantage in reliability for DNA fingerprinting. In practice, 

however, DNA fingerprinting by RAPD markers remains an easier and 

less expensive technique for individual clonal characteriza tion. 

Acknowledgments 

This study was supported by the Genetic Improvement Activity, Task 

Vill, Bioenergy Agreement, International Energy Agency, and the Natura l 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Figure 1 . DNA polymorphism of poplar clones 

amplified with primer Deca-1 0. 

T = P. balsamifera 

ON = P. dettoides x P. nigra 

J P. xjackii 

EU P. x euramericana 

0 = P. deltoides 

N = P. nigra 

OT = P. de/toides x P. trichocarpa 

NM = P. nigra x P. maximowiczii 

TO P. trichocarpa x P. deltoides 

Ma P. maximowiczii 

M Molecular Markers VI from Boehringer Mannheim 

Corporation 

~ species differences 

Table 4. Identification of poplar species and 

hybrids by primers and molecular marker. 

Primers Marker size Species and hybrids' 

Deca-10 922 J, D, TD, DT, DN, EU, 

N, NM, M 

807 

600 

583 

M 

ON, EU, N, NM 

J, D, TD, DT 

Deca-2 11 02 M 

953 

884 

T 

Chl-1 1710 

DN, EU, N, NM 

N, NM, M 

1035 

77 

TD 

DT 

Deca-7 1649, 1474 

272 

DN,EU 

NM,M 

T 

ON 

J 

EU 

D 

N 

DT 

NM = TO 

M = 

P. balsamifera 

P. deltoides x P. nigra 

P. xjackii 

P. x euramericana 

P. deltoides 

P. nigra 

P. deltoides x P. trichocarpa 

P. nigra x P. maximowiczii 

P. trichocarpa x P. deltoides 

P. maximowiczii 

11 9 
Section Ill Molecular Biology 

T J D TO --=D::....:T'--- --=D::...::..N-=-- M 

u 1 2 3 4 5 6 7 8 9 10 1112 13 14 151617 18 19 

Figure 2. DNA polymorphism of poplar 

clones amplified with primer 

Deca-2. 

T = P. balsamifera 

ON = P. deltoides x P. nigra 

J P. xjackii 

EU = P. x euramericana 

0 = P. defloides 

N = P. nigra 

DT = P. deltoides x P. trichocarpa 

NM = P. nigra x P. maximowiczii 

TO = P. trichocarpa x P. defloides 

Ma = P. maximowiczii 

M Molecular Markers VI from Boehringer Mannheim 

Corporation 

-+ = species differences 

M 

L 

Sciences and Engineering Research Council of Canada 

(1 SERC) operating grant to L. Zsuffa. We gratefully acknowledge the technical support of Mr. K. Vitols in the PCR work. 

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T J D TD DT ----=D::.....:.. N-=----- M 

u 1 2 3 4 5 6 7 8 9 10 1.112 1314 1516 17 18 19 

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J P. x jackii 

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Section Ill Molecular Biology 

T J D TD DT ---=0::...=.-N~ M 

u 1 2 3 4 5 6 7 8 9 10 1112 1314 15 16 17 18 19 

Figure 4. DNA polymorphism of poplar clones 

amplified with primer Deca-7. 

T = P. balsamifera 

ON = P. deltoides x P. nigra 

J P. xjackii 

EU = P. x euramericana 

0 = P. delloides 

N = P. nigra 

OT = P. del/oides x P. trichocarpa 

NM = P. nigra x P. maximowiczii 

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123 
Chapter 16 

Seasonal Vegetative Storage Proteins of Poplar1 

Gary D. Coleman 

Introduction 

The availability of nitrogen often limits forest tree 

growth. Many tree species have evolved physiological 

strategies to reduce the impact of limited nitrogen availability. One strategy is to resorb nitrogen from leaves during autumn, and store it in perennial tissues during 

overwintering. During spring, this stored nitrogen is remobilized and used to support spring growth (Ryan and 

Bormann 1982; Taylor and May 1967). Besides reducing 

nitrogen loss from leaf fall, nitrogen resorption and storage may also reduce dependence on soil supplies during 

periods of high demand such as spring growth (Ryan and 

Bormann 1982). This review focuses on the molecular 

physiology of seasonal vegetative storage proteins in poplar. Advances in the biochemistry, physiology, and molecular biology of poplar vegetative storage proteins are 

reviewed. The general topic of woody plant vegetative storage proteins was reviewed in 1994 by Stepien et al. Poplar 

seed storage proteins are reviewed in this volume by 

Beardmore et al. 

Seasonal Nitrogen Cycling 

Nitrogen cycling in forest ecosystems is a complex process that involves nitrogen deposition, leaching, plant and 

soil uptake and return, and internal plant redistribution 

(Attiwill and Adams 1993). Internal nitrogen redistribution occurs when: 1) nitrogen is redistributed from source 

to sink tissue during growth, according to sink demands 

(Dickson et al. 1985; Vogelman et al. 1985); and 2) nitrogen 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

124 

is resorbed from senescing leaves, overwinter storage in 

perennial tissues, and spring mobilization to new growth. 

Fall nitrogen resorption is a complex physiological process that is not well understood in woody perennials. Generally from 40 to 80 percent of leaf nitrogen is resorbed 

from fall senescing leaves before abscission. Leaf nitrogen 

resorption is associated with leaf protein hydrolysis and 

retranslocation of amino acids (Boerner 1984; Chapin and 

Kedrowski 1983; Luxmoore et al.1981; Ostman and Weaver 

1982). Protein hydrolysis and amino acid transport can 

account for 80 to 90 percent of leaf nitrogen removal 

(Chapin and Kedrowski 1983). Although factors that control fall leaf senescence and abscission are poorly understood in trees, it is know that nitrogen resorption can vary 

among species (Boerner 1984) and according to leaf position within a tree (Staaf and Sljemquist 1986). Nitrogen 

recycling is rapidly re-established during forest development (Ryan and Bormann 1982). 

Seasonal nitrogen recycling has been extensively studied in tree fruit crops and is reviewed by Titus and Kang 

(1982). Considerable information is available for apple 

(Malus), which illustrates some general concepts of seasonal nitrogen recycling in deciduous trees. In apple, fall 

leaf senescence is accompanied by a decline in leaf nitrogen levels. This is associated with the translocation of leaf 

nitrogen to overwintering perennial storage sites, in particular the shoot bark (O'Kennedy et al. 1975; Oland 1963). 

During leaf nitrogen translocation, leaf protein levels decline by almost SO percent while bark protein levels increase by over 200 percent (Kang and Titus 1980). Most of 

the decline in leaf protein is due to the preferential loss of 

ribulose 1,5-bisphosphate carboxylase/ oxygenase 

(RUBISCO) (Kang and Titus 1980; O'Kennedy and Titus 

1979). About 90 percent of the nitrogen in overwintering 

bark is in protein (Kang and Titus 1980; O'Kennedy and 

Titus 1979). 

Studies indicate that spring growth is correlated with 

the level of stored nitrogen (Taylor and May 1967). Recent 

studies using 15N have more precisely determined the importance of stored nitrogen to growth. Sanchez et al. (1991) 

showed that in pear trees, 45 percent of stored nitrogen 

was partitioned into new spring growth, and sto~ed nitrogen accounted for 48 percent of the nitrogen in new growth. 
'V4i!JI 

Similar results have also been reported for walnut trees, 

where stored nitrogen accounted for 75 percent of the nitrogen in xylem sap during spring flowering and spur leaf 

development (Deng et al. 1989). Partitioning of stored nitrogen to new growth also seems independent of soil nitrogen supplies (Millard 1994; Millard and Neilsen 1989; 

Millard and Proe 1991; Millard and Thomson 1989). 

Although nitrogen cycling and storage in Populus are 

not as well studied as apple, some information is available. In an aspen-mixed-hardwood forest, it was estimated 

that about 40 to 60 percent of foliar nitrogen was resorbed 

during fall leaf senescence (Pastor and Bockheim 1984). 

Killingbeck et al. (1990) estimated that in aspen over 50 

percent of foliar nitrogen was resorbed and stored in per~nnial tissues during fall, and that the rate of resorption 

was dependent on leaf senescence since resorption rates 

were reduced during premature abscission. Total leaf nitrogen content declined nearly 50 percent during fall leaf 

senescence in P. deltoides and coincided with a reduction 

in salt-extractable proteins and an increase in free amino 

acid concentration (Cote and Dawson 1986; Cote et al. 

1989). Bark nitrogen levels increased by as much as 97 

percent during this period and correlated with increased 

salt-extractable proteins (Cote and Dawson 1986; Cote et 

al. 1989). Hollwarth (1976) showed that 95 percent of the 

nitrogen in the overwintering bark of P. balsamifera was 

contained in the protein fraction. Bark protein levels declined during spring growth while levels of soluble nitrogen compounds, including amino acids, increased 

(Hollwarth 1976). Similar patterns of seasonal nitrogen 

partitioning have been reported for poplar hybrids (P x 

euramericana cv. 'Eugenei' and P. tristis x P. balsamifera cv. 

'Tristis #1') where it was shown that about 75 percent of 

tree nitrogen is in the foliage during growth, but 80 percent of the nitrogen is retained in the tree after leaf abscission (Pregitzer et al. 1990). 

Seasonal nitrogen cycling occurs in a wide-range of temperate deciduous species and is an important component 

of tree nitrogen budgets.· Seasonal nitrogen storage involves fall resorption of leaf nitrogen before abscission 

providing a mechanism for conserving plant nitrogen. 

Although nitrogen can be stored in different chemical compounds, the predominate form is protein. 

Poplar Vegetative Storage Proteins 

Definition 

Vegetative storage proteins (VSPs) are a heterogeneous 

class of proteins, that when accumulated, temporarily store 

carbon and nitrogen during periods of excess availability, or 

by resorption of nutrients that might otherwise be lost durUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Seasonal Vegetative Storage Proteins of Poplar 

ing tissue senescence. Stored carbon and nitrogen are available for later use by the plant (see Staswick 1994 for review). 

O'Kennedy and Titus {1979) proposed that in apple shoot 

bark a VSP should accumulate to high levels in dormant 

shoots and decline to low levels or disappear when growth 

resumes. Seasonal VSPs have been identified in several 

angiosperm and gymnosperm species (table 1). In poplar, 

specific VSPs have been identified that, because of their 

predominance in the bark, are referred to as bark storage 

proteins (BSP). 

Accumulation and Localization 

A major 32-kDa (kilodalton) BSP accumulates in bark 

phloem parenchyma and xylem ray cells during the fall 

and disappears during spring regrowth (Sauter et al. 1988; 

Wetzel et al. 1989a). During BSP accumulation, the large 

central vacuoles of bark phloem parenchyma and xylem 

ray cells are replaced by small spherical protein storage 

vacuoles that range from 0.5 to 0.8 11m in diameter (Sauter 

et al. 1988; Sauter et al. 1989; Sauter and van Cleve 1990; 

van Cleve et al. 1988; Wetzel et al. 1989b ). Sauter and van 

Cleve (1990) demonstrated that morphological changes in 

these protein storage vacuoles were correlated with 

changes in BSP content. Immunochemicallocalization has 

demonstrated that these protein storage vacuoles are the 

sites for 32-kDa BSP accumulation (Sauter and van Cleve 

1990; van Cleve et al. 1988). 

BSP accumulation is not uniform throughout the tree, is 

greatest in the bark of 1-year-old shoots and wood, declines basipetally in bark and wood, and then increases in 

roots (Sauter et al. 1989). Therefore, BSP storage is greatest 

in tissues adjacent to sites of subsequent spring growth. 

Storage of nutrients near growing points would reduce 

the transport distance for these reserves and may improve 

nutrient reuse efficiency. 

Biochemical Characterization 

In addition to the 32-kDa BSP (Coleman et al. 1991; 

van Cleve et al. 1988; Wetzel et al. 1989a), a 36-kDa 

(Langheinrich and Tischner 1991; Stepien and Martin 1992) 

and a 38-kDa protein (Stepien and Martin 1992) were reported to accumulate in poplar bark. All of these different 

molecular weight BSPs are glycosylated (Langheinrich and 

Tischner 1991; Stepien and Martin 1992). Stepien and Martin (1992) showed that the neutral sugar content of BSP 

ranged from 10 to 20 percent; galactose-(1-3)-N-acetylgalactosamine and galactose-(1-4)-N-acetylglucosamine 

were present, but terminally linked mannose and sialic acid 

were absent. Results from deglycosylation analysis 

(Langheinrich and Tischner 1991) and peptide mapping 

(Stepien and Martin 1992) indicate that these different BSPs 

represent glycolytic isoforms of the "core" 32-kDa protein. 

Native BSP appears to be a heterodimer composed of 2 

125 
Section Ill Molecular Biology 

Table 1. Occurrence of bark storage proteins in trees. 

? 

Species Tissue 

Angiosperms 

Acer saccharum bark 

Betula papyrifera bark 

Populus deltoides bark 

bark 

Populus x euramericana wood 

bark 

Prunus persica bark 

wood 

Robinia pseudoacacis bark 

Salix x smithiana bark 

Sambucus nigra bark 

Sophora japonica bark 

Gymnosperms 

Larix decidua bark 

Metasequoia wood 

glyptostroboides 

Pinus strobus bark 

Pinus sylvestris bark 

Tax odium distichum wood 

isoforms with an estimated molecular weight of 58 kDa 

(Langheinrich and Tischner 1991). Immunological and 

peptide mapping studies indicate that the 32-kDa protein 

in bark and wood is likely the same protein (Stepien et al. 

1992). Although BSPs are known to be glycosylated, it is 

unknown if they are modified in any other manner. Unlike soybean VSPs, which also have phosphatase activity 

(DeWald et al. 1992}, it is unknown if poplar BSP has any 

enzyme activity. 

Molecular Cloning 

Complementary DNA (eDNA) clones were isolated and 

sequenced for the 32-kDa BSP (Clausen and Apel 1991; 

Coleman et al. 1992). Comparison of the predicted amino 

acid sequence with the NH2-terminal amino acid sequence 

from the purified 32-kDa protein suggests that the 32-kDa 

BSP is synthesized as a preprotein with NH2-terminal signaling sequences for endoplasmic reticulum transport and 

vacuolar targeting (Coleman et al. 1992). Analysis of the 

predicted amino acid sequence indicates that the mature 

processed protein consists of 288 residues with a calculated molecular weight of 31,502 (Coleman et al. 1992). A 

potential glycosylation site (ASN-XXX-SER) is present in 

the mature peptide and is probably involved in the generation of glycolytic isoforms (Coleman et al. 1992). Based 

upon the predicted amino acid sequence, the protein is 

rich in the essential amino acids Leu, Lys, and Phe (Clausen 

and Apel 1991; Coleman et al. 1992). 

126 

Molecular mass (kDa) Reference 

16,24 Wetzel et al. 1989a 

24 Wetzel and Greenwood 1991 

32 Wetzel et al. 1989a 

32 Coleman et al. 1991 

32 Sauter et al. 1988 

32 Stepien and Martin 1992 

16, 19 Arora et al. 1992 

19 Arora et al. 1992 

29,31 Nsimba-Lubaki and 

Peumans 1986 

32 Wetzel et al. 1989b 

34.5, 37.5 Greenwood et al. 1986 

30-35 Hankins et al. 1987 

25,27, 32 Wetzel and Greenwood 1989 

32,34 Harms and Sauter 1991 

15 

15 

35 

Wetzel and Greenwood 1989 

Wetzel and Greenwood 1989 

Harms and Sauter 1991 

Poplar BSP is encoded by a small multigene family 

(Coleman et al. 1992) consisting of approximately 2 to 3 

genes (Coleman unpublished results). The BSP multigene 

family segregates as a single locus and is likely to be clustered (Davis et al. 1993). In addition, the BSP gene family 

is linked to a related gene family of wound-inducible 

cDNAs (WIN4) from poplar leaves (Davis et al. 1993). A 

number of bsp genomic clones were isolated (Coleman 

unpublished results) and 1 member, bspa, was sequenced 

(Coleman and Chen 1993 ). 

Bspa is contained within a 3.9-kb EcoRV I Afllll fragment 

that includes a 1,646-bp (base pair) coding region, 1,246 

bp upstream of the coding region, and 1,022 bp downstream of the coding region. The open reading frame is 

interrupted by 4 introns of 152, 262, 175, and 144 nucleotides in length. The transcriptional start site is located 71 

nucleotides upstream of the translation start site (Coleman 

and Chen 1993). A number of potential regulatory sequence 

motifs are located in the upstream region of bspa including a putative TATA box that is located 25 nucleotides upstream of the transcriptional start site. Approximately 50 

bases upstream of the TATA is a potential basic leucine 

zipper protein binding site (b-zip) that consists of the Gbox core sequence ACGT (Armstrong et al. 1992; Oeda et 

al. 1991). Further upstream, approximately 300 nucleotides 

from the G-box core sequence, is a palindromic TGAC 

motif of 2 identical half-sites forb-zip binding that are identical to the cAMP response element CRE (Katagiri et al. 

1989). Between these 2 sites are located several A/T rich 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
regions that may have a role in bsp transcription, since similar sequences had a role in regulating plant gene transcription (Bustos et al. 1989; Datta and Cashmore 1989). The 

significance of these sequences in regulating bspa gene 

expression is unknown, but current research suggests that 

transcriptional and post-transcriptional regulation are 

important in bspa gene regulation (Coleman unpublished 

results). 

BSP Gene Expression 

Several environmental factors including photoperiod 

(Coleman et al. 1991; Coleman et al. 1992; Langheinrich 

and Tischner 1991), nitrogen availability (Baftados 1992; 

Coleman et al. 1994; van Cleve and Apel1993), temperature (van Cleve and Apel1993), and wounding (Davis et 

al. 1993; Stepien and Sauter 1994) induce bsp expression. 

BSP accumulation is also tissue-specific, occurring primarily in stem bark parenchyma and xylem ray cells. However, regulatory factors governing tissue-specific 

expression remain undefined (Coleman et al. 1994; Davis 

et al. 1993). 

Overall, the relationship between the above mentioned 

environmental factors that induce bsp expression is unknown. The exception is photoperiod and nitrogen availability, which interact during bsp induction and BSP 

accumulation. Although photoperiod is an important signal for bsp expression, nitrogen availability can interact 

with photoperiod to modulate gene expression and protein accumulation (Coleman et al. 1994). For example, reduced nitrogen availability delays photoperiod induced 

increases in steady state levels of bsp messenger RNA 

(mRNA). However, this delay is transitory since continued short-day exposure leads to similar levels of bsp mRNA 

in both nitrogen deficient plants and plants with optimal 

levels of nitrogen (Coleman et al. 1994). Furthermore, although both nitrogen deficient plants and those with optimallevels of nitrogen eventually have similar levels of bsp 

mRNA under short-day conditions, nitrogen deficient 

plants have reduced levels of BSP (Coleman et al. 1994). In 

severely defoliated plants, which have reduced levels of 

foliar nitrogen available for resorption and mobilization, 

short-day exposure still results in increased bsp mRNA levels (Coleman unpublished results). These results suggest 

that nitrogen availability and photoperiod both influence 

bsp expression, but this may be achieved by different 

mechanisms. Under short-day conditions, the major influence of nitrogen availability is on protein accumulation, 

while photoperiod is more closely associated with increases 

in mRNA levels. This might be explained if short-day photoperiod acts at the level of gene transcription and I or 

mRNA stability, while nitrogen availability influences 

some form of translational or post-translational control. 

Under certain conditions, however, nitrogen availability 

must also influence other levels of bsp gene regulation beUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Seasonal Vegetative Storage Proteins of Poplar 

cause both bsp mRNA and BSP accumulate to high levels 

in long-day plants treated with high levels of nitrogen 

(Coleman et al.1994). Clearly, bsp gene regulation involves 

complex interactions between photoperiod and nitrogen 

availability. 

VSP gene expression in soybean involves an interaction 

between nitrogen sinks and sources (Staswick 1994). Carbon source-sinks change during short-day induction of 

dormancy in poplar (Dickson and Nelson 1982). Under 

long-day conditions, poplars translocate carbon from mature source-leaves to young sink-leaves. During short-day 

conditions, carbon translocation changes from young sinkleaves to stem and root tissues (Dickson and Nelson 1982). 

Besides changes in source-sinks, short-day dormancy induction also leads to reduced carbon translocation from 

mature source-leaves (Dickson and Nelson 1982). Shortday photoperiod significantly alters source-sink relations 

in poplar, which probably are important in bsp induction. 

Environmental regulation likely involves alteration of nitrogen source-sink relations associated with bsp expression. 

One mechanism could involve short-day alteration of nitrogen source-sinks and coincident induction of leaf senescence. 

Because of altered source-sink relations, available nitrogen 

from leaf senescence would be mobilized from the leaves to 

the stems, instead of from leaf to leaf. This increased level of 

stem nitrogen would then result in bsp induction. Although 

this mechanism provides a system for regulating bsp expression, it does not account for bsp induction in nitrogen, deficient or severely defoliated plants. An alternative mechanism 

might involve induction of both bsp expression and leaf senescence by photoperiod. Short-day induction of bsp would 

provide a sink for nitrogen mobilization from leaves. Sink 

relations would be altered through reduced leaf sinks, while 

BSP synthesis and accumulation would increase stem sink 

strength. This increase in stem sink strength would be accompanied by increased nitrogen availability (nitrogen 

source) from photoperiod induced leaf senescence. Thus, 

short-days would induce both bsp expression and leaf senescence, thereby leading to source-sink changes. This might 

be reflected by the differences that nitrogen availability has 

on short-day accumulation of bsp mRNA and BSP (Coleman 

et al. 1994). 

The molecular mechanisms controlling bsp expression 

are not well characterized. Results from nuclear run-off 

transcription assays suggest that transcriptional and posttranscriptional processes contribute to short-day and nitrogen induced bsp expression (Coleman unpublished 

results). Because BSP is encoded by a small multigene family, it is also possible that different environmental factors 

(i.e., photoperiod, nitrogen, or wounding) may result in 

differential gene expression. However, experiments involving 51 nuclease protection assays have failed to detect any 

evidence for differential gene expression during both shortday photoperiod and nitrogen induced gene expression 

(Coleman unpublished results). 

127 
Section Ill Molecular Biology 

Clonal Variation in BSP Accumulation 

Pauley and Perry (1954) showed that stem growth cessation in P. deltoides and P. trichocarpa varied by ecotype, and 

that this variation was inversely correlated with the latitude 

of origin. Similar to growth cessation, maximum bsp mRNA 

levels of different ecotypes of P. deltoides vary inversely with 

the latitude of origin when grown in natural conditions. The 

latitude of origin accounted for nearly 70 percent of the variation in the date of bsp mRNA accumulation in different 

ecotypes (Coleman unpublished results) 

Besides variation in gene induction, the bark protein content (mg/ g fresh weight) of different poplar clones also varies among clones (Coleman et al. 1991; Langheinrich 1993). 

It seems probable that different poplar clones vary in their 

ability to resorb and store nitrogen from senescing leaves. 

The significance of this variation and its influence on resource 

use are undetermined. If BSP accumulation level is a component of overall nutrient-use efficiency, then its manipulation could be an important component in production. 

BSP Degradation and Nitrogen Mobilization 

Since BSP stores reduced nitrogen during overwintering for later use, then the plant must have mechanisms for 

mobilizing the stored BSP nitrogen. For BSP to be used 

during spring shoot growth the protein must be degraded 

to either individual amino acids or small polypeptides, so 

that the nitrogen can be loaded into the xylem and transported to supply new growth. BSP degradation and nitrogen mobilization are controlled by the sink demand from 

newly elongating buds and shoots (Coleman et al. 1993). 

Environmental factors, such as day length and temperature, appear to influence BSP degradation indirectly by 

influencing new shoot growth and nitrogen sink demand 

(Coleman et al. 1993). Since BSP degradation and nitrogen 

mobilization are dependent on new shoot growth, newly 

growing shoots must somehow communicate with BSP 

storage sites and initiate protein degradation. The nature 

of this communication is unknown, but it may involve 

phytohormones, such as gibberellins (GAs) or cytokinins, 

since these are correlated with bud break (Domanski and 

Kozlowski 1968; Powel11987). 

Nitrogen mobilization from BSP probably involves protein hydrolysis similar to the hydrolysis that occurs during seed storage protein utilization (Miintz et al. 1985; 

Shutov and Vaintraub 1987). The nature of the proteases 

involved in BSP degradation are unknown; however, activity by an endoprotease may be involved since 2 polypeptides of about 12 and 14 kDa appear to be the initial 

hydrolysis products (Baiiados 1992). Furthermore, Sauter 

and van Cleve (1992) reported that glutamine is the predominant amino acid in the spring xylem sap of poplar, 

and that it can account for 75 percent of the amino acid 

sap content. Given this predominance of glutamine, BSP 

128 

degradation must involve protein degradation to the constituent amino acids that are then used for glutamine synthesis and nitrogen transport. 

Final Comments 

The ability of poplars to resorb nitrogen from senescing 

leaves and store it as protein during overwintering makes 

them an excellent model to investigate the physiology, 

genetics, and molecular biology of seasonal nitrogen cycling. The ability to manipulate the growth cycle, the ease 

.of vegetative propagation, and the ability to be genetically 

transformed adds to the value of poplars as a model for 

woody plant molecular physiology. 

Although advances have been made in understanding 

the physiology and molecular biology of seasonal nitrogen cycling in poplars, significant questions remain. An 

interdisciplinary research approach that combines physiology, genetics, and molecular biology is required to increase the understanding of seasonal nitrogen cycling. The 

generation of transgenic poplars for studying the regulation of gene expression and the role of BSP offers exciting 

opportunities. Such research will further our understanding of seasonal nitrogen cycling in poplars and advance 

our knowledge of the molecular physiology of woody 

plants. 

Acknowledgments 

This research was supported in part by the National 

Research Initiative Competitive Grants Program/U.S. 

Department of Agriculture. 

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for the spring growth ~f apple trees. J. Exp. Bot. 40: 1285-

1289. 

Miintz, K.; Bassiiner, R.; Lictenfeld, C.; Scholz, G.; Weber, 

E. 1985. Proteolytic cleavage of storage proteins during 

embryogenesis and germination of legume seeds. 

Physiol. Veg. 23: 75-94. 

Nsimba-Lubaki, M.; Peumans, W.J. 1986. Seasonal fluctuations of lectins in barks of elderberry (Sambucus nigra) 

and black locust (Robinia pseudoacacia). Plant Physiol. 

80: 747-751. 

O'Kennedy, B.T.; Hennerty, M.J.; Titus J.S. 1975. Changes 

in nitrogen reserves of apple shoots during the dormant 

season. J. Hortic. Sci. 50:321-329. 

O'Kennedy, B.T.; Titus, J.S. 1979. Isolation and mobilization of storage proteins from apples shoot bark. Physiol. 

Plant. 45: 419-424. 

Oeda, K.; Salinas, J.; Chua, N.-H. 1991. A tobacco b-zip transcription activator (TAF-1) binds to a G-box-like motif 

conserved in plant genes. EMBO J. 10: 1793-1802. 

Oland, K. 1963. Changes in the content of dry matter and 

major nutrient elements of apple foliage during senescence and abscission. Physiol. Plant. 16: 682-684. 

Ostman, N.L.; Weaver, G.T. 1982. AutUmnal nutrient transfers by retranslocation, leaching, and litter fall in a 

chestnut oak forest in southern Illinois. Can. J. For. Res. 

12:40-51. 

Pastor, J.; Bockheim, J.G. 1984 . .Distribution and cycling of 

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22: 845-850. 

Pregitzer, K.S.; Dickmann, D.J.; Hendrick, R.; Nguyen, P.U. 

1990. Whole-tree carbon and nitrogen partitioning in 

young hybrid poplars. Tree Physiology. 7: 79-93. 

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northern hardwood forests. BioScience. 32: 29-32. 

Sanchez, E.E.; Righetti, T.L.; Sugar, D.; Lombard, P.B. 1991. 

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Hort. Sci. 66: 479-486. 

Sauter, J.J.; van Cleve, B.; Apel, K. 1988. Protein bodies in 

ray cells of Populus x canandensis Moench 'robusta'. 

Planta. 173: 31-34. 

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130 

Planta. 183: 92-100. 

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acids in the xylem sap of Populus x canadensis and its 

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1557-1566. 

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303-322. 

Stepien, V.; Sauter, J.J.; Martin, F. 1992. Structural and immunological homologies between storage protein in the wood 

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Stepien, V.; Martin, F. 1992. Purification, characterization 

and localization of the bark storage proteins of poplar. 

Plant Physiol. Biochem. 30: 399-407. 

Stepien, V.; Sauter, J.J. 1994. Ringing induces the accumulation of vegetative storage proteins in poplar bark. 

Trees. 9: 88-92. 

Stepien, V.; Sauter, J.J.; Martin, F. 1994. Vegetative storage 

proteins in woody plants. Plant Physiol. Biochem. 32: 

185-192. 

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peach tree II. Storage and mobilization of nitrogen in 

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van Cleve, B.; Clausen, S.; Sauter, J.J. 1988. Immunochemicallocalization of a storage protein in poplar wood. J. 

Plant Physiol. 133: 371-374. 

van Cleve, B.; Apel, K. 1993. Induction by nitrogen and 

low temperature of storage-protein synthesis in poplar 

trees exposed to long days. Planta. 189: 157-160. 

Vogelmann, T.C.; Dickson, R.E.; Larson, P.R. 1985. Comparative distribution and metabolism of xylem-born 

amino compounds and sucrose in shoots of Populus 

deltoides. Plant Physiol. 77: 418-428. 

Wetzel, S.; Greenwood, J.S. 1989. Proteins as a potential 

nitrogen compound in bark and leaves of several softwoods. Trees. 3: 149-153. 

Wetzel, S.; Demmers, C.; Greenwood, J.S. 1989a. Seasonally fluctuating bark proteins are a potential form of 

nitrogen storage in three temperate hardwoods. Planta. 

178: ~75-281. 

Wetzel, S.; Demmers, C.; Greenwood, J.S. 1989b. Spherical 

organelles, analogous to seed protein bodies, fluctuate 

seasonally in parenchymatous cells of hardwoods. Can. 

J. Bot. 67:3439-3445. 

Wetzel, S.; Greenwood, J .5. 1991. A survey of seasonal bark 

proteins in eight temperate hardwoods. Trees. 5: 153-

157. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Chapter 17 

Poplar Seed Storage Proteins1 

'w """ Tannis L. Beardmore, Suzanne Wetzel, and Sharon M. Regan 

"-' 

Introduction 

Seed storage proteins have been actively studied since 

the mid 1700s (Beccari 1745) particularly in agricultural 

plant species. These studies can be attributed to the historic role of agricultural plants as a primary nutrition 

sourc~ for both humans and livestock. Recently, the study 

of a~cultural pl:u'ts has shifted toward genetically improvmg crops to mcrease stored protein content and enhance the nutritive value of the protein through 

manipulation of amino acid content (Blechl and Anderson 1996; Ceoloni et al. 1996; Hoffman et al. 1988; Kjemtrup 

et al.1994; Lawrence et al. 1994). At a basic scientific level, 

seed storage protein (SSP) synthesis provides a particularly interesting model for examining gene regulation since 

storage protein expression is restricted to specific developmental times and cell types. This research area has 

yielded important information regarding the regulation 

and control of developmental events in embryogenesis and 

protein targeting. Seed research has been neglected in 

woody plants until recently. Although seeds of woody 

species are not always important for consumption, the 

study of seed storage }'roteins is necessary to understand 

topics such as tree biology, species distribution, 

phylogenetics, and systematics. 

The following review consists of 3 sections. The first 

section is a general discussion of the current information 

regarding seed storage proteins. In the second section, 

woody plant work is discussed with a focus on the Populus 

seed storage proteins. The final section presents future 

prospects in storage protein research in Populus. 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

Seed Storage Proteins 

Importance of Storage Proteins 

Storage proteins provide a source of reduced nitrogen, 

carbon, and amino acids to growing tissues. When present 

in the seed, these proteins are called seed storage proteins 

(SSPs). When present in vegetative tissues, they are referred 

to as vegetative storage proteins (VSPs). Seed storage prote~ and ~SPs are 2 different forms of plant storage protem (Stasw1ck 1988, 1990). For a discussion of the Populus 

VSPs, see the chapter by Gary Coleman (this volume). Seed 

storage proteins accumulate in the latter stages of seed 

development and are mobilized during germination and 

seedling growth (Higgins 1984). Until seeds can conduct 

sufficient photosynthesis to become fully autotrophic, storage proteins in combination with other stored reserves (i.e., 

lipids and starch) provide an essential food source as the 

seedling initiates heterotrophic growth. 

Storage proteins were once thought to be metabolically 

inert and lacking in enzymatic activity (Larkins 1981). 

However, recently several storage proteins were found to 

function as storage compounds of nitrogen and carbon and 

as metabolically active enzymes. For example, 

phytohemaglutinins, ureases (Casey et al. 1986), ribulose 

bisphosphate carboxylase (Wittenbach et al. 1980), 

lipoxygenase (Tranbarger et al. 1991), lectins (Rudiger 1984; 

Spilatro et al. 1996), trypsin inhibitors, and Kunitz inhibitors are often classified as storage proteins. Many of these 

proteins function in stress responses, specifically in plant 

defense mechanisms. This could be advantageous for the 

germinating seed if a requirement develops for the in vivo 

enzymatic function. 

Characterization of Seed Storage Proteins 

Seed storage proteins are distinguished from other identified proteins based on 2 criteria they: 1) accumulate in 

the latter stage of seed development and are mobilized 

during germination and seedling growth; and 2) are 

present in relatively large quantities in mature seed 

131 
Section Ill Molecular Biology 

(Higgins 1984). These proteins can account for approximately 10 to 40 percent of the dry weight in mature seeds 

(Larkins 1981). Storage proteins are a complex of individual 

proteins that can be bound together by a combination of 

disulfide, hydrogen, ionic, and hydrophobic bonds. Storage proteins are primarily oligomeric, possessing multiple 

subunits within which are polypeptide chains that are usually disulfide bonded (reviewed in Larkins 1981). 

Seed storage proteins are a diverse group of proteins 

because they are encoded by multigene families and have 

different post-translational processing (Bevan et al. 1993). 

This polymorphism can exist both within and among genotypes of the same species (Gonzalez and Henriques-Gil 

1994; Hager et al. 1995; Horstmann et al. 1993). Despite 

this diversity, there are constraints on protein structure, 

probably related to their function, which result in similarities among the storage proteins. For example, the conservation of specific domains or amino acid motifs may be 

necessary for their processing, transport, and packaging 

during development and for their proteolysis during germination. Thus, selection pressure on amino acid sequence 

is perhaps less important than that on conservation of overall amino acid composition. 

Traditionally, SSPs were classified on the basis of their 

solubility (Osborne 1924). Based on sequential extraction 

of the proteins, this classification system still provides the 

primary framework for current SSPs studies. This system 

is composed of 4 storage protein classifications: 1) albumins (soluble in water and dilute buffers with a neutral 

pH); 2) globulins (soluble in sa~t solutions but insoluble in 

water); 3) glutelins (soluble in dilute acids or alkaline solutions); and 4) prolamins (soluble in aqueous alcohols). 

Recently storage proteins were classified on the basis of 

conserved elements in the promoter regions of storage 

proteins genes (Morton et al. 1995). Interestingly, classification based on conserved elements corresponds closely 

to classification of storage proteins based on the solubility 

system of Osborne's (1924). Other parameters often used 

in classifying SSPs include sedimentation coefficients of 

the proteins (in Svedburg units, S), amino acid composition, molecular weight, and subunit and polypeptide composition (Larkins 1981). Unfortunately, these diverse 

classification criteria have increased the complexity of determining phylogenetic relationships. 

Seed storage proteins were characterized in many species throughout the angiosperms and to a lesser extent in 

the gymnosperms. Although generalizations regarding 

SSPs are difficult, the major seed storage protein groups 

found in angiosperms are the globulins, prolamins, and 

albumins, with the globulin SSPs being the most widely 

distributed storage proteins (Derbyshire et al. 1976; Luthe 

1992). The 7S (vicilin type) and llS {legumin type) proteins are the major types of globulin SSPs based on the 

sedimentation coefficient of their aggregated forms 

(Derbyshire et al. 1976). Prolamin storage proteins are pri132 

marily restricted to monocotyledonous plants, while albumin storage proteins are found predominantly in dicotyledonous plants (Derbyshire et al. 1976). 

Synthesis, Deposition, and Mobilization of 

Seed Storage Proteins 

Synthesis of SSPs primarily occurs during the maturation stage of seed development (Larkins 1981). This stage 

in seed development is characterized by cell expansion and 

the deposition and accumulation of stored reserves (i.e., 

protein, lipid, and carbohydrate). Expression of SSPs is 

primarily restricted to seed tissue that is terminally differentiated. In angiosperms, sites of SSP synthesis and accumulation are in the embryo, primarily in the storage 

parenchyma cells of the cotyledons (Panitz et al. 1995; 

Weber et al. 1978) and in the persistent endosperm (Shewry 

et al. 1993). These sites of SSP expression and subsequent 

SSP accumulation provide nutrients for the germinating 

embryo. Other more transient sites of SSP expression, including the suspensor (Nagai et al. 1991; Newcomb 1973; 

Panitz et al. 1995) and liquid (nonpersistent) endosperm 

(Czako et al. 1991; Harris et al. 1989; Quatrano et al. 1986), 

can also occur during very early stages of seed development to sustain embryogenesis. 

Transcription and translation of storage protein messenger RNA(mRNA) occurs during the maturation stage. Storage protein gene expression is regulated mainly at the 

transcriptional level (reviewed by Bevan et al. 1993; Morton 

et al. 1995). Several classes of DNA sequence motifs and 

associated transacting factors are recognized as having a 

role in SSP gene regulation (reviewed by Morton et al. 

1995). Identifying regulatory sequences has been difficult, 

since these elements appear to reside far upstream from 

the SSP gene (e.g., greater than 1 kilobase). Also, the regulation of these genes appears to involve the interaction of 

multiple DNA elements. 

Translational regulation of SSP synthesis also can occur. 

For example, in alfalfa somatic embryogenesis SSP, mRNA 

is present in the early stages of embryo development, but 

restricted to the nontranslated ribonucleoprotein particles 

(RNP) pool (Pramanik et al. 1992). When these embryos 

reach the early maturation stage, the SSP mRNA are shifted 

to polysomes. Furthermore, differential transcript stability was suggested as an important regulatory component 

in SSP mRNA accumulation in soybean embryos (Walling 

et al. 1986). 

Storage proteins are synthesized with a signal polypeptide that is cleaved when the protein is translocated into 

the lumen of the endoplasmic .reticulum (ER) (Muntz 

1989). Some storage proteins also contain propeptides that 

must be cleaved to produce the physiologically active form 

(Ceriotti et al. 1991). Many storage proteins are synthesized as long polypeptides that are co- or post-translationally cleaved to generate 2 or more subunits (Croy et 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
al. 1980). Proteases responsible for these cleavages were 

identified in certain species (reviewed by D'Hondt et al. 

1993). 

Subsequent processing of these proteins is not well understood. Many processing events occur in the ER; disulfide bond formation, cleavage of the carboxy-terminal 

peptide, chaperone-mediated folding, isomerization, and 

N-glycoslyation (reviewed by Shewry et al. 1995). From 

the ER, most proteins are processed via the Golgi apparatus and are targeted to the vacuole or protein bodies (reviewed by Chrispeels 1991). 

Little is known about the regulatory factors involved in 

the cessation of SSP synthesis. Repression or down regulation of storage protein synthesis occurs toward the end 

of the maturation stage just before seed desiccation. Chern 

and coworkers (1996) recently identified a regulatory protein that is apparently involved in repressing SSP transcription in Phaseolus vulgaris. 

Overall, available information regarding storage protein hydrolysis is limited. It was suggested that multiple mechanisms are responsible for SSP hydrolysis, 

involving transcriptional and post-transcriptional regulation (Callis 1995). Briefly, the hydrolysis of the storage proteins into their constituent amino acids is 

induced by proteases (Fincher 1989). The amino acids 

are reused for protein synthesis or they are deaminated, 

providing carbon skeletons for respiratory oxidation 

(Callis 1995). Proteases involved in SSP hydrolysis are 

apparently synthesized de novo during germination and, 

to a lesser extent, are present in an inactive form in the 

dry seed, which is activated upon germination (Wilson 

1986). 

Table 1. Seed storage proteins in angiosperm woody species. 

Native 

protein Subunits 

Species Family (kDa)1 (kDa) 

Cercis siliquastrum Fabaceae 

Castanea sativa Fabaceae 260 to 240 62 to 56, 

48 to 41 

Castanea crenata Fabaceae 260 to 240 34 to 32 

Quercus ilex Fabaceae 62 to 50, 

46 to 40, 

35 to 32 

Quercus robur Fabaceae 58 to 50, 43, 

34 to 31 

Populus Salicaceae 120 to 100 60,58 

grandidentata 

1 kilodaltons 

2 not reported 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Poplar Seed Storage Proteins 

Seed Storage Proteins in 

Populus and Other Woody 

Plants 

Woody Plants 

Seed research in woody plants is problematic due to 

the time required for these plants to obtain reproductive 

maturity, their long reproduction cycle, and irregular flowering processes. Nonetheless, research on seed storage proteins of woody plants has originated from 2 areas. One 

area is the development of somatic embryogenesis procedures for tree species of commercial importance (i.e., primarily gymnosperms). In this area of study, SSPs are used 

as markers to determine the embryogenic potential (Roberts et al. 1989; Sotak et al. 1991) and competency of the 

embryogenic system to produce a somatic embryo that is 

biochemically similar to its zygotic counterpart (Flinn et 

al. 1991b; Hakman 1993; Hakman et al. 1990). The second 

area of study examines the phylogenetic origin and molecular evolution of woody species (Luthe 1992). 

To date, most information regarding characterization 

of SSPs in woody plant species is related to their accumulation patterns during embryo development (Flinn et al. 

1991b; Hakman 1993; Hakman et al. 1990). Such information is determined by electrophoretic and immunological 

analysis. Alternatively, DNA and amino acid sequence information have been more limited. Current knowledge of 

SSPs in woody species is in tables 1 and 2. Storage proPolypeptides Protein 

(kDa) family Reference 

168, 144, 104, globulin Gonzalez and 

100, 72, 68, 54, Henriques-Gil 1994 

52,36,34 

36 to 31, 23 to 22, globulin Collada et al. 1991 

20 to 19, 18 

globulin Collada et al. 1991 

40 to 30, 24 to 22, glutelin Collada et al. 1991 

22 to 21 

34 to 31, 23 to 22, glutelin Collada et al. 1991 

22 to 21, 17 

36,32,22, 18,14 albumin Beardmore et al. 1996 

133 
Section Ill Molecular Biology 

Table 2. Seed storage proteins in gymnosperms. 

Native 

protein Subunits 

Species Family (kDa)1 (kDa) 

Ginkgo biloba Ginkgoaceae 400 50, 31 

Macrozamia Zamiaceae 260 126 

communis 

Abies koreana Pinaceae 4 55 

Cedrus atlantica Pinaceae 4 55 

Larix Pinaceae 

occidental is 

Picea abies Pinaceae 

Piceae g/auca Pinaceae 44 

Picea glauca x Pinaceae 4 

P. engelmanii 

Picea glauca x Pinaceae 4 

P. enge/manii 

Pinus monticola Pinaceae 4 55 to 51 

Pinus pinaster Pinaceae 190 47,27,22 

175 47,27,22 

Pseudotsuga Pinaceae 63 to 55 35 to 32, 

menziesii 25 to 22 

Biota orienta/is Cupressaceae 60 to 58 

Calocedrus Cupressaceae 4 

decurrens 

Chamaecyparis Cupressaceae 60 to 56 

lawsoniana 

1 kilodaltons 

2 relative molecular weight 3 seed storage protein 4 not reported 

teins from such families as Pinaceae, Cupressaceae, 

Ginkgoaceae, Fabaceae, and Salicaceae have been characterized (tables 1 and 2). 

The presence of SSPs homologous to the US and 7S 

globulin proteins were identified in several gymnosperms 

(Allona et al. 1994a, 1994b; Flinn et al. 1991a; Gifford 1987; 

Hakman et al. 1990; Leal and Misra 1993a) and a few of 

the woody angiosperms (Arahira and Fukazawa 1994; 

Collada et al. 1991). Amino acid sequences of various 

woody species, such as Pinus pinaster (Allona et al. 1994a), 

Calocedrus decurrens (Hager and Dank 1996 ), Picea glauca 

x P. engelmanii (Newton et al. 1992), Pseudotsuga menziesii 

(Leal and Misra 1993a), and Ginkgo biloba (Arahira and 

Fukazawa 1994), show that SSPs are highly conserved 

plant proteins and that the amino acid identity levels follow taxonomic groupings among closely related species. 

134 

Polypeptides SSP3 

(kDa)2 family Reference 

28,20,13 globulin Jensen and Berthold 1989 

44 globulin Blagrove et al. 1984 

43,28,22,16 globulin Jensen and Lixue 1991 

32,21 globulin Allona et al. 1994b 

45,33,22 globulin Allona et al. 1994b 

41,35,22 globulin Misra and Green 1994 

42,33,28,22 globulin Hakman et al. 1990 

globulin Newton et al. 1992 

35, 33, 24, 22, globulin Flinn et al. 1991a, 1991b 

17, 16 

35 to 31,22 4 Gifford 1987 

41 albumin Flinn et al. 1991a, 1991b 

globulin Allona et al. 1994a 

globulin Allona et al. 1994a 

globulin Leal and Misra 1993a 

23 to 22 globulin Allona et al. 1994b 

45, 25 to 24, globulin Allona et al. 1994b 

21 

34 to 30, globulin Hager and Dank 1996 

25 to 23 

34 to 31, globulin Allona et al. 1994b 

24 to 21 

In gymnosperms, SSP gene expression can be regulated 

by the plant growth regulator abscisic acid and the osmotic environment (Dunstan et al. 1988; Misra et al. 1993; 

Roberts 1991; von Arnold and Hakman 1988). In gymnosperm embryos and megagametophytes, temporal 

changes in the SSP accumulation are similar to that found 

in angiosperms, with accumulation occurring in the midto late-maturation stage (Flinn et al. 1991a, 1991b; Hakman 

1993; Hakman et al. 1990; Misra and Green 1994; Roberts 

et al. 1989). Protein bodies are most evident in the cotyledons of gymnosp~rm embryos (Durzan et al. 1971; 

Hakman 1993; Johnson et al. 1987), suggesting that this is 

the predominant site of accumulation in the embryo. Leal 

and Misra (1993b) observed transient accumulation of SSP 

transcripts very early during development in the embryo 

and megagametophyte of Picea glauca. Possibly, these tranUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
sient accumulation sites supply nutrients to the developing embryo. 

Populus 

Seed storage protein analysis has been conducted for 

only 1 Populus species, P. grandidentata. In Populus, this 

area of study is tedious due to difficulties in effectively 

working with very small seeds and the necessity of working quickly due to the short duration of seed viability (i.e., 

loss of viability can occur as soon as 5 days after tree collection) (Brinkman 1974; Helium 1973). 

Storage protein composition can be elucidated using 

two-dimensional, polyacrylamide gel electrophoresis to 

allow for the selective and sequential disassembly of the 

storage proteins into their constituent polypeptides. Using this approach, P. grandidentata SSP was found to be a 

120 to 100 kilodalton (kDa) albumin storage protein (i.e., 

water soluble) containing 60 and 58 kDa subunits 

(Beardmore et al. 1996). Refer to table 1 and figures 1A 

and 1B for a summary of storage protein composition. 

When these subunits are reduced (i.e., disulfide bonds are 

broken), they yield polypeptides with molecular masses 

of 36, 32, 22, 18, and 14 kDa (figure 1B). Stoichiometrically the protein is a dimer, containing 2 hydrogen-bonded 

subunits of 60 and 58 kDa. 

The 36 and 32 kDa polypeptides are glycosylated and 

occur in approximately 8 to 12 isomeric forms (pi 7.0 to 

8.2) (figure 1C). The 18 and 14 kDa polypeptides do not 

exist as isomers and have a pi of 7.0. The 22 kDa polypeptide is suggested to be either very acidic (< 5.0) or very 

basic (>8.0), since it does not resolve during conventional 

isoelectric focusing (Beardmore et al. 1996). 

This research has illustrated similarities between the 

seed and vegetative storage proteins in Populus 

(Beardmore et al. 1996 ). Protein fingerprint pa ttems of the 

36 kDa polypeptide isolated from seed (sexual) and stem 

tissue (vegetative) yielded polypeptides of identical molecular masses. However, it is important to note that while 

the VSP and SSP may be similar with regard to constitutive polypeptides (i.e., both contain 36 and 32 kDa 

polypeptides), they differ with respect to their native proteins and subunit composition. Specifically, the SSP 36 and 

32 kDa polypeptides are disulfide bonded, while the VSP 

36 and 32 kDa polypeptides are hydrogen bonded (figures 2A, 2B ). 

Physiology of Populus Seed Storage 

Protein: Accumulation, Deposition, and 

Mobilization 

Poplar seed consists of an embryo approximately 3 mm 

in length surrounded by the testa (Schreiner 1974). The 

embryo is the main area of deposition for storage protein 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Poplar Seed Storage Proteins 

(Beardmore et al. 1996; Breton et al. 1993). Soluble protein 

content of P. deltoides zygotic embryos significantly increased from 25 days after pollination (DAP) to 40 DAP, 

when the embryo was mature (Breton et al. 1993). Breton 

and coworkers (1993) suggest that this increase was due 

in part to storage protein deposition. Polypeptides with a 

similar molecular mass (approximately 60 to 58, 36, 32, 18, 

14 kDa) to those identified in P. grandidentata embryos were 

detectable relatively early (15 DAP) in P. deltoides globular 

embryos (Breton et al. 1993), suggesting that storage protein synthesis may occur early in embryo development. 

Storage protein mobilization in P. grandidentata occurs 

while the seeds germinate. The P. grandidentata embryos 

germinated by 72 h after imbibition, coinciding with the 

disappearance of the storage proteins from SDS-PAGE gels 

(Beardmore et al. 1996 ). 

Homology of Populus Seed Storage 

Proteins 

Putative storage proteins were identified on the basis of 

their relative abundance in P. deltoides and P. trichocarpa 

seeds (Breton et al. 1993 ). In P. delta ides and P. trichocarpa 

seeds, these storage proteins were identified with molecular masses in the range of 36 to 32, 22, and 20 kDa (Breton 

et al. 1993 ), similar to the molecular masses of the P. 

grandidentata SSPs. 

A survey of the major polypeptides present in selected 

Salicaceae family members revealed that these seeds contained large quantities of polypeptides with similar or the 

same molecular masses as the P. grandidentata storage proteins (Beardmore et al. 1996 ). Such similarities suggest that 

Populus species and other Salicaceae family members may 

have homologous storage proteins. However, more studies are required for proper identification of the SSPs in these 

various species. 

In addition, the SSPs in yellow poplar (Liriodendron 

tulipifera L.) zygotic and soma tic embryos (Sotak et al. 1991) 

also appear to be very similar to those identified in P. 

grandidentata. The putative storage protein in L. tulipifera 

has a molecular mass of approximately 55 kDa (Sotak et 

al. 1991). Polypeptides with molecular mass in the mid-30 

kDa range and in the 22 to 14 kDa range were also present 

in large quantities and at the same relative abundance as 

the 55 kDa polypeptide, suggesting that these polypeptides may also be SSPs (Sotak et al. 1991 ). SSPs in seeds of 

the Fagaceae family also appear to contain polypeptides 

with similar molecular masses (table 1). 

Homology of the Seed Storage Protein to 

the Vegetative Storage Protein in Populus 

The poplar 36 kDa VSP and SSP exhibit extensive homology, based on proteolytic digestion patterns, antibody 

cross-reactivity, and chemical characteristics (Beardmore 

135 
Section Ill Molecular Biology 

A. i) Protein was separated in the first dimension with 

nondissociating conditions using polyacrylamide gel 

electrophoresis (PAGE). 

A. ii) Protein was separated in the second dimension with 

dissociating sodium dodecyl sulphate (SDS) and 

reducing conditions (dithiothreitol, DTI) (SDS-PAGE 

+ DTI). 

B. i) Protein was separated in the first dimension with 

dissociating conditions (SDS-PAGE). 

B. ii) Protein was separated in the second dimension with 

dissociating and reducing conditions (SDS-PAGE + 

DTI). 

C. Protein was separated in the first dimension by 

isoelectric focusing (IEF) and in the second 

dimension by dissociating and reducing conditions 

(SDS-PAGE + DTI). 

A. i) 

ii) 

SOS 

+ 

OTT 

j 

B. 

ii) 

SOS 

+ 

OTT 

c. 

SOS 

+ 

OTT 

1 

i) 

116 106 kOa ...... 

NO 

v v 

-60 

-sa 

- 36 koa 32 

= 22- 14 

SDS 

6 kDa 32 

a.s IEF 

V VV"il -

--

• 

=22-14 

s.p PH 

60 - sa 

- 36 _ 32 koa 

-22- 14 

Figure 1. A 2-dimensional, polyacrylamide gel analysis of the seed storage proteins in Populus grandidentata. Molecular 

mass markers in kilodaltons (kDa). 

136 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
A)seeo 

116~ 

106~ 

B)STEM 

500~ 

I . 

NO 

60 

58-

36-

32-

... 

SOS 

<: [ 

iii. 

-~~ 

-36 

-32 

-22 

-18 

-14 

SOS 

+ 

OTT 

36 

-32 

Figure 2. Summary of the seed storage proteins and 

vegetative storage protein in Populus. Onedimensional electrophoresis of: i) nondissociated 

protein (NO); ii) dissociated protein (SOS-PAGE); 

and iii) dissociated and reduced protein (80SPAGE + OTI). A) Seed storage protein isolated 

from P grandidentata. B) Vegetative storage 

protein isolated from P alba stems. 

et a!. 1996). Such homology indicates tha t these proteins 

may be encoded by the same gene. This situa tion contrasts 

with the relationship of the VSP and SSP in soybean w he re 

the storage proteins a re expressed by sepa rate vegetativespecific and seed-specific genes (Staswick 1988). However, 

differences in post-translationa l modification of VSP and 

SSP in popla r (i.e., disulfide bond formation) could result 

from tissue-specific diffe rentia l processing of the proteins. 

Such regulation was suggested for the alte rna tive oxidase 

in pota to, where protein analysis of leaves, roots, and tubers indicated diffe rential processing (Hiser and Mcintosh 

1995). Isolation of the complementa ry D A (eDNA) of the 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Poplar Seed Storage Proteins 

SSP wou ld help cla rify the relationship between the VSP 

and SSP in popla r. 

The hypothesis that the same gene codes for VSP and 

SSP is supported by recent transgenic work with the 

Populus VSP promoter (Gary Coleman preliminary and 

unpublished results). When the VSP promoter was linked 

to ~-glu ur ase (GUS) gene and transferred into tobacco, GUS expression occurred in the seed and also the 

vegetative tissues. Whether this expression can account 

for all or just a portion of the SSP in the seed is unknown. 

Despite the potential relationship be tween the VSP and 

SSP genes, at least 3 possibilities for the organization and 

regu la tion of the SSP genes exist with poplar. One possibility is that the SSP 36 and 32 kDa p olypepti des are encoded by sepa ra te genes. These polypeptides are likely 

related in sequence to each othe r and to the VSP, since 

immunogenic ana lysis cou ld not d ifferentiate these proteins (Beardmore eta!. 1996). Because the SSP 36 and 32 

kDa polypeptides ap parently occur in equal amounts, the 

respecti ve genes must be expressed at a simila r rate. Association of the 2 polypep tides to form the complete native sto rage pro te in wo uld, however, d epend o n 

inte rmolecula r d isulfide bonds. Althou gh identified disulfide bridge forma tion in p lant proteins is usually intramolecular, several examples of intermolecula r d isulfide 

bridges a re d ocumented . Examples of intermolecula r d isulfid e bridges in storage p roteins include castor-seed 2S 

albumin protei n (Shin eta!. 1993), wheat high mole.cular 

mass glutinin (Shani et a!. 1994), soybean seed 34 kDa 

protein (Komatsu e t a!. 1992), and lectin from £ranthis 

hyemalis (Kuma r e t a!. 1993). Based on these examples, 

the SSP 36 and 32 kDa polypeptides could possib ly be 

encoded by sepa rate genes, with their products associated by inte rmolecu lar disulfide bonds. 

Alternatively, the 36 and 32 kDa SSP could be encoded 

by 1 large gene, the product of wh ich contains intramolecula r disulfide bonds. Similar to many other seed storage proteins, the 2 subunits a re subsequently formed as 

separate 36 and 32 kDa peptides by a specific proteolytic 

cleavage. The sequence of this la rger gene may be related 

to the VSP gene. Finally, the 36 and 32 kDa proteins could 

be diffe rentially-processed products of the same gene. This 

appa ren tly occurs with the VSP; the 36 and 32 kDa proteins a re g lycoforms, differing only in the extent of 

glycosylation (Langheinrich and Tishner 1991; Stepien and 

Martin 1992). A review of this topic is in the chapter by 

Ga ry Coleman (this volume). 

Currently, evidence supports the hypothesis that 1 gene b 

diffe rentially processed to p rod uce the 36 and 32 kDa 

polypeptides that are temporally and spatially regulated to 

form VSP in vegetative tissues and SSP in seeds. Thus, the 

poplar storage protein system could be an excellent model 

to analyze d ifferential regulation at the transcriptional, translational, and post-translational levels. The answer may be 

resolved by analyzing the gene corresponding to SSP in pop137 
Section Ill Molecular Biology 

lar seeds. Isolation of the putative SSP gene(s) could be 

achieved with either a lambda gtll expression library with 

the SSP antisera or by using the VSP gene to isolate related 

cDNAs in the seed. Future efforts may also include analysis 

of the VSP and SSP promoter(s) to determine if tissue-specific elements are responsible for their expression patterns. 

Approaches to Understand Seed Storage 

Protein Gene Expression 

Despite many empirical observations about embryogenesis, the molecular and biochemical basis of this developmental pathway is poorly understood. An important 

limiting factor is the difficulty in obtaining experimental 

populations of developing seeds that are synchronized in 

their development and viable year round. Recent innovations in Populus, such as the development of somatic embryogenesis protocols (Michler and Bauer 1991) and the 

successful expression of the Arabidopsis LEAFY (LFY) gene 

in poplar transformants (Sundberg et al. this volume; 

Weigel and Nilsson 1995), provide potentially useful experimental systems that promote laboratory study of poplar SSP gene expression. Somatic embryogenesis protocols 

are useful because they allow mass quantities of developmentally synchronized embryos to be produced through 

manipulation of tissue culture conditions. Transgenic poplars expressing the developmental regulator gene LEAFY, 

might provide a convenient source of reproductively mature material for year round controlled pollinations because these plants produce reproductive structures 

independent of seasonal cues. Until these new approaches 

prove useful, other model systems will likely serve as surrogate hosts for poplar SSP transgenes. Because tobacco 

and Arabidopsis are easily transformed and have short juvenile phases, they will likely be the species of choice for 

transgenic work. 

Production of an engineered seed storage protein requires 

accurate processing at all levels of regulation, from tissuespecific expression in the seed, to transport to the appropriate vacuole I protein body. To ensure success of future genetic 

engineering efforts, significant research has occurred on various aspects of seed storage protein production. These studies have included research on identification of suitable 

proteins (Mehta et al. 1994), stability of the mRNA (Stayton 

et al. 1991), stability of the protein (Bagga et al. 1995), posttranslational processing of the protein including 

glycosylation (Sturm et al. 1988), assembly of subunits 

(Beachy et al. 1985), and other modifications (Ellis et al. 1988). 

Accurate targeting and assembly of storage proteins were 

studied by Greenwood and Chrispeels (1985), DeClercq et 

al. (1990), Kjemtrup et al. (1994), and Zheng et al. (1995). 

Most transgenic research has involved the production of 

desirable proteins, but reverse genetics was used in 1 study 

to alter protein composition of seeds (Kohno-Murse et al. 

138 

1995). Several in-depth reviews of these and other related 

studies have focused on genetic engineering to alter the nutritional value of cereals (McKinnon and Henry 1995; Shewry 

et al. 1994) and legumes (Tabe et al. 1993, 1995). 

Conclusion and Future Prospects 

At a fundamental level of scientific interest, considerable knowledge can be gained by continued study of storage proteins in Populus. The physiology, biochemistry, and 

regulation of the SSP have received little attention, and 

the relationship of the poplar VSP to the SSP must be clarified. Populus is an excellent experimental system to study 

nitrogen metabolism in woody plants. The similarities of 

the SSP and VSP raise important questions regarding how 

different storage forms of nitrogen are regulated in Populus. 

Seed storage proteins in poplar are potentially useful in 

diverse areas of study. For example, information about the 

poplar SSP can contribute to phylogenetic studies. Such 

information could provide a valuable marker for assessing climate change and the effect of tree species distribution particularly for members of the Salicaceae family that 

are on the rare and endangered lists for Canada and the 

United States (Argus and Pryer 1990). These species are at 

the outer range of their distribution and such information 

could prove vital in monitoring changes in species distribution. Study of poplar seed SSP could also enhance insights into seed development necessary for extending seed 

viability particularly for many members of the Salicaceae 

family whose existence may be threatened without effective management and knowledge of their seed. 

Acknowledgments 

This research was supported in part by PERD panel and 

ENFOR. The authors would like to thank Linda De Verno 

and Carrie-Ann Whittle for their review of this manuscript, and John Davis for review of the manuscript and 

valuable discussions concerning the relationship of the 

VSP to SSP. 

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von Arnold, 5.; Hakman, I. 1988. Regulation of somatic 

embryo development in Picea abies by abscisic acid. J. 

Plant Physiol. 132: 164-169. 

141 
Section Ill Molecular Biology 

Walling, L.; Drews, G.N.; Goldberg, R.B. 1986. Transcriptional and post-transcriptional regulation of soybean 

seed protein mRNA levels. Proc. Natl. Acad. Sci. USA. 

83: 2123-2127. 

Weber, E.; Manteuffel, R.; Neumann, D.1978. Isolation and 

characterization of protein bodies of Vicia Jaba seeds. 

Biochem. Physiol. Pflanzen. 172: 597-614. 

Weigel, D.; Nilsson, 0. 1995. A developmental switch sufficient for flower induction in diverse. plants. Nature. 

377: 495-500. 

Wilson, K.A. 1986. Role of proteolytic enzymes in the mobilization of protein reserves in the germinating dicot 

142 

seed. In: Dallin, M.J., ed. Plant proteolytic enzymes. Boca 

Raton, FL, U.S.A.: CRC Press: 19-48. 

Wittenbach, V.A.; Ackerson, R.C.; Giaquinta, R.T.; Hebert, 

R.R. 1980. Changes in photosynthesis, ribulose 

bisphosphate carboxylase, proteolytic activity, and ultrastructure of soybean leaves during senescence. Crop 

Sci. 20: 225-231. 

Zheng, Z.; Sumi, K.; Tanaka, K.; Murai, N. 1995. The bean 

seed storage protein beta-phaseolin is synthesized, processed, and accumulated in the vacuolar type-11 protein bodies of transgenic rice endosperm. Plant Physiol. 

109: 777-786. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Chapter 18 

Molecular Genetic Analysis of Populus Chloroplast DNA 1 

Eun W. Noh and Jae S. Lee 

Introduction 

Molecular techniques have been applied to differentiate closely related species and varieties or to study genetic 

variation of wild populations. Diverse methods have been 

applied to generate different genetic markers. Therefore, 

information obtained by i:he markers varies in amount and 

type. 

Cloned DNA sequences are useful to probe specific 

regions of genomes for polymorphisms at the DNA sequence level. After DNA digestion with restriction enzymes; these polymorphisms are detected as variation 

in the length of DNA fragments homologous to alabeled probe. Such variation is termed restriction fragment length polymorphism (RFLP). The RFLP 

technique was successfully employed in many tree species including poplars. D'Ovidio et al. (1990) distinguished different Populus species by analyzing 185 and 

255 ribosomal DNA (rONA) with RFLP. Faivre-Rampant et al. (1992) also reported a simple method of differentiating poplar clones by RFLP, and a donor species 

in putative hybrids was identified by fragment profiles. 

Although RFLP generates many polymorphic markers 

that are useful to differentiate clones and individuals, 

the technique is time consuming, labor intensive, and 

expensive. 

Another kind of DNA marker called random amplified 

polymorphic DNA (RAPD) has been developed based on 

polymerase chain reaction (PCR). In this method, polymorphisms are detected by differential amplification of 

DNA fragments. This technique provides an advantage 

, Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

over RFLP in its simplicity, rapidity, and requirement for 

only small amounts of crude DNA. Estimates of genetic 

similarity based on RAPD profiles in poplar have demonstrated consistency with the present taxonomy of the 

genus Populus (Castiglione et al. 1993; Liu and Fumier 

1993). However, a major limitation of this technique is 

that markers are usually dominant rather than codominant. Reproducibility of RAPD banding patterns also 

presents a problem because the pattern is affected by 

different concentrations of reaction components and 

cycle conditions. 

PCR techniques are also used to produce genetic 

markers based on site-specific amplication. For this 

technique, known or anticipated sequence information is used to design primers to amplify a specific 

site. For site-specific PCR, ribosomal RNA (rRNA) 

genes are widely used because they comprise domains that are conserved across diverse taxa (Sogin 

1990). A further derivation of this technique uses 

restriction endonucleases to cut the amplified fragments (Liston 1992; Nishio et al. 1994). This PCRRFLP method also locates genomic regions 

containing variation. When specifically designed 

primers are used, banding patterns are usually more 

reproducible than random primers. 

Organelle genomes like chloroplast DNA (cpDNA) 

and mitochondrial DNA (mtDNA) offer strong potential to provide markers for tree species. The small size 

of organelle genomes provides a major advantage. Although the size varies from organism to organism, 

cpDNA is circular and 135 to 160 kilobases (kb) in size 

for most land plants (Olmstead and Palmer 1994). Another merit associated with cpDNA is its conserved 

nature. Evolution of cpDNA has occurred by mutations 

such as inversions, deletions, insertions, and substitutions. However, comparisons from different taxa have 

revealed that the cpDNA nucleotide sequences were 

well conserved during the evolution of land plants. In 

most angiosperms, including poplars, cpDNA and 

mtDNA are maternally inherited. Since little variation 

occurs within cpDNAs and mtDNAs of a species, any 

polymorphisms in these genomes could be a useful 

marker. 

143 
Section Ill Molecular Biology 

Review of RFLP Analysis of 

Populus Chloroplast DNA 

With the availability of many gene probes from wellstudied plants, cpONAfrom any plant species can be compared by the RFLP technique. Comparisons among species 

within a genus have shown only a few differences that are 

often confined to 1 or 2 restriction site polymorphisms. 

Such comparisons were demon·strated in Medicago (Rose 

and Schlarbaum 1988), Nicotiana (Salts et al. 1984), Solanum 

(Kawagoe and Kikuta 1991), and Picea (White et al. 1993). 

Salts et al. (1984) studied cpDNA of 7 American and 4 

Australian species of Nicotiana by RFLP. With the RFLP 

profiles, more than 120 polymorphic sites were distinguishable on cpDNA of N. tabacum. 

With Populus, previous results have confirmed maternal inheritance of cpDNA in the genus. However, maternal or paternal inheritance is detectable only when a 

marker specific to an individual is available. Clones and 

species were also readily differentiated by the markers 

developed by RFLP. In addition, Populus cpDNAs were 

characterized by the RFLP technique. Mejnartowicz {1991) 

analyzed cpONAof Populus species (P. trichocarpa), hybrids 

(P. maximowiczii x P. trichocarpa and P. maximowiczii x P. nigra), and F1 progeny to study inheritance of cpDNA. With 

each of 5 restriction enzymes (Pstl, EcoRI, Hindiii, BamHI, 

and Xbal) tested, differences were observed between the 2 

parental clones. All test progeny showed the characteristic pattern of the female parent and no paternal or mixed 

patterns were observed. Rajora and Dancik (1992), studying 2 Populus species (P. deltoides and P. nigra) and their F1 

hybrid progeny, confirmed uniparental maternal inheritance of the cpDNA. Using 13 different enzymes in combination with 4 different probes to detect polymorphisms 

between the 2 species, they found 36 species-specific restriction fragment polymorphisms. Between P. deltoides and 

P. maximowiczii, 16 species-specific restriction fragment 

polymorphisms were observed. Using RFLP analysis, Lee 

et al. (1994a) compared cpDNAs of Populus spp. (P. alba, P. 

glandulosa, P. alba x P. glandulosa, P. davidiana, and P. nigra) 

and reported little intrageneric variation. They used 5 different restriction enzymes (Pstl, EcoRI, BamHI, Bglll, and 

Kpnl) to find polymorphisms among 5 Populus species. With 

the exception of P. nigra, which showed a different fragmentation pattern when cut with the enzyme EcoRI, fragmentation patterns appeared similar among the Populus species. 

However, Southern hybridization with the tobacco rbcL 

gene probe produced identical patterns among Populus 

species for all the enzymes tested; suggesting that gene 

order among Populus species is conserved. With all the 

enzymes tested, theN. tabacum reference plant showed 

different restriction profiles than those of Populus species. 

144 

PCR-RFLP with Populus cpDNA 

Recent advances in PCR technology have allowed examination and comparison of specific DNA regions in 

higher plants. In many DNA regions, variation was frequently observed in noncoding spacer regions rather than 

in coding regions. Nuclear rONA spacers exist between 

genes encoding large and small subunit rRNAs that are 

ubiquitous among various organisms. This spacer region 

has been extensively studied in many different organisms 

by RFLP. Recent RFLP analysis of intergenic spacers (IGS) 

between the 2 rRNA subunit genes has revealed polymorphisms among various Populus species or individuals 

(Faivre-Rampant et al. 1992). However, little work has focused on the nuclear genome except for the rONA spacer 

region perhaps because of the complexity of the nuclear genome and limited sequence information for nuclear DNA. 

Unlike nuclear genomic DNA, cpDNA is well studied 

in higher plants. Sequence information on the chloroplast 

genome is, therefore, relatively abundant. Complete nucleotide sequences of cpDNAs are published for several plant 

species (Hiratsuka et al. 1989; Shinozaki et al. )986). Because many coding regions in cpDNA are highly conserved 

among higher plants, spacers can be amplified by PCR 

using primers deduced from coding regions (Liston 1992). 

This PCR-RFLP method has 2 advantages over conventional RFLP: 1) labeling is unnecessary because a probe is 

not required and 2) specific DNA regions can be compared 

in detail using restriction enzymes that recognize 

tetranucleotides. Employing the PCR-RFLP method, we 

have analyzed 3 different regions of cpONA in Populus 

species: 1) 165-235 rONA spacer; 2) rpoC1C2 region; and 

3) rpl2-psbA region. 

Populus Materials 

Our studies of 3 different regions of cpDNA used the 

following Populus materials: 1) 7 clones of P. davidiana (6 

clones from diverse regions of Korea and 1 clone from 

China); 2) 1 clone of P. glandulosa; 3) 2 clones of P. nigra; 4) 

2 clones of P. alba; 5) 3 samples of P. maximomiczii; 6) 3 

samples of P. alba x P. glandulosa; 7) 3 samples of P. nigra x 

P. maximowiczii; and 8) 3 samples of P. koreana x P. nigra. In 

addition, Nicotiana tabacum, Salix pseudolasiogyne, and 

Quercus accutissima were used as reference plant material. 

Analysis of 16S-23S rONA Spacer 

Ribosomal RNA genes of chloroplasts are arranged as: 

165 rDNA-spacer-235 rONA-spacer-55 rONA. The rrn 

operon is located within the inverted-repeat region of cp 

DNA, and 2 transfer RNA (tRNA) genes are contained in 

the spacer between the 165 rONA and 235 rONA. In 

Euglena gracilis, the spacer is 258 base pairs (bp) long, 

whereas the spacer exceeds 2 kb in maize and tobacco. The 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
tRNA genes in the 165-235 rONA spacer of higher plants 

contain introns, and the difference in spacer length is due 

to intron size. In maize, the intron in tRNN'• is 949 bp and 

in tRNAAia is 806 bp (Koch et al. 1981). Corresponding intrans in N. tabacum are 707 and 710 bp, respectively 

(Takaiwa and 5ugiura 1982). 

To amplify the 165-235 rONA spacer in Populus cpDNA, 

primer sequences were deduced from cpONA genes in 

N. tabacum (5hinozaki et al. 1986). The primer sequences 

are: 5'-GGA AGG TGG GGC TGG ATC CAC C-3' (from 

the 3' end of 165 rONA) and 5'-CCT CGT CTC TGG GTG 

CCT AGG-3' (from the 5' end of 235 rONA). In all the 

Populus species and Salix pseudolasiogl;ne (reference plant), 

the spacer spanned 2.3 kb. However, the spacer appeared 

smaller (2.1 kb) in N. tabacum suggesting that Populus spp. 

have a larger intron than tobacco in the 2 tRNA genes 

within the spacer. Restriction digestions of the spacer 

with several different enzymes are in table 1. As table 1 

shows, enzymes that recognize and cut tetranucleotides 

typically generate more bands than those that recognize 

hexanucleotides. 

No variation among Populus species was observed with 

the 7 enzymes tested (Hinfl, Hpaii, Haeiii, Taql, Kpni, Sacl, 

and BamHI). N . tabacum showed different fragmentation 

patterns for all the enzymes tested except Sacf. In addition, no restriction sites were observed in the spacer for 

Table 1. Restriction fragmentation patterns of amplified 

16S-23S rONA spacer in Populus spp. and Nicotiana 

tabacum. No variation was observed among Populus 

species. 

Enzyme Species Fragment size (bp)' 

Kpnl 

Sacl 

BamHI 

Taql 

Haelll 

Hpall 

Hinfl 

Populus spp. 970; 420; 380; 350; 200 

N. tabacum 1 ,500; 6002 

Populus spp. 1 ,300; 1,000 

N. tabacum 1 ,200; 9602 

Populus spp. 1 ,000; 41 0; 360; 340; 190 

N. tabacum 1 ,575; 5752 

Populus spp. 1 ,250; 380; 230; 200; 140 

N. tabacum 1,128; 346; 145; 1422 

Populus spp. 500; 430; 390; 320; 290; 180 

N. tabacum 764; 553; 302; 295; 2462 

Populus spp. 530; 430; 300; 290; 220; 160 

N. tabacum 985; 880; 495; 1 002 

Populus spp. 530; 400; 220; 160 

S. pseudolasiogyne 530; 400; 220; 160 

N. tabacum 540; 310; 300; 200; 160; 

120; 120; 120; 1002 

1 Approximate estimation. Bands less than 100 bp are not 

shown. 

2 The fragment size of N. tabacum was calculated from 

the published map (Shinozaki et al. 1986). 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Molecular Genetic Analysis of Populus Chloroplast DNA 

the enzymes Hpai, Psti, Xbai, Oral, Hind III, EcoRI, and Smal. 

With Populus, no variation is apparent in the spacer between 165 and 235 rONA (figure 1). Therefore, the sequence 

of the chloroplast rONA spacer region seems highly conserved among Populus spp. 

Analysis of rpoC1 C2 Region 

The rpoC1 and C2 genes encode RNA polymerase subunits and are located within the large single copy region 

of the chloroplast genome. The rpoC1 gene contains an 

intron (absent in monocots) and is separated from rpoC2 

by an intergenic spacer (IG5) (5hinozaki et al. 1986). The 

intron and the IG5 regions are expected to show higher 

levels of sequence divergence than the coding regions. 

Primers for rpoC1C2 amplification were synthesized according to Liston (1992) and contained the sequences: 5'-

AAG CGG AAT TTG TGC TTG T-3' (from the antisense 

strand of the rpoC1 gene) and 5'-TAG ACA TCG GTACTC 

CAG TGC-3' (from the sense strand of the rpoC2 gene). 

These primers have successfully amplified 3.7 kb to 4.3 kb 

products from pine and other dicots (Liston 1992). 

Amplifica tion of the rpoC1C2 region produced a 4.3 kb 

band with several Populus species (P. alba, P. glandulosa, P. 

alba x P. glandulosa, P. davidiana, and P. nigra) and 5. 

pseudolasiogyne; a smaller band (4.1 kb) was produced with 

N. tabacum. Liston (1992) reported that amplification of the 

region with the same p rimers resulted in a 4 kb band from 

Astragalus (Fabaceae). Restriction fragmentation patterns 

of the amplified rpoC gene region in Populus spp., 5. 

pseudolasiOgljlle, and N. tabacum are in table 2. Restriction 

digestion of the amplified fragment with the enzymes Kpni, 

Figure 1. BamHI restriction 

digestion of the amplified 

16S-23S rDNA spacer. 

Lanes 1 to 7 (from the 

left) are: pGEM DNA 

marker, Nicotiana 

tabacum, Populus nigra, 

P. davidiana, P. 

glandulosa, P. alba x P. 

glandulosa, and P. alba. 

145 
Section Ill Molecular Biology 

Table 2. Restriction fragmentation patterns of amplified 

rpoC1 C2 region in Populus spp., Salix pseudolasiogyne, 

and Nicotiana tabacum. No variation was observed 

among Populus species. 

Enzyme Species Fragment size (bp)1 

Kpnl Populus spp. 2,600; 1 ,550; 180 

S. pseudolasiogyne 2,600; 1 ,550; 180 

N. tabacum 2,590; 1 ,520 2 

Sacl Populus spp. 4,300 

S. pseudolasiogyne 4,1 00; 200 

N. tabacum 2,700; 1 ,4102 

BamHI Populus spp. 2,400; 1 ,000; 900 

N. tabacum 2, 130; 1 ,9802 

Taql Populus spp. 520; 450; 390; 340; 240 

S. pseudolasiogyne 520; 500; 450; 430; 290; 240 

N. tabacum 675; 41 0; 360; 350; 335; 

220; 170; 170; 160; 135; 1352 

Haelll Populus spp. 1 ,450; 1 ,430; 1 ,000; 420 

S. pseudolasiogyne 1 ,450; 1 ,000; 700; 690; 450 

N. tabacum 1 ,080; 890; 500; 450; 400; 

370; 230; 1902 

Hpall Populus spp. 2,200; 950; 350; 280; 270; 180 

N. tabacum · 920; 760; 660; 540; 340; 

270; 250; 2002 

Hinfl Populus spp. 720; 485; 450; 350; 330; 

220; 170 

S. pseudolasiogyne 720; 490; 450; 340; 330; 

280; 220; 170; 165 

N. tabacum 600; 390; 365; 335; 330; 

235; 175; 175; 170;165; 135; 

1052 

Hindlll Populus spp. {300 

S. pseudolasiogyne 4,300 

N. tabacum 3,640; 4702 

EcoRI Populus spp. 2,000; 1 ,550; 750 

S. pseudolasiogyne 2,000; 1 ,550; 750 

N. tabacum 1 ,530; 1 ,380; 1 ,2002 

1 Approximate estimation. Bands less than 100 bp a re not 

shown. 2 The fragment size of N. tabacum was calculated from 

the published map (Shinozaki et al. 1986). 

Sad, Taql, Haeiii, EcoRl, BamHI, Hpall, Hindll l, and Hi11 fl 

did not discriminate among Populus species. However, 

Salix was distinguishable from Populus species with the 

enzymes Sacl, Taql, and Haelll. In all the Populus spp. tested, 

no Hi11diii, Pstl, or Sad restriction sites were detected in 

the region, but Sad and Hi11dlll restriction sites do occur 

in the comparable region of N. tabacum. In most examples, 

N. tabacum exhibited a totally distinct banding pattern from 

Populus species. An exception was evident when cpDNA 

was cut with Kpnl; 1 Kpni site was apparently conserved 

among Populus, Salix, and even N. tabacum. 

146 

Figure 2. -Haelll restriction digestion of the amplified 

rpoC1 C2 region. Lanes 1 to 12 (from the left) 

are: pGEM DNA marker, Populus alba, P alba x 

P glandulosa, P glandulosa, P davidiana, P 

maximowiczii, P nigra x P maximowiczii, P 

nigra, P koreana x P nigra, Salix 

pseudolasiogyne, N. tabacum, and lambda 

phage (Hindlll cut) . . 

Two Haelll sites in the rpoC1C2 region are apparently 

conserved and flank the IGS in tobacco, spinach, rice, and 

maize (Liston 1992). The distance between the 2 Haeiii sites 

was estimated to be 210 bp in Astragalus, 193 bp in tobacco, 

and 234 bp in rice. Because the fragment contains a 35-bp 

coding region, IGS length in Astragalus, tobacco, and rice 

is 175, 158, and 199 bp, respectively. In Populus, the smallest Haeill fragment observed was 420 bp. Thus, Populus 

spp. may have a larger IGS since the coding region is highly 

conserved among organisms. This 220 bp difference could 

account for the difference in the total length of amp lified 

fragments between Populus and tobacco (Populus produced 

a band length of 4.3 kb, whereas tobacco showed 4.1 kb in 

total length of the amplified fragment). Based on these 

studies, this region appears very highly conserved among 

Populus species, a lthough some va riation occurs be tween 

Populus and Salix. 

Analysis of the Region Around the Spacer 

Between rp/2 and psbA Genes 

In many higher plants, the psbA gene is located within 

the large single copy region close to 1 end of the inverted 

repeat. The gene encodes a herbicide-binding protein, Qb 

(Shinozaki et al. 1986). The rp/2 gene codes for a ribosomal protein and is loca ted within the inverted repeat region. Although these 2 genes are adjacent to each other, 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
their transcription directions are opposite in most plants 

studied. Additionally, the tmH gene is located in the spacer 

between the 2 genes. 

Two primers were designed to flank the psbA gene, 

spacer, and rpl2 gene: 5'-CCT TGG GGT TAT CCT GCA 

CT-3' (from the sense strand of rpl2 gene) and 5'-ACT GCA 

ATT TTA GAG AGA CGC G-3' (from the sense strand of 

psbA gene). In Populus, observed amplification products 

of the primers are 2.07 kb. No spacer length variation was 

evident among Populus species and 5. pseudolasiogyne. With 

N. tabacum and Querws acutissima (reference plant), however, the spacer appeared smaller (approxima tely 1.73 kb ). 

Restriction fragmentation patterns of the s pace r from 

Populus s pp., 5. pseudolasiogyne, Q. acutissima, and N. 

tabacum are in table 3. Usually, fragmentation patterns were 

identical among Populus species and a Salix species. Restriction digestions with the enzymes J-laellJ, Hpnll, and 

Taql do not discriminate among tested species of Populus. 

However, Salix is separable from Populus species with the 

enzyme Hinfl (figure 3). The Hinfl enzyme a lso allows differentiation of P. davidiana and P. glandulosa from othe r 

Populus species. Also of note is that P. nigra exhibited different fragment sizes than other Populus species, and no 

Bam HI, Kpnl, or Sacl restriction sites were detected in the 

Table 3. Restriction fragmentation patterns of amplified 

rp/2-psbA region in Populus spp., Quercus acutissima, 

and Nicotiana tabacum. 

Enzyme Species 

Taql Populus spp. 

S. pseudolasiogyne 

Q. acutissima 

N. tabacum 

Haelll Populus spp. 

S. pseudolasiogyne 

Q. acutissima 

N. tabacum 

Hpall Populus spp. 

S. pseudolasiogyne 

Q. acutissima 

N. tabacum 

Hinfl all Populus except 

P. g/andulosa and 

P. davidiana 

P. glandulosa 

P. davidiana 

S. pseudolasiogyne 

Q. acutissima 

N. tabacum 

Fragment size (bp)' 

1 ,035; 1 ,030 

1 ,035; 1 ,030 

1,040; 700 

1,040; 415; 1872 

960; 765; 300 

960; 765; 300 

970; 765 

768; 668; 138; 1002 

1 ,650; 270; 130 

1 ,650; 270; 130 

1 ,560; 180 

1 ,500; 2382 

675; 670; 440; 167 

680; 670; 440; 167 

680; 670; 440; 167 

650; 550; 400; 167 

540; 520; 180; 180; 160 

747; 527; 167; 1202 

1 Approximate estimation. Bands less than 1 00 bp are not 

shown. 2 The fragment size of N. tabacum was calculated from 

the published map (Shinozaki et al. 1986). 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Molecular Genetic Analysis of Populus Chloroplast DNA 

Figure 3. Hinfl restriction digestion of the amplified rpf2.-

psbA region. Lanes 1 to 12 (from the left) are: 

pGEM DNA marker, Populus alba, P. alba x P. 

glandulosa, P. glandulosa, P. davidiana, P. 

maximowiczii, P. nigra x P. maximowiczii, P. 

nigra, P. koreana x P. nigra, Salix 

pseudolasiogyne, Quercus accutissima, and N. 

tabacum. 

spacer region. Again, N. tabacum exhibited totally different fragmentation profiles from Populus species with all 

the enzymes tested. Among the 3 regions analyzed, only 

this spacer region is polymorphic among Populus species. 

The conserved nature of Populus cpD 'A revealed in our 

work apparently contrasts with data obtained by other labs. 

Mejnartowicz (1991) and Rajora and Dancik (1992) reported much variation in cpDNA. This difference in results may be due to genera l d ifferences in techniques. 

Whereas their results represented variation in whole 

cpDNA, our approach was limited to small, localized regions of cpDNA. Therefore, it appears that variability may 

be found in Populus by applying PCR-RFLP to other regions of cpDNA. 

PCR Amplification of Populus 

Chloroplast DNA 

Many workers have used total DNA to analyze organelle 

genomes. Major advantages of using total 0 A may be 

the yield and simplicity of extraction procedures. Total 

0 lA also allows flexibi li ty because it can be used for the 

RFLP analysis of the nuclea r, mitochondrial, and chloroplast genome. However, each analysis requires specific 

147 
Section Ill Molecular Biology 

DNA probes that hybridize to the target DNA after fractionation by restriction digestion and electrophoresis. 

At a lower annealing temperature, amplification of 

cpDNAwith primers specific to the rDNA pacerprodu~es 

a thick single band of 2.3 kb for all the Populus tested (figure 4). Several other bands also appear, among them are 2 

bands of approximately 680 and 690 bp that appear in all 

Populus test species. Primers deduced from the rpoC1C2 

coding region also produced amplification products that 

varied among the species (figure not shown), and some 

species-specific bands were apparent. 

If both chloroplast and mitochondrial DNA can be obtained in a purified form, a RAPD technique could be 

employed even with small amounts of DNA. Although 

these genomes are small and relatively conserved, current 

results with Populus cpDNA indicate that variation is sufficient for detection by RAPD. For the RAPD method, chloropla sts are isolated from leaf tiss ue (young or 

tissue-cultured leaves). After extraction from the chloroplasts, cpDNAs are subjected to PCR amplification using 

random primers or sequence-specific primers. To generate more bands, a lower annealing temperature is applied. 

Depending on the primers used, up to 10 bands can be 

amplified (Lee et al. 1994b). In our studies, arbitrary oligomers (10-mers) generated RAPD patterns when annealing was performed at 35 oc. These RAPD patterns allowed 

Figure 4. Amplification of Populus cpDNA with primers 

specific to the 16S-23S rDNA region. Annealing 

was done at 43 oc. Lanes 1 to 9 (from the left) 

are: pGEM DNA marker, Populus davidiana 1, P 

davidiana 2, P glandulosa 1, P glandulosa 2, P 

alba x P glandulosa 1, P alba x P glandulosa 2, 

P alba 1 , and P alba 2. 

148 

the differentiation of all test species. Overall, RAPD techniques on cpD A can provide effective markers for tree 

species when intact chloroplasts are obtainable. 

Conclusion 

Polymorphisms detected in cpDNA have been used as 

markers to stud y geneti c variation in wild populations or 

identify clones of higher plants. Most p revious work on 

cpDNA of tree species including Populus has used RFLP. 

However, the RFLP technique has been used only sparingly for tree species because it is laborious and time consuming. The recently developed technique of PCR-RFLP 

allows analysis of specific genomic regions. With abundant sequence data ava ilable from well-known plants, 

specific regions of Populus cpD A can be amplified. Using PCR-RFL~ 3 different cpD1 A regions were compared 

among 8 species and hybrids of Populus. Among the 

Populus species tested, some polymorph isms were detected 

in 1 region (rp/2-psbA), but no variation was detected in 

the other 2 regions (165-235 rONA spacer and rpoC1C2 

region). Based on these results, Populus cpDNA appears 

highly conserved. However, data from other RFLP work 

indicate that many polymorphic sites occur in Populus 

cpDNA. If PCR-RFLP is used to examine other cpD TA 

regions, variable regions may be identified in the future. 

Literature Cited 

Castiglione, S.; Wang, G.; Damiani, G.; Bandi, C.; Bisoffi, 

S.; Sala, F. 1993. RAPD fingerprints for identification and 

for taxonomic studies of eli te popla r (Populus spp .) 

clones. Theor. Appl. Genet. 87: 54-59. · 

D'Ovidio, R.; Mugnozza, G.S.; Tanza rella, O.A. 1990. Ribosomal RNA gene structure in some Populus species 

(Salicaceae) and their hybrids. Pl. Syst. Evol. 173: 187-

196. 

Faivre-Rampant, P.; }eandroz, S.; Lefvre, F.; Leminee, M.; 

Villar, M.; Serville, A. 1992. Ribosomal Dt A studies in 

poplars: Populus deltoides, P. 11igra, P. trichocarpa, P. 

maximowiczii, and P. alba. Genome. 35: 733-740. 

Hiratsuka, J.; Shimada, H.; Whittier, R.F.; Ishibashi, T.; 

Sakamoto, M.; Mori, M.; Kondo, C.; Honji, Y.; Sun, C.P.; 

Meng, B.Y.; Li, Y.; Kanno, A.; ishizawa, Y.; Hirai, A.; 

Shinozaki, K.; Sugiura, M. 1989. The complete sequence 

of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tR A genes accounts for a major plastid D A inversion during the 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
evolution of the cereals. Mol. Gen. Genet. 217: 185-194. 

Kawagoe, Y.; Kikuta, Y. 1991. Chloroplast DNA evolution 

in potato (Solanum tuberosum L.). Theor. Appl. Genet. 

81: 13-20. . 

Koch, W.; Edward, K.; Kassel, H. 1981. Sequencing of the 

16S-23S spacer in a ribosomal RNA operon of Zea mays 

chloroplast DNA reveals two split tRNA genes. Cell. 

25: 203-213. 

Lee, J.S.; Noh, E.W.; Lee, S.K.; Kwon, K.W. 1994a. Analysis 

of Populus cpDNA by restriction fragment length polymorphism (RFLP) techniques. J. Kor. For. Soc. 83: 20-24. 

Lee, J.S.; Noh, E.W.; Jang, S.S.; Lee, S.K.; Noh, E.R.; Lee, O.K. 

1994b. Development of species specific RAPD markers 

using Populus cpDNA. Kor. J. Breed. 26: 335-339. 

Liston, A. 1992. Variation in the chloroplast genes rpoC1 

and rpoC2 of the genus Astragalus (Fabaceae): evidence 

from restriction site mapping of a PCR amplified fragment. Amer. J. Bot. 79:953-961. 

Liu, Z.; Fumier, G.R. 1993. Comparison of allozyme, RFLP, 

and RAPD markers for revealing genetic variation 

within and between trembling aspen and bigtooth aspen. Theor. Appl. Genet. 87: 97-105. 

Mejnartowicz, M. 1991. Inheritance of chloroplast DNA in 

Populus. Theor. Appl. Genet. 82: 477-480. 

Nishio, T.; Sakamoto, K.; Yamaguchi, J. 1994. PCR-RFLP 

of S locus for identification of breeding lines in cruciferous vegetables. Plant Cell Rep. 13: 546-550. 

Omstead, R.G.; Palmer, J.D. 1994. Chloroplast DNA systematics: A review of methods and data analysis. American Journal of Botany. 81: 1205-1224. 

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Molecular Genetic Analysis of Populus Chloroplast DNA 

Rajora, O.P.; Dancik, B.P. 1992. Chloroplast DNA inheritance in Populus. Theor. Appl. Genet. 84: 280-285. 

Rose, R.J.; Schlarbaum, S.E. 1988. Chloroplast genomic 

variation and phylogeny in Medicago section Intertextae. 

Can. J. Bot. 66: 1352-1358. 

Salts, Y.; Hermann, R.G.; Peleg, R.G.; Lavi, U.; Izhar, 5.; 

Frankel, R.; Beckmann, J.S. 1984. Physical mapping of 

plastid DNA variation among eleven Nicotiana species. 

Theor. Appl. Genet. 69: 1-14. 

Shinozaki, K.; Tanaka, M.; Wakasugi, T.; Hayashida, N .; 

Matsubayashi, T.; Zaita, N.; Chunwongse, J.; Obokata, 

J .; Yamaguchi-Shinozaki, K.; Ohto, C.; Torazawa, K.; 

Meng, B.-Y.; Sugita, M.; Dena, H.; Kamogashira, T.; 

Yamada, K.; Kusuda, J.; Takaiwa, F.; Kato, A.; Tohdoh, 

N.; Shimida, H.; Sugiura, M. 1986. The complete nucleotide sequence of the tobacco chloroplast genome; its 

gene organization and expression. EMBO J. 5: 2043-2049. 

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D.H.; Sninsky, J.J.; White, T.J., eds. PCR protocols: A 

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restriction site maps of chloroplast DNA of Picea abies, 

Picea glauca, Picea mariana, and Picea sitchensis. Can. J. 

For. Res. 23: 427-435. 

149 
Chapter 19 

Molecular Marker Techniques to Study Variability of 

Populus Pathogens1 

Girma M. Tabor and Harold S. McNabb, Jr. 

Introduction 

Molecular genetic techniques are commonly used in 

many laboratories and many have been developed that 

address diverse problems in plant pathology. In recent 

years, tremendous advances have occurred concerning the 

molecular genetics of plant/parasite interactions including the cloning of plant genes for (Jones et al. 1994; Martin 

et al. 1993; Whitham et al. 1994) and corresponding 

avirulence genes in pathogens (Huynh et al. 1989; Kearney 

and Staskawicz 1990; Kobayashi et al. 1989, 1990; 

Staskawicz et al. 1984). Studies on the molecular genetic 

basis of in poplar are also underway (Cervera et al. 1996; 

Goue-Mourier et al.1996; Newcombe and Bradshaw 1996). 

However, poplar pathogen populations must be characterized before ecological interactions of pathogenesis and 

host resistance are fully understood. 

The crown gall pathogen (Agrobacterium tumefaciens) 

and its host interactions are genetically and biochemically well described (Beneddra et al. 1996; Binns 1990; 

Stachel et al. 1985, 1986; Yusibov et al. 1994; Zupan and 

Zambryski 1995). However, molecular genetic studies 

of other poplar pathogens and their associated diseases 

are just developing and are primarily tailored toward 

basic questions on taxonomy, epidemiology, and population genetics. An understanding of these basic qu estions is urgently needed so that critical processes 

involved in host-pathogens interactions will be defined 

and used to establish coherent selection and breeding 

programs for Populus spp. 

' Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.G.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

150 

Many molecular marker techniques are available to generate diverse genetic information for various purposes. 

Molecular marker techniques used to study Populus pathogens are the same as those used to study the host. Because 

these techniques are well described elsewhere in this volume (Cervera et al.; Lin et al.; Noh and Lee), a detailed 

description is not presented here. However, various attributes of these techniques are discussed within the context of this chapter. 

Polymerase chain reaction (PCR) methods are the most 

widely used molecular genetic techniques to study Populus 

pathogens. Although PCR ·offers a virtually limitless 

source of genetic information, its use is curtailed because 

basic information on the taxonomy, etiology, and epidemiology of the pathogens is lacking. 

In this chapter, we present some preliminary studies 

conducted to gain understanding about 2 economically 

important pathogens of Populus, Septaria spp. and 

Melampsora spp. Presently, random amplified polymorphic 

DNA (RAPD) and site-directed PCR are used in Populus 

pathology laboratories to provide basic molecular genetic 

information of Populus pathogens. 

Random Amplified Polymorphic DNA 

{RAPD) 

Diseases that are serious problems to many Populus 

clones are leaf and stem diseases caused by Septaria spp. 

In the north central and northeastern United States, 5. 

musiva (teleomorph: Mycosphaerella populorum) is considered a serious pathogen. Besides leaf spot, S. musiva causes 

lethal stem cankers on Populus triclzocarpa hybrids. In the 

Pacific Northwest (PNW) of North America, S. populicola 

(teleomorph: M. populicola) causes leaf spot without lethal 

stem cankers. Of current debate is whether these 2 Septaria 

populations are different species or whether canker development is limited by the environmental conditions in 

the PNW. Using RAPDs, Ward and Ostry (1996) showed 

that bulked collections from these 2 pathogen populations 

exhibit molecular polymorphism, suggesting that these 

populations may be isolated and distinct. They also found 
that Septaria population from the PNW did not exhibit 

much polymorphism. In contrast, molecular polymorphism was observed within populations from the North 

Central United States (figure 1). 

RAPD markers can be reliable for typing an individual. 

In contrast, typing a population using RAPDs is d ifficult 

because collected samples must accurately represent a ll 

individua ls of a population. However, obtaining a representative sample from wild populations is form idable and 

often impossible. In addition, RAPD markers a re not easy 

to reproduce. The problem of obtaining representa ti ve 

samples of a p opulation, coupled w ith the re prod ucibility 

problems o f RAPD markers, can rende r these markers 

unreliable for population typing. 

RAPD ma rkers linked to plant genes of interest can be 

successfull y used for selection, breeding, and other gene tic 

manipulations (Bradshaw and Stettler 1995; Goue-Mourier 

et a l. 1996; Ha ley et a l. 1993; Miche lmore et a l. 1991; 

ewcombe and Bradshaw 1996). Similarly, RAPD markers may be important when working w ith pathogen isolates that are inbreds or isogenic. 

Site-Directed Polymerase Chain Reaction 

(PCR) 

Site-directed PCR requires some p rior knowledge of target Dt A sequences to be amplified. The funga l ribosomal 

R A (rR A) genes are wid e ly used for site-d irected PCR 

because they comprise domains that are h ighly conserved 

Figure 1. Random amplified polymorphic DNA (RAPD) of 

Septaria spp. isolates from the North Central 

states, USA. From the left, lane 1 =1-kb DNA 

ladder; lane 2=RAPD control (no template DNA); 

and lanes 3 through 20=RAPDs various Septaria 

spp. isolates from the North Central states using 

Operon primer F-1 0, 5' to 3' - GGAAGCTIGG. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Molecular Genetic Analysis of Populus Chloroplast DNA 

among a wide range of taxa. The conserved nature of these 

domains has allowed the synthesis of genera l p rimers that 

can amplify several regions of the rRNA operon from various fungi. Consequently, this has allowed the synthesis of 

taxa-specific primers (Gard es and Bruns 1993; Tisserat et 

a!. 1994; White et al. 1990). 

A typica l eukaryotic nuclear rRNA is polycistronic; coding regions for 185, 5.85, and 285 rR 'As are g rouped (in 

the order listed) within a single transcription unit. An entire transcribed unit includes 2 noncoding regions, termed 

the internal transcribed spacers (ITS), which separate the 

3 coding regions from each other. Sequences in the rRNA 

coding. regions a re h ighly conserved among related taxa. 

Little or no variation is evident among rRNA ~od in g sequences of closely re la ted taxa at the class, family, genus, 

or species level. This lack of variation in the rRNA coding 

regions among related taxa limits their use as a diagnostic 

tool. 

Besides the conserved regions, the rRN A operon of several fungi consists of regions that can vary among species 

in a genus and among subspecies within a species. These 

variable regions can have practical importance to a Populus 

pathologist. Species, subspecies, and hybrids potentia lly 

can be identified based on their differences in the variable 

regions of the rR TA op eron. Depending on the taxa, the 

ITS regions of fungi may vary in length and ON A sequence 

(Ga rd es and Bruns 1993; Tisserat e t a l. 1994; Wh ite et al. 

1990) und muy be used to identify and classify funga l 

pathogens o f Populus. 

The other region of the rRI A that may be of practical 

value to a p lant pathologist is the intergenic spacer (IGS) 

region. In most organisms, rRNA genes occur as multip le 

copies clustered in long tandem a rrays on several different chromosomes. A single transcription uni t w ithin a cluster is separated by an IGS that is not transcribed. In some 

fungi, the IGS regions con tain 55 genes, producing 2 IGS 

regions (Kim e t a l. 1995). IGS lengths and sequences are 

known to va ry among related taxa, wh ich can be exp loited 

for practical identification purposes. 

Coding a nd noncoding regions of rR 'A genes from several fungi can be readily PCR amplified with genera l primers that target rRNA genes from diverse organisms. PCR 

products then may be sequenced using automated sequencing machines, and these DNA sequences can be used 

for diagnostic and phylogenetic studies. 

When ITS and IGS lengths are the same from separate 

pathogen isolates, seque ncing may reveal some d ifferences 

in the DNA. Based on the DNA sequence diffe re nces, 

unique enzyme restriction sites may be identified and used 

to generate Dt A restriction patterns that are unique to 

these pathogens. In addition, based on D A sequence differences, specific PCR p rimers can be designed for selecti ve a mplifi ca ti o n of D NA fr agments from specific 

pathogen races or species (Gardes and Bruns 1993; Tisserat 

et a!. 1994). 

151 
Section Ill Molecular Biology 

In our laboratory, ITS regions and the 5.8S rRNA 

genes of the 3 major Melampsorn species on ~opul s w~ e 

amplified using gene ra l prime rs. Derive~ mf~rmat10n 

will be used to ide ntify species and p ossible mte rspecific hybrids among M. medusae, M. occidental is, a nd M. 

larici-popul ina. 

Materials and Methods 

Template DNA 

Template DNA was obta ined by eithe r adding a few 

(10 to 30) ured iospores directly to the PCR mixture or by 

extracting genomic 0 'A from urediospores. To extract 

genomic DNA, fresh d ry u rediospores were mixed with 

an equal volume of d iatoma ceous earth product and 

ground with a plastic pestle operated by an electric dri~l. 

The lysis buffer containing 50 mM Tris-HCI (pH 7.2), ::JO 

mM EDTA (pH 8.0), and 3 percent sodium dodecyl sulfate (SDS) was a utoclaved for 15 min. The grinding slurry 

was centrifuged at 800 x g and incuba ted at 65 oc for 1 h. 

After incubation, standa rd p henol/ chloroform extraction 

and ethanol precipitation were cond ucted to obtain the 

temp late DNA. 

Primers and PCR Amplification 

Primers u sed in this procedure we re 

ITS4 (TCCTCCGCTTATTG AT ATGC) a n d ITS5 

(GGAAGTAAAAGTCGTAACAAGG). These primers 

a re uni versa l because they amplify rRNA genes from 

wide range of fun gi (Ga rd es and Bruns 1993; Tissera t 

et a l. 1994; White et al. 1990). 

The amplification reaction was performed in 100 1t1 

volumes containing 10 mM Tris-HCI (pH 8.3), 50 mM 

KCI, 200 11M of each dNTP (dATP, d TTP, d GTP, and 

dCTP), 0.5 11M of each primer, 2.5 units of Taq polyme rase, and 1.5 mM MgCI

2

• Amplifica ti on was conducted for 35 cycles of d enaturation (1 m in) at 93 °C, 

annealing (35 sec) at 58 °C, and ex ten sion (2 min) a t 72 

oc. Fin a l extensional 72 oc was pe rformed fu r 10 min. 

All reagents were obtained from the Perkin-Elmer Corporation (Norwa lk, CT, USA). 

Restriction Enzyme Digestion 

The PCR product was electrophoresed in 1.4 percent 

agarose gel, stained with ethidium bromide (0.5Jig / l), and 

visualized under UV light. The PCR product then was digested with severa l restriction enzymes in attempts to detect any restriction fragment length polymorphism. 

152 

Sequencing of PCR Product 

The PCR products were purified with spin columns 

(Amicon, Inc., Beverly, MA, USA). Product concentration 

was measured using a fluorometer, and the concentration 

was adjusted for a utomated sequencing. 

Results 

Amplified produ cts from intact spores and genomic 

DNA were approximately 700 base pairs and no difference in product size was observed among tested species 

of Melampsorn (fig ure 2). Restriction digestion products of 

several enzymes did not produce any de tectable polymorphism for use in distinguishing the 3 species. This also 

was supported by DNA sequence data in that ITS and 5.8S 

rRNA gene sequences from all 3 species showed a very 

high degree of similarity. These ITS and 5.8S seque ~es 

from Melmnpsom spp. were also highly homologous With 

those from othe r rust fungi. 

Discussion 

Our results indicate that DNA sequences of ITS regions 

from the 3 Melampsorn species a rc identical. Thus far, these 

sequences ha ve exhibited no distinguishing characteristics for d iagnostic purposes. As expected, the 5.8S genes 

were also identica l. Although the ITS and 5.8S sequences 

cannot be used for disting uishing these 3 species, these 

DNA sequences a re useful for phylogenetic comparisons 

with othe r orga nisms. The ability to amplify rRNA genes 

without ex tracting genomic DNA grea tly simplifies studies on rRt A genes of Me/am psora species. 

Figure 2. Polymerase 

chain reaction (PCR) 

products showing the 2 

ITS regions and the • 

5.8S rRNA gene from 3 

species of Melampsora 

with primers ITS4 and 

ITS5. Intact spores 

provided as template 

DNA. From the left, 

lanes 1 through 3=M. 

larici-populina; lanes 4 through 6=M. occidentalis; lanes 

7 through 9=M. medusae; lane 1 O=control (no template); 

and lane 11 =1 00 bp ladder. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Acknowledgments 

This research was supported under Subcontract No. 19X43391C with Oak Ridge National Laboratory under Martin Marietta Energy Systems, Incorporated contract 

DE-ACOS-840R21400 with the U.S. Department of Energy. 

We thank Dr. Michael E. Ostry for providing a gel photograph used in this manuscript (figure 1 ). 

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USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Section IV Biotic and Abiotic Resistance 
Chapter20 

In Vitro Screening and Selection for Disease Resistance1 

Michael E. Ostry 

Introduction 

Poplar improvement efforts are directed at improving 

tree traits such as rapid early growth, yield, and fiber quality. The goal of improvement programs is to increase resistance to diseases that have the potential to reduce yields 

or kill trees. Selection, breeding, and testing for poplar 

disease resistance are hampered by the long generation 

time of trees, the difficulty in identifying and capturing 

desired traits using classical breeding techniques, and the 

limited knowledge of juvenile-mature correlations of desired traits. 

Screening for disease resistance in the field is time-consuming, costly, and dependent upon natural fluctuations 

in inoculum abundance and weather factors that influence 

pathogen spread, infection, disease development, and disease expression. Trees in field tests are also subject to many 

pathogens and insect pests that periodically confound test 

results (Ostry et al. 1989). 

Various cultural and chemical control strategies have 

been suggested for most of the major poplar pathogens. 

If available, the use of resistant clones is the best longterm management practice. Selection of adapted, highly 

productive clones with disease resistance is not easily 

achieved. Host-parasite interactions are complex, dynamic, and affected by many environmental variables and 

the developmental stage and general health of the host. 

These factors can significantly affect disease resistance or 

tolerance. 

Application of new techniques in molecular biology and 

plant biotechnology to poplar improvement may decrease 

the time necessary to introduce new traits and increase 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

efficiency of new genotype selection and screening (Daub 

1986; Helgeson 1983; Miller and Maxwel11983; Ostry and 

Michler 1993; Ostry and Skilling 1992). Cell and tissue 

culture techniques are essential to many of these approaches and most have been successfully applied to poplars (Ostry and Ward 1991). Techniques range from a tissue 

culture method for eliminating viruses from poplar clones 

(Berbee et al. 1976) to using aspen root cultures for screening isolates of the fungus l.Accaria bicolor for their ability to 

form ectomycorrhizae, which may improve tree survival 

and growth (Ostry et al. 1994). 

The availability of a wide array of in vitro techniques for 

manipulation and regeneration of poplars offers new approaches to study host-parasite interactions. In vitro techniques may provide methods to rapidly screen and select 

poplars for resistance or tolerance to disease with gx:eater 

efficiency than traditional field tests. Various in vitro techniques, including disease resistance screening, can greatly 

contribute to successful poplar breeding programs 

(Frohlich and Weisgerber 1985). 

In vitro techniques to screen poplars for disease resistance has many important advantages over screening intact plants in the field, growth room, or greenhouse. In 

addition to reducing the time and cost for testing, perhaps 

the greatest advantages of in vitro techniques are the precise control of the physical and chemical environmental 

conditions, the ability to rapidly screen a large number of 

genotypes in a small space, and the exclusion of other microorganisms. In vitro techniques take advantage of simplified experimental host-parasite systems where 1 or a 

few host cell types can be uniformly challenged by a pathogen or host-specific toxin. 

In vitro approaches to disease resistance screening also 

have some important disadvantages. Resistance is probably determined by multiple factors, and may be governed 

by a series of biochemical reactions influenced by many 

host, pathogen, and environmental factors not present 

during in vitro testing. The general health of the host and 

accompanying stress factors may differ from intact plants 

in the field. The potential absence of preformed defensive 

barriers, induced inhibitory compounds, and organized 

tissues may limit the usefulness of in vitro screening techniques, especially if cell and tissue culture systems are 

155 
Section IV Biotic and Abiotic Resistance 

used. While these limitations may not exist when plant 

parts are used, wounds made to the plant part before or 

during screening may alter the expression of resistance. 

Major differences may also exist in the physiology and 

ploidy levels of plant cells or plant parts compared with 

intact plants. Cells and tissues in culture are actively growing and their reactions to pathogens may differ from mature tissues in intact plants. Also, plant hormones used in 

tissue culture media may influence host-parasite interactions, altering responses from those normally expressed 

ex vitro. 

TISsue cultures are not necessarily free from pathogens; 

these organisms can occur in cultures and regenerated 

plants (Cousin et al. 1990). Additionally, in vitro selection 

for a specific trait may inadvertently select undesirable 

traits. In vitro selection techniques may enhance, but not 

necessarily replace, traditional methods for poplar improvement. 

Examples of in vitro techniques for screening of poplars 

are briefly reviewed in this chapter. Some examples include 

tissue and cell culture techniques where the plant cells or 

plant cultures are challenged by the pathogen, and others 

include laboratory challenges of plants or plant parts that 

were grown in the field or greenhouse. The examples are 

arranged by the type of stress or pathogen that was studied. Examples of techniques used for screening for resistance tp damaging abiotic agents are also provided. In all 

cases, a brief description of the methodologies and summaries of the results are included. 

Abiotic Stress Agents 

There are few reports of in vitro techniques used to study 

the effects of abiotic agents on poplars. A tissue culture 

assay in which the osmotic potential of the media was 

modified with polyethylene glycol (PEG) was used to determine if poplar callus culture responses to drought stress 

were similar to assays with whole plants (Tschaplinski et 

al. 1995). The callus cultures failed to display osmotic adjustment to water stress. The authors concluded that callus cultures could not substitute for assessing water stress 

at the whole plant level. 

Callus cultures of Populus maximowiczii were used to 

compare levels of exogenously supplied lead with culture 

morphology and anthocyanin accumulation, and to determine the deposition and concentration of lead within 

the cultured cells (Ksiazek et al. 1984). Cultures were 

grown under dark or light conditions for 2 weeks, then 

exposed to aqueous lead solutions at various concentrations for 24 h. Lead reduced both the biosynthesis of anthocyanins and total fresh weight; perhaps because of cell 

division disturbances. Lead accumulated predominantly 

in the intercellular spaces and plant cell walls of the cultured tissues .. 

156 

Callus and cell suspension cultures were employed to 

study the aluminum tolerance of a hybrid poplar clone 

(Choi et al. 1987). Fresh weight of callus cultures and dry 

weight of cell cultures were significantly reduced by increasing aluminum concentrations. 

Callus and adventitious shoot cultures were used to 

screen poplar clones for salt (NaCI) tolerance (Li and Chen 

1984). Similarly, shoot tip and bud cultures were used to 

screen hybrid poplar clones for salt tolerance (Lee et al. 

1986). Increasing salt concentrations decreased culture 

growth. Large differences in growth responses were detected among the clones. Variations in salt tolerance were 

also detected among individual plants within the clones. 

The authors suggested that although tolerant clones were 

identified, they required further testing under field conditions to provide planting recommendations for saline 

soils. 

Screening for Antimicrobial Activity 

Callus cultures of aspen (P. tremuloides) produced bactericidal substances that stimulated and then inhibited an 

Agrobacterium bacteria species in vitro (Mathes et al.1971). 

This activity depended upon how long the callus cultures 

were grown before inoculation with the bacterium 

(Mathes et al. 1971). Callus was produced from sterilized, 

stem internodal sections incubated on a basal medium in 

the dark. The antimicrobial substance was secreted into 

the medium and was active in the absence of host cells. 

This type of assay may be useful to screen poplar genotypes for host-specific compounds that may have roles in 

ex vitro. Compounds in the bark of aspen were fungistatic, 

inhibiting the Hypoxylon mammatum pathogen (Hubbes 

1966; Kruger and Manion 1994) and other organisms 

(Mathes 1963). Similar investigations should contribute 

to the study of. host responses and production of preformed and induced defense compounds. 

The possible roles of abscisic acid (ABA) in defense reactions were studied using callus cultures of a P. x 

euramericana clone (Hrib and Rittich 1992). Callus cultures 

obtained from stem internodes and leaf blades were cocultured with the fungus Phaeolus schweinitzii in Petri 

dishes, and the fungal growth was monitored. Stem-derived callus had a higher ABA content and a greater inhibition of fungal growth than leaf-derived callus. The 

authors suggested that ABA intensifies the defense reaction and plays a role in. In contrast, Stopiska (1994) determined that leaves of a poplar clone, found to have the 

most resistance to Ceratocystis fimbriata, had less ABA 

content than a susceptible clone. It was concluded that 

the levels and/ or ratios of the plant growth regulators 

that were measured were responsible for the levels of 

among the poplar clones studied. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Bacterial Canker 

The interactions of Xanthomonas populi and poplar tissues were studied by inoculating stem explants of oneyear-old branches (Lange 1968}. Growing the explants 

under sterile conditions allowed detailed study of the effects of a-naphthaleneacetic acid (NAA}, tissue development, and wounding on infection and cell division. Results 

showed that actively metabolic plant cells and wounding 

were necessary for canker formation. Bacterial growth 

stimulated by NAA occurred in the intercellular spaces 

of callus tissues. The authors suggested that bacterial levels and the developmental state of host tissues were important factors for symptom expression. Callus cultures 

were used to study the effects of X. populi on cell division 

and growth of callus tissues (Krawiarz and Przybyl1980). 

Results confirmed earlier reports that this bacterium induced poplar cell division and stimulated callus tissue 

formation. In another attempt to develop a technique for 

disease resistance screening, Kechel and Boden (1985a, 

1988) inoculated several clones of in vitro grown plants 

with X. populi. Clonal response to inoculation was similar 

to those of trees inoculated in the field and to young, 

rooted plants regenerated from tissue culture (Kechel and 

Boden 1985b ). 

Melampsora Leaf Rust 

Leaf rust, caused by several species of Melampsora fungi, 

is a potentially serious worldwide poplar disease. Investigators have relied on in vitro techniques using detached 

leaves and leaf disks for detailed studies on the interactions of these highly variable rust fungi with many poplar clones and species. Detached leaf cultures offer many 

important advantages in studying obligate pathogens 

(Chandrashekar 1982; Shain 1974). Such studies have focused on the: infection process (Shain and Jarlfors 1987}, 

pathogen variation (Chandrashekar and Heather 1980; 

Hsiang and van der Kamp 1985; Hsiang and Chastagner 

1993; Pinon et al. 1987; Pinon and Peulon 1989; Shain 1988}, 

effects of ozone on leaf rust interaction (Coleman et al. 1987), 

and resistance among poplar species and clones (Hamelin 

et al. 1994; Heather et al. 1980; Lefevre et al. 1994; Prakash 

and Heather 1986a, 1989; Singh and Heather 1982a). 

Races within isolates of M. medusae collected from natural stands of P. deltoides were identified using a leaf disk 

assay to reveal differences in latent period, uredial production, and isolate x cultivar interactions (Hamelin et al. 

1992; Prakash and Thielges 1987). Inoculation of leaf disks 

from a set of host differentials also revealed the presence 

of races of M. larici-populina, the Eurasian rust fungus first 

reported in the United States in 1991 on various species of 

poplars in California and Washington (Pinon et al. 1994). 

Temperature and light were identified as critical factors 

in disease expression of rust on inoculated leaf disks. This 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

In Vitro Screening and Selection for Disease Resistance 

supports the hypothesis that environment plays an important role and emphasizes the importance of field screening (Chandrashekar and Heather 1981; Prakash and 

Heather 1986b; Prakash and Thielges 1989a; Singh and 

Heather 1982b, 1982c). Tissue culture and a leaf disk bioassay were employed to demonstrate somaclonal variation in P. deltoides for race-specific resistance to leaf rust 

caused by M. medusae (Prakash and Thielges 1989b). 

Marssonina Leaf Spot 

A laboratory technique was developed that uses excised 

leaf disks to compare the resistance of poplar clones to 

Marssonina fungi (Spiers 1978). This technique is a modification of earlier methods used to screen poplar clones to 

M. brunnea and leaf rust. A cork borer was used to punch 

leaf disks from tree leaves and make wells in Petri dishes 

of 2 percent water agar. Leaf disks were placed in the agar 

wells, inoculated with conidia of Marssonina fungi, and 

incubated in natural light at room temperature for 8 to 12 

days. The level of resistance among test clones was classified using a disease rating scale based on the number of 

lesions per unit area. Disease resistance varied with clone, 

and clonal resistance varied with the applied concentration of conidia. This technique is useful to evaluate the 

relative resistance among clones; however, its use to evaluate field resistance is limited unless the level of natural 

inoculum is known. 

Septoria Leaf Spot 

An excised leaf disk bioassay was also used to screen 

hybrid poplar clones for resistance to Septaria musiva; results were compared with the known field reactions of 

these clones (Ostry et al. 1988). The modified method of 

Spiers (1978) allowed for separation of clones with similar 

field resistance to S. musiva. The segregation was based on 

disease progress curves obtained by monitoring progressively enlarging areas of necrosis on inoculated leaf disks 

that were incubated in the light over 32 days. Results correlated well with those obtained under natural field conditions. Spore concentration was not a factor in the 

classification of clones and the results were repeatable. 

However, the authors suggested that this technique should 

be considered only as a preliminary screen for clones before field tests. This method was also used to identify increased resistance to Septaria leaf spot in poplar plants 

that were regenerated from tissue cultures of a previously 

susceptible clone (Ostry and Skilling 1988). 

Hypoxylon Canker 

Several investigators have utilized tissue cultures to 

study Hypoxylon canker caused by the fungus Hypoxylon 

157 
Section IV Biotic and Abiotic Resistance 

mammatum (Ostry et al. 1990; Race and Manion 1994; Valentirle et al. 1988). To screen and propagate aspen resistant to the canker, Wann (1985) challenged aspen 

hypocotyls and cotyledon explants with a toxin from H. 

mammatum. Surviving explants were elongated, rooted, 

and transferred to soil. These plantlets were tested for toxin 

resistance using a leaf puncture bioassay. The result was 

that toxin resistance expressed in vitro was also expressed 

by intact plants. It was proposed that this resistance trait 

was not induced by the tissue culture system but was natural in all families tested (Einspahr and Wann 1985}. The 

investigators suggested that this technique has potential 

value for selecting aspen with canker resistance. 

Cambial activity of various poplar clones and species in 

the presence of H. mammatum culture filtrates was investigated (Pinon 1986). Effects on callus proliferation from 

challenged stem internode sections were compared with 

the responses of intact trees after field inoculations. Results indicated that the filtrate assay could select among 

susceptible and nonhost poplar species, but was not sufficiently specific for use in early selection of resistant aspen 

clones. A toxin-tolerant line of callus was obtained from 

Leuce (currently termed Populus) poplars and a leaf bioassay indicated that plants regenerated from this line retained the toxin resistance (Antonetti and Pinon 1993). 

Culture filtrates of H. mammatum and aspen shoot cultures were used to examine the correlation of a toxin bioassay to disease incidence in the field (Belanger et al. 

1989a). Although clonal differences in reactions to culture 

filtrates were detected, the bioassay results did not correlate with disease incidence in the field. This suggests that 

other variables are involved in this pathosystem. Further 

investigations using shoot cultures revealed that moisture 

stress and related changes in amino acids may be important to the susceptibility of aspen clones to infection by H. 

mammatum (Belanger et al. 1989b, 1990). 

Using shoot cultures from parent trees and their progeny, 

Kruger and Manion (1993a) demonstrated that sensitivity 

to H. mammatum culture filtrates was under genetic control. 

The authors suggested that the demonstrated resistance 

was horizontal and controlled by a small number of genes. 

However, the in vitro response of selected aspen clones to 

culture filtrates was not related to their response when 

inoculated with ascospores (Kruger and Manion 1993b). 

Summary 

Reports are increasing on the potential of in vitro screening for disease resistance in poplars; however, because of 

technique limitations, more research is needed before they 

can be practically and reliably applied to poplar improve158 

ment efforts. We need a better understanding of the genetics of host-pathogen interactions, the role of environmental variables, and juvenile-mature correlations to host 

resistance. Many investigators strongly suggest that in vitro 

screening can provide preliminary information on host 

responses to pathogens, but these techniques should not 

be considered as suitable substitutes for field tests. Although there have been several reports of strong correlations with in vitro screening to field results, field verification 

is frequently lacking. 

The examples reviewed include in vitro techniques to 

screen entire plants or detached plant parts for disease resistance. A more powerful technique would involve selection at the cellular level with characterized, host-specific 

toxins. Thus far, the limited work with crude or partially 

purified toxins from poplar pathogens has had only partial success. 

Advances in biotechnology, the use of molecular genetics, and the availability of poplar pedigrees will provide 

future opportunities to study host defense mechanisms. 

These new tools will provide great assistance to develop 

and refine disease resistance screening and selection techniques to increase yields and reduce poplar production 

costs. 

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USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
'*" 

'-' 

~ 

~ 

Chapter21 

Transformation of Hybrid Aspen for Resistance to 

Crown Gall Disease1 

~ Hiroyasu Ebinuma, Etsuko Matsunaga, Keiko Yamada, and Mikiko Yamakado 

Introduction 

'Kitakami Hakuyou' are elite clones of hybrid aspen 

produced by Nippon Paper Industries (NPij for commercial use. These elite clones were obtained by crossing 5 

selected female trees of Populus sieboldii with a male elite 

tree of Canadian P. grandidentata, exhibit better growth features than other species or interspecific hybrids belonging 

to the section Leuce (currently termed Populus), and express significant heterosis (Takayama 1968). These characteristics make them well suited for afforestation and pulp 

wood in Japan. However, in some geographic areas, 

'Kitakami Hakuyou' clones are extremely sensitive to 

crown gall disease, caused by Agrobacterium tumefaciens. 

This disease can cause considerable economical loss to 

nurseries growing rosaceous plants, Rubus species, grapevines, or various nut-bearing trees. The phytopathogenic 

bacterium, A. tumefaciens, infects a wide variety of dicotyledonous plants and induces tumors on the infected plants 

(Moore and Warren 1979). Because this bacterium provides 

a useful method to introduce desirable genes into plants, 

we have used biotechnology in an attempt to improve characteristics of 'Kitakami Hakuyou.' Our objective was to 

use an antisense DNA method to improve crown gall disease resistance in aspen clones (Ebinuma et al. 1991, 1992). 

In this paper, we report on the construction of plasmids 

containing antisense DNA, and on a highly efficient transformation procedure for hybrid aspens. 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.~ Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

Construction of Vectors 

Containing Resistance Genes 

to Crown Gall Disease 

A. tumefaciens introduces genes located on the transfer 

DNA (T-DNA) of the tumor-inducing plasmid (TI-plasmid; 

Kim et al. this volume) into the plant genomic DNA, and 

induces tumors in the infected regions (Bevari and Chilton 

1982). In the tumors, plant hormones, auxin and cytokinin, are over produced due to the expression of 3 integrated 

oncogenes (iaaH, iaaM, and ipt) in the plant genome. We 

used the 1i plasmid of the pathogenic A. tumefaciens strain 

P022, which was isolated from a natural crown gall on 

hybrid aspen, to make antisense DNA constructs (Wabiko 

et al. 1989). We constructed the binary vector based on the 

plasmid pBI121, which contained the 1.1-kilobase (kb}, 

EcoRI fragment 34 of the iaaH gene and the 0.7-kb, Hindiii 

fragment 41 of the iaaM gene in the sense (S) or antisense 

(A) orientation under the control of the cauliflower mosaic virus 355 promoter (figure 1). The plasmid was transferred to A. tumefaciens strain LBA4404 and used to 

transform tobacco. 

A dramatic inhibition of gall formation was observed 

after infection by a virulent A. tumefaciens strain in 1 of the 

5 transgenic tobacco plants expressing a part of iaaM gene 

in the sense orientation (41s4} (table 1, figure 2). Another 

of the 5 transgenic tobacco plants with the antisense orientation (41a3) showed weak, but distinct, resistance. We 

investigated the copy number and expression of the integrated genes in transgenic tobacco plants by the Southern 

and northern hybridization analyses (Sambrook et al. 

1989). Southern analysis revealed that the resistant 

transgenic plant 41s4 contained 4 copies of the sense insert, the nonresistant plant 41s2 had 1 copy, and 41s3 had 

6 copies. The amount of transcript in the resistant plant 

(41s4} was lower than that in the nonresistant plants (41s2 

and 41s3); however, no correlation between transcriptional 

activity and resistance was detected. Although the molecu161 
Section IV Biotic and Abiotic Resistance 

iaaH iaaM 

E E H H 

pBI121 GUS345 iaaH34A -Haai 

415 iaaM -

41A -Maai 

Figure 1. Plasmid construction. iaaH=indoleacetamide 

hydrolase gene; iaaM=tryptophan monooxygenase gene; GUS=glucuronidase gene; 

NPT //=neomycin phosphotransferase gene; 

35S=35S promoter of cauliflower mosaic virus; 

N=promoter of nopaline synthase gene; 

T =polyadenylation signal of nopaline synthase 

gene; E=EcoRI restriction site; H=Hindll l restriction sites. 

Table 1. Bioassay of tumorigenicity using leaf discs of 

transgenic tobacco. 

Tobacco Number of Percentage of discs 

clones' leaf discs inoculated2 producing tumors2 

C1 15 93 

Lc2 15 73 

41a3 15 80 

41s43 15 0 

1 C1 =normal tobacco plants 

Lc2 =transgenic tobacco plants transformed by p81121 

41 a3 = transgenic tobacco plants transformed with antisense DNA fragment 34 

41 s4 = transgenic tobacco plants transformed with 

sense DNA fragment 41 

2 Leaf discs were inoculated with pathogenic 

Agrobacterium tumefaciens and placed on agar plates (5 

leaf discs/plate, total of 3 plates) and gall development was 

examined on the 15 leaf discs. 

3 Nine little shooty teratomas developed on leaf discs. 

162 

Control 41s4 

Figure 2. Leaf disc tests of transgenic tobacco. 

Control=stem segments test; 

41 s4=stem infection test. 

lar mechanism is not fully understood, our findings indicate that crown gall disease in ~ri d aspen may be red uced by the 0.7-kb, Hindiil fragment 41 of the iaaM gene 

in the sense orientation under the control of the 355 promoter. 

Highly Efficient Transformation 

Procedures for Hybrid Aspens 

To improve the genetic transformation o f hybrid aspens, we developed improved regenera tion procedures. 

Beca use shoot regene ration frequencies can vary from 

0 to 100 percent d epending on the variety of Populus 

sp p., commercia lly useful trees in the gen us Populus 

frequently exhib it lower regeneration frequencies than 

model trees used in resea rch. To help overcome this 

problem, we applied an Agrobacterium transformation 

method, developed for tobacco, to ou r commercial hybrid aspen clones (Matsunaga et al. 1992). This tra nsfo rmation procedure requires 2 steps, induction and 

growth of transgenic ca llus from infected stem segments, and shoot regen eration from the callus. This 

method has 3 potential problems: 1) the ca llus growth 

stage presents a high risk of generating genetic mutants; 

2) a relative ly long time is required for shoot regeneraUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
tion; and 3) the efficiency of obtaining transgenic trees 

with the desired gene is very low. To alleviate these 

problems, we developed an improved tissue culture 

method that increases the frequency of ad ventitious 

shoot differentiation of our commercial hybrid aspen 

clones. 

By investigating the composition of the nitrogen 

source in the tissue culture medium, we found tha t concentrations of both nitrogen sources, ammonium and 

nitrate, influence the physiological state that induces 

differentiation of adventitious buds. Molar ratios from 

1:2 to 1:5 of ammonium to nitrate were tested in the 

cult~re medium. The ratio of 1:3 was particularly effective for highly efficient regeneration of adventitious 

buds. By using a culture medium with this nitrogen 

composition, we could directly diffe rentiate adventitious buds from infected internodal segments of hybrid 

aspen clones, without employing a prelimina ry step for 

inducing and growing callus, and subsequently regenerate plantlets. This method should reduce risks o f genetic mutations during culturing, and requires a shorter 

regeneration time because extra steps are not required 

to induce and grow callus. 

The binary vector plasmid, pBI121, which contained the 

0.7-kb, Hindiii fragment 41 of iaaM gene in the sense (S) 

orientation under control of the 355 promoter, was transferred to A. tumefaciens strain LBA4404. Under aseptic conditions,.stems of flask-grown plants of hybrid aspen clone 

'Y63' (P. sieboldii x P. grandidentata) were cut into internodal segments of 5 mm in lerygth. Stem segments were 

further cut longitudinally into 2 pieces, then inoculated 

with the A. tumefaciens vector strain. Infected stem segments were transferred to modified Murashige and Skoog 

(MS) (Murashige and Skoog 1962) agar solid medium (2 

percent w I v sucrose, 0.5 mg /1 zeatin, 500 mg/1 ca rbenicillin, 100 mg / 1 kanamycin, and 0.8 percent w /v agar) in 

which nitrogen was supplied as 10 mM ammonium and 

30 mM nitrate. After culturing for 2 months at 25 oc under 3,000 lux, 20 to 40 percent of the inoculated stem segments regenerated adventitious buds. After culturing an 

additional month under similar conditions, the foliage 

frcm each bud had grown to a length of 2 to 3 em. These 

stems were aseptically excised and subcultured to a modified 2/3-strength MS medium, in which zeatin was replaced by 0.05 mg /1 indole-3-butyric acid (IBA) for 

rooting. The rooting culture continued and 85 plantlets 

were obtained after 1 month. 

Resistance of transgenic aspens to crown gall disease 

was evaluated by bioassay tests of tumorigenicity. Stem 

segments of transgenic and control hybrid aspens were 

inocula ted with a pathogenic A. tumefaciens strain P022 

with the same method used for transformation. Inoculated 

stem segments were placed on MS agar medium to 

observe gall formation. In resistant clones, we observed 

the development of teratoma tous shoots (figure 3a). For 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Transformation of Hybrid Aspen for Resistance to Crown Gall Disease 

a) stem segments test 

Control Transgenic Aspen 

b) stem infection test 

Control Transgenic Aspen 

Figure 3. Bioassays of transgenic hybrid aspen. 

Transgenic aspen with sense DNA fragment 41 . 

a) Internodal stem segments were infected with 

pathogenic Agrobacterium tumefaciens strain 

P022 and placed on agar plates. b) Internodes 

of stems were wounded with a needle and 

inoculated with A. tumefaciens strain P022. 

another bioassay, we wounded 5 internodes of greenhouse-grown, transgenic aspens with a needle a t 1 point 

pe r inte rnod e, and inoculated with A. tumefaciens strain 

P022. In approximate ly 10 percent of the transgenic 

plants, chara cteristic symptoms of crown gall disease 

were inhibited (figure 3b); however, considerable variability in gall development was apparent. Although 

several aspects need in tensive investi gation, these results provide interesting p rospects for use of an 

antisense Dt A method to improve crown gall disease 

resistance in aspen clones. 

163 
Section IV Biotic and Abiotic Resistance 

Acknowledgments 

The authors want to thank Drs. H. Sano and H. Wabiko 

for their collaborative works to produce a transgenic tobacco resistant to crown gall disease. 

Literature Cited 

Bevan, M.W.; Chilton, M.-D. 1982. T-DNA of the 

Agrobacterium Ti and Ri plasmids. Ann. Rev. Genet. 16: 

357-384. 

Ebinuma, H.; Wabiko, H.; Ohsima, K.; Hata, K.; Sano, H. 

1991. The genetic engineering of poplar trees.- Construction of resistant genes to crown gall disease. Forest Breeding. 4: 14-17. 

Ebinuma, H.; Wabiko, H.; Ohshima, K.; Hata, K.; Sano, H. 

1992. The genetic engineering of poplar trees.- A first 

practical application of a homology-based interaction 

(Matzke effect) for protection against the plant disease. 

In: Proceedings, 1992 Fifth international conference on 

164 

biotechnology in the pulp and paper industry: Tokyo, 

Japan. Uni Publishers Co., LTD.: 467-472. 

Matsunaga, E.; Ebinuma, H.; Yamada, K.; Ohsima, K.; Hata, 

K. 1992. Introduction of resistant genes into hybrid aspen and analysis. In: Abstract of Papers, 1992 Second 

tree molecular biology symposium: 72-77. 

Moore, L.W.; Warren, G. 1979. Agrobacterium radiobacter 

strain 84 and biological control of crown gall. Annu. Rev. 

Phytopathol. 17: 163-179. 

Murashige, T.; Skoog, F. 1962. A revised medium for rapid 

growth and bioassays with tobacco tissue cultures. 

Physiol. Plant. 15: 473-497. 

Sambrook, J.; Fritsch, E.F.; Maniatis, T., eds. 1989. Molecular cloning: A laboratory manual. 2nd edition. Cold 

Spring Harbor, NY, U.S.A.: Cold Spring Harbor Laboratory Press. 

Takayama, Y. 1968. Studies on the breeding of aspens (I). 

-Height growth in the early stage of F1 seedlings of 

Populus sieboldii Miq. x P. grandidentata Michx. Nichirinshi. 50(9): 267-273. 

Wabiko, H.; Kagaya, M.; Kodama, I.; Masuda, K.; Kodama, 

Y.; Yamamoto, H.; Shibano, Y.; Sano, H. 1989. Isolation 

and characterization of diverse nopaline type Ti plasmids of Agrobacterium tumefaciens from Japan. Arch. 

Microbial. 152: 119-124. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Chapter22 

Designing Small Antimicrobial Peptides and 

Their Encoding Genes1 

William A. Powell and Charles A. Maynard 

Introduction 

This chapter describes an ongoing process to genetically 

engineer durable pathogen resistance into poplar and other 

tree species. The process involved designing, testing, and 

selecting antimicrobial peptide sequences to use as 

transgenic plant gene products. The synthetic peptide sequences were then encoded into DNA. Gene promoters, 

translation initiation and termination sequences, codon 

preferences, messenger RNA (mRNA) secondary structure, 

peptide stability, and peptide targeting were considered 

when de5igning gene constructs to test in transgenic plants. 

At this writing, the first of several gene constructs are being used to transform hybrid poplar. 

Identifying Opportunities for 

Genetic Engineering 

Modifying the genotype of a plant through conventional 

breeding is a slow process, especially if several cycles of 

breeding and selection are necessary to achieve the desired objectives. If the plant is a long-lived woody species 

that may take years to flower and may not express the 

character of interest until it reaches maturity, the problems and expenses can be astronomical. Genetic engineering techniques have the potential to eliminate years from 

conventional development time. 

The preceding argument has been made many times, 

and in broad measure it is true. However, modifying gena1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

types using genetic engineering may be at least as costly 

as conventional breeding. Armed with a few breeding 

lines, a small plot of land, a few hundred pollination bags, 

and a handful of artist's brushes, the conventional plant 

breeder can assemble an incredible range of genotypes in 

a few years. In contrast, the biotechnological alternative 

of direct gene transfer requires a laboratory equipped for 

plant tissue culture and molecular biology, and a team of 

tissue culture and molecular biology specialists. With 

model plant species such as tobacco and petunia, the process of creating transgenic plants and testing them can be 

completed in a year or less. However, in many agronomically useful crop species or in tree species, the process may 

require several years. Given the equipment, supplies, and 

personnel involved, total costs per gene transferred can 

be hundreds of thousands of dollars. When contemplating the molecular biology approach to genotype modification, it is important to carefully choose the plant species 

and variety, the trait of interest, and the genes used to 

convey that trait. 

Identifying Suitable Plant Species and Traits 

When engineering genes encoding several antimicrobial 

peptides for use as pathogen-resistance genes, the model 

plant species should be one that has high economic value, is 

susceptible to Agrobacterium infection, and is easily regenerated from single cells or small pieces of tissue. The ideal trait 

is one that cannot be obtained through conventional breeding and is controlled by a single genetic locus. The ideal gene 

is fully characterized, produces a gene product or products 

that can be easily assayed, and is expressed only in the desired tissue (e.g., anthers, fruit, roots, cambium, leaves) or 

under the desired developmental or environmental stimulation (e.g., flowering, wounding, insect attack, heat stress). 

There are several other constraints to consider. For example, if the plant species will be grown as a food crop, 

the gene product should be nontoxic and nonallergenic. 

Also, if there is any question of human toxicity, the gene 

product should only be expressed in portions of the plant 

that are not consumed. 

165 
Section IV Biotic and Abiotic Resistance 

Field Testing and Release of Engineered 

Woody Plants 

For more than a decade, the regulatory requirements for 

field testing and eventual commercial use of engineered 

organisms have been debated (OAT 1989). The consensus 

of the scientific community is that transgenic plants should 

be evaluated by the same sets of procedures and the same 

criterion as any new variety developed through conventional breeding (Miller et al. 1990; Miller 1994). Although 

the debate continues, the trend is toward relaxing oversight (Hoyle 1996). For instance, in 1993 the USDA Animal and Plant Health Inspection Service (APHIS) changed 

from a petition and permit process to a notification and 

acknowledgment process. Under the old system, all field 

trials were reviewed in advance and had to comply with 

National Institute of Health guidelines (NIH 1994 and previous versions) before a permit was granted. Under current guidelines, researchers who want to field test 

transgenic organisms must still comply with NIH fieldtesting guidelines, but must only notify the USDA of the 

field test. 

As of September 1995, APHIS has approved or acknowledged 1,818 field trails at 6,762 locations (Schechtman 

1995). More than 40 different species have ongoing or completed field trials. The process has become so routine that 

it can now be conducted through the Internet using electronic mail (Reding 1995). 

The final step in successful development of a transgenic 

plant is approval for commercial use, or receiving what 

APHIS refers to as nonregulated status. As of January 1996, 

14 transgenic crop lines had been granted nonregulated 

status. Approximately 30 more have gone through the fieldtesting phase and are awaiting release. Most relevant to 

the eventual release of transgenic poplar or other tree species is the virus-resistant squash developed by Asgrow 

Seeds, which is fertile and will be grown in regions known 

to have native interbreeding wild relatives. APHIS granted 

the petition because the reviewers concluded that virusresistance traits have "no potential to increase the 'weediness' of either the crop or its relatives" (Medley et al. 1994). 

Although the trend could reverse if unforeseen problems arise with ongoing field tests or releases, we feel that 

by the time transgenic poplar trees are ready for field testing and release, the guidelines will have relaxed further 

and compliance will be uncomplicated. However, if genetic isolation is required for nonregulated status or to 

avoid problems with public acceptance, there are several 

possible approaches. Sterile genotypes may be chosen for 

transformation or the ploidy level of the transgenic plants 

may be altered, rendering them unable to cross with local 

wild populations. In addition, other researchers are actively working on sterility-inducing genes to incorporate 

in the transgenic plants along with the primary genes of 

interest (Strauss et al. 1995). 

166 

Among tree species, the genus Populus is close to being 

a model system because it has several species and species 

hybrids that have been transformed. Most reports deal with 

hybrids between P. alba and P. grandidentata ( Chun et al. 

1988a, 1988b; Chung et al. 1989; Fillatti et al. 1987; 

Klopfenstein et al. 1991, 1993; McCown et al. 1991; McNabb 

et al. 1990). Several other species and hybrids have also 

been transformed, including P. tremula (Lee et al. 1989), P. 

tomentosa (Wang et al. 1990), P. tremula x P. tremuloides hybrids (Nilsson et al. 1992), P. koreana (Kim et al. 1989), P. 

nigra (Tian et al. 1993), and P. alba x P. tremula (Brasileiro et 

al. 1992; De Block 1990; Devillard 1992; Leple et al. 1992). 

One phenotypic modification that has received attention from conventional plant breeders and molecular biologists is increased pathogen resistance (Cornelissen and 

Melchers 1993). In the United States, it is estimated that 

$9.1 billion in agricultural crops are lost each year to pathogens (Agrios 1988). Among trees, chestnut blight, Dutch 

elm wilt, and cottonwood leaf spot, caused by Cryphonectria 

parasitica (formerly Endothia parasitica), Ceratocystis ulmi 

(Ophiostoma ulmi), and Septaria musiva (teleomorph: 

Mycosphaerella populorum), respectively, have caused major damage to important timber and ornamental tree species. All tree species represent a $19 billion annual crop in 

the United States (Echt 1995). Thus, studying antimicrobial peptide expression as a means of disease resistance is 

a high priority. 

Antimicrobial Peptides 

Various groups of membrane-active peptides found 

throughout the plant and animal kingdoms have useful 

antimicrobial properties. Since there have been several recent reviews describing the properties and uses of antimicrobial peptides (Cornelissen and Melchers 1993; 

Destefano-Beltran et al. 1993; Raikhel et al. 1993; Rao 1995), 

we will not attempt to cover the whole topic. Reports of 

transgenic tobacco expressing antimicrobial peptides, although promising, demonstrate the need for more experimentation. Increased disease resistance was reported in 

transgenic tobacco expressing a-thionin (Carmona et al. 

1993) and a modified cecropin B peptide, Shiva-1 (Jaynes 

et al. 1993). However, other reports showed no significant 

increase in disease resistance in transgenic plants expressing other cecropin peptides (Hightower et al. 1994; 

Montanelli and Nascari 1991). Considering these reports, 

in vitro antimicrobial activity does not necessarily mean in 

vivo pathogen resistance; therefore, genes must be carefully designed to ensure that the encoded peptides are 

stable, expressed at effective levels, and localized in the 

proper plant cell parts. 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 
.. 

... 

.. 

• 

In this cha~ter, we emphasize the process of selecting 

promising antimicrobial peptides and designing genes 

encoding them for use in tree species. We limit our discussion to pept\des that are small ( <60 amino acids in length), 

nonenzymatic, linear, without any unusual or modified 

amino acids, and to those that demonstrate an inhibitory 

activity to the growth of a plant pathogen. These peptides 

are interesting because their small size: 1) allows synthesis of analogs with amino acid sequence changes that can 

be analyzed for antimicrobial activity change; 2) reduces 

the expense of constructing genes that encode them; and 

3) allows possible construction of a single gene tha t wou ld 

encode 2 or more peptide products. 

The strategy for antimicrobial peptide use is to have 

transgenic p lants express these inhibitory peptides at 

wound or infection sites and stop the spread of the pathogen. The use of these peptides can be envisioned as infection court-targeted plant pesticides. There are 2 advantages 

of this disease control strategy over current pesticide use: 

1) only a small fraction of the compounds wou ld end up 

in the food chain or in the environment; and 2) these peptides breakdown rapidly. 

Selecting and Designing Antimicrobial 

Peptides 

When selecting antimicrobial peptides for usc in pathogen-resistance genes, we looked for those that differed in 

structure and in mode of action. The first peptide chosen, 

ESF12 (Powell et al. 1995),.is a synthetic analog of PGLa, 

which is a natural antimicrobial peptide produced in the 

skin of the African clawed frog (Xenopus /nevis) (Saravia et 

al. 1988). ESF12 is an 18-amino acid peptide that at micromolar concentrations inhibits conidial germination of 3 

se lected p lant-pathogeni c fungi: Sep taria musiva, 

Cryphonectria parasitica, and Fusarium oxysporum. These 

organisms cause Septaria leaf spot and Septaria cankers 

on poplars (Populus spp.), chestnut blight on many chestnu t species (Castanea spp.), and Fusa rium wilt of .ton:a.to 

(Lycopersicon esculentum), respectively. ESF12 a lso mh1~1ts 

the growth of the bacterial p lant pathogens Agrobact.emiiii 

tumefaciens, Erwinia amylovora, and se udomo~ws ym~gae. 

ESF12 is structura lly simila r to the lyt1c peptJdes, 

magainins and cecropins. These contain an amphipathic 

a-helix thought to form channels in biological membranes 

(Christensen et al. 1988; Matsuzaki et al. 1994; Vaz Gomes 

et al. 1993). Genes expressing analogs of cecropin B enhanced resistance of transgenic tobacco to bacteria l wilt 

caused by Pseudomonas solanacearum (Jaynes et al. 1993). 

We expect that properly constructed genes codi~g and 

expressing ESF12 will also enhance pathogen reststance 

in poplars and other tree species. 

The second antimicrobial peptide chosen, Ac-AMP1.2, 

is an analog of Ac-AMP1 (Broekaert et al. 1992) with the 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 · 

Designing Smalll Antimicrobial Peptides and Their Encoding Genes 

amino terminal Va l replaced with Met to facilitate expression. The Ac-AMP1 peptide was originally identified in 

the grain amaranth (Amarmzthus spp.) where it is expressed 

in the seed coat and apparently helps to protect the seed 

from microbial degradation. Ac-AMP1 inhibited funga l 

growth at micromolar levels and inhibited the growth of 

Gram-positive but not Gram-negative bacteria (Broekaert 

et al. 1992). Ac-AMP1 is a defensin-like peptide containing 3 disulfide bonds, and is homologous to the cysteine/ 

glycine rich domains of many chitin-binding proteins. AcAMP1's ability to bind to chitin suggests that it has a different mode of inhibitors from magainins and cecropins, 

and is therefore different from the synthetic peptide ESF12. 

Testing Antimicrobial Peptides for 

Phytotoxicity 

An antimicrobial peptide wou ld be of little use as a 

pathogen-resistance mechanism if it was toxic to the plant 

expressing it. For this reason we also used a germination 

assay with pollen of Castanea mollissimn, Salix Iucida, Malus 

domesticn, and Lycopersicon esculentum. ESF12 and several 

other peptides assayed were nontoxic at 50-fold higher 

concentrations than those inhibiting fungal spore germination (Powell et al. 1995). Ac-AMP1 was not tested in vitro 

due to the cost of its synthesis and, since it is naturally 

found in plant tissue, it is expected to be nontoxic to plan ts. 

This hypothesis will be tested when it is cloned and expressed in transgenic poplar. 

Designing Genes for Correct 

Expression in Plants 

All of the pep tides we eva luated were synthesized from 

chemica l components, not extracted from biologi ca l 

samples. Synthesiz ing and testing different amino acid 

sequences allowed us to experiment widely. We made several amino acid changes from the original model before 

a rriving at ESF12; therefore, there was no gene ava.ilable 

that coded for the peptide and a new gene was des1gned 

and constructed. 

Codon Choices 

Since no natural source of ESF12 exists, a gene capable 

of expressing it in a transgenic p lant ha~ to be designed 

and constructed. Several items were cons1dered to ensure 

that design flaws would not interfere with optimal expression. Because of redundancy in the genetic code, 1 to 6 

codons may specify the same amino acid. For example, 6 

167 
Section IV Biotic and Abiotic Resistance 

different codons specify arginine but only one specifies 

methionine. Although the genetic code is common among 

all eukaryotes, there are differences in frequency of use 

among organisms. Since the fina l destination of our gene 

constructs will be 1 or more tree species, we chose from a 

set of 44 dicot-preferred codons (Campbell and Gowri 

1990) to encode the peptides ESF12 and Ac-AMP1.2 into 

DNA sequences. 

Each open reading frame includes an efficient translation initiation site. In most plant mRNA, the 5' proximal 

AUG is the initiation codon Uoshi 1987), but initiation is 

also determined by the context of the AUG (Kozak 1986a, 

1989). The anima l initiation consen su s sequen ce 

(CCACCAUGG) and the p lant initiation consensus sequence (UAACAAUGGC) initiate translation equally well 

in tobacco mesophyll cells (Guerineau e t a!. 1992). The 

animal consensus sequence has the advantage of containing a Nco! restriction endonuclease recognition site 

(CCATGG) in the genes DNA sequence. The location of 

this restriction site can be used in future modifications, 

such as the addition of leader sequences that target the 

gene product to various parts of the cell. 

ext, a termination codon was chosen. In dicots, the 

mRNA termination codon UAA is preferred (46 percent) 

over UGA (36 percent) or UAG (18 percent) (Angenon et 

a!. 1990). Data suggest tha t efficient termination in plants 

might require a tetranucleotid e sequence of UAAA, 

UGAA, or UAGA where A is preferred (41 percent) and C 

is avoided (6 percent) in the last position (Angenon eta!. 

1990). Plants contain transfer R As (tRl As) capable of 

misreading UAG and therefore the UAGAsequence might 

be used to prevent this stop codon from producing an oversized product. 

Lastly, the resulting nucleotide sequences were tested 

using computer modeling and modified to produced minimal mRNA secondary structure. Folding of mRNA into 

hairpin loops or other secondary structures can inhibit 

translation (Kozak 1986b). Minimizing seconda ry structures also aids the synthesis of the D TA used to produce 

the gene construct. 

Post-Translation Factors 

Post-translationa l processes, regulating peptide stability and targeting for cellu la r export, must be considered 

in addition to optimizing factors involved with translation. If the peptides need to be stable within the cell, the 

amino acid sequence should follow the N-End Rule. Proteins containing Met, Thr, Ser, Gly, or Va l at the Lterminus are relatively stable. Proteins containing Lys, Arg, Glu, 

Asp, Gin, or Asn are rapidly degraded by the ubiquitin 

system (Va rshavsky 1992). Another post-translation process to consider is the targeting of the pep tides. If it is desirable to have the peptides exported out of the cell, there 

168 

are several leader sequences identified. PR-l a, b, and c 

leaders from tobacco (Pfitzner and Goodman 1987), sCEC 

leader from the insect Hyalophora cecrovta. (c.ec.ropia moth) 

(Denecke et a!. 1990), and the ap24 leader from tobacco 

(Melchers et al. 1993) are examples of leader sequences 

that direct the export of fused proteins in transgenic plant. 

We are presently evaluating the ap24 leader for promoting export of our antimicrobial peptides. 

Promoter Evaluation 

To ensure proper expression of the gene construct, several promoters are being tested. First, the constitutive cauliflower mosaic virus (CaMV) 355 promoter is being used 

in the vector pCEA 1 (figure 1 ). The 355 is a strong constitutive promoter and will be used primarily to determine 

the effects on the plant tissues to express pep tides. If high 

concentrations of the peptides damage or kill plant cells, 

we would be unable to regenerate transformants. If the 

gene is sublethal, but still toxic, we would expect to see 

severely altered phenotypes among the transgenics. Existing transformants of petunia, willow, and poplar have 

been obtained. The only consistent effect observed was an 

increased difficulty in rooting the transgenic plants. This 

work is still in progress. 

Constitutive expression of the peptides will probably 

be unnecessary for protection against pathogens. We are 

examining a set of wound-inducible promoters that should 

confine expression of the antimicrobial peptides to the tissues most susceptible to pathogen a ttack. The poplar 

wound-inducible promoters we are eva luating include 

W/N3.12 and WIN6.39b (Bradshaw eta!. 1989; Davis et a!. 

1991; Hollick and Gordon 1993). 

Creating Multiple-Gene Cassettes 

for Durable Resistance 

Plant breeders, geneticists, and pathologists have been 

combating pathogens for more than a century and have 

prod uced many hundred s of disease-resistant varieties. 

In many cases, within a few years to a few decades of 

commercial release, the resistant variety is successfully 

attacked by a new strain of the pathogen. The length of 

time a variety retains its resistance is called durability. 

Many variab les affect durability, but one of the most frequently cited causes of low durability is resistance based 

on a single gene product. This problem may be more pronounced in woody perennials than in annual crops beca u se individual plants a re exposed to a pathogen 

population for yea rs to decades. This long-term exposure 

enhances the probability of selecting pathogens that can 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Designing Small! Antimicrobial Peptides and Their Encoding Genes 

BamHI Sail Sspl Xhal Neal 

GGATCCGTCGACAATATTCTCGAGGCCGCCACCATGGC 

• M e tA l 

AAGTAGAGCCGCAGGTCTTGCCGCACGGCTTGCCAGAC 

•a S e r A r gA I aA I aG !y L eu AI a Al aA r gLeuA l aA r gL 

Neal 

TTGCACTTCGGTAACCATGGGTGAATGTGTTAGAGGTA 

• e u A I a L e u A r g • • • • M e t G I y G 1 u C y s V a I A r g G 1 y A 

GATGCCCAAGTGGTATGTGTTGCTCCCAATTCGGTTAC 

• rgCys P roSer Gl yMe t Cy s Cy s S e r Gl n P h e G l yT y r 

Sstl 

TGTGGGAAAGGTCCCAAATACTGCGGTTAAGAGCTC 

•c ys Gl yLy s G l y Pr o L ysTyrCy s Gly • •• 

Bam HI 

promoter 

Sst I 

l'b; 

Termin ator 

pCEA1 

(11 059 bp) 

Figure 1. DNA and encoded amino acid sequences of the ESF12/ACAMP1 .2 gene 

construct in the pCEA 1 plant binary vector. 

gation of the mutated pathogen. For 

a pathogen to overcome 2 or more 

d iffe ren t antimi cro bia l peptid es 

would require multiple muta tions to 

simultaneously overcome the effects 

of each. It is highly improbable tha t 

such multiple mutations would simultaneously occur in a single microorganism. The p robability decreases 

w ith each additional a ntagonistic 

gene product. Therefore, a genetic engineering stra tegy using severa l antimic robia l gene products sho uld 

produce highly durable resistance. 

Multiple-Gene Transfer 

There arc at least 4 options for gene tica lly engineering plants to produce multiple gene products. The 

first option is to include multiple 

genes in a single construct w ith separa te promoters to drive the production of each gene. This is a difficult 

and complex task. The final construct 

size may be so la rge tha t it strains the 

limit of the Agrobncterium vector system. The la rge size also increases the 

chance for rearrangement and inactivation of 1 or more of the genes. 

A second option would be to transfo rm d iffe re n t pla nts, gene ti cally 

cross them, and eva lua te the progeny 

to find 1 o r more individua ls expressing both genes. For annual plants, this 

approach holds promise, but for longlived woody perennials, the time required is prohib itive. In addition, this 

approach does not genetically link the 

genes, and allows for offspring to 

contain only 1 of the genes. This could 

jeopa rdize resistance d urability by 

a llowing the propagation of pathogens that have mutated and can overcome 1 of the gene products. The 

overcome the single-gene resistance. Antimicrobial pep tides 

a re the simplest of single-gene traits. Therefore, it could be 

expected that pathogens could evolve quickly to overcome 

resistance based on a single antimicrobial peptide. 

presence of these pa thogens in the popu lation would effectively reduce the layers of resistance in plants still p roducing multiple pathogen-resistance gene products. 

To genetica lly engineer d urable resista nce, a multi-laye red defense system should be employed using 2 or more 

gene products that employ diffe rent inhibitory mechanisms. A single mutation overcoming the effects of just 1 

antimicrobial peptide would have no selective ad vantage 

because the plant's other peptide would prevent propaUSDA Forest Service Gen. Tech. Rep. RM-GTR-297 1997 

As a third option, after recovery of a w ho le p lan t exp ressing the fi rst gene, a second round of transformation and selection is ini tiated to incorporate the second 

gene of in terest. This ap proach could be used on poplar o r other tree species if the genes of interest can be 

eva lu ated early and if different selectab le ma rke rs are 

availab le for each round of transformation and sclcc169 
Section IV Biotic and Abiotic Resistance 

tion. This a pproach has the disadvantage o f not genetically linking the genes. . 

The fourth option is to express mulhple gene products 

from a single gene construct under the control of a single 

promoter. This option has the advantage of linking _th_e 

coding regions, keeping the gene construct small, and ~mmmizing the transformation procedures. We are testmg 2 

approaches to this option. The first approach takes dv

tage of a second initiation in translation. If 2 open readmg 

frames are located within a few base pairs of one another 

and the first open reading frame is small, the second open 

reading frame can be expressed at a reduced rate (Putterill 

and Gardner 1989). We have designed a gene construct in 

which the 2 open reading frames encoding the antimicrobial peptides are next to one another and contain efficient 

translational initiation sites (figure 1). We are testing this 

construct for expression of both peptides. 

A second approach is to encode both antimicrobial peptides into 1 open reading frame that includes the self-cleaving protease TEV- Ia (Marcos and Beach y 1994; 

vonBodman et al. 1995). This will produce a polyprotein 

that will self-cleave in the transgenic plant, releasing the 2 

peptides. The disadvantage to this approach is that residual 

amino acids are left on the cleaved peptides. There fore, it 

must be determined if these added amino acids will have 

any effect on the antimicrobial activity of the pep tides. The 

advantage of this approach is that the peptidcs are linked 

and would be released in essentially equal molar quantities. We are presently evalua ting this approach for 

transgenic poplar. 

Summary 

In this chapter we have described a process of using 

genetic engineering to develop durable pa thogen resistance for poplar and other tree species. Toxicity tests of 

various amino acid sequences, designed on the basis of 

structural properties of natural antimicrobial peptides, 

provided information on which substitutions enhanced or 

reduced activity against various plant pathogens. Simila r 

tests using pollen showed that the peptides appea r to be 

nontoxic to plant cells even at 50 times the concentration 

needed to inhibit fungi. Initiation and termination sequences, codon preferences, secondary structure, peptide 

stability, and multiple product expression were considered 

in designing a gene to express the antimicrobial peptides 

in vivo. 

Present efforts focus on transformation and recovery of 

nonchimeric whole p lants expressing the peptide(s) of interest under the control of either the 355 constituti ve promote r or a wound-inducible promoter. The goal is to 

170 

develop a gene cassette to express 2 or more nti~icr i~\ 

peptides tha t will confer durable at o~~Q reststance 111' 

transgenic plants. 

Acknowledgments 

The authors thank Ms. Catharine Ca tranis for participating in the research and Dr. David Griffin, and Ms. Ma ry 

Beth Ritter for critical reading of this manuscript. This work 

was supported by Mr. William Heckrod t through the Community Foundation for the Fox Valley Region, the Research 

on Energy and Material Conservation in the Forest Industry (REMCO) Project, the USDA Cooperative State Research Service, Mcintire-Stennis Research Program, and 

the ew York State Chapter of the American Chestnut 

Foundation. 

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USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Chapter23 

Potential of Proteinase Inhibitors for Enhanced 

Resistance to Populus Arthropod and Pathogen Pests1 

Scott A. Heuchelin, Lise Jouanin, Ned B. Klopfenstein, and Harold S. McNabb, Jr. 

Introduction 

Proteinase inhibitors have recently been the focus of considerable research and speculation about their potential 

role as plant defense chemicals in host-pest interactions. 

These small molecular weight proteins, and the genes that 

code for them, may soon be a source of resistance to arthropod and pathogen pests for tree improvement programs. Detailed information on proteinase inhibitors and 

their origins can 'be obtained from the reviews of Ryan 

(1981, 1990) and Barrett and Salvesen (1986). In general, 

proteinase inhibitors interfere with the active sites of proteolytic enzymes by slowing or halting the rate of hydrolysis by a protease on its protein substrate. Proteases cleave 

proteins into smaller polypeptides and their constituent 

amino acid residues by hydrolyzing the peptide bonds between amino acids. To help understand the different groups 

of proteinase inhibitors and the types of proteolytic activity 

they suppress, we will consider the diverse proteases potentially used by biotic antagonists in the environment. 

Classification of Proteinases 

and Proteinase Inhibitors 

Two types of proteases are used in the degradation of a 

typical protein. Endopeptidases, specific for peptide bonds 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

between distinct amino acid groups (residues), are responsible for the initial stages of protein breakdown and subsequent degradation of polypeptides. Exopeptidases are 

not specific for the internal bonds that hold together an 

amino acid chain, but rather for the bonds of the terminal 

amino or terminal carboxyl residues. Cleavage of terminal residues by exopeptidases generates free amino acids 

from peptides. For protease nomenclature, the term "proteinase" is used for proteolytic enzymes possessing endopeptidase activity (Barrett 1985). 

Proteinases and their inhibitors are classified according to the mechanism by which the peptide bonds are 

hydrolyzed. Six proteinase families have been determined and are named according to specific amino acid 

residues that are active in the catalytic mechanisms or 

compounds crucial to the catalytic activity (Neurath 

1984). The 6 families can be placed into the following 4 

groups: 1) aspartic, 2) serine, 3) cysteine, and 4) metalloproteinases. For detailed information on proteinases, 

reviews by Beynon and Bond (1989), Dalling (1986), and 

North (1982) are informative. 

Aspartic proteinases are active in acidic environments 

and typically have optimal enzyme activity in the range 

of pH 3.5 to 5.5. Acid aspartic proteinases are produced in 

most eukaryotic organisms and have been found in many 

fungi. Inhibitors of aspartic proteinases include the protein pepstatin, epoxy propane, and diazoacetyl-compounds such as the diazoacetyl-norleucine methyl 

ester-copper ion complex (Beynon and Bond 1989; Dalling 

1986; North 1982). 

Serine proteinases are active in neutral to alkaline environments and typically have optimal activity in the 

pH range of 7.5 to 9.0. Alkaline serine proteinases are 

in almost all prokaryotic and eukaryotic organisms including pathogenic fungi and bacteria and phytophagous insects. Specific inhibitors of serine proteinases 

include the: 1) proteinase inhibitor (PI) I and PI II proteins of the Solanaceae; 2) soybean trypsin inhibitor and 

its relatives in the Leguminosae; 3) protein aprotinin; 

and 4) irreversible chemical inhibitors such as phenylmethylsulfonyl fluoride {PMSF) and diisopropyl173 
Section IV Biotic and Abiotic Resistance 

phosphofluoridate (DIFP) (Beynon and Bond 1989; 

Dalling 1986; North 1982). 

Cysteine proteinases are a diverse group and are active 

in environments with widely ranging pH values. Optimal activity has been observed from pH 4.0 to 8.0, depending on the organism and its environment. Cysteine 

proteinase activity is typically activated or enhanced in 

the presence of cysteine, low molecular weight thiol 

groups, and other reducing agents. This group of proteinases has been found in almost all organisms including 

phytophagous insects. To date, reports of cysteine proteinases in fungi are few. Specific inhibitors of cysteine 

proteinases include the OCI (oryzacystatin) protein of rice, 

iodoacetamide, iodoacetate, N-ethyl maleimide, and 

heavy metals (Beynon and Bond 1989; Dalling 1986; North 

1982). 

Metallo-proteinases are typically active in near neutral pH environments; however, some metallo-proteinases have optimal activity in pH conditions as low as 

pH 5. Most metallo-proteinases contain a crucial zinc 

atom within their catalytic site. These proteinases are 

widespread in prokaryotic and eukaryotic organisms 

including some pathogenic fungi. Inhibitors of metalloproteinases include phosphoramidon, hydroxamic acid 

derivatives, mercaptoacety ldipeptides, ethylenediaminetetraacetic acid (EDTA), and other metal-chelating agents 

(Beynon and Bond 1989; Dalling 1986; North 1982). 

Of these inhibitors, the small molecular weight proteins are of particular interest to researchers. Usually 

these inhibitor proteins are tight-binding reversible inhibitors of their proteinase class that have little reactivity with other types of proteinases. Since they are 

reversible inhibitors and follow an equilibrium dissociation constant, their activity is dependent on their 

concentration in the system studied. Because they have 

exhibited almost no toxicity to nontarget organisms, and 

their affect on the target organism is rarely acute, they 

are ideal for biological systems. 

Of even greater interest, is the potential to create 

transgenic trees that can express inhibitor proteins in 

their leaf, root, and I or cambial tissues. Once the gene 

for a particular inhibitor protein is cloned, it can be incorporated into a transformation vector and used to 

create transgenic trees that may possess higher resistance to a particular arthropod or pathogen pest 

(Klopfenstein and Hart 1997). Research in this direction was recently initiated using Populus as a model 

transformation system for woody plants. Overall, such 

research has focused on 2 inhibitor proteins; PI II, a 

serine proteinase inhibitor from potato (Heuchelin et 

al. 1997; Klopfenstein et al. 1991, 1993, 1997) and OCI 

(Cornu et al. 1996; Leple et al. 1995). A summary of the 

research conducted with these inhibitors, including 

transformation and expression studies, pest susceptibility assays, and in vivo resistance bioassays follows. 

174 

Expression of the Serine 

Proteinase Inhibitor, PI II, 

in Poplar 

The serine proteinase inhibitor, PI II, is a low molecular 

weight protein (monomer Mr 12,300) (Plunkett et al. 1982) 

that exists in dimeric form and inhibits the proteolytic activity of trypsin and chymotrypsin (Ryan 1981, 1990; Ryan and 

An 1988). PI II is the gene product of the proteinase inhibitor 

II (PIN2) gene, which was first cloned from tomato and potato (Graham et al. 1985; Sanchez-Serrano et al. 1986). 

In theory, PI II could effectively reduce the vigor of fungi 

by depriving them of necessary amino acids (Ryan 1990). 

Lorita et al. (1994) proposed the use of proteinase inhibitors as a novel class of fungicides after observing deleterious effects of serine proteinase inhibitors on fungi. A similar 

study at Iowa State University (I.S.U.) using PI II, supports 

the fungistatic effects of serine proteinase inhibitors 

(Heuchelin and McNabb 1995). Proteinase inhibitors have 

reduced the activities of fungal proteinases in 

Colletotrichum lindemuthianum (Mosolov et al. 1979) and 

Fusarium so/ani (Mosolov et al. 1976 ). Our studies show 

that Mycosphaerella populorum (Septoria musiva anamorph) 

produces proteinases in vitro that are inhibited by PI II 

(Heuchelin and McNabb 1995). Proteinases also have a 

significant role in the pathogenesis of host tissues. 

Movahedi and Heale (1990) concluded that proteinases 

may act synergistically with the cell wall degrading enzymes in pathogenesis. Inhibition of these proteinases may 

reduce infective capacity, colonization, and sporulation of 

the fungus. Reductions in any of these areas could slow 

fungal spread decreasing disease pressure. 

Insect growth, development, and reproduction are also 

potentially influenced by PI II. PI II in the diet of insect 

species reduced growth in Heliothis zea and Spodoptera 

exiqua (Broadway and Duffey 1986), and Manduca sexta 

(Johnson et al. 1989). A potential role of PI II in resistance 

to arthropod pests is apparent. 

Hybrid poplar clones cv. 'Ogy' (Populus x euramericana) 

and cv. 'Hansen' (P. alba x P. grandidentata) were used in 

studies at I.S.U. and the USDA Forest Service National 

Agroforestry Center (NAC). In North America, the major 

defoliating insect pest of 'Ogy' and other Populus hybrids 

of Aigeiros parentage is the cottonwood leaf beetle, 

Chrysomela scripta (Coleoptera: Chrysomelidae). Although 

C. scripta is not a major pest of 'Hansen' and other hybrids 

of the Leuce (currently termed Populus) section, the imported willow leaf beetle, Plagiodera versicolora (Coleoptera: 

Chrysomelidae), can cause considerable defoliation and 

is a major pest of white poplar, aspens, and their hybrids 

(Ostry et al. 1989). 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Potential of Proteinase Inhibitors for Enhanced Resistance to Populus Arthropod and Pathogen Pests 

To study PIN2 gene expression, poplar clones were transformed with an Agrobacterium binary vector system. Initially, 'Hansen' was transformed with the binary vector 

plasmid pRT45 to test the wound-inducible PIN2 promoter 

with the chloramphenicol acetyl transferase (CAT) reporter 

gene. Using northern analysis, these studies demonstrated 

inducible expression of PIN2-CAT when leaves were artificially wounded (Klopfenstein et al. 1991). 

To test the importance of proteinases in host-parasite 

interactions, transgenic 'Ogy' and 'Hansen' were produced containing a chimeric fusion of the PIN2 structural 

gene with the cauliflower mosaic virus 35S or bacterial 

nopaline synthase (NOS) promoter (Heuchelin et al. 1997; 

Klopfenstein et al. 1993, 1997). An Agrobacterium binary 

vector system, containing either the pRT102 or pRT104 

transfer DNA (T-DNA) vector and a disarmed tumor-inducing (Ti) plasmid (EHA 101) as the helper plasmid, was 

used to transform poplars with PIN2 gene constructs. The 

T-DNAalso included a NOS promoter linked to a neomycin phosphotransferase (NPTII) gene as a selectable 

marker (Heuchelin et al. 1997; Klopfenstein et al. 1997). 

The PIN2 coding region is regulated by a NOS promoter 

in vector plasmid pRT102 and by the CaMV 35S promoter 

in vector plasmid pRT104. Transgenic plantlets were regenerated under kanamycin selection. Putative transgenic 

lines were analyzed for transgene incorporation by Southern analysis and I or polymerase chain reaction. PIN2 expression was verified by enzyme-linked immunosorbent 

assays (ELISA) and/ or western blot analysis. 

Three lines of transformed '<?gy' and the untransformed 

material ('Ogy' control) were used in an in vitro feeding 

assay with C. scripta. In addition, 11lines of transformed 

'Hansen' and the untransformed material ('Hansen' control) were used in a separate in vitro feeding assay with 

larvae of P. versicolora. C. scripta larvae grown on the 3 

transgenic 'Ogy' lines consumed less leaf area and had 

lower larval weights than those reared on control material (Kang et al. 1997). On 1 transgenic 'Hansen' line 

(Tr665), P. versicolora larvae consumed significantly less 

tissue than larvae on the control material. Overall, some 

PIN2-expressing 'Hansen' lines also showed trends for 

lower pupal weights and longer development times for P. 

versicolora (Klopfenstein et al. 1997). 

Expression of a Cysteine 

Proteinase Inhibitor in Poplar 

Poplars, important trees in western Europe and France, 

are sensitive to many pathogens and pests including the 

poplar leaf beetles, Chrysomela tremulae and C. populi, which 

are 2 important defoliating insects (Augustin et al. 1993). 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

In nurseries and short-term plantations, these insects can 

completely defoliate the plants and drastically reduce 

growth. During wide outbreaks on young plantations, larvae and adults have attacked the bark and caused plant 

mortality. Chemical treatments must be applied in the 

nursery to reduce attacks, but these treatments are expensive and potentially hazardous to the environment. Some 

natural resistance mechanisms are known, but selecting 

these traits in classical improvement programs is difficult. 

Genetic engineering is a new strategy being explored at 

the Institut National de Ia Recherche Agronomique (INRA), 

France, to introduce into poplar a gene conferring resistance to these beetles. 

Major proteolytic activity in the gut of C. tremulae was 

used to define a strategy for controlling larval growth 

and development using an inhibitor of digestive proteinases. Proteinases from midguts of third instar larvae were extracted and characterized. Basal proteinase 

activity showed optima at pH 7 and pH 9. In the presence of cysteine proteinase activators (dithiothreitol or 

2-mercaptoethanol), a strong increase (approximately 7 

fold) in the proteinase activity at pH 7 was observed. The 

efficiencies of a serine proteinase inhibitor (soybean Bowman-Birk Inhibitor, BBI) and a OCI to inhibit the proteinase activity were tested. Results indicate that cysteine 

proteinases sensitive to OCI constitute the major pool of 

gut proteinases of C. tremulae larvae (Leple et al. 1995). 

Consequently, OCI gene expression in transgenic poplars 

should produce a deleterious affect on C. tremulae survival 

in planta. 

The eDNA of OCI (Abe et al. 1987) was introduced in a 

transformation vector under the control of the CaMV 35S 

promoter with a double enhancer sequence (Kay et al. 

1987). An NPTII gene was used for selection of the transformed cells on kanamycin containing medium. Transformation was performed on a Leuce hybrid poplar (P. 

tremula x P. tremuloides) clone 'INRA 353-38,' which is very 

sensitive to C. tremulae. Two Agrobacterium-mediated transformation procedures, co-inoculation (Brasileiro et al. 

1991) and co-cultivation (Leple et al. 1992), previously established for a P. tremula x P. alba hybrid, were adapted to 

the P. tremula x P. tremuloides hybrid. Twelve independent 

lines were obtained and characterized at the molecular 

levels (Southern and northern analyses). These experiments determined which lines were expressing the OCI 

messenger RNA (mRNA) at a high level and which contained a low (1 or 2) T-DNA copy number. Western blot 

analysis was performed on leaf extracts using an antisera 

raised against OCI. In the best expressing lines, the OCI 

protein represented 2 percent of the total soluble proteins. 

Selected clones were micro-propagated and transferred 

to the greenhouse for C. tremulae feeding tests (Cornu et 

al. 1996; Leple et al. 1995). Feeding tests were conducted 

from egg hatch to the pupation of larvae on young leaves. 

From the third day of feeding, we observed significant 

175 
Section IV Biotic and Abiotic Resistance 

differences in larval mortality on the selected transformed 

lines compared to the control plants. These differences 

increased until the pupation stage (table 1) (Cornu et al. 

1996; Leple et al. 1995). 

A deleterious effect was observed after a long delay, 

which allowed the larvae to cause considerable damage to the leaves. However, development and growth 

of larvae were delayed and adults emerging at the end 

of the pupae instar were often abnormal. Therefore, a 

longer development time will influence the number of 

generations and promote parasitism in natural conditions, and lower weight of egg-laying parents may adversely affect fecundity resulting in less offspring. Even 

if the protection conferred by OCI in transgenic poplars is not absolute, the addition of these different effects should decrease the size of future C. tremulae 

populations. A field trial of selected OCI-expressing 

lines and control plants was planted in 1995 at INRA, 

Orleans, France, which will allow further experimentation under natural conditions. · 

Conclusions 

Proteinase inhibitors show great promise as a tool to 

help manage arthropod and pathogen pests of Populus. 

However, interpreting ecological interactions of proteinase inhibitors is complex. More studies are needed to 

evaluate the role of specific proteinase inhibitors in managing specific pests using intact plants under field conditions, where interactions with other pest management 

practices, other host resistance mechanisms, natural enemies, and less-favorable environments exist. Deployment 

strategies must be devised to maximize resistance stability and minimize adverse environmental effects. Populus 

is well suited for addressing these issues. 

Table 1. Percentage of mortality of Chrysomela tremu/ae 

feeding on oryzacystatin ( OCI) transgenic poplar 

(Populus tremula x P. tremu/oides) foliage. 

Feeding day 3rd 7th 15th 25th 

Larval stage 1.1 1.2 1.3 Pupae 

Control 0.25 1.75 3.75 4.50 

OCI-4 3.50 7.25 21.50 29.75 

OCI-33 1.00 11.75 27.00 42.50 

OCI-51 0.75 17.00 38.00 43.50 

Source: Cornu et al. 1996; Leph~ et al. 1995 

176 

Acknowledgments 

Researchers at I.S. U. and NAC thank Robert Thornburg 

for contributing the PIN2 gene and its vector plasmids 

pRT45, pRT102, and pRT104, and Vic Steenackers (Poplar 

Research Center, Geraardsbergen, Belgium) for providing the I.S.U. research project with 'Ogy.' Special thanks 

to Woody Hart and Rick Hall for collaborative assistance. 

Research at I.S.U. was supported under Subcontract No. 

19X-43391C with Oak Ridge National Laboratory under 

Martin Marietta Energy Systems, Incorporated contract 

DE-AC05-840R21400 with the U.S. Department of Energy, 

cooperative research agreement #28-C2-637 from the 

USDA Forest Service, Consortium for Plant Biotechnology Research, Inc. (DOE I CPBR Subagreement No. 

OR22072-17), and Hatch Act and State of Iowa funds (Iowa 

Agriculture and Home Economics Experiment Station 

Projects No. 2210, 2748, and 3088). Use of trade names in 

this paper does not constitute endorsement by the USDA 

Forest Service. 

Researchers at INRA are grateful to Drs. Abe and Arai 

for providing OCI eDNA. Research at INRA was supported 

by grants from the Ministere de I' Agriculture et de Ia Peche 

(DFF-CTPS, OGM peupliers) and from the Commission 

of the European Communities (AIR-IRPI). 

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1989. A Guide to the insect, diease and animal pests of 

poplars. Agriculture Handbook 677. Washington, DC: 

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Proteinase inhibitors I and II from leaves of wounded 

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177 
Chapter24 

Expression of Transgenic Bacillus thuringiensis 

o-endotoxin in Poplar1 

David D. Ellis and Kenneth F. Raffa 

Introduction 

Bacillus thuringiensis (Bt) is one of the most widely used 

pesticides, with more than 2.3 million kg of spores applied 

worldwide (Rowe and Margartis 1987). Bt represents 74 

percent of the total pesticides applied to Canadian forests 

for spruce budworm and 75 percent of the pesticides 

sprayed for gypsy moth (Lyman tria dispar) control in North 

America. As a pesticide, it is considered environmentally 

friendly due to: 1) a high specificity of the toxin proteins 

against target insects; 2) the natural occurrence of the bacterium; 3) a lack of polluting residues; 4) safety to nontarget insects, humans, or other animals; and 5) the short-lived 

nature of the toxin. The short-lived nature of Bt provides a 

margin of application safety by decreasing residual buildup. However, this feature hinders proper administration 

and partly contributes to a history of insect control failures. 

The high specificity of Bt is due to the production of 

different 8-endotoxins, or crystal proteins, that display activity against certain groups of insects. Bt strains, loosely 

divided into 4 major classes based on toxin structural similarities and activity against certain insect pests, are: 1) cry I, 

that exhibit activity against lepidopterans; 2) cryll, with 

activity against lepidopterans and dipterans; 3) cry III, with 

activity against coleopterans; and 4) cryiV, with activity 

against dipterans (Hofte and Whiteley 1989). Insecticidal 

activity of these classes is not absolute. For example, cryiB 

genes show activity against coleopterans and lepidopterans. 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja. M.A.. eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G .• tech. eds. 1997. 

Micropropagation, genetic engineering. and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins. CO: 

U.S. Department of Agriculture. Forest Service, Rocky Mountain 

Research Station. 326 p. 

178 

Isolation of new Bt strains is critical because of large 

variations in the toxicity of each Bt strain to different groups 

of forest pests (Macintosh et al. 1990; Ramachandran et al. 

1993b; van Frankenhuyzen et al. 1991, 1993). In 1 study 

(van Frankenhuyzen et al. 1991), 3 subclasses of cryiA endotoxin were tested and were equally toxic against the 

forest tent caterpillar (Malacosoma disstria). However, in 3 

species of budworm (Choristoneura fumiferana, C. 

occidentalis, C. pinus), cryiA(a) and cryiA(b) were 5 times 

more toxic than cryiA(c). Further, in gypsy moth, cryiA(a) 

and cryiA(b) were 100 times more toxic than cryiA(c). These 

toxicity tests are complicated because toxicity varies with 

the insect developmental stage. With cottonwood leaf 

beetle (Chrysomela scripta), susceptibility to purified cryiB 

toxin decreases with increasing larval age. Specifically, the 

first instar larvae are 2 to 4 times more susceptible than 

third instar larvae (Ramachandran et. al. 1993a). In addition, only 30 percent mortality occurred in adult leaf 

beetles, even with the highest Bt dose. 

Bt 0-endotoxins disrupt the midgut after insects ingest 

Bt spores containing a high concentration of the crystal 

protein. The crystal protein is solubilized in the midgut 

into a protoxin that is activated by a protease to form a 

toxin that binds a specific receptor in the midgut (Adang 

1991). Toxin binding causes the midgut adenosine triphosphate (ATP) level to decrease within 1 min of ingestion, and 78 percent of potassium ion transport is inhibited 

within 10 min. The resulting osmoregulatory imbalance 

causes cells to imbibe water, burst, and form lesions in the 

midgut (Reichelderfer 1991). 

Specificity of the toxin to different insect orders is due 

to different toxins that bind to distinct receptors in the 

midgut. Defining the receptor domain of each toxin is an 

active area of research to increase toxin specificity and 

engineer toxins for multi-pest activity (Feitelson et al. 1992). 

Specificity of receptor binding is an important factor because mutations that alter the receptor binding region can 

render individuals resistant to the toxin. Therefore, engineering toxins with multiple receptor binding sites is one 

strategy to decrease the evolution of resistance in insect 

populations. However, the evolution of resistance is not 
'\&I 

eliminated by mixtures of Bt toxins (McGaughey and 

Whalon 1992). 

A major factor in the short-lived nature of Bt spray effectiveness is that the toxin in Bt spores is photo labile. 

Several strategies have been tested to increase the effective life of the pesticide in the field, such as the expression 

of the active toxin in other bacteria like Pseudomonas 

fluorescens {Feitelson et al. 1992). 

Another option for increasing field effectiveness of Bt is 

by genetically engineering plants to express Bt 8-endotoxin. This is effective in several plant species (Adang et al. 

1993; Barton et al. 1987; Ellis et al. 1993; Fischoff et al. 1987; 

Koziel et al. 1993; Perlak et al. 1991, 1993) including poplar (McCown et al. 1991). However, the problem with this 

approach is insufficient transgene expression and I or instability of the 8-endotoxin RNA or protein in genetically 

engineered plants (Adang et al. 1993; Perlak et al. 1991; 

Wong et al. 1992). While the endotoxin comprises up to 50 

percent of the total protein in sporulating Bt cultures, it is 

difficult or impossible to detect the 8-endotoxin in genetically engineered plants with an unmodified 8-endotoxin 

gene. This frequently occurs even in plants where 

transgene expression produces toxicity in insect feeding 

trials. 

Truncation of the gene, leaving the N-terminal half of 

the protein unaltered, results in protein expression at barely 

detectable levels. A comparison of the Bt 8-endotoxin sequences with other plant genes reveals significant differences in G I C content, A IT rich regions that resemble plant 

introns, the presence of potential polyadenylation sequences, -ATTTA- motifs sho"wn in other systems to destabilize plant messenger RNA (mRN A), and the presence 

of codons rarely used in plants. Resynthesis of the gene to 

eliminate these sequences increased Bt toxin levels in the 

plants up to 100-fold (Perlak et al. 1991). 

Currently, several crop plants genetically engineered 

with a Bt 8-endotoxin gene have received or are close to 

receiving regulatory clearance for commercial production. 

Field tests with transgenic poplar containing and expressing the Bt 8-endotoxin gene are ongoing in North America 

and are planned for in Europe. 

Bt in Transgenic Poplars 

The first report of Bt 8-endotoxin gene expression in poplar was the over expression of a partially modified cryiA(a) 

gene controlled by an enhanced cauliflower mosaic virus 

(CaMV) 355 promoter in Populus alba x P. grandidentata cv. 

'Crandon' (cl. 'NC5339') (McCown et al. 1991). The cryiA(a) 

gene was truncated so that the gene encoded approximately 55 percent of the amino-terminus of the original 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Expression of Transgenic Bacillus thuringiensis o-endotoxin in Poplar 

protein. This truncation allowed the entire toxin region to 

remain intact. In addition, the amino-terminal141 codons 

were synthetically changed to correspond to preferred 

plant codons. In this study, 1 of 4 transformed lines expressed the cryiA(a) gene at a level high enough for significant effects on the survival and larval weight gain of 

forest tent caterpillar. However, plants from this transgenic 

line (NOD 11) did not significantly affect gypsy moth survival, although larval weight gain and leaf area consumed 

were significantly reduced. Interestingly, the 25 percent 

level (1 in 4) of transgenic poplar lines that expressed Bt 8-

endotoxin at lethal levels is similar to the percentage of 

transformed tobacco plants that displayed insect toxicity 

after transformation with the same construct (Barton et al. 

1987). 

Plants from the NOD 11 transformed poplar line were 

used for several other studies, including greenhouse 

(Robison et al. 1994) and field (Kleiner et al. 1995) testing 

for Bt expression. In the greenhouse study, the transformed 

NOD 11 poplars were grown for several asexual generations, throughout which they maintained constant morphology and growth. The Bt-expressing transformed line 

(NOD 11) continued to cause growth reductions in forest 

tent caterpillar and gypsy moth, and mortality in forest 

tent caterpillar. Gypsy moths showed a higher ability to 

recover from Bt exposure following feeding on the NOD 

11 plants. Forest tent caterpillars consumed a lethal dose 

after 5 days, yet a 2-day exposure was not lethal. In both 

cases, development was delayed; however, the gypsy moth 

could completely recover relative to control insects after 

20 days (Robison et al. 1994). 

In the field, NOD 11 maintained expression of cryiA(a) 8-

endotoxin at toxic levels, even after winter dormancy. Insect 

bioassays with forest tent caterpillar and gypsy moth during June, July, and August of the second field-growing season showed uniform inhibition of feeding and decreased 

growth. Mortality of the forest tent caterpillars in the June 

bioassay was higher than that of the gypsy moth in either of 

the subsequent assays (Kleiner et al. 1995). Observed differences between control and transformed plants in the field 

were less than that in the previous greenhouse study. These 

differences are perhaps attributable to reduced feeding on 

the field-grown, control leaves due to increased toughness. 

In France, a cryiiiA gene was inserted and tested in a P. 

tremula x P. tremuloides hybrid. The toxin encoded by this 

gene is effective against Coleoptera such as the leaf beetle 

Chrysomela tremulae. Detection of cryiiiA gene expression 

was difficult and reverse transcriptase-polymerase chain 

reaction (RT-PCR) provided the only reliable detection 

method other than insect feeding assays. Non-choice feeding assays with leaves from transformed plants showed 

increased mortality of C. tremulae larvae after 18 days. 

(Cornu et al. 1996). Current work with a synthetic cryiiiA 

gene is expected to yield higher expression (G. Pilate, personal communication). 

179 
Section IV Biotic and Abiotic Resistance 

Several transformed lines of a Populus nigra x P. 

maximowiczii hybrid d. 'NM6' that contain a partially modified cryiB gene were regenerated (Francis 1996). This gene 

is derived from Bt strain HD-290-1, which was toxic to 

cottonwood leaf beetle and forest tent caterpillar when 

painted on leaves (Ramachandran et al. 1993a). Two transformed lines were planted in the field and monitored for 

transgene expression throughout 2 growing seasons. Feeding assays with cottonwood leaf beetles revealed increased 

mortality, although the differences were not significant 

when compared to the controls. However, a significant 

decrease in beetle weight gain was observed in 1 transgenic 

line suggesting decreased larval feeding (Francis 1996). 

Managing Biotype Evolution 

One concern with the genetic engineering of Bt o-endotoxin in plants is the evolution of resistant insect biotypes. 

With poplar, and other forest trees, an additional concern 

is long-term nature of the crop. However, these long-lived 

perennials also offer alternative options for managing biotype evolution, which may be unavailable or uneconomical with agricultural crops. Studying agricultural crops can 

be valuable when evaluating these management strategies 

because insects have historically evolved resistance to agricultural chemical pesticides. This experience has shown that 

resistant biotypes will evolve, if selection pressures are 

strong. Further, if selection pressures are strong, every physiological mode of insecticide action can be overcome by insect populations (Raffa 1989). Based on this, it is reasonable 

to assume that genetically engineered resistance can be overcome by biotype evolution, as has occurred with chemical 

pesticides under conditions of strong selection pressures. 

A simplistic view of biotype evolution is that resistance 

to an insecticidal agent arises in an individual insect by a 

single spontaneous genetic mutation that gives that individual a selective advantage when exposed to that insecticide. Such mutations may occur whether the insecticide is 

present or not, but only confer a selective advantage when 

the insecticide is present. Due to this selective advantage, 

this individual's resistant genes are passed on to the next 

generation at a higher incidence. These resistant genes 

become dominant in the insect population as long as they 

continue to confer a selective advantage. This process could 

render an entire population resistant because only resistant individuals will survive to pass on their genes to the 

next generation. With genetically engineered poplar, the 

continual pressure to give resistant genotypes a selective 

advantage will favor evolution of resistant populations. 

Resistance to Bt has been documented in more than 500 

species of insects (Tabashnik et al. 1994). The most studied case is that of the diamond back moth (Plutella 

180 

xylostella) in Hawaii, where strains with up to a 2,800-fold 

resistance to Bt were isolated. Resistance in the diamond 

back moth is due to decreased binding of the toxin in the 

midgut, which is inherited as an autosomal recessive trait 

(Tabashnik et al. 1994). When selection pressure is lifted, 

by removal of the Bt, the moths become susceptible after 

several generations. This may be partly due to the resistant alleles conferring decreased fitness in the absence of 

Bt, compared to the wild-type moths. 

The diamond back moth is an excellent experimental 

case to study biotype evolution to Bt and factors that influence this resistance, such as the continual and frequent 

application of Bt. Because resistance decreases with removal of Bt, management strategies using temporal, spatial, or other crop refugia can be used to decrease the 

selective pressures for resistant insect populations to 

evolve. Several other management strategies to decrease 

the prevalence of resistant biotypes dominating in the 

population were proposed (Alstad and Andow 1995; 

Coombs 1993; Raffa 1989) and are discussed below as they 

pertain to the genetic engineering of poplar for the expression of a Bt o-endotoxin gene. 

It is important to review some evidence for the evolution of field resistance to Bt (Koziel et al. 1993). Usually, 

biotype evolution: 1) resulted in only moderate resistance 

levels; 2) was developed under spraying conditions that 

provided variable toxicity exposure due to rapid breakdown of the Bt o-endotoxin between applications; 3) involved a single insecticidal agent over several generations; 

and 4) involved relatively little immigration between resistant and wild populations. This last factor is crucial to 

the interpretation of lab results because relatively high 

immigration between populations of most poplar pests 

would be expected. Thus, many studies may only demonstrate the genetic potential for resistance to arise, not 

the rate at which such resistance would occur. However, 

these studies show that insect behavior, insect physiology, delivery mode of the Bt toxin, availability of refugia, 

and many other factors play a major role in determining 

the actual degree and speed at which resistant biotype 

populations evolve. 

A discussion on risk assessment by Raffa et al. (this volume) addresses some management and deployment strategies for lessening or changing selection pressures on 

insects to evolve resistance to Bt. These and other strategies are detailed below because they pertain specifically 

to the genetic engineering of poplar for the expression of a 

o-endotoxin gene. 

Multiple Resistances 

As previously discussed, the lethal effect of a Bt o-endotoxin is due to toxin binding of a single receptor in the 

midgut. Therefore, a single mutation that changes thereceptor binding site would alter or diminish binding and 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
affect toxicity. An alteration in the binding site was a cause 

of resistance in the diamond back moth. One strategy to 

overcome the selective advantage of a single mutation is 

to mix different forms of insecticidal activity in plants. If, 

for example, plants are genetically engineered with 2 or 

more Bt o-endotoxin genes that had different and distinct 

binding sites in the midgut, spontaneous mutations must 

occur at both sites in the same insect for resistance to occur. This strategy, however, may not be as effective as sequential spraying or exposure to individual Bt strains 

where simultaneous exposure to multiple Bt endotoxins 

is minimal (McGaughey and Whalon 1992). 

One consideration with forest trees, such as poplar, is 

whether multiple resistances in a single tree are as effective as a mosaic of trees with different resistances. Both 

strategies potentially delay biotype evolution by exposing insect populations to more than one form of toxin. 

However, if sequential spraying and I or the sequential 

exposure of different Bt toxins are preferred, using genetically engineered trees in a mosaic planting design is advantageous. With this strategy, sequential exposure to 

different Bt o-endotoxins occurs in a spatial mosaic as insects move from 1 tree to another, whereas a temporal 

mosaic exists with sequential spraying. The outcome is 

similar in that insects are exposed to only a single toxin at 

a time; however, the extent of exposure depends on the 

degree of insect movement between trees within a forest 

mosaiC·. 

Management strategies influence whether single tree or 

stand management for insect resistance is practical. Usually a resistance based on stand management is favorable. 

However, depending upon the availability and difficulty 

of transforming different commercially important poplar 

hybrids, deployment of single transgenic trees with multiple resistances may currently be the only economically 

feasible strategy. 

One pitfall to the genetic engineering of different Bt oendotoxins is that cross resistance to different o-endotoxins has been documented in numerous insects (McGaughey 

and Whalon 1992). For example, a strain of Heliothis 

virescens resistant to cryiA(c) was cross resistant to crylA(a), 

cryiA(b), crylB, crylC, and cryiiA toxins. This suggests that 

mixtures, rotations, or mosaics of different Bt o-endotoxin 

may not be the most effective biotechnological tool to control resistant biotypes. However, other reports (Koziel et 

al. 1993) note that such resistance is only marginal, and 

that resistant biotypes are unable to survive under continual Bt exposure or when feeding on a transgenic plant 

expressing Bt. 

An alternative strategy is to combine Bt o-endotoxins 

with other heterologous insecticidal genes, such as a proteinase inhibitor gene. With this strategy, cross resistance 

to different Bt o-endotoxins is not an issue. An example of 

this strategy would be genetic engineering with a Bt 8-

endotoxin gene in combination with a proteinase inhibiUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Expression of Transgenic Bacillus thuringiensis o-endotoxin in Poplar 

tor gene. By inserting these 2 insecticidal genes, the expression of the proteinase inhibitor would decrease nutrient uptake in the insect, weakening it, while Bt would 

be lethal. This might work with marginally Bt-resistant 

individuals by increasing their susceptibility to the o-endotoxin. Further discussion on the use of a proteinase inhibitor gene for insect resistance in poplar is presented 

by Heuchelin et al. (this volume). 

A final strategy in the use of multiple resistances is the 

stacking of Bt o-endotoxin with natural insect resistance. 

This strategy is mentioned (Raffa et al. this volume; 

Robison et al. 1994) for management of multiple pest complexes with a mosaic of genetically engineered and natural resistance, using a genetically engineered trait directed 

against 1 pest and the natural resistance against another. 

This strategy could alter selection pressures on insects to 

evolve resistance to an engineered trait by targeting the 

same pest with both natural and engineered resistance. 

An example would be to mix a trait for high phenolic glycoside levels, a deterrent to many lepidopterans, with the 

genetic engineering for cryiA o-endotoxin. 

Again, the question of the benefit from single-tree versus stand resistance can be asked. What must be 

avoided is imposing a selection pressure on the insect 

population so that the insects must overcome both resistances to achieve reproductive success. Raffa (1989) 

used a similar scenario when arguing that using genetically engineered Bt to target bark beetles might lead to 

a physiological immunity to both the natural and engineered protection, thereby eliminating natural resistance as a mode of protection. 

Altered Expression Patterns of the 

Bt Insecticide 

Selection pressures for the evolution of resistant insect populations are greatest when all insects in a population are subjected to very high and constant 

insecticidal levels. Spraying Bt on poplars when insects 

are present can be accomplished, with only limited success, and selective spraying of only specific tissues on 

impacted trees is impractical. With genetic engineering, 

the opportunity exists to express the endotoxin either 

in an inducible manner or only in the tissues affected 

or most valuable to the tree. These 2 strategies are referred to as temporal and spatial refugia, respectively 

(Gould 1988). In both cases, some insects within the 

population would escape toxin exposure providing individuals on the same tree that are not subjected to selection pressures for the evolution of resistant biotypes. 

This is a similar strategy to that occurring naturally in 

a tree for the production of secondary compounds that 

inhibit insect feeding (Gould 1991; Ramachandran et 

al. 1994). 

181 
Section IV Biotic and Abiotic Resistance 

The effectiveness of inducing Bt o-endotoxin for insect 

protection in plants was demonstrated with a chemically 

induced PR-la promoter controlling a partially modified 

cryiA(b) gene (Williams et al. 1992). In this study, insect 

tolerance in plants was induced by spraying a chemical 

inducer that activated the PR-la promoter. While no one 

is advocating the induction of Bt o-endotoxin expression 

in plants with the use of a chemical spray, this report shows 

the potential and efficacy of inducible expression of insecticidal proteins in plants. 

Wound inducible promoters were used in poplar 

to express chloramphenicol acetyltransferase (CAT) 

(Klopfenstein et al. 1991) and P-glucuronidase (GUS) (Ellis 

et al. 1994) using a proteinase inhibitor (PIN2) promoter 

from potato (Thornburg et al. 1987). The degree and 

method of mechanical wounding profoundly affected the 

level of GUS induction, confirming that this promoter is 

induced by wounding after a given injury threshold level 

is reached. This is important because trees can tolerate a 

mild level of insect attack. By not exposing feeding insects to Bt o-endotoxin during low infestation periods, a 

subpopulation of insects would be maintained that were 

never exposed to selection pressures to evolve resistance. 

These individuals could then serve to dilute resistant alleles in the population. 

However, it is unknown whether the PIN2 promoter 

can be sufficiently induced in poplar for Bt o-endotoxin 

expression to impart adequate insect protection. However, our studies found significant increases in GUS expression following feeding by forest tent caterpillar on 

greenhouse-grown transgenic 'Crandon' (data not 

shown). Field tests with these PIN2-GUS transformants 

have maintained wound-inducible expression of GUS 

even after 3 years in the field; however, wound-inducible expression levels may vary during the year. To date, 

we know of no studies where this or other inducible 

promoters were used to control expression of Bt o-endotoxin in poplar. 

A good example of the efficacy of spatial refugia with 

Bt was provided with the cottonwood leaf beetle 

(Ramachandran et al. 1994). Susceptibility of the cottonwood leaf beetle to cryiB decreases with age; first 

instars have a 4-fold lower LC50 than third instars 

(Ramachandran et al. 1993a). Cottonwood leaf beetle 

eggs are oviposited on the lower rna ture leaves and the 

young larvae are relatively immobile. Only as the larvae mature, do they feed on the upper younger leaves. 

This distinction is important because loss of mature 

leaves has a significantly lower impact on growth than 

loss of younger leaves. Furthermore, the stage of the 

beetle life cycle that is the most susceptible to Bt o-endotoxin, and hence most vulnerable to selection pressures for the evolution of resistant biotypes, feeds 

almost exclusively on the mature tree leaves. By expressing Bt o-endotoxin only in the younger leaves, the 

182 

most susceptible stage of the beetle life cycle would not 

be exposed to the o-endotoxin and the selection pressure on the insects to evolve resistance would decrease. 

The lower leaves would be sacrificed, but tree productivity may not be substantially affected. 

To test protection afforded by Bt expression in only 

the younger leaves of poplar, a Bt spore solution (Mone) was applied to only the young leaves of poplar 

hybrid d. 'NM6.' This application compared untreated 

plants to those with Bt treatment of only the mature 

leaves or the whole plant. The whole-plant treated control was superior because it had the greatest height 

growth, lowest number of egg masses, and lowest larval survival. However, treatment of only the younger 

leaves offered significantly greater protection than untreated plants. Further, plants with only the young 

leaves treated had greater growth than either the untreated controls or plants in which only the lower leaves 

were treated (Ramachandran et al. 1994). These results 

suggest that spatial refugia within the same tree can be 

an effective management strategy for decreasing theselection pressure on insects to evolve resistance to Bt, 

while not substantially sacrificing tree production. 

Precise tissue-I development-specific expression of Bt 

o-endotoxin necessary for spatial refugia in poplar has 

not been demonstrated. However, several promoters 

that offer such expression are being tested. An 

Arabidopsis ubiquitin (UBQ-1) promoter (Callis et 

al.1990) is expressed significantly higher in the younger 

leaves of transgenic tobacco than in the older leaves 

and is one candidate promoter for testing in the above 

study with transgenic Bt o-endotoxin. Another promoter 

that would offer a pattern of spatial refugia is the ribuIose-1,5-bisphosphate carboxylase (RUBISCO) small 

subunit promoter that could offer differential expression in different aged leaves, and could target Bt endotoxin specifically to photosynthetically active tissues 

(Manzara and Gruissem 1988). In addition, the use of 

the Arabidopsis Rubisco small subunit, atslA promoter, 

with its transit peptide sequence, increased cryiA(c) 

mRNA 10- to 20-fold compared with a CaMV 355 promoter. Further, increases in cryiA(a) were observed 

when the untranslated leader and transit peptide sequences were used with CaMV 355 (Wong et al. 1992). 

Results with the untranslated leader and transit peptide sequences are significant because many tissue-I 

development-specific promoters express genes relatively weakly compared with CaMV 35S. Therefore, 

unenhanced promoters may not provide expression at 

high enough levels for use with Bt. Modification of the 

coding sequence as with the synthetic cry genes are crucial; however, the use of chimeric constructs, as demonstrated with CaMV 355 and the atsl sequences, will 

also be important tools for enhancing expression with 

weaker promoters. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Lethal vs. Sublethal Bt Expression 

Some lepidopteran larvae, such as the spruce bud worm, 

avoid Bt-treated leaf discs in feeding assays when given a 

choice of Bt-treated versus non-treated discs 

(Ramachandran et al. 1993b ). With genetically engineered 

spruce, the bud worm detected the presence of Bt o-endotoxin and stopped. feeding on tissue that expressed a 

cryiA(a) gene at sublethal levels (Ellis et al. 1993). This 

avoidance of Bt by insect pests could be used as a management strategy for lessening selection pressures for resistant biotypes to evolve (Gould 1988). Avoidance 

decreases feeding, delaying the time to pupation, and rendering the larvae more susceptible to predators, desiccation, and parasites. Sublethal Bt expression would lessen 

selection pressures on the insect populations to evolve resistance; however, it would not prevent resistant biotypes 

from evolving, and resistance at the population level would 

inevitably evolve. Therefore, interplanting of the stand 

with non-Bt expressing trees, or the availability of ample 

other food sources, is also important to maintain a non-Bt 

exposed insect population. 

Continued exposure to a sublethal Bt level may accelerate the evolution of tolerance in a population so that a 

higher level of Bt is required for mortality. This conclusion 

is based on experimental and modeling data from Bt and 

chemical insecticides where it is crucial to avoid fixing resistan.ce alleles in homozygotes (Koziel et al. 1993 ). Because 

resistant genes are generally recessive, and dominant resistant genes for Bt o-endotoxins have not been identified, 

a high-dose approach would kill nearly all of the population and eliminate the heterozygotes. Also, this strategy 

would target the most susceptible or earliest larval stage. 

Thus, this principle argues against the sublethal Bt approach and the use of spatial refugia where the target is 

not the most susceptible stage of the insect, as mentioned 

above for the cottonwood leaf beetle. 

Bt Interaction With Plant Secondary 

Compounds 

Plant allelochemicals play a large role in the interaction 

of plants and insects. These effects can be either positive 

or negative and may be toxic or inhibitory such as with 

steroidal glyco-alkaloids and protein binding of tannins. 

Raffa et al. (this volume) discuss the preference of cottonwood leaf beetle versus the forest tent caterpillar to different poplar hybrids based on the foliar content of phenolic 

glycosides. Several studies have looked at the interaction 

of Bt endotoxin with plant allelochemicals. Similar to the 

effects of the allelochemicals on insects, both positive and 

negative effects occur. For example, the phenolics resorcinol and gallic acid increase the activity of Bt (Sivamani et 

al. 1992), while tannins and nicotine decrease the effect of 

Bt (Krischik et al. 1988; Luthy et al. 1985). Effects of differUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Expression of Transgenic Bacillus thurlngiensis o-endotoxin in Poplar 

ent plant extracts were also compared. Compared with 

alder extract, less mortality occurred with a Bt-supplemented diet when Douglas-fir extract was added. This 

agrees with the reports above as Douglas-fir is higher in 

tannins and alder is higher in phenolics (Moldenke et al. 

1994). 

Allelochemical interactions have management implications for poplar plantations genetically engineered with 

the Bt o-endotoxin because these plant secondary compounds are strongly influenced by the environment. For 

example, poplars are typically higher in tannins when 

grown under stress conditions, such as lack of water or 

nutrients. Under these stressed conditions, a higher tannin content may actually decrease the efficacy of genetically engineered Bt, even when expression levels are 

unchanged. Although the trees are also more susceptible 

to insect attack under weakened conditions, these 

allelochemical interactions can be useful tools for managing insect biotype evolution. 

Most poplar stands have traditionally relied on relatively 

few inputs, although management practices are changing. 

In stands where water and fertilizer are supplied sporadically throughout the year, applications could be timed to 

increase or decrease growth. Such management practices 

could change the allelochemical profile of tissues to coincide with and manage yearly insect cycles. The increase 

and decrease of tannins in slow growing versus rapidly 

growing tissues, respectively, is a good example of how 

this might work. Add to this, the expression of a Bt o-endotoxin whose efficacy can be modulated by the management of tannins and other allelochemicals in the leaf. 

During periods of high insect populations, rapid growth 

could be encouraged by water and fertilizer applications 

to decrease tannin levels and increase the efficacy of the Bt 

o-endotoxin. During periods when insects populations are 

not threatening, water and fertilizer resources could be 

withheld. Although this would decrease growth, a 

multidisciplinary management program for sustained fiber production may interpret this strategy as being in the 

long-term interest of the stand. Such precise manipulation 

of plant chemistry currently is impossible, however, with 

proper monitoring it might be possible in the future. 

What is currently possible, is using our knowledge of 

plant secondary compounds to aid in understanding the 

spatial refugia set up by the natural plant allelochemicals. 

For example, if higher Bt o-endotoxin expression in the 

younger leaves is desired, one option is to use a promoter 

construct to express Bt in only the young leaves as suggested with the UBQ-1 promoter. Depending on the poplar hybrid used, such a pattern of expression may be 

inherently established with the natural chemistry of the 

plant. For example, in poplar hybrid cv. 'Crandon,' the level 

of phenolic glycosides in young leaves is relatively high 

compared with older leaves, and the reverse is true with 

condensed tannins (McCown et al. 1996 ). These changes 

183 
Section IV Biotic and Abiotic Resistance 

correlate with the mortality observed with gypsy moth in 

leaf disc assays. 

Refugia 

The idea of refugia where non-exposed individuals in 

the population can develop, is perhaps the most important element in the management of biotype evolution on a 

population level. Because resistant alleles are recessive, 

constant interbreeding with individuals not carrying the 

resistant alleles helps ensure that the alleles do not become 

dominant in the population. For this process to occur, the 

resistant allele must provide no other selective advantage. 

Therefore, maintenance of nearby plant populations that 

allow the development of non-exposed individuals is crucial. These non-Bt expressing plants could be interspersed 

throughout the stand by interplanting genetically engineered trees with non-genetically engineered trees, or by 

establishing nonproduction stands surrounding production stands. 

For control of codling moth on walnuts, a variation of 

this strategy was proposed where apple trees genetically 

engineered with Bt are interplanted within walnut plantations (Dandekar et al. 1994). Since apple is the preferred 

host of the codling moth, these will be "bait trees" in the 

orchard and will control most of the codling moth population. A certain level of codling moth will attack the walnut, but hopefully walnut damage will be small and the 

Bt-expressing apple trees will sufficiently reduce the codling 

moth population. Any resistant biotypes that survive from 

the apples will interbreed with the individuals that survived 

on the walnut. Although this strategy has not been implemented, it could also work in poplar where insect-preferred 

hybrids are genetically engineered with the Bt o-endotoxin 

and interspersed among the production clones. However, if 

target insects avoid Bt exposure, like the spruce budworm, 

this strategy might attract insects that feed on desirable production clones within stands. 

As previously mentioned, the use of temporal or spatial 

refugia is another approach. Theories on application and 

utility of these refugia are the same as that for non-engineered trees. The important aspect is to provide an area 

where non-exposed individuals can complete their life 

cycle to interbreed with individuals that were subjected to 

the selection pressure of Bt toxicity. 

Conclusion 

Genetic engineering, whether for insect resistance, herbicide resistance, sterility, lignin modification, or increased 

growth, is a powerful and valuable tool for the forest man184 

ager. However, each genetically engineered trait will require modifications to the management of poplar plantations. Use of genetic engineering for insect resistance with 

Bt o-endotoxin gene(s) in poplar is no exception. Incorporation of genetic engineering with an "Integrated Pest 

Management'' (IPM) strategy for controlling biotype evolution is crucial for the success of this technology. An overriding management objective is to lessen selection 

pressures so that resistant biotypes do not dominate in the 

insect population. A constant influx of non-selected individuals is important to dilute resistant alleles and lessen 

the impact of resistant biotypes. 

Several different genetically engineered Bt o-endotoxins were tested in transgenic poplar, and a few were tested 

in the field. Improvements in the expression of Bt o-endotoxins in crop plants through resynthesis of the coding region, improved expression constructs, combining of 

multiple toxin domains, and identification of new strains 

have dramatically improved the insecticidal activity in 

plants. Soon these advances will be incorporated into trees, 

including poplar, and commercial plantations will be established, much like the Bt-containing maize and potatoes 

currently available. 

Acknowledgments 

We thank Margarita Gilbert, Stephanie Mcinnis, Dr. 

Krystyna Klimaszewska, and Dr. Jan Klimaszewski for 

comments on the chapter. Funding for our work with Bt 

in poplar was from the Consortium for Plant Biotechnology Research, the University of. Wisconsin-Madison College of Agricultural and Life Sciences, and Graduate 

School, and HATCH grants. 

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Section IV Biotic and Abiotic Resistance 

Perlak F.J.; Stone, T.B.; Muskopf, Y.M.; Petersen, L.J.; Parker, 

G.B.; McPherson, S.L.; Wyman, J.; Love, S.; Reed, G.; 

Biever, D.; Fischoff, D.A. 1993. Genetically improved 

potatoes: Protection from damage by Colorado potato 

beetles. Plant Mol. Bioi. 22: 313-321. 

Raffa, K.F. 1989. Genetic engineering of trees to enhance resistance to ·insects: Evaluating the risks of biotype evolution and secondary pest outbreak. BioScience. 39: 524-534. 

Ramachandran, R.; Raffa, K.F.; Bradley, D.; Miller, M.; Ellis, 

D.; McCown, B. 1993a. Activity of an insecticidal pro-: 

tein from Bacillus thuringiensis HD-290-1 strain to coleopteran and lepidopteran defoliators of poplars. 

Environ. Entomol. 22: 190-196. 

Ramachandran, R.; Raffa, K.F.; Miller, M.J.; Ellis, D.O.; 

McCown, B.H. 1993b. Behavioral responses and sublethal effects of spruce budworm and fall webworm larvae to Bacillus thuringiensis cryiA(a) toxin in diet. 

Environ. Entomol. 22: 197-211. 

Ramachandran, R.; McCown, B.H.; Ellis, D.O.; Raffa, K. 

1994. Biotechnology and host-plant resistance: Benefits, 

risks, strategies for development, and deployment. In: 

Ananthakrishnan, T. N .; Raman, A., eds. Chemical ecology of phytophagous insects. New York: International 

Science Pub.: 275-287. 

Reichelderfer, C.F. 1991. Interactions among 

allelochemicals, some Lepidoptera, and Bacillus 

thuringiensis. In: Barosa, P.; Krischik, V.A.; Jones, C.G., 

eds. Microbial mediation of plant-herbivore interactions. 

New York: John Wiley & Sons, Inc.: 507-524. 

Robison, D.J.; Raffa, K.F. 1989. Hybrid poplar productivity and suitability for the forest tent caterpillar: a 

frame work for evaluation. Gen. Tech. Rep. NC-140. 

St. Paul, MN, U.S.A.: U.S. Dept. of Agriculture, Forest Service, North Central Forest Experiment Station: 

155-162. 

Robison, D.J.; McCown, B.H.; Raffa, R.F. 1994. Responses 

of gypsy moth and forest tent caterpillar to transgenic 

poplar containing a Bacillus thuringiensis a-endotoxin 

gene. Environ. Entomol. 23: 1030-1041. 

186 

Rowe, G.E.; Margaratis, A. 1987. Bioprocess developments 

in the production of bioinsecticides by Bacillus 

thuringiensis. CRC Critical Review of Biotechnology. 6: 

87-127. 

Sivamani, E.; Rajendran, N.; Senrayan, R.; 

Ananthakrishnana, T.N.; Jayaraman, K. 1992. Influence 

of some plant phenolics on the activity of 0-endotoxin 

of Bacillus thuringiensis var. galleriae on Heliothis armigera. 

Entomol. Exp. Appl. 63: 243-248. 

Strauss, S.H.; Howe, G.T.; Goldfarb, B. 1991. Prospects for 

genetic engineering of insect resistance in forest trees. 

Forest Ecol. & Management. 43: 181-209. 

Tabashnik, B.E.; Pinson, N.; Groeters, F.R.; Moar, W.J.; 

Johnson, M.W.; Luo, K.; Adang, M.J. 1994. Reversal of 

resistance to Bacillus thuringiensis in Plutella xylostella. 

Proc. Natl. Acad. Sci. USA. 91: 4120-4124. 

Thornburg, R.W.; An, G.; Cleveland, T.E.; Johnson, R.; Ryan, 

C.A. 1987. Wound-inducible expression of a potato inhibitor II chloramphenicol acetyl transferase gene fusion 

in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA. 

84: 744-748. 

van Frankenhuyzen, K.; Gringorten, J.L.; Gauthier, D.; 

Milen, R.E.; Masson, L.; Perferon, M. 1991. Specificity 

of activated cryiA proteins from Bacillus thuringiensis 

HD-1 for defoliating forest Lepidoptera. Appl. & Envir. 

Microbial. 57: 1650-1655. 

van Frankenhuyzen, K.; Gringorten, J .L.; Mil en, R.E.; 

Gauthier, D.; Pusztai, M.; Brousseau, R.; Masson, L. 1993. 

Toxicity of activated cryl proteins from Bacillus 

thuringiensis to six forest Iepidoptera and Bombyx mori. 

J. Invert. Path. 62: 295-301. 

Williams, S.; Friedrich, L.; Dincher, S.; Carozzi, N .; 

Kessman, H.; Ward, E.; Ryals, J. 1992. Chemical regulation of Bacillus thuringiensis a-endotoxin expression in 

transgenic plants. Bio/Technology 10: 540-543. 

Wong, E.Y.; Hironaka, C.M.; Fischhoff, D.A. 1992. 

Arabidopsis thaliana small subunit leader and transit peptide enhance the expression of Bacillus thuringiensis proteins in transgenic plants. Plant Mol. Bioi. 20: 81-93. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
\uiv 

Chapter25 

Genetic Engineering for Air-Pollutant Resistance in 

Hybrid Aspen 1 

~ Saori .Endo, Etsuko Matsunaga, Keiko Yamada, and Hiroyasu Ebinuma 

~ 

Introduction 

Preservation of the natural environment, a growing 

worldwide concern, is being approached in various ways. 

Forests are useful because they continuously eliminate air 

pollutants while remediating and preserving the natural 

environment. Forest trees have a high capacity to absorb 

air pollutants and detoxify toxic substances produced by 

air pollution, but they do not adequately resist harmful 

air pollutants and other environmental stresses. An effective way to preserve the natural environment is to improve 

the resistance of forest trees to air pollutants and other 

environmental stresses. 

Damage to plant cells ~a used by environmental stresses, 

including herbicide exposure, high intensity light with low 

temperature, or air pollutants such as ozone and sulfur 

dioxide, is related to the over production of active oxygen 

in the cells. Active oxygen, such as singlet oxygen {1

0 2), 

superoxide (0;), and hydrogen peroxide (H20 2), are the 

by-products of many biological oxidations. For example, 

the electron transport chain of mitochondria and chloroplasts is a well-documented source of active oxygen. In 

plant cells, detoxification systems for active oxygen have 

evolved to facilitate immediate removal of active oxygen. 

However, under stressful situations, the equilibrium between the oxidative and antioxidative capacity can change 

within the plant cells, so that over production of active 

oxygen is induced. As the oxygen detoxification system of 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.R., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

higher plants has become better understood, we have attempted to improve the detoxification system by using 

genetic engineering to increase resistance to environmental stress. 

Detoxification System of Active 

Oxygen 

The production and destruction of active oxygen species, such as singlet oxygen (102

), superoxide (0;), and 

hydrogen peroxide (H20 2), occur during normal plant cell 

metabolism and are regulated processes. In plant cells, 

photosynthesis is the major source of active oxygen species. Chloroplasts generate highly active oxygen species 

by direct donation of excitation energy, or electrons, to 

oxygen from the photosynthetic electron transport chain. 

To counteract the toxicity of active oxygen species, plants 

possess an efficient antioxidative defense system composed of both nonenzymatic and enzymatic constituents. 

The typical detoxification system for active oxygen is described in figure 1. Superoxide, which is produced via the 

electron transport chain, is catalyzed to H2

0 2 by superoxide dismutase (SOD; Enzyme Commission number, EC 

1.15.1.1). The H20 2 is destroyed in the ascorbate peroxidase (APX; EC 1.11.1.11) reaction, and ascorbate is regenerated either directly by the electron transport chain or by 

the ascorbate-glutathione cycle. 

Increased activities of these antioxidative enzymes in 

the detoxification system may be related to additional 

stress-defense capacity. Thus, several research efforts are 

seeking to improve the natural antioxidative defense capacity by over expression of an antioxidative enzyme in 

transgenic plants. We are interested in improving the 

antioxidative defense capacity in woody plants by producing transgenic hybrid aspen with over expression of 

an antioxidative enzyme, glutathione reductase (GR; EC 

1.6.4.2). 

187 
Section IV Biotic and Abiotic Resistance 

2 H + 0 2 

w-U ,~2AsAXNAD ( P) + 

lsoo l IAPx l 

H 2 0 2MDA NAD (P) H+H + 

OHA 2GSH NADP + 

lGR I 

GSSG NADPH+H+ 

Figure 1. Detoxification system of active oxygen species in 

higher plants. SOD=superoxide dismutase; 

APX=ascorbate peroxidase; AsA=ascorbate; 

MDA=monodehydroascorbate; 

DHA=dehydroascorbate; GSH=reduced glutathione; GSSG=oxidized glutathione. 

Glutathione reductase, wh ich exists in chloroplasts 

and cytoplasm of plant cells, catalyzes the reduction of 

oxidized glutathione (GSSG) to reduced glutathione 

(GSH) by accompanying oxidation of ADPH (Foyer 

and Halliwe ll 1976; Gillhan and Dodge 1986; 

Rennenberg 1980). Furthermore, GR can be either a limiting -factor in the recycling capacity of an antioxidant, 

such as glutathion e, or responsible for the actua l 

amount of antioxidant in the cell. Transgenic tobacco 

plants conta ining a transferred GR gene have exhibited 

elevated GR activity and improved resistance to environmental stress associated with active oxygen (Aono 

eta!. 1991, 1993). 

Production of Transgenic Plants 

With the E. coli GR Gene 

Two derivatives of the binary vector plasmid pBI121, 

which contained the E. coli GR gene under control of 

the 355 promoter (pEGR4 and pEGR6), were constructed. In the pEGR6 construct, the ON A fragment 

coding for a chloroplastic transit-peptide was inse rted 

between the 355 promoter and the GR gene to transport GR protein from the cytosol to the chloroplast (figure 2A). Using our transformation me thod (Ebinuma 

188 

A 

pEGR4 HPT II CallY 355 NOS 

RB LB 

E. coli GR uene 

pEGR6 TP 

B 

0.24 

0.2 

0.16 

0.12 

0.08 

0.04 

0 

wt GR4-1 GR4-2 GR6-1 GR6-2 

Figure 2A. Construction of the T-DNA region of pEGR4 and 

pEGR6. RB=right border; LB=Ieft border; NPT ll=a 

chimeric gene for neomycin phosphotransferase; · 

CaMV 35S=35S promoter of cauliflower mosaic 

virus; NOS=nopaline synthase terminator; 

TP=chloroplasttransit peptide. 

Figure 2B.GR activity of transgenic aspen (GR4-1 , GR4-2, 

GR6-1 , and GR6-2) and nontransgenic aspen 

(wt). GR activity of leaf extracts was measured 

in a reaction mixture, which contained 0.1 M 

potassium phosphate (pH 7.8), 0.2 mM GSSG, 

and 0.2 mM NADPH in a final volume, and was 

monitored by the decrease in absorbance of 

NADPH at 340 nm. 

et a!. this volume), we introduced these genes into a 

hybrid aspen c lone 'Y63' (Populus sieboldii x P. 

grandidentata). We subsequently obtained 86 transgenic 

plants, 28 lines transformed with GR4 and 58 lines 

transformed with GR6. Expression of the E. coli GR gene 

was confirmed by immunochemical analysis. 

The g luta thione red uctase activity of transgenic 

plants was assayed in leaf extracts by monitoring absorbance decreases of NADPH at 340 nm. The GR activity of transgenic aspen, GR4 and GR6, ranged from 

1- to 3-fold higher than that of nontransgenic plants. 

Seven lines of GR4 transgenic aspen and 16lines of GR6 

transgenic aspen showed significantly higher GR activity in comparison with non transgenic aspen. The GR 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
activity of the transgenic plants, used in tolerance experiments, is shown in figure 2B. Elevated GR activity 

in the transgenic plants confirms expression of the introduced E. coli GR gene. These results indicate that 

increases in GR activity were caused by GR over expression. 

Resistance to Environmental 

Stress Caused by Over Production 

of Active Oxygen Species 

When plants are exposed to stressful conditions, such 

as a high intensity light, air pollutants, some herbicides, 

and metals, the generation of active oxygen species is increased. Increase in active oxygen production under stressful conditions results from either the inhibition of the 

photosynthesis pathway to decrease C02 assimilation, or 

the direct involvement in free radical production. So, plants 

under stressful conditions have increased susceptibility to 

photoinhibition with the subsequent development of chlorosis or necrosis. 

Transgenic aspens with elevated GR activity (figure 2B) 

should exhibit higher resistance to active oxygen when 

compared to nontransgenic aspens. To examine the resistance of transgenic aspens to environmental stresses, we 

exposed transgenic aspens to paraquat, sulfur dioxide 

(S02), and high intensity light at low temperature. 

Paraquat 

Several herbicides may cause production of active oxygen species, either by direct involvement in radical production or by inhibition of the biosynthetic pathways. 

Paraquat (methyl viologen; 1,1-dimethyl-4,4-bipyridium 

dichloride) is a herbicide that produces active oxygen in 

plant cells under light. Upon accepting an electron from 

Photosystem I, paraquat forms a free radical. The paraquat 

radical is recycled to paraquat by reacting with 0 2 to produce active oxygen, which then causes the disruption of 

cellular membranes and visible leaf damage. 

To assess the resistance to paraquat, leaf discs from aspen were incubated with paraquat and illuminated. This 

treatment caused visible damage to leaf discs of 

nontransgenic plants, as exemplified by a disruption in 

the cellular membranes and a color change to dark brown. 

The extent of cellular damage was quantified by solute 

leakage, which is a measurement of membrane disruption. 

Leaf discs of transgenic and nontransgenic aspen were 

incubated with various concentrations of paraquat (0, 1, 

10, and 100 J.!M) for 1 hat 25 oc in darkness. After paraquat 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Genetic Engineering for Air-Pollutant Resistance in Hybrid Aspen 

treatment, they were washed and floated on H20 under 

light (6,000 lux). Solute leakage was assessed by conductivity of the floating solution. Increased solute leakage of 

cellular components is indicative of cellular membrane 

disruption. Leaf discs of nontransgenic aspen showed a 

dose-dependent increase in membrane damage at 1 J.!M 

paraquat, and were nearly completely disrupted at 10 J.!M. 

Transgenic aspen leaf discs showed no damage at 1 ~-tM 

paraquat (figure 3); however, at 10 J.!M, the extent of damage in transgenic leaf discs was not significantly different 

from that of nontransgenic aspen. In addition, after the 1 

~-tM paraquat treatment, leaf discs of nontransgenic aspen 

showed visible damage, whereas those of transgenic aspen did not. 

These results indicate that leaf tissues of the GR 

transgenic aspen are resistant to paraquat when compared 

to nontransgenic aspen plants. Using paraquat treatment, 

we tested 2lines of cytosolic transgenic aspen (GR4-1 and 

GR4-2) and 2lines of chloroplastic transgenic aspen (GR6-

1 and GR6-2). We obtained similar results in 4 independent experiments. However, we were unable to detect any 

significant difference in tolerance to paraquat between 

cytosolic GR transgenic aspen (GR4) and chloroplastic 

transgenic aspen (GR6). 

ll "> 0.8 

·~ (,) 

;::, 

"'C 

c 8 0.6 

G) 

> "i 

~ 0.4 

0.2 

0 

Figure 3. 

wt GR4-1 GR4-2 GR&-1 GR&-2 

Electrolyte leakage from paraquat-treated leaf 

discs. Electrolyte leakage was assessed by 

conductivity of the floating solution. Conductivity 

was measured after illumination for 20 h, and 

relative values at 1 ~-tM (filled columns) and 10 

~-tM (unfilled columns) were determined against 

conductivity at 1 00 J.tM paraquat. 

189 
Section IV Biotic and Abiotic Resistance 

Sulfur Dioxide 

Air pollutants, such as ozone (03) a nd sulfur dioxide 

(S02), are thought to be major factors influencing modern forest de cline. Sulfur dioxide injury, which may 

occur in many plant species, is potentially severe under light or daytime conditions. Damage by S0 2 to biological systems is probably caused via the generation 

of radicals, such as 0 2·, OH·, and S03·, during S0 2 oxidation to sulfate. In the chloroplasts, S02 oxidation can 

be initiated by superoxide generated from the photosynthe tic electron transport chain, this process can induce the production of active oxygen species and sulfur 

trioxide radi ca ls. 

We exposed transgenic and nontransgenic aspen 

grown in a growth chamber for 4 months to 1 ppm S02 

at 25 oc unde r light (550 J.!E · m·

2 • s· 1

) with 70 percen t 

rela tive humidity. Three samples of each non transgenic 

and transgenic (GR4-1, GR4-2, GR6-1, a nd GR6-2) aspen line we re used for the S02 fumigation expe riment. 

After fumi ga tion for 7 h, all nontransgenic p la nts 

showed symptoms of foliar damage such as necrosis 

and dehy dration (figure 4A); howeve r, leaves of 

transgenic plants with elevated GR activity (GR4 and 

GR6) exhibited lowe r damage levels (figure 48). Similar results were obtained from independent rep lica tions. 

Transgenic plants with GR activity levels simila r to 

those of nontransgenic plants also sh owed severe damage. The S02 resistance of transgenic plants is apparently dependent on the increased GR activity from ove r 

expression of the E. coli GR gene. 

We quantified the extent of cellular damage caused 

by S02 fumigation by solute leakage, which is an indicator of membrane disruption. Leaves a t the same position were removed, cut into lea f discs, and incubated 

in Hp at 30 oc for 1 h. Solute lea kage wa s then measured. After fumigation fo r 4 h, leaf discs of 

nontransgeni c p lants showed increased solute lea kage. 

Nontransgenic leaves fumigated for 7 h showed comple te disrup tion. Leaves of nontransgenic aspen had 

S02-dependent membrane damage, whereas leaves of 

transgenic plants had less d amage. Damage exhibited 

by cytosolic GR transgenic plants was not significantly 

diffe rent from that of chl oroplasti c GR transgenic 

plants. These resu lts indica te that over expression of 

the E. coli GR gene contributes to 502 resistance. 

High Intensity Light With Low Temperature 

When plants are exposed to high intensity light, active 

oxygen species are over produced. Exposure of plants to 

high intensity light can exceed the capacity of photosynthesis, leading to over production of active oxygen. Injury 

resulting from high intensity light is potentially increased 

with low temperature. 

190 

A 

B 

Figure 4. Effect of S02 fumigation. Leaves from the same 

position in transgenic and nontransgenic aspen 

after exposure to 1.0 ppm S02, 25 °C, light 

(550 J.lE . m 2 . s·1) and 70% relative humidity for 

7 h. A) Leaf of nontransgenic aspen. B) Leaf of 

transgenic aspen (GR6-1) with elevated GR 

activity. 

To induce photoinhibition, we expose d leaves of 

transgenic and non transgenic aspen to high intensity light 

(10,000 lux) with low tempera ture (5 oc). Leaves at the 

same position in transgenic (GR4 a nd GR6) and 

non transgenic aspen (2 clones of every line) were exposed 

to the stress conditions that cause photoinhibition. After 

treatment for 6 h, leaves of the nontransgenic plants 

showed an increase in solute leakage. In contrast, leaves 

of transgenic aspen with over-expressed GR showed less 

(ca. 50 percent) d amage. These results are similar to those 

from transgenic tobacco with over-expressed SOD. We 

believe that transgenic aspen with over-expressed GR are 

resistant to photoinhibition. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Conclusion 

The results presented here demonstrate that GR 

transgenic aspens exhibit higher tolerance to active oxygen. GR transgenic aspens may also exhibit resistance to 

other environmental stresses, such as ozone and metal, that 

result in the production of active oxygen species. We believe that "environmental stress-resistant" trees produced 

by genetic engineering represent a very promising approach to help preserve the natural environment. 

Acknowledgments 

We thank Dr. N. Kondo, H. Saji, and M. Aono (National 

Institute for Environmental Studies) for their collaborative studies. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Genetic Engineering for Air-Pollutant Resistance in Hybrid Aspen 

Literature Cited 

Aono, M.; Kubo, A.; Saji, H.; Natori, T.; Tanaka, K.; Kondo, 

N. 1991. Resistance to active oxygen toxicity of 

transgenic Nicotiana tabacum that expresses the gene for 

glutathione reductase from Escherichia coli. Plant Cell 

Physiol. 32: 691-697. 

Aono, M.; Kubo, A.; Saji, H.; Tanaka, K.; Kondo, N. 1993. 

Enhanced tolerance tophotooxidative stress of transgenic 

Nicotiana tabacum with high chloroplastic glutathione reductase activity. Plant Cell Physiol. 34: 129-135. 

Foyer, C. H.; Halliwell, B. 1976. The presence of glutathione 

and glutathione reductases in chloroplasts: a proposed 

role in ascorbic acid metabolism. Planta. 133: 21-25. 

Gillham, D.J.; Dodge, A.D. 1986. Hydrogen-peroxide scavenging system within pea chloroplasts. A quantitative 

study. Planta. 167: 246-251. 

Rennenberg, H. 1980. Glutathione metabolism and possible 

roles in higher plants. Phytochemistry. 21: 2771-2781. 

191 

Section V Biotechnological Applications 
Chapter26 

Genetic Modification of Lignin Biosynthesis in 

Quaking Aspen and Poplar1 

Wout Boerjan, Marie Baucher, Brigitte Chabbert, Michel Petit-Conil, 

Jean-Charles Leple, Gilles Pilate, Daniel Cornu, Bernard Monties, Dirk lnze, 

Jan Van Doorsselaere, Lise Jouanin, and Marc Van Montagu 

with Chung-Jui Tsai, Gopi K. Podila, C.P. Joshi, and Vincent L. Chiang 

Introduction 

lignin is a complex aromatic heteropolymer and, second to cellulose, the most abundant organic polymer in 

the biosphere. In different tree species, lignin content is 

between 15 and 36 percent of the dry wood weight 

(Higuchi 1985). During plant development, lignin is deposited in secondary cell walls of mainly xylem cells, providing rigidity and structural support to the cell walls and 

enabling solute conductance in the vascular system 

(Higuchi 1985; Monties 1989). Moreover, lignin plays a role 

in by providing a barrier to prevent the spread of invading pathogens (Walter 1992). 

Despite its important biological role, lignin is undesirable in paper manufacturing (Dean and Eriksson 1992; 

Whetten and Sederoff 1991). For instance, during kraft 

pulping, the most widely used process for chemical 

pulping, lignin is separated from the cellulose by cooking 

the wood chips in NaOH (sodium hydroxide) and Na2

S 

(sodium sulfide). To remove 1000 kg of lignin, 800 kg of 

NaOH and 300 kg of N~S are required (Chiang et al. 1988). 

Subsequently, the cellulose fraction containing residual lignin is bleached. Bleaching conditions are determined by 

how much lignin remains linked to the cellulose after the 

cooking step. Cooking and bleaching partially depolymer1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

ize the cellulose fibers. Wood with a high lignin content 

requires longer cooking and bleaching times and different 

bleaching sequences; hence, a lower pulp yield is obtained. 

Due to the high lignin content in the raw material, chemical pulping results in low biomass use and pulp yield. In 

addition, residual wastes are hazardous to the environment (Biermann 1993). Reduction of lignin content or 

changing its composition or reactivity to facilitate lignin 

extraction would produce economical and environmental 

benefits through lower pulping costs, higher pulp yields, 

and less pulp-mill waste. 

During mechanical pulping, wood chips are ground and 

most of the biomass is effectively converted to pulp. Disadvantages of this procedure are yellowing of the paper 

after sunlight exposure, reduced paper strength, and imperfect paper surface structure (Biermann 1993). Following mechanical pulping, it would be beneficial to reduce 

the lignin amount and/or change the lignin composition 

to produce paper with an improved quality. 

Lignin is not always undesirable. The lignin content in 

wood is an important factor determining its intrinsic heat 

content for use as fuel (Whetten and Sederoff 1991) because the carbon content of lignin is 50 percent higher than 

that of polysaccharides (Brown 1985). This feature is particularly relevant to poplars and willows because they are 

cultivated as nonfossil, energy crops (Pearce 1995). Poplars and willows are excellent energy crops because they 

are fast-growing, easily coppiced, and grow well on diverse soils. Burning 1000 kg of dried coppice can generate 

as much electricity as 650 kg of coal, but avoids the release of up to 500 kg of carbon into the atmosphere (Pearce 

1995). Therefore, increasing the intrinsic heat content of 

trees by increasing their lignin content might be a useful 

goal for tree genetic engineering. 

Furthermore, the amount and structure of lignin may 

determine some mechanical properties of wood. Mechanical defects of wood, such as deformation after sawing or 

drying, were correlated with reaction wood that is fre193 
Section V Biotechnological Applications 

quently observed in some poplar clones. These defects are 

associated with variations in cellulose patterns and lignin 

deposition in the cell walls (Bailleres et al. 1995). 

Lignin is derived from the oxidative polymerization of 

3 different hydroxycinnamyl alcohols or monolignols. 

These monolignols, p-coumaryl alcohol, coniferyl alcohol, 

and sinapyl alcohol (figure 1), differ from each other only 

by their degree of methoxylation and generate the phydroxyphenyl (H), guaiacyl (G), and syringyl (S) units of 

the lignin polymer, respectively. Angiosperm (e.g., poplar) lignin consists of coniferyl and sinapyl alcohol and is 

classified as G-S lignin. Gymnosperm lignin contains 

mainly coniferyl alcohol and is classified as G lignin; 

whereas, lignin of grasses also has significant amounts of 

H-type lignin (Higuchi 1985). 

Lignin biosynthesis was recently reviewed in considerable detail by Boudet et al. {1995) and Whetten and Sederoff 

(1995). In this chapter, we present a brief description of 

the lignin biosynthesis pathway and an update on the 

complementary DNAs (cDNAs) and genes isolated from 

poplar that code for enzymes involved in lignin biosynthesis. Furthermore, we will focus on several examples in 

which lignin structure modification was obtained by genetic engineering. 

Lignin Biosynthesis 

Biosynthesis of the monolignols in angiosperms (figure 1) starts with the deamination of phenylalanine, derived from the shikimate pathway, to cinnamic acid, and 

the hydroxylation of cinnamic acid to p-coumaric acid. 

These enzymatic conversions are catalyzed by phenylalanine ammonia-lyase (PAL) and cinnamate-4-hydroxylase 

(C4H), respectively. As listed in table 1, cDNAs for PAL 

were isolated from P. x interamericana (Populus trichocarpa 

x P. deltoides) (Subramaniam et al. 1993) and P. kitakamiensis 

(P. sieboldii x P. grandidentata) (Osakabe et al. 1995a). Genomic sequences for PAL (Osakabe et al. 1995a, 1995b) and 

C4H (Kawai et al. 1994) were isolated from P. kitakamiensis. 

The acid p-coumaric is hydroxylated at its C-3 position by 

4-coumarate-3-hydroxylase (C3H) to produce caffeic acid. 

No eDNA or genomic sequences encoding C3H have been 

isolated from poplar or from any other plant species. This 

compound is methylated at the C-3 hydroxyl group to form 

ferulic acid by bispecific caffeic acid I 5-hydroxyferulic 

acid-0-methyltransferase (COMT). Ferulic acid is then 

hydroxylated at its C-5 position by ferulate-5-hydroxylase 

(FSH) to form 5-hydroxyferulic acid, then the C-5-hydroxyl 

group is methylated by the same bispecific COMT to create sinapic acid. cDNAs for COMT were cloned from P. 

tremuloides (Bugos et al. 1991) and P. trichocarpa x P. deltoides 

194 

6

0 ::t +--- phenylalanine 

I 

PAL 

o;r;OMT o;ro:5H o;ro:OMT o;roH 

6°~ OoH-+ Ooc;-:HOocH:-:.oOocH, OH OH OH OH OH 

p-coumaric caffeic ferulic 5-hydroxyferulic sinapic 

acid acid acid acid acid 

! 4CL ! 4CL ! 4CL ! 4CL ! 4CL 

&

o s~c~:A3H&o ,:::AOMT6o ,.s-co: -6:0 ,.::::DMT6: ,.S·CoA 

__. I --+ I --+ --+ I OH OCH, HO OCH, CH,O OCH, OH OH OH OH OH 

! CCR ! CCR ! CCR 

6" &~ .. CH,06.:H, OH OH OH 

p-coumaraldehyde coniferaldehyde sinapaldehyde 

! CAD ! CAD ! CAD 

60H 

~::. CH,06:::. OH OH OH 

p-coumaryl coniferyl sinapyl 

alcohol alcohol alcoho~ 

CHI (GI (5) 

Figure 1. Lignin biosynthesis pathway. PAL=phenylalanine 

ammonia-lyase; C4H=cinnamate-4-hydroxylase; 

C3H=4-coumarate-3-hydroxylase; COMT =bispecific caffeic acid/5-hydroxyferulic acid-0-

methyltransferase; F5H=ferulate-5-hydroxylase; 

CCoA3H=coumaroyi-CoA-3-hydroxylase; 

CCoAOMT =caffeoyi-CoA-0-methyltransferase; 

4CL=4-hydroxycinnamate-CoA ligase; 

CCR=cinnamoyi-CoA reductase; CAD=cinnamyl 

alcohol dehydrogenase. 

(Dumas et al. 1992). Genes encoding COMTwere isolated 

from P. tremuloides (Tsai et al. 1995) and P. kitakamiensis 

(Hayakawa et al. 1996). In addition, a eDNA for FSH was 

recently isolated from Arabidopsis thaliana (C. Chapple, 

personal communication). 

Subsequently, p-coumaric acid, ferulic acid, and sinapic 

acid are converted to the corresponding coenzyme A (CoA) 

esters by 4-hydroxycinnamate-CoA ligase (4CL). cDNAs 

encoding 4CL were isolated from P. trichocarpa x P. deltoides 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Genetic Modification of Lignin Biosynthesis in Quaking Aspen and Poplar 

Table 1. cDNAs and genes from Populus spp. Involved in lignin biosynthesis. Numbers refer to the different cDNAs or 

"4Viii genes that have been cloned. 

~ eDNA/gene 

Gene number Species Reference 

PAL cDNA2 P. trichocarpa x P. deltoides Subramaniam et al. (1993) 

eDNA 11 P. kitakamiensis Osakabe et al. (1995a) 

( P. sieboldii x P. grandidentata) 

gene 2 P. trichocarpa x P. deltoides Subramaniam et al. (1993) 

gene 52 P. kitakamiensis Kawai et al. (1994) 

Osakabe et al. (1995a, 1995b) 

C4H gene 13 P. kitakamiensis Kawai et al. (1994) 

COMT eDNA 1 P. tremuloides Bugos et al. (1991) 

eDNA 1 P. trichocarpa x P. deltoides Dumas et al. {1992) 

gene 1 P. tremuloides Tsai et al. (1995) 

gene 2 P. kitakamiensis Hayakawa et al. (1996) 

4CL cDNA2 P. trichocarpa x P. deltoides Allina and Douglas (1994) 

CCR eDNA 1 P. trichocarpa Leple et al. (1995) 

gene 1 P. trichocarpa J. Van Doorsselaere (unpublished) 

CAD eDNA 1 P. trichocarpa x P. deltoides Van Doorsselaere et al. (1995a) 

gene 1 P. trichocarpa 

Peroxidase cDNA3 P. kitakamiensis 

cDNA4 P. trichocarpa 

gene4 P. kitakamiensis 

gene4 P. trichocarpa 

CCoAOMT eDNA 1 P. tremuloides 

cDNA4 P. trichocarpa 

gene 2 P. trichocarpa 

1 1 partial PAL eDNA was isolated. 2 2 independent genes or gene fragments were isolated. 3 4 independent clones were isolated, 1 was sequenced. 

by Allina and Douglas (1994). These CoA esters are converted through 2 successive reductions to the corresponding alcohols by cinnamoyl-CoA reductase (CCR) and 

cinnamyl alcohol dehydrogenase (CAD). Recently, at the 

University of Gent (Gent, Belgium), a eDNA and a genomic 

clone encoding CCR were isolated from P. trichocarpa (Leple 

et al. 1995; J. Van Doorsselaere, unpublished results). Also, 

cloning of a eDNA for CAD from P. trichocarpa x P. deltoides 

was reported recently (Van Doorsselaere et al. 1995a), and 

a corresponding CAD genomic clone was isolated from P. 

trichocarpa (J. Van Doorsselaere, unpublished results). Resulting monolignols are subsequently dehydrogenated 

with the formation of mesomeric free phenoxy radicals that 

polymerize and produce the complex lignin heteropolymer. Several cDNAs and a gene encoding peroxidases 

with potential roles in lignification were isolated from P. 

kitakamiensis (Kawai et al. 1993, 1994; Osakabe et al. 1994, 

1995c) and P. trichocarpa (Christensen et al. 1995). PotenUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

J. Van Doorsselaere (unpublished) 

Kawai et al. (1994) 

Osakabe et al. ( 1994) 

Christensen et al. (1995) 

Kawai et al. (1993) 

Kawai et al. (1994) 

Osaka be et al. ( 1995c) 

J. Christensen (unpublished) 

Meng and Campbell (1995) 

Meyermans et al. (1995) 

Chen et al. (1995) 

tial roles of peroxidases, laccases, and coniferyl alcohol 

oxidases in lignification were reviewed and discussed by 

Bourlet et al. (1995). 

Recently, more attention has focused on alternative pathways involved in the biosynthesis of the monolignols. One 

such pathway involves the hydroxylation of p-coumaroylCoA to form caffeoyl-CoA by p-coumaroyl-CoA-3-hydroxylase (CCoA3H), and the subsequent methylation of 

caffeoyl-CoA to produce feruloyl-CoA by caffeoyl-CoA0-methyltransferase (CCoAOMT). It was strongly suggested that in Zinnia, CCoAOMT is as important in 

lignification as is COMT (Ye and Varner 1995; Ye et al. 1994). 

Recently, CCoAOMT cDNAs were isolated from P. 

tremuloides (Meng and Campbell 1995) and P. trichocarpa 

(Meyermans et al. 1995), and genomic clones were isolated 

from P. trichocarpa (Chen et al. 1995.). 

Matsui et al. (1994) and Daubresse et al. (1995) focused 

attention on a previously unknown pathway in lignin bio195 
Section V Biotechnological Applications 

synthesis. These groups performed feeding experiments 

on several angiosperm and gymnosperm plants with radioactively labeled p-glucocoumaryl alcohol, coniferin, and 

syringin, which are the P-glucoside storage ·forms of pcoumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, 

respectively. They found evidence for an interconversion 

of these compounds at the monolignol, glucoside, or lignin level. Moreover, this pathway exists in gymnosperms 

and angiosperms, in which the conversion from H to G 

and H toG to S was observed, respectively (figure 1). Since 

the level at which the hydroxylation and methylation reactions occur is not fully established, these reactions are 

not shown in figure 1. 

Strategies to Modify Lignin Biosynthesis 

Two broad categories of linkages between building units 

in the lignin polymer are the ether linkages and the carbon-carbon linkages (figure 2). Ether linkages, such as the 

P-0-41inkage, are easily broken during the de lignification 

~-0-4 

5-5 

Figure 2. Schematic representation of the p-0-4 ether 

linkage and 5-5 carbon-carbon linkage. The 

structures are represented as dimers. For an 

overview of the different linkages refer to Adler 

(1977). 

196 

process. In contrast, C-C linkages, such as the 5-5 linkage, 

are very resistant to chemical attack (Boudet et al. 1995; 

Chiang and Funaoka 1990a, 1990b; Chiang et al. 1988). 

Gymnosperm lignin is composed mainly of G units that 

are methoxylated only once; therefore, the C-5 aromatic 

position is available for a C-C linkage during the polymerization process (e.g., with a C-5 or C-P from another 

coniferyl alcohol). Due to S units that have no free C-5 

positions, angiosperm lignin has less 5-5 and P-5 linkages 

than gymnosperm lignin and is, therefore, less condensed 

than gymnosperm lignin. This could be one reason why 

it is easier to delignify angiosperm wood (hardwood) by 

kraft pulping than gymnosperm wood (softwood). However, the methoxy groups that are more abundant in 

syringyllignin are converted to air-polluting mercaptanes 

by the reaction with sulfides during chemical pulping 

(Biermann 1993). A genetically engineered change in the 

S:G unit ratio of the lignin polymer could potentially 

modify extraction properties of lignin for chemical 

pulping. 

Two genetic approaches have been proposed to modify 

the degree of methoxylation in lignin. In gymnosperm 

wood, a genetically induced methoxylation at the C-5 position could render the lignin less condensed. In angiosperm wood, substitution of -OCH3 groups by -OH 

groups could result in the incorporation of 5-hydroxyguaiacyl (5-0H-G) units, thereby increasing lignin solubilization in the pulping liquor (Boudet et al. 1995). Because 

COMT catalyzes methylation reactions in lignin biosynthesis, manipulation of the COMT activity by genetic engineering would likely result in a modified degree of 

methylation. Increased COMT activity might result in an 

elevated S content. Manipulation of COMT has also been 

proposed to reduce lignin content since brown-midrib 

mutations in maize (bm) and sorghum (bmr) were associated with a reduction of COMT activity and of COMT and 

CAD activity, respectively (Grand et al. 1985a; Pillonel et 

al. 1991). In both cases, the mutation conferred lower lignin content. Moreover, the maize bm3 mutant was shown 

to have a defective COMT gene (Vignols et al. 1995). Both 

mutants have an altered lignin composition with the incorporation of the 5-0H-G unit into the lignin polymer 

(Chabbert et al. 1993; Lapierre et al. 1988). These mutants 

are characterized by a reddish-brown coloration of the 

midrib. 

Down-regulation of the COMT gene expression was 

.obtained in transgenic tobacco (Atanassova et al. 1995; 

Dwivedi et al. 1994; Ni et al. 1994) and transgenic poplar 

(Van Doorsselaere et al. 1995b ); the most marked results 

were·a drastic reduction of the S:G ratio and an incorporation of the 5-0H-G residue in the lignin polymer. For more 

details, see below. 

CAD was also targeted to manipulate the lignification 

process because a reduced CAD activity might limit 

monolignols available for polymerization. Evidence for this 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
hypothesis was derived from chemical-inhibitor studies 

and mutant analysis. First, inhibition of CAD activity in 

poplar stems by 2 CAD activity inhibitors resulted in a 

45 percent reduced incorporation of 14C:labeled cinnamic 

acid into lignin (Grand et al. 1985b). Second, the sorghum 

bmr6 mutant with reduced CAD and COMT activities, 

exhibits lower lignin content with an increased incorporation of cinnat:naldehydes (Pillonel et al. 1991). In addition, 

the maize bml mutant has a lower lignin content and 

strongly reduced CAD activity (Holt et al. 1995). 

Modifying CAD gene expression using an antisense 

strategy was described for tobacco (Halpin et al. 1994; 

Hibino et al. 1995; Higuchi et al. 1994). Lignin content and 

composition were not significantly modified, but a different lignin containing cinnamaldehydes was obtained. 

Moreover, this change in lignin structure impacted lignin 

extractability. 

Other enzymes from the lignin biosynthesis pathway 

were up- or down-regulated. Transgenic tobacco plants 

with decreased PAL activity (20 percent residual activity) 

had a reduced lignin content and severely affected development (Elkind et al. 1990). An anionic peroxidase, reportedly involved in lignification, was overproduced and 

down-regulated in tobacco (Lagrimini 1991 ). However, no 

significant changes in lignin content or composition were 

observed in plants with a down-regulated peroxidase level 

(Chabbert et al. 1992). In addition, plants with an overproduced·peroxidase synthesized more lignin. 

In this chapter, genetic engineering of quaking aspen 

with antisense COMT, and poplar with antisense COMT 

and antisense CAD constructs are discussed along with 

the effects of down-regulation of these enzymes on the lignin content, composition, and structure. 

Manipulation of Lignin 

Biosynthesis in Transgenic 

Aspen and Poplar via an 

Antisense COMT Strategy 

Manipulation of COMT Gene Expression in 

Transgenic Aspen 

In a study at Michigan Technological University 

(Houghton, MI, USA) transgenic tobacco (Nicotiana 

tabacum) plants expressing an antisense aspen COMT gene 

(Ptomtl) driven by the cauliflower mosaic virus (CaMV) 

355 promoter, showed a pronounced inhibition of COMT 

activity in the developing xylem tissue. These plants exhibited a marked reduction (up to 20 percent) inS lignin 

units with no significant reduction in V lignin building 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Genetic Modification of Lignin Biosynthesis in Quaking Aspen and Poplar 

units (Dwivedi et al. 1994). The S (amount of 

syringaldehyde):V (amount of vanillin and vanillic acid) 

ratio is a measure for the monomeric composition of lignin as determined by nitrobenzene oxidation (Adler 1977; 

Monties 1989). Down-regulation of COMT gene expression in these transgenic plants also reduced lignin content 

from 5 to 12 percent when compared to the wild type. The 

antisense approach was subsequently applied to quaking 

aspen (P. tremuloides) to investigate the effects of antisense 

down-regulation of the COMT gene on lignin biosynthesis in a woody angiosperm species. For this purpose, efficient Agrobacterium tumefaciens-mediated gene 

transformation and a whole-plant regeneration system was 

developed using greenhouse plants as explant material 

(Tsai et al. 1994). 

A 5' 470-bp (base pair) fragment from the aspen COMT 

eDNA (Bugos et al. 1991) was cloned in antisense orientation behind the CaMV 355 promoter (figure 3), and this 

gene construct was introduced into aspen according to Tsai 

et al. (1994). More than 100 transgenic aspen plants were 

obtained, most of which were phenotypically indistinguishable from the wild-type aspen. These plants were 

analyzed by polymerase chain reaction (PCR) to investigate whether the T-DNA was present. For all transformants, positive amplification of the neomycin 

phosphotransferase II (NPTII) gene was observed. From 

these plants, 31 were randomly selected for further analysis. Using the NPTII gene, 355 promoter, or COMT gene as 

a probe, DNA gel blot analysis demonstrated T-DNAintegration in most of the plants, and further showed that the 

31 plants were derived from 17 independently transformed 

lines. 

Expression of the antisense COMT gene in the transgenic 

plants was demonstrated by RNA gel blot analysis. Individual plants derived from 13 independent transformed 

lines were selected for the analysis. A sense COMT RNA 

probe was synthesized to detect only antisense transcript 

derived from the introduced antisense COMT gene. Seven 

lines (numbers 7, 15, 17, 18 19, 23, and 30) showed a hybridization signal of 0.7 kb, the size expected for the 

antisense COMT mRNA. No steady-state antisense COMT 

mRl'\JA was detected for the other transgenic lines, perhaps 

indicating an effective interaction with the native sense 

transcript. Based on the observations of several other studies, such plants might display the strongest antisense effect (Atanassova et al. 1995; Van Doorsselaere et al. 1995b ). 

COMT enzyme assays were conducted on xylem tissue 

from individual plants derived from the 13 independent 

transformants. Both caffeic acid and 5-hydroxyferulic acid 

were used as substrates. In most of the transgenic plants 

assayed, COMT activity toward caffeic acid and 5-

hydroxyferulic acid was reduced by 5 to 50 percent and 

10 to 60 percent, respectively. 

However, lignin content of these transgenic plants, as 

determined by the Klason method (Effland 1977) and the 

197 
Section V Biotechnological Applications 

RB LB 

NPTII 355-E -4 bi-QMT 

6'-470bp 

eDNA 

05-T~-----< 

Figure 3. Diagram of the aspen antisense COMT gene construct. RB and LB=right and left border of the T-DNA, respectively. N08-P and NOB-T =promoter and terminator of the nopaline synthase gene, respectively; NPTII=coding 

sequence of the neomycin phosphotransferase II gene; 358-E and 358-P=enhancer and promoter of the cauliflower 358 gene, respectively; AMV-RNA4=alfalfa mosaic virus RNA 4; bi-OMT=bi-specific caffeic acid/5-

hydroxyferulic acid-0-methyltransferase. 

acetyl bromide method (liyama and Wallis, 1988), did not 

differ markedly from wild-type lignin content. It is possible that expression of the antisense COMT gene may affect the lignin structure with respect to methylation. The 

antisense COMT plants are currently being analyzed for 

lignin composition. 

Manipulation of COMT Gene Expression in 

Transgenic Poplar (P. tremula x P. alba) 

A poplar COMT eDNA plasmid (pPCL4) (Dumas et al. 

1992) was used to create several constructs under the control of the CaMV 355 promoter, which drives constitutive 

expression in poplar (Leple et aL 1992). Different constructs 

with 5' and 3' COMT fragments in sense and antisense orientation were made. Results of poplar transformation with 

the pASB plasmid, containing a 0.9-kb 3' fragment of the 

COMT eDNA cloned in antisense orientation, are previously reported (Van Doorsselaere et al. 1995b). 

The plasmid pASB and the control plasmid pBI121 (355-

GUS construct; Jefferson et al. 1987) were introduced into 

hybrid poplar (P. tremula x P. alba) by Agrobacterium 

tumefaciens-mediated transformation (Leple et al. 1992). 

Fifteen independent transformants were generated for each 

construct. All antisense and pBI121 transformants were 

analyzed for stable integration of the T-DNA by DNA gel 

blot analysis. The T-DNA copy numbers for the 

transformants were between 1 and 4. No morphological 

abnormalities were observed in the different 3-month-old, 

greenhouse-grown, transgenic lines. However, 2 ASB 

transformants (ASB2B and ASB10B) appeared to have a 

different wood color when the phloem was peeled off. The 

xylem of these trees was pale rose, whereas the xylem of 

wild-type poplars was white I yellow. As previously mentioned, a red-brown coloration of the xylem occurred in 

198 

brown-midrib mutants from maize and sorghum with an 

altered lignin content. 

RNA gel blot analysis of xylem tissue was performed to 

investigate whether expression of the antisense COMT 

transcript reduced the sense steady-state COMT mRNA 

level. In 4 ASB transgenic poplars (ASB4A, ASB6A, ASB2B, 

and ASB10B), steady-state COMT transcript was significantly reduced with the lowest levels occurring in ASB2B 

and ASB10B. No antisense transcript was detected in these 

4 ASB lines. 

All ASB transformants and controls were analyzed for 

their COMT activity toward caffeic acid in xylem tissue. 

Plants ASB4A and ASB6A had a 50 percent reduction in 

COMT activity compared with the control (figure 4). In 

xylem of ASB2B and ASB10B plants, COMT activity was 

reduced by 95 percent (5 percent residual activity). On 

protein gel blots of ASB2B and ASB10B, where xylem proteins bound tobacco anti-COMT-11 antibodies (Hermann 

et al. 1987), COMT protein level was very low when compared to the control, thereby confirming the enzyme assay data. 

Xylem tissue was harvested from controls and ASB 

plants and subjected to thioacidolysis followed by gas chromatography (Lapierre et al. 1986) to investigate the effect 

of a decreased COMT activity on the monomeric composition of lignin. This technique allows analysis of the 

noncondensed lignin fraction (i.e., determination of those 

units linked by arylglycerol aryl ether linkages) (figure 2). 

Due to decreased S units and increased G units in xylem 

from plants ASB2B and ASB10B, the S:G ratio was significantly reduced (3- to 4-fold) when compared to the control plants (table 2). In plants ASB4A and ASB6A, the S:G 

ratio was also reduced but only by 1.3- to 2-fold (data not 

shown). Transgenic poplars with no reduction in COMT 

activity exhibited an S:G ratio comparable to that of conUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
ASB4A ASB6A ASB2B ASBIOB 

Figure 4. COMT activities, in percent compa red to the 

control value, towards caffeic a cid in stem xylem 

from transforma nts ASB4A, ASB6A, ASB2B, 

ASB1 08 , a nd control (C). 

trol p lants (d ata not shown}. Considerable variation existed in the amou nt and composition of the lignin among 

d ifferent samp ling dates, perhaps ind icating differentia l 

expression of the lignin biosynthesis genes throughout 

d evelopment (Van Doorsselae re eta!. 1995b). In p lants 

ASB2B and ASB10B, the partially methoxyla ted 5-0H-C 

unit was detected among the thioacidolysis products. No 

significant differences between control p lants and the different transgenic Jines were detected when lignin amount 

was determined by the Klason method and the acetyl bromide method (table 2). 

Genetic Modification of Lignin Biosynthesis in Quaking Aspen and Poplar 

Discussion 

Two independ ent studies to modify lignin con tent and 

composition in aspen and poplar were conducted to investigate the in vivo role o f b ispecifi c caffeic acid / 5-

hyd roxyferulic acid 0 -methyltransferase (COMT) in lignin 

biosynthesis. The eDNA sequences of COMT genes isola ted from P tremuloides and P trichocnrpn x P. deltoides show 

99 percent identity at the amino acid level (Dumas eta!. 

1992). Transgenic aspen and poplar p la nts were obtained 

that constitu tively express COMT gene fragments in an 

antisense orientation. This provides a un ique opportuni ty 

to compare a nd examine effects of simila r gene constructs 

in 2 d istinct species of Populus. 

At Mic h iga n Technological Un ive rsity, more than 

100 transgenic aspen trees were produced that constitutively expressed the 5' 470-bp of the aspen COMT gene in 

antisense orien tation. A s ubset of these transgenic plants 

was ana lyzed for the presence and exp ression of the COMT 

gene fragment in antisense orientation. Several lines had 

a reduced COMT activity {up to 50 percent) using both 

caffe ic acid and 5-hyd roxyferulic acid as substra te. This 

reduced COMT activity did not affect the morphology, 

g rowth, or overa ll lignin content of these plants. Curre ntly, 

the e transgenic aspen trees a re being analyzed for their 

lig nin composition. 

At the University of Cent (Cent, Belg ium), in collaboration with French laboratories of the lnstitut Na tional de Ia 

Recherche Agronomique (1 1 RA) and as part of a Eu ropean project (OPLICE), transgenic poplar trees expressing a 3' 900-bp part of th e COMT gene in antisense 

orientation were obtained. Fou r transgenic lines showed 

a reduced transcript level of endogenous COMT and reduced COMT activity. The most d istinct resu lts were that 

an inhibition of COMT activity d id not reduce lignin quanTable 2. Klason lignin content and lignin composition of xylem from control and antisense COMTplants. Data are 

expressed as mole g·' of cell wall residue (CWR}, which is that left after s uccessive extractions of the xylem with toluene 

ethanol, ethanol, and water. Lignin composition was analyzed by thioacidolysis. The data a re the mean of two independent 

measurements of 1 or 2 plants. Standard deviations for KL, G, S, a nd 5-0H-G were 3% and 6%, at the most, res pective ly. 

June 1994 

PBI121 

ASB2B 

ASB10B 

October 1994 

PBI1 21 

ASB2B 

ASB108 

' = not detected 

PBI121 =control 

KL G 

(Kiason lignin) (guaiacyl unit) 

19.0 133 

16.8 150 

17.1 160 

19.6 132 

19.3 197 

19.2 179 

ASB2B and ASB1 08 =plants with 95% reduced COMT activity 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

s 5-0H-G 

(syringyl unit) (5-hydroxyguaiacyl unit) S:G 

189 1.42 

34 16 0.23 

51 13 0.31 

207 1.57 

50 16 0.25 

84 6 0.47 

199 
Section V Biotechnological Applications 

tity. However, the overall 5:G composition was dramatically reduced, and incorporation of the 5-0H-G unit was 

reduced in the lignin of A5B2B and A5B10B. In wild-type 

plants, 5-hydroxyferulic acid is converted into sinapic acid, 

and the corresponding 5-0H-G monomeric unit cannot be 

detected via thioacidolysis. Our data show that this unusual monomeric unit can be incorporated into the 

arylglycerol aryl ether lignin structures of the transformed 

poplars. In addition, 5-hydroxyferulic acid is not incorporated as such but rather as 5-hydroxyconiferyl alcohol. 

These results suggest that 5-hydroxyferulic acid is converted into the corresponding alcohol in the same manner 

as p-coumaric acid, ferulic acid, and sinapic acid. Remarkably, the G content in plantsA5B2B and ASB10B was higher 

than the controls. 

A similar relative variation in content of noncondensed 

5 and G monomeric units was previously observed with 

brown-midrib bm3 mutants of maize (Barriere et al. 1994). 

Indeed, the maize bm3 mutant has reduced COMT activity and synthesized modified lignin with a 4- to 5-fold reduced 5:G ratio (due to an increase in G and a decrease in 

5 units) when compared to the wild-type (Lapierre et al. 

1988). Moreover, an accumulation of the 5-0H-G unit is 

observed in this mutant. 

Recently, Dwivedi et al. (1994), Ni et al. (1994), and 

Atanassova et al. (1995) reported on down-regulation of 

COMT frorr\ tobacco by expressing aspen, alfalfa, and tobacco COMT gene fragments, respectively. Dwivedi et al. 

(1994) observed a 45 percent reduction in COMT activity 

that resulted in a slightly lower content of 5 units. The 5:V 

ratio was reduced by 20 percent. In contrast, Ni et al. (1994) 

showed that the expression of an alfalfa COMT antisense 

RNA in tobacco reduced lignin content without modifying lignin composition (5:V). No significant amounts of 

the 5-0H-G unit were found by these authors. This can be 

explained either by the absence of such monomers in lignin, or more likely, by the oxidative degradation of the 

corresponding product during nitrobenzene oxidation 

(Rolando et al. 1992). Partially methoxylated monomeric 

units containing ortho-diphenolic functionality, such as the 

5-0H-G monomers, are lost after nitrobenzene oxidation 

under standard conditions (Lapierre et al. 1988). 

Atanassova et al. (1995) showed that antisense COMT tobacco plants with a strongly reduced COMT activity, are 

significantly affected in the proportion of syringyl units in 

their lignin and accumulate 5-0H-G units, whereas the 

total lignin content was not modified. These results support those obtained in poplar. 

Clearly, reduced 5 content in the transgenic poplars was 

due to the down-regulation of COMT. However, lignin 

quantity was not modified and G unit content was increased, suggesting that G units in the noncondensed lignin fraction are synthesized by an alternative route (e.g., 

via CCoAOMT). We hypothesize that the CCoAOMT enzyme plays an important role for lignification in poplar, 

200 

as in Zinnia (Ye and Varner 1995; Ye et al. 1994). Involvement of more than 1 OMT activity in lignin biosynthesis 

was also suggested by Dwivedi et al. (1994) and Matsui et 

al. (1994). 

Down-regulated COMT poplars showed a discolored 

xylem that may be comparable to the phenotype of the 

bm3 maize mutant. The bmr6 sorghum mutant and the 

CAD down-regulated tobacco plants also showed a reddish xylem discoloration (Halpin et al. 1994; Hibino et al. 

1995; Higuchi et al. 1994; Pillonel et al. 1991). When the 

CAD activity in xylem tissue of A5B2B and A5B10B trees 

was measured, no differences were observed compared 

with the control indicating that the red coloration was not 

correlated with a reduced CAD activity. Further investigations are needed to determine whether the red coloration in down-regulated CAD and COMT plants is 

attributable to similar biochemical alterations. 

Kraft cooking (pulping) experiments on wood of 1-yearold A5B2B and A5B10B trees were recently performed and 

showed, consistent with the lower level of S units, a higher 

kappa number after the cooking step. This observation 

indicates that the lignin is more difficult to remove from 

poplars with down-regulated COMT activity, and that the 

expected positive effect of the 5-0H-G unit on lignin solubilization does not counterbalance the negative effect of 

the lower 5:G ratio on lignin extraction. 

Manipulation of Lignin 

Biosynthesis in Transgenic 

Poplar (P. tremula X P. alba) via 

an Antisense CAD Strategy 

Manipulation of CAD Gene Expression in 

Transgenic Poplar (P. tremula x P. alba) 

As previously mentioned, cinnamyl alcohol dehydrogenase (CAD) is considered an important target enzyme 

for reducing lignin content by genetic engineering because 

it catalyzes the conversion of cinnamaldehydes to 

cinnamyl alcohols, the last step in monolignol biosynthesis. Therefore, a study was initiated at the University of 

Gent, in collaboration with the INRA, to modify CAD gene 

expression in poplar and investigate its affect on lignin 

biosynthesis. 

A eDNA for CAD from P. trichocarpa x P. deltoides was 

isolated by heterologous screening with a tobacco CAD 

eDNA (Van Doorsselaere et al. 1995a). The full-length 

eDNA was expressed under the control of the CaMV 355 

promoter in sense and antisense orientation. These constructs were introduced into P. tremula x P. albausing an 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Agrobacterium-mediated transformation technique (Leple 

et al. 1992). For both constructs, 15 independent 

transformants were isolated for which T-DNA copy numbers were between 1 and 5. 

To investigate transgene effectiveness in overproducing 

or down-regulating CAD, CAD activity measurements and 

RNA gel blot analyses were performed. For the poplars 

transformed with the antisense construct, the CAD activity in xylem was reduced by a maximum of 70 percent 

(30 percent residual activity). This response was observed 

for lines ASCAD14, ASCAD21, and ASCAD52. In these 

lines, a very low steady-state sense CAD transcript level 

was observed by RNA gel blot analysis. For the poplars 

transformed with the sense construct, the maximum overproduction was about 30 percent more than the control. 

Two lines, CAD1 and CAD4, showed a 70 percent reduction in CAD activity. This reduced activity was due to a 

co-suppression phenomenon because only very low levels of steady-state sense CAD mRNA were detected by 

RNA gel blot analysis. All these transformed plants displayed normal growth and development. 

Interestingly, transformants with a minimum 50 percent 

reduction in CAD activity displayed a red coloration of 

the xylem tissue. In 2-year-old transgenic poplars, red coloration was observed in the outer, young xylem tissue not 

in the internal, older xylem. A reddish-brown coloration 

was observed for brown-midrib mutants with a reduced 

lignin content, and for genetically engineered tobacco 

plants with reduced CAD activity (Halpin et al. 1994; 

Hibino et al. 1995). When the red poplars and controls were 

Genetic Modification of Lignin Biosynthesis in Quaking Aspen and Poplar 

stained with phloroglucinol (indicative for aldehyde end 

groups), a different red-brown coloration was seen in the 

down-regulated CAD plants when compared to the controls, which stained pink. This result suggests an altered 

level of aromatic aldehyde groups in the lignin of CAD 

down-regulated plants. 

No lignin reduction (Klason method) was observed in 

the xylem from poplars with reduced CAD activity 

(table 3). Despite the obvious phenotypic difference, no 

modification in the lignin composition {S:G ratio) was detected. As for COMT, a considerable and significant variation in the lignin content and composition was observed 

among different sampling dates. We speculate that this 

variation is due to differences in the activities of lignin 

biosynthesis enzymes over the different growing seasons 

and years. To determine the origin of the red color, dioxane-soluble lignin fractions were isolated from red and 

white poplar xylem, then analyzed by spectrophotometry 

(250-500 nm). Dioxane was used to extract lignin without 

ionizing phenolic groups. The absorption spectrum of the 

dioxane lignin from red poplars showed a higher absorbance in the range from 300 to 340 nm when compared to 

the spectrum of dioxane lignin from white poplar. This 

absorbance was decreased by sodium borohydride 

(NaBH) reduction. Such UV spectral behavior is indicative of increased amounts of conjugated aromatic aldehydes in the lignin polymer (Tollier et al. 1995). 

Clear differences between red and white plants were 

obtained after alkali treatment of the xylem cell wall residue (CWR). CWR is the residue corresponding to insoluble 

Table 3. Klason lignin content and lignin composition of xylem from control and antisense CAD poplars. Data are 

expressed as mole g-1 of cell wall residue (CWR), which is that left after successive extractions of the xylem with toluene 

ethanol, ethanol, and water. Lignin composition was analyzed by thioacidolysis. The data are the mean of two independent 

measurements of 1 to 2 plants. Standard deviations for KL, G, S, and S+G were 3% and 6%, at the most, respectively. 

KL G s 

(Kiason lignin) (guaiacyl unit) (syringyl unit) S:G S+G 

June 1993 

PBI121 18.7 133 270 2.03 403 

ASCAD3 18.7 113 259 2.29 372 

ASCAD14 16.6 133 286 2.15 419 

ASCAD21 16.4 136 277 2.05 413 

ASCAD52 17.5 107 217 2.03 324 

June 1994 

PBI121 18.4 153 261 1.65 413 

ASCAD3 19.4 133 200 1.50 333 

ASCAD14 19.5 136 233 1.70 369 

ASCAD21 18.5 131 173 1.32 304 

ASCAD52 20.3 126 196 1.55 323 

PBI121 =control 

ASCAD3 = wild-type CAD activity 

ASCAD14, ASCAD21, ASCAD52 = 70% reduction of CAD activity 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 201 
Section V Biotechnological Applications 

cell wall structure remaining after successive xylem extractions with toluene ethanol, ethanol, and water. Analkali treatment of the CWR ionizes free phenolic functions 

and partially hydrolyzes polysaccharides resulting in partial solubilization of a lignin fraction from the cell wall. 

This alkali treatment allows the recovery of a saponified 

residue and an alkali fraction. Saponified residue of the 

red poplars contained significantly less Klason lignin 

when compared to the white poplars. Alternatively, substantial quantities of lignin were isolated by acid precipitation of the alkali fraction from the red poplars, which 

was not so for the white poplars. Spectral characterization of the nonprecipitated alkali fraction revealed higher 

levels of phenolic compounds for the red poplars when 

compared to the white poplars. High Pressure Liquid 

Chromatography (HPLC) analysis of these compounds 

showed increased amounts of benzaldehydes, mainly 

syringaldehyde (up to 10-fold) and vanillin (2- to 4-fold). 

These data show that lignin amount and composition were 

not significantly modified; however, reactivity of the lignin polymer toward alkali was significantly altered resulting in a higher extractability of lignin. 

Discussion 

In our European Union-funded project, transgenic poplars (P. tremula x P. alba) were produced with reduced CAD 

. enzymatic activity. Both cosuppressed and antisense downregulated CAD plants were obtained. Enzymatic inhibition was correlated with a red coloration of the wood. 

Although the lignin levels of these transgenic lines were 

similar to the control, lignin characteristics and structure 

were modified. Evidence for increased amounts of aromatic 

aldehydes in the lignin from red poplars was found, and 

more lignin was extracted upon alkali treatment. No modification of the S:G ratio was observed. 

Comparable results were obtained by Halpin et al. (1994), 

who analyzed antisense CAD tobacco plants. CAD activity 

in these transgenic tobacco plants was reduced to 7 percent 

of the control value. CAD down-regulated red tobacco plants 

had no lower lignin content; however, evidence for an alteration in lignin composition was demonstrated in that more 

phenolic material was extracted by NaOH and thioglycolic 

acid. Pyrolysis mass spectrometry demonstrated increased 

amounts of coniferaldehyde but especially syringaldehyde. 

From the red poplars and the antisense CAD tobacco 

plants, more alkali lignin was released in comparison to 

the wild type. This result is interesting because alkali treatment together w~th sulfonation of the wood chips provide 

the basis for chemical pulping. As previously mentioned 

by Higuchi et al. (1994), ~-0-4 linkages could be broken 

more easily via the kraft process when the terminal alcohol groups were replaced by aldehyde groups. 

In the transgenic poplars, as in the CAD transgenic tobacco plants, lignin content was unchanged (Halpin et al. 

202 

1994; Higuchi et al. 1994). It is possible that the CAD activity 

must be further reduced to lower the lignin content. Indeed, 

Grand et al. (1985b) showed that lignin content in poplar 

can be reduced with specific inhibitors of the CAD enzyme, 

and the bmr6 mutants also have a lower lignin content 

(Pillonel et al. 1991 ). It is possible that the 30 percent residual 

CAD activity in the down-regulated poplars is caused by a 

different enzyme also having CAD activity. 

The chemical cause of the red coloration of the stem has 

not been clarified. Higuchi et al. (1994) and Tollier et al. 

(1995) have shown that the polymerization of 

coniferylaldehyde in vitro produces a wine-red synthetic 

dehydrogenation polymer, whereas the polymerization of 

cinnamyl alcohols produces a pale-brown polymer. Furthermore, using thioacidolysis, thest:! authors provided 

evidence for the incorporation of coniferaldehyde units in 

the synthetic polymer. However, no evidence for the occurrence of these units was found when the same analytical procedures were applied to the reddish CWR from CAD 

down-regulated poplars. This suggests either the occurrence of coniferaldehyde derivatives not depolymerized 

by thioacidolysis or, more probably, their rearrangement 

into benzaldehyde derivatives that exist at increased levels in these plants (Tollier et al. 1995). Red coloration might 

be caused by an extended conjugated system generated 

by incorporating aldehydes into the polymer. In the study 

by Halpin et al. (1994), the red stem coloration was visible 

during plant growth but disappeared at the flowering 

stage. In poplar, the red color also disappeared with wood 

maturation and was only associated with young xylem. A 

disappearance of the red color during maturation was also 

observed in the brown midrib mutants. Because the red 

color is probably due to the presence of conjugated aldehydes in the lignin, this conjugated system may become 

reduced by an unknown mechanism during aging resulting in the disappearance of the red color. 

Kraft pulping experiments are being performed on the 

transgenic poplars with the lowest CAD activity. Preliminary experiments on young trees indicate that the red poplars are slightly easier to delignify, providing a pulp whose 

residual lignin content can be significantly lower according to kappa number determinations. Field trials are being established to investigate whether these promising 

results are reproducible over successive growth years. 

Perspectives 

Different strategies to modify lignin content in plants 

have been proposed by several authors (Boudet et al. 1995; 

Dean and Eriksson 1992; Whetten and Sederoff 1991 ). Besides CAD and COMT, several other genes involved in lignin biosynthesis are interesting to manipulate. Such genes 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
include those encoding laccase and peroxidases involved 

in the polymerization of the monolignols, CCR possibly 

encoding a flux-regulating enzyme, and CCoAOMT encoding the enzyme that is possibly involved in the biosynthesis of the G units. Furthermore, since many cDNAs 

encoding lignification enzymes have been cloned (e.g., 

COMT, CCoAOMT, CAD, CCR), the altered expression of 

more than 1 gene (e.g., COMT in combination with 

CCoAOMT) might be a realistic goal. Eliminating several 

of these genes simultaneously may achieve a modification 

of the lignin composition and a reduction in lignin content. It may be possible to identify new proteins involved 

in lignin biosynthesis by comparative two- dimensional, 

protein-gel. analysis between wild-type and transformed 

poplars with altered lignin biosynthesis (e.g., antisense CAD 

plants). Similarly, comparative HPLC analysis of wild-type 

and transformed plants may uncover new routes in lignin 

biosynthesis. Another strategy to modify lignin structure 

might be altering expression of transcription factors that interact with 1 or several genes involved in lignin biosynthesis. Finally, these gene constructs could be transferred to 

economically important poplar hybrids (e.g., P. trichocarpa x 

P. deltoides), or other tree species such as eucalyptus, which 

are very important for the pulp and paper industry. 

Acknowledgments 

CJT and coworkers are grateful to Dr. Raju S.S. Datla for 

providing the BinSynGus vector and to NORAM Chemical Co. (Wilmington, DE) for thidiazuron. The research was 

supported by a grant (#92-37301-7598) from the Plant Genetic Mechanisms and Molecular Biology Program of the 

USDA National Research Initiative Competitive Grants 

Program to VLC and GKP, and from CPBR-DOE Energy 

from Biomass Program to GKP and VLC. 

WB and coworkers thank Martine De Cock, Christiane 

Germonprez, and Karel Spruyt for help with the manuscript and figures, Frank Jajik and Marie-Claude Sourie 

for technical assistance, Catherine Lapierre for critical 

views on thioacidolysis, and Patrick Zimmer and Gerard 

Vastra for plant maintenance in the greenhouse. 

This work was supported by grants from the Commission of the European Communities (ECLAIR-OPLIGE No 

AGRE-0021-C(EDB), TIMBER-FAIR-RT95-0424, and AIRCT93-1661), and the Flemish Government (VLABUL 2321 

95-01.:.04 I 28). JVD is indebted to the "Vlaams lnstituut voor 

de Bevordering van het Wetenschappelijk-Technologisch 

Onderzoek in de lndustrie" and J-CL to the "Conseil 

Regional de Ia Region Centre" for predoctoral fellowships. 

JVD and MB thank the European Molecular Biology Organization and the Commission of European Communities for short-term fellowships. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Genetic Modification of Lignin Biosynthesis in Quaking Aspen and Poplar 

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205 
Chapter27 

Potential Use of Populus for Phytoremediation of 

Environmental Pollution in Riparian Zones1 

Mary Ellen Dix, Ned B. Klopfenstein, Jian-Wei Zhang, Sarah W. Workman, and 

Mee-Sook Kim 

Introduction 

Environmental pollution is a serious threat to human 

life and to our ecosystems. Riparian zones, the narrow band 

of land between terrestrial and aquatic systems, are especially vulnerable to environmental pollution because many 

pollutants are transported through these systems via surface or subsurface runoff. Pollutants include fertilizers (e.g., 

nitrates), pesticides, agrichemical by-products, heavy metals, trichloroethylene, halogenated phenolics, and other 

waste products (Schoeneberger 1994). Because agricultural 

and industrial pollutants are widespread, there is increasing interest on organisms that accumulate, detoxify, or 

degrade these substances. While it is known that plants 

and microorganisms modify their environments, their potential use as mitigative tools to clean pollutants has only 

recently gained acceptance (Brown 1995). Woody perennial plants are ideal for remedial purposes because they 

can be planted over large areas at low cost and can concentrate or degrade environmental pollutants over several 

years (Moffat 1995), while also providing other economic 

or ecological services. As metabolic pathways for pollutant detoxification, uptake, and/ or degradation are described, woody plants can be selected or engineered to 

remediate specific environmental pollutants. 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

206 

Why Populus? 

Populus is well suited for use inphytoremediation (the 

use of specially selected and engineered plants for environmental remediation) plantings. Populus is easy to establish and grows quickly. Its high transpiration rate and 

wide-spreading root system make it ideal to intercept, absorb, degrade, and/ or detoxify contaminants, while reducing soil erosion. Historically, this widely distributed genus 

has naturally grown in riparian areas, thus many genotypes are adapted for growth on potential remediation 

sites. Populus plantings are amenable to coppicing and 

short-rotation harvest, thereby helping to maintain sustained root vigor. Further, if a biofuels or fiber market is 

available, harvests can generate additional income that 

helps offset establishment costs (Strauss and Grado this 

volume). Although Populus is not part of the human food 

chain, many vertebrates and invertebrates use the trees 

for food, shelter, and reproductive sites. Such increases in 

biodiversity can contribute to sustained productivity of 

adjacent aquatic habitat and crop land (Dix et al. in press). 

Populus is well studied, with established silvicultural, 

vegetative propagation, breeding, and harvesting protocols (Stettler et al. 1996). In addition, Populus is amenable 

to tissue culture manipulation, genetic engineering, and 

genetic mapping (various chapters this volume). Thus, 

Populus is an ideal candidate for genetic engineering and 

selection for absorption, detoxification, and/ or degradation of environmental pollutants such as heavy metals, 

nitrates, pesticide residues, and other waste products. 

Pollutant-Neutralizing Trees 

Plants have many mechanisms for neutralizing toxic 

pollutants including immobilization, absorption, and ac-
Potential Use of Populus for Phytoremediation of Environmental Pollution in Riparian Zones 

cumulation or sequestration of contaminants (except cytoplasmic toxins). Plants also support symbiotic, root-associated microorganisms that can contribute greatly to 

contaminant neutralization (Stomp et al. 1994). One example of phytoremediation is using metal accumulating 

plants to remove heavy metals from the soil. All plants 

can accumulate essential heavy metals from the environment and some accumulate nonessential metals such as 

cadmium {Cd}, lead (Pb}, and cobalt (Co) (Baker and 

Brooks 1989; Ernst et al. 1992; Ostry this volume; Salt et al. 

1995). Tolerance to aluminum has been reported for several poplar and other plant species {Baker and Brooks 1989; 

Chung and Chun 1990; Ernst et al1992). In addition, studies on landfills and municipal sludge recycling systems 

demonstrate the ability of Populus and other plants to take 

up and tolerate heavy metals and other potential pollutants (Salt et al. 1995; Schultz et al. 1995; Shrive et al. 1994). 

Populus designed for use in riparian remediation plantings 

also must be able to take up, translocate, and I or resist toxic 

pollutants. An ideal tree may have densely packed roots 

for inactivating toxins or removing them from the soil for 

subsequent translocation to the leaves and storage in roots 

or stems (Schoeneberger 1994; Stomp et al. 1994). 

Nitrates 

One of the largest fertilizer pollutants from both agricultural and urban sources is nitrates (NO). Large amounts 

of nitrates have already entered ground and surface waters, adversely affecting the health of aquatic organisms, 

humans, and other components of the ecosystem (Duda 

1982; Komar and Magner 1996; Lowra.nce 1992). Nitrates 

are also indirect by-products of livestock operations 

(Welsch 1991). Large root systems of certain Populus genotypes have an affinity for nitrates. For example, in simulated groundwater depletion studies Populus x canadensis 

roots substantially reduced the concentration of nitrates 

continuously over a 2-month period (O'Neill and Gordon 

1994). Furthermore, wide-spread root systems of Populus 

are more effective than confined root systems in uptake of 

these nutrients (Licht 1992). Clonal and species differences 

in root growth and root physiology are common in Populus 

(Pregitzer et al. 1990). Nitrate uptake, although it varies 

with environmental conditions, is apparently heritable in 

selected Populus spp. (Nguyen et al. 1990; Pregitzer et al. 

1990; Pregitzer and Friend 1996). Thus, planting Populus 

clones selected for their large root masses on sites with 

potential for nitrate runoff could increase absorption of 

nitrates. Nitrate uptake in these poplars could perhaps be 

further enhanced by genetic engineering or selective breeding of poplar for larger root mass or increased protein storUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

age (Coleman this volume). For example, root system 

growth was stimulated in P. alba x P. glandulosa (Chung et 

al. 1989), P. davidiana Dode (Lee et al. 1989), P. deltoides x P. 

nigra (Charest et al. 1992) and P. nigra x P. maximowczii 

(Charest et al. 1992) by transformation with Agrobacterium 

rhizogenes. 

Chemical Tolerance and Detoxification 

Herbicides are commonly sprayed on crops to eliminate 

competing weeds. However, herbicides and their by-products that reach streams by direct runoff, leaching, erosion, 

and other processes can be toxic to aquatic plants and animals. For planting between the crops and streams, herbicide-tolerant poplars could be produced or selected to 

remove, detoxify, degrade, or tolerate selected pollutants 

including herbicides. In vitro selection, genetic engineering, 

genetic screening (e.g., marker- assisted selection), and other 

molecular techniques have potential for producing or selecting genotypes with improved remediation efficiency. 

Chemical tolerance 

In vitro techniques were developed for detecting 

soma clonal variation in the tolerance of Populus to herbicides (Michler and Haissig 1988). Using such in vitro techniques, 4 hybrid Populus lines were selected for increased 

tolerance to glyphosate and sulfometuron methyl (Michler 

and Haissig 1988). In addition, several Populus variants 

selected for tolerance to sulfometuron methyl were found 

to have increased acetolactate synthase (ALS) activity 

(Michle·r 1993; Riemenschneider and Haissig 1991; 

Riemenschneider et al. 1988}. 

Genetic engineering techniques are available for transferring tolerance mechanisms into Populus. Mutant acetolactate 

synthase genes, crs1-1 and als, that confer resistance to sulfonylurea or the herbicide chlorsulfuron were individually 

used to transform P. tremula x P. alba (Brasileiro et al. 1992; 

Chupeau et al. 1994). Transgenic Populus cells containing the 

mutant als gene grew on a selective media containing 200 

nM chlorsulfuron (Chupeau et al. 1994}, and transgenic 

plants expressing the mutant crs1-1 gene were completely 

resistant to high doses of chlorsulfuron (Brasileiroet al. 1992). 

P. alba x P. grandidentata was genetically transformed with 

the mutant aroA gene for 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase that confers tolerance to the herbicide glyphosate (N-(phosphonomethyl)-glycine) (Donahue 

et al. 1994; Fillatti et al. 1987; Karnosky et al. this volume). 

Transgenic Populus plants expressing the mutant aroA gene 

demonstrated more herbicide tolerance than the control 

Populus (Donahue et al. 1994). 

In work to increase tolerance to air pollution, hybrid 

aspen (P. sieboldii x P. grandidentata) was transformed with 

an E. coli glutathione reductase (GR) gene (Endo ~tal. this 

volume). GR-expressing, transgenic aspen displayed re207 
Section V Biotechnological Applications 

sistance to oxidative stress caused by the herbicide 

paraquat (methyl viologen: 1, 1-dimethyl-4,4-bipyridium 

dichloride) or sulfur dioxide (SOJ This approach could 

potentially confer tolerance to oxidative stresses caused 

by other environmental pollutants. Pollutant-tolerant 

Populus could be further selected or engineered with 

remediatory functions, or used to support growth of 

remediatory microorganisms. 

Chemical detoxification 

Other remediatory approaches are aimed toward direct 

degradation or detoxification of toxic pollutants. P. alba x 

P. tremula, P. tremula x P. alba, and P. trichocarpa x P. deltoides 

hybrids were transformed with a bar gene that codes for 

the enzyme phosphinotricin acetyl transferase (PAT) 

(Chupeau et al. 1994; De Block 1990; Devillard 1992). PAT 

inactivates the commercial herbicide phosphinotricin 

(glufosinate, Basta) by acetylation (De Block 1990). 

Ongoing work at the University of Washington and 

Washington State University demonstrated that Populus 

hybrids (P. trichocarpa x P. deltoides) can oxidize trichloroethylene (TCE) to produce carbon dioxide and other metabolites. Further experiments are underway to determine 

the capacity of Populus to remove and degrade TCE from 

groundwater (Strand et al. 1995). Pioneering work was 

initiated to enhance environmental detoxification by genetically engineering trees with genes that encode 

remediatory functions (Stomp et al. 1994). Two genes from 

Alcaligenes eutrophus, tfdB and tfdC, were isolated and cloned 

in an attempt to detoxify halogenated phenolics. One gene, 

tfdB, encodes a chlorophenol hydroxylase, and the other gene, 

tfdC, encodes a chlorocatecol 1,2-dioxygenase. Chlorophenols are sequentially hydroxylated by these 2 enzymes 

to form chlorocatechol. Subsequently, the ring is cleaved to 

create chloro-cis-cis-muconate. Initial tests are underway 

using 2,4-dichlorophenol, a breakdown product of 2,4-dichlorophenoxyacetic acid (2,4-D), and trichloroethylene. Gene 

constructs have been made with tfdB and tfdC under the control of a cauliflower mosaic virus (CaMV) 355 constitutive 

promoter for transformation of Populus, black locust (Robinia 

pseudoacacia), and sweet gum (Liquidambar styracijlua). Subsequent studies are assessing active enzyme levels, uptake, 

and fate ofTCE in these trees (Stomp et al. 1994). Such studies in direct detoxification further demonstrate the potential 

of Populus for phytoremediation. 

Soil Conditions and Microorganisms 

. S~ccess of poplar plantings in remediating a riparian 

sit~ Is de~endant on soil conditions and microorganisms. 

Sod chemistry plays a pivotal role in this process with soil 

208 

pH and chelating agents affecting uptake of metals. For 

example, many metals in soils are bound to oxides. Plants 

can di.ssolve these oxides and enhance their solubility by 

~eleasmg reductants from the roots. However, soil pH can 

mfluence metal bioavailablity and uptake. Plants growing in soils with low pH typically display higher metal toxicity because of decreased metal adsorption to soil particles. 

This, in tum, can increase concentrations of metals in the 

soil solution and subsequent leaching (Salt et al. 1995). 

Trees support a diverse population of soil microorganisms, including bacteria, ecto- and endomycorrhizal fungi, 

actinomycetes, and blue green algae. In tum, many of these 

microorganisms help tree establishment and growth by 

greatly increasing the uploading capacity of roots. These 

soil microorganisms are instrumental in the processes of 

remediation, stabilization, and filtration of water and soil. 

L~ke tre:s, soil microorganisms participate directly or indirectly m these processes. Direct remediation occurs when 

organisms take up, store, detoxify, or degrade toxic compounds and their derivatives. Indirect remediation occurs 

through beneficial effects on associated organisms directly 

involved in remediation. Thus, overall effectiveness of 

remediation processes is based on interactions among the 

plant species, the type(s) of pollutants, and the soil microflora (Stomp et al. 1994). 

Mycorrhizae,.symbiotic associations between soil fungi 

and roots, can greatly increase the root surface area and 

pro:ide a low-resistance pathway for water transport 

(K~~de 1990). These symbioses can influence the plant's 

abihty to take-up metals, and possibly influence plant tolerance to heavy metals. However, site conditions can influence the development of mycorrhizal associations. In 

natural conditions and with advanced stand age, Populus 

roots generally form ectomycorrhizae. When these roots 

are flooded, in the early stages of stand establishment, or 

in very fertile soils, they may form vesicular arbuscular 

mycorrhizae or no mycorrhizal associations (Heilman et. 

al. 1996). 

Genetic .selection and manipulation of rhizosphere microor~a~Isms c~n potentially improve biological 

reme.d~ahon ~f. sotl and water. Several ectomycorrhizal 

fung1 Immob1hzed the herbicide chlorpropham, while 

other ectomycorrhizal fungi degraded chlopropham to 3-

chloroaniline (Rouillon et al. 1989). Paxillus involutus, an 

e~tomycorrhizal colonizer of conifer and hardwood speCies, was transformed by particle bombardment with the 

hygromycin phosphotransferase gene (HPT) as a selectable marker and the P-glucuronidase (GUS) gene as a reporter gene. The transgenes were actively expressed after 

stable integration into the fungal genome, and the ability 

to form ectomycorrhizal roots was unaffected (Bills et al. 

19?5). Thu~, t~e potenti~l to genetically engineer mycorrhizal fungi with remedtatory functions is demonstrated. 

Populations of rhizosphere microflora could be increased 

indirectly by genetically increasing Populus root mass 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Potential Use of Populus for Phytoremediation of Environmental Pollution in Riparian Zones 

through transformations such as with Agrobacterium 

rhi~ogenes. !~creased root mass could support larger populations of rhtzosphere microorganisms that could also be 

~enetically engineered with improved remediatory functions (Stomp et. al. 1994). However, ethical concerns must 

be thoroughly addressed before such strategies can be 

implemented in the field (Yang et al. this volume). 

Strategies and Considerations 

for Plantings 

For efficient and sustained remediation, Populus planted 

at remediation sites must tolerate prevailing site conditions such as excess nitrates and herbicides, as well as damage by insect pests and diseases. Accumulation of 

pollutants is toxic to many plants; thus, these plants must 

tolerate existing pollution levels and higher concentrations 

than normally exist within the plant. However, many native poplars traditionally recommended for riparian zones 

are relatively slow growers and may be intolerant of pol-

~utants. Genetic engineering and selection can potentially 

tmprove the tolerance of Populus trees to various pollutants. Such pollution tolerance could amplify potential biomass benefits of Populus plantings. 

Pest outbreaks are common in riparian Populus plantings 

(Ostry et al. 1988), and can threaten remediation activity 

of the planting. Genetic engineering, genetic selection, and 

in vitro selection can facilitate the development of Populus 

clones with enhanced pest resistance (Cervera et al. this 

volume; Ebinuma et al. this volume; Ellis and Raffa this 

volume; Heuchelin et al. this volume;. Ostry this volume; 

Powell and Maynard this volume). Eventually, techniques 

developed by this research will be used to develop a variety of Populus cultivars and clones with improved resistance I tolerance to insects and disease. In addition, 

planting establishment and maintenance guidelines should 

include strategies for integration with other pest management techniques (e.g., enhancing natural controls). Such 

approaches could minimize the need for additional pesticide application to Populus plantings. 

Because Populus biomass plantings usually require intensive management, they should be established at least 1 

planting zone away from the stream. These plantings could 

serve as an effective intermediate buffer zone for absorption and degradation of environmental pollutants. The 

plant zones adjacent to the stream could be designed to 

delay or absorb excess chemicals and soil from Populus 

plantings. As mentioned earlier, soil microorganisms found 

in poplar plantings have a primary role in site remediation. 

These microorganisms also must tolerate the pollutants 

and other conditions at the site. 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 

. Fi~ally, potential applications of this technology beyond 

npanan fores.t buffer are numerous. Similarly designed 

Populus plantmgs could be used to remediate industrial 

waste sites, agricultural waste water, sewage, and mine 

land. F~r e.xample, in the Pacific Northwest, bioengineers 

are destgnmg systems that use Populus biomass plantings 

for recovering nitrates and other fertilizers from irrigated 

waste water, or removing urea and heavy metals from 

dairy waste, human sewage, and landfill leachate. Most 

o~ these practices are exploiting the nitrogen affinity and 

htgh water consumption of hybrid Populus (Gary Kuhn, 

USDA Natural Resource Conservation Service, personal 

communication). 

Conclusion 

Planting poplar near riparian zones and toxic waste sites 

has generated considerable interest as an economical 

method to remediate toxic sites while providing income 

and environmental benefits. Planting Populus in riparian 

zones may provide unique opportunities for remediation 

of multiple toxins. Populus has high potential for environmental remediation because its biology is well studied, and 

its management, production, genetic engineering, genetic 

selection, and in vitro manipulation techniques are well 

developed and readily available. Demonstration plantings 

have been established in several communities to limit 

movement of potential ground-water contaminants. Such 

plantings are used to remediate leachate from contamin~ted landfill and waste water systems while producing 

bwmass and providing wildlife habitat. However, 

phytoremediation of pollution in urban and rural landscapes is a long process and is primarily effective only on 

pollutants near the surface. It is a relatively environmentally safe process that can be used for large areas. Removal 

of pollutants by this method does not necessarily require 

much energy (Stomp et al. 1995). Additional research, 

development and field trials are needed before the specific biochemical processes involved in pollutant uptake, 

transport, and accumulation are fully understood. Environmental impacts of using Populus remediation plantings 

must also be thoroughly evaluated before such plantings 

can be fully utilized. 

Acknowledgments 

The authors thank David P. Anderson, Stefanie G. 

Aschmann, Richard C. Carman, Richard A. Cunningham, 

209 
Section V Biotechnological Applications 

Gary A. Kuhn, and Michele M. Schoeneberger for reviewing earlier drafts of this manuscript. Use of trade names in 

this chapter does not constitute endorsement by the USDA 

Forest Service. 

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211 
Chapter28 

Poplar Genetically Engineered for Reproductive Sterility 

and Accelerated Flowering1 

Richard Meilan and Steven H. Strauss 

Introduction 

High-yielding clones of hybrid poplars (Populus) were 

recently developed in the northwestern United States. Although many genes are currently available (e.g., those affecting insect and herbicide resistance) for enhancing the 

usefulness of these clones (Strauss 1995), a key obstacle to 

commercialization of genetically engineered trees is the environmental safety of transgenes. Federal regulators will 

likely require development of a strategy to mitigate environmental impacts from release of transgenes into wild 

populations. Engineered sterility would help satisfy this 

requirement (reviewed in Strauss et al. 1995). 

In addition to gene containment, sterility offers other 

benefits. Reproductive growth is an energy-requiring process. By interrupting floral development, energy may be 

diverted into biomass production. Sterility also reduces 

genetic pollution. Selected hybrid cottonwood clones are 

planted on a very large scale in the Pacific Northwest of 

the United States. Their pollen clouds may potentially af-

.fect the genetic structure of surrounding wild stands. Planting sterile versions of these clones wo~ld alleviate this 

concern. Finally, engineered sterility would reduce the level 

of airborne allergens and undesired litter from reproductive tissues. 

Because trees have long juvenile periods, accelerating 

flowering is essential to verify the effectiveness of introduced sterility-causing transgenes within a reasonable time 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

212 

period. To our knowledge, there are no published methods for inducing precocious flowering and seed production in Populus. We include discussion of several techniques 

used to promote flowering in other woody angiosperms. 

Floral Genes 

Flower development occurs in 3 phases: 1) conversion 

of a vegetative meristem to an inflorescence meristem; 2) 

conversion of an inflorescence meristem to a floral meristem; and 3) emergence of floral organs from the floral 

meristem (reviewed in Weigel1995; Yanofsky 1995). Many 

genes involved in the initiation of flowering were identified through mutational analysis. Flowering-time genes 

from Arabidopsis have been divided into early- and lateflowering categories. Two late-flowering genes that were 

recently cloned are CONSTANS (CO) (Putterill et al. 1995) 

and LUMIDEPENDENS (Lee et al. 1994). A model showing control of flowering by time-of-flowering genes was 

recently proposed by Martinez-Zapater et al. (1994). 

Floral homeotic genes, which encode transcription factors and control many aspects of floral development, were 

isolated from Arabidopsis, Antirrhinum, and other plant 

species. These genes are usually divided into 3 functional 

classes: 1) floral meristem identity genes; 2) floral organ 

identity genes; and 3) spatial regulators of organ identity 

genes. 

LEAFY (LFY) is an example of a meristem identity gene 

from Arabidopsis. It is involved in controlling the transition from an inflorescence to a floral meristem. Its homolog 

in Antirrhinum is referred to as FLORICAULA (FLO). Strong 

mutations at this locus cause inflorescence initiation within 

bract axils resulting in a branchy phenotype with substantial or complete disruption of flower production (Coen et 

al. 1990; Huala and Sussex 1992; Weigel et al. 1992). 

A second floral meristem identity gene, APETALA1 

(AP1), encodes a putative transcription factor containing 

the MADS box, which is a highly homologous domain 
Poplar Genetically Engineered for Reproductive Sterility and Accelerated Flowering 

shared by the yeast transcription factor MCM1, the llGAMOUS gene from Arabidopsis, the DEFICIENS gene from 

Antirrhinum, and the human serum response factor S.RF. 

APETALA1 is involved in controlling the switch from inflorescence to floral meristems, and is also required for 

sepal and petal development (Mandel et al. 1992). 

Floral organ identity genes are expressed in the floral 

meristem just before the emergence of the primordia whose 

development they regulate. These genes are divided into 

3 functional groups that are designated A, B, and C. Each 

of the 4 floral organs emerges from concentric whorls of 

cells in the floral meristem (figure la). The different classes 

of floral organ identity genes are expressed in 2 adjacent 

whorls (figure lb). The location of their expression dictates the type of floral organ produced. For example, when 

an A-function gene is expressed in whorl 1, sepals form; 

the combined expression of A- and B-function genes in 

whorl 2 produces petals; when B- and C-function genes 

are expressed in whorl 3, stamens are produced; and Ca 

b 

Gene 

Function: 

B 

Whorl: 

Organ: 

Figure 1. 

A c 

1 2 3 4 

Sepals Petals Stamens Carpels 

a) Arrangement of concentric whorls of cells in 

the floral meristem. b) The A-8-C model showing the overlapping zones of expression of the 

floral organ identity genes (adapted from Coen 

and Meyerowitz 1991 }. 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 

function gene expression in whorl4 results in pistil development. This general model appears valid for a variety of 

plant species; however, there are many exceptions and 

elaborations on this basic scheme including action of partially redundant genes (e.g., AP1 and CAL in zone A) 

(Yanofsky 1995). 

The AGAMOUS (AG) gene is an example of a C-function floral organ identity gene. Mutations in AG cause the 

conversion of stamens to petals and carpels into new flowers (Bowman et al. 1989). AG and AP1 are also examples 

of spatial regulators. AP1 is involved in activating AG 

expression and is subsequently down-regulated by AG, 

which results in a loss of AP1 expression in whorls 3 and 4. 

Because mutations in some floral homeotic genes impair fertility at early stages of floral differentiation in male 

and female flowers, they are attractive targets for engineering sterility. Recently, 3 floral homeotic genes were 

cloned from Populus trichocarpa and identified as putative 

homologs of FLO/LFY (PTFL), DEFICIENS (PTD), and AG 

(PTAG) (Sheppard et al.1996). These genes are being used 

in our ongoing efforts to engineer sterile poplars. 

Several floral-specific genes were isolated from highly 

differentiated floral tissues such as anthers and pistils. Most 

encode structural or catalytic proteins essential for pollen 

or carpel function. Examples include stylar transmitting 

tissue-specific genes (Wang et al. 1993), anther tapetumspecific genes (Goldberg 1988), and genes expressed in both 

pistils and anthers (Gasser et al. 1989). Although these promoters can be used to engineer sterility for transgene containment, they may not enhance vegetative growth because 

they are expressed late in flower development. 

Engineering Sterility 

One primary strategy for engineering sterility involves 

the expression of cytotoxin genes in a floral tissue-specific 

fashion resulting in organ-specific ablation or disruption 

of tissues. Another primary strategy relies on suppression 

of floral genes that are essential to produce fertile gametes. 

Tissue Ablation 

Promoters 

Many tissue-specific promoters currently in use were 

isolated from tobacco. The most commonly employed male 

reproductive tissue-specific promoter is TA29 (Goldberg 

1988). This promoter, which directs expression exclusively 

in the tapetum of the anther, was used to engineer sterility 

in tobacco and oilseed rape (Mariani_ et al. 1990). Without 

the tapetum, microspore development is arrested. In a similar approach, female sterility was achieved using the 

213 
Section V Biotechnological Applications 

stigma-specific STIG1 promoter, also from tobacco 

(Goldman et al. 1994). 

Other useful promoters were characterized from genes 

involved in gametophytic self-incompatibility. These include promoters from SLG, an S-locus glycoprotein gene 

(Thorsness et al. 1991), and SLR, an S-locus-related gene 

(Hackett et al. 1992). Both promoters are expressed principally in stigmatic and stylar tissues but are also expressed 

in pollen to variable degrees. These provide organ-specific ablation when fused to cytotoxin genes. 

Some occurrences of instability in transgene expression 

are reported. For example, Denis et al. (1993) demonstrated 

that when male-sterile rapeseed (Brassica napus) containing a tapetal-specific promoter was fused to a ribonuclease 

gene, it frequently reverted to a fertile state. This instability was associated with temperatures greater than 25 °C. If 

sterility is an important aspect of gene containment for 

regulatory purposes, it is critical to demonstrate that sterility is maintained under a range of conditions and developmental stages. This could be achieved by carefully 

selecting transformed lines for stable expression, using 

redundant constructs (discussed below), or using 

trans genes flanked by expression stabilizing matrix-attachment regions (reviewed in Spiker and Thompson 1996 ). 

Many floral homeotic genes are floral-specific, required 

for floral meristem/ organ development, and expressed 

either as the floral meristems emerge or before floral organ primordia develop. Because of their early and frequent 

floral-specific expression, floral homeotic gene promoters 

are probably best suited for engineering sterility while also 

enhancing vegetative growth. The APETALA3 promoter 

fused to DTA (see below) gave organ-specific ablation of 

petals and stamens in Arabidopsis (Day et al. 1995). 

Cytotoxins 

Two widely used cytotoxin genes are Bamase, an extracellular ribonuclease isolated from Bacillus amyloliquefaciens 

(Hartley 1988) and the diphtheria toxin subunit A (DTA) 

gene from Corynebacterium diphtheriae (causative agent of 

diphtheria) (Palmiter et al. 1987). The latter encodes an 

ADP ribosyl transferase, which interrupts protein synthesis by covalently modifying eukaryotic elongation factor 

2 (EF-2). With the B subunit of the toxin deleted, it is unable to enter cells autonomously. Although it is a very effective cytotoxin used successfully to engineer 

reproductive sterility in plants (Day et al. 1995; Koltunow 

et al. 1990; Mariani et al. 1990; Thorsness et al. 1991 ), its 

commercial application may be controversial because of 

its origin. However, the use of Barnase is technically complicated because, unlike DTA, its encoded protein is toxic 

to eukaryotic cells. Therefore, it is necessary to include a 

gene encoding an inhibitor in gene constructs that contain 

Bamase. Barstar encodes a protein that specifically forms 

a complex with the Bamase gene product to inactivate it 

214 

to protect the transformation vectors (e.g., Agrobacterium 

tumefaciens) from its ribonuclease activity (Hartley 1988). 

Many other cytotoxin genes are available that can be 

used to avoid some of these obstacles. For example, the 

taxA gene from Pseudomonas aeruginosa produces an exotoxin that, at the molecular level, has the same mode of 

action as DTA (Lory et al. 1988). Another class of cytotoxin, 

ribosome inactivating proteins (RIPs), interferes with protein synthesis by altering the large subunit of ribosomal 

RNA (rRNA). Castor bean seeds (Ricinus communis) are a 

rich source of ricin, which is a representative member of a 

large class of plant-derived RIPs. This protein catalyzes 

the cleavage of an adenine base from an exposed loop near 

the 3'-end of 28S eukaryotic rRNA (position 4,324 in rat). 

This loop is known as the sarcin loop because a-sarcin, a 

cytotoxin from Aspergillus, cleaves the phosphodiester linkage between a guanosine and adenosine directly adjacent 

to the site where ricin acts (Stripe et al. 1992). 

Suppression 

Known as co-suppression, transgenes can inhibit their 

own expression and that of homologous native genes 

(Flavell 1994). Recently, it was shown that untranslatable 

versions of transgenes are more efficient at suppression 

(Smith et aL 1994). Gene silencing can also be achieved by 

constitutively expressing the transgene in antisense orientation (reviewed by Mol et al. 1994). A disadvantage of 

the suppression strategy is that the native gene or a highly 

homologous equivalent is usually needed for efficient suppression, whereas the promoters employed in the ablation 

approach typically function across diverse classes of plants. 

To help ensure that sterility remains stable throughout 

the life of the plant, building in some redundancy would 

be prudent (Strauss et al. 1995). This approach could involve targeting multiple genes for suppression. For example, a transcriptional fusion of a floral meristem identity 

gene, such as PTFL, with a floral organ identity gene, such 

as PTAG, could provide strong suppression. Because PTAG 

is a C-function floral homeotic gene, it controls both pistil 

and stamen development. If PTFL function is some how 

restored in the transgenic plants or allows occasional production of fertile organs, as in strong LEAFY mutants of 

Arabidopsis, both male and female flowers will remain sterile by virtue of PTAG suppression. 

Reversible Sterility 

Restoring fertility may be desirable so that valuable 

transgenic lines could be used in further breeding. For 

completely sterile trees, it will be necessary to use an inducible promoter (Mett et al. 1993; Weinmann et al. 1994) 

that stimulates production of a cytotoxin inhibitor, such 

as Barstar (Mariani et al. 1992), a site-directed recombinase 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Poplar Genetically Engineered for Reproductive Sterility and Accelerated Flowering 

to remove or deactivate sterility transgenes (Kilby et al. 

1993 ), or antisense versions of cytotoxin genes. 

Early Flowering 

The long juvenile period of trees severely impedes study 

of their reproductive biology. Confirmation that engineered 

trees are sterile requires an examination of their flowers. 

Surprisingly, little effort has been directed toward developing methods to induce early flowering in Populus. However, several techniques were used to accelerate flowering 

in other woody angiosperms, which hopefully can be 

adapted for poplars. In addition, ectopic expression of floral regulatory genes was reported to accelerate flowering 

in several species including poplar. 

Chemical Approaches 

The most successful inductive treatments for woody 

angiosperms have been the triazole growth retardants, 

which includes compounds such as paclobutrazol and 

uniconazole. These chemicals specifically inhibit the enzymatic conversion of ent-kaurene to ent-kaurenoic acid 

in the gibberellic acid biosynthetic pathway (Rademacher 

1989). Besides producing short-statured plants, triazole 

also accelerates flowering. Paclobutrazol, the most widely 

used triazole, stimulated flowering in apple (Malus spp.) 

(Tukey 1983; Volz and Knight 1986), pear (Pyrus nivalis) 

(Williams and Edgerton 1983), cherry (Prunus cerasus) 

(Edgerton 1986), clove (Syzygium aromaticum) (Martin and 

Dabek 1988), and eucalypts (Eucalyptus nitens and E. 

globulus) (Griffin et al. 1993; Moncur and Hasan 1994; 

Moncur et al. 1994). 

Other growth inhibitors, such as daminozide (Alar®, 

B-9®, SADH) and chlormequat (CCC, Cycocer~), can stimulate flowering. Daminozide has been used for this purpose 

in apple (Batjer et al. 1963; Luckwill and Silva 1979). There 

is also an unpublished report of daminozide causing precocious flowering in aspen (Li 1995, personal communication, North Carolina State University, Raleigh, NC 27695 

USA). Chlormequat has led to enhanced yields in grape 

(Vitis vinifra) (Bravdo et al. 1992). 

Physical Methods 

Phloem Restriction 

Girdling (removing a swath of bark around the entire 

stem) stimulated flowering in certain tree species (Eris and 

Barut 1993; Wesoly 1985), but this treatment ultimately 

results in tree death. Incomplete, overlapping girdles provide a less severe alternative that was effective for stimuUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

lating flowering in white spruce (Picea glauca) (Pharis et 

al. 1985) and loblolly pine (Pinus taeda) (Wheeler and 

Bramlett 1991). A related technique, scoring (cutting completely through the bark around the trunk with a sharp 

knife without removing any bark), has been used to stimulate flowering, especially in apple (Veinbrants 1972). Wire 

girdles were also used .with some success to stimulate flowering in aspen. This method involves encircling selected 

branches or the main stem with tightly twisted, steel wire. 

The wire is adjusted as the plant grows to avoid killing 

parts distal to the wire. This technique is best applied in 

the spring or early summer (Li 1995, personal communication, North Carolina State University, Raleigh, NC 27695 

USA). 

Root Growth Control 

Confining root growth can lead to precocious flowering 

in peach trees (Prunus persica) (Richards and Rowe 1977). 

Copper treatment of pots or of fabric used to envelop roots 

prevents root circling and penetration. This form of restriction leads to development of more root tips, which frequently seems beneficial, especially in conjunction with 

nitrogen fertilization (Proebsting 1995, personal communication, Horticulture Dept., Oregon State University, 

Corvallis, OR 97331 USA). A similar effect may be achieved 

by root pruning or wrenching, which can also lead to a 

more densely branched root system. Similarly, Li (1995, 

personal communication, North Carolina State, Raleigh, 

NC 27695 USA) has stimulated aspen flowering by growing seedlings in small pots and cutting the roots of potted 

plants with a large knife in 2 or 3 directions tangential to 

the stem. 

Shoot Training 

Fruit tree growers have induced early flowering by training the shoots to grow horizontally. With this technique, it 

is important to keep the whole shoot horizontal including 

the leader. If the leader is removed, damaged, or allowed 

to resume upright growth, the shoot reverts to a juvenile 

state (Pharis 1995, personal communication, Biology Dept., 

University of Calgary, Calgary, Alberta, Canada T2N 1N4). 

After testing a variety of shoot training techniques, Denby 

et al. (1988) showed that 2 techniques, angle training and 

spindle training, increase yield and induce precocious 

flowering in pear (cv. 'Anjou'). 

Cultural Conditions 

Photoperiod 

Eucalyptus occidentalis End I. flowers precociously at less 

than 1 year of age when grown under a photoperiod of at 

least 16 h (Bolotin 1975). In addition, photoperiod may 

modify responses to other environmental factors. For ex215 
Section V Biotechnological Applications 

ample, the stimulation of flower bud formation in Camellia japonica by exposure to high temperatures is enhanced 

by long photoperiods (Bonner 1947). Birch (Betula 

verrucosa) (Longman and Wareing 1959), Japanese larch 

(Larix leptolepis), and blackcurrant (Ribes nigrum) (Robinson 

and Wareing 1969) were all stimulated to flower by long 

photoperiods. 

Nutritional Status 

Improved nutritional status is often considered a flowerinducing treatment. With tea crabapple (Malus hupehensis) 

seedlings, Zimmerman (1971) reduced the time to first 

flowering from 3 years to 9lh months by growth under 

continuously "favorable conditions" in a greenhouse. This 

method included weekly treatments with 20-20-20 (N-PK) water soluble fertilizer. Aldwinckle (1975) obtained 

nearly identical results with apple watered weekly with 

15-6.4-12.4 fertilizer supplemented with trace elements, 

in addition to the incorporation of a slow release 17-10.8-

7.3 fertilizer (Osmocote®) in the upper layer of the soil 

every 2 to 3 months. Also, the form of supplied nitrogen 

is apparently important. Grasmanis and Edwards (1974) 

showed that when ammonium was supplied to liquidcultured apple, a significantly greater flowering response 

occurred than when trees received nitrate as their sole 

nitrogen source. 

Moisture Stress 

Considerable evidence shows that water stress can 

enhance flower initiation in conifers, and that hot, dry 

summers generally induce abundant seed crops in conifers and broadleaf species (Philipson 1990). Water 

stress was recently used to stimulate aspen flowering 

in a greenhouse setting. Potted plants were periodically 

allowed to dry down to predawn moisture-stress readings between -6.9 and -9.7 bars (-100 to -140 psi) before 

rewatering. This treatment is apparently effective oRly 

when applied during shoot elongation (Li 1995, personal communication, North Carolina State University, 

Raleigh, NC 27695 USA) 

Temperature 

Low temperatures were used to induce precocious flowering in several tree species (reviewed by Jackson and 

Sweet 1972) including peach (Prunus japonica) (Lammerts 

1943), olive (Olea europaea) (Hackett and Hartmann 1963), 

sweet orange (Citrus sinensis) (Moss 1976), and lychee (Litchi chinensis) (Menzel1983). More recently, Moncur (1992) 

showed that transferring Eucalyptus lansdowneana seedlings 

from a heated greenhouse (24/19 oc, day /night temperatures) to a cold regime (15/10 °C) for 5 to 10 weeks before 

returning them to warm conditions (24 I 19 °C) was sufficient to induce floral buds. 

216 

Molecular Techniques 

Recently, over expression of regulatory genes early in 

reproductive development induced early flowering. When 

AP1 or LFY (reviewed above) is expressed constitutively 

under control of the 355 promoter from cauliflower mosaic virus (CaMV), transgenic Arabidopsis plants flower in 

vitro within weeks instead of nearly 2 months (Mandel and 

Yanofsky·1995; Weigel and Nilsson 1995). In addition, the 

Arabidopsis-derived LFY gene expressed under the control 

of 35S in hybrid aspen (Populus tremula x P. alba) also simulated flowered in vitro within several weeks (Weigel and 

Nilsson 1995). 

A MADS box containing a gene of unknown function, 

OsMADS1, was recently isolated from rice (Oryza sativa). 

When expressed under control of the 355 promoter in tobacco, transgenic plants flowered 8 days earlier than their 

wild-type counterparts (Chung et al. 1994). CO, a flowering-time gene (discussed above) from Arabidopsis, is a zinc 

finger-containing transcription factor. Transgenic 

Arabidopsis plants containing extra copies of CO under the 

control of its native promoter flowered significantly earlier than wild-type plants (Putterill et al. 1995). Finally, a 

member of the SQUAMOSA binding protein-like gene family (SPL-3) was recently shown to bind to the APl promoter. Constitutive expression of SPL-3 in Arabidopsis 

resulted in early flowering (Saedler et al. 1996). 

These early flowering genes have 2 important applications. First, they can be used to retransform existing 

transgenic plants that contain constructs expected to cause 

sterility. If the constructs function properly, induced flowers should be sterile. Second, early flowering genes can be 

used to generate tester lines that are maintained in vitro. 

These lines can then be transformed with newly developed sterility-causing constructs to rapidly indicate their 

use in trees. 

Conclusion 

Research on floral development over the last decade has 

provided a wealth of genes and knowledge on which to 

base strategies for modifying plant reproductive development. With the diversity of approaches available, a means 

can be established to genetically engineer reproductive 

sterility. The primary issues are determining the effectiveness of constructs for inducing stable, bisexual sterility in 

a variety of genotypes and environments, and identifying 

a reliable method to accelerate flowering for expedient 

research. Within the next 5 to 10 years, constructs should 

become available that can induce highly stable, male and 

female sterility in poplars and other tree species. Introduction of such constructs into commercially valuable genoUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Poplar Genetically Engineered for Reproductive Sterility and Accelerated Flowering 

types will simplify regulatory approval of transgenic varieties and reduce environmental impacts of large-scale tree 

farming using engineered clones. 

Acknowledgments 

We thank Dr. Ray Seidler and the U.S. Environmental 

Protection Agency for grant support (CR822880-1), and 

Will Rottmann, Amy Brunner, Lorraine Sheppard, and 

Caiping Ma for their collaborative aid on our poplar sterility project at Oregon State University. 

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219 
Chapter29 

Integration of Molecular and Classical Genetics: 

A Synergistic Approach to Tree lmprovement1 

Ronald J. Dinus and Gerald A. Tuskan 

Introduction 

Commercial tree improvement (i.e., application of classical genetics to tree breeding) involves selection, mating, 

testing, and propagation of genotypes having desirable 

growth, yield, wood quality, fiber quality, and biotic and 

abiotic stress tolerance. Despite significant improvement 

of many species, progress is constrained by long-generation intervals, poor juvenile-mature trait relationships, and 

difficulty in identifying and accumulating valuable genes. 

Advances are occurring in molecular genetics that can 

promote tree improvement programs. In this chapter, molecular genetics includes vegetative propagation, DNAbased marker technologies, and genetic transformation. 

TlSsue culture is included because of its effectiveness in 

genetic transformation and expediting delivery of improved materials, whether manipulated by other molecular techniques or not. These molecular techniques offer 

much promise to offset or reduce constraints that limit classical tree breeding. 

This chapter addresses integration of molecular and classical genetics, with emphasis on the potential impacts of 

molecular techniques on breeding progress. The discussion builds on writings of other authors (e.g., Cheliak and 

Rogers 1990; Chun et al. 1988; Ostry and Michler 1993; 

Riemenschneider et al. 1988; Tuskan 1992) by explaining 

individual molecular techniques, giving examples of recent accomplishments, highlighting advantages of poplars 

as model species, and describing critical research needs. 

1 Klopfenstein. N.B.; Chun. Y. W.; Kim. M.-S.; Ahuja. M.A .• eds. 

Dillon. M.C.; Carman. R.C.; Eskew. L.G •• tech. eds. 1997. 

Micropropagation. genetic engineering. and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins. CO: 

U.S. Department of Agriculture. Forest Service. Rocky Mountain 

Research Station. 326 p. 

220 

The first section describes molecular techniques and their 

parallels to classical breeding practices. Several potentially 

useful techniques (e.g., chromosome transplantation) are 

not discussed because the research is in the most basic 

phases. The second section reviews classical breeding strategies for intra and interspecific breeding. The third section describes means for integrating molecular techniques 

and classical breeding practices, how they complement 

each other, and how molecular techniques affect genetic 

gain and breeding efficiency. The last section summarizes 

and discusses future implications. 

Background 

Molecular techniques present opportunities but also create challenges for classical tree breeding. The many opportunities and challenges are best addressed by first 

working with model species; the genus Populus is uniquely 

suited. Using poplars as a model is also appropriate because of their increasing use as a commercial source of fiber and energy. 

As is common for most tree species, poplars generally are 

outcrossing and heterozygous. Most members of the genus 

are dioecious, which further enhances outcrossing and diversity (Stanton and Villar 1996). Poplars are prolific pollen 

and seed producers, and wind dispersal ensures wide 

gennplasm distribution. Many species inhabit diverse environments across broad geographic ranges. Variation has been 

further enhanced by interspecific hybridization. 

Breeding methods are available for species and hybrids 

of Populus. Pollen can be collected, stored, and handled 

with relative ease. Cross pollination can be conducted on 

branches brought into greenhouses or laboratories (Stanton 

and Villar 1996). In contrast with many other tree species, 

Populus seed ripens within a few months of pollination. 

Difficult crosses (e.g., among species in the same or different sections of Populus) can be assisted via mentor pollen 

and/ or embryo rescue. 
Integration of Molecular and Classical Genetics: A Synergistic Approach to Tree Improvement 

.Elite geno~pes from individual Populus species or hyb.nd populations can be propagated vegetatively by a vanety of means, and commercial planting of some selections 

is done routinely with dormant, nonrooted cuttings (Land 

and Cunningham 1994). Ease of vegetative propagation 

has fostered establishment of replicated clonal plantations 

for precise evaluation of genetic value. Research in such 

pla~tations has yielded a wealth of knowledge about genetics, anatomy, physiology, biochemistry, growth, yield, 

and ~col~gy of Populus. Most Populus species and hybrids 

are dtplotd, have 38 chromosomes, and a relatively small 

genome (C = 0.55 pico grams and approximately 550,000 

base pairs) (Bradshaw and Stettler 1993). For all of these 

reasons, poplars are well-suited for investigation and manipulation via molecular techniques. 

Molecular Techniques 

Several examples of molecular techniques that are 

promising for classical tree breeding are discussed in this 

section. Vegetative propagation by in vitro and more conventional means can shorten the time required and increase genetic gains. DNA markers and genetic 

fingerprinting can· be used to discern and verify identity 

of breeding materials. Markers can also be used to accelerate breeding via reliable early selection. Genetic transformation offers opportunities for creating and exploiting 

variants that might otherwise be expensive and time consuming or unavailable. 

Vegetative Propagation 

Embryo Rescue 

Interspecific hybridization is an important tactic in poplar breeding strategies. Many crosses are productive, but 

some critical combinations have reduced seed yields. In 

vitro culture methods to rescue embryos and increase seedling recovery have been developed (Raquin et al. 1993; 

Stanton and Villar 1996). 

Cell and Protoplast Fusion 

This in vitro technique can provide a means to enhance 

genetic variability by creating somatic hybrids among 

sexually incompatible species and by combining nuclei 

and cytoplasmic organelles (Kikkert this volume). Various symmetrical and asymmetrical combinations can be 

produced that may have value to breeders. Efficient methods to regenerate plants from protoplasts of diverse taxa 

can also facilitate genetic transformation (Charest et al. 

this volume). 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Somaclonal Variation and Selection 

Somaclonal variation often occurs spontaneously in tissue culture systems, sometimes yielding plants with considerable variation, phenotypically and I or genotypically, 

frox_n the donor plant (Fry et al. this volume). If useful genetic ch~nge occurs, variants can be added to breeding 

populations. Stable, lasting variation, even if not heritable, 

can be exploited via vegetative propagation. 

Exposing cell or tissue cultures to selective agents can 

be used to screen prospective parents for traits such as resistance to biotic and abiotic stress. Coupling somaclonal 

variation, as previously described, with selective screening is promising since valuable variants may arise under 

pressure of the selective agent (Ostry this volume). 

Dihaploids 

Heterozygosity, which is typical of forest trees, has both 

advantages and disadvantages for breeding. Although 

breeders have access to many genes, they are hindered in 

concentrating valuable genes into manageable numbers 

of genotypes. Creation of dihaploid (homozygous) plants 

may help overcome such obstacles. Anthers or ovules from 

elite individuals can be used to establish haploid tissue 

cultures. Plants regenerated from such cultures, after natural or artificial chromosome doubling, could be used as 

pure lines. Uddin et al. (1988) reviewed research on anther culture and demonstrated regeneration from P. 

deltoides anther cultures. Further advances were reported 

by Mofidabadi et al. (1995). 

Mass Propagation 

Many poplar species can be propagated by rooted or 

nonrooted cuttings. Although widely used, this approach 

has the following limitations: 1) relatively small numbers 

of cuttings can be produced from individual donors during a growing season; 2) rooting ability varies among donors and frequently declines with donor age; and 3) genetic 

transformation is not feasible. Research has improved cutting yields, but further improvement in rooting efficiencies is needed (Ostry and Michler 1993). Examples of 

applied programs are provided by Lambeth et al. (1994). 

As noted in the ehapters of this volume on in vitro culture, because poplars are relatively easy to manipulate in 

culture, significant advances have been recently achieved. 

In vitro propagation can be accomplished by any of 3 approaches: 1) regeneration from existing meristems; 2) organogenesis from adventitious meristems; or 3) somatic 

embryogenesis. 

Regeneration from existing meristems is a common 

method and is noted for its simplicity and reduced risk of 

somaclonal variation. Multiplication rates are limited, 

however, and the approach is not especially amenable to 

genetic transformation. 

221 
Section V Biotechnological Applications 

Propagation from adventitious meristems (i.e., organogenesis) can be achieved with a large array of explants 

(Ernst 1993). This approach can produce high multiplication rates and is useful for genetic transformation. Despite 

significant advances, however, procedures must be tailored 

to individual genotypes and risk of unwanted soma clonal 

variation is relatively high. · 

Somatic embryogenesis offers higher multiplication rates 

than other in vitro methods, generally has less unwanted 

somaclonal variation, and is ideal for genetic transformation. High frequency embryogenesis has been obtained 

with various tissues from several poplar species and hybrids (Michler 1995). The approach has also been used for 

in vitro selection for resistance to Septaria musiva (Ostry et 

al. 1994) and tolerance to herbicides (Michler and Haissig 

1994). 

DNA-Based Marker Techniques 

A variety of recombinant DNA techniques can be used 

to identify DNA markers (i.e., selectively neutral, polymorphic DNA sequences). Such markers can be applied 

in various phases of classical breeding to fingerprint natural populations, candidates for breeding, and propagules 

for commercial planting. Additional applications include 

creation of genetic maps, identification of markers for valuable traits, and isolation of desirable genes. 

DNA Fingerprinting 

DNA fingerprinting, in the broadest sense, includes any 

procedure that characterizes genetic variation at the DNA 

level. In its narrowest form, fingerprinting is a statistical 

process for identifying individual genotypes (Weising et 

al. 1995). Several techniques are available for detecting 

differences in DNA sequences among related and 

nonrelated individuals. Analysis of restriction fragment 

length polymorphisms (RFLP) was one of the first such 

techniques. In this method, a probe or short segment of 

DNA isolated from a related (homologous) or nonrelated 

(heterologous) genotype is hybridized to genomic DNA 

following digestion with restriction endonuclease enzymes 

(Southern 1975). Overall differences in fragment lengths 

and I or in homologies at hybridization sites are used to create a unique DNA fingerprint (Cervera et al. this volume). 

Recently, polymerase chain reaction (PCR) fingerprinting techniques were developed. These include randomly 

amplified polymorphic DNA (RAPD) markers (Welsh and 

McClelland 1990; Williams et al. 1990), minisatellite markers ijeffreys et al. 1985), simple sequence repeat (SSR) 

markers (Tautz and Renz 1984), and amplified fragment 

length polymorphism (AFLP) markers (Zabeau 1993). 

These approaches use DNA primers in conjunction with 

DNA polymerase enzymes to create numerous fragments 

of DNA having lengths dependent upon sequences in the 

222 

genomic template. Since amplified DNA fragments from 

populations within species and individuals within populations vary in size, such differences can be used to develop a characteristic fingerprint (Cervera et al. this 

volume; Lin et al. this volume). 

Choices of fingerprinting technique and genotypes to 

fingerprint vary with breeding objectives. Polymerase 

chain reaction techniques are faster and less expensive to 

apply than other approaches, but many PCR markers are 

codominant and therefore less informative. Even so, PCRbased RAPD markers were used to characterize population differences in several poplar species; for example, 

aspen (P. tremuloides) (Tuskan et al. 1996) and black cottonwood (P. trichocarpa) (Reed 1995). Similarly, RAPD and 

SSR markers were used to fingerprint commercial poplar 

clones developed in breeding programs around the world 

(Lin et al. this volume). 

Linkage Analysis and Genetic Maps 

Linkage analysis is a method for determining the linear 

arrangement of genes or genetic markers on a single 

segregational unit (i.e., a chromosome). Recombination 

frequency is used to establish the extent of crossing over 

between genes at 2 separate loci. Genes that assort independently are not linked, and the genetic distance is 

deemed the maximum measure of 100 centiMorgans (eM). 

The closer that 2 loci are located to each other, the smaller 

the recombination value. Genetic maps are created by testing the relative colinearity of multiple loci in sequential 

linkage analyses. Physical and morphological traits, 

isozymes, RFLP markers, and PCR-based markers can be 

positioned on genetic maps. Several computer software 

programs are available to construct genetic maps such as 

LINKAGE-I (Suiter et al. 1983), GMENDEL (Liu and 

Knapp 1990), and MAPMAKER (Lander et al. 1987). 

Genetic maps can be used to determine taxonomic relationships among potential breeding candidates, indirectly 

select candidates based on presence or absence of linked 

markers, and isolate genes from rare phenotypes (Weising 

et al. 1995). Creating or acquiring usable populations is 

the first step in constructing genetic maps. Backcross or F 2 

populations typically are used to maximize recombination 

frequencies. In addition, parents with extreme phenotypes 

(e.g., those susceptible or resistant to pest attack) can be 

used to ensure abundant variation. Ideally, 400 to 500 offspring should be available from each generation to ensure 

precise estimates of crossing over in linkage analyses. Poplars are well-suited as models for genetic mapping because 

they have a relatively small genome, can be manipulated 

to produce large numbers of offspring per parent, have 

relativeiy short generation intervals, and can be evaluated 

at early ages. 

The relationship between a genetic and physical map 

depends on the amount of DNA in the haploid genome, 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Integration of Molecular and Classical Genetics: A Synergistic Approach to Tree Improvement 

number of chromosomes, and recombination frequency of 

the particular species. As noted previously, poplars match 

such criteria well and have exhibited a physical and genetic ratio of approximately 200 kilobase pairs per eM 

(Bradshaw et al. 1994). Given these advantages, a moderately saturated genetic map was created for poplars, and 

500 genetic markers from RFLP and PCR techniques were 

linked to30 phenotypic traits (Bradshaw and Stettler 1995). 

Bradshaw (1996) has proposed reconciling the genetic map 

with the physical one. 

Genetic Markers and Marker-Aided Selection 

Research on genetic markers in poplars has advanced 

rapidly in recent years. Markers were associated with traits 

governed by 1 or a few genes, for example, disease resistance (Newcombe and Bradshaw 1996; Newcombe et al. 

1996), and also with quantitative trait loci (QTLs) that govern characteristics such as height and diameter growth 

(Bradshaw 1996; Bradshaw and Stettler 1995). Since most 

commercially important traits in forest trees are quantitative, linkage relationships between genetic markers and 

QTLs allows indirect selection at early ages. For reliable 

and economical use, however, such relationships must be 

verified across families in breeding programs, within varying genetic backgrounds, and across environments (Strauss 

et al. 1992). Given the time and expense required to identify. useful markers, traits for application of marker-aided 

selection should possess high value, have low heritabilities, and I or be difficult to measure. Some examples are 

disease resistance, particularly when pathogenic variation 

exists (Newcombe 1996), complex wood properties (Williams and Neale 1992), and resistance to abiotic stresses 

that are difficult to replicate (Tauer et al. 1992). Markeraided selection schemes are being tested with hybrid poplars (Bradshaw 1996). 

Genetic Maps and Gene Isolation 

Genetic maps can also be used to isolate desirable genes. 

In this approach, map-based gene cloning flanking genetic 

markers around a gene of interest are used to "walk" toward the expressed DNA sequence (Tanksley et al. 1995). 

For greatest effectiveness, markers should be within 1 to 2 

eM, or approximately 200 to 400 kilobases, of the gene in 

question. Thus, high density genetic maps are a prerequisite. Ultimately, genes isolated by this means can be verified and I or used via genetic transformation. Although 

valuable, map-based gene cloning will probably only be 

used in the near future for exceptionally rare or valuable 

traits. Nevertheless, poplars and related genera should be 

useful for developing and evaluating the approach. For 

example, the technique is being used to isolate a gene associated with femaleness in Salix viminalis (AlstromRapaport et al. 1996). Widespread application will require 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

development of genetic maps, reliable gene transfer techniques, and established associations of specific genes with 

desirable traits. 

Genetic Transformation 

Genetic transformation, or nonsexual addition of specific genes, permits acquisition of traits otherwise unavailable. This technique can also be used to incorporate 

additional copies of indigenous genes that are closely 

linked to undesirable genes circumventing the need for 

generations ofbackcrossing. Bypassing the sexual process 

offsets time and cost difficulties posed by long generation 

intervals. 

As discussed (Charest et al. this volume; Han et al. 1996; 

Kim et al. this volume), a variety of gene transformation 

procedures have been tested and used with poplars. The 

first successful achievement of such transformation involved a gene conferring herbicide tolerance on a hybrid 

poplar (Fillatti etal. 1987). Poplars are noted as models for 

transformation because of the ease, relative to most other 

tree species, with which they can be manipulated and regenerated in cell and tissue culture (Chandler 1995). Poplar transformation is becoming more routine and is being 

expanded to include a widening array of genotypes and 

genes. For example, recovery of transgenic plants of P. 

deltoides was recently reported (Dinus et al. 1995). 

Transformation can also be adapted to reduce expression of existing genes that encode undesirable traits. This 

antisense technology involves inserting a reverse copy of 

the gene in question. Messenger RNA (mRNA) transcribed 

from the reverse copy is thought to bind with that from 

the normal gene inactivating the mRNA and preventing 

synthesis of a gene product. Early research on antisense 

technology with forest trees was performed with poplars; 

for example, control of lignin biosynthesis and deposition 

(Boerjan et al. this volume; Chiang et al. 1994). Successful 

deployment of antisense technology will require a better 

understanding of the extent, stability, and underlying 

mechanisms of suppressed expression. 

Introduced genes are not always expressed, may not be 

expressed in spatially and temporally desirable manners, 

and may not persist across the long life cycle of forest trees 

(Ahuja this volume). Ensuring proper expression is a major technical challenge. In addition, gene insertion may provoke undesirable genetic and I or epigenetic changes; for 

example, the co-suppression phenomenon. Currently, longterm testing is required to ~nsure the presence of desirable changes or absence of undesirable changes. Better 

methods are needed to target gene delivery and verify 

transformation, gene expression, and genetic fidelity. As 

noted earlier, cell and tissue culture methods are also 

needed that are less genotype specific. Research with poplars is helping to fill these knowledge gaps, as discussed 

in Stettler et al. (1996a) and this volume. 

223 
Section V Biotechnological Applications 

Classical Genetics and Breeding 

Early poplar breeders achieved substantial improvement 

by exploiting variation existing in native populations. 

Promising individuals were selected from natural stands 

(i.e., base populations), vegetatively propagated, and 

evaluated in clonal trials (figure 1). In turn, the best individuals were vegetatively propagated for commercial 

planting. 

Short-Term Strategies 

A first step beyond the opportunistic approach involves 

wide crosses among desirable individuals from the same 

and/ or different species, evaluating offspring in clonal 

trials, then multiplying the best offspring for commercial 

planting (figure 1). Matings, intra or interspecific, are repeated and I or adjusted to maintain or increase in accordance with performance of cloned offspring. New parents 

and crosses are added as needed. Heterosis and I or acquisition of specific traits have been a justification for using 

interspecific matings. Clonal deployment remains the 

norm since it captures both additive and nonadditive genetic variation, enhances gain by shortening the time between selection and commercial planting (Matheson and 

Lindgren 1985), and typically is less expensive than producing and planting seedlings (Foster 1986; Lambeth et 

al. 1994). 

Our informal survey of poplar breeders found that the 

short-term strategy of wide crossing within and among 

species, selection, and clonal deployment (figure 1) will 

be widely used until more sophisticated strategies are 

deemed necessary and economical. This strategy is lucrative, and the advantages are adequate, especially in view 

of the associated low cost. 

Long-Term Strategies 

Most breeders acknowledge that short-term strategies 

will not pay returns forever and that greater gains are possible from simple recurrent selection within or among species or from reciprocal recurrent selection using 2 or more 

species. Long-term strategies using such approaches have 

been advanced by Lambeth et al. (1994), Bisoffi and 

Gullberg (1996), Stanton {1995), and Li (1995, pers. comm.) 

and emphasis gradually is shifting toward their implementation. Theoretical arguments favoring such strategies have 

been presented by Foster {1986, 1993a) and White (1995). 

Simple recurrent selection involves selection, mating, 

and testing of a limited number of parents within a single 

breeding population (figure 2). Breeding cycles are completed by selecting parents for subsequent generations 

224 

based on progeny tests of each preceding generation. 

Simple recurrent selection is easier and less expensive to 

implement than reciprocal recurrent selection and is wellsuited for capturing additive genetic variation. Reciprocal 

recurrent selection involves selection, mating, and testing 

a limited number of parents from 2 or more parallel populations; often different species within the genus (figure 3). 

Parents are selected within populations to produce subsequent generations, while propagation populations are created by matings among populations in each cycle of 

improvement. Reciprocal recurrent selection is well-suited 

for creating interspecific hybrids, capturing both additive 

and nonadditive variation, and reducing inbred materials 

in commercial populations. 

Long-term strategies (figures 2 and 3) call for amassing 

large, but not overly burdensome, base populations selected from natural stands, earlier breeding work, and I or 

collections from other breeders. Parents, regardless of generation, may be divided into sub lines. Productivity is typically emphasized across generations, but individual 

sublines can be manipulated to maximize improvement 

in specific traits, develop complementary phenotypes, and 

maintain overall genetic diversity (Stanton 1995). Individual organizations undoubtedly will have differing 

needs and constraints, and are therefore likely to pursue 

divergent objectives and strategies (Bisoffi and Gullberg 

1996). Traits most likely emphasized within sublines include wood properties, disease resistance, bole form, 

canopy architecture, and reproductive potential. Sexual 

reproductive capabilities can be stressed in programs relying on reciprocal recurrent selection as recovery of hybrid seedlings is sometimes limited (Stanton 1995; Stanton 

and Villar 1996; Stettler et al. 1996b). Ease of vegetative 

propagation can also be emphasized. 

Matings within sub lines can be arranged as disconnected 

factorials, with provisions for assortative mating (Foster 

1986; Stanton 1995). Parents within factorials and sublines 

can be grouped according to breeding values estimated 

from clonal trials of offspring, the best being mated with 

best in descending order until all selected parents are used. 

Factorial mating schemes are preferred ·since they yield 

reasonable numbers of families from many parents and 

require relatively few crosses per parent. Inbreeding can 

be tolerated within sub lines but propagation populations, 

with some exceptions, are derived from crossing among 

sublines (Bisoffi and Gullberg 1996). 

Early and step-wise testing and selection can also be 

used to reduce numbers of clones tested in field trials. Thus, 

a several step screening process can be implemented, with 

each trial eliminating some families from further testing. 

Overall costs would be reduced since many families would 

be eliminated in the earliest and least expensive tests (van 

Buijtenen and Lowe 1989). Poplar breeders are likely to 

use an initial screening for survival and growth in seedling nurseries. Subsequent evaluation for rooting ability, 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Integration of Molecular and Classical Genetics: A Synergistic Approach to Tree Improvement 

P1: 

In vitro Selection 

DNA 

Fingerprinting 

P1: 

Selection 

DNA 

DNA Fingerprinting ~ 

F1: 

In vitro Selection 

Marker-aided Selection 

Genetic 

DNA Fingerprinting ' 

F1: 

In vitro Selection 

Marker-aided Selection 

Genetic 

--------

In vitro Selection 

Marker-aided Selection 

DNA 

Fingerprinting 

Figure 1. Short-term, de novo selection and wide crossing among diverse base populations and/or species. Solid 

lines=pl.ant material movement; dashed lines=information flow; curved lineS=application of molecular genetics 

techniques. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 225 
Section V Biotechnological Applications 

DNA Fingerprinting 

P1: 

Embryo Rescue 

F1: 

In vitro Selection 

Genetic 

DNA 

Fingerprinting 

......... ·--············ 

QS) 

X 

DNA Fingerprinting t 

F2: 

In vitro Selection 

Marker-IJUkd Selection 

DNA 

Fingerprinting 

~-···················· 

~ 

~ X 

t 

DNA Fingerprinting 

F3: 

DNA Fingerprinting 

Figure 2. Long-term simple recurrent selection, where initial base populations may consist of one or more species. After 

the first selection cycle, breeding or parental populations generally are closed, with further selections derived 

from advanced generation progeny sets. Solid lines=plant material movement; dashed lines=information flow; 

curved lines=application of molecular genetics techniques. 

226 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
P1:( 

DNA Fingerprinting I 

F1: 

DNA 

Fingerprinting 

F2: 

X 

X 

DNA Fingerprinting I 

Integration of Molecular and Classical Genetics: A Synergistic Approach to Tree Improvement 

Embryo Rescue 

L I 

Fingerprinting 

Selection 

X 

DNA 

Fingerprinting I DNA 

Selection 

DNA X 

I DNA 

Fingerprinting 

Selection 

Transformation 

Figure 3. Long-term reciprocal recurrent selection, where inital base populations may consist of one or more species. In 

each generation, the alternate populations serve as tester parents for creation of new F, hybrids. Each commercial population contains newly created F, hybrid progeny. Solid lines=plant material movement; dashed 

lines=information flow; curved lines=application of molecular genetics techniques. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 227 
Section V Biotechnological Applications 

growth, and other traits (e.g., pest resistance) could oc~ur 

during greenhouse or field multiplication. L~ter testmg 

for growth, pest resistance, and propagule yte_Jds could 

occur in cutting nurseries. Only the few clones JUdged as 

superior in such tests would be evaluated in more costly 

growth and yield trials. . . · . 

Parental or breeding populations and contmued cyclmg 

are intended to exploit additive genetic variation, although 

correlations between average seedling and clonal performance may be imperfect. Added precision offered by clonal 

trials, however, can partially offset discrepancies. Quantifying the extent of such correlations remains a vita~ research 

need. Individual parents for subsequent generations may 

be identified via combined family and within family selection. This approach should improve average breeding 

population quality (i.e., exploit additive genetic variation), 

while capturing new combinations associated with nonadditive variation. 

Propagation populations can be drawn from this. same 

composite of families, seedlings, and clones. Selection of 

the most outstanding individuals, despite family background, exploits both additive and nonadditive variation. 

Thus, individuals moved to propagation and commercial 

populations are selected for their total genetic worth and 

are not necessarily the same as those selected for breeding. For commercial use, large numbers of related individuals are unlikely if parental or breeding populations 

are of sufficient size. Should significant relatedness occur, 

diversity can be maintained by deploying related materials in different planting areas or years. 

Strategies for deploying clones in commercial plantings 

are beyond the scope of this chapter, but are nevertheless 

a critical issue. Debates continue over the adequate clone 

number for protection against biotic and abiotic stresses, 

along with advantages and disadvantages of pure versus 

mixed plantings. Poplars, whether manipulated by classical breeding and I or molecular techniques, seem ideal for 

performing this much needed research. Some recent findings can be found in papers by Foster (1993a, 1993b ), 

Knowe et al. (1994), Foster and Knowe (1995), Bishir and 

Roberds (1995), and DeBell and Harrington (1997). 

Integration of Molecular and 

C·lassical Genetics 

Molecular techniques, ranging from vegetative 

propagation through genetic markers, genetic mapping, 

and gene transfer, have parallels and counterparts in 

classical breeding. Integration is best discussed by emphasizing parallels, then describing how techniques can 

be applied in each classical breeding phase, what ben228 

efits can be expected, and how the approaches are likely 

to benefit each other. 

Acquiring and Infusing Genetic Variation 

Acquiring a broad genetic base at the outset of breeding 

is necessary to minimize reductions in genetic variability 

over subsequent generations. Additional variability can 

be infused later by introducing new materials from natural populations, but this will dilute gains captured in preceding generations. Thus, later introductions should be 

helpful primarily to breeders using short-term strategies 

such a~ those in figure 1. 

Molecular techniques (e.g., genetic transformation) can 

produce or ensure new variation but are best employed in 

short-term strategies. Long-term strategies (figures 2 and 

3) could benefit most from application to already improved 

materials in the mating phase. Potential applications are 

discussed below. · 

Breeders spend considerable time and resources defining the genetic architecture of candidate species. While 

molecular techniques (e.g., DNA fingerprinting) will not 

replace provenance and progeny tests, they can yield useful information faster, more powerfully, and more precisely 

than the isozyme methods once used to study genetic architecture (Burdon 1994). Thus, DNA fingerprinting can 

be used to maximize variation at the initiation of breeding 

(figures 1 to 3). This technique can also confirm that prospective parents are of the preferred species, provenance, 

hybrid background (Lin et al. this volume), or gender. With 

further research, such techniques might be used to determine capacity for sexual and vegetative propagation and 

for the genetic basis of heterosis. Also, the parents likely 

to yield offspring with high levels of heterosis. 

Gauging impacts on efficiency is difficult, but most 

molecular applications should raise the quality of initial 

breeding materials. Also, avoiding errors (e.g., 

misidentification) will save considerable time and effort. 

Selection 

Molecular approaches that parallel selection in classical 

breeding programs, whether following short- or long-term 

strategies, include direct measures such as inducing and 

selecting somaclonal variation and indirect ones based on 

genetic maps and markers (figures 1 to 3 ). 

Selection can be performed in cell and tissue cultures, if 

the desired trait is expressed in vitro and outcomes are directly correlated with field performance. Cultures representing thousands of genotypes can be exposed to a 

selective agent or environment in modest laboratory facilities. Candidates identified as possessing the trait can 

be moved quickly into ~reeding populations. Given reliable culture ·systems, useful variants could also be transUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Integration of Molecular and Classical Genetics: A Synergistic Approach to Tree Improvement 

ferred directly to propagation populations. Recent advances 

in somaclonal variation and selection of Populus are reviewed 

by Fry et al. (this volume) and Ostry (this volume). 

Indirect approaches, such as DNA fingerprinting, might 

be used during the selection phase for some of the same 

purposes described above (i.e., confirming identity, sex, 

and reproductive potential). Genetic maps and markers 

can promote selection of superior genotypes. As previously noted, a genetic map of hybrid poplars is being constructed to locate genes for various traits. Genetic markers, 

includin~ QTLs, are being developed in the process 

(Bradshaw 1996; Bradshaw et al. 1994). Advantages of 

genetic markers include production in large numbers, 

Mendelian inheritance, and limited influence of environmental conditions or age. Numerous offspring could be 

evaluated early in life, with the best parents used for 

breeding and the best individuals transferred, quickly and 

directly, to propagation populations (Cervera et al. this 

volume). 

Impacts of these several techniques on selection efficiency should be significant. Large-scale, in vitro selection 

can magnify heritabilities and selection differentials and 

yield time and cost savings. Instead of selecting the best 

10 of 100 entries in a field trial, in vitro selection could yield 

similar numbers (from 10 or 100 times more candidates) 

at earlier ages and in less time. Genetic markers could have 

similar, possibly greater, impacts. Increased heritabilities 

and. selection differentials from either of these approaches 

could magnify genetic gain per generation. Gains per unit 

time would be increased by evaluating more individuals 

earlier than possible with classical testing procedures. Finally, savings in time and expense should result from culling undesirable or misidentified candidates before they 

are used for mating. 

Marker-aided selection clearly holds much promise, but 

widespread application awaits verification trials. One to 

several generations of full-sib offspring are required to 

develop markers, and extensive collaboration among molecular and classical geneticists is necessary to create useful populations. Since development is costly, sound 

information on economic advantages compared with classical selection is also needed. Commercial application may 

occur only if costs and risks are shared via regional, national, 

and I or international consortia, or if costs of accepting an 

undesirable phenotype exceed those of marker development 

and application. Poplar species, with their relatively short 

times to reproductive maturity and harvest, seem ideal for 

demonstrating the merits of such techniques. 

Mating 

Classical breeders recombine and I or add useful genes 

via intra and interspecific matings. Several molecular techniques parallel and complement these activities, while ofUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

fering significant advantages in the mating phase. Techniques for gene addition and recombination, however, may 

provide the greatest advantage. With these new tools, 

breeders can actually enhance variability rather than 

merely work to reduce its decline over generations. 

Somaclonal variation represents a potentially useful 

technique for increasing variation. Whether by actual 

mutation, gene and chromosome rearrangements, or activation of repressed genes, variation has been identified 

under a variety of cell and tissue culture conditions. After 

appropriate testing, useful variants can be incorporated 

into breeding and I or propagation populations. 

Although the somaclonal variation approach has potential for enhancing variability and saving time, it also 

has several drawbacks. Behavior in culture may or may 

not correlate with that in commercial plantings. Somaclonal 

variation is viewed as essentially random, thus it may not 

occur reliably for traits of interest. Useful genes may be 

linked to undesirable traits and epigenetic effects may accrue. Eliminating such unwanted effects could require 

generations of selection and breeding. In addition, considerable time and expense may be required to confirm 

utility, stability, and heritability (Stack 1987). Application 

to poplars, however, seems more feasible than with other 

forest tree species. Many poplar variants can be produced 

in culture and evaluated within 3 to 5 years, which is half 

the harvest rotation age for many poplars and within which 

reproductive maturity can sometimes occur. 

Somaclonal variation seems a useful addition to classical 

breeding, and more research on its causes and control seems 

warranted. For the near term, however, application seems 

best during selection of propagation populations. Exposing 

cells and I or tissues from desirable individuals to directed 

and stringent in vitro selection to induce variation is potentially more efficient than large and lengthy field trials. 

Several other cell and tissue culture applications that parallel the mating function also have promising potential. Reliable procedures for embryo rescue can enhance seedling 

recovery from interspecific crosses increasing genetic variability and selection differentials (figures 1 to 3). 

In vitro production of homozygous or dihaploid plants 

would promote selection for native and inserted recessive 

genes and yield pure lines or true-breeding hybrids more 

quickly than repeated backcrossing. Research on this promising technique seems worth pursuing. 

Protoplast or cell fusion might be used to combine genes 

and I or cytoplasms from unrelated or sexually incompatible species in special purpose hybrids. Asymmetrical derivatives regenerated from culture may serve as bridges 

for wide crosses among distantly related species. The dioecious nature of poplars and limitations on crossing between members of different subgenera encourage the use 

of such techniques. While not presently routine, protoplast and ceH fusion are promising for future application. 

Given their ease of manipulation in culture, poplars can 

229 
Section V Biotechnological Applications 

serve as models for developing methods and demonstrating use. 

When genetic variation is minimal or nonexistent, useful traits sometimes cannot be acquired, let alone improved. In such situations, genetic transformation (figures 

1 to 3) may enable breeders to enhance genetic variation 

via gene transfer from other genotypes, tree species, or 

unrelated organisms. Bypassing the sexual process allows 

change to occur in a directed manner and in less time than 

required for reproductive maturity. 

Exploiting gene transfer technology during the mating 

phase requires collaboration between classical and molecular geneticists to ensure that transferred genes are expressed in the acceptable tissue or organ at appropriate 

times during the life cycle. In addition, collaboration is 

needed to monitor spread of the gene throughout recipient populations and stability across generations. Transformation using antisense technology may require even 

longer and more expensive testing. 

As noted by Meilan and Strauss (this volume), the most 

significant drawback of gene transfer is the potential for 

genetic contamination of native germplasm via pollen or 

seed cast by transgenic plants. Indeed, current and foreseeable regulations governing release of transgenic plants 

into the environment may prohibit sexual reproduction of 

transgenic trees. Thus, sterility should be among the first 

traits modified via gene transfer. 

Application also requires availability of genotypes with 

proven silvicultural value. Availability of improved genetic 

backgrounds depends on a thriving classical breeding program. Additionally, the ultimate use of transformants requires a population with sufficient genetic diversity to 

minimize vulnerability to the many risks in commercial 

plantations. Transferring genes into all breeding materials 

could be prohibitively expensive. Until more is known 

about gene expression, stability, and safety, transformation of individuals only for propagation populations may 

be the best way to use this particular technology. This approach could also yield significant side benefits; eliminating reproductive structures could divert more 

photosynthate toward vegetative growth (Meilan and 

Strauss this volume). 

Genetic marker and DNA fingerprinting techniques 

could also be helpful in the mating phase (figures 1 to 3). 

As in other phases, these techniques could confirm that 

prospective parents are of the desired origin, identity, sex, 

and reproductive capacity. In long-term programs, important applications include ensuring balanced representation of males and females in sublines, monitoring 

inbreeding within sublines, tracking overall diversity 

across generations, and verifying hybrids. When genetic 

transformation is inv~lved, such techniques could be used 

to verify transformation, confirm persistence of inserted 

genes across generations, and monitor fidelity for other 

traits. 

230 

Genetic markers might also further interspecific hybridization and backcrossing. While desirable genes can often 

be obtained via hybridization, backcrossing usually is necessary to concentrate them while diluting or eliminating 

linked genes that are unwanted. Backcrossing forest trees, 

however, is an extremely long and costly process. Genetic 

markers could ease the task by identifying individuals 

possessing the desired trait and perhaps eliminating less 

desirable features at early ages (Bernateky and Mulcahy 

1992). Matings could then be restricted to individuals 

known to possess the trait. This application combined with 

measures to hasten reproductive maturity could increase 

the benefit of backcrossing in tree breeding. 

Despite some drawbacks, molecular techniques clearly 

have value in the mating phase. First, they have potential 

for increasing variability and ensuring continued diversity across generations. Second, novel genes not otherwise 

available in forest trees can be acquired independently of 

the sexual process preventing undesirable genes from accompanying desirable ones during mating and/ or backcrossing. In addition, genetic markers can be used to reduce 

errors and improve efficiency. 

Impacts of introducing previously unavailable genes are 

difficult to quantify in terms of genetic gain. Outcomes, 

undoubtedly, will vary greatly with affected traits. Qualitative assays, however, can be made by comparing projected before and after performances. As one example, 

genetic gain would approach or exceed 100 percent if individuals altered by somaclonal variation or gene transfer survived pest attack while unaltered individuals 

succumbed. Alternatively, transfer of genes affecting lignin quality and I or quantity (Boerjan et al. this volume; 

Chiang et al. 1994) may produce relatively small changes. 

Nevertheless, overall impact could be considerable because measurable change would be obtained within a 

single generation. Obtaining similar changes via classical 

breeding would require many generations since. genetic 

variation in lignin content is small and heritabilities are 

modest at best. 

Propagation and Production 

Efficient systems for vegetative propagation are essential for using molecular genetics and classical breeding (figures 1 to 3). A variety of propagation methods, ranging 

from nonrooted cuttings through micropropagation or 

somatic embryogenesis, can work with poplars. Each approach, however, has specific applications for which it is 

best suited. For example, nonrooted cuttings are useful for 

establishing commercial plantings, but are not suited for 

genetic transformation. In contrast, somatic embryogenesis is useful for transformation, somaclonal variation and 

selection, and even cell and protoplast fusion. The high 

cost of organogenesis and embryogenesis, however, may 

limit their use for mass propagation. Currently, their use 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
'~ 

Integration of Molecular and Classical Genetics: A Synergistic Approach to Tree Improvement 

is associated with genetic transformation and regeneration of a few transgenic plants for subsequent multiplication by other methods. As a practical example, regeneration 

from existing and adventitious meristems is often used to 

multiply improved materials for testing and further expansion in rooted cutting nurseries. Somatic embryogenesis techniques might also be used to salvage immature 

embryos that are not amenable to routine embryo rescue. 

If workable, this application would ensure that more offspring and parents are represented in clonal tests. 

Genetic markers and related techniques also have important roles in propagation and production (figures 1 to 

3). For selection and mating, genetic markers can be used 

to identify parents and offspring with high propensities 

for vegetative propagation. This would encourage moving improved materials into propagation and commercial 

populations and gradually improve capabilities in breeding populations. In addition, DNA fingerprinting can be 

used to confirm and maintain identities in propagation and 

commercial populations, monitor persistence of added 

genes, and prevent theft of valuable germplasm. Indeed, 

such techniques may stimulate breeders and growers to 

create and market "brand name" clones. 

Besides the indirect benefits, vegetative propagation can 

produce at least 2 direct impacts on breeding efficiency and 

genetic gain. First, vegetative propagation can raise selection efficiency; more individuals from more families can be 

tested more precisely in less time. Second, all genetic variation, additive arid nonadditive, is captured raising heritabilities. With greater heritabilities and selection differentials, 

gains and returns per generation can exceed those derived 

from programs relying solely on the sexual process. Breeders using either short- or long-term strategies (figures 1 to 3) 

can rely on vegetative propagation to generate materials for 

propagation and commercial populations. 

As noted, poplars are easy to propagate, and propagation technology has improved in recent years. Nonetheless, problems persist, primarily for in vitro methods, as 

exemplified by unwanted physiological or genetic variation. As a result, derived plants may require lengthy and 

expensive field testing to ensure accurate reflection of donor qualities. Additionally, many procedures remain genotype-specific, thereby limiting selection efficiency and 

raising costs. When only a fraction of elite individuals can 

be propagated for commercial use, breeders must select, 

breed, and test more individuals or relax selection differentials. Improved techniques that are less genotype-specific, yet maintain genotypic and phenotypic fidelity, 

remain an important research goal. 

Because capability for vegetative propagation is apparently heritable to some extent, selection and breeding to 

enhance such capability could yield significant dividends 

to both classical and molecular geneticists. Breeders would 

benefit greatly from improved efficiency, and contributions 

from their molecular counterparts would also be accelerUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

ated. Also, genes controlling ease of vegetative propagation may be transferable from other organisms into recalcitrant individuals. Collaboration could lead to synergistic 

outcomes. 

Vegetative propagation is also limited by certain.factors 

that restrict breeding efficiency. Selection in classical breeding programs typically requires lengthy and costly field 

trials. By the time genotypes are certified as superior, rooting frequencies can decline to inadequate levels. Though 

not as critical for poplars as for many other trees, such 

effects can be restrictive. Further research on rejuvenation 

would benefit poplar breeding efforts. 

Discussion and Conclusion 

Notable advances have occurred in development of molecular techniques. Application of molecular techniques may 

offset or reduce many biological and economic constraints 

on classical breeding. Nonetheless, molecular techniques 

present challenges and opportunities. Integration with classical breeding will require decisions concerning research 

priorities, applicability of particular techniques, and resource 

allocation. Implementing these decisions requires understanding the techniques and conditions under which they 

can be integrated advantageously and safely. 

DNA fingerprinting will help characterize populations, 

estimate genetic variability within and among populations, 

and monitor diversity and structure of populations generated, maintained, and manipulated in the breeding process. Discerning and maintaining identity during selection, 

breeding, and propagation will improve overall reliability. Identification could open markets for and prevent theft 

of proprietary genotypes. Developing and using such techniques, however, is expensive. Generating populations 

with appropriate pedigrees requires suitable genetic resources· and collaboration with classic breeders. 

Genetic markers should lend new efficiency to selection. 

Large-scale, early selection would help offset constraints 

imposed by long generation intervals and harvest rotations providing greater genetic gain per unit time. Efficiency of interspecific hybridization could also be 

improved, since markers can be used to guide matings 

among parent species. Building requisite genetic maps and 

identifying useful markers, however, requires that breeders have resources to produce appropriately pedigreed 

populations. Validating marker value entails precise and 

lengthy field trials. In view of the associated high costs, 

research must expand use to more diverse, less specialized pedigrees, and find markers strongly correlated with 

traits of interest. Viewed from another perspective, costs 

could be prohibitive unless improved materials are used 

in large numbers over multiple planting years (Teasdale 

231 
Section V Biotechnological Applications 

1995). Regulations governing permissible minimum numbers of genotypes for commercial planting could worsen 

the cost-benefit outlook. Poplars were among the first species for which genetic maps and markers were developed; 

their continued usc as models will aid refinement and extension lo other tree species. 

Genetic transformation offers potential improvement, 

both quantitatively and qualitatively, beyond classical 

breeding. Sexual sterility is a trait likely to be added via 

genetic transformation, since it could become a condition 

for commercialization of transgenic trees. Regulatory considerations aside, possession of this trait could divert energy from reproductive activities to biomass accumulation 

and further manipulation of improved materials on a scale 

larger than possible with classical breeding methods. 

Ongoing research on transformation, especially with 

poplars, will probably yield useful genotypes. Meaningful exploitation, however, remains limited by a delay in 

identifying valuable genes. Most past efforts relied on 

genes used in agricultural crops. Valuable lessons were 

learned, but genes unique to trees may have greater value. 

This research is critical, especially with regard to organization, structure, and control of genetic systems. Existing 

knowledge of poplar biology emphasizes the value of poplars for such research. 

Determining commercial potential of transgenic trees, 

sterile or nonstcrilc, will require extensive, long-term field 

trials to ensure that desired outcomes are realized and that 

undesirable effects arc minimal or absent (Stack 1987). 

Transformation with antisense genes will require further 

research and possibly even more stringent testing. Whatever the approach, materials used for transformation must 

first be selected and bred for traditional silvicultural values. Having suitably improved and diverse materials available for transformation requires continued maintenance 

and manipulation of genetic variability. 

New and improved systems for vegetative propagation 

could greatly assist classical breeding. Mass propagation 

hastens delivery of genetic gains and encourages application of techniques such as genetic transformation. Ill vitro 

embryo rescue and somaclonal variation and selection 

have additional potential for supplying new germplasm. 

Expanding these outcomes, however, depends on developing systems with minimal side effects and maximum 

applicability to the large array of materials routinely used 

in breeding programs. Exploiting improved methods, 

nonetheless, requires existence of genetic variability, and 

the ability to manipulate it. 

Effective integration of molecular techniques requires the 

existence and maintenance of active classical breeding programs. Molecular techniques are unlikely to replace classical breeding, but instead will enhance the capabilities of 

breeders to solve practical problems. The opposite side of 

this issue must not be overlooked, however, since intensive 

collaboration among disciplines is essential. Integration de232 

mands recognition of common problems, raising relevant 

questions, and understanding the biological, genetic, and 

economic consequences. Provisions should be established 

to maintain or expand breeding efforts while also funding 

efforts to develop and implement useful molecular techniques. DevCioping individual techniques is costly, but overall costs will be magnified because usc of one technique may 

demand application of others. For example, genetic transformation depends on vegetative propagation. Organizations 

seeking to exploit molecular techniques should be prepared 

to provide increased funding for both classical and molecular genetic programs, at least until molecular techniques produce cost savings and increased returns from practical 

applications in classical breeding. 

Support of both classical and molecular efforts is wise. 

Gains from classical breeding have been substantial and 

further progress is certain. Reducing breeding efforts could 

offset the realization of future genetic gains over the ncar 

term, diminish potential for future gains, and preclude 

exploitation of molecular techniques. The more sophisticated the breeding strategy, the more benefit gained from 

technical integration. Alternatively, failure to fund development and application of molecular techniques could be 

equally ill fated. Integration holds such high promise that 

cooperative efforts could capture considerable and lasting advantage. Especially critical is the increasing likelihood that competitors will protect methods and products 

by securing intellectual property rights (Chandler 1995). 

Balanced investments and research arc required, with 

adequate support and funds for ongoing breeding efforts 

and the development of promising molecular techniques. 

Clear priorities, consistent goals, and stable support are 

essential to avoid one area of research outpacing another, 

with applications hindered because needed techniques arc 

not ready at the appropriate time. With proper foresight and 

balanced support, useful molecular techniques can be devised and drawn into classical breeding, just as other disciplines (e.g., quantitative genetics) were incorporated earlier. 

Likelihood of enhanced returns from technical integration seems more than sufficient to attract support within 

individual organizations. Prospects for lower overall costs 

may foster formation and maintenance of centers of excellence via research and development consortia (Griffin 

1995). Prudent organization and astute management of 

cooperative endeavors could hasten research, development, and application. Open, flexible management is essential so that the pursuit of intellectual property rights 

by individual organizations does not interfere with technical integration or existing cooperative tree improvement 

programs. Hopefully, involved organizations will realize 

that overly aggressive pursuit of intellectual property 

rights can jeopardize collaborative efforts, and that costs 

of developing molecular techniques, properly integrating 

them, and covering risks of failure are beyond the capabilities of most individual organizations. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Integration of Molecular and Classical Genetics: A Synergistic Approach to Tree Improvement 

Wise investments coupled with collaboration within and 

among organizations are necessary to foster productive 

integration of genetic technologies. Classical and molecular genetics have many parallels and melding the best of 

each into a new synergistic system depends on mutual 

support. Only by increased commitment to overall tree 

improvement can both enterprises contribute to better 

management of and greater returns from forest tree resources. Individuals working with model species, such as 

poplars, can contribute much toward these important ends. 

Acknowledgments 

Written contributions to this chapter were partially supported by Lockheed Martin Energy Research Corporation 

for the U.S. Department of Energy under Contract #DEAC05-960R22464. The submitted manuscript has been 

authorized by a contractor of the U.S. Government under 

Contract #DE-AC05-960R22464. Accordingly, the U.S. 

Government retains a nonexclusive, royalty-free license to 

publish or reproduce the published form of this contribution, or allow others to do so, for U .5. Government purposes. In addition, the authors thank Bail ian Li, R.J. 

Rousseau, and B.J. Stanton for their helpful thoughts on 

breeding strategies. 

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235 
Chapter 3D 

Application of Tissue Culture Systems for 

Commercial Plant Production1 

Kathryn A. Louis and LoriAnn E. Eils 

Introduction 

Tissue culture propagation or micropropagation in the 

commercial production of Populus species for large scale 

clonal plantings has limited use due primarily to the high 

cost per plantlet, especially when compared to species that 

are readily cloned using cutting propagation. Howe,·er, 

two more practical applications of tissue culture propagation of Populus species are: 1) propagation of those species 

that do not readily propagate via other less costly methods (e.g., aspen and hybrid aspen); and 2) multiplying stock 

plants of newly developed or released genotypes with limited availability. This chapter discusses methods used to 

clone Populus species including: 1) traditional shoot-tip/ 

axillary bud culture; 2) recutting or hedging mini-plants; 

3) root suckering in vitro and in situ; and 4) leaf micro-cross 

section (MCS) technology. 

Traditional Shoot-Tip/ Axillary 

Bud Culture 

An excellent bibliography about tissue culture and cell 

culture of Populus species was compiled by the USDA Forest Service (Ostry and Ward 1991). Readers are encouraged to review papers listed in that document. A brief 

review of the method used at Minn vitro, Inc. is described 

below and illustrated in figure 1. 

' Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

236 

Stock plants, which provide explants, are obtained as 

small plants or dormant branches. Plants are grown indoors under a cool-white, fluorescent (CWF) photoperiod 

of 16 h until new shoot growth occurs. Dormant branches 

are stripped of existing leaves, and proximal branch ends 

are recut and placed in water under a 16 h photoperiod. 

Branch ends are recut every 2 to 3 days at the time of water replacement. After 2 to 4 weeks, new forced growth 

occurs. In Minnesota, dormant branches can be successfully forced beginning in March through normal bud break 

(early May) and again in August through early November. Explant material is not usually field collected because 

of difficulties eliminating fungal spores and other contaminants on field-grown material. 

To initiate Populus species cultures, shoot-tip stem cuttings (approximately 2.5 to 7.5 em long) are collected from 

actively growing plants or forced branches. Existing leaves 

are removed and stems with terminal and axillary buds 

are surface disinfested using a standard commercial bleach 

~~, ~·iJ 

LABORATORY \I I 

ttHJ 4w,Ks 

~ \ "----~ .· 

4 WEEKS 

~ 

Figure 1. Representation of a traditional shoot-tip/axillary 

bud culture method used at Minn vitro, Inc. 
solution (10 percent for 10 min), followed by 3 sterile water rinses. Explants are aseptically cultured on Murashige 

and Skoog (MS) (1962) basa l medium supplemented with 

3 percent sucrose, 0.1 to 0.5 mgl I benzyladenine (BA), and 

5 g/1 agar with a preautoclave pH of 5.75. The culture cycle 

is 4 weeks with a CWF photoperiod of 16 h. High BAconcentrations are used for multiple shoot production during 

the proliferation cycles, followed by reduced BA concentrations for shoot development before harvest. Although 

most literature reports the use of Woody Plant Medium 

(WPM) (Lloyd and McCown 1980) as the basal medium, 

we have found that MS medium prod uces superior 

microshoots (data not shown). During the process of harvesting microshoots from the culture vessel, small 

microshoots (less than 2.5 em) and proliferating clusters 

are transferred to fresh medium for later harvest. 

For ex vitro rooting, microshoots that are 2.5 em or 

longer are h arvested a nd placed in a standard 288 seed 

germination plug covered tray (trimmed to fit into a 

standard 1020 flat). The plug tray and fl at are covered 

with a clea r dome and placed under a CWF 16 h photoperiod. The plug tray is filled with a rooting medium 

composed of peat:perlite:vermiculite (1:1:1 ). All preformed 

in vitro roots are removed before ex vitro rooting. (In our 

experience, in vitro formed roots of Populus species did not 

survive after transplanting into peat-based rooting medium. New adventitious roots developed at a slower rate 

than from microshoots without p reformed roots.) Visible 

roots usually form within 10 days. After 3 weeks of rooting, we recommend tha t weekly fertilization begin at 200 

ppm nitrogen, using a 20:20:20 (N:P:K) formulation. At approximately 4 weeks, acclimation is accomplished by propping open the clear dome slightly for the first day, then 

increasing the opening each day, until day 7 when the dome 

is completely removed. Plantlets are then transplanted to 

larger containers and placed in the greenhouse or nursery 

bed with 70 percent shade for the first few days. The 

amount of shade is gradually decreased over a 7-day period, then fertilizer applications can be doubled. 

This traditional shoot-tip I axillary bud culture procedure 

is based on a plant biology that allows existing shoot tips 

and axillary buds to develop and continue initiation of new 

shoot tips and axillary buds. These shoot tips and axillary 

buds then elonga te to produce microshoots. This cycle can 

be repeated indefinitely, provided that transfers occur in a 

timely manner. Reducing the detrimental effects of systemic bacteria that can become evident over time can be 

accomplished by a more rapid transfer cycle (every 2 to 3 

weeks), using only shoot tips (1 to 2 em), and I or the addition of an tibiotics to the medium. (Based on research by 

Young et a!. (1984) and our in-house research, we found 

that a combination of 25 mg/1 cefotaxime, 25 mgl l tetracycline, and 6 mg/1 rifampicin will successfully suppress 

bacterial growth in most Populus species cultures.) Other 

problems are low tooting rates and poor acclimation for 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Application of Tissue Culture Systems for Commercial Plant Production 

some genotypes. Our overall success rate has been that approximately one-third of the genotypes are readily cloned, 

one-third of the genotypes are cloned with difficulty, and 

one-third of the genotypes are not amenable to this method. 

Recutting or Hedging of Plantlets 

Although this method is not an in vitro method, it is 

worth mentioning because it is often very successful when 

MEDIA 'L-------------'-------' 

EXCHANGE 

c 

• 

. 

. 

. 

MICROSECTION BUDS SHOOTS ROOTS 

Figure 2. Schematic representation of Micro-Cross 

Section (MCS) Technology (courtesy of Minn 

vitro, Inc.). A) Leaf tissue is placed on a tape 

carrier and sliced into strips. B) The leaf tape 

strip is placed on a support substrate. The 

culture medium is liquid (no agar) and the 

culture vessel has flow-through medium exchange as required. No physical transfer of the 

explant is required. C) Leaf explant initiates and 

develops adventitious buds that form shoots, 

followed by rooting. For details of this method, 

see Louis and Eils (1994). 

237 
Section V Biotechnological Applications 

tissue cultured plantlets are used. Plantlets that arc produced as previously described can be grown in a greenhouse or growth chamber until their main shoots are 8 to 

10 em in height. Cuttings are then collected from these 

mini-stock plants and rooted as previously d escribed. The 

remaining portions of the mini-stock plants continue to 

grow via axillary shoots, which can also be used as minicuttings once the axillary shoots are 6 to 8 em in length. 

Mini-cuttings produced in this manner usually root at a 

higher percentage than those from tissue culture (data not 

shown). A likely reason for this rooting increase is that 

mini-cuttings have a more developed cuticle. Thus, although mini-cuttings are less fragile, they apparently maintain the juve nile characteristics of tiss ue-c uI tu red 

microcuttings. This hedging me thod can be successfully 

performed for several flushes. 

Root Suckering 

Cloning Populus species using root suckers has been 

successful for species that naturally propagate in this manner (e.g., P. tremuloides). The field I greenhouse method begins with digging up roots (1 to 3 em in diameter and 15 to 

20 em in length). These roots are cleaned with soa p and 

water, followed by a bleach trea tment (full-strength for 10 

min), and a tap-water rinse. Root segments are then buried in sand and maintained under moist conditions. Within 

2 to 4 weeks, shoots develop from preformed and adventitious buds within the roots. When the shoots are 4 to 6 

em in length, they are removed from the parent root segment and trea ted as mini-cuttings (as previously described ). Papers by Schier (1974, 1976, 1981) provide more 

detail regarding preformed and adventitious root-derived 

buds. In our work, this method seems most successful in 

the spring and is apparently limited by the inherent 

suckering ability of the parent plant. A major problem is 

correctly identifying which roots belong to what tree. This 

p roblem can be alleviated by cloning the identified plant 

with the usual methods (e.g., cuttings, tissue culture, etc.) 

then growing the cloned plants in large containers until 

roots of sufficient size are obtained. 

An in vitro version of this method was developed at 

the University of Minnesota (Hanson e t al. 1992; Louis 

et al. 1992a, 1992b) in which in vitro growing shoots 

were treated to induce roots (i.e., BA was removed from 

the medium). These in vitro roots were then cut into 0.5 

em or longer segments and returned to culture. Adventitious shoots were initiated and developed from the root 

explants cultured on MS medium supplemented with 0.01 

to 1.0 mg /1 thidiazuron (TDZ). Our attempts to produce 

large qualities of roots by growing roots without stem tissue were unsuccessful. Although in situ root suckering 

238 

has been used to clone some aspen genotypes, to our 

knowled ge, the use of in vitro root suckering as a commercia l method to clone plants has not yet occurred. 

Micro-cross Section Technology 

Micro-cross section (MCS) Technology is a method 

used to clone plants based on adventitious bud initiation and d evelopment from leaf tissue. This method was 

initia lly d eveloped by esearch~rs at the University of 

Minnesota (Lee-Stadelmann et al. 1989). Very small (400 

J.lm to 3 mm) leaf segments are placed onto MS medium 

supplemented with 3 percent sucrose, 0.8 mg/1 BA, and 

0.01 mg/1 a-naphthaleneacetic acid (NAA). After 4 weeks, 

adventitious buds begin to form. For microshoot development, the explant is transferred to medium supplemented 

with a reduced BA concentration (0.1 mg / 1) and without 

AA. As microshoots develop, they are harvested and 

rooted as previously described. Because very small pieces 

of lea f tissue can be used, a large number of explants can 

be generated for adventitious shoot production. Thus, a 

proliferation stage is hardly needed and a large number of 

plants can be produced in a short time. 

The uniform size and shape of the explants makes this 

process amenable to robotics. Continued refinement of this 

technique along with development of automated handling 

equipment would probably close the cost gap be tween 

tissue culture propagation and seedling propagation. Additionally, the semi-solid medium can be replaced with a. 

liquid medium if a support substrate is used . Five "offthe-shelf" substrates can be used to replace agar: 1) cotton balls; 2) cotton cosmetic rounds; 3) Grodan® rock wool; 

4) lsolite® soil amendment; and 5) Sorbarod® cigarette filters. This has led to the envisioned MCS Technology illustrated in figure 2. The results of our continued effort to 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
develop MCS Technology are summarized in a TAPPI Biological Symposium paper (Louis and Eils 1994). Forty-five 

different Populus genotypes were tested, of which P. 

tremuloides x P. tremula (hybrid aspen) genotypes were the 

most responsive. These results are most promising because 

hybrid aspen plants do not readily propagate using other 

asexual methods. 

The first commercial trial of MCS Technology was a 10-

times scale-up of our research size (i.e., 10 times our standard 100 micro-cross sections), which was replicated for a 

total of 2,000 micro-cross sections. This scale-up resulted 

in reduced microshoot numbers per leaf section and extensive fungal growth during acclimation (data not 

shown). This reduced production was likely caused by 

using leaves of a less than optimal developmental stage. 

Fungal growth during acclimation was apparently caused 

by the residual medium in the liquid support substrates. 

Even though the substrates were flushed with water before acclimation, adequate sucrose/nutrients apparently 

remained to support fungal growth. 

Discussion 

Populus species tissue culture propagation can be classified as either preformed (terminal or axillary) or ad ventitious meristem culture methods. Preformed meristem 

culture is the traditional method in which shoot tips with 

existing meristems are grown in an environment that enhances the production of more meristems at a much faster 

rate than occurs on a whole-plant basis. 

Adventitious bud initiation and subsequent development 

of microshoots captures the plant's totipotent biology. For 

example, using in vitro root suckering and MCS Technology, 

a cell, or a small group of cells, divides and differentiates 

into a new meristem that subsequently develops into a 

microshoot. Adventitious bud initiation has been used only 

on a research basis at our laboratory. MCS Technology has 

shown the greatest potential for producing hybrid aspen at 

a lower cost than traditional shoot-tip culture. 

The most commercially successful method is the traditional shoot-tip I axillary bud method. We have used this 

method to mass produce 45,000 hybrid aspen for a pilot 

field planting and 10,000 aspen for horticultural use. We 

have also used traditional.shoot-tip culture to clone unique 

or rare plants to increase stock plant numbers for cutting 

propagation. 

Generally, regardless of the tissue culture propagation 

method used, differences occur among Populus species and 

genotypes within a species. Our results have followed the 

one-third:one-third:one-third "rule" of success; approximately one-third worked very well, one-third worked 

moderately well, and one-third did not work at all. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Application of Tissue Culture Systems for Commercial Plant Production 

Acknowledgments 

This research was supported in part by the U.S. Department of Agriculture under Grant No. SBIR, Phase II 92-

33610 and by funding approved by the Minnesota 

Legislature ML 1991, Chapter 254, Art. 1, Sec. 14, Subd. 7g 

as recommended by the Legislative Commission on Minnesota Resources from the Minnesota Future Resources 

Fund. 

The authors thank M. Ostry, D. Skilling, K. Ward, and B. 

Bucciarelli (USDA Forest Service); N. Anderson, W. 

Hackett, W. Johnson, and C. Mohn (University of Minnesota); G. Wyckoff and B. Li (University of Minnesota, 

Aspen-Larch Cooperative); B. Pajala (Minnesota Dept. of 

Natural Resources); R. Schantz-Hansen, R. Settergren, L. 

Hubbel, M. Fasteland, and J. Eaton (Potlatch Corporation); 

J. McCoy and L.C. Peterson (Blandin Paper Company); K. 

Wearstler and C. Wierman (Boise Cascade); D. Ostlie (Energy Performance Systems); and G. Larson and D. Langseth 

. (WesMin RC&D) for their generosity in supplying plant 

material for our studies, and G. Betts and B. Berguson 

(NRRI) for technical and engineering assistance. 

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and roots. Can. J. For. Res. 6: 253-261. 

Young, P.M.; Hutchins, A.S.; Canfield, M.L. 1984. Use of 

antibiotics to control bacteria in shoot cultures of woody 

plants. Plant Science Letters. 34: 203-209. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Chapter31 

Economics of Producing Populus Biomass for 

Energy and Fiber Systems1 

"WW Charles H. Strauss and Stephen C. Grado 

~ 

Introduction 

The economics of producing Populus biomass is a fairly 

uncomplicated concept. Some set of inputs, usually land, 

labor, and capital, is required in the biomass output production. Tune is an integral part of this process, and the 

use of these inputs over time must be considered an investment proposition. Variability in the types and amounts of 

inputs, length of time, and quantity of output complicates 

the analysis. Much of the information presented by this book 

should be factored into the economic analysis of Populus. 

This chapter provides a general model of Populus production and its use in ethanol manufacture. Major components of the model were developed from research 

sponsored by the U.S. Department of Energy's Short Rotation Woody Crop Program (SRWCP). The model considers the financial and energy costs of producing Populus 

biomass at the plantation sites, and the added costs of harvesting, transporting, and storing the biomass. The resulting raw material costs apply to the conversion of Populus 

biomass for either energy or fiber products. 

An expanded model on the conversion of Populus biomass to ethanol is in the final section of this chapter and includes all of the production costs, from the plantation site to 

the final product. Overall, these models are more important in identifying key components of the production process than they are in specifying particular costs. They 

underscore the critical dimensions of the process and identify where further research and development is needed. 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

Basic Design for Populus 

Plantations 

Populus models by Strauss and Wright (1990) and Strauss 

a:'d Grado (1992) were developed to consider a plantation system established on high quality agricultural sites 

~t a densi~ of 2,100 trees/ha (approximately 2.2 m2 spacmg). Rotation length was from 5 to 8 years, with 2 or 3 

rotations ~ticipated from any given planting. The opti-

~um rotation was selected on a least-cost pasis using a 

discounted cash-flow analysis (Strauss et al. 1990). 

Similar to agricultural row crops, plantations were established with a fall and spring planting site preparation 

and a spring planting of the poplar cuttings. At the onset 

of the fall season, the site received a total-kill herbicide 

and mowing operation to remove old field vegetation, followed by plowing (table 1). Lime was added at this time, 

depending on soil acidity. In the spring, the soil was harrowed, with a pre-emergent herbicide to counter residual 

weeds. To supply an adequate nutrient base for tree 

growth, phosphorus and potassium were added before 

planting. However, nitrogen was not applied until the third 

and fifth growing seasons to avoid augmenting weed 

growth in the first 2 growing seasons. Machine planting 

of the poplar cuttings was assumed within the cost structure of the model. 

Additional herbicides were scheduled at the beginning 

of the first 2 growing seasons to counter potential weed 

growth. Protection from insect and canker damage was 

thr~ugh a biennial insecticide/fungicide spray program. 

This was a preventive cost against infestations, such as cottonwood leaf beetle (Chrysomela scripta) and Septaria canker. 

Annual charges were assessed for land rent, property 

taxes, and managerial supervision of the production effort. These requirements represented specific inputs to the 

production function. 

Harvesting and transportation strategies were developed from previous studies (Stokes et al. 1986; Strauss et 

241 
Section V Biotechnological Applications 

Table 1. Financial and energy costs for the establishment and maintenance of SRWC Populus plantations. 

Costs in U.S. dollars for 1995. 

Establishment 

Fall/spring herbicides (3.3 kg/ha) 

Mowing/brushing 

Plowing/harrowing 

Liming (1.0 Mglha) 

Fertilization (60 kg/ha each P&K) 

Planting (2, 1 00 cutting/ha) 

Summer herbicides, year 1 and 22 (2.2 kg/ha) 

Total establishment costs 

Maintenance 

Insecticide/fungicide (1.6 kg/ha/appl.) 

Fertilization (120 kg of N/ha/appl.) 

Land rent 

Land taxes 

Managerial 

Approximate average annual maintenance costs 

1 Mega joule per hectare. 2 Year 2 costs discounted at 5%. 

al. 1988; Stuart et al. 1985). Harvesting equipment was 

designed for the small-diameter, closely--spaced SRWC 

plantations. After harvest, the biomass was either chipped 

on-site or at the processing site. Transportation used was 

either tractor-trailers for delivery of chipped material or 

flatbed units for delivery of bundled tree stems. 

Financial Cost Structure 

The cost for each operation was developed from the 

SRWCP data base (table 1). Charges for contracting for 

the establishment and operation of commercial-sized plantations compared favorably to previous models of SRWC 

systems (Lothner et al. 1985; Per lack et al. 1986; Strauss et 

al. 1988). Cost differences within particular operations 

were attributed to the type of equipment and amount of 

material used. All establishment and maintenance costs 

reflected an agricultural site with good aspect and soil 

quality. 

Establishment totaled $682/ha, including the discounted 

charge for second-year herbicides (table 1). Herbicide application represented 51 percent of establishment costs; the 

materials constituted over 90 percent of this expense. Land 

preparation (mowing /brushing, plowing /harrowing) and 

planting were 34 percent of the establishment costs; 54 

percent of this expense was tied to the poplar cuttings. 

First-year liming and fertilization was the final 15 percent. 

242 

$/ha 

210 

24 

56 

49 

51 

152 

140 

682 

36 

42 

102 

16 

42 

199 

1,760 

748 

2,547 

289 

1,729 

2,156 

1,277 

10,506 

878 

7,593 

49,820 

7,620 

261 

61,936 

Maintenance included the application of nitrogen during 

the third and fifth growing season and the use of insecticides/ fungicides on an alternate year basis. In addition, an 

annual managerial cost was assessed for administrative and 

operational needs (table 1). Land rent represented the opportunity costs, or annual net return, of a good corn production site (7.8 Mg of grain/ha/yr). The capitalized net 

value of the site at a 5 percent real rate of return was 

$2,040 /ha. This was consistent with the land values used 

in previous SRWC studies and was 20 percent above the 

U.S. average for farm real estate (USDA ERS 1988). Annual property taxes were 0.75 percent of this land value. 

Energy Cost Structure 

The energy accounting system for the proposed SRWC 

plantations was patterned after other agricultural cropping 

systems (Pimentel1980; Roller et al. 1980) and related studies of commercial-sized SRWC plantations (Strauss et al. 

1989; Zavitikovski 1979). Equipment costs included the 

energy: 1) embodied in the equipment's basic materials; 

2) employed in equipment fabrication; and 3) embodied 

in repair parts. Net energy consumption over an equipment unit's life span was 82 percent of its total energy 

(Pimentel 1980). The division of a unit's net energy consumption by its life span, in hours, provided an hourly 

equipment cost. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Machinery costs for any given task were the product of 

operating times and hourly equipment costs. Energy costs 

for fuel, related material inputs, and labor were added to 

their respective equipment tasks. Material input costs included the energy embodied in materials such as fertilizer, and the added energy required in its manufacture. 

Labor's energy cost was based on a net analysis of the energy required by humans as agricultural laborers (Fluck 

1981) and was 75 MJ /h (75 mega joule per hour). Although 

this charge was higher than previous estimates, the energy cost of labor in most operations was small. 

The energy cost of land was also organized as an opportunity cost, based upon the net energy secured from corn 

production (49,820 MJ /ha/yr). This energy value compared favorably to previous estimates of corn production 

in several U .5. regions (Pimentel1980 ). The energy charge 

for property taxes was based upon the energy to financial 

cost ratio of land rent and the cost of taxes. 

The energy cost of establishing SRWC plantations was 

10,506 MJ /ha (table 1). Site preparation and planting was 

52 percent of this expense, herbicides were 29 percent, and 

fertilization/liming was the final 19 percent. Site preparation and planting had proportionally larger energy costs 

than financial costs due to the higher energy charges for 

machinery and poplar cuttings. Annual maintenance was 

substantial because of the energy budgeted for land rent 

and taxes. The energy cost of poplar cuttings also reflected 

th~ land's energy potential, with 40 percent of the cutting 

cost tied to land use. 

Plantation Yields and Unit Costs 

A proposed yield was developed from the SRWCP data 

sets (Strauss and Wright 1990). The 2,100 trees/ha plantation was projected to have a maximum mean annual increment of 16 metric tons, oven dry, per hectare per year 

(Mg(OD)/ha/yr) by the sixth year. . 

Unit costs were estimated on a financial and energy basis 

using an investment analysis approach developed for SRWC 

plantations (Strauss et al. 1990). Under ~his approa~, ~it 

costs are estimated as a function of the discounted fmancial 

costs (or energy costs) of establishing and maintaining the 

plantation and the discounted plantation yields. 

Establishment ($/ha) + Maintenance ($/ha) 

discounted discounted Unit cost 

($/Mg(OD}}=-----------

Yield (Mg(OD)/ha) 

discounted 

To avoid the time bias associated with longer rotations, 

the individual rotation lengths were analyzed as a perUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Economics of Producing Populus Biomass for Energy and Fiber Systems 

petual series. A 5 percent real rate of return was used 

throughout the analysis, with the optimum rotation length 

selected on a least-cost basis. 

Plantation Costs 

Analyses of a two-rotation system placed the least-cost 

solutions at the sixth year, with unit costs of $19.06/ 

Mg(OD)and 4,381 MJ/Mg(OD)(tables2and 3). If the plantations could sustain 3 rotations per planting, the leastcost solutions were still at the sixth year, with costs of 

$17.71 /Mg(OD) and 4,360 MJ /Mg(OD). These reductions 

were attributed to the extended prorate of the establishment costs over an additional rotation. However, from an 

energy cost standpoint, the reduction was small due to the 

large, fixed expense ~f land in the production model. 

Stratification of the financial costs by major inputs 

(equipment, fuel, materials, labor, and land) showed that 

the plantation operations were land intensive, with 44 percent of the plantation costs tied to land, 31 percent to material inputs, 18 percent to labor, and 8 percent to 

equipment and fuel (table 2). Basically, the opportunity 

cost of good quality land dominated the cash-flow aspects 

of the biomass production system. 

On an energy cost basis, the plantations were again land 

intensive, with 93 percent of the costs originating from land 

(table 3). As previously identified, this cost represented 

the net energy gain available to land from corn production. Although this amount of energy was not actually used 

by the system, it did represent a minimum energy payment to land from the SRWC system. As for actual energy 

used by the system, the materials used in establishment, 

protection from insects, etc., and fertilization were the major expenses, representing 74 percent of these energy costs. 

Fuel, equipment, and labor were the remaining energy 

inputs (table 3). 

Total Delivered Cost 

Unit costs for the harvesting/ transportation function 

were developed from auxiliary studies of these operations 

and were added to the plantation costs on a current, 

nondiscounted basis (Stokes et al. 1986; Strauss et al. 1988; 

Stuart et al. 1985). These amounted to $24/Mg(OD) and 

977 MJ /Mg(OD}, based upon a SRWC-designed harvesting system and a 40-km one-way truck haul. A 15 percent 

loss of material was assessed against the harvesting/ transportation function (tables 2 and 3). 

The total delivered cost on a financial basis, including 

harvest/ transportation and a 15 percent material loss, was 

$46.42/Mg(OD) (table 2). Nearly 60 percent of the total cost 

originated from harvesting/ transportation and material loss. 

In reviewing the entire system, equipment was the primary 

input, representing 31 percent of all costs (table 2). 

243 
..._ 

Section V Biotechnological Applications ...-

~ 

Table 2. Financial costs by input type and operation for SRWC Populus biomass. Costs in U.S. dollars for 1995. ...-

~ 

Unit cost Percentage of cost by input 

Operation ($/Mg(OD))1 Equipment Fuel Materials Labor Land ~ 

Establishment 5.47 13.3 6.8 72.1 7.6 0.2 ...s) 

lnsect./fung. 1.30 13.3 3.3 81.4 1.9 0.0 .,g 

...._; 

Fertilization, 1.02 11.4 2.9 81.0 4.7 0.0 

land rent and V:liil 

taxes 8.30 0.0 .0.0 0.0 0.0 100.0 ~ 

Managerial 2.97 0.0 0.0 0.0 100.0 0.0 ~ 

Plantation ....., operations2 19.06 5.3 2.3 30.6 18.1 43.7 

~ 

Harvesting and 

Vlf) transportation3 24.00 55.0 22.1 0.0 22.9 0.0 

Material loss \;8) 

(15%) 3.36 5.3 2.3 30.6 18.1 43.7 -.81 

Total delivered \31 

cost 46.42 31.0 12.5 14.8 20.6 21.1 ~ 

1 Dollars per metric ton, oven dried. 2 Operation costs compounded to the end of the rotation at an interest rate of .05. ....., 

3 Strauss et al. (1988), revised for inflation to 1995. ~ 

-..:1 

Table 3. Energy costs by input type and operation for SRWC Populus biomass. ~ 

~ 

Unit cost Percentage of cost by input 

Operation ($/Mg(OD))' Equipment Fuel Materials Labor Land ~ 

Establishment 84.1 9.4 48.2 34.1 2.3 6.0 \;JJ/1 

olnsect./fung. 31.9 8.7 ° 19.0 72.0 0.3 0.0 v.sl 

Fertilization 183.3 1.2 2.4 96.3 0.1 0.0 .... 

~ 

Land rent and 

taxes 4,069.9 0.0 0.0 0.0 0.0 100.0 ..... 

Managerial 11.4 0.0 0.0 0.0 100.0 0.0 ...-

Plantation ~ 

operations2 4,380.6 0.3 1.2 5.2 0.3 93.0 ~ 

Harvesting and ~ 

transportation3 976.9 15.1 82.3 0.0 2.6 0.0 ..., Material 

loss (15%) 773.0 0.3 1.2 5.2 0.3 93.0 

V:liil 

\:!ill 

Total delivered 

cost 6,130.5 2.7 14.1 4.4 0.7 78.2 ~ 

1 Mega joules per metric ton, oven dried. ...- 2 Operation costs compounded to the end of the rotation at an interest rate of .05. 3 Strauss et al. (1988), with revised labor energy costs. .... 

.....-

\/Q;il 

244 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 0 

~ 

'vel 
The total energy cost was 6,130 MJ /Mg(OD) (table 3). 

As a major input, land was 78 percent of the energy cost. 

Exclusive of land, 65 percent of the energy was for fuel, 20 

percent for materials, 12 percent for equipment, and 3 percent for labor. 

Sensitivity Analysis 

A review of the financial impact of various inputs on 

the total delivered cost of SRWC biomass showed equipment having the greatest impact, with a 10 percent change 

in its cost causing a 3.2 percent change in the delivered 

cost of biomass (figure 1). Again, over 90 percent of the 

equipment costs originated from the harvest/ transportation. As expected, inputs representing a s~aller portion 

of total cost had a lesser affect on the delivered cost of 

biomass. 

Plantation yields had the greatest affect on unit costs. A 

10 percent change in output would shift delivered costs 

by approximately 5 percent. This key relationship underscored the importance of maintaining or exceeding the targeted production level of 16 Mg(OD)/ha/yr. 

Ethanol ·Manufacturing Inventory 

Control Model 

SRWC research has focused on genetic, silvicultural, and 

economic evaluations of plantation strategies. Research on 

the conversion of woody biomass into energy products 

represents another major endeavor. Inherent to the linkage between raw materials and final products is the need 

to manage the inventories of inputs and outputs. 

Forests are unique because trees represent the capital 

input and product output. As a product, they represent a 

financial commitment to inventory. Moreover, trees are 

perishable and may be limited by the duration of the harvest period. The storage of harvested biomass involves 

additional capital and operating costs, along with fluctuation in the inventory over time. Overall, matching the 

cyclical and finite nature of plantations with the steady 

state demands of a .processing plant is a complex inventory problem. 

General Structure of an Inventory Control 

Model 

An inventory control model was developed by Grado 

and Strauss (1993) to determine the least costly approach 

for supplying biomass to a processing plant. A dynamic 

programming format was used to evaluate the plantation, 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Economics of Producing Populus Biomass for Energy and Fiber Systems 

Cl) 

~ 

E 

0 

:c 

0 

~ u 

"0 

! 

.~ a; 

"C 

g 

Q) 

C) 

c 

ns 

.r; 

u 

E 

CD 

e 

CD 

a. 

6.0 

5.0 

4.0 

3.0 

2.0 

1.0 

0.0 

·1.0 1-..-~-=----

-4.0 

-5.0 

·6.0 L_ ___ .J..._ ___ ...L... ___ _.__ ___ _ 

-10.0 -5.0 0.0 5.0 10.0 

Percent change to input costs and plantation yields 

Figure 1. Sensitivity of total delivered cost of Populus 

biomass to changes of input costs and 

plantation yields. 

harvest, and manufacturing components of an ethanol 

supply system. 

The plantation component was based upon Populus 

grown under 4 to 8 year rotations using a strategy similar 

to that proposed by Strauss and Grado (1992). Harvesting 

covered a 6-month period that followed the growing season, and employed technologies similar to Stokes et al. 

(1986) and Stuart et al. (1985). The ethanol manufacturing 

process was based upon enzymatic hydrolysis of woody 

biomass as presented by Wright (1989) and Bergeron et al. 

(1989). The proposed facility could process 10,000 Mg(OD) 

of biomass per month to meet a maximum output of just 

over 3 million liters per month. Each component of the 

model generated particular inventories; various inventories were held during any given time period. 

The model identified all costs associated with the plantation, harvest, and manufacturing components. Each component used an accounting format similar to that identified 

by Strauss and Grado (1992). Establishment and maintenance costs for the plantation were added as capitalized 

expenses to the harvest and transport costs, which were 

then added to the manufacturing costs. All costs are reported in U.S. dollars for 1995 on a per metric ton, oven 

dry (Mg(OD)), or per liter (L) basis. 

Model solutions were for an annual operating cycle and 

provided: 1) the minimum cost combination for plantation, harvest, and manufacturing strategies; 2) the harvesting schedule within a year; 3) a recommended inventory 

policy for standing trees, harvested raw materials, and final product; and 4) net comparisons of existing supplies 

to operating demands for each component. 

245 
Section V Biotechnological Applications 

Model Results and Cost Comparisons 

The use of inventory control policies as solutions to the 

model provided more finished product at a lower unit cost 

under any given rotation. Usually, the optimum harvesting policy provided substantial cost savings for any given 

rotation. Although longer rotations could provide more 

raw material and related finished product, there was an 

increase in the inventories of raw material and product 

and in raw material deterioration (table 4). Inventory control reduced these expenses for any given rotation. For 

example, the least costly solution for a 6-year rotation was 

$0.410/L (table 4), which was 38 percent lower than the 

highest cost solution for the same rotation. · 

Among all rotation strategies, the least costly solution 

was 6 years (table 4). This was consistent with the solution 

found in the previous model. However, note that this cost 

was not significantly lower than for the 7- or 8-year rotations. The highest cost items within the system were the 

manufacturing process, harvest/ transportation, lost sales 

(penalty costs assessed when the supply of final product 

could not meet market demand), and plantation maintenance. The lowest cost items within the system were plantation establishment, raw material and final product 

storage, and storage deterioration. 

The model solution provided storage policies for standing trees, harvested materials, and finished products, along 

with a plantation harvest schedule. Average storage time 

for harvested biomass over an operating year was 2.5 

months, with nearly 16,500 Mg(OD)/mo held in storage. 

Ethanol inventories were influenced by monthly manufacturing, final product demand, and the inventory policy. 

Storage of the final product was less costly than for harvested biomass (table 4). For the 6-year rotation, the average storage time for ethanol was only 1 month, with 1.9 

million liters held per month. The upper storage level for 

ethanol was 3.0 million liters, which also represented a 

minimum long-term storage plant capacity. 

The objective of the inventory policies was to coordinate 

plantation inventories, wood chip storage, and ethanol storage. When viewed in the context of the dynamic market 

demand for the finished product, these policies were successful in establishing efficient harvesting schedules and in 

lowering the total costs of the production system. 

Conclusions 

What have we learned from over 15 years of SRWC research? First, if we only consider the production costs at 

the plantation site, land is the dominant factor. This is particularly true in cases where plantations are placed on productive soils. Land rent and taxes can represent from 40 to 

SO percent of the preharvest biomass cost. The cost of using good quality land is an alternative net return available 

from other agricultural pursuits. For SRWC systems to 

compete for land, comparable or higher net returns must 

Table 4. Financial costs for an ethanol manufacturing system under alternate Populus rotation lengths. Cost in U.S. 

dollars for 1995. 

Costs ($/liter) 

Rotation length (years) 

4 5 6 7 8 

Plantation 

Establishment 0.015 0.013 0.010 0.011 0.010 

Maintenance 0.068 0.058 0.048 0.046 0.046 

Harvest 

Harvest/transport 0.079 0.075 0.074 0.074 0.074 

Storage 

Wood chips 0.009 0.013 0.017 0.020 0.020 

Penalty 

Deterioration loss 0.002 0.004 0.008 0.009 0.009 

Manufacturing 

Processing 

Ethanol 0.242 0.242 0.242 0.242 0.242 

Storage 

Ethanol 0.007 0.007 0.010 0.010 0.010 

Penalty 

Market loss 0.140 0.060 0.001 0.000 0.000 

Total 0.562 0.472 0.410 0.412 0.411 

246 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
'\J:V 

be provided by woody biomass. Lower priced, marginal 

lands would reduce these costs, but would probably result in lower yields. · 

Second, the best means for lowering the unit cost of 

woody biomass at the plantation is to increase plantation 

yields. Although 16 Mg(OD)/ha/yr was suggested as an 

average annual yield, sustaining this net average may be 

difficult. If this annual yield was reduced by 10 percent, 

preharvest costs would increase by over 12 percent, thereby 

increasing delivered costs by over 5 percent. A comparison of small-plot research yields to commercial field yields 

by Hansen (1988) suggested that the current field potential for SRWC plantations may be more in the range of 10 

to 12 Mg(OD)/ha/yr. Further increases were considered 

possible, but these future gains will depend on cultural 

and breeding research. Higher yields would also require a 

more precise matching of clonal hybrids to growing sites 

and the successful implementation of cultural strategies 

on these sites. 

Biotechnology will largely determine the future competitive position of Populus. Research efforts should be 

directed to 2 key areas; growth and yield, and maintenance costs. One method for decreasing costs is developing genetically superior stock with higher yields and lower 

maintenance requirements. Higher yields are achievable 

through increased survival rates of planting stock and the 

overa.ll plantation, improved coppicing between rotations, 

and increased growth rates. Associated with these enhancements, would be the adaptation of Populus to lower 

quality sites, thereby reducing land costs. Further cost reductions are possible by developing varieties that require 

less maintenance in terms of site preparation, soil amendments, herbicides, and pesticide sprays. Potentially, these 

improvements might involve certain compromises such 

as faster-growing varieties that require higher maintenance costs. Under final analysis, the most cost-effective 

opportunities for biotechnology will be those that address 

yield and the major cost components of the production 

equation. 

Separate from the biological forces of growing Populus 

are the technical factors required in the harvest, transport, 

and storage of this raw material. These items more than 

doubled the cost of the delivered and stored raw material. 

Woody biomass is not a convenient material to handle or 

move and, as such, further innovations are needed. Associated, many harvesting and transportation strategies are 

adaptations of technologies used in domestic forests. Although further cost reductions are possible, they may require the development of commercial SRWC markets; this 

precondition could stalemate the development process. 

The manufacture of woody biomass into a liquid fuel 

introduced another set of production costs. As identified 

in the expanded model, over 50 percent of the final output 

cost was tied to the conversion process, with the raw material at the plantation site representing less than 15 perUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Economics of Producing Populus Biomass for Energy and Fiber Systems 

cent of the total production costs. In the context of today's 

energy markets, the use of woody biomass for either liquid fuels or direct-bum electric generation cannot compete with abundant and relatively cheap fossil fuels. 

However, over the past 15 years, Populus has been a viable 

fiber stock for the paper and paperboard industry in selected regions of the U.S. In the Pacific Northwest, Populus 

plantations were established in a relatively short time and 

have provided a dependable fiber supply to certain niche 

markets. 

One potential shortcoming in this review of Populus has 

been a disregard for the inherent qualities of the raw material. Basically, this woody biomass was viewed as a l?ase 

of simple sugars or fiber stock for 2 general types of final 

products. However, inherent to Populus is considerable 

genetic variation for fiber length, chemical composition, 

and other qualities affecting overall product. Optimization of these qualities for specific applications deserves 

further attention and should serve as a focus for biotechnology research. 

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Bergeron, P.W.; Wright, J.D.; Wyman, C.E.1989. Dilute acid 

hydrolysis of biomass for ethanol production. In: Energy from biomass and wastes XII. Chicago, IL, U.S.A.: 

Institute of Gas Technology: 1277-1297. 

Fluck, R.C. 1981. Net energy sequestered in agricultural 

labor. In: Transactions of the American Society of Agricultural Engineers- 1981. 24: 1449-1455. 

Grado, S.C.; Strauss, C. H. 1993. An inventory control model 

for supplying biomass to a processing facility. Applied 

Biochemistry and Biotechnology. 39 I 40: 310-317. 

Hansen, E.A. 1988. SRIC yields: A look to the future. In: 

Proceedings: Economic evaluations of short-rotation 

biomass energy systems. Duluth, MN, U.S.A. International Energy Agency: 197-207. 

Lothner, D.C.; Hoganson, H.M.; Rubin, P.A. 1985. Examining short-rotation hybrid investments using stochastic simulation. Duluth, MN, U.S.A.: U.S .. Department of 

Agriculture, Forest Service, North Central Forest Experiment Station. 31 p. 

Perlack, R.D.; Ranney, J.W.; Barron, W.F.; Cushman, J.H.; 

Trible, J.L. 1986. Short-rotation intensive culture for the 

production of energy feedstocks in the U.S.: A review 

of experimental results and remaining obstacles to commercialization. Biomass. 9: 145-59. 

Pimentel, D. 1980. Handbook of energy utilization in agriculture. Boca Raton, FL, U.S.A.: CRC Press. 475 p. 

Roller, W.L.; Kenner, H.M.; Kline, R.D.; Mederski, H.J.; 

Curry, R.D. 1980. Grown organic matter as a fuel raw 

247 
Section V Biotechnological Applications 

material source. NASA Cr-2608. Dayton, OH, U.S.A.: 

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Stokes, B.J.; Frederick, D.J; Curtin, D.T. 1986. Field trials of 

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Strauss, C.H.; Grado, S.C.; Blankenhorn, P.R.; Bowersox, 

T.W. 1988. Economic evaluations of multiple rotation 

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Strauss, C.H.; Grado, S.C.; Blankenhorn, P.R.; Bowersox, T.W. 

1989. Developing financial and energy accounting models for woody biomass systems. Solar Energy. 42: 379-86. 

Strauss, C.H.; Grado, S.C.; Blankenhorn, P.R.; Bowersox, 

T.W. 1990. Financial selection of rotation age for SRIC 

plantations. Biomass. 23: 5-69. 

Strauss, C.H.; Wright, L.L. 1990. Woody biomass production costs in the United States: An economic summary 

of commercial Populus plantation systems. Solar Energy. 

45: 105-10. 

248 

Strauss, C.H.; Grado, S.C. 1992. Input-output analysis of 

energy requirements for short rotation intensive culture 

woody biomass. Solar Energy. 48: 45-51. 

Stuart, W.B.; Markey, D.S.; Teel, J.B. 1985. A prototype short 

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USDA Economic Research Service. 1988. Agricultural land 

values and markets: Situation and outlook report. ARlO. Washington D.C., U.S.A.: U.S. Department of Agriculture, Economic Research Service. 30 p. 

Wright, J.D. 1989. Evaluation of enzymatic hydrolysis 

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1247-1277. 

Zavitikovski, J. 1979. Energy production in irrigated, intensively cultured plantations of Populus tristis #1 and 

Jack Pine. Forest Science. 25: 383-392. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Chapter32 

Environmental Risk Assesslnent and Deployment Strategies 

for Genetically Engineered Insect-Resistant Populus1 

Kenneth F. Raffa, Karl W. Kleiner, David D. Ellis, and Brent H. McCown 

Introduction 

Most studies on genetically engineered plants have concentrated on efficacy; few have focused on environmental 

safety {Seidler and Levin 1994). This emphasis reflects the 

rapid increase in our technological capabilities over the last 

15 years and reflects an uncertainty over how best to scientifically generate relative rankings of the likelihood and severity of possible adverse effects. Environmental risk 

assessment is not an exact science and can only provide direct comparisons between treatments and checks under the 

most controlled, and therefore least realistic, conditions. 

Moreover, risk assessment inevitably raises questions that 

are at least partially subjective, value-laden, and contextual. 

This chapter attempts to ·identify the more significant 

issues of environmental risk, weigh their relative impacts, 

and suggest possible strategies for reducing adverse effects. We limit our discussion to potential environmental 

effects. Impacts on social and economic systems, while 

important, are beyond our expertise. 

Approaches to Environmental 

Risk Assessment 

An appropriate definition of environmental risk is debatable (Morgan 1993; Wilson and Crouch 1987). In this 

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman. R.C.; Eskew. L.G., tech. eds. 1997. 

Micropropagation. genetic engineering. and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

chapter, environmental risk means: "the likelihood that 

release of a novel material will cause adverse effects such 

as mortality or reduction in populations of nontarget organisms due to acute, chronic, or reproductive effects, or 

disruption of community or ecosystem function" {Urban 

and Cook 1986). Predicting consequences becomes increasingly complex as the scale expands from individual- to 

community-level interactions. For example, negative effects 

on individuals do not necessarily translate into reduced 

populations. Natural and managed systems provide many 

instances of compensatory feedback where removing 

substantial numbers of individuals does not affect population density. Conversely, the prospect of indirect effects 

from community-level interactions, even when no individual effects appear important, is a serious concern. Basic ecological studies provide a wealth of examples. 

Indirect interactions, across multiple trophic levels, incorporating biotic and abiotic environmental factors, and 

mediated by a broad range of symbionts, competitors, and 

alternate hosts, can exert enormous influences and generate unpredicted outcomes {Angle 1994; Bergelson 1994; 

Ehler 1990; Holt 1977; Price et al. 1980; Simberloff 1985). 

Similarly, the history of applied ecologies, such as agriculture and forestry, shows that indirect interactions often yield the least predictable yet most damaging 

consequences. 

The issue of ecological complexity is a paradox to risk 

assessment. Controlled evaluations of acute effects on isolated individuals generate the least variable and seemingly 

most "reliable" results. Studies that attempt to unravel 

more diffuse and incipient effects are ultimately more important, yet unfortunately they are less likely to provide 

definitive answers {Angle 1994). Ironically, current approaches to training, funding, and productivity are 

strongly biased toward the former approach. 

Comprehensive risk assessment must also weigh anticipated benefits against risk. Genetic engineering of Populus 

offers several potential benefits, such as reduced pesticidal 

inputs, improved carbon sequestration, alleviation of pressures to exploit unmanaged systems, and improved 

sources of alternatives to fossil fuels {Kleiner et al. 1995; 

249 
Section V Biotechnological Applications 

McCown et al. 1991; Raffa 1989; Raffa et al. 1993; Robison 

et al. 1994). These are potentially enormous benefits; however, they are beyond the scope of this paper. 

Potential risks can be categorized based on their spatial 

and temporal scales. For example, an effect could be limited to the treated site, or it could impact neighboring ecosystems. Effects can be short-term, such as the release of a 

toxic gene product into the environment, or self-replicating, such as the escape of viable germplasm. Such distinctions can be somewhat blurred and need to be assessed as 

part of the complete risk evaluation process. Still, there is 

general agreement that the most serious concerns arise 

when genetically engineered organisms could cause selfperpetuating injury to commercial or natural ecosystems 

beyond the immediate area of release. 

Criteria for Risk Assessment 

Different individuals, agencies, and organizations have 

advocated different criteria, burdens of proof, and levels 

of evidence governing the planned release of genetically 

engineered organisms. Probably the most helpful guidance 

is provided by a National Academy of Sciences committee headed by Arthur Kelman (NAS 1987). In the opinion 

of NAS, what matters is the product not the process. According to this perspective, introducing genetically engineered organisms "poses no risks different from the 

introduction of unmodified organisms and organisms 

modified by other methods." Therefore, "assessment of risk 

should be based on the organism, not the method of engineering." Subsequent authors have delineated some important differences between g~netic engineering and plant 

breeding, and hence the need for limits in applying this 

equivalency (Dale and Irwin 1995; Giampietro 1994; Regal 1994). However, this starting point has proven useful 

and has withstood the test of time. Similar conclusions are 

stated by Tiedje et al. (1989) in an Ecological Society of 

America report: "transgenic organisms should be evaluated and regulated according to their biological properties (phenotypes), rather than according to the genetic 

techniques used to produce them." 

Emphasizing phenotypes over the methods by which 

they arise has proven useful because it rebuts scientifically 

unfounded criticisms and focuses on interactions between 

gene products and their environment. Rather than dismissing environmental concerns, this approach highlights the 

importance and complexity of predicting responses to gene 

products at the community level, and the need for ecological expertise in devising scientifically based policies. 

If the product not the process is critical, then expertise in 

the methods of genetic engineering is not directly relevant 

to predicting how novel organisms will interact with eco250 

systems. Molecular expertise is invaluable, however, in 

protecting against unintended changes in the genome, incorporating methods of sterility, and controlling and evaluating patterns of expression. The criteria for estimating and 

the approaches to alleviating environmental concerns require interdisciplinary efforts (Raffa 1989). 

Raising every imaginable hazard that could arise from 

genetically engineered organisms is neither difficult nor 

helpful. This approach could hinder the enormous value 

of biotechnology and dilute needed emphasis on legitimate concerns. At the other extreme, the view is sometimes expressed (or implied) that all concerns arise merely 

from a lack of scientific understanding or breadth. This 

view seriously underestimates the complexity of scaling 

from molecular- through ecosystem- level processes. Failure to consider such complexity invariably detracts from 

the long-term sustainability of new technologies; a costly 

lesson already appreciated by agrichemical companies. The 

issue needs to be one of reasonable probability (de Zoeten 

1991; Frederick and Egan 1994; Hubbes 1993; NAS 1989; 

Raffa 1989; Strauss et al. 1991; Tiedje et al. 1989). For example, Tolin and Vidaver (1989) propose that "restrictions 

should be based on demonstrated, not conjectural risks." 

However, we would substitute "realistic" for "demonstrated" to promote a more proactive approach· to risk 

management. In our view, the likelihood of risk may be 

realistic if 2 conditions are met: 1) a clear mechanism, based 

on known biological processes and verified assumptions, 

can be delineated; and 2) there is relevant precedent. 

Few specific risks meet the above criteria. Those that do 

can be classified into 3 general categories: 1) escaped plants 

or genes, 2) evolution and consequences of resistant pest 

biotypes, and 3) alteration of multi-trophic processes. We 

first describe how Populus systems relate to these questions and then address each risk. Biotype evolution will 

be developed as a more detailed case study, as this is our 

primary area of interest. We conclude with an overall synthesis of environmental risk assessment in Populus. 

For each concern, the potential risk can be addressed 

by asking 3 questions: 1) "Is there assurance that the proposed event (i.e., gene escape, biotype evolution, altered 

multi-trophic process) will not occur?"; 2) "Is there assurance that the effects will be harmless if this event does 

occur?"; and 3) "Are there ways for reducing the likelihood and impact of harmful effects?" These questions 

place the burden of proof on the novel gene product to be 

consistent with ~ow other novel products such as biological control agents, introduced plant materials, and pesticides are evaluated (Caltagirone and Huffaker 1980; 

Charudattan and Browning 1992; FIFRA 1978; Fuester 

1993; Harris 1985; Hinkle 1993; Hutton 1992; Upholt 1985; 

USDA FS 1991; White et al. 1992). During actual experimentation, however, the null hypothesis is one of no effect. Also note that questions 1) and 2) are evaluated in 

the absence of any ameliorating steps, but the availability 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Environmental Risk Assessment and Deployment Strategies for Genetically Engineered Insect-Resistant Populus 

and implementation of such tactics (question 3) could 

greatly reduce resulting concerns. 

Populus Growing Systems 

The safety of releasing a genetically engineered organism is determined by the gene product and the environment into which it will be introduced (Abbott 1994; Falk 

and Bruening 1994;}epsonet al. 1994; Pimentel et al. 1989). 

In general, risks are lower for intensively cultured short 

rotation tree crops than for large forested expanses of longlived species (Raffa 1989). Populus occupies a relatively 

broad range of growing conditions along this continuum. 

For example, trembling aspen, Populus tremuloides, is one 

of the most widely distributed naturally occurring species in North America. It is a valued forest tree with anumber of uses such as soil quality improvement, watershed 

maintenance, C02 sequestration, and wildlife habitat. 

When used for timber, Populus is harvested from self-regenerating forests and grown in commercial stands. 

Populus is also a major component of rapid rotation systems such as biofuel plantations. These intensively managed systems, more closely resembling agricultural than 

forest production, have short growing intervals, are based 

on carefully derived clonal material, and are subjected to 

intensive cultural and chemical inputs. 

Populus has also become the focus of intense basic research by molecular biologists, plant physiologists, and 

ecologists. Populus is the preeminent tree model for tissue 

culture, molecular mapping, and transgenic technology. 

Concurrently, Populus has become a key model for basic 

ecophysiological and plant-herbivore interaction studies. 

Thus, Populus provides an ideal system for evaluating the 

role of plant community structure in the efficacy and environmental safety of various deployment strategies and 

for integrative studies from the molecular- through ecosystem- level scales. 

Movement of Transgenes Into 

Native Populations 

The most direct form of proposed environmental harm 

is that genes encoding novel traits might become established in feral populations and subsequently exert a weedy 

effect. A variety of mechanisms for gene escape have been 

proposed such as hybridization into gene pools of wild 

relatives, crop abandonment, movement of cuttings by 

animals or water, etc. Escape of transgenes into the enviUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

ronment could occur on several levels, and the risk associated with each level of escape varies. A commonly proposed level of escaped transgenes is via pollen or seed. 

Another level is the physical movement of plant parts into 

the surrounding environment. Because virtually all the 

poplars used in short rotation plantations are clones multiplied by vegetative cuttings, small branches can be moved 

off site by animals or overland water flow. The root sprouting habit of several poplars poses a similar concern. 

Precedents by which to evaluate possible consequences 

of escaped material are offered by the literature on accidental or injudicious introduction of exotic species. Following the N AS (1987) rationale that introducing 

genetically engineered organisms should not be treated 

differently from other unmodified organisms, the literature on exotic introductions provides useful precedents to 

evaluate the likelihoods and consequences of escapees 

(Williamson 1994). A large and well-documented data base 

details numerous instances in which accidentally or deliberately introduced microorganisms, arthropods, nematodes, plants, and vertebrates became established in 

non-native ecosystems, and subsequently exerted severe 

economic and environmental consequences (e.g., Liebhold 

et al. 1995; Lodge 1993; Mooney and Drake 1986; US Congress OTA 1993 ). 

A number of molecular biologists, agronomists, ecologists, and plant protection specialists have 'cautioned that 

some escape is likely. For example, Strauss et al. (1995) 

stated unambiguously, uGene flow within and among tree 

populations is usually extensive, which makes the probability of transgene escape from plantations high." 

Timmons et al. (1995, 1996) expressed a similar conclusion 

for Brassica. Likewise, the ecologists Kareiva et al. (1994) 

concluded that "the escape of transgenic pollen is inevitable." Williamson's (1994) analysis of historical records 

of deliberately introduced organisms, concludes that 

nearly all escape, and of these 10 percent become established. 

Our ability to address whether there is sufficient assurance that escaped genes would be harmless is considered 

case by case (Dale and Irwin 1995). Various authors, including molecular biologists (Strauss et al. 1995), ecologists (Kareiva et al. 1994; Regal 1994; Seidler and Levin 

1994; Tiedje et al. 1989), and crop protection specialists 

(Dale 1994; Williamson 1994), have identified possible 

adverse effects of escaped transgenes. Some examples include creation of new (or enhanced) pests, harm to nontarget species, and disruptions to biotic communities, 

natural food webs, and ecosystem processes. 

In each of these cases, there are well established mechanisms by which such adverse consequences might arise, 

and substantial literature providing precedents from analogous introductions. Examples of possible mechanisms include: 1) enhanced competitiveness of a genetically 

engineered organism (due to pest resistance or physiologi251 
Section V Biotechnological Applications 

cal environmental tolerance of stress) that displaces existing or subsequent beneficial organisms (Ellis et al. 1984; 

Moamad et al. 1984); 2) reductions in seed dispersal, pollination, or biodiversity by insecticidal transgene products 

(Simmonds 1976; McGranahan et al.·1988); or 3) acquisition of traits that enhance competitive status by existing 

weed species (Windle and Franz 1979). Again, the historical record with traditional introductions is of some value. 

Williamson (1994) reports that 10 percent of the 10 percent 

of escaped species that establish become problematic. It 

must be emphasized,. however, that such figures do not 

reflect refined deployment strategies (using specific information about target species, transgenes, rotation cropping 

system, and location) that could accompany planned releases of transgenic poplars. And, although the historical 

record of planned releases of genetically engineered organisms is still relatively small, to date there have been no 

known adverse effects. 

Despite the potential for adverse effects, a number of 

attributes of the transgene, the parent organisms, phenotypic expression, and target pest-environment system 

could reduce risk (Tiedje et al. 1989). For example, risk 

analysis must consider whether pollen from transgenic 

hybrids is compatible with surrounding populations and 

also whether the timing of pollen release occurs when stigmatic surfaces in surrounding populations are receptive. 

Any impact of escaped genes will likely vary with the novel 

gene product as well. For example, risks associated with 

the escape of a Bacillus thuringiensis (Bt) endotoxin gene 

may be different than those for a gene modifying lignin. 

Differences in plant species and growing systems are also 

pertinent. In agronomic food crops such as soybean, maize, 

potato, and tomato, measures such as sterility have not 

been a requirement for registration. Conversely, a case-bycase analysis of each transgene-species-planting site, combinations may be needed. 

The third question, whether the risk or impact of escape 

can be ameliorated, is currently the subject of intense effort. Risk from escape by vegetative material could be reduced by management practices that minimize root 

sprouting outside the plantation and the distance plant 

material is moved. This can be achieved by planting buffer 

strips that are routinely cultivated and/ or planted with 

an annual crop so that escapes can be readily identified 

and treated with herbicide. One management strategy for 

contending with pollen or seed dispersal might be to identify late flowering clones for a breeding population, such 

that harvesting occurs before sexual maturity in the 

transgenic trees. Such an approach would offer functional, 

while not physiological, sterility. Another strategy might 

be the use of sterile triploids. 

Physiological approaches to reproductive sterility in 

genetically engineered trees have recently been reviewed 

(Strauss et al. 1995) and are not be treated extensively here. 

Basically, these include using floral promoter-cytotoxin' to 

252 

ablate floral tissues and disrupting expression of essential 

floral genes. In the former approach, cytotoxic genes regulated by reproductive-specific promoters kill all cells committed to reproductive development. The latter approach 

uses antisense RNA, sense suppression, or promoter-based 

suppression to impair the expression of genes required for 

fertility. These approaches can be deployed with varying 

levels of gender specificity, have relative advantages and 

disadvantages (Meilan and Strauss this volume; Strauss 

et al. 1995), and have yielded some successes (Mariani et 

al. 1990). The current obstacles relate to our lack of basic 

information about reproductive gene sequences and expression in clonally propagated species such as Populus. 

Very little is known about the long-term stability of 

transgene expression in woody perennials. In 1 test examining the expression of a marker gene in field poplars, some 

level of seasonal variability in transgene expression was 

observed, but in general this variation was predictable, and 

relatively continuous expression levels occurred from year 

to year (Ellis et al. 1994). Of greater concern, however, is 

the variation in expression levels and patterns that occur 

between individual transformants containing the same 

construct. In addition to the variation in the overall levels 

and patterns of transgene expression, current molecular 

understanding of transgene regulation in plants is at a relatively elementary level. Genetic engineering for sterility 

requires very precise control over a transgene to interrupt 

and terminate flowering. There currently is no way to ensure that this transgene will function in all the plants over 

the 5 to 10 years a poplar plantation requires to mature. 

Additional research is needed to devise such capabilities. 

Evolution of Resistant Pest 

Biotypes and Emergence of 

Secondary Pests 

General Considerations 

the evolution of insect and microbial biotypes in response to genetically engineered plants has been an area 

of concern since the early development of plant transformation technologies (Gould 1988). The same 3 questions 

posed for gene escape, with emphasis on insects, will be 

addressed here: 1) Is there assurance that resistant pests 

will not evolve? 2) Is there assurance that resistant pests 

will not cause environmental harm if they do arise? and 3) 

Are there ways for reducing the likelihood and impact of 

resistant biotypes? 

The potential of pest-biotype evolution is well established by a strong mechanistic foundation and historical 

precedent (Brattsten et al. 1986; Forgash 1984; Georghiou 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Environmental Risk Assessment and Deployment Strategies for Genetically Engineered Insect-Resistant Populus 

and Lagunes-Tejeda 1991; Georghiou and Saito 1983; 

McGaughey 1985; Roush and Tabashnik 1990; Tabashnik 

1994). Extensive insecticide application, deployment of 

resistant cultivars, and even cultural practices have repeatedly selected for resistant insect biotypes (Via 1990). Moreover, the underlying mechanisms have been well 

characterized at the molecular, biochemical, physiological, and population levels (Cohan and Graf 1985; EggersSchumacher 1983; Flexon and Rodell1982; Kulkarni and 

Hodgson 1984; Muggleton 1982; Mullin and Scott 1992; 

Oppenoorth 1984; Roush 1987; Ryan and Byrne 1988; 

Skylakakis 1982). Intraspecific differences among gypsy 

moths and forest tent caterpillars from different geographic 

sources to poplars transgenically expressing Bt have been 

observed (figure 1). Variation among these and other forest lepidopterans to exogenously applied Bt is also well 

documented (Rossiter et al. 1990; Van Frankenhuyzen et 

al. 1995). Adaptations by insects to altered sources of food 

plant quality, quantity, and distribution are well documented in natural and managed systems (Singer et al. 1993; 

1.0 

.. M. DISSTRIA ~ L. DISPAR 

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0 0.8 

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.... 

z 

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a:: 

0.6 

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u. 

w 

(.) 

z 0.4 w 

a:: 

w 

u. 

w 

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0.0 

ONT WI NS F51 

POPULATION 

Figure 1. Intraspecific variation among gypsy moth and 

forest tent caterpillar populations to transgenic 

poplars expressing a Bt endotoxin gene. Two 

forest tent caterpillar, Malacosoma disstria, 

populations showed different levels of aversion 

from transgenic relative to control foliage. A 

similar difference was observed between 2 

gypsy moth, Lymantria dispar, populations 

(Robinson et al. 1994 )_. ONT =Ontario, Canada; 

WI=Wisconsin, USA; NS=Nova Scotia, Canada 

and USDA APHIS; F51 =gypsy moths from 

laboratory culture at USDA Beneficial Insects 

Laboratory, Newark, DE, USA; ns=nonsignificant. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Singer and Parmesan 1993; Via 1990). Thus, herit~ble variation required for gene frequency alteration in response to 

selection is sufficient. 

It does not matter whether an insect toxin is deployed 

through spray application, traditional breeding, or genetic 

transformation. For example, the introduction of new plant 

varieties is sometimes followed by a population increase 

of previously innocuous herbivores to pest levels. Attention should not be limited to plants specifically engineered 

for pest resistance, as development of presumably unrelated plant qualities can also alter selective pressures (Raffa 

1989). For example, development of new rice varieties for 

various agronomic properties led to the emergence of new 

pest complexes (Oka and Bahagiaivani 1984). In addition, 

engineering plants for altered, increased, or novel secondary products could have multiple consequences. Even 

chemical groups that are typically considered "defensive" 

can directly benefit adapted herbivores (Bernays and 

Woodhead 1982), or indirectly benefit them by reducing 

the efficacy of beneficial predators (Codella and Raffa 

1995), parasitoids (Campbell and Duffey 1979), and 

entomopathogens (Andrews et al. 1980). 

Selective pressures imposed by transgenic trees could 

be higher than those resulting from insecticide treatments. 

Insecticide-resistant biotypes have been rare among forest pests, despite a rather extensive history of synthetic 

chemical and Bt application. Tree-feeding herbivores are 

not physiologically unique in this regard. Rather, tree-feeding insects are exposed to more variable exposure patterns, 

less complete spray deposition, and less frequent treatment, than are agronomic insects. These sources of variability could be lost using transgenics. Depending on 

migration rates, neighboring untreated forests could provide refugia for susceptible genotypes, when they are near 

to transgenic plantings. In this regard, transgenic poplars 

could pose less of a threat of biotype evolution than many 

transgenic agronomic crops. Where poplars are planted in 

isolation from native refugia, however, such as in irrigated 

deserts, this benefit would be minimal. 

Usually, the impact of evolved resistance against pesticides, resistant cultivars, and genetically engineered trees, 

is limited to loss of efficacy (Raffa 1989). However, 2 general categories of adverse effects could extend beyond 

treated plantations: 1) induced insect emigration into 

neighboring non-transgenic stands; and 2) lost efficacy of 

previously useful tools in non-transgenic stands. 

The possibility of between-stand movement raises the 

ethical concern that a grower who chooses not to plant 

genetically engineered trees may be subjected to immigrants from engineered stands (the same could be true of 

most traditionally bred forms of resistance and chemical 

pesticides). This risk may be greater in tree than agronomic 

crops, because airborne larval dispersion is on average 

more important in forest insect life histories. A critical need 

in assessing this impact is understanding the relationship 

253 
Section V Biotechnological Applications 

between toxicity and repellency, and in particular, whether 

repellency occurs pre- or post- ingestion (Hoy and Head 

1995; Ramachandran et al. 1993a, 1993b). This could affect, for example, whether emigrating individuals possess 

a slightly higher level of physiological tolerance than the 

general population. The available evidence suggests. that 

these parameters vary markedly with the insecticidal product, its interaction with plant allelochemicals, and insect 

species. We currently lack information on how best to balance the value of behavioral aversion as a resistance-delaying tactic versus its impact on non-treated stands (Gould 

1988; Hoy and Head 1995; Johnson and Gould 1992). 

The possibility of transgenic plants reducing the utility 

of an externally sprayed biopesticide, such as Bt endotoxin, to growers who use Integrated Pest Management 

(IPM) approaches based on economic injury levels, raises 

a similar ethical concern. This is analogous to neighboring growers applying insecticides on a calendar rather than 

density-activated basis. Likewise, integrated plant protection programs could be compromised when biotypes 

evolve against transgenic resistances that are based on elevated or altered allelochemicals (again, the same could 

be true of some traditional breeding). 

Loss of efficacy can be compounded when the mechanism of evolved resistance confers cross-resistance to other 

insecticides or resistant-cultivar allelochemicals (Brattsten 

et al. 1986; Brattsten 1991). Cross-resistance is a widely 

occurring phenomenon that can arise by a number of wellcharacterized physiological mechanisms. For example, the 

introduction of the synthetic pyrethroids, derived analogues of Chrysanthemum spp. extracts, encountered rapid 

biotype evoiution in regions where the synthetic organochlorine DDT had been widely used. Similarities in the 

pharmacological properties of these 2 groups provide a 

physiological explanation for cross-resistance, but a priori 

considerations based on the unrelatedness of their molecular structures failed to predict these consequences. 

Mitigation Strategies 

Although resistant biotypes are likely to evolve if 

transgenic poplars are deployed without preconceived 

resistance management programs, a variety of ameliorating strategies can be used. There are many examples of 

effective pest control tactics providing satisfactory control 

over many decades. Likewise, naturally evolved plant 

defenses provide many examples of long-term stability. 

Even among trees, in which host-generation times exceed 

those of insects and pathogenic microbes by orders of 

magnitude, most members of the host population are protected most of the time (e.g., Edmunds and Alstad 1978; 

Whitham 1983 ). A major principle to emerge independently 

from toxicology, plant breeding, and ecology is that the 

pattern and intensity of selection, more than the actual 

mode of toxicity, most strongly affect biotype evolution 

254 

rates (Brattsten et al. 1986; Tabashnik and Croft 1982). The 

rate, impact, and extent can be greatly reduced by considering features of the target system and by incorporating 

heterogeneity at multiple levels of scale. Preconceived resistance management programs now accompany the introduction of many pesticides, as agrichemical 

corporations recognize the economic value of protecting 

their investments. Likewise, deployment of transgenic 

cotton and corn is now accompanied by guidelines prescribing inclusion of non-engineered seed. 

Some features of the tree-insect system that can accelerate or retard biotype evolution include the availability of 

refugia for susceptible insect genotypes, attributes of the 

major pests' physiology, behavior, and ecology, and compatibility of the novel trait with other management tactics. Thus, intensively cultured, short-rotation Populus 

plantations pose less risk than forests of long-lived species such as Pseudotsuga. In the latter case, the enormous 

differences between pest and host generation times would 

greatly reduce the efficacy of any biotype-delaying tactics. 

Likewise, the defoliator guild that most strongly impacts 

Populus poses less risk than, for example, the scolytids associated with Pinus and Picea. In the latter case, beetle preference for stressed trees limits them to such hosts during 

lengthy nonoutbreak periods. Conferring a novel resistance 

that was expressed regardless of tree vigor would greatly 

alter the selective pressures on bark beetles and possibly 

result in more pestiferous behavior (Raffa 1989). No such 

relationship appears to regulate population dynamics of 

most folivores. 

Diversifying tactics, such as mixed block plantings and 

crop rotation, are well suited for Populus. A wide range of 

hybrid poplar clones are available for deployment, including some that provide rapid growth and resistance against 

some key pests (Robison and Raffa 1990, 1994, 1997a, 

1997b). Mixed block plantings can also incorporate host 

plant tolerance. For example, some of the less resistant 

clones against Malacosoma disstria feeding can withstand 

considerable defoliation without experiencing severe 

growth losses (figure 2). 

Protection against multiple pest complexes can be 

achieved by integrating traditional and transgenic resistances. The need and potential for this approach are illustrated by the lepidopteran and coleopteran feeding guilds. 

Strong resistances against both groups have been identified, but no clones are highly resistant to both (Robison 

and Raffa 1994). Continued hybridization or characterization are unlikely to improve this relationship because the 

same allelochemicals, specific phenolic glycosides, which 

inhibit lepidopterans benefit coleopterans (Bingaman and 

Hart 1993; Lindroth and Bloomer 1991; Ramachandran et 

al. 1994; Smiley et al. 1985) (figure 3). Understanding these 

relationships can help guide molecular strategies. For example, inserting only a coleopteran-active Bt (crylllA, 

cryiB) into 'NM6' (P. nigra x P. maximowiczii), and only the 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Environmental Risk Assessment and Deployment Strategies for Genetically Engineered Insect-Resistant Populus 

14-

~ 

i?ii 12-

t5 

~~ 10 

~~ 8 

c:li: 

~~ 6 

~--

~ 4 

c;; 

w 2 a: 

0 

0 

NC11505 

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NC5331 

NC11432 

2 

NC11445 

NC11382 

NC5262 

NC11396 

NC11004 

NC5377 

NC527t 

I 

4 6 8 10 

TOLERANCE TO DEFOLIATION 

(RANK ORDER) 

12 14 

Figure 2. Variation in host resistance against defoliation by 

forest tent caterpillar (FTC) larvae and tolerance 

to a controlled level of artificial defoliation among 

hybrid poplar clones. Some clones are relatively 

unable to prevent defoliation but are highly 

tolerant if it occurs (from Robison & Raffa 

1997a). cl. 'DTAC2' (Populus deltoides var. 

angulata x P. x berolinensis); cl. 'NC5262' (cl. 

'NE387') (P. balsamifera var. subcordatal 

candicans x P. x berolinensis); cl. 'NC5271 (cl. 

'NE19') (P. nigra var. charkowiensis x P. nigra 

var. caudina); cl. 'NC5331' (cl. 'NE299') (P. nigra 

var. betulifolia x P. trichocarpa); cl. 'NC5377' (cl. 

'Wisconsin #5') (P. deltoides x P. nigra); cl. 

'NC11004' (P. deltoides cv. 'Siouxland'); cl. 

'NC11382' (cl. 'NE27') (P. nigra var. 

charkowiensis x P. x berolinensis); cl. 'NC11396' 

( cl. 'NE49') (P. maximowiczii x P. x bero/inensis); 

cl. 'NC11432' (cl. 'NE252') (P. deltoides var. 

angulata x P. trichocarpa); cl. 'NC11445' (cl. 

'NE280'; cl. 'NE157') (P. nigra x P. /aurifolia); cl. 

'NC11505' (cl. 'NE388'; cl. 'NE88') (P. 

maximowiczii x P. trichocarpa); cl. 'NM6' (cl. 

'Max-5') (P. nigra x P. maximowiczil). 

lepidopteran-active Bt (cryiA(a)) into 'NC5271' (P. nigra var. 

charkowiensis x P. nigra var. caudina), can cut in half the 

number of genetically engineered trees needed to express 

any 1 trait, yet still provide full protection against both 

pests (table 1). Different forms of resistances can also be 

combined with transgenic traits. For example, the clones 

'NE332' (P. simonii x P. x berolinensis) and 'NC11382' (P. nigra var. charkowiensis x P. x berolinensis) show resistances to 

M. disstria, but these defenses are based on foliar phenolic 

glycosides and bud resins, respectively (table 2) 

(Ramachandran etal.1994; Robison and Raffa 1997a). Such 

combinations can increase heterogeneity because Bt interUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

80 A b 

60 B ...J 

< ~ > ~ 

> B -------0 ~ 

~ 

a: 40 ~ ·o---- :::) 

en 

oe 

20 

0 

50 60 70 80 90 

~ Chrysomela scripta PHENOLICS (ug/mg) 

--o-- Ma/acosoma disstria 

22 55 

a B 

:E. 20 B ______ ..o 50 ~ w ------ w ::!! 45 :::E 

i=S 18 i= .! 

!z·@- !z;; w t) 40 w .~ ~VI 

16 ~"tl 

&ti n. . 

m 35 g::e 

w Gj 14 b Gj Q 30 c 

12 25 

50 60 70 80 90 

NC5271 NE332 NMS 

PHENOLICS (uglmg) 

Figure 3. Opposing effects of foliar phenolics on 2 defoliating pests attacking hybrid poplars. The cottonwood leaf beetle, Chrysomela scripta, exhibits 

high survival and rapid development on clones, 

such as 'NM6' (Populus nigra x P. maximowiczit), 

which have high phenolic contents. Survival and 

development rates are poor on clones, such as 

'NC5271' (P. nigra var. charkowiensis x P. nigra 

var. caudina), which have low foliar phenolic 

concentrations. Conversely, the forest tent 

caterpillar, M. disstria, experiences high survival 

and rapid growth on 'NC5271 ,' and poor survival 

and growth on 'NM6.' (Ramachandran et al. 

1994). 

acts with different phytochemical groups differently 

(Appel and Schultz 1994; Hwang et al. 1995). 

Heterogeneity can be further enhanced by linking expression to wound-inducible promoters. Wound-inducible 

expression of inserted genes could simulate the" economic 

injury levels" that trigger pesticide applications in Integrated Pest Management systems. That is, a certain level 

of insect feeding would be tolerated before expression was 

elicited. Opportunities for increasing heterogeneity at this 

level are supported by existing variation in inducibility 

among poplar clones (table 3). However, preliminary evidence suggests that the sensitivity of existing wound-in 

255 
Section V Biotechnological Applications 

Table 1. Proposed integrated use of traditionally bred and natural plant resistance with genetic engineering to confer 

protection against multiple pest complexes. An example using hybrid poplar. 

Level of protection against pest group 

Source of resistance Example Lepidoptera Coleoptera 

Heritable plant defense Clone 

{Current) 'NM6'1 

'NC5271'2 

Transgenic trait Bt Endotoxin 

(Current) cry lilA 

cryiA(a) 

Integrated combination Clone+ Bt 

(Proposed) 'NC5271 '

1 + cryiA(a) 

'NM6'2 + cryii/A 

1 'NM6' =Populus nigra x P. maximowiczii 2 'NC5271' = P. nigra var. charkowiensis x P. nigra var. caudina 

Table 2. Surces of resistance against forest tent caterpillar, Malacosoma disstria, in 2 hybrid poplar clones 

(Robison & Raffa 1997a). Foliage of 'NE332' is less 

suitable for forest tent caterpillar larvae than is foliage of 

'NC11382.' Bud resins in 'NC11382' are more effective at 

immobilizing larvae and preventing access to foliage. 

Ratio of M. disstria 

performance in tissue 

Insect parameter 'NC11382'1fNE332'2 

Foliage Growth rate (mg/day) 

Development time 

Feeding (2nd instar) 

Survival 

Weight (mg) 

Buds Larval mobility 

Weight (mg) 

6.5 

1.5 

19.0 

2.3 

1.7 

0.4 

0.7 

1 'NC11382' = Populus nigra var. charkowiensis x P. x 

berolinensis 2 'NE332' = P. simonii x P. x berolinensis 

Table 3. Clonal variation in Populus inducibility in response 

to forest test caterpillar, Ma/acosoma disstria, feeding 

Clone 

'NC11382' 

'NE332' 

Percent forest test caterpillar survival 

Constitutive tissue Damaged tissue 

90 

85 

85 

49 

Source: Robison & Raffa (1997a) 

ducible promoters may need to be increased before this 

strategy can provide field-level efficacy (Ellis et al. 1996}. 

Further heterogeneity could arise from tissue- and temporally-specific expression. Protecting only favored leaves 

256 

High Low 

Low High 

Low High 

High Low 

High High 

High High 

can sometimes provide protection nearly equivalent to 

treating entire plants (table 4). This approach is most suitable when insects that prefer productive tissues can also 

tolerate other foliage. Within-plant spatial variation in 

allelochemistry occurs commonly among naturally coevolved plant defenses. For example, in Populus, phenolics are concentrated in the youngest leaves, with the result 

that lepidopteran defoliators feed mostly on older tissue. 

The underlying physiological basis for uneven phytochemical distribution is complex, but among the benefits incurred by the host are protection of the most 

photosynthetically active tissue, retained ability to translocate carbon to the growing tip, and reduced likelihood 

of complete defoliation (Coleman 1986; Meyer and Montgomery 1987). Likewise, pines allocate diterpene resin acids to new not old foliage. Thus, pine sawflies feed on the 

older foliage, a habit that only removes photosynthetically 

less valuable tissue. At first glance, this might suggest a 

high potential for these herbivores to overcome such defenses, but most herbivore species have not evolved this 

ability. The evolutionary "choice" in this case is not between overcoming a biochemical barrier and starvation. 

Rather, those larvae that did feed on new foliage would 

grow less and be less fecund than those on old foliage, 

and hence be less competitive. 

Table 4. Performance of cottonweed leaf beetle, Chrysomela 

scripta, on trees completely or partially treated with Bt. 

Treatment 

Control 

All foliage 

Young only 

Mature only 

No. egg masses 

produced 

10.25 

5.75 

3.50 

9.75 

Source: Ramachandran et al. (1994) 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Environmental Risk Assessment and Deployment Strategies for Genetically Engineered Insect-Resistant Populus 

Alteration of Multi-Trophic 

Processes 

Experiences with pesticides, introduced organisms, and 

other xenobiotic inputs suggest several means by which 

products of genetically engineered plants could affect ecosystem-level processes. Adverse impacts include reduced 

populations of predators, parasites, scavengers, pollinators, and endangered or aesthetically valued species. Both 

direct and indirect mechanisms have been delineated for 

impacts on each of the above, and strong historical data 

provides examples for each (e.g., see Caltigirone and 

Huffaker 1980; Ehler 1990; Findlay and Jones 1990; Holt 

1977; Pimentel1980, 1991; Pimentel and Warneke 1989). 

Direct effects of xenobiotics on natural enemies can occur by acute toxicity or biomagnification. In general, genetically engineered trees should be less directly damaging 

to natural enemies than are traditional pesticides. One of 

the major advantages of transgenic plants is that toxins 

can be delivered directly to the herbivore, without broadcast application. The likelihood of biomagnification depends on the gene product. To date, most traits engineered 

into plants involve gene products that are rapidly broken 

down within the target insects. For example, we are unaware of any instances where predators were directly affected from ingesting prey killed by Bt. Other more stable 

gene products, however, could be problematic. 

Evaluating potential effects on parasites is more difficult. A major concern is that parasitoids will oviposit in 

insects in which they cannot complete development before host death. This could drastically reduce parasite 

populations, and thereby release secondary pests. For example, negative effects of plant allelochemicals on parasitoid success are well documented (Barbosa and Saunders 

1984; Campbell and Duffey 1979). However, xenobiotics 

can sometimes benefit parasitoids. For example, Bt application can enhance performance and population densities of the gypsy moth larval parasitoid, Cotesia melanoscela 

(Weseloh et al. 1983). In this example, the delayed growth 

rates caused by Bt apparently increase the period during 

which surviving early instars are vulnerable to parasitism. However, interpreting such results is complicated. 

For example, Johnson and Gould (1992) have argued that 

synergism between genetically engineered resistance and 

parasitoids could accelerate biotype evolution and its resultant hazards. That is, if exposure to a particular product increases the likelihood of ultimate mortality, selection will 

more strongly favor tolerance against the predisposing agent. 

But in some cases, increased parasitoid densities resulting 

from synergism could subsequently exert mortality independent of the xenobiotic. More research is required to 

better quantify and partition these multiple effects. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Som.e indirect effects following the introduction of any 

new organism are inevitable. In some cases, an introduced 

biological control agent may competitively displace other 

parasitoids, yet provide less overall control (Ehler and Hall 

1982). This most commonly occurs where a non-host factor (e.g., nectar) is limiting. In the case of transgenic poplars, we see no readily apparent mechanism by which this 

could occur. Likewise, major effects on aesthetically valued 

species outside the genetically engineered plantations seem 

unlikely. Threats to endangered invertebrates arise primarily from habitat destruction, so environmental concerns 

would be better addressed if they included potential consequences of extensive Populus cultivation per se, rather than 

just the transgenic approaches taken to protect them. 

Perhaps the least understood component of risk assessment concerns potential adverse affects on soil organisms. 

Risk assessment in this area is especially difficult because 

our basic understanding of nutrient cycling, detritivore taxonomy and ecology, and soil biochemical and biophysical 

processes is limited. Historical precedents of canopy inputs affecting soil processes include anthropogenic inputs, 

such as pesticides and other pollutants, and natural products such as foliar nutrients and lignins, allelochemicals 

from induced foliage, and insect frass (Aber et al. 1990; 

Cates et al. 1995; Horner et al. 1988; Mattson and Addy 

1975; Melillo et al. 1982; Sugai and Schimel1993). 

Whether the effects of transgenic leaf litter are nonexistent, short- or long- term, and have point or non-point effects, depends on the gene product and soil properties. 

Products most commonly suggested for transformation into 

Populus have relatively high specificity, which reduces risk. 

For example, various Bt endotoxins are specific to Lepidoptera, Diptera, or Coleoptera. The first 2 groups do not 

appear to pose significant concerns; Lepidoptera exert a relatively minor role in litter decomposition, and genes conferring protection against Diptera are unnecessary in Populus. 

Beetles, however, are important components of the soil fauna, 

functioning as decomposers, vectors of beneficial microbes, 

and predators on a variety of potentially injurious arthropods 

and fungi. Thus, introduction of Coleoptera-active Bt or proteinase inhibitor poses some concern. Gene products with 

relatively general activity could be more problematic. For 

example, proteinase inhibitors can sometimes affect relatively 

diverse taxa and require more detailed evaluation. 

Novel gene products could be altered by soil biochemical 

and biophysical processes, as occurs with synthetic materials (Angle 1994). Consideration of genetically engineered 

organisms must extend beyond the actions of the gene products themselves and include studies of breakdown products. 

The consequences of a stable gene product must also be considered. Although a protein may not be toxic to soil organisms at the levels present in a single leaf, buildup in the soil 

over a season or many years may pose a problem. Such residual effects are difficult to predict because stability is affected by factors such as soil pH, nutrient content, rainfall, 

257 
Section V Biotechnological Applications 

and temperature. The stability of proteins may also be altered within the plant and vary during the year. 

Tactics for reducing potential risks to natural enemies and 

detritivores relate to the inherent properties of the gene products themselves and their expression. In general, environmental risk can be minimized when these products and their 

derivatives are specific to the target insect, of short duration, and exposed in a spatially and temporally limited pattern. Existing approaches to toxicological evaluation are 

available for such analyses. However, further theoretical 

development is required before optimal relationships between transgenic plants and parasitoids can be devised. 

Conclusion 

Some serious environmental concerns must be weighed 

against the potential benefits of genetically engineered 

Populus. Risk assessment can be improved by focusing on 

the most likely sources of environmental harm as opposed 

to generic listings of all hypothetical outcomes. As stated 

by previous authors from a broad range of backgrounds, 

emphasis should be placed on how gene products will 

interact with ecosystems not how these products arose. 

Conversely, the notion that genetic engineering has somehow been singled out for unique environmental scrutiny 

should be dispelled because there is a long history of 

guidelines and regulations lim.iting other insect control. 

tactics including synthetic and naturally derived insecticides, biological control agents, insect growth regulators, 

antifeedants, and even cultural control (e.g., Charudattan 

and Browning 1992; Coulson and Soper 1989; Howarth 

1991; Mcevoy 1996; Miller 1990; Samways 1988; Upholt 

1985; USDA FS 1995). 

A specific risk merits concern where its potential is supported by established mechanisms and relevant precedent. 

These criteria are met for several potential threats arising 

from 3 general categories of risk: 1) escape of engineered 

germplasm; 2) evolution of resistant biotypes; and 3) alteration of multi-trophic processes. Underlying mechanisms for 

each of these have been well established from multiple disciplines and across molecular through community levels. 

Risks can be prioritized as to whether: 1) they would be localized or affect adjacent ecosystems; 2) environmental harm 

would depend on continued deployment or be self-perpetu- · 

ating; and 3) potential ameliorating tactics are available. 

Table 5 summarizes the major anticipated risks, general 

mechanisms by which they might occur, historical precedents 

from which valuable lessons can be applied, and possible 

preventative strategies. Four points emerge from this overview. First, there is a need for proactive research on the likelihood of various environmental hazards and tactics for 

offsetting them. Second, interdisciplinary approaches are 

essential. Many of the challenges associated with plant genetic engineering may be identified from ecological perspectives, yet have fundamentally molecular solutions and vice 

versa (Raffa 1989).It is especially important that integrative 

collaborations function throughout the entire discovery and 

development process, rather than in sequential fashion. Sequential approaches fail to fully synergize the expertise that 

enhances efficacy and environmental safety and are likely 

to generate rivalries from differing vested interests. Third, 

none of the risks appears unmanageable if appropriate molecular, physiological, ecological, and management strategies are employed in a cohesive fashion. Fourth, Populus 

provides a particularly suitable model for research and deployment. There is a strong knowledge base from genetic, 

physiological, ecological, and production perspectives, and 

a need for traditional and emerging forest products that can 

be economically produced by this genus. 

Table 5. Summary of environmental risks, mechanisms, precedents, and preventative strategies for genetically. engineered 

insect resistant Populus. · 

Risk 

Escape 

Resistant biotypes 

Altered tritrophic 

processes 

258 

Mechanisms 

Pollen transfer; hybridization 

vegetative materials 

Altered selection pressures; 

release from competitors, 

dispersion, cross resistance 

Direct & indirect affects on 

beneficial species; effects of 

gene products on nutrient cycling 

Precedents 

Introduced 

pests 

Pesticides; 

resistant cultivars 

Introduced pests; 

pesticides; 

pollutants 

Prevention 

Sterility; site management 

early harvest 

Variable & opposing selective 

pressures; between- & within-plant 

mosaics, tissue-, temporal- & woundspecific expression 

Specificity of gene products and 

breakdown products; rapid 

environmental turnover of gene 

products; monitoring 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
''W 

Environmental Risk Assessment and Deployment Strategies for Genetically Engineered Insect-Resistant Populus 

Acknowledgments 

This work was supported by the University of Wisconsin-Madison College of Agricultural and Life Sciences, 

Hatch, the UW-Madison Graduate School, McintireStennis, and the Consortium for Plant Biotechnology. 

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263 
Chapter33 

Biotechnology Risk Assessment: 

A View From Plant Pathology1 

X. B. Yang 

Introduction 

Agricultural science has established a framework for biological impact assessment through past work. Various risk 

assessments for diverse areas of agriculture, resulted in an 

accumulation of considerable information. Baseline information for biological impact assessment dates from the last 

century (Fulkerson 1987). One type of biological impact 

assessment is the environmental impact assessment on the 

release of transgenic poplar. Plant pathologists played a 

major role in an early experimental release of transgenic 

poplar (McNabb et al. 1991). Plant pathology has provided 

much of the information for developing biological impact 

assessment (Teng and Yang 1993) with a focus on complex 

microbe-related risk assessments. This paper discusses biological risk assessment for woody plants compared to herbaceous crops, specifically: 1) the need for and status of 

risk assessment; 2) risk assessment concepts; and 3) risk 

assessment methodology. 

Need for and Status of 

Risk Assessment 

Biotechnology has changed some sectors of agriculture 

into industries perceived as high risk and high return. The 

increasing ability to manipulate genetic components in 

, Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

264 

biological systems elevates the potential for ecosystem 

impacts. Some propose that genetic engineering could create ecologically high-risk, super-competitive organisms 

that could alter ecosystem function. Biotechnology has 

generated new concepts and potentials for modifying biological systems. Biological scientists are asked to design 

predictable biological systems for human needs while sustaining natural resources (Fulkerson 1987). Many biological science disciplines assess potential ecosystem impacts 

from genetic engineering technology. Forestry initially focused on poplar, an agricultural crop grown worldwide 

for fiber, energy, and wood production, to assess the impact of biotechnology on the environment (Klopfenstein 

et al. 1991, 1993; McNabb et al. 1991). 

Biological impact assessment estimates the potential or 

actual impact, including hazards and benefits, of the presence, introduction, or entrance of specific organisms into 

a biological system. Impacts can arise from the introduction of any new technology into a natural ecosystem, 

whether a physical product or knowledge-based process. 

All impacts should be assessed. 

In microbial ecology, issues regarding testing and application of biotechnological products are frequently discussed (GilJett 1986; Lindow et al. 1989; Turgeon and Yoder 

1985). Because of the nature of plant pathogens, and because microbes form the lower trophic levels of most ecosystems, genetic engineering of organisms has been 

considered a risk to ecosystem stability. This potential for 

environmental risk is associated with the: 1) creation of a 

new pest; 2) enhancement of existing pathogens through 

gene transformation; 3) harm to nontarget species; or 4) 

any other ecosystem disruption. Early environmental impact assessment of transgenic microbes involved the potential application of non-ice-nucleating bacteria to prevent 

frost injury in California (Andow et al 1989; Lindow et al 

1983; Lindow and Panopoulus 1988). Currently, some epidemiological studies concern horizontal gene transfers in 

microbes such as those using Colletotrichum spp. for biological weed control (TeBeest et al. 1992). 

The environmental impact of transgenic woody plants 

is a major issue in the development and application ofbio-
technology. A concern of risk assessment is the potential 

for transgenic plants to displace native species (Duchesne 

1993; Pimentel et al. 1990; Rogers and Parkes 1995; Teng 

and Yang 1993; Tiedje et al. 1989). Similarly, the potential 

of transgenic woody plants to replace wild flora is also a 

major concern. The need to assess the biotechnological 

impacts on nontargeted pests and nonpests, and the potential threat this poses to forest ecosystems has been discussed (Duchesne 1993). Unfortunately, assessing the 

environmental fate and impact of transgenic trees lags 

behind the ability to create them. Methods are needed to 

assess the environmental impact of transgenic trees to 

safely incorporate them into forestry research and silviculture. 

Risks associated with transgenic plants have been extensively discussed (Gould 1988; Pimental et al. 1990; 

Tiedje et al. 1989). In 1987, transgenic tomato plants were 

the first field-tested, genetically engineered, food crop 

(Muench 1990). A gene encoding the coat protein of tobacco mosaic virus (TMV) was introduced into tomato 

plants for virus resistance. Risks associated with such a 

release are the potential formation of a virus with altered 

vectors and new host ranges and the possibility of new 

gene combinations (Zoeten 1991).Alternatively, such new 

combinations will likely occur less than that in nature (Falk 

and Bruening 1994). In other countries, various transgenic 

agricultural plants are at different stages leading up to 

field tests (e.g., tungro-resistant rice and virus X-resistant 

potato). 

In the scientific community, a consensus is emerging on 

how much biosafety is needed. However, until a common 

policy is agreed upon, countries will continue to have differing guidelines. Transgenic hybrid poplar trees were used 

as an early first field test to assess the risk of transgenic 

woody plants (McNabb et al. 1991). The risk of harmful 

gene transfer via pollen dispersal was considered 

(McPartlan and Dale 1994; Sawahel1994; Williamson 1993). 

A procedure to assess such risk was proposed in the Netherlands (Evenhuis and Zadoks 1991). However, methodology has not been developed. Teng and Yang (1993) 

proposed an outline for assessing the microbe-related risk 

on herbaceous crops. A conceptual risk assessment outline includes the common principles of impact assessment 

from different specializations, many of which are applicable to risk assessment for a woody crop such as poplar. 

~c~ Concepts of Risk Assessment 

l...tiv 

The National Academy of Sciences (1983) defines "risk 

assessment" as the use of scientific methods, models, and 

data to develop information about specific risks. More reUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Biotechnology Risk Assessment: A View From Plant Pathology 

cently, risk assessment was defined as a process to determine and evaluate potential risks, and the magnitude and 

probability of those risks occurring (Teng and Yang 1993). 

There are 2 steps in risk assessment: 1) risk determination, 

which is identifying and characterizing the risk source; and 

2) risk estimation, which is estimating the probability and 

magnitude of adverse effects from an introduced organism in an ecosystem (figure 1). Biotechnology-related impact assessment is concerned with the estimation of 

potential or actual consequences after introduction of 

transgenic products into an ecosystem. Risk assessment 

before field testing may be premature unless the risk is 

known (Teng 1991). 

Biotechnology-related risk concerns hazards that are 

negative acts or events in quantitative terms with the probability of risk. In this concept, risk assessment includes: 1) 

identifying the hazard; 2) characterizing the risk; and 3) 

managing the risk. The first 2 steps are similar to the risk 

determination step previously mentioned. Risk; in both of 

the above contexts, is used in plant pathogen-related assessments; however, the concept of risk changes with the 

discipline or situation. For example, risk is also considered the product of probability and the impact of a hazard, where hazard is any undesirable event (Evenhuis and 

Zadoks 1991). 

"Monitoring," repetitive measurements made to specify 

the state of a system over time, does not include the data 

interpretation. Monitoring can provide a "time series," 

Risk Determination 

+ 

Data and Information "-----_.( Database J Generation 

+ 

System Synthesis 

+ y 

Prediction ( - GIS ~ 

i_ 

Risk Interpretation 

Figure 1 . Schematic of risk assessment process in the 

context of biological impact assessment. 

l 

265 
Section V Biotechnological Applications 

which is a collection of observations made sequentially in 

time to obtain "background" or "baseline information" 

(Duniker 1989). The background or baseline information 

is a description of conditions or dynamics existing in an 

ecosystem before an intervention, such as introducing a 

genetically engineered microbe (GEM), and serves as a 

check for any assessment. Statistically, a system baseline 

may be the inherent variability of an ecosystem. By using 

different statistical techniques, such as a time-series analysis, variability of system output can be partitioned into 

inherent variability (regular pattern) and noise. For example, Jacobi and Tainter (1988) reported a time series of 

drought affects on loblolly pine growth measured as reduced annual increment compared with the healthy status. The pine trees, measured annually for 60 years, showed 

a declining growth rate in the first 30 years, followed by a 

growth rate increase then a decrease again. Tune-series 

analysis detected a 3-year periodic pattern in the data 

(Yang, unpublished). Patterns from such long-term data 

are baselines for comparison with predicted values, and 

the noise level provides information to estimate uncertainties of the system and its perturbations. 

A "marker" is critical for quantitative assessment of recombinant DNA (recDNA) released into the environment. 

A good marker is sensitive, specific, efficient, and low cost. 

Several major techniques exist for DNA marker development: genetic-engineering, radioactivity, fluorescence, and 

immunology. Currently, antibiotic resistance is the primary 

marker for quantitative monitoring of GEM populations 

(Kluepfel et al. 1991). Markers of this type generally affect 

the competitive ability of an organism and therefore, are 

less useful for monitoring programs that are aimed at survival prediction. In the transgenic poplar, a chloramphenicol acetyltransferase (CA1) gene is used as reporter and 

marker gene (McNabb et al. 1991). When detected by a 

DNA probe, specific sequences of genetic code provide a 

sensitive marker, but associated cost and time limitations 

may restrict quantitative analysis. 

The "endpoint" is a value used to characterize change 

at a particular level of interest (Suter 1990). Any change in 

an ecosystem arises from changes at the individual, population, community, or ecosystem level. To assess impact 

efficiently, an endpoint is defined to determine assessment 

output clearly. Suter (1990) proposed 2 types of endpoints 

in environmental risk assessment: 1) "assessment endpoint," which is a formal expression of the actual environmental values to be protected; and 2) "measurement 

endpoint," which is an expression of an observed or measured response to the hazard. Suter suggested that all risk 

assessments must have assessment endpoints based on 

assumptions; however, a measurement endpoint is notalways possible because it must be based on observations. 

Suter (1990) proposed that a good assessment endpoint 

should be: 1) socially and biologically relevant; 2) have 

clear a operational definition, measurability and accessi266 

bility to prediction; and 3) be sensitive to hazard. The early 

field-test of transgenic poplars at Iowa State University 

had a well defined operational endpoint, which was to 

measure growth of transgenic poplars in comparison to 

nontransgenic poplars under field conditions (McNabb et 

al. 1991). 

Assessment Methodology 

Risk Determination 

To efficiently assess a risk, ecosystem components at the 

individual, population, or community level must be outlined to define outputs and endpoints. Well-defined outputs and endpoints sharpen research objectives by 

allowing identification of knowledge gaps and data collection. There are direct and indirect effects from the introduction of transgenic plants into an environment. Direct 

effects are predictable by well-conducted risk assessment 

studies. Indirect effects, which are less predictable than 

direct effects, occur on nontargeted organisms feeding on 

the targeted organisms. For example, many lepidopteran 

(butterfly and moth) species were destroyed by parasites 

imported into Hawaii for moth control, causing the extinction of several important predators including wasps 

and insect-eating birds (Howarth 1980). Similarly, using 

fungi to control weeds can adversely affect animals that 

depend on the weeds. Fuxa (1988) estimated that about 8 

percent of introductions for biological control, including 

insects and fungi, have caused extinction of native species. Sometimes, the physical environment could also be 

affected by introduced organisms. For example, large-scale 

application of non-ice-nucleating bacteria could alter normal precipitation patterns. Gene flow of an introduced 

transgene into nontransgenic populations is also a major 

concern because it could result in unwanted evolutionary 

consequences within an ecosystem (McPartlan and Dale 

1994; Morris et al. 1994, 1994; Sawahel1994). Assessing indirect effects from ecological and evolutionary consequences 

on the ecosystem is, however, difficult (Williamson 1993). 

In plant pathology, 2 types of potential risk occur. If genetically modified crops are introduced, there is a risk that 

low durability resistance (Miller 1993; Rogers and Parkes 

1995) or a change in pathogens, such as plant virus alteration will be introduced (Zoeten 1991). If modified microbes are introduced, there is a risk that they could 

become a new pathogen. Methods to identify this type of 

risk are available and commonly involve host-range testing of microbes before experimental release. Currently, to 

determine host range a close phylogenetic relationship between plants and their coevolved pathogens is assumed. 

For example, recombinant ice-nucleating bacteria were 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
tested on 75 plant species for pathogenicity and survival. 

Often, assessment results vary with the testing scale. Two 

pathogen-based biological control studies used the same 

fungus, Colletotrichum gloeosporioides f. sp. aeschynomene 

(TeBeest 1988; Weidemann and TeBeest 1990), but a different number of species to determine the host range. In 

these studies, the potential number of hosts increased 

as the number of tested species increased. Host range 

testing is helpful to avoid nontarget effects; however, 

Hollander (1991) considered that a pathogen's host 

range can change to permit exploitation of new food 

sources. For example, about 30 percent of pathogens 

attacking U.S. crops, originally fed on native vegetation before evolution allowed feeding on crop plants. 

While much has been learned about pathogen-host coevolution, our ability to predict potential genetic variability of many pathogens remains limited (Weidemann 

and TeBeest 1990). 

Information is lacking on the general risks of transgenic 

woody plants because data on the environmental impacts 

of genetically modified, perennial crops, which are allowed 

to undergo reproductive processes in the field, is limited. 

Because woody plants are part of natural forests, their risk 

management is less controllable than annual/biannual 

crop systems. Studies are needed to directly address the 

environmental impacts of transgenic woody plants. Because of diversity in forest ecosystems, risk assessment of 

transgenic forestry crops is distinct from that of agronomic 

crops. Forest trees have undergone relatively little domestication and often have re~ated wild relatives growing in 

the same area, which increases the likelihood of hybridization between introduced and native trees. For example, 

some researchers used genetic engineering to improve 

herbicide resistance of woody plants (Brasileiro et al. 1992; 

De Block 1990; Devillard 1992; Fillatti et al. 1987). Hybridization between transgenic and wild plants can produce a 

means of escape for engineered herbicide-resistance genes. 

Klinger and Ellstrand (1994) demonstrated with Raphmws 

sativus that an advantageous transgene introduced into 

natural populations tends to remain. Raybould and Gray 

(1994) suggested a possible invasion of hybrids produced 

by crossing genetically modified crops with natural communities. 

Risk associated with transgenic trees involves the following questions: 1) Would the engineered gene be transmitted to other trees in the wild? 2) How does transgenic 

pollen compete with wild-type pollen in the environment? 

3) What is the fitness of the developing progeny resulting 

from transgenic pollen compared with that of progeny resulting from wild-type pollen? 4) What is the stability and 

potential spread of the transgene in wild species? and 5) 

What are the overall effects of the transgene on the ecosystem? Another chapter (Raffa et al.) in this volume provides a detailed analysis of determining the potential risk 

associated with releasing transgenic poplar. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Biotechnology Risk Assessment: A View From Plant Pathology 

Information Collection and Generation 

Data and information from previous research have been 

used in almost every study on impact assessment. 

MacKenzie and Henry (1991) stated, in the National Biological Impact Assessment Program (NBIAP), that analysis of existing knowledge would identify information gaps 

and determine the biosafety research needed. Before it is 

analyzed, data cannot provide the basis for a decision. 

Published results appropriately interpreted are considered 

information and are acceptable for use in decision-making. However, because information can be misleading, 

knowledge should be considered an abstraction of validated information. Knowledge is useful for reliable assessment, but assumptions are often required when knowledge 

is unavailable. 

Because of the perceived or unknown nature of an introduced organism, genetically engineered organisms are 

tested in microcosms to collect data for a risk assessment 

project. A preliminary step is to use a microcosm analogous to field conditions for collecting data in the laboratory. A microcosm is a powerful tool for collecting primary 

data under· relatively realistic, albeit restricted, conditions, 

which allows diverse plant pathogens to be safely and efficiently studied. 

In plant pathology, microcosms are used to test an 

organism's response to specific environmental factors, 

which allows quantitative extrapolation of research results 

to the field environment for model development. There 

are 2 types of microcosms: 1) a simple design such as a 

growth chamber, which answers a few specific questions; 

and 2) a subunit of a natural system that contains selected 

sensitive abiotic and biotic components (Fournie et al. 1988; 

Pritchard et al. 1988). Data collection from a microcosm 

depends on the specific endpoint(s) of an assessment. For 

transgenic herbicide-resistant soybeans, many tests can be 

conducted within a microcosm. Plant pathologists can use 

microcosms to assess the survival of microbes under various simulated environments. Transgenic poplar plantlets 

were tested in a microcosm greenhouse at Iowa State University before field release (Klopfenstein et al. 1991). 

Recent studies show that microcosm tests are useful to 

predict outcomes of many field trials of transgenic agronomic crops or microbes. Lindow and Panopoulos (1988) 

compared survival of non-ice-nucleating Pseudomonas 

syringae in microcosms to survival in field tests and concluded that microcosm results can closely predict field 

outputs. Work by Kluepfel et al. (1991) and Cook et al. 

(1991) provides additional evidence that microcosm studies can predict the spread and survival of genetically engineered bacteria in the field. 

However, no microcosm study can replace field trials. A 

field test provides data on the population dynamics of an 

organism under natural conditions. After microcosm studies show that potential hazards are controllable in the field, 

267 
Section V Biotechnological Applications 

field tests of introduced organisms may be needed after 

approval by the appropriate government agencies. In a 

microcosm, simulating an entire system with all possible 

environmental conditions is impossible. Only field experiments provide data that comprehensively reflect theresponse of a released organism. 

Most early field experiments examined the development 

of released transgenic plants. For example, the field test of 

transgenic poplar in Iowa determined if introduction posed 

an environmental risk (McNabb et al. 1991). Little information is available on competition between transgenic and 

wild-type woody plants. Information on competition between introduced and native organisms is urgently needed 

for regulatory agencies associated with risk assessment. 

Such information can only be confirmed in the field. An 

engineered trait without any adaptive advantage will probably not persist in the population. The selective advantage of an engineered trait must be great enough to improve 

the fitness of the transgenic plants in comparison to 

wild-type plants. Apart from their value in the impact assessment process, data from field studies also validate results from models and confirm conclusions from 

microcosm studies. 

Synthesizing Information 

Mechanisms for rationally synthesizing information are 

essential to the assessment process (Gillett 1986). This synthesis allows organization of data by ecological and statistical methods to reflect the behavior of the ecosystem after 

introduction of the genetically modified organism (GMO). 

By re-examining old data and synthesizing results, new 

information is often derived because the entire ecosystem 

is being considered. Models, expert panels, and expert 

systems synthesize information. Expert panels are useful 

when quantitative data is unavailable, while models are 

valuable when enough quantitative data exist to estimate 

the parameters of an assessed system. Models also provide information about the behavior of an introduced organism under ranging weather conditions, which expert 

panels and expert systems do not. These projections depend on a model's ability to reflect interactions among host, 

pathogens, and environment (Teng and Yuen 1991). 

Expert panels, groups of subject matter authorities, assess risk. Experience with traditional unmodified organisms provides the basis for risk analysis of genetically 

modified organisms (MacKenzie and Henry 1991). The U.S. 

National Biological Impact Assessment Program (NBIAP) 

(MacKenzie 1991) used this approach to solicit consensus 

protocols for field testing GEMs and genetically altered 

plants. Expert opinion was used to estimate disease impact on crops and the potential of biotechnology to reduce 

impact (Herdt 1991). 

An expert system, composed of a knowledge base and a 

268 

procedure to infer an answer, is a computer program designed to simulate the problem-solving mechanism used 

by subject matter experts (Latin et al. 1987). Until general 

knowledge on the entire impact assessment process is 

available for field tests with GMOs, the only available system for impact assessment is the electronic bulletin board 

used by the National Biological Impact Assessment Program (MacKenzie and Henry 1991). In comparison with 

expert panels, expert systems are better for information 

delivery rather than improving assessment reliability. 

When quantitative information is available, a model 

provides a positive or negative answer and an assessment 

of risk magnitude. Empirical and me~hanistic models are 

available for prediction. Empirical models use statistical 

methods such as regression models, with limited predictive ability for biotechnology-related assessment. Mechanistic models use an understanding of underlying 

processes within an ecosystem. Most models used for risk 

assessment are simulations that, when coupled with databases and extrapolation algorithms, are useful to assess 

different ecosystem change strategies (Teng 1991). Simulation modeling, considered the most reliable technique 

for risk assessment (Duniker and Baskerville 1986; Teng 

1991 ), has been used to assess chemical risks to the environment (Barnthouse and Suter 1984; O'Neill et al. 1982), 

impacts of plant pathogens in biocontrol (DeJont et al. 

1990), plant quarantine programs (Yang et al. 1991), and 

biotechnology benefits (Andow et al. 1989). A simulation 

model was used with historical weather data to determine 

risk of several diseases to wheat (Luo and Zeng 1990; Luo 

et al. 1995). In global warming studies, simulation models 

are frequently used to assess the future impact on 

biodiversity under different climate scenarios. 

The CLIMEX computer program developed in Australia exemplifies computer techniques for generating predictive models. The program, which compares climates in 

ecology, was applied to modeling microbes, arthropods, 

and plants (Sutherst and Maywald 1985; Worner 1988). 

Concerns about global climate change, have focused attention on the theory and methodology of climatic requirements of organisms as tools for predictive ecology. If 

transgenic poplar grows over large areas, the CLIMAX 

program may quantitatively assess potential damage from 

new insects, pathogens, and weeds. 

Prediction and Evaluation 

Risk prediction uses data-based predictive models to 

estimate chances of undesirable events. Prediction can be 

over time, which is assessing a future event or risk based 

on present information, or can be over space, which is regionally assessing the event or risk over a defined area 

based on information from multiple sites. Prediction over 

time is feasible if baseline information is available; howUSDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
ever, accuracy decreases as the predictions extend further 

into the future. 

Impact prediction over space and time relies on a quality database; prediction value is enhanced by data used in 

conjunction with geographic inform~tion systems (GIS) 

and geostatistical techniques. A database is critical for any 

regional assessment and should contain spatial data sets 

of vegetation, hosts, pathogens, weather, soil, and other 

variables that represent ecosystem attributes over time. 

Data on each variable is a descriptive information layer 

covering the region of interest. Because most ecosystem 

variables are interrelated, the population magnitude can 

be calculated if such relationships are available. Several 

computerized databases are being developed for impact 

assessment in plant pathology. 

Geographic information systems, computer programs 

that use specific mathematical algorithms to enable input, 

management, analysis, and display of geographic point 

data (Berry 1987), are powerful tools for environmental 

risk assessment. A quantitative regional assessment requires analyses of extensive spatial data. In a GIS, each 

data set is a layer over a geographic area; layers are physically and biologically correlated. Knowing the relationships among layers allows assessment of regional impact. 

A regional impact map can be composed from spatially 

interrelated maps of individual layers. Because maps are 

visual evidence, they frequently help managers assess 

impacts and expedite the decision-making process. 

GISs have been used in biological impact assessment in 

plant pathology. To predic.t the occurrence of potato-late 

blight in Pennsylvania, Royer et al. (1990) used a disease 

model with inputs from a relative moisture and temperature map. Royer and Yang (1991) used similar methods to 

generate a potential epidemic map of soybean rust in Pennsylvania and Maryland. GIS techniques allow generation 

of hazard maps in different forms depending on data set 

availability. However, if data are available from only limited locations, extrapolation across these locations may 

produce an overlay map that generates inaccurate conclusions (Berry 1987). An impact map with distinct symbols 

is appropriate when location number is limited. Using data 

from remote sensing for GIS analysis, Steven et al. (1991) 

predicted the potential invasion of a weed, Dyers woad 

(lsatis tinctoria), iri northern Utah. 

Although tools exist to project system behavior into the 

future, risk evaluation is still an art (Gillett 1986). Because 

risk is the probability of the magnitude of a hazard 

(Duniker and Baskerville 1986), some assessment uncertainty and subjectivity will always exist. Risk management 

depends on the extent that uncertain elements in an assessment have been identified. In microbe-related risk assessment, uncertainty can arise from every ecological 

process and failure becomes a function of these events 

(Alexander 1985). Prediction of survival and spatial dispersal produces the most uncertainty in microbe-related 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Biotechnology Risk Assessment: A View From Plant Pathology 

risk assessment because survival and dispersal are the least 

known aspects of epidemiology. For example, in DeJont et 

al.'s (1990) assessment for a mycoherbicide, the distance 

of spore dispersal was calculated by a Gaussian model with 

limited observations. Predictions with Gaussian models 

for long-distance dispersal (greater than 1,000 m) have not 

peen validated for many pathosystems. Furthermore, 

socio-political factors exert a strong influence on risk interpretation for decision making. As recommended by the 

Gaussian model, it was decided that the safe distance between an applied area and an orchard was 5,000 m. However, the Dutch government determined that the risk of 

using Chondrostereum purpureum to control Prunus serotina 

is acceptable when an application area is less than 500 m 

from an area of commercial fruit production (DeJont et al. 

1990). This illustrates that risk assessment is distinct from 

interpretation. 

Conclusion 

Hundreds of field tests with GMOs have been completed; the results demonstrate that. behaviors of GMOs 

are predictable (Casper and Landsmann 1992; MacKenzie 

and Henry 1991). Currently, no generally acknowledged 

methodology for biological impact assessment exists; most 

research efforts use their own techniques. As discussed 

previously, biological risk assessment includes risk determination and risk estimation. Risk determination results 

in evaluation options, but often is the main assessment 

activity. This activity consists of system definition, risk 

identification, and determination of endpoints and knowledge gaps. Risk estimation generate's data and information from microcosm and field studies, and synthesizes 

ecosystem-w~de information into a prediction system capable of generating options. Results from risk determination and risk estimation are subject to risk evaluation. 

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USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Chapter34 

Biotechnology, Biodiversity, and Bioethics1 

M. Raj Ahuja 

Introduction 

Recent advances in plant biotechnology have enabled 

genetic improvement of forest trees. Basic techniques in 

tissue culture, genetic engineering, and molecular biology, 

developed in herbaceous crops, have been applied to forest tree species with varying successes. Although this suggests that biotechnology methods need furthe r 

investigation and adaptation for application to long-lived 

and highly heterozygous forest trees, several recent advances are promising. These include rapid clonal propagation, germplasm preservation, gene transfer, molecular 

markers, genome mapping, and the isolation, cloning, and 

expression of genes (Ahuja 1991a, 1993a; Ahuja et al. 1996; 

Bonga and Durzan 1987). Although considerable progress 

was achieved in some of these areas during the past decade, others are in the embryonic stage. 

Most of the advances mentioned above are discussed 

elsewhere in this book. I will address clonal propagation 

(in vitro regeneration) and gene transfer (genetic engineering) in relation to biodiversity and bioethics. Although 

somewhat philosophical, the pros and cons of new technologies should be examined in the framework of their 

impact on biodiversity, long-term adaptation, and evolutionary survival of forest trees. Pertinent questions include: 

What rules and ethics should people follow for long-term 

biodiversity conservation? Is the application of biotechnology, involving clonal propagation, likely to substantially 

reduce genetic variation in trees? Would the transfer of 

chimeric genes into forest trees increase the risk of undesirable genetic instability? Would new or foreign genes 

escape from commercial plantations, become established 

'Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. 

Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. 

Micropropagation, genetic engineering, and molecular biology 

of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: 

U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 326 p. 

in natural populations, and later become maladapted? 

Would transgenic plants generate new diseases and pose 

a threat to the host ecosystem? These and related questions are discussed in this chapter. 

Forest Biotechnology 

Forest biotechnology development, although similar to 

that of herbaceous plants, has subtle differences including 

the longevity, heterozygous nature, life cycle, and environment of forest trees. Forest trees have long generation 

cycles, with the vegetative phase ranging from 1 to several decades. Once trees are germinated in nature or transplanted to plantations, they generally remain anchored in 

I location where they are exposed to changing environments and other vagaries of nature. Some of these factors 

may influence their physiology and alter complex morphogenetic processes. During the long life cycle of 

trees, many genetic and epigenetic changes are probably 

important to maintain within-tree and within-population 

genetic diversity, and to assure long-term survival of individuals and populations. Therefore, application of biotechnology to woody perennials should be considered 

long-term goals, as opposed to short-term annual benefits 

appropriate to crop plants that are harvested and replaced 

each year. This suggests a strategy shift for genetic improvement and modification, and for maintenance of forest biodiversity. Two promising methods in biotechnology, 

involving in vitro regeneration and genetic engineering, 

are discussed in relation to forest tree biodiversity. 

In Vitro Regeneration and Genetic ~iversity 

In vitro regeneration of plants may be accomplished by 

exploiting one of the differentiation pathways, either from 

haploid or somatic tissues. The pathway from haploid tissues involves regeneration by organogenesis, which is the 

sequential differentiation (usually not simultaneously) of 

shoots and roots on tissues. The pathway from somatic 

tissues may proceed via embryogenesis, which is when 

273 
Section V Biotechnological Applications 

both shoot and root poles differentiate more or less simultaneously on the embryogenically competent cells. 

Somatic embryos are structurally similar to zygotic embryos and, following germination, produce somatic 

seedlings. 

In vitro regeneration by organogenesis has been achieved 

with many woody plant species. With most, juvenile explants (e.g., embryos, cotyledons, or shoots from young 

plants) were used for clonal propagation (Ahuja 1988, 

1991a, 1993a; Bonga and Durzan 1987; Pardos et al. 1994). 

Rejuvenation by tissue culture from mature tissues remains 

challenging in many forest tree species, although a degree 

of in vitro-conditioned rejuvenation was demonstrated in 

some species, including Populus (Ahuja 1983, 1986, 1987, 

1993b; Ahuja et al. 1988; Chun 1993; Ernst 1993). 

Since the first report by Winton (1968) on plantlet regeneration from callus cultures of triploid quaking aspen 

(P. tremuloides), several other species and hybrids of Populus 

were clonally propagated by tissue culture technology 

(Ahuja 1988, 1991a, 1993a; Bonga and Durzan 1987). By 

employing bud explants from 23~ to 40-year-old trees of 

European aspen (P. tremula) and hybrid aspen(P. tremula x 

P. tremuloides), clonal propagules were regenerated for 

multiple genotypes (Ahuja 1983, 1986, 1993b). Micropropagation varied among the aspen trees. Bud explants 

from some mature aspens were unresponsive to tissue 

culture milieu, while in others they exhibited successful 

differentiation of microshoots for plant regeneration. 

Among other factors, regeneration potential of tissue depends on the explant source, tissue physiological state, time 

of year when tissue collection occurs, and age and genotype of the donor tree. In forest trees, maturation state and 

genotype largely determine the regeneration potential. In 

aspens, tissues from mature-tree buds apparently do not 

require special rejuvenation treatments because bud explants can grow and differentiate on relatively simple culture media. The genotype apparently is predominant in 

determining the in vitro morphogenetic response in aspens. 

In many other hardwoods, such as beech, oak, and most 

conifers, growth and differentiation from tissues of mature trees are strongly influenced by the maturation state. 

Theoretically, maturation state may also indirectly affect 

genetic diversity by modifying propagation qualities. In 

this situation, elite and mature genotypes may not contribute to the overall genetic diversity of the population. 

Regeneration by somatic embryogenesis is another 

promising technique for large-scale, clonal multiplication 

of woody plant species. In most studies, somatic embryos 

were differentiated from the embryonal axes of zygotic 

embryos. Clonal propagation by somatic embryogenesis 

in several conifer and hardwood species was reported 

(Becwar 1993; Gupta et al. 1993; Gupta and Grob 1995; 

Redenbaugh and Ruzin 1989). Since somatic embryos can 

be grown in liquid media, it should be possible to increase 

their production in bioreactors for mass cloning of elite 

274 

genotypes (Becwar 1993; Gupta et al. 1993; Gupta and Grob 

1995). For an efficient delivery system to plant somatic 

embryos in soil, somatic embryos were encapsulated to 

produce "artificial seeds" (Redenbaugh and Ruzin 1989). 

This technology should be available for commercial forestry by the year 2,000 (Gupta et al. 1993). 

In spite of optimism for somatic embryogenesis-mediated clonal forestry, several basic problems must be resolved (Chen and Ahuja 1993). Inducing somatic embryos 

is difficult for many commercially important forest tree 

species and genotypes, or the frequency of induced somatic embryos may be too low for practical applications. 

In several forest tree species, there are also problems with 

maturation and germination of somatic embryos and development of encapsulation protocols leading to somatic 

seedlings. Since somatic embryos usually go thorough a 

callus development phase, the relative genetic stability of 

somatic embryos, somatic seedlings, and somatic plants 

(Ahuja 1991b; Ahuja and Libby 1993) should be investigated at the morphologic and molecular levels before introduction into clonal forestry programs. 

There are 2 main genetic concerns with tissue culturederived plants, whether regenerated by organogenesis or 

somatic embryogenesis. The first relates to genetic instability in plant tissue cultures. Plant cells grown in an artificial environment of tissue culture are often under stress, 

and are likely to generate genetic and epigenetic mistakes; 

rapid mitotic divisions in the callus phase may contribute 

to this instability. Such genetic changes are manifested as 

single gene mutations, chromosome aberrations, and modifications of DNA methylation patterns (Kaeppler and 

Phillips 1993; Phillips et al. 1994). Callus cells are especially vulnerable to tissue-culture induced mutations. One 

possible cause of genetic variation in cultured tissues is 

the use of high concentrations of phytohormones. In particular, the auxin 2,4-dichlorophenoxyacetic acid (2,4-D) 

induces a high frequency of changes in chromosome structure (Pavlica et al. 1991) and variation in DNA methylation patterns (Phillips et al. 1994). New genetic variation 

in micropropagated plants may be reduced by using only 

low concentrations of hormones and excluding 2,4-D. Our 

two-step micropropagation method (Ahuja 1984), using 

an aspen culture medium (ACM) (Ahuja 1983), produced 

over 10,000 regenerants from bud explants of more than 

100 European aspen and hybrid aspen mature trees. In our 

cultures, minimal callus formation occurred, and 

microshoots differentiated directly on the bud explants. 

Very few gross phenotypic variants were observed in these 

micropropagated aspens; however, undetectable gene 

mutations may have been present. Likewise, new genetic 

variation could be minimized in other woody perennials 

by using organogenesis or somatic embryogenesis for regeneration. Although variability will always occur, it depends on how much and what types are acceptable for 

clonal forestry programs. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
A second concern relates to the potential loss of genetic 

diversity by clonally propagating forests. A common belief is that clonal forestry leads to genetically uniform forests, which is a possibility but not a certainty (Libby 1982). 

Through use of sufficient numbers of pedigreed clones, 

genetic diversity can be effectively maintained, better controlled, and even enhanced in clonal forests, as compared 

with management options for zygotic or seedling forestry 

(Libby and Ahuja 1993). Through effective education and/ 

or regulation, common management errors in testing and 

deployment can be avoided in a clonal forestry program. 

Genetic Engineering and Biodiversity 

Although genetic engineering and hybridization by conventional breeding can augment genetic variation in 

plants, there are important differences between these 2 

processes. Not only are different techniques used, but the 

type of genes transferred and the time required also differ substantially. In terms of quick returns, the time needed 

to produce a new genotype can be a critical factor for its 

commercial exploitation. Producing and using genetically 

stable woody plants may require a long time span; therefore, a conflict exists between the basic research required 

to generate and test reliable new genotypes, and the commercial desire for quick returns. 

A breeder uses a backcross program to transfer desirable genes from 1 species to another. Following hybridization, the hybrid and _its progeny are backcrossed 

repeatedly for several generations to 1 parent species so 

that a single dominant gene or a small set of desirable genes 

is transferred. Essentially, this introgressive hybridization 

procedure selects for a single or a few gene(s) from 1 parent in the genetic background of the recurrent backcross 

parent. By using this approach, several useful genes for 

disease resistance or other traits were transferred in plants 

(Ahuja 1962; Ahuja and Hagen 1967; Knott 1961; Sears 

1956). Transfer of such genes may involve intergeneric, 

interspecific, or intraspecific hybridizations. The F1 intergeneric and interspecific hybrids are usually sterile; however, male sterility is more common than female. Some 

viable eggs may be formed in these hybrids between 

widely divergent species or genera; these can be used for 

backcrosses to the recurrent male parent. The hybrid and 

first backcross generations are highly heterozygous and 

exhibit considerable morphological and chromosomal 

variation. The backcross program is accompanied by selection of phenotypes with the desired trait(s) from the vast 

array of genetic variants in the backcross generations. Intraspecific hybridizations involving the transfer of a dominant or a recessive allele from the donor to the recurrent 

parent may similarly involve backcross or selfing cycles 

to achieve the desired goal. However, released genetic 

variability would be relatively less compared with the 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Biotechnology, Biodiversity, and Bioethics 

larger variation that accompanies transfer of genes by the 

interspecific or intergeneric hybrid route. 

Gene transfer by genetic engineering is a one-step process that bypasses the time-consuming hybridization procedure. However, to establish the mode of inheritance and 

transmission of transgenes from male and female gametes, 

hybridization between transgenic and control plants is 

necessary. Genetic engineering uses chimeric or recombinant genes constructed by DNA recombinant technology. 

The chimeric genes are placed between the transferred 

DNA (T-DNA) borders of a disarmed tumor inducing (Ti) 

plasmid from Agrobacterium and transferred to plants 

mainly by an Agrobacterium-mediated gene transfer 

method or a biolistic DNA delivery system. Because a few 

genes are usually located between the T-DNA borders, at 

least 2 or more chimeric genes are transferred to plants 

during genetic transformation. In most studies, 1 selectable marker and 1 reporter gene are included. Other DNA 

sequences are usually included in the cassette but are typically ignored for the analysis. The reporter genes may be 

attached to promoters from viruses, bacteria, or plants. For 

example, the gene encoding neomycin phosphotransferase 

(NPTII), which confers kanamycin resistance from bacteria, is put under the regulatory control of a NOS (nopaline 

synthase) or OCS (octapine synthase) promoter from 

Agrobacterium. A proteinase inhibitor II (PIN2) gene from 

potato conferring pest tolerance under control of 35S, a 

promoter region derived from the cauliflower mosaic virus, or NOS was expressed in transgenic hybrid poplar 

(Klopfenstein et al. 1993). As mentioned, besides the hybrid chimeric genes, other genetic sequences are often 

present between the T-DNA borders; these usually remain 

cryptic or uninvestigated. Therefore, an array of other 

"hitchhiking" DNA sequences in the T-DNA are transferred to the host plant along with the marker and reporter 

genes. 

Transfer of genes by hybridization is a slow process that 

can require years for crop plants and perhaps decades for 

forest trees. Introgressive hybridization involving transfer 

of genes from 1 species to another has been important in 

plant speciation and evolution. Genes that displayed adaptive fitness were retained, while those with low fitness were 

gradually eliminated.· Traditional breeding has been employed for genetic improvement between individuals in a 

species or between related species, where the genetic differences are minimal. Alternatively, genetic engineering can 

move functional genetic traits between widely divergent 

organisms in a relatively short time. Transgenic plants, including Populus, were produced with functional genes from 

insects (e.g., luciferase gene from the firefly) and bacteria 

(e.g., herbicide-resistance genes), and transgenic pigs and 

rodents were produced with functional human genes. This 

phylogenetic leapfrogging offers many unique opportunities to create populations with novel combinations of 

adaptive (Regal1994) and nonadaptive genes. In ecologi275 
Section V Biotechnological Applications 

cally competent organisms, such as forest tree species, introduced genes of low fitness may be eliminated by natural selection over the course of several generations. Most 

new trait combinations in transgenic plants do not seem 

ecologically adaptive, as with the firefly luciferase gene 

in tobacco or Populus. However, genes conferring resistance against diseases, pests, frost, or salinity might be 

more adaptive in selected ecosystems. 

Although genetic engineering offers options for generating new gene combinations that may be adaptive, an inherent risk of genetic instability associated with trans genes exists 

in herbaceous annual plants and forest trees (see chapter 

titled "Transgenes and Genetic Instability" in this volume). 

Not only is transgene expression unpredictable, but 

transinactivation of ectopic or endogenous genes are more 

frequent in transgenic plants than was anticipated. This implies that transgenic plants may be genetically unstable when 

they possess certain transgenes. Long-term testing of 

transgenic trees is recommended before widespread use. 

.Biodiversity is defined as variety and variability among 

living organisms and the ecosystems in which they exist 

(Woodruff and Gall1992). Biodiversity may be considered 

at 3 hierarchial levels: 1) genetic diversity; 2) species diversity; and 3) ecosystem diversity (Smitinand 1995). In 

this chapter, biodiversity is the genetic variation within 

populations of genetically engineered or aggressively bred 

species in a pl~tation-generated ecosystem. In a changing environment, populations with diverse pools of genetic variation should be more ecologically competent, 

with increased response and survival during large and/ 

or rapid environmental change. Tree species belong to this 

category and have survived the vagaries of nature over 

millions of years. Cross-hybridization between individuals within populations or between species and genera has 

enabled natural gene transfer, which has enriched gene 

pools and contributed to survival and evolution. 

Transfer of genes between species and genera by .introgressive hybridization is a slow process, but natural selection allows adequate time to incorporate adaptive gene 

combinations and reject those of inferior fitness. Phylogenetic leapfrogging by genetic engineering, which bypasses 

the traditional genetic tradeoffs (Regal 1994), presents a 

new dimension in biodiversity for long-lived forest trees. 

Whether genetic engineering-mediated gene transfers contribute to adaptive biodiversity in forest tree species or 

generate more genetic instability should be examined on 

a long-term global basis. 

Bioethics in Biotechnology 

Advances in biotechnology, and especially genetic engineering of plants and animals, inspire reflection on their 

276 

short- and long-term affects on the global ecosystem. Bioethical concerns in forestry originated with conservationally minded foresters and environmentalists who had an 

interest in maintaining biodiversity and ecosystem stability (Coufal and Spuches 1995). Disturbances in 1 component of the forest ecosystem can cause devastating affects 

on other ecosystem elements. Certain ethical rules and 

biosafety regulations should be followed during the application of biotechnological research. Conservation of the 

genetic diversity of tree populations and the biodiversity 

of the ecosystems in which they grow is imperative. Because of differences in their value judgements, those interested in quick returns of biotechnological applications 

are often in conflict with "go-slow" traditionalists. 

Forest trees are long-lived, usually well adapted, and 

have relatively long generation cycles with vegetative 

phases that can extend from 1 to several decades. Most 

genetic changes in their genome structure, whether by classical mutation or phylogenetic leapfrogging, may generate instability. However, some genetic changes may have 

adaptive value over time. According to Tiedje et al. (1989), 

evaluation and regulation of transgenic organisms should 

be based on their biological properties, including phenotypes, rather than on the genetic techniques used to produce them. Transgenic trees should be evaluated at 

different stages of their development by morphological, 

biochemical, and molecular methods to monitor transgene 

expression and other unexpected genetic changes. Since 

the potential risks of releasing transgenic poplars are discussed in chapters by Raffa et al. (this volume) and Yang 

(this volume), they are only briefly discussed here. 

The release of transgenic plants into natural ecosystems 

raises several questions: 1) Will transgenic crops generate 

new viruses and new diseases? (Falk and Bruening 1994); 

2) What are the potential risks of cross-fertilization between 

transgenic crops and their wild relatives? (Baranger et al. 

1995; Kareiva et al. 1994; Paul et al. 1995); and 3) Will 

transgenic trees be genetically stable on a long-term basis 

under field conditions? Whether transgenic crops could 

create new viruses and diseases is a small concern for 

woody plants. Some viruses may infect forest trees, while 

others may exist as systemic entities. Unless trees are engineered for resistance against a specific virus, the risk of 

creating a virulent new virus by genetic recombination 

seems minimal. Alternatively, interactions between a 

transgene or its product with an endogenous tree virus 

might produce a mutant virus that may be harmful to the 

host. Therefore, it is difficult to predict if transgenic plants 

could become a source of new viruses. 

The second concern, escape of transgenic pollen into the 

environment and cross-fertilization of wild relatives, poses 

serious biosafety and regulatory problems. In the worst 

scenario, the engineered plants or their hybrid derivatives 

may become invasive and eliminate original untransformed populations. Pollen can travel long distances or 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
can be spread by pollinating insects. Forest trees are generally cross pollinated; therefore, the spread of transgenic 

pollen to related species is considered a genuine biological concern. A recent study shows that containment of recombinant pollen or transgenes is unlikely if physical 

isolation is the only strategy (Kareiva et al. 1994). Genetic 

engineering of reproductive sterility in trees is another 

option (Strauss et al. 1995) for containing transgenes. Previously, it was argued that engineering of male sterility 

could stimulate faster tree growth and wood production, 

reduce production of allergic pollen in the atmosphere, and 

offer new options for hybrid breeding (Strauss et al. 1995). 

Currently, it is thought that some of these suggestions are 

based on speculation. Studies are needed to determine 

whether wood production can be increased by decreasing 

floral tissues through manipulating floral-specific genes, 

toxin encoding genes controlled by floral-specific promoters, or other approaches. However, manipulating floralspecific genes, or introducing the rolC gene from 

Agrobacterium rhizogenes with an appropriate promoter, 

may produce growth abnormalities in trees. The rolC gene 

under control of a 355 promoter causes a degree of male 

sterility in tobacco and a multitude of morphological and 

physiological changes (Schmiilling et al. 1993). The rolC 

gene under control of 2 different promoters, 355 and rbcS, 

has also been transferred to aspen (Fladung et al. 1996). In 

aspen, 355-rolC transgenics exhibited much smaller, 

nonlanceolate, pale-green leaves than rbcS-rolC transgenic 

aspens, which produced pale-green leaves that were only 

slightly smaller than the controls. In tobacco, the 355-rolC 

transgene induces lanceolate and pale-green leaves. A 

transgene may act differently in an annual tobacco plant 

and a perennial Populus tree. The effects of rolC in tobacco 

or Populus have only been studied under greenhouse conditions. Therefore, whether this pleiotropic gene will behave similarly under the 2 sets of environments cannot be 

predicted. Nevertheless, all strategies toward containment 

of transgenic pollen or lessening of floral tissues in 

transgenic trees should be encouraged. 

Gene silencing has developed into a challenging field 

of study that will hopefully contribute toward understanding trans genes and native gene stability. Recently, instances 

of transgene instability were reported (Finnegan and 

McElroy 1994; Jorgensen 1995; Matzke and Matzke 1995). 

The bioethic principle requires that all research in genetic 

engineering of plants or animals be reported for administrative authorities to conduct proper risk assessment for 

biosafety regulation of transgenic organisms (Giampierto 

1994; Miller et al. 1995). More than 800 applications have 

been filed for testing genetically modified organisms in 

the environment, most of them in the United States (The 

Gene Exchange 1993). Nearly all of these tests were conducted on small plots, which may not reflect the entire 

spectrum of transgene-environment interactions. Since 

commercial application of transgenic crops would occur 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Biotechnology, Biodiversity, and Bioethics 

in large-scale field facilities, extrapolations from small plots 

may not provide adequate risk assessment information 

(Seidler and Levin 1994). 

Value Judgement 

Recent advances in biotechnology have ushered in a new 

era for the genetic improvement of forest tree species. 

Micropropagation through organogenesis and somatic 

embryogenesis has been achieved in many woody plants. 

Biotechnological procedures have also been effectively 

used to preserve woody plant germplasm (Ahuja 1989, 

1994; Chun 1993). Various approaches have been used to 

transfer foreign genes to forest trees. Genetic engineering 

can provide exceptional genotypes for integration into 

clonal forestry and zygotic forestry programs. However, 

concerns have been raised regarding decreased 

biodiversity by clonal propagation, increased risk of genetic instability in genetically engineered plants, and the 

potential escape of transgenes into natural populations. 

Genetic variation can be maintained and better managed 

by employing appropriate numbers of genetically diverse 

pedigreed clones in clonal forestry programs (Ahuja and 

Lil:?by 1993; Libby 1982). Multiclonal blocks of clones are 

recommended for conservation of ecosystem biodiversity. 

The highest risk regarding transgenic crops is the escape of transgenic pollen into nature and hybridization of 

wild relatives (Abbot 1994;Angle 1994; Kareiva et al. 1994; 

Regal1994; Seidler and Levin 1994; Williamson 1994 ). Since 

transgenic pollen would be difficult to contain under natural conditions, especially when transgenic crops are grown 

commercially, other strategies, including genetic manipulation of reproductive sterility (Strauss et al. 1995), should 

be explored for forest trees. Presently, more information is 

needed about dispersal, and the ability of transgenic pollen to preferentially pollinate wild relatives (Baranger et 

al. 1995; Lefol et al. 1995; Paul et al. 1995); or possibly 

transgenic pollen may pose no more risk than normal pollen from untransformed trees. Because of the relative ease 

of in vitro regeneration and genetic transformation, Populus 

can serve as a valuable model system to evaluate questions regarding the ecological risks of transgenic trees. 

Nevertheless, environmentally compatible applications of 

biotechnology must be developed (Frederick and Egan 

1994), and biosafety regulations and directives must be 

followed until the risk factors associated with genetically 

engineered woody plants are assessed and the adequate 

means to address them are found. 

The relevance of bioethics in forestry is emphasized in 

the following quote from a recent paper by Coufal and 

Spuches (1995). "The value of ethics in forestry is unlimited, although not always plain to see. At best, ethics com277 
Section V Biotechnological Applications 

mands the power of moral authority, an authority that can 

often be flouted with few visible consequences, unlike the 

authprity of the laws of nature. If we can believe that ethics allows us to view the world more clearly and not be 

limited by our scientific perceptions of it; that good forestry management will not be possible unless ethical dimensions are given the same consideration as scientific 

and economic factors; and thus that ethics should be an 

integral part of all forestry curricula .... " 

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279 

Section VI Appendices 
Authors and Affiliations 

M. Raj Ahuja 

Federal Research Centre for Forestry and Forest Products, 

Institute of Forest Genetics 

Sieker Landstrasse 2 

22927 Grosshansdorf 

Federal Republic of Germany 

Tel: 49 4102 696156 

Fax: 49 4102 696 200 

Marie Baucher 

Laboratorium voor Genetica 

Department of Genetics 

Flanders Interuniversity Institute for Biotechnology 

Universiteit Gent 

K.L. Ledeganckstraat 35 

9000 Gent 

Belgium 

Tannis L. Beardmore 

Natural Resources Canada 

Canadian Forest Service 

Hugh John Fleming Forestry Complex 

Regions St. S. 

P.O. Box 4000 

Fredericton, New Brunswick 

Canada E3B 5P7 

Tel: 506 452 3881 

Fax: 506 452 3525 

E-mail: tbeardmore@fcmr.forestry.ca 

Wout Boerjan 

Laboratorium voor Genetica 

Department of Genetics 

Flanders Interuniversity Institute for Biotechnology 

Universiteit Gent 

K.L. Ledeganckstraat 35 

9000 Gent 

Belgium 

Tel: 32 9 2645202 

Fax: 32 9 2645349 

E-mail: woboe@gengenp.rug.ac.be 

Richard C. Carman 

USDA Forest Service 

Rocky Mountain Forest and Range Experiment Station 

National Agroforestry Center 

East Campus - University of Nebraska 

Lincoln, NE 68583-0822 

USA 

Tel: 402 437 5178 ext 17 

Fax: 402 437 5712 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Authors and Affiliations 

Maria Teresa Cervera 

Laboratorium voor Genetica 

Department of Genetics 

Flanders Interuniversity Institute for Biotechnology 

Universiteit Gent 

K.L. Ledeganckstraat 35 

9000 Gent 

Belgium 

Tel: 32 0 9 264 53 70/71 

Fax: 32 0 9 264 53 49 

E-mail: mamon@gengenp.rug.ac.be 

Brigitte Chabbert 

Laboratoire de Chimie Biologique 

Institut National de la Recherche Agronomique (INRA) 

F-78850 Thiverval-Grigon 

France 

Pierre J. Charest 

Natural Resources Canada 

Canadian Forest Service 

580 Booth Street, 7th floor 

Ottawa, Ontario 

Canada KIA OE4 

Tel: 613 947 9011 

Fax: 613 947 9090 

E-mail: pcharest@am.ncr.forestry.ca 

Vincent L. Chiang 

Institute of Wood Research 

Department of Forestry 

Michigan Technological University 

1400 Townsend Drive 

Houghton, MI 49931-1295 

USA 

Tel: 906 487 2454/2959 

Fax: 906 487 2915 

E-mail: vchiang@mtu.edu 

Young Woo Chun 

College of Forestry 

Kookmin University 

861-1, Chongnung-dong, Songbuk-gu 

Seoul, 136-702 

Republic of Korea 

Tel: 82 2 910 4812 

Fax: 82 2 910 4809 

E-mail: ychun@kmu.kookmin.ac.kr 

281 
Section VI Appendices 

Gary D. Coleman 

Deparbnent of Horticulture and Landscape Architecture 

122 Holzapfel Hall 

University of Maryland at College Park 

College Park, MD 20742-5611 

USA 

Tel: 301 405 4371 

Fax: 301 314 9308 

E-mail: GC76@umail.umd.edu 

Daniel Cornu 

Station d' Amelioration des Arbres Forestiers 

Institut National de Ia Recherches Agronomique (INRA) 

F-45160 Ardon 

France 

Yvonne Devantier 

Molecular Genetics and Tissue Culture Group 

Petawawa National Forestry Institute 

Canadian Forest Service 

Chalk River, Ontario 

Canada KOJ 1JO 

Madelyn C. Dillon 

USDA Forest Service 

Rocky Mountain Forest and Range Experiment Station 

3825 East Mulberry Road 

Fort Collins, CO 80524 

USA 

Tel: 970 498 2344 

Fax: 970 498 1660 

E-mail: FSWA/S=M.Dillon/ ou=S28A 

@MHS.ATIMAIL.COM 

DG address: M.Dillon:s28a 

Ronald J. Din us 

Deparbnent of Wood Sciences 

Faculty of Forestry 

University of British Columbia 

Vancouver, B.C. 

Canada V6T 124 

Mary Ellen Dix 

USDA Forest Service 

Rocky Mountain Forest and Range Experiment Station 

National Agroforestry Center 

East Campus - University of Nebraska 

Lincoln, NE 68583-0822 

USA 

Tel: 402 437 5178 ext 25 

Fax: 402 437 5712 

DG address: M.Dix:S28L04a 

282 

Gerry C. Douglas 

Agricultural and Food Development Authority 

Kinsealy Research Centre 

Malahide Rd. · 

Dublin 17 

Ireland 

Hiroyasu Ebinuma 

Central Research Laboratory 

Nippon Paper Industries 

Oji 5-21-1, Kita-ku 

Tokyo 114 

Japan 

Fax: 81 3 3914 3350 

LoriAnn E. Eils 

Minn vitro, Inc. 

15 West 36th Street 

Minneaplois, :MN 55408 

USA 

David D. Ellis 

BC Research Inc. 

Conifer Biotechnology Group 

3650 Westbrook Mall 

Vancouver, BC 

Canada V6S 2L2 

Tel: 604 224 5331 

Fax: 604 224 0540 

E-mail: Elvis@bcr.bc.ca 

Saori Endo 

Central Research Laboratory 

Nippon Paper Industries 

Oji 5-21-1, Kita-ku 

Tokyo 114 

Japan 

Patricia Faivre-Rampant 

Station d' Amelioration des Arbres Foretiers 

Institut National de la Recherche Agronomique (INRA) 

F-45160 Ardon 

France 

D. John Fry 

Department of Environmental Resource Management 

Faculty of Agriculture 

University College, Belfield 

Dublin4 

Ireland 

Tel: 353 1706 7777 

Fax: 353 1706 1102 

Telex: 32693 EI 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Marie-Christine Goue 

Institut National de Ia Recherche Agronomique (INRA) 

Station d' Amelioration des Arb res Foretiers 

F-45160 Ardon 

France 

Stephen C. Grado 

College of Forest Resources 

Mississippi State University 

Box 9681 

Mississippi State, MS 39762-9681 

USA 

Richard B. Hall 

Department of Forestry 

235 Bessey Hall 

Iowa State University 

Ames, lA 50011 

USA 

Tel: 515 2941453 

Fax: 515 294 2995 

E-mail: rbhall@iastate.edu 

Simon Hawkins 

Station d' Amelioration des Arbres Forestiers 

Institut National de Ia Recherches Agronomique (INRA) 

F-45160 Ardon 

France 

Scott Heuchelin 

Department of Plant Pathology 

Russell Laboratories 

University of Wisconsin - Madison 

1630 Linden Drive 

Madison, WI 53706 

USA 

Tel: 608 263 2093 

Fax: 608 263 2626 

E-mail: sah@plantpath.wisc.edu 

Martin Hubbes 

University of Toronto 

Faculty of Forestry 

33 Willcocks Street 

Earth Sciences Centre 

Toronto, Ontario 

Canada M5S 383 

Tel: 416 978 6831 

E-mail: hubbes@larva.forestry. utoron 

Dirk Inze 

Laboratoire Assode de l'Institut National de la Recherche 

Agonomique 

Universiteit Gent 

K. L. Ledeganckstraat 35 

B-9000 Gent 

Belgium 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 

Authors and Affiliations 

Cathy Jones 

Molecular Genetics and Tissue Culture Group 

Petawawa National Forestry Institute 

Canadian Forest Service 

Chalk River, Ontario 

Canada KOJ 1]0 

C. P.Joshi 

Institute of Wood Research 

Department of Forestry 

Michigan Technological University 

1400 Townsend Drive 

Houghton, MI 49931-1295 

USA 

Lise J ouanin 

Laboratoire de Biologie Cellulaire 

Institut National de la Recherche Agronomique (INRA) 

Centre de Versailles 

F-78076 Versailles Cedex 

France 

Tel: 33 1 30 83 30 63 

Fax: 33 1 30 83 30 99 

E-mail: jouanin@versailles.inra.fr 

Hoduck Kang 

Tree Molecular Biology Laboratory 

Division of Forest Microbiology 

Forestry Research Institute 

Chongryangri-Dong, Tongdaemun-Ku 

Seoul130-012 

Republic of Korea 

Tel: 82 2 961 2625 

Fax: 82 2 967 5101 

David F. Karnosky 

Michigan Technological University 

School of Forestry and Wood Products 

1400 Townsend Drive 

Houghton, MI 49931-1295 

USA 

Tel: 906 487 2898/2454 

Fax: 906 487 2897/2915 

E-mail: Karnosky@mtu.edu 

Julie Russell Kikkert 

Cornell University 

New York State Agricultural Experiment Station 

Department of Horticultural Sciences 

Hendrick Hall 

Geneva, NY 14456-0462 

USA 

Tel: 315 787 2221 

Fax: 315 787 2216 

E-mail: jrk2@cornell.edu 

283 
Section VI Appendices 

Mee-Sook Kim 

Department of Forestry, Fisheries & Wildlife 

101 Plant Industry 

University of Nebraska -Lincoln 

Lincoln, NE 68583-0814 

USA 

Tel: 402 437 5178 ext 28 

Fax: 402 437 5712 

E-mail: fofw047@unlvm.unl.edu 

Karl W. Kleiner 

Department of Entomology 

University of Wisconsin 

345 Russell Lab/ 1630 Linden Drive 

Madison, WI 53706 

USA 

Tel: 608 262 4755 

E-mail: kkleiner@facstaff. wisc.ed u 

Ned B. Klopfenstein 

USDA Forest Service 

Rocky Mountain Forest and Range Experiment Station 

National Agroforestry Center 

East Campus -University of Nebraska 

Lincoln, NE 68583-0822 

USA 

Tel: 402 437 5178 ext 18 

Fax: 402 437 5712 

DG address: N.Klopfenstein:S28L04a 

E-mail:FSWA/S=N.Klopfenstein/ ou=S28L04A 

®MHS.ATIMAIL.COM 

Jae S. Lee 

Biotechnology Division 

Forest Genetics Research Institute 

Forestry Administration 

P. 0. Box 24, Suwon 

Kyonggido 441-350 

Republic of Korea 

Tel: 82 331 2921187 

Fax: 82 331 292 4458 

Jean-Charles Leple 

Station d' Amelioration des Arb res Forestiers 

Institut National de la Recherches Agronomique (INRA) 

F-45160 Ardon 

France 

Dolly C. Lin 

Faculty of Forestry 

University of Toronto 

Toronto, Ontario 

Canada M5S 3B3 

E-mail: j.lin@utoronto.ca 

284 

C. H. Anthony Little 

Natural Resources Canada 

Canadian Forest Service 

P. 0. Box 4000 

Fredericton, New Brunswick 

Canada E3B 5P7 

Kathryn A. Louis 

Sun Gro Horticulture 

110-110th Ave. NE, #490 

Bellevue, WA 98004 

USA 

Tel: 206 450 4447 

Etsuko Matsunaga 

Central Research Laboratory 

Nippon Paper Industries 

Oji 5-21-1, Kita-ku 

Tokyo 114 

Japan 

Charles A. Maynard 

Faculty of Forestry 

State University of New York 

College of Environmental Science and Forestry 

1 Forestry Drive 

Syracuse, NY 13210-2787 

USA 

Tel: 315 470 6560 

Fax: 315 470 6956 

E-mail: C.Maynard@Mailbox.syr.edu 

Brent H. McCown 

Department of Horticulture 

University of Wisconsin 

1575 Linden Drive 

Madison, WI 53706 

USA 

Tel: 608 262 0574/0517 

Fax: 608 262 4743 

E-mail: bhmccown@facstaff.wisc.edu 

Harold S. McNabb, Jr. 

Deparbnents of Plant Pathology and Forestry 

251 Bessey Hall 

Iowa State University 

Ames, lA 50011 

USA 

Tel: 515 294 3120/2350 

Fax: 515 294 9420/2995 

E-mail: snabb18@iastate.edu 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 
Richard Meilan 

Department of Forest Science 

A039E Peavy Hall 

Oregon State University 

Corvallis, OR 97331-7501 

USA 

Tel: 503 737 6097 

Fax: 503 737 2668 or 541 737 5814 

E-mail: meilanr@ccmail.orst.edu 

meilan@fsl.orst.ed u 

Bernard Monties 

Laboratoire de Chimie Biologique 

Institut National de Ia Recherche Agronomique (INRA) 

F-78850 Thiverval-Grignon 

France 

Thomas Moritz 

Department of Forest Genetics and Plant Physiology 

The Swedish University of Agricultural Sciences 

S-90183 Umea 

Sweden 

Ove Nilsson 

The Salk Institute for Biological Studies 

10010 North Torrey Pines Road 

La Jolla, CA 92037 

USA 

Eun WoonNoh 

Biotechnology Division 

Forest Genetics Research Institute 

Forestry Administration 

P. 0. Box 24, Suwon 

K yonggido 441-350 

Republic of Korea 

Tel: 82 331 292 1187 

Fax: 82 331 292 4458 

Olof Olsson 

Department of Molecular Biology 

Medicinaregatan 9C 

Goteborg University 

S-413 90 Goteborg 

Sweden 

Tel: 46 31 773 3268 

Fax: 46 31 773 3801 

E-mail: Olof.Olsson@genfys.slu.se 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Authors and Affiliations 

Michael E. Ostry 

USDA Forest Service 

North Central Forest Experiment Station 

1992 Folwell Ave. 

St.Paul, MN 55108 

USA 

Tel: 612 649 5231/5113 

Fax: 612 649 5285 

DG address: M.Ostry:s23a 

Young Goo Park 

Department of Forestry 

College of Agriculture 

Kyungpook National University 

San K yng-dong, Seo-gu 

Daegu, 702-701 

Republic of Korea 

Tel: 82 53 950 5747 

Fax: 82 53 950 6701 

E-mail: ygpark@bh.kyungpook.ac.kr 

Michel Petit-Conil 

Centre Technique du Papier 

F-38044 Grenoble Cedex 

France 

Gilles Pilate 

Station d' Amelioration des Arbres Forestiers 

Institut National de Ia Recherches Agronomique (INRA) 

F-45160 Ardon 

France 

E-mail: pilate@orleans.inra.fr 

GopiK.Podila 

Department of Biological Sciences 

Michigan Technological University 

1400 Townsend Drive 

Houghton, MI 49931-1295 

USA 

William A. Powell 

Faculty of Environmental and Forest Biology 

State University of New York 

College of Environmental Science and Forestry 

1 Forestry Drive 

Syracuse, NY 13210-2787 

USA 

285 
Section VI Appendices 

Kenneth E Raffa 

Departments of Entomology and Forestry 

University of Wisconsin 

345 Russell Lab I 1630 Linden Drive 

Madison, WI 53706 

USA 

Tel: 608 262 1125/4775 

Fax: 608 262 3322 

E-mail: raffa@macc. wisc.ed u 

Sharon M. Regan 

Department of Forest Genetics and Plant Physiology 

Swedish University of Agricultural Sciences 

5-90183 Umea 

Sweden 

Don E. Riemenschneider 

USDA Forest Service 

North Central Forest Experiment Station 

Forestry Sciences Laboratory 

5985 Highway K 

Rhinelander, WI 54501 

USA 

Tel: 715 362 7474 

DG address:D.Riemenschneider:r09f06a 

N.Th. Saieed 

Department of Forestry 

Mosul University 

Mosul 

Iraq 

Goran Sandberg 

Department of Forest Genetics and Plant Physiology 

The Swedish University of Agricultural Sciences 

5-90183 Umea 

Sweden 

James C. Sellmer 

University of Wisconsin-Extension 

Racine County, 14200 Washington Avenue 

Sturtevant, WI 53706 

USA 

Dongill Shin 

Department of Plant Genetics & Breeding 

Hyosung Catholic University 

Hayang, Kyungsan, Kyungpook 7130-702 

Republic of Korea 

286 

Sung-Ho Son 

Research Institute 

Forestry Administration 

P. 0. Box 24, Suwon 

Kyonggido 441-350 

Republic of Korea 

Tel: 82 331292 4458 

Fax: 82 331 290 1188 

Charles H. Strauss 

School of Forest Resources 

Pennsylvania State University 

104 Ferguson Building 

University Park, PA 16802-4302 

USA 

Tel: 814 865 7541 

Fax: 814 865 4574 

Steven H. Strauss 

Department of Forest Science 

020 Forestry Sciences Lab 

Oregon State University 

Corvallis, OR 97331-7501 

USA 

Tel: 541 737 6578 

Fax: 541 737 1393 

E-mail: strauss@fsl.orst.ed u 

Bjorn Sundberg 

Department of Forest Genetics and Plant Physiology 

The Swedish University of Agricultural Sciences 

S-90183 Umea 

Sweden 

Tel: 46 90 166303/166301 

Fax: 46 90 165901 

E-mail: Bjom.Sundberg@genfys.slu.se 

Girma Melesse Tabor 

Department of Forestry 

251 Bessey Hall 

Iowa State University 

Ames, lA 50011 

USA 

Tel: 515 294 1221 

E-mail: gtabor@iastate.edu 

Chung-Jui Tsai 

Institute of Wood Research 

Department of Forestry 

Michigan Technological University 

1400 Townsend Drive 

Houghton, MI 49931-1295 

USA 

Tel: 906 487 2959 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Hannele Tuominen 

Department of Forest Genetics and Plant Physiology 

The Swedish University of Agricultural Sciences 

5-90183 Umea 

Sweden 

Gerald A. Tuskan 

Biofuel Feedstock Development Program 

Building. 1503 

Oak Ridge National Laboratory 

Bethal Valley Road 

Oak Ridge, TN 37830-6352 

USA 

Tel: 423 576 8141 

Fax: 423 576 8143 

E-mail: gtk@ornl.gov 

Jan Van Doorsselaere 

Laboratorium voor Genetica 

Department of Genetics 

Flanders Interuniversity Institute for Biotechnology 

Universiteit Gent 

K.L. Ledeganckstraat 35 

9000 Gent 

Belgium 

E-mail: jadoo@genwetl.rug.ac.be 

Marc Van Montagu 

Laboratorium voor Genetica 

Department of Genetics 

Flanders Interuniversity Institute for Biotechnology 

Universiteit Gent 

K.L. Ledeganckstraat 35 

9000 Gent 

Belgium 

Marc Villar 

Station d' Amelioration des Arb res Foretiers 

Institut National de la Recherche Agronomique (INRA) 

F-45160 Ardon 

France 

Tel: 33 38 41 78 74 

Fax: 33 38 41 78 79 

E-mail: villar@orleans.inra.fr 

Suzanne Wetzel 

Natural Resources Canada 

Canadian Forest Service 

P. 0. Box 490, 1219 Queen St. E. 

Sault Ste. Marie, Ontario 

Canada P6A 5M7 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Authors and Affiliations 

Sarah W. Workman 

Department of Forestry, Fisheries & Wildlife 

University of Nebraska - Lincoln 

National Agroforestry Center 

Lincoln, NE 68583-0814 

USA 

Tel: 402 437 5178 ext 40 

Fax: 402 437 5712 

Keiko Yamada 

Central Research Laboratory 

Nippon Paper Industries 

Oji 5-21-1, Kita-ku 

Tokyo 114 

Japan 

Mikiko Yamakado 

Central Research Laboratory 

Nippon Paper Industries 

Oji 5-21-1, Kita-ku 

Tokyo 114 

Japan 

X. B. Yang 

Department of Plant Pathology 

351 Bessey Hall 

Iowa State University 

Ames, lA 50011 

USA 

Tel: 515 294 8826 

Fax: 515 294 9420 

E-mail: XBYang@iastate.edu 

Jianwei Zhang 

Environmental Resources Research Inst. 

Pennsylvania State University 

220 Forest Resource Building 

University Park, PA 16802-4704 

USA 

Tel: 814 865 4092 

Louis Zsuffa 

Faculty of Forestry 

University of Toronto 

Toronto, Ontario 

Canada M5S 383 

ph: 416 978 6512 

fax: 416 978 3834 

E-mail: louis.zsuffa@utoronto.ca 

zsuffa@larva.forestry. u to ron t 

287 
Section VI Appendices 

Reviewer acknowledgment 

David P. Anderson 

USDA-Natural Resources Conservation Service 

Watershed Science Institute 

Carlos M. Araya 

University of Nebraska- Lincoln 

Stefanie G. Aschmann 

USDA-Natural Resources Conservation Service 

Watershed Science Institute 

J. Michael Bowker 

USDA Forest Service, Southern Research Station 

Harvey D. Bradshaw, Jr. 

University of Washington 

Zong Ming Cheng 

North Dakota State University 

Gary D. Coleman 

University of Maryland at College Park 

Joe P. Colletti 

Iowa State University 

Richard A. Cunningham 

USDA-Agricultural Research Service (retired) 

John M. Davis 

University of Florida 

Mary Ellen Dix 

USDA Forest Service, Rocky Mountain Forest and Range 

Experiment Station, National Agroforestry Center 

Raymon A. Donahue 

Washington State University 

Luc C. Duchesne 

Forestry Canada, Great Lakes Forestry Centre 

Craig S. Echt 

USDA Forestry Service, North Central Station 

Stephen G. Ernst 

University of Nebraska- Lincoln 

Glenn R. Fumier 

University of Minnesota 

288 

Barry Goldfarb 

North Carolina State University 

Michael S. Greenwood 

University of Maine 

Richard B. Hall 

Iowa State University 

Kyung-Hwan Han 

Oregon State University 

Elwood R. Hart 

Iowa State University 

Patricia L. Herman 

University of Nebraska - Lincoln 

Glen T. Howe 

University of Minnesota 

Kyung-Moon Kim 

University of Nebraska - Lincoln 

RudyM. King 

USDA Forest Service, Rocky Mountain Forest and Range 

Experiment Station 

Bohun B. Kinlock, Jr. 

USDA Forest Service, Pacific Southwest Station 

Gary A. Kuhn 

USDA-Natural Resources Conservation Service 

William J. Libby 

New Zealand Forest Research Institute 

Carol A. Loopstra 

Texas A&M University 

Charles A. Maynard 

State University of New York- Syracuse, College of 

Environmental Science and Forestry 

Amitava Mitra 

University of Nebraska - Lincoln 

Margaret T. Mmbaga 

Tennessee State University 

David Neale 

USDA Forest Service, Pacific Southwest Station 

Eun-Gyu No 

Texas A&M University 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Michael E. Ostry 

USDA Forest Service, North Central Station 

SoonOhPark 

University of Nebraska - Lincoln 

Nagmani Rangaswamy 

USDA Forest Service, Alabama A&M University 

Paul E. Read 

University of Nebraska - Lincoln 

Don E. Riemenschneider 

USDA Forest Service, North Central Station 

Peter R. Schaefer 

South Dakota State University 

Michele M. Schoeneberger 

USDA Forest Service, Rocky Mountain Forest and Range 

Experiment Station, National Agroforestry Center 

Carol M. Schumann 

USDA-Agricultural Research Service 

Appalachian Soil & Water Conservation Laboratory 

Ronald R. Sederoff 

North Carolina State University 

Robert M. Skirvin 

University of Illinois 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 

Paul E. Staswick 

University of Nebraska- Lincoln 

Loren G. Stephens 

Iowa State University 

Steven H. Strauss 

Oregon State University 

Charles G. Tauer 

Oklahoma State University 

Francine M. Tremblay 

Universite Laval 

Anne K. Vidaver 

University of Nebraska- Lincoln 

James A. Walla 

North Dakota State University 

F. William Whitmore 

Ohio State University 

X.B. Yang 

Iowa State University 

Jianwei Zhang 

Reviewer Acknowledgment 

University of Nebraska- Lincoln (subsequently: 

Pennsylvania State University) 

289 
Scientific and Common Names of Biological Organisms 

Associated With Populus Biotechnology 

Madelyn C. Dillon, Richard C. Carman, and Mee-Sook Kim 

Populus Sections 

Abaso section Eckenwalder - poplar section containing 

Populus mexicana Wesmael, categorized by morphology, 

geographical localization, and crossability 

Aigeiros section Duby- cottonwood and black poplar section, categorized by morphology, geographical localization, and crossability 

Leuce section Duby (currently termed Populus section) -

aspen and white poplar section, categorized by morphology, geographical localization, and crossability 

Leucoides section Spach- Himalayan poplar, Wilson poplar, and swamp cottonwood section, categorized by morphology, geographical localization, and crossability 

Populus section (formerly Leuce section) - aspen and 

white poplar section, categorized by morphology, geographical localization, and crossability 

Tacamahaca section Spach - balsam poplar section, categorized by morphology, geographical localization, and 

crossability 

Turanga section Bunge - Euphrates poplar section, categorized by morphology, geographical localization, and 

crossability 

Populus Species and Varieties 

Populus adenopoda Maxim. (P. suaveolens, s.l.)- Chinese 

aspen; Populus (formerly Leuce) section 

Populus alba L. - white poplar; Populus (formerly Leuce) 

section 

Populus angulata Ait. (P. deltoides var. angulata Ait.) -

Eastern cottonwood, southern form; Aigeiros section 

Populus angustifolia James- narrow-leaf or yellow cottonwood; Tacamahaca section 

290 

Populus balsamifera L. (P. tacamahaca) -balsam poplar; 

Tacamahaca section 

Populus ciliata Wall./Royal - Himalayan poplar; 

Tacamahaca section 

Populus davidiana (Dode) Schneid. (P. tremula, s.l.) -

Korean aspen; Populus (formerly Leuce) section 

Populus deltoides Bartr. ex Marshall- Eastern cottonwood; 

Aigeiros section 

Populus euphratica Oliver- Euphrates poplar; Tzlranga section 

Populus fremontii S. Watson - Fremont cottonwood; 

Aigeiros section 

Populus glandulosa Uyeki. (P. davidiana var. glandulosa) 

- Suwon poplar; Populus (formerly Leuce) section 

Populus grandidentata Michaux- bigtooth aspen; Populus 

(formerly Leuce) section 

Populus koreana Rehder (P. suaveolens, s.l.) - Korean 

poplar; Tacamahaca section 

Populus lasiocarpa Oliver - Leucoides section 

Populus laurifolia Ledebour - laurel poplar; Tacamalzaca 

section 

Populus maximowiczii A. Henry (P. suaveolens, s.l.) -

Japanese poplar; Tacamahaca section 

Populus mexicana Wesmael -Mexican poplar; Abaso sec- ~ 

tion 

Populus nigra L. - black poplar; Aigeiros section 

Populus nigra var. betulifolia Torr. - Aigeiros section 

Populus nigra var. charkowiensis Schroed. (P. nigra var. 

plantierensis Schneid.) - Aigeiros section 

Populus nigra L. var. italica Duroi- Lombardy poplar; 

Aigeiros section 

Populus serotina (P. x canadensis Moench) (P. x 

euramericana (Dode) Guinier)- Aigeiros section 

Populus sieboldii Miquel -Japanese aspen; Populus (formerly Leuce) section 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Scientific and Common Names of Biological Organisms Associated With Populus Biotechnology 

Populus simonii Carriere- Simon poplar; Tacamahaca section 

Populus suaveolens Fisher - Tacamahaca section 

Populus szechuanica Schneider- Tacamahaca section 

Populus tacamahaca Mill. (P. balsamifera, s.l.)- balsam 

poplar, collectively; Tacamahaca section 

Populus tomentosa Carr. (P. alba var. tomentosa Carriere) 

(P. x tome~ttosa) (P. alba x P. adenopoda) (P. alba x P. 

davidiana) - Chinese white poplar; Populus (formerly 

Leuce) section 

Populus tremula L.- European aspen; Populus (formerly 

Leuce) section 

Populus tremuloides Michaux- quaking aspen; Populus 

(formerly Leuce) section 

Populus triclzocarpa Torr. & Gary - black cottonwood; 

Tacamahaca section 

Populus tristis Fisch. - Himalayan balsam poplar; 

Tacamahaca section 

Populus wislizenii Sargent (P. fremontii var. wislizenii 

Wats.) (P. deltoides, s.l.) - Rio Grande cottonwood; Aigeiros 

section 

Populus xiaohei T.S. Hwang ex C. Wang et Tung (P. x 

xiaolzei) - Chinese small black poplar 

Populus yunnanensis Dode-species in the Tacamahaca section 

Populus Hybrids 

Populus x acuminata Rydb. (P. x andrewsii Sarg.) - P. 

angustifolia x P. deltoides hybrid, lance leaf cottonwood 

Populus x bernardii Boivin - P. deltoides x P. tremuloides 

hybrid, Bernard poplars 

Populus x berolinensis Dippel (P. x rasumowskyana Schr.) 

(P. x petrowskyana Schr.) - P. laurifolia x P. nigra hybrid, 

Berlin or Russian poplars 

Populus x canade1zsis Moench (P. x euramericana (Dode) 

Guinier)- P. deltoides x P. nigra hybrid 

Populus x canescens Sm.- P. alba x P. tremula hybrid, gray 

poplars 

Populus x dutillyi Lepage- P. balsamifera x P. deltoides hybrid 

Popzdus x euramericana (Dode) Guinier (P. x canadensis 

Moench)- P. deltoides x P. nigra hybrid, Euramerican poplars 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Populus x generosa Henry (P. x interamericana Brockh.) -

P. deltoides x P. trichocarpa and reciprocal hybrid, 

interamerican poplars 

Populus x heimburgeri Boivin- P. alba x P. tremuloides hybrid 

Populus x interamericana Brockh. (P. xgenerosa Henry)-

P. deltoides x P. trichocarpa and reciprocal hybrid, 

interamerican poplars 

Populus xjackii Sarg.- P. balsamifera x P. deltoides hybrid, 

Jackii poplars 

Populus x kitakamiensis- P. sieboldii x P. grandidentata hybrid 

Populus x parryi Sarg.- P. fremontii x P. trichocarpa hybrid, 

Parry cottonwood 

Populus x rouleauiana Boivin- P. alba x P. grandidentata 

hybrid 

Populus x sennii Boivin- P. angustifolia x P. tremuloides hybrid 

Populus x smithii Boivin- P. grandidentata x P. tremuloides 

hybrid 

Other Trees and Plants 

Abies spp. - genus of fir trees 

Abies koreana E.H. Wils. - Korean fir tree 

Acer saccharum Marsh. - sugar maple tree 

Albizia lebbeck (L.) Benth. - siris tree 

Amaranthus spp.- genus of several species characterized 

by a lasting red pigment in the stems and leaves 

Antirrhinum spp. L.- genus of snapdragon 

Arabidopsis thaliana (L.) Heynh. - mouse-ear cress 

Astragalus spp.- genus of milk vetch (herbs) 

Bambusa spp. - genus of bamboo 

Betula papyrifera Marsh.- paper birch tree, white birch 

tree 

Betula platyphylla Sukachev - birch tree 

Betula verrucosa Ehrh. - European white birch tree 

Biota orienta lis (L.) Endl.- oriental arborvitae 

Brassica spp. - genus of coles and mustards 

Brassica napus L. - rapeseed 

291 
Section VI Appendices 

Calocedrus decurrens (Torr.) Florin -California incense cedar tree 

Camellia japonica L. - common camellia 

Carica spp. - genus of papaya 

Castanea spp. - genus of chestnut trees 

Castanea crenata Sieb. & Zucc. -Japanese chestnut tree 

Castanea mollissima 81. - Chinese chestnut tree 

Castanea sativa Mill. - European chestnut tree 

Cedrus atlantica Manetti - Atlas cedar tree 

Cercis siliquastrum L.- redbud tree 

Chamaecyparis lawson ian a (A. Murr.) Pari.- Port-Orfordcedar tree 

Chrysanthemum spp. - genus of chrysanthemum 

Citrus spp. -genus of citrus, including grapefruit, lemon, 

and orange 

Citrus sinensis (L.) Osbec - sweet orange tree 

Coffea arabica L. - arabic coffee, common coffee 

Cupressaceae - Cypress family 

Elaeis guineensis Jacq.- oil palm tree, American oil palm 

tree 

Eranthis hyemalis (L.) Salish. - winter aconite (for 

Eranthis) 

Eucalyptus spp.- genus of severalAustralian tall trees with 

wood valued as timber and having aromatic leaves that 

yield oils used in medicine 

Eucalyptus globulus Labill. - bluegum eucalyptus tree 

Eucalyptus grandis Hill ex Maiden. - rosegum eucalyptus tree 

Eucalyptus lansdowneana F.J. Muell. & J.E. Br.- crimson 

mallee box eucalyptus tree 

Eucalyptus nitens Maiden. - shining eucalyptus tree 

Eucalyptus occidentalis Endl. - flat-topped yate eucalyptus tree 

Eucalyptus regnans F. Muell. - mountain-ash eucalyptus 

tree 

Eucalyptus urophylla S. T. Blake - ampupu 

Euglena spp.- genus of single-celled, freshwater organisms characterized by the presence of chlorophyll, a reddish eyespot, and a single anterior flagellum 

Fabaceae - beech family 

Ginkgoaceae - ginkgo family 

292 

Ginkgo biloba L. - ginkgo tree, maidenhair tree 

Glycine max (L.) Merr. - sojabean, soybean 

Hibiscus sabdariffa L. - Indian sorrel 

Isatis tinctoria L. - dyers woad 

Juglans spp. - genus of walnut 

Larix decidua Mill. - European larch tree 

Larix laricina (Du Roi) K. Koch -eastern larch tree, tamarack tree 

Larix leptolepis (Sieb. & Zucc.) Gord.-Japanese larch tree 

Larix occidentalis Nutt.- western larch tree 

Liquidambar styraciflua L. - sweetgum tree 

Liriodendron tulipifera L. - yellow poplar tree 

Litchi chinensis Sonn. - lychee 

Lycopersicon spp. - genus of tomatoes 

Lycopersicon chilense Dun. - wild tomato 

Lycopersicon esculentum Miller - goldapple, loveapple, 

tomato 

Macrozamia communis L.A.S. Johnson- palmlike plant 

of Australia 

Malus spp. - genus of apples 

Malus domestica Borkh. - common apple tree 

Malus hupehensis (Pamp.) Rehd. - tea crabapple tree 

Medicago spp. - genus of alfalfa or lucern 

Metasequoia glyptostroboides Hu & Cheng - dawn redwood tree 

Musa spp. - genus of bananas 

Nicotiana spp. - genus of tobacco 

Olea spp.- genus of olive trees 

Olea europaea L. - common olive trees 

Oryza sativa L. - rice 

Paulownia spp.- genus of several Chinese deciduous trees 

having large, heart shaped, opposite leaves and pyramidal panicles of purplish or white flowers with a spotted 

interior 

Paulownia tomentosa (Thunb.) Siev. & Zucc. - foxglove 

tree 

Petunia x hybrida Hort. Vilm. -Andr. - common garden 

petunia 

Phaseolus vulgaris L.- kidney bean, French bean, snap bean 

Picea spp. - genus of spruce trees 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Scientific and Common Names of Biological Organisms Associated With Populus Biotechnology 

Picea abies (L.) Karst- Norway spruce tree 

Pice a engelmanii Parry ex Engelm.- Engelmann spruce tree 

Picea glauca (Moench) Voss - white spruce tree 

Pinaceae - pine family 

Pinus spp. - genus of pine trees 

Pinus lambertiana Dougl.- sugar pine tree 

Pinus monticola Dougl. ex D. Don- western white pine 

tree 

Pinus palustris Mill. - longleaf pine tree 

Pinus pinaster Ait. - maritime pine tree 

Pinus radiata A.D. Don- Monterey pine tree 

Pinus rigida Mill.- pitch pine tree 

Pinus strobus L. - eastern white pine tree 

Pinus sylvestris L.- Scots' pine tree 

Pinus taeda L. - loblolly pine tree 

Pisum sativm L. - garden, common, English, and green pea 

Prunus spp. - genus of cherry, peach, and plum trees 

Prunus cerasus L. - pie cherry, sour cherry 

Prunus japonica Thunb. -peach tree 

Prunus persica Batsch - peach tree 

Prunus serotina Ehrh.- black cherry tree 

Pseudotsuga spp. - genus of Douglas-fir trees 

Pseudotsuga menziesii Franco. - Rocky Mountain Douglas-fir tree, coast Douglas-fir tree 

Pyn1s spp. - genus of pear trees 

Pyrus nivalis Jacq.- snow pear tree 

Quercus accutissima Carruthers- sawtooth oak tree 

Quercus ilex L. - holly oak tree, Holm oak tree 

Quercus petraea (Mattushka) Lieblein.- durmast oak tree, 

sessile oak tree 

Quercus robur L. - English oak tree 

Raphanus sativus L. - radish plant 

Ribes nign1m L. - black currant 

Ricinus communis L. - castor bean 

Robinia pseudoaca~ia L.- black locust tree 

Rubus spp. - genus of raspberry and blackberry 

Salicaceae - willow family 

Salix spp. - genus of willow trees 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Salix babylonica L. - weeping willow tree 

Salix caprea L. - European willow tree, goat willow tree 

Salix Iucida Muhl.- shining willow tree 

Salix x smithiana Willd. - hybrid of 5. caprea and basket 

willow tree 

Salix viminalis L. - basket willow tree 

Sambucus nigra L. - European elder tree 

Santalum album L. - sandal wood tree 

Solanum spp. - genus of cosmopolitan herbs and shrubs, 

including potato, eggplant, and nightshade 

Sophora japonica L.- Japanese pagoda-tree 

Syzygium aromaticum (L.) Merrill & L.M. Perry- clove tree 

Taxodium distichum (L.) Rich - bald cypress tree 

Ulmus spp. - genus of elm trees 

Vitis vinifra L.- European grape, wine grape 

Zinnia spp. - genus of zinnia 

Microorganisms and Insects 

Agrobacterium rhizogenes Conn -bacterial pathogen; 

cause of hairy root disease 

Agrobacterium tumefaciens (Smith & Townsend) Conn -

bacterial pathogen; cause of crown gall disease 

Alcaligenes eutrophus Davis in Davis, Doudoroff, Stanier 

and Mandel - Gram-negative eubacteria 

Aspergillus spp. P. Mich. ex Link: Fr.- genus of fungi that 

causes black mold of cotton bolls, fruits, vegetables 

Bacillus amyloliquefaciens a (sic) Fukumato -soil bacterium that breaks down proteins into polypeptides and resembles trypsin in its action 

Bacillus thuringiensis Berliner - pathogenic bacteria for 

insects; produces a crystalline product that is toxic tolarvae of Lepidoptera 

Ceratocystis fimbriata Ellis & Haist. - fungal pathogen; 

cause of aspen canker 

Chondrostereum purpureum (Pets.: Fr.) Pouzar - fungal 

pathogen; cause of silver-leaf disease of fruit trees 

Choristoneura fumiferana (Clemens) - spruce bud worm 

Choristoneura occidentalis Freeman- western spruce budworm 

293 
Section VI Appendices 

Choristoneura pinus Freeman -jack pine bud worm 

Chrysomela populi L. - poplar leaf beetle 

Chrysomela scripta Fabr.- cottonwood leaf beetle 

Chrysomela tremulae E - poplar leaf beetle 

Colletotrichum spp. Corda - genus of broad-host-range, 

fungal pathogens; cause of anthracnose, root rot, and seedling blight; also used as a biocontrol agent 

Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. in 

Penz. - conidal state of Glomerella cingulata; fungal pathogen causing anthracnose of citrus, banana, and other plants 

Colletotrichum lindemuthianum (Sacc. & Magnus) 

Scribb.-fungal pathogen causing anthracnose of bean and 

other plants 

Corynebacterium diphtheriae (Kruse) Lehmann and 

Neumann- bacterial pathogen; gram-positive, rod-shaped 

cause of diphtheria 

Cotesia melanoscela Ratzeburg - gypsy moth parasitoid 

Cryphonectria parasitica (Murrill) Barr - fungal pathogen; cause of chestnut blight 

Erwinia amylovora (Burrill) Winslow et al. - bacterial 

pathogen; cause of fire blight disease on cottoneaster, pear, 

and apple · 

Fusarium oxysporum Schlechtend.: Fr. f. sp. lycopersici 

(Sacc.) W.C. Snyder & H.N. Hans- fungal pathogen; cause 

of tomato and mimosa wilt 

Fusarium solani (Mart.) App. et Wr. emend. Snyd. et 

Hans. - broad host range, fungal pathogen causing root 

and stem rot, damping off, and vascular wilt 

Glomerella cingulata (Stoneman) Spauld. & H. Schrenk 

- broad-host-range, fungal pathogen; cause of blight, leaf 

spot, rot, cankers, and dieback 

Heliothis virescens (Fabricius)- tobacco budworm 

Heliothis zea (Broddie) - bollworm 

Hyalophora cecropia (Linnaeus) - cecropia moth 

Hypoxylon mamma tum (Wahl.) Miller- fungal pathogen; 

cause of hypoxylon canker of aspen 

Laccaria bicolor (Maire) Orton. - Ectomycorrhizal 

basidiomyceteous fungus 

Lymantria dispar (Linnaeus) - gypsy moth 

Malacosoma disstria (Hubner) - forest tent caterpillar 

Manduca sexta (Linnaeus) - tobacco homworm 

Marssonina brunnea (Ell. & Ev.) Sacc. - fungal pathogen; 

cause of poplar and cottonwood leaf spot 

294 

Melampsora spp. - genus of rust fungus 

Melampsora larici-populina Kleb. - rust fungus; cause of 

rust disease on Populus spp. and Larix spp. 

Melampsora medusae Thuem. - fungal pathogen; cause 

of cottonwood leaf rust 

Melampsora occidentalis H. Jacks. - fungal pathogen; 

cause of poplar rust 

Mycosphaerella populorum G.E. Thompson- (anamorph: 

Septoria musiva)- fungal pathogen; cause of poplar leaf spot 

and canker 

Ophiostoma ulmi (Buisman) Nann f. (formerly 

Ceratocystis ulmi Busim. C. Moreau) - fungal pathogen; 

cause of elm wilt disease 

Paxillus involutus (Fr.) Fr. - ectomycorrhizal colonizer 

Phaeolus schweinitzii (Fr.: Fr.) Pat. - fungal pathogen; 

cause of conifer butt rot 

Plagiodera versicolora (Laicharting) - imported willow 

leaf beetle 

Plodia interpunctella (Hubner) - Indianmeal moth 

Plutella xylostella (Linnaeus) - diamondback moth 

Pseudomonas aeruginosa (Schroeter) Migula - bacterial 

pathogen of animals and plants 

Pseudomonas fluorescens (Trevisan) Migula -bacteria existing in soil and water that is the cause of soft rot disease 

Pseudomonas solanacearum Smith- bacterial wilt pathogen 

Pseudomonas syringae van Hall- bacterial spot and blight 

pathogen 

Salmonella spp. - genus of bacterial pathogen; various rodshaped bacteria, causes diseases in humans and animals, 

some are infectious 

Septaria musiva Peck (teleomorph: Mycosphaerella 

populorum) - fungal pathogen; cause of poplar leaf spot 

and canker 

Septaria populicola Peck (teleomorph: Mycosphaerella 

populicola G.E. Thompson) -fungal pathogen; causes leaf 

spot of poplar (e.g., Tacamahaca section) 

Spodoptera exiqua (Hubner) -beet armyworm 

Tobacco Mosaic Virus- positive sense, single strand RNA 

virus with a broad host range 

Xanthomonas populi (Ride) Ride & Ride- bacterial pathogen; cause of bacterial canker and dieback of Populus spp. 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 
~ 

~ Glossary of Populus Tissue Culture and 

~ Molecular Biology ~ 

'-' Mee-Sook Kim, Madelyn C. Dillon, and Richard C. Carman 

ABAXIAL- the side or on the side away from an axis such 

as on the lower surface of a leaf. 

ABIOTIC FACTORS- nonliving factors such as light, temperature, wind, fire, soil, water, air. 

ADAXIAL- the side or on the side toward an axis such as 

on the upper surface of a leaf. 

ADDITIVE GENETIC VARIATION - the proportion of 

genetic variation that occurs in response to natural, mass, 

or 11 pick-the-winner" selection; the basis of a parent's 

breeding value or general combining ability. 

ADVENTITIOUS- produced in an abnormal or unusual 

position, at an unusual time of development, or away from 

the natural habitat such as when plant organs develop on 

callus or when nonzygotic embryos develop without an 

ovary. 

AFLP - see amplified fragment length polymorphism. 

ALLELE - any of the alternative forms of a gene that may 

occur at a given locus. 

ALTERNATE HOST - 1 of 2 kinds of plants on which a 

parasitic fungus must develop to complete its life cycle. 

AMIDE- an inorganic compound derived from ammonia 

by replacement of a hydrogen atom with another element. 

AMINO ACID - any of a class of organic molecules having an amino (-NH2

) group and a carboxyl (-COOH) group; 

20 amino acids are the usual components of protein. 

AMPHIDIPLOID - a polyploid from hybridization of 2 

species or genera with resulting duplication of chromosome complement. 

AMPLIFIED FRAGMENT LENGTH POLYMORPHISM 

- a powerful DNA fingerprinting technique based on selective polymerase chain reaction (PCR) amplification of 

restriction fragments from a complete digestion of genomic 

DNA; this technique shows the reliability of the RFLP technique but avoids Southern blotting by detecting restriction fragments with specific PCR amplification. 

ANEUPLOIDY - a condition in which the chromosome 

number is not an exact multiple of the basic number in the 

genome. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

ANGIOSPERM- a division of the plant kingdom that includes all flowering plants; vascular plants in which double 

fertilization occurs resulting in development of fruit containing seeds; composed of 2 classes, the Monocotyledonae 

and Dicotyledonae. 

ANNUAL NET RETURN- the difference between gross 

annual income and annual expense. 

ANTAGONISM - the opposition or counteraction that occurs between organisms such as a mold-inhibiting bacteria. 

ANTHER- the part of a stamen in which pollen develops. 

ANTHOCYANIN - one of a group of water soluble pigments (glycosides) that vary in color and are present in 

plant cell sap and flowers. 

ANTIBIOTIC- a chemical compound produced by 1 microorganism that inhibits or kills other microorganism. 

ANTIBODY- a protein produced in a warm-blooded animal in reaction to a foreign antigen that is capable of reacting specifically with that antigen. 

ANTIGEN - foreign proteins, and occasionally complex 

lipids, carbohydrates, and some nucleotides, which induce 

the production of antibodies in warm-blooded animals. 

ANTIMICROBIAL- destroying or inhibiting the growth 

of microorganisms. 

ANTISENSE RNA -see antisense gene. 

ANTISENSE GENE- a gene constructed in vitro by reversing the orientation of a portion of transcribed DNA, 

usually including the region encoding a protein, and placing this next to a transcription-control sequence (i.e., the 

promoter); this gene cassette can be delivered to the target 

cell, resulting in genetic transformation and inhibition of 

expression of the target gene (antisense RNA). 

ANTISERUM (ANTISERA)- the blood serum of a warmblooded animal that contains antibodies. 

APEX - the most extreme point of growth of a plant; the 

apical shoot and root tips are located at the apexes (apices) and contain the apical meristem. 

295 
Section VI Appendices 

APICAL DOMINANCE- the terminal bud influence; a 

widespread phenomenon in the plant kingdom that is related to the auxin content of apical buds and is exerted on 

lateral buds resulting in their growth suppression. 

ASCOSPORE- sexual spores produced in an ascus (sac) 

by ascomycetes (sac fungi). 

AUG (Adenine, Uracil, Guanine)- see initiation codon. 

AUTOTROPHIC-self-nourishing; organisms that arecapable of synthesizing nutrition from inorganic compounds; 

all chlorophyll-containing plants. 

AUXIN - one of a large class of plant growth regulators 

that regulates growth, especially one that induces root and 

shoot growth through cell elongation rather than through 

cell division; the commonly used auxins in plant tissue 

culture are indole-3-acetic acid (IAA), indole-3-butyric acid 

(IBA), cx-naphthaleneacetic acid (NAA), and 2,4-

dichlorophenoxy acetic acid {2,4-D). 

AVIRULENCE GENE- plant resistance (or incompatibility) is governed by the interaction of a single dominant 

host gene for resistance (R gene) and the corresponding 

pathogen genes for avirulence (avr gene). 

AXIL- the angle between the stem axis and a leaf, branch, 

or other appendage attached to it; the site of axillary buds 

(shoots). 

AXILLARY SHOOT- see axil. 

BACILLUS THURENGIENSIS-soil bacteria that produces 

endotoxins that are highly toxic to insects; a type of biological control. 

BACKCROSS - a cross between a progeny and either of 

its parents; usually used to incorporate a single desirable 

trait from 1 species or variety with several desirable traits 

from the species or variety used as the recurrent parent; 

repeated backcrossing is used to create near isogenic lines. 

BACTERICIDE - a chemical compound that kills bacteria. 

BASE PAIR (bp)- 2 nitrogenous bases that pair in doublestranded DNA or RNA molecules; the amount of adenine 

(A)- thymine (T) and guanine (G)- cytosine (C) pairs; in 

RNA, thymine is replaced by uracil (U); see nucleotide. 

BASIPETAL - proceeding from the apex toward the base 

or from above downward. 

BINARY VECTOR - T-DNA is located on a separate vector plasmid instead of the Ti-plasmid; T-ON A includes the 

gene(s) of interest and a selectable marker gene;see Ti-p lasmid. 

BIOASSAY- a biological assessment procedure performed 

on living cells or on a living organism that is sometimes 

used to detect minute amounts of substances that influence or are essential to growth. 

296 

BIOLISTICS -particle bombardment or microprojectile 

bombardment used to carry foreign DNA into living cells. 

BIOLOGICAL CONTROL -pest control using agents of 

biological origin including parasites, predators, and 

pathogens. 

BIOREACTOR- a container, such as a large fermentation 

chamber, for growing living organisms for industrial-scale 

production. 

BIOTIC FACTORS - living factors such as those related 

to presence, behavior, and/ or interactions of neighboring 

organisms (e.g., predators, parasites). 

BIOTYPE - a subgroup within a species usually characterized by the possession of a single or few characteristics 

in common. 

BRACT- a small, specialized leaf in the axil from which a 

flower or floral axis develop. 

C - see C value. 

C VALUE- the total amount of DNA in a haploid genome. 

CAAT BOX - part of a conserved sequence located upstream of the startpoints of eukaryotic transcription units; 

see startpoint and leucine zipper. 

CALLI CLONES- clones derived from callus that may possess a high degree of genetic and phenotypic variability. 

CAL LOG ENESIS - the process of callus formation. 

CALLUS (CALLI) - disorganized tumor-like masses of 

plant cells that form in culture. 

CALYX - the sepals of a flower considered a group. 

CAMBIUM - a thin layer of longitudinally dividing cells 

between the xylem and phloem that produces secondary 

growth. 

CANKER - a defined, relatively localized necrotic lesion 

primarily on the bark and cambium. 

CANOPY - in a forest, the uppermost layer of branches, 

twigs, and leaves. 

CAPITALIZED EXPENSES -expense on capital goods by 

firms, government agencies, households, etc.; may replace 

depreciated capital or create new capital. 

CARPEL- a simple pistil or a division of a compound pistil; one of the innermost whorl of modified leaves that bear 

the megaspores. 

CATKIN - a special type of spikelike inflorescence that 

bears either pistillate or staminate flowers and falls as a 

whole from the plant. 

eDNA -see complementary DNA. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
CECROPINS - a family of basic proteins with activity 

against a broad range of Gram-positive and Gram-negative bacteria. 

CELLULASE- an enzyme that hydrolyzes cellulose. 

CELLULOSE- a complex carbohydrate composed of glucose units that form the main constituent of the cell wall 

in most plants. 

CENTIMORGAN (eM) - a measure of genetic distance; 

the distance that separates 2 genes between which there is 

a 1 percent chance of genetic recombination (1 eM). 

CENTROMERE- the heterochromatic region of a eukaryotic chromosome associated with spindle fibers that participates in normal chromosome movement in mitosis and 

meiosis; it divides just before replicated chromosome separation and holds chromatids together. 

CHAPERONE- see molecular chaperones. 

CHIMERA - an individual, organ, or part consisting of 

components of diverse genetic constitution. 

CHITIN - a polysaccharide (polymer of N-acetylglucosamine residues) that forms part of the hard outer integument especially of insects, arachnids, and crustaceans; also 

a component of some fungal cell walls. 

CHITINASE- an enzyme that catalyzes the hydrolysis of 

chitin. 

CHLOROPLAST DNA (cpDNA)- see organelle DNA. 

CHLOROPLAST - a plastid that contains chlorophyll, is 

abundant in the cells of green plants, and functions in photosynthesis. 

CHLOROSIS - yellowness in normally green plants resulting from a lack of chlorophyll. 

CHROMATIDS - the longitudinal subunits produced by 

chromosome replication and joined at the centromere. 

CHROMATIN - the fibrous aggregate of DNA and histone protein that makes up a eukaryotic chromosome. 

CHROMOSOME- a discrete unit of the genome carrying 

many genes; each chromosome is a very long molecule of 

duplex DNA and an approximately equal mass of proteins; 

it is microscopically visible as a thread-like, morphological entity only during cell division. 

CHROMOSOME LANDING- a technique based on the 

isolation/ development of DNA marker(s) at a close physical distance to the genetic sequences (e.g., gene) of interest; the DNA marker(s) serves as a probe for screening 

genomic libraries allowing the isolation of the DNA clone 

containing the genetic sequences of interest; this technique 

eliminates the need for chromosome walking. 

CHROMOSOME REARRANGEMENT- a chromosomal 

USDA Forest Service Gen. Tech. Rep. RM-GTA-297. 1997. 

Glossary of Populus Tissue Culture and Molecular Biology 

aberration in which chromosomal segments are rearranged 

by inversion and translocation. 

CHROMOSOME WALKING - a procedure used for the 

systematic isolation and cloning of overlapping and contiguous regions of chromosomal DNA contained in a genomic library of cloned DNA; this procedure, which can 

be difficult to apply in plant species with complex genomes, allows the progressive development of genetic 

markers, each closer to the gene or genetic sequences of 

interest. 

CIS-ACTING DNA SEQUENCE - in eukaryotes, short 

(e.g., 10 to 12 bp) nucleotide sequences found in the 5'-

flanking regions or within genes; they represent recognition elements that interact with specific transcription 

factors and are required for eukaryotic gene regulation. 

CLADISTICS- a system of classification based on the phylogenetic relationships and evolutionary history of groups 

of organisms. 

CLONAL PROPAGATION - see clone; see vegetative 

propagation. 

CLONE - a group of vegetatively propagated organisms 

consisting of an ortet and its ramets; a cell line of single 

cell origin; a gene or piece of DNA replicated in a host 

bacterium; all definitions imply genetically identical material and reproduction by mitotic division. 

CO-CULTIVATION - the joint culture of 2 or more types 

of cells such as a plant cell and a microorganism (includes 

most Agrobacterium spp.). 

CO-INTEGRATION VECTOR - a transformation vector 

in which the T-DNA includes the gene(s) of interest with a 

selectable marker gene instead of oncogenes on the li-plasmid; see li-plasmid. 

CODING REGION- see coding sequence. 

CODING SEQUENCE- the part of a gene that directly 

specifies the amino acid sequence of its protein product; 

noncoding sequences of genes include control regions such 

as promoters, operators, and terminators, and intron sequences of certain eukaryotic genes. 

CO DOMINANT - the products of both genes at a locus 

that are expressed nearly equally. 

CODON -a specific sequence of 3 consecutive nucleotides 

that is part of the genetic code and that specifies a particular 

amino acid in a protein or starts or stops protein synthesis. 

COLLAR SHOOT- a shoot arising from a line of junction 

between a root and its stem. 

COMPATIBILITY- the ability of pollen to affect fertilization; a susceptible plant and virulent pathogen capable of 

causing disease. 

297 
Section VI Appendices 

COMPETENCE- in development, the physiological state 

or condition of a tissue that enables it to react morphologically in a specific way to stimuli such as inducers. 

COMPLEMENTARY DNA (eDNA) - a single-stranded 

DNA that is complementary to RNA and is synthesized 

from it by reverse transcription of RNA in vitro. 

CONIDIOPHORE - a specialized fungal hypha-bearing 

conidia. 

CONIDIUM (CONIDIA)- an asexual fungal spore usually at the tip of a special hypha called a conidiophore. 

CONSTITUTIVE EXPRESSION- gene expression exemplified by any gene that encodes a product continuously 

required in the maintenance of basic cellular processes or 

cell architecture, as opposed to a regulated gene that typically encodes products required for growth under specific 

physiological conditions or in particular cell types. 

COPPICE- a forest originating mainly from shoots or root 

suckers rather than from seed or tree cuttings; harvesting 

to promote new shoot growth. 

COPY NUMBER- the number of genes per genome, plasmids, or genes contained in a cell. 

COROLLA- the petals of a flower considered as a group 

or unit. 

COSUPPRESSION - a process by which transgenes can 

inhibit their own expression and that of homologous (e.g., 

native) genes. 

COTYLEDON - the seed leaf or leaves of an embryonic 

plant (i.e., 1 in a monocotyledon, 2 in a dicotyledon, and a 

variable number in gymnosperms). 

CROSS-POLLINATION- the transfer of pollen from one 

plant to a female flower of another plant or clone. 

CROSS-RESISTANCE - protection given to a plant by a 

pathogen (e.g., virus) infection to subsequent infection by 

strains of related pathogens. 

CROSSING OVER - a process of exchange between 

nonsister chromatids of a pair of homologous chromosomes that results in the recombination of linked genes. 

CRYOPRESERVATION - storage of cells, tissues, seeds, 

etc. at extremely low temperatures. 

CULTIVAR- an assemblage of cultivated individuals distinguished by any useful reproducible character and designated by 'cv.' 

CYTOKININ- a large class of plant growth regulators that 

promote cell division and enlargement in vitro and in vivo 

in the presence of auxins and have other effects such as 

control of organ differentiation, influence of auxin transport, breaking of dormancy, and apical dominance in buds; 

298 

commonly used cytokinins in plant tissue culture are 6-

benzyladenine (BA), 2-isopentyladenine (2-iP), zeatin, and 

kinetin; thidiazuron (TDZ) has cytokinin-like activity. 

CYTOLOGY- a branch of biology dealing with the structure, function, and life history of cells. 

CYTOPLASM - the protoplasm (or "cell sap") of a cell 

surrounding the nucleus; it includes the cytoplymph, organelles, inclusions, and the plasma membrane. 

CYTOTOXIN- a substance having a specific toxic affect 

on certain cells or cellular components. 

DE NOVO- anew; once more; again. 

DEAMINATION- the removal of an amino (-NH2

) group 

from an organic compound. 

DEFENSIN - one class of antimicrobial peptide like 

cecropins and magainins; see cecropins and magainins. 

DEHYDROGENATE- the removal of hydrogen (H). 

DEMETHYLATION - the removal of a methyl (-CH3

) 

group. 

DENATURATION - the loss of the native configuration 

of a macromolecule from specific treatment (e.g., heat treatment) that usually results in loss of biological activity. 

DEOXYRIBONUCLEIC ACID (DNA) - the macromolecule, usually composed of 2 polynucleotide chains in a 

double helix, which is the carrier of genetic information in 

all cells and many viruses; see nucleotide. 

DEPOLYMERIZE - the degradation of a polymer to oligomers and/ or monomers. 

DICOT (DICOTYLEDON)- see cotyledon. 

DIHAPLOID - possessing a diploid number of chromosomes from a doubling of the haploid genome. 

DIMER- a compound formed from 2 molecular subunits 

(i.e., monomers). 

DIOECIOUS- producing male and female flowers on different plants. 

DIPLOID- a cell or organism with 2 complete sets of homologous chromosomes (i.e., 2N). 

DISCOUNTED CASH FLOW- appraising projects based 

on discounting future costs and benefits to their present 

values. 

DNA- see deoxyribonucleic acid. 

DNA FINGERPRINTING- electrophoretic identification 

of individuals using DNA probes for highly polymorphic 

regions of the genome so that the genome of virtually every individual exhibits a unique pattern of bands. 

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DNA MICROINJECTION - see microinjection. 

DNA POLYMERASE- an enzyme that synthesizes a 

daughter (complementary) strand(s) of DNA under the 

direction of a DNA template strand. 

DOMINANT- an allele or the corresponding phenotypic 

traits that are expressed in a heterozygote. 

DORMANT - in a resting or nongrowing state such as 

buds, seeds, spores. 

DOWN-REGULATED- causing reduced gene expression. 

DOWNSTREAM- the direction in which a nucleic acid 

or protein molecule is synthesized or on the C-terminal 

side of any site within a polypeptide. 

EARLYWOOD - the wood formed early in the growing 

season (earlywood) that has larger cells with thinner walls 

and a lower density than the wood formed later in the season (latewood). 

ECOSYSTEM- an ecological system; a natural unit of living and nonliving components that interact to form a system in which cyclic interchange of materials takes place 

between living and nonliving units such as in an aquarium, 

lake, or forest. 

ECOTYPE - within a species having a wide geographical 

distribution, a subgroup that has developed specific 

adaptions to local conditions such· as temperature, light, 

and humidity. 

ECTOMYCORRHIZAE -·see mycorrhizae. 

ECTOPIC - occurring in an abnormal position or in an 

unusual manner or form. 

ELECTROPHORESIS- a technique used to separate molecules on the basis of their molecular size and charge by 

differential movement through a liquid or a gel in response 

to an applied electric field. 

ELECTROPORATION - introduction of DNA fragments 

into a cell by means of an electric migration. 

ELISA- see enzyme-linked immunosorbent assay. 

ELONGATION FACTOR - proteins that associate with 

ribosomes cyclically during addition of each amino acid 

to the polypeptide chain during protein synthesis. 

EMBRYO - that portion of the seed resulting from the 

union of male and female gametes that can develop into a 

mature plant. 

EMBRYOGENESIS - the process of embryo or embryoid 

formation, whether by sexual (zygotic) or asexual (somatic) 

means. 

ENDOGENOUS- produced or synthesized within the organism or system. 

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Glossary of Populus Tissue Culture and Molecular Biology 

ENDOMYCORRHIZAE- see mycorrhizae. 

ENDOPLASMIC RETICULUM (ER)- a complex set of 

membrane lamellae and tubules in eukaryotic cells that is 

the site of major biosynthetic processes such as protein 

secretion and membrane assembly. 

ENDOSPERM- food storage tissue contained in the seed 

and surrounding the embryo. 

ENDOTOXIN- a poisonous substance present in bacteria 

but separable from the cell body only on its disintegration. 

ENERGY CROP - a crop (e.g., poplar, willow, or switch 

grass) grown and used as a renewable energy source. 

ENHANCER- a cis-acting sequence that increases the activity of some eukaryotic promoters and can function in either 

orientation and in any location relative to the promoter. 

ENZYME- a protein or ordered aggregate of proteins that 

catalyzes a specific biochemical reaction and is unaltered 

after the process. 

ENZYME-LINKED IMMUNOSORBENT ASSAY 

(ELISA)- a serological test in which one antibody carries 

with it an enzyme that catalyzes a colorimetric reaction. 

EPIDEMIOLOGY- a branch of plant and animal pathology that studies the incidence, distribution, and control of 

disease in a population. 

EPIGENETIC- environmentally induced variations in the 

phenotype perpetuated by cloning but not involving permanent changes in the genotype resulting from the interaction of genetic factors that occurs during the normal 

development of an individual plant; epigenetic phenomena including habituation, determination, and juvenile/ 

adult phase changes. 

EPISTASIS- the interaction between nonallelic genes such 

that one gene interferes with or prevents expression of the 

other. 

ESTER -any class of often fragrant compounds formed 

by the reaction between an acid and an alcohol. 

ETIOLOGY- the assigned underlying cause or reason; the 

predisposing causes of either abiotically or biotically induced events, especially disease. 

EUCHROMATIN - a region of a chromosome having normal staining properties and undergoing the normal cycle of 

condensation; relatively uncoiled in the interphase nucleus 

and apparently containing most of the structural genes. 

EUKARYOTE - a cell or an organism composed of cells 

with true nuclei (chromosomal DNA enclosed in nuclear 

envelopes) and membrane-bounded cytoplasmic organelles (e.g., chloroplasts and/ or mitochondria) in which 

nuclear division is by mitosis or meiosis. 

299 
Section VI Appendices 

EX SITU - somewhere other than the natural or original 

position or place. 

EX VITRO- organisms removed from culture and transplanted, usually to soil or potting mixture. 

EXOGENOUS- introduced from or produced outside the 

organism or system. 

EXON - the sequences in a gene that are retained in the 

mRNA after the introns are removed from the primary 

transcripts. 

EXOTOXIN - a toxic substance excreted by organisms. 

EXPLANTS - the excised plant portion used to initiate a 

tissue culture. 

F1 - see F1 generation. 

F1 GENERATION- the first (filial) generation produced 

by crossing 2 parental lines. 

5'-P GROUP- the end of a DNA or RNA strand where the 

phosphate is attached to the number 5 carbon of the nucleotide sugar. 

FLANKING DNA SEQUENCE -segments of DNA immediately adjacent to the 2 ends of a DNA region under 

consideration; any DNA sequence flanking close to the 5' 

(upstream) end or 3' (downstream) end of a gene. 

FLANKING REGION -see flanking DNA sequence. 

FLUOROMETRY- measuring fluorescence and related 

phenomena (e.g., intensity of radiation). 

FOREIGN DNA- foreign gene; see transgenic plant. 

FOREIGN GENE- transgene, chimeric gene, and alien 

gene; see transgenic plant. 

FULL-SIBS -progeny (sibling) with both parents in common. 

FUNGICIDE- a compound toxic to fungi. 

FUNGISTATIC- a compound that prevents fungal growth 

without killing the fungus. 

G-BOX- a ubiquitous, cis-acting DNA regulatory element 

(G-box motif: CACGTG or CCACGTGG) found in plant 

genomes; proteins known as G-box factors bind toG-boxes 

in a context-specific manner, mediating a wide variety of 

gene expression patterns. 

GAMETE - a haploid generation male sperm cell or female egg cell capable of developing into a zygote (embryo) 

after fusion with a germ cell of the opposite sex. 

GAMETOCLONE- a plant regenerated from cell cultures 

originating from gametic culture. 

GAMETOPHYTE- the few-celled, haploid generation portion of a seed plant from meiotic division that through 

mitosis produces male or female gametes. 

300 

GAS CHROMATOGRAPHY- a chromatographic technique in which an inert gas is run through a column to 

separate materials. 

GENE - the hereditary unit containing genetic information that is transcribed into RNA and processed into an 

RNA molecule that functions directly or is translated into 

a polypeptide chain. 

GENE EXPRESSION- the multistep process by which a 

gene is regulated and its product synthesized. 

GENE FREQUENCY- the proportion of one type of allele 

to the total of all alleles at this genetic locus in a breeding 

population; the probability of finding the specific gene 

under consideration when a gene is randomly chosen from 

the population. 

GENE SILENCING- alteration of a structural gene or 

regulatory region in a DNA sequence so that the gene is 

not expressed. 

GENE TRANSFER - see genetic transformation. 

GENERATION TIME - the average time between 2 successive generations; the time required for a cell population to double. 

GENETIC GAIN -the increase in productivity following 

a change in gene frequency. 

GENETIC MAP- a chromosome map showing the relative locations of the known genes or markers on the chromosomes of a given species. 

GENETIC MARKER- any region used as an experimental probe to mark a nucleus, chromosome, gene, or DNA 

region. 

GENETIC TRANSFORMATION- the unidirectional gene 

transfer and incorporation of foreign DNA by prokaryotic 

and eukaryotic cells, and the subsequent recombination 

of part or all of that DNA into the cell's genome. 

GENOME- the total complement of genes contained in a 

cell or virus; commonly used to refer to all genes present 

in 1 complete set of chromosomes in eukaryotes. 

GENOMIC LIBRARY- a gene bank consisting of a set of 

independent clones that statistically contains the entire 

genome of an organism among the recombinant DNA 

molecules; construction and screening of a complete library 

of clones for any genome is a starting point for isolating 

specific genes. 

GENOTYPE- the genetic constitution of an organism or 

virus as distinguished from its appearance or phenotype; 

the allelic composition of 1 or more genes of interest. 

GEOGRAPHIC INFORMATION SYSTEMS (GIS)- computer software programs that use mathematical algorithms 

to enable input, management, analysis, and display of geographic point data. 

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GERMPLASM- the genetic material that forms the physical basis of inherited qualities that are transmitted from 

generation to generation by the germ cells. 

GLUCOSIDE- a glycoside that yields glucose on hydrolysis. 

GLYCOSIDE- a compound in which 1 or more sugars are 

involved in a linkage to another molecule. 

GOLGI APPARATUS (DICTYOSOME)- a subcellular organelle consisting of individual stacks of membranes near 

the endoplasmic reticulum; it is involved in glycosylating 

proteins and sorting them for transport to different cellular locations. 

GRAM-POSITIVE/GRAM-NEGATIVE BACTERIA-see 

Gram's staining reaction. 

GRAM'S STAINING REACTION - the specific staining 

techniques used to differentiate bacteria into Gram-positive and Gram-negative based on cell wall characteristics. 

GYMNOSPERMS- any class or subdivision of woody vascular seed plants that produces naked seeds not enclosed 

in an ovary that sometimes have motile spermatozoid (e.g., 

conifers). 

HAPLOID - a cell or organism having only 1 set of chromosomes (e.g., 1N). 

HERITABILITY- the ratio of genetic variance to phenotypic variance; narrow-sense heritability: the ratio of additive genetic variance to phenotypic variance that is useful 

in predicting the respons~ of a population to natural selection or to "pick-the-winner" selection; broad-sense heritability: the ratio of total genetic variance to phenotypic 

variance that is used to estimate the degree of genetic control of a trait in a population and to predict response to 

clonal selection. 

HETEROCHROMATIN - chromatin that remains condensed, is not transcribed into mRNA, and can be heavily 

stained during interphase; usually adjacent to the centromere and in the telomeres of chromosomes. 

HETERODIMER- a chemical structure consisting of 2 dissimilar subunits. 

HETEROGENEITY- a preparation of macromolecules in 

which the macromolecules differ in size, charge, structure, 

or other properties. 

HETEROGENEOUS- dissimilar of diverse components 

or constituents. 

HETEROPOLYMER - a high molecular weight chemical 

compound consisting of different repeated molecules 

(monomers). 

HETEROSIS - the marked vigor or capacity for growth 

often exhibited by crossbred animals or plants; hybrid 

vigor. 

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Glossary of Populus Tissue Culture and Molecular Biology 

HETEROTROPHIC - designating a type of nutrition in 

which an organism requires preformed carbohydrates or 

other organic compounds for energy. 

HETEROZYGOUS- having dissimilar alleles of 1 or more 

genes; not homozygous. 

HISTOCHEMISTRY - a science that combines the techniques of biochemistry and histology in the study of the 

chemical constitution of cells and tissues. 

HOMEOSTASIS- the tendency of a system to be specified by context (individual, population, process, etc.) to 

maintain a dynamic equilibrium and, in case of disturbance, to restore the equilibrium by its own regulatory 

mechanisms. 

HOMEOTIC GENES- genes that determine fundamental patterns of development. 

HOMOLOGOUS- in reference to DNA, having the same 

or similar nucleotide sequence; in reference to chromosomes, those that pair in meiosis, have the same morphology, and contain genes that govern the same traits. 

HOMOZYGOUS- having the same allele of a gene in homologous chromosome. 

HOST- an organism (e.g., host plant) that is invaded by a 

parasite and from which the parasite obtains its nutrition. 

HYBRIDIZATION- the crossing of different races or species, also sometimes used to denote the crossing of individuals; the pairing of complementary RNA and DNA 

strands to give an RNA-DNA hybrid. 

HYDROXYLATION- the introduction of a hydroxyl (-OH) 

group into an organic compound. 

HYPHA (HYPHAE) - one of the structural threads that 

make up the mycelium of a fungus. 

HYPOCOTYL- the part of the embryo or embryonic seedling stem between the cotyledons and radicle. 

ICE-NUCLEATING BACTERIA - certain bacteria (e.g., 

Pseudomonas syringae, P. fluorescens, and Envinia herbicola) 

whose presence on the surface of plant tissue may result 

in increased sensitivity to frost injury at temperatures that 

would not otherwise cause damage, 

IN PLANT A - occurring within the plant. 

IN SITU- in the natural or original position or place. 

IN VITRO- experimentation on whole organisms or portions thereof, in glassware or culture; growing under artificial conditions as in tissue culture. 

IN VIVO - in the living body of a plant or animal. 

INBREEDING- mating between genetically related individual organisms. 

301 
Section VI Appendices 

INCOMPATIBILITY- the inability of pollen to affect fertilization because growth of the pollen tube is arrested in 

the style; also see avirulence gene. 

INITIATION CODON- any codon in mRNA that directs 

initiation of genetic translation by stimulating the binding of initiator tRNA and by phasing the translation of 

mRNA; in eukaryotes, AUG is the usual initiator and is 

translated as methionine. 

IN STAR - the stage of development or the form assumed 

by an insect between successive molts. 

INTERCELLULAR SPACE- the space between or among 

cells. 

INTERGENIC SPACER (IGS)- any of the DNA sequences 

(sequence, region, spacer, intercistronic region) flanking 

the eukaryotic genes on the 3' and/ or 5' side. 

INTERNAL TRANSCRIBED SPACERS (ITS)- a variable, 

noncoding region between the highly conserved nuclear 

small and large subunit rONA that is useful for distinguishing closely related species. 

INTROGRESSION - the incorporation of genes from 1 

species into the gene pool of another species by hybridization and backcrossing. 

INTRON - a noncoding segment of DNA that is transcribed but removed from the transcript by splicing together the sequences (exons) on either side of it. 

INVERTED REPEAT- a pair of base sequences present in 

the same molecule that are identical or nearly identical 

but oriented in opposite directions; often found at the ends 

of transposable elements. 

ISOENZYME -see isozyme. 

ISOGENIC - any group of individuals that possess the 

same genotype, irrespective of their being homozygous 

or heterozygous 

ISOLATE- a culture or subpopulation of microorganisms 

separated from its parent population and maintained under controlled conditions; to affect such separation and 

control such as isolating a pathogen from diseased plant 

tissue. 

ISOMER- any of 2 or more substances that are composed 

of the same elements in the same proportions but differ in 

properties because of differences in the arrangement of 

atoms (structure). 

ISOTONIC- having the same osmotic potential; the same 

molar concentration of a solution. 

ISOZYME- any of 2 or more chemically distinct but functionally similar enzymes. 

KB - see kilobase. 

302 

KILOBASE - 1,000 bases (nucleotides) of DNA or 1,000 

bases of RNA. 

LARVA (LARVAE)- the wormlike, wingless, immature, 

feeding form that hatches from the egg of insects and undergoes complete metamorphosis; called caterpillar, maggot, grub, or wiggler. 

LATEWOOD -see earlywood. 

LEADER SEQUENCE - a segment of nucleotides at the 

5'-end of mRNA that precedes the AUG initiation codon 

where translation begins; the leader is an untranslated segment of mRNA that varies in length but always contains 

part or all of a unique nucleotide sequence (e.g., ShineDalgano sequence). 

LEAF PLASTOCHRON INDEX - stages of vegetative 

(physiological) development of leaves in relation to the 

shoot apex. 

LENTICEL- an opening on the stem of a plant through 

which air is admitted to underlying tissues. 

LEUCINE ZIPPER - in DNA-binding proteins, an amino 

acid sequence (30 residues with leucine at every seventh 

amino acid in the region of similarity) that binds specifically to the CAAT box and transcriptional enhancer. 

LIGAND- a small molecule (e.g., activators, substrates, 

and inhibitors of enzyme activity) bound to a protein by 

noncovalent forces; an ion, a molecule, or a macromolecule 

group that binds to another chemical entity to form a larger 

complex. 

LIGNIFICATION- the process of deposition of lignin in 

cells walls. 

LIGNIN- a complex substance present in the cell walls of 

woody plants that, with cellulose, forms wood. 

LINKAGE - any association of genes in inheritance that 

exceeds what is expected from independent assortment 

and is due to their location on the same chromosome; linkage is assessed by the tendency of 2 markers to remain 

together during recombination. 

LOCUS (LOCI) - the site or position of a particular gene 

on a chromosome. 

MAGAININS- 1 of 2 closely related peptides (23 amino 

acids each) isolated from frog skin that kill a variety of 

bacteria, fungi, and protozoa; believed to be part of a defense system in vertebrates that is different from the immune system. 

MARKER ASSISTED SELECTION (MAS) - an efficient 

approach to identify linkage between genetic markers and 

quantitative trait loci (QTL); an indirect, early selection 

based on the DNA markers. 

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MARKER GENE - a gene, usually dominant, with a large, 

recognizable effect that can identify recombinant organisms 

or 1 parent in offspring from open or mixed pollination. 

MATING PAIR- a male and female brought together for 

breeding and reproduction. 

MATRIX ATIACHMENT REGIONS (MAR) - a region 

of DNA that attaches to the nuclear matrix; known as SAR 

for scaffold attachment site. 

MEGAGAMETOPHYTE - the female gametophyte; the 

plant that develops from a megaspore. 

MEGASPORE - the larger of 2 types of spores that produce a female gametophyte; also called macrospore. 

MEIOSIS- the process of nuclear division in gametogenesis or sporogenesis in which 1 replication of the chromosomes is followed by 2 successive divisions of the nucleus 

to produce 4 haploid nuclei. 

MERISTEM- plant tissue composed of dividing cells and 

producing organs such as leaves, flowers, roots, etc. 

MESSENGER RNA (mRNA)- RNA produced by transcription that carries the code for a particular protein from 

the nuclear DNA to a ribosome in the cytoplasm and acts 

as a template for the formation of that protein. 

METHYLATION - the modification of a molecule (e.g., 

DNA or RNA base) by the addition of a methyl (-CH3

) 

group. 

MICROMOLAR (JlM) - one millionth of a mole. 

MICROBE -a microscopic, possibly pathogenic, organism or microorganism. 

MICROCOSM- a community, institution, or other unit 

serving as a small representation of a larger unit. 

MICROINJECTION- direct physical injection into individual cells, particularly for DNA transfer. 

MICROPARTICLE BOMBARDMENT- see biolistics. 

MICRO PROJECTILE BOMBARDMENT- see biolistics. 

MICROPROPAGATION - refers to propagation in culture by axillary or adventitious means; a general term for 

vegetative (asexual) in vitro propagation. 

MICROSHOOT- a shoot that is produced and grown under in vitro conditions such as for micropropagation. 

MICROSPORE- a haploid male cell that ripens into a pollen grain; immature pollen grain. 

MITOCHONDRIAL DNA (mtDNA) - see organelle 

DNA. 

MITOSIS - the process of nuclear division in which the 

replicated chromosomes divide and the daughter nuclei 

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Glossary of Populus Tissue Culture and Molecular Biology 

have the same chromosome number and genetic composition as the parent nucleus. 

MOLAR- of or relating to a mole of a substance; containing 1 mole of solute in 1 liter of solution. 

MOLE- in the International System of Units, the base unit 

amount of pure substance that contains the same number 

of elementary entities as atoms in exactly 12 grams of the 

isotope carbon 12. 

MOLECULAR CHAPERONES - a family of unrelated 

classes of protein that mediate the correct assembly and 

conformation of other polypeptides but are not components of the functional assembled structures; one important class of molecular chaperones is the 70-KDa family of 

heat shock proteins or stress-related proteins. 

MONOCOTS (MONOCOTYLEDON) -see cotyledon. 

MONOLIGNOLS- the building blocks of lignin (e.g., pcoumaryl alcohol, coniferyl alcohol, and sinapyl alcohol). 

MONOMER- an individual structural unit that undergoes polymerization. 

MORPHOGENESIS - the formation and differentiation 

of tissues and organs. 

mRNA -see messenger RNA. 

MUTANT - any variant strain, individual, cell, or gene 

resulting from mutation. 

MUTATION -any heritable alteration, whether a single 

base change in DNA (or RNA) or a large-scale rearrangement, in the genetic material of a living cell or virus. 

MYCELIUM (MYCELIA) - the mass of interwoven filamentous threads (hyphae) that forms the vegetative body 

of a fungus. 

MYCORRHIZAE- the symbiotic association of the mycelium of ct fungus with the roots of a seed plant in which 

the mycelium forms a thick web over and between the 

outer surface of roots (ectomycorrhizae) or the mycelium 

hyphae colonize the root cells (endomycorrhizae or vesicular arbuscular mycorrhizae). 

NECROSIS - the death of cells usually resulting in tissue 

darkening. 

NODULE- independent, spherical, dense cell clusters that 

form a cohesive unit and display consistent internal cell/ 

tissue diffentiation, derived from plant cell suspension 

culture. 

NONADDITIVE GENETIC VARIATION - the proportion of genetic variation that does not respond to simple 

mass selection and that causes specific pairwise crosses to 

depart from performance values predicted by the breeding values of the parents. 

303 
Section VI Appendices 

NORTHERN ANALYSIS -see northern blotting. 

NORTHERN BLOTTING- a technique, analogous to 

Southern blotting, used for transferring RNA from agarose gel to a membrane where it can be hybridized to a 

labeled, complementary, single-stranded DNA or RNA 

probe. 

NUCLEAR SCAFFOLD ATTACHMENT REGIONS 

. (SAR) - see matrix attachment site. 

NUCLEOTIDE - any of several compounds that consist 

of a ribose or deoxyribose sugar joined to a purine or pyrimidine base and to a phosphate group; the basic structural units of nucleic acids. 

OLIGOMER- a chemical compound composed of a few 

to several monomers (structural units). 

OLIGONUCLEOTIDE - a short polymeric chain of multiple (e.g., 2 to 20) nucleotides. 

ONCOGENE - a gene whose product has the ability to 

transform eukaryotic cells so that they grow in a manner 

analogous to tumor cells. 

OPEN READING FRAME (ORF) - a stretch of triplet 

codons with an initiator codon at one end and termination codon at the other, identifiable by nucleotide sequences 

coding for protein. 

OPERATOR - a regulatory region in DNA that interacts 

with a specific repressor protein in controlling the transcription of adjacent structural genes. 

OPERON -a collection of adjacent structural genes regulated by an operator and a repressor. 

OPINE- a family of amino acids (e.g., octopine, nopaline, 

agropine, and agrocinopine) that are catabolized by 

Agrobacterium strains. 

OPPORTUNITY COSTS- the value of the foregon~ alternative action; the most favorable price that a factor of production (land, labor, or capital) can command. 

ORGANELLE DNA - extranuclear DNA (cpDNA, 

mtDNA) of eukaryotes localized in cellular organelles 

other than the nucleus and usually inherited uniparentally 

(frequently maternal) in plants and animals. 

ORGANELLE GENOME - see organelle DNA. 

ORGANOGENESIS- the initiation and growth of organs, 

usually roots and shoots, from cells or tissue; organs may 

form on the surface of explants (direct organogenesis) or 

upon an intervening callus phase (indirect organogenesis). 

ORTET- the initial individual that is vegetatively propagated to produce a clone; see ramet. 

OSMOTIC POTENTIAL (OSMOTIC PRESSURE) - the 

pressure exerted by the flow of water through a semiper304 

meable membrane separating 2 solutions with different 

concentrations of solute. 

OSMOTICUM- an agent such as mannitol, sugars, polyethylene glycol, and salts used to maintain the osmotic 

potential of the surrounding medium. 

OVIPOSIT- to discharge eggs from the body. 

OVULE- an oval body in the ovary of a flower that develops into a seed. 

PALINDROME - a sequence of DNA that is the same 

when a strand is read left to right and the other complementary strand is read right to left; consists of adjacent 

inverted repeats. 

PALISADE PARENCHYMA - the undulating lower epidermis of plant leaves with meandering vascular bundles; 

larger cells of the lower epidermis. 

PARAMUTATION - directed genetic change caused. by 1 

allele on the other. 

PARASITE- an organism living on or in another organism (host) from which it obtains nutrition. 

PARENCHYMA- a tissue of higher plants that consists of 

thin-walled, living photosynthetic or storage cells capable 

of division that, even when mature, makes up much of the 

substance of leaves and roots, pulp of fruits, and parts of 

stems and supporting structures. 

PARTICLE ACCELERATION DNA DELIVERY SYSTEM 

- see biolistics. · 

PARTICLE BOMBARDMENT- see biolistics. 

PATHOGEN- an agent (parasite) capable of causing disease. 

PATHOGENESIS -the phase of the pathogen life cycle 

when it is associated with the host. 

PATHOSYSTEM - a particular host and pathogen interaction. 

PCR - see polymerase chain reaction. 

PECTINASES- an enzyme that degrades pectin (the adhesive material that cements cells together) and is used 

alone or with other enzymes to digest the polygalacturonic 

acid component of plant cell walls for protoplast isolation. 

PEPTIDE- a compound formed by the union of 2 or more 

amino acids such as a dipeptide or polypeptide. 

PETAL- a modified leaf, usually colored, forming a part 

of the corolla of a flower. 

PETIOLE- the slender stalk that supports the blade of a 

leaf. 

PHASE CHANGE- the developmental change frotp one 

maturation state to another. 

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PHENOTYPE - the observable properties of a cell or an 

organism resulting from the interaction of the genotype 

and the environment. 

PHENYLPROPANOID PATHWAY- a metabolic pathway 

that produces phenolic compounds (e.g, phytoalexins) 

with a significant range of biological functions including 

defense of plants against pests; see phytoalexin. 

PHLOEM- food-conducting tissue in vascular plants consisting principally of sieve tubes and companion cells. 

PHOTO LABILE- unstable in light. 

PHOTOPERIOD- the duration of the light portion in an 

alternating light-dark sequence. 

PHOTOSYNTHESIS- the process by which plant chloroplasts make carbohydrates from carbon dioxide and water in the presence of chlorophyll by using light energy 

and releasing oxygen. 

PHOTOSYSTEM I- a photochemical reaction system in 

photosynthesis that produces NADPH+ but does not emit 

oxygen. 

PHYLLOTAXY- the leaf arrangement around the stem of 

a plant that is genetically determined and species characteristic. 

PHYLOGENETIC- of or relating to the evolu~on of agenetically related group of organisms as distinguished from 

the development of the individual organism. 

PHYLOGENY - the evolutionary history of an organism 

or taxonomic group. 

PHYTOALEXIN - a substance that fuhibits the development of fungi on hypersensitive tissue that is formed when 

host plant cells come in contact with the parasite. 

PHYTOHORMONE- see plant growth regulator. 

PHYTOREMEDIATION- the use of specially selected and 

engineered plants for environmental remediation. 

PHYTOTOXICITY- poisonous to plants. 

PISTIL - a gynoecium or a unit of a gynoecium; the female 

structure of a flower consisting of an ovary, style, and stigma; 

a female organ composed of 1 or more carpels. 

PLANT GROWTH REGULATOR - any organic substance, other than a nutrient, which in small amounts induces a physiological, developmental, timing, or size 

change response in plant cell, tissues, or organs; this body 

of substances includes plant hormones, growth regulators, many herbicides, substances from nonplant sources, 

and laboratory-synthesized substances; see auxin and cytokinin. 

PLANT GROWTH HORMONE- see plant growth regulator. 

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Glossary of Populus Tissue Culture and Molecular Biology 

PLANT GROWTH SUBSTANCE-see plant growth regulator. 

PLANT HORMONE- see plant growth regulator. 

PLANT TRANSFORMATION-see genetic transformation. 

PLASMA MEMBRANE- the cell membrane that surrounds the cellular organelles and cytoplasm. 

PLASMID - an autonomous, self-replicating, extrachromosomal, circular DNA. 

PLEIOTROPY - the condition in which a single mutant 

gene affects 2 or more distinct and seemingly unrelated 

traits. 

PLOIDY- the number of chromosome sets per cell (e.g., 

haploid, diploid, or polyploid). 

POINT MUTATION -a mutation caused by the substitution, deletion, or addition of a single nucleotide pair. 

POLAR TRANSPORT- directional transport within a 

plant (e.g., movement of an endogenous plant growth regulator). 

POLLEN - the partly developed male gametophyte produced by the anthers of seed plants, a germinated microspore; these male spores often appear as yellow dust. 

POLYADENYLATION SEQUENCES- the 3' ends of most 

eukaryotic mRNAs containing long stretches of 

polyadenine (A) that are enzymatically added after transcription. 

POLYCISTRONIC- a prokaryotic mRNAcarrying the information for the synthesis of more than 1 protein. 

POLYETHYLENE-GLYCOL-MEDIATED GENE TRANsFER-introduction of DNA fragments into a cell by the chemical substance polyethylene-glycol (PEG); treatment of a plant 

protoplast with PEG promotes absorption of DNA. 

POLYMER- a chemical compound or mixture of compounds formed by polymerization and consisting essentially of repeating structural units. 

POLYMERASE- an enzyme that catalyzes covalent joining of nucleotides. 

POLYMERASE CHAIN REACTION PRIMERS -see polymerase chain reaction; see primer. 

POLYMERASE CHAIN REACTION -repeated cycles of 

DNA denaturation and renaturation with primer oligonucleotide sequences, and replication that results in exponential growth in the copy number of the target DNA 

sequence located between the primers. 

POLYMORPHISM- the presence in a population of 2 or 

more relatively common form's of a gene, chromosome, or 

genetically determined trait. 

305 
Section VI Appendices 

POLYPEPTIDE- a polymer of amino acids linked together 

by peptide bonds (e.g., protein). 

POLYSOME - a multiribosomal structure representing a 

linear array of ribosomes held together by mRNA. 

POSITION EFFECT- a change in the expression of a gene 

depending on its position within the genome. 

PREDATOR - any organism that preys on other organisms. 

PRIMER- a short sequence (e.g., RNA or DNA oligonucleotide) that is paired with 1 strand of nucleic acid and provides a free 3'-0H end at which a polymerase starts 

synthesis of a complementary strand. 

PRIMORDIUM (PRIMORDIA)- the earliest, rudimentary 

developmental stage of an organ or cell such as leaf primodia, 

which are barely visible on a dissected shoot tip. 

PROBE- a defined (labeled) DNA or RNA sequence used 

to detect complementary sequences by hybridization techniques (e.g., Southern blotting, northern blotting). 

PROGENY- the offspring of a particular parent or a particular combination of 1 female and 1 male organism. 

PROKARYOTE - an organism in which a nuclear envelope is lacking and the genetic material does not divide by 

mitosis or meiosis (e.g., bacteria and blue-algae). 

PROMOTER- a specific DNA sequence where RNA polymerase binds and initiates transcription of a gene or operon; in an operon, the promoter is usually located at the 

operator end, adjacent but external to the operator; the 

nucleotide sequence of the promoter determines the nature of the enzyme that attaches to it and the rate of RNA 

synthesis. 

PROTEASE - any of numerous enzymes that hydrolyze 

proteins and are classified according to the most prominent functional group at the active site. 

PROTEIN - a molecule composed of 1 or more polypeptide chains. 

·PROTEINASE - an enzyme the catalyzes the hydrolytic 

breakdown of internal (peptide) bonds of protein (polypeptide). 

PROTOCLONE- distinct phenotypic regenerants from a 

plant protoplast; a clone initiated from a protoplast or 

protoplast-fusion. 

PROTOPLAST -a cell without a cell wall but with a 

plasma membrane. 

PROTOPLAST FUSION - the uniting of 2 protoplasts. 

PUPA (PUPAE) - in insects with complete metamorphosis, a dormant, inactive stage between the larva and the 

adult. 

306 

PURINE - an organic base in nucleic acids; the predominant purines are adenine (A) and guanine (G). 

PYRIMIDINE - an organic base in nucleic acids; the predominant pyrimidines are cytosine (C), uracil (U, in RNA 

only), and thymine (T, in DNA only). 

QTLs - see quantitative trait loci. 

QUALITATIVE RESISTANCE (VERTICAL RESISTANCE)- many plant varieties are resistant to some pathogen races while susceptible to other races of the same 

pathogen; such resistance is sometimes called specific, 

qualitative, or differential resistance but is usually referred 

to as vertical resistance; vertical resistance is usually controlled by 1 or a few genes. 

QUANTITATIVE RESISTANCE (HORIZONTAL RESISTANCE)- all plant have a certain level of unspecific resistance against each of their pathogens; such resistance is 

sometimes called nonspecific, general, quantitative, adult 

plant, field, or durable resistance but is usually referred to 

as horizontal resistance; horizontal resistance is usually 

controlled by many genes. 

QUANTITATIVE TRAIT- a trait typically measured on 

a continuous scale, such as height or weight, which results from the combined action of multiple genes in conjunction with environmental factors. 

QUANTITATIVE TRAIT LOCI- multiple discrete chromosomal sites (loci) that control quantitatively inherited 

polygenic traits. 

RACE - a genetically and often geographically distinct 

mating group within a species; a group of pathogens that 

infect a given set of plant varieties. 

RADICLE- the lower part of the axis of a plant embryo or 

seedling. 

RAMET - all vegetative propagules of an ortet; a clone is 

composed of the ortet and its ramets. 

RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) 

- random amplified polymorphic DNA markers are generated by polymerase chain reaction (PCR) amplification 

using arbitrary primers (e.g., oligonucleotides or 10mers); 

amplified DNA fragments are electrophoretically separated on agarose gel and can be viewed under UV light 

when stained with ethidium bromide. 

RAPD - see random amplified polymorphic DNA. 

RAY CELL- a radiating, linear group of cells, as a pith ray. 

rONA -see ribosomal DNA. 

REACTIONWOOD - tilting of trees by wind results in 

redistribution of the amount and nature of cambial growth 

on the leeward and windward sides of the stem and in 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
'\(V 

formation of abnormal reactionwood that occurs on the 

lower sides of tilted stems. 

RECALCITRANT- resistant to directed change (e.g., tissue that is difficult to manipulate in vitro). 

RECEPTOR - transmembrane protein located in the 

plasma membrane that binds a ligand in a domain on the 

extracellular side and has a change in activity of the cytoplasmic domain. 

RECESSIVE - obscured in the phenotype of a heterozygote by the dominant gene, often due to inactivity or absence of the product of the recessive gene. 

RECOMBINANT DNA-see recombinant DNA technology. 

RECOMBINANT DNA TECHNOLOGY- the techniques 

by which genetic recombination is carried out in vitro; the 

breakage and rejoining of DNA molecules from different 

organisms, and the production and isolation of the modified DNA or fragments thereof. 

RECOMBINANT GENES - genes prqduced by recombinant DNA technology. 

RECOMBINASE - any enzymes that recognize specific 

DNA sequences, introduce single-strand breaks, and are 

involved in general recombination. 

RECOMBINATION FREQUENCY- a measure of the frequency of exchange (crossing over) occurring between 2 

specific loci. 

REFUGIA- an area that provides protection/ escape from 

ecological consequences occurring elsewhere. 

REGENERATION- to give or gain new life or to renew 

by a new growth of tissue such as regenerants (e.g., shoots, 

roots, or plantlets}, which form from explants in plant tissue cultures. 

· REGULATORY GENE- a gene with the primary function 

of controlling the rate of synthesis of the products of 1 or 

more other genes. 

REJUVENATION- a change in a tissue or an organism 

from a more mature state to a more juvenile one. 

REMEDIATION - to improve the ecosystem by removing, detoxifying, or absorbing harmful substances. 

REPEAT-INDUCED POINT MUTATION (RIP) - point 

mutations that occur frequently in the newly methylated 

sequences resulting from deamination of methylated 

cytosines. 

REPORTER GENE- a coding unit whose product is easily assayed such as chloramphenicol acetyltransferase 

(CAT), ~-glucuronidase (GUS}, and luciferase (luxF2); it 

may be connected to any promoter of interest so that gene 

expression can be used to assay promoter function. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Glossary of Populus Tissue Culture and Molecular Biology 

REPRESSOR- a protein that binds specifically to a regulatory sequence adjacent to a gene and that blocks transcription of the gene. 

RESTRICTION ENDONUCLEASE- a nuclease that recognizes a short specific nucleotide sequence (restriction 

site) in a DNA molecule and cleaves the molecule relative 

to that site; also called restriction enzyme. 

RESTRICTION ENZYME- see restriction endonuclease. 

RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) - genetic variation in a population associated with the size of restriction fragments containing 

sequences homologous to a particular probe DNA; the 

polymorphism results from the positions of restriction sites 

flanking the probed fragment, each variant is essentially a 

different allele. 

REVERSE TRANSCRIPTASE - see reverse transcription. 

REVERSE TRANSCRIPTION - synthesis of DNA on a 

template of RNA accomplished by reverse transcriptase 

enzyme. 

RFLP- see restriction fragment length polymorphism. 

RHIZOSPHERE - the soil region that surrounds and is 

influenced by plant roots. 

Ri-PLASMID- in Agrobacterium rhizogenes, a large rootinducing plasmid that transfers a specific segment of DNA 

(the T-DNA) into plant cells where it integrates into the 

nuclear genome and induces hairy root disease. 

RIBONUCLEASE (RNase) - any enzyme that cleaves 

phosphodiester bonds in RNA. 

RIBONUCLEIC ACID (RNA) - a nucleic acid in which 

the sugar constituent is ribose. 

RIBONUCLEOPROTEIN PARTICLE (RNP) - any of the 

ribonucleoprotein complexes involved in gene expression 

of eukaryotes. 

RIBOSOMAL DNA (rONA)- the family of chromosomal 

or extrachromosomal DNA sequences encoding ribosomal 

RNA. 

RIBOSOMAL PROTEIN - any of the essential proteins 

that are synthesized individually and assembled into ribosome. 

RIBOSOMAL RNA (rRNA) - RNA that is linked 

noncovalently to the ribosomal proteins in the 2 ribosomal 

subunits and that constitutes about 80 percent of the total 

cellular RNA; the transcription product of ribosomal DNA. 

RIBOSOME - a subcellular structure consisting of 2 subunits each composed of RNA and proteins on which the 

codons of mRNA are translated into amino acids in protein synthesis. 

307 
Section VI Appendices 

RIBOSOME BINDING SITE- in mRNA, a site for binding ribosomes that is required for initiation of genetic translation; in E. coli, 3 basic elements constitute the ribosome 

binding site: the Shine-Dalgarno sequence, the initiator 

codon (usually AUG), and an intervening spacer region, 

normally 5 to 9 nucleotides long. 

RNA- see ribonucleic acid. 

RNA BLOT ANALYSIS- see northern blotting. 

RNA POLYMERASE - an enzyme that makes RNA by 

copying the base sequence of a complementary DNA 

strand. 

RNase- see ribonuclease. 

RNP- see·ribonucleoprotein particle. 

rRNA - see ribosomal RNA. 

SCAFFOLD ATTACHMENT REGION (SAR) - see matrix attachment region. 

SDS-PAGE ANALYSIS-see sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 

SEGREGATION- separation of the members of a pair of 

alleles into different gametes during meiosis. 

SELECTABLE MARKER- used in marker-assisted selection or to select a transformed genotype from a mixture of 

transformed and nontransformed cells; antibiotic-resistance and herbicide-resistance genes are examples, where 

cells that lack the marker are killed on a selective medium. 

SELF-INCOMPATIBILITY- in diverse angiosperm families, the failure of self-fertilization; the inability to produce 

seed following self-pollination. 

SEPAL- a modified leaf forming one of the components 

of the calyx of a flower. 

SEQUENCE DIVERGENCE- the percent difference in a 

nucleotide sequence between 2 related nucleic acid sequences or, in an amino acid sequence, between 2 proteins. 

SHIKIMATE- a hydroxylated, unsaturated acid that is a 

key intermediate in the biosynthesis of the aromatic amino 

acids. 

SHIKIMATE PATHWAY- a pathway for the synthesis of 

sh~ate and its conversion to phenylalanine, tyrosine, 

and tryptophan. 

SODIUM DODECYL SULFATE-POLYACRYLAMIDE 

GEL ELECTROPHORESIS (SDS-PAGE) - electrophoresis in polyacrylamide gels containing SDS; this electrophoretic technique separates denatured macromolecules 

(e.g., proteins) and provides estimates of molecular weights 

by reference to the mobility of marker proteins of known 

molecular weight. 

308 

SOMACLONAL VARIATION - variation occurring in 

plants regenerated from cell cultures that originated from 

somatic tissue. 

SOMATIC HYBRID - a cell or plant product of somatic 

cell fusion that results from cell or protoplast fusion and 

implies genomic integration. 

SOMATIC EMBRYOGENESIS - a nonsexual developmental process leading to the differentiation of zygotic embryo-like structures from somatic cells. 

SOUTHERN BLOTTING- transfer of single-stranded, restriction-cut DNA fragments, separated by agarose gel electrophoresis to a membrane that is then analyzed by 

hybridization to labelled, single-stranded DNA or RNA 

probes. 

SOUTHERN HYBRIDIZATION- see Southern blotting. 

SPACER SEQUENCE- any DNA sequence separating 

neighboring genes; spacer sequences may or may not be 

transcribed. 

SPONGY MESOPHYLL- a leaf tissue consisting of loosely 

arranged, chloroplast-bearing usually lobed cells; also 

called spongy parenchyma. 

SPONTANEOUS MUTATION - a mutation occurring in 

the absence of any known mutagenic agents. 

STALK - the main supporting axis or stem of a plant or 

embryo. 

STAMEN- the pollen-producing structure of a flower consisting of an anther on the tip of a filament. 

STARTPOINT - the position on DNA corresponding to 

the first base incorporated into RNA. 

STERILITY - any complete or partial failure of an individual to produce functional gametes or viable zygotes 

under a given set of environmental conditions. 

STIGMA - the portion of the pistil that receives the pollen; the expanded apex of the style. 

STOMATE (STOMATA)- a minute opening in a leaf or 

stem through which gases pass. 

STORAGE PROTEIN - a protein stored in specific tissue 

under specific environmental conditions that can be metabolized at a later time. 

STRUCTURAL GENE- a gene that encodes the amino 

acid sequence of a polypeptide chain (protein). 

STYLE- the slender, elongated portion of a pistil with the 

stigma at its tip. 

SUBCULTURE - a culture. derived from another culture 

or the aseptic division and transfer of a culture or~ portion of that culture to fresh nutrient medium. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
SUCKER- a shoot originating from the basal stem region 

or roots of a plant. 

T-DNA- transferred DNA; see TI-plasmid. 

TANDEM ARRAY- multiple copies of the same DNA sequence lying in series. 

TATA BOX- a conserved A-T rich septamer about 25 bp 

before the startpoint of each eukaryotic RNA polymerase 

II transcription unit. 

TELOMERES- the tips of a chromosome that contain DNA 

sequences required for stability of chromosome ends. 

TEMPLATE - a nucleic acid strand whose base sequence 

is copied in a polymerization reaction. 

TERATOMA- a tumor derived from embryonic tissue consisting of a disorganized mass of many tissue types; many 

cells representing a variety of differentiated tissues. 

TERMINATION CODON - 1 of the 3 mRNA codons 

(UAG, UAA, and UCG) at which polypeptide synthesis 

stops. 

3'-0H (hydroxy) GROUP - the end of a DNA or RNA 

strand that terminates in a sugar and has a free hydroxyl 

group on the number 3 carbon. 

TI-PLASMID- inAgrobacterium tumefaciens, a large tumorinducing plasmid that transfers a specific segment of DNA 

(the T-DNA) into plant cells where it is integrated into the 

nuclear genome and induces crown gall tumors. 

TOTIPOTENCY- the capacity of a cell to express all of its 

genetic information under appropriate conditions and to 

develop into a complete and fully differentiated organism. 

TRACHEID- in vascular plants, an elongated, dead cell 

with a pronounced cavity or lumen forming an element of 

xylem. 

TRANS-ACTING FACTOR - any of the protein factors 

that mediate transcription of eukaryotic genes by transcriptional activation and bind to cis-acting DNA sequence elements. 

TRANSCRIPTION - the process by which the information contained in the coding strand of DNA is copied into 

a single-stranded RNA molecule with a complementary 

base sequence. 

TRANSCRIPTION FACTOR- any of the multiple ancillary DNA-binding proteins that interact with the cis-regulatory DNA sequences to control gene expression. 

TRANSFER RNA (tRNA)- a small RNA molecule that 

translates a codon into an amino acid in protein synthesis. 

TRANSFORMANT - any cell that has either stably integrated foreign DNA into its chromosomal DNA or transiently harbors foreign DNA. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 

Glossary of Populus Tissue Culture and Molecular Biology 

TRANSGENE- see transgenic plant. 

TRANSGENE EXPRESSION - the multistep process by 

which a transgene is regulated and its encoded product 

synthesized. 

TRANSGENIC PLANT- an individual plant containing 

a foreign gene (a trans gene) that was introduced by a gene 

transfer technique. 

TRANSIT PEPTIDE- in the case of post-translationally 

imported mitochondrial and chloroplast protein, an amino 

terminal sequence that contains targeting information and 

is removed during or shortly after import into the organelles. 

TRANSLATION - the process of forming a protein molecule at a ribosomal site of protein synthesis from information contained in mRNA. 

TRANSLATION INITIATION SITE-see ribosome binding site. 

TRANSLOCATION - the interchange of parts between 

chromosomes (e.g., nonhomologous chromosomes); the 

movement of mRNA with respect to a ribosome in protein 

synthesis. 

TRANSPOSABLE ELEMENT- a DNA sequence capable 

of moving from 1 location to another in a genome. 

TRANSPOSON- a transposable element especially when 

it contains genetic material controlling functions other than 

those related to its relocation. 

TRIPLOID -a cell or individual organism with 3 complete sets of chromosomes. 

tRNA - see transfer RNA. 

UBIQUITIN- a small protein (76 amino acid residues) in 

nearly all prokaryotic and eukaryotic cells with a high 

degree .of amino acid conservation between groups of organisms. 

UPSTREAM - on the 5' side of any given site in DNA or 

RNA and on the N-terminal side within a polypeptide; it 

identifies sequences proceeding in the opposite direction 

from expression. 

UREDIOSPORES- binucleate resting spores produced in 

the life cycle of rust fungi that are capable of infecting the 

host species on which they were produced. 

UREDIUM (UREDIA)- a fruiting structure formed by the 

rust fungi in which urediospores are produced. 

VACUOLE - a space or cavity within a cell such as the 

cavity of a plant cell containing cell sap. 

VASCULAR CAMBIUM- the cambium of a vascular cylinder that produces secondary xylem and phloem. 

309 
Section VI Appendices 

VECTOR -an organism that carries and transmits a 

pathogen to another organism (e.g., plant) particularly 

inoculum and especially a fungus, insect, mite, or nematode that transmits a mollicute or a virus; the plasmid 

or nucleic acid vector u~ed in the inserti,on of genes into 

cells in genetic engineering; see binary vector and cointegration vector. 

VEGETATIVE PROPAGATION- the propagation or multiplication of plants by asexual means. 

VITRIFICATION - a condition of cultured tissue with 

leaves and sometimes stems that have a glassy, transparent, or wet and often swollen appearance; eventually shoot 

tip and leaf necrosis occurs. 

WESTERN BLOTIING - the transfer of proteins from a 

polyacrylamide gel onto a suitable immobilizing matrix 

where the proteins may be probed with a specific antibody 

to identify particular proteins. 

XENOBIOTIC- components of the chemical environment 

that are foreign to the metabolic network of an organism. 

XYLEM - 1 of 2 components of vascular tissue; the wood 

portion of woody plants; complex tissue made up of fibers, vessel members, tracheids, and parenchyma cells; it 

conducts water and mineral salts and lends structural support to plant organs. 

zyGOTE - the product of the fusion of a female and a 

male gamete in sexual reproduction; a fertilized egg. 

310 

Sources of Terminology 

Ammer, C.;Ammer, D.S.1984. Dictionary of business and 

economics. New York: The Free Press: 507 p. 

Donnelly, D.J.; Vidaver, W.E. 1988. Glossary of plant tissue 

culture. Advances in Plant Sciences Series, Vol 3. Portland, Oregon: Dioscorides Press: 141 p. 

Hartl, D.L. 1991. Basic genetics. Boston: Jones and Bartlett: 

447-467. 

King, R.C.; Stansfield, W.D. 1990. A dictionary of genetics. 

Oxford: Oxford University Press: 406 p. 

Lewin, B. 1994. Genes V. Oxford: Oxford University Press: 

1235-1257. 

Libby, W.J. 1994. Genetic terms for silviculturists. _The Inland Empire Tree Improvement Cooperative: 22 p. 

Rieger, R.; Michaelis, A.; Green, M.M. 1991. Glossary of 

genetics: Classical and molecular. Berlin: SpringerVerlag: 553 p. 

Steen, E. B. 1971. Dictionary of biology. New York: Barnes 

& Noble Books: 630 p. 

Stenesh, J. 1989. Dictionary of biochemistry and molecular biology. New York: John Wiley & Sons, Inc.: 525 p. 

Wright, J.W. 1976. Introduction to forest genetics. New 

York: Academic Press: 439-455. 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 
Index of Populus 

Populus sections 

Abaso 290 

Aigeiros 8, 13,35-37, 106, 107, 112, 114, 

121, 290, 291 

Leuce 7, 10, 13, 35-38, 53, 106, 107, 114, 121, 

175,290,291 

Leucoides 13, 106, 107, 121, 290 

Tacamahaca 8, 13, 35-37, 53, 106, 107, 112, 

114,121,290,291,294 

Turanga 13, 106, 290 

Populus species and hybrids 

Populus 

alba 6, 14, 26-29, 31, 32, 35, 38, 41, 42, 

106,107,112,114,123,137,144-148,159, 

166,290,291 

'5972-S38' 35 

alba x P. glandulosa 26-29, 31, 32, 55, 57, 

144-148,171,207,210 

alba x P. grandidentata 8, 10, 14, 15, 18, 

20-23,25-29,31,35,43,45,46,49,52-56, 

61,62,70,85,91,94, 166,174,179,200, 

202, 207, 291 

'Crandon' ('NC5339') 10, 15, 18, 20, 

21, 35, 61, 62, 70, 72, 73, 85-88, 91, 93, 

94, 179, 182, 183 

'Favorit' 35 

'Hansen' 15, 20, 21, 91, 174, 175 

alba x P. tremula 52, 55, 166,_ 208, 291 

alba x P. tremuloides 35,291 

'802-36-7' 35 

angulata 1, 255,290 

angustifolia 34, 35, 106, 107, 290 

angustifolia x P. fremontii 35 

balsamifera (P. tacamahaca) 106, 107, 117-

122, 125, 290, 291 

'TACS' 118 

'TAC21' 118 

'TAC51' 118 

balsamifera var. subcordata/candicans x P. 

x berolinensis 255 

'NC5262' ('NE397') 255 

ciliata 10, 12, 14, 21, 23, 26, 31, 37, 40, 48, 

106,107,290 

davidiana 15, 22, 26, 55, 58, 106, 107, 144-

148,171,207,210,290 

deltoides 1, 14, 15, 17, 19, 22, 23, 26, 32, 

35, 55, 57, 66, 106-110, 112, 114, 117-123, 

125, 126, 1~130, 135, 144, 148, 157, 160, 

221,223,233,235,255,290,291 

'035' ('W-1-2') ('Larsson') ('039') 

118 

'0196' ('Northwest') 118 

'0391' (Mixoploid) (Brockville#C136) 

118 

'K417' 35 

'NC11004' ('Siouxland') 255 

deltoides var. angulata x P. x berolinesis 

255 

'DTAC2' 255 

deltoides var. angulata x P. trichocarpa 255 

'NC11432' ('NE252') 255 

deltoides x P. maximowiczii 107 

deltoides x P. trichocarpa (P. 

interamerica1Ul) 66,108,109, 117-122 

291 

'DT AC9' ('Donk') 118 

'DT ACtO' ('Barn') 118 

fremontii 34, 35, 106, 107, 290, 291 

glandulosa 14, 15, 22, 26-29, 31, 46, 47, 

144-148,171,290 

grandidentata 105,106, 133, 135-137,161, 

166,290 

koretma 106, 166, 171, 290 

koretma x P. nigra 26-31, 144, 146, 147, 

159 

lasiocarpa 14, 106, 107, 290 

laurifolia 26, 35, 106, 107, 255, 290, 291 

maximowiczii 15, 19, 22, 23, 49, 106, 107, 

112,114,117-123,144,146-148,156,290 

'M900' 118 

'M901' 118 

'M908' 118 

maximowiczii x P. x berolinensis 255 

'NC11396' ('NE49') 255 

maximowiczii x P. nigra 144 

maximowiczii x P. trichocarpa 14, 35, 144, 

255 

'NC11390' 35 

'NC11505' ('NESS') ('NE388') 255 

'Androscoggin' 35 

nigra 1, 26, 28, 29, 31, 34, 35, 48, 55, 57, 

98, 106, 107, 112, 114, 117-123, 144-148, 

166,172,290 

'N75' ('#113') 118 

'N84' (1talica') ('#555/50) 118 

'N91' ('Purkersdorf') (#44/62(10)) 

118 

'N100' ('Kunovice')( 312/65(002/66)) 

118 

'R1029' 35 

1talic' 31,34,35,118,~90 

1talica' 31,34,35,118,290 

var. caudi111l 14, 26, 30, 35, .255, 256 

var. charkowiensis 14, 26, 30, 35, 255, 

256,290 

var. italica 31, 34, 35, 118, 290 

nigra x P. deltoides (P. x interamericana) 

66 

nigra x P. laurifolia 26, 35, 255 

'NC5272' 35 

'NC11445' ('NE157') ('NE280') 255 

nigra x P. maximowiczii 8, 10, 12, 14, 15, 

19, 23, 26-29, 32, 49, 55, 62, 63, 85, 88, 94, 

117-122,144,146,147,180, 185,207,~ 

256 

'NM1, 63, 118 

'NM2' ('Max-1') 118 

'NM6' ('Max-5') 8, 62, 63, 85, 88, 94, 

180, 182,185,~256 

'NMS' ('#62-7') 118 

nigra x P. trichocarpa 26-29,35,62,255 

'NC5331' 35,62,255 

nigra var. betulifolia x P. nigra 14 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 

nigra var. betulifolia x P. nigra cv. 'Volga' 

35 

nigra var. betulifolia x P. trichocarpa 14, 

35,37,255 

'NC5331' 35, 255 

'NE299' 35, 37, 255 

nigra var. dulrkowiensis x P. x berolinensis 

255, 256' 

'NC11382' ('NE27'). 255, 256 

nigra var. charkowiensis x P. deltoides 14, 

35 

nigra var. charkowiensis x P. nigra var. 

caudi1Ul 14, 26, 30, 31, 35, 255, 256 

'NC5271' ('NE19') 35,255,256 

seroti1Ul 1, 290 

sieboldii 26, 106, 161, 290 

sieboldii x P. grandidentata (P. 

kitalalmiensis) (P. x kitalalmiensis) 55, 66, 

68,161,163,164,188,194,195,204,205, 

207,291 

'Kitakami Hakuyo' 161 

'Y63' 163, 188 

simonii 26, 28, 29, 32, 38, 43, 106,107,291 

simonii x P. nigra 36 

simonii x P. x berolinensis 255,256 

'NE332' 255, 256 

szechuanica 106, 107, 291 

tacamahaca (P. balsamifera) 106 

tacamahaca x P. trichocarpa 26 

tomentosa (P. x tomentosa) 26-29, 55, 106, 

107, 166, 172, 291 

tremula 5, 8, 14, 15, 26-29, 35, 43, 55, 92, 

93,106,107,166,171,274,290,291 

'Erecta' 5 

tremula x P. alba 26-31, 35, 36, 42, 52-55, 

57, 61, 63, 64, 84, 98, 171, 175, 176, 198, 

200,202,207,208,210,216,270 

'706-8' 35 

'7171-B4' 35, 63, 84,85 

'INRA 7171B4' 35, 63, 84, 85 

tremula x P. tremuloides 37, 55, 74,86, 

92-94, 100, 166, 175, 176, 179, 274 

'333-4' 37 

'INRA 353-38' ('353-38') 86, 175 

tremuloides 14, 15, 19, 23, 26, 35, 43, 54, 

55, 58,59, 66, 69, 73, 100, 105-107,111, 

129, 156, 158-160, 194, 195, 197, 199, 205, 

222,233,238,239,251,274,291 

tremuloides x P. tremula 239 

trichocarpa 1, 14, 19, 49, 106-110, 112, 

121, 128, 135, 144, 148, 150, 195, 213, 222, 

291 

trichocarpa x P~ balsamifera 35 

'TI32' 35, 37, 39, 41 

trichocarpa x P. deltoides (P. x 

interamerica111l) 14, 35, 41, 53, 55, 66, 69, 

108,117-122,194,195,199,200,203,205, 

208,291 

'OTAC7' ('Unal') 118 

'~TAC8' ('Beaupre') 118 

'OTAC26' ('Boelare') 118 

'Hunnegem' 35, 41 

trichocarpa x P. tacamahaca 15, 26 

tristis 291 

tristis x P. balsamifera 125 

311 
'Tristis #1, 125, 248 

wislizenii 106, 107, 291 

xiaohei 36, 42, 291 

yunnanensis 106, 107, 291 

x balsamifera 117 

x berolinensis 36, 255, 256, 291 

x canadensis 114, 122, 123,130,207,290, 

291 

x euramericana (P. deltoides x P. nigra) 1, 

3, 14-16, 19, 21, 22,26-31,34,35, 37, 42, 

55, 58, 63, 107-109, 117-122,125, 126, 156, 

174,204,207,255,290 

'DN5' ('Gelrica') 118 

'DN14' ('Harff) 118 

'DN16' ('Batarde d'Hauterive') 118 

'DN17' ('Robusta') 118 

'DN21' ('I-78B') ('LW42') 118 

'DN30' ('Canada Blanc') 15, 118 

'DN93' ('Triplo') ('1-37 /61') 118 

'DN106' 63 

'DN173' ('Dorskamp') ('Koster 925') 

118 

'DN111' ('Spijk') ('Koster 2195') 118 

'1-45/51' 15, 118 

'1-214' 31, 118 

'1-455' 118 

'NC5311' 255 

'Beaupre' 37 

'Carpaccio' 37 

'Eugenei' 15, 125 

'Guariento' 37 

'L Avanzo' 37 

'Ogy' 3, 16, 35, 58, 174-111 

'Robusta' 35, 42, 118, 130 

'Wisconsin #5' 15, 255 

xgenerosa (P. generosa) 1, 291 

x interamericana (P. deltoides x P. 

trichocarpa) (P. trichocarpa x P. deltoides) 

35,112,194,291 

'Bam' 35,118 

'Rap' 35 

xjackii 117-122,291 

'JAC4' 118 

'JAC7' 118 

1AC28' 118 

x kitakamiensis (P. kitakameiensis) (P. 

sieboldii x P. grandidentata) 55, 66, 68, 

161,163,164,188,194,195,204,205,207, 

291 

312 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 
General Index 

A 

10-mer (see: oligonucleotide) 

355 (CaMV) (see: CaMV35S promoter) 

70S (double enhancer sequence) (see: 

CaMV) 63,67 

A1 95 

A4a 66 

abaxial 15, 40, 295 

Abies 33, 34, 98, 291 

Abies koreana (A. koreana) 134, 291 

ablate 252 

abscisic acid (ABA) 156 

abscission 77, 124, 125 

Ac (Activator) 53, 55 

Ac-AMP1 (Amaranthus defensin-like 

protein) 167 

Ac-AMP1.2 167, 168 

ACC (1-aminocyclo-propane-1-carboxylic acid) 21 

accelerated flowering 212, 215, 216 

acclimation (acclimate) (acclimatization) 

7, 75, 237, 239 

Acer 129 

saccharum 126, 291 

acetolactate synthase gene (ALS) 55, 61, 

67,207 

acetosyringone (AS) 56 

active oxygen 187-189 

adaptive advantage 86, 268 

adaxial 20, 295 

additive genetic variation (additive and 

nonadditive) 224, 228 

adenine 214,296,306 

S-adenosyl-L-methionine synthase 

(SAM) 66 

ADP ribosyl transferase 214 

adult phase 5, 299 

adventitious 5, 7, 13-15, 33, 47, 62, 156, 

163, 221, 231, 237-239, 295, 303 

AFLP (amplified fragment length 

polymorphism) 102, 104-106, 108, 109, 

119, 222, 295 

AGAMOUS (AG) 213 

agarose 27, 103, 104, 116, 117, 152, 304, 

306,308 

Agrobacterium 55, 156, 165, 175, 275, 

293,297,305 

Agrobacterium-mediated transformation (gene transfer) 24, 51-57, 68, 70, 

90, 91, 97, 162, 169, 175, 197, 201, 275 

rhizogenes 51, 55, 76, 78, 91, 93, 207, 

209, 277, 293, 307 

tumefaciens 51, 53-56, 67, 70, 74, 76-

78, 85, 150, 161-163, 167, 197, 198, 

214, 293, 309 

A281 52,53,56 

A348 53,56 

C58 53, 56, 74 

EHA101 56 

LBA4404 56, 161, 163 

P022 161, 163 

air pollution 44, 187, 207 

air-pollutant resistance 187 

albinism 35 

Albizzia lebbeck 34 

albumin 132-135, 137 

Alcaligenes eutrophus 208, 293 

alfalfa mosaic virus 63, 198 

allele 96, 102, 104, 110, 184, 275, 295, 

299-301, 304, 307 

allelochemical 183 

allergen 165, 212 

allozyme 102, 105, 107, 108 

ALS (acetolactate synthase) 55, 61, 67, 

207 

alternate host 295 

Amaranthus 167, 291 

amide 295 

1-aminocyclo-propane-1-carboxylic acid 

(ACC) 21 

_amphidiploid 95, 295 

amplification 101-104, 116, 117, 143, 

145,147,148,151,152,197,295,306 

amplified fragment length polymorphism (AFLP) (see: AFLP) 

amplified polymorphic DNA (RAPD) 

103, 116, 143, 148, 150, 151, 222, 306 

(see: RAPD) 

amplified sequence polymorphism 

(ASP) 105 

analog 167 

aneuploid 38 

angiosperm 65, 67, 125, 133, 194, 196, 

197, 215, 295, 308 

anionic peroxidase 66, 197 

annealing 103, 104, 117, 148, 152 

annual net return 242, 295 

annual yield 247 

anther 14, 15, 33, 165, 213, 295, 308 

anther culture 14, 15, 33, 36, 221 

anther tapetum-specific gene 213 

anthocyanin 96,295 

antibiotic-resistance gene 91 

antifeedant 258 

antigen 295 

antimicrobial 295 

antimicrobial peptide 298 

Antirrhinum 212, 213, 291 

antisense 67, 75, 87, 145, 197-203, 215 

antisense DNA 161-163 

antisense gene 232, 295 

antisense orientation 67, 87, 197-

200, 214 

antisense RNA 67, 200, 252, 295 

antisense technology 223 

antiserum (antisera) 295 

AP1 212, 213, 216 

ap24 leader 168 

APETALAl (AP1) 212, 213, 216 

APET ALA3 214 

aphidicolin 62 

apical dominance 7, 77, 296, 298 

apical meristem 13, 76, 78, 295 

Arabidopsis 74-78, 80, 81, 103, 138, 182, 

212-214, 216 

thaliana 52, 67, 194, 291 

aroA (modified EPSP synthase) 70-72, 

91, 92,207 

ASCAD (antisense CAD) 87 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 

ascorbate peroxidase 187, 188 

ascorbate.:.glutathione cycle 187 

ascospore 296 

aspartic proteinase 173 

Aspergillus 214, 293 

assessment endpoint 266 

Astragalus (Fabaceae) 145, 146, 291 

AtALS (see also: acetolactate synthase) 

67 

ats1A (Arabidopsis RUBISCO small 

subunit promoter) 71, 182 

AUG 296,302, 308 

autoradiography 104 

auxin (see also: 2,4-D; IAA; IBA; and 

NAA) 10-12, 30, 47, 51, 77-80, 274, 296, 

298,305 

avirulence 150, 296, 302 

axillary bud 7, 13, 15, 16, 18, 76, 236, 

237,239 

axillary shoot 13, 22, 296 

B 

b-zip (leucine zipper protein binding 

site) 126 

BA (benzyladenine) 11, 14-16, 19, 29, 

30, 38, 39, 46, 62, 92, 298 

Bacillus 

amyloliquefaciens 214, 293 

thuringiensis (see: Bt) 

thuringiensis -endotoxin (see: Bt 

endotoxin, Bt endotoxin genes) 

backcross 103,110,222,274,296 

backcross breeding 103, 110, 275 

backcross population 222 

bacterial canker 108, 294 

bacterial wilt 294 

bactericide 296 

Bambusa 34, 291 

bar 52, 55, 56, 208 

bark cambium 13 

bark phloem parenchyma 125 

bark storage protein (BSP) 66, 124-128 

Barnase 214 

Barstar 214 

basipetal 296 

beetle 86, 241, 254-256, 294 

benzaldehyde 202 

benzyladenine (6-benzyladenine) (BA) 

(see: BA) 

6-benzylaminopurine (see: BA) 

Betula 43, 83 

papyrifera 126,291 

platyphylla 291 

populifolia 5 

verrucosa 216, 291 

binary vector 56, 70, 93, 296, 310 

bioassay 158,296 

biochemistry 124, 138, 221, 301 

biocontrol 268, 294 

biodiversity 44, 206, 252, 268, 273, 275-

277 

bioethics 273, 276, 277 

biofuel 251 

biolistic (microparticle bombardment) 

(microprojectile-mediated DNA 

delivery)(particle bombardment/ 

313 
acceleration DNA delivery system) 7, 

12, 51, 60-63, 85, 90, 91, 208, 275, 296, 

303, 304 

biological control 250, 257, 258, 266, 

267,296 

biological impact assessment 264, 265, 

269 

biological risk assessment 264, 269 

biomagnification 257 

biomass 13, 40, 116, 193, 203, 209, 212, 

232, 241-247 

biopesticide 254 

bioreactor 12, 296 

biosafety 265, 267, 270, 276, 277 

Biota 

orienta/is 134, 291 

biotype 250, 252-254, 257, 296 

bm (brown-midrib) 196, 197, 200 

bmr (brown-midrib) 196, 197, 200, 202 

bole form 224 

bombardment (see: biolistic) 

Bowman-Birk Inhibitor (BBI) (see also: 

proteinase inhibitor) 175 

bract 212, 296 

Brassica napus 214, 291 

BSP (bark storage protein) 66, 124-128 

Bt (Bacillus thuringiensis) 9, 52, 55, 62, 

86, 87, 172, 178-184, 252, 253, 257, 259, 

293 

endotoxin 9, 177-184 

endotoxin genes (endotoxin) 2, 55, 

252,253 

cryiA 9, 87, 178, 179, 181-183, 255, 

256, 261 

cryiB 178, 180-182, 253, 254 

cryiC 181 

cryll 178 

cryiiA 181 

crylll 178 

cryiiiA 87, 179, 184, 254, 256 

cryiV 178 

bud resin 255, 256 

bud set 5, 77 

budworm 293, 294 

c 

C value 296 

C-function 213, 214 

CAATbox 302 

CAD (cinnamyl alcohol dehydrogenase) 

65-67, 86-88, 194-197, 200-203 

cadmium 207 

caffeic acid 194, 199 

caffeic acid/5-hydroxyferulic acid-0-

methyltransferase (COMT) (omt) 65-

67, 87, 194-203 

caffeoy 1-CoA -0-methyl transferase 

(CCoAOMT) 194, .195, 200, 203 

CAL 213 

calliclone 35 

callogene;;is 12, 296 

callus (calli) 7, 10-12, 14, 19, 23, 25-27, 

29-34, 36-39, 41-43, 45-49, 53, 54, 56, 61-

63, 67, 92, 156-160, 163, 234, 274, 279, 

295,296,304 

Calocedrus 134 

decurrens 134, 292 

314 

calyx 296, 308 

cambium 13, 15, 16, 74, 77, 79, 94, 165, 

296 

cambial tissue 46, 174 

Camellia japonica 216, 292 

cAMP response element (CRE) 126 

CaMV 35S promoter (35S) (CaMV 

promoter) (cauliflower mosaic virus) 

62, 63, 67, 70-72, 76-78, 85, 86, 88, 91, 93-

95, 161, 162, 168, 175, 179, 182, 188, 197, 

198,200,208,216,275,277 

CaMV 70S promoter (70S) 63, 67 

CaMV promoter (35S) (70S) (see: CaMV 

355 promoter) 

canker 108, 150, 157, 158, 241, 293, 294, 

296 

capitalized expense 245, 296 

capitalized net value 242 

carbenicillin 56, 163 

carbon metabolism 81 

carbon sequestration 249 

Carica 33, 34, 292 

carpel 213, 296 

casamino acids 28 

Castanea 133, 167, 292 

crenata 133, 292 

mollissima 167, 292 

sativa (C. sativa) 133, 292 

CAT (chloramphenicol 

acetyltransferase) 52, 54-56, 67, 175, 

182, 266, 307 

catkin 13, 19 

cauliflower mosaic virus (CaMV)(see: 

CaMV 35S promoter) 

CCoAOMT (caffeoyl-CoA-0-

methyltransferase) 194, 195, 200, 203 

CCR (cinnamoyl-CoA reductase) 194, 

195, 203, 205 

eDNA (complementary DNA) 65, 66, 

~1~1a1~u~1~1~1~1~ 

200,204,296,298,308 

cecropin 166, 167, 171 

Cedrus 

atlantica 134, 292 

cefotaxime 56, 237 

cell culture 10, 61, 92, 156, 236 

cell division 28, 30, 61-63, 77, 78,·156, 

157, 296-298 

cell fusion 229, 308 

cell suspension 10-12, 14, 19-26, 61-63, 

156,303 

cell synchronization 61 

cellulase 26, 27, 297 

cellulose 193, 194, 297 

centiMorgan (eM) (see: eM) 

centromere 297, 301 

Ceratocystis fimbriata (aspen canker) 

156,293 

Cercis 

siliquastrum 133, 292 

chalcone synthase (CHS) 65, 96 

Chamaecyparis 134, 292 

lawsoniana 134, 292 

chaperone 297 

chaperone-mediated folding 133 

chemical composition 247 

chemical tolerance 207 

chestnut blight (Cryphonectria parasitica) 

(C. parasitica) 166, 167, 233, 294 

chimera 297 

chimeric gene 55, 60, 67, 90-92, 94, 96, 

97, 

chitin 167, 297 

chitin-binding protein 167 

chitinase 66, 297 

chloramphenicol acetyltransferase (see: 

CAT) 

chlormequat 215 

chloro-cis-cis-muconate 208 

3-chloroaniline 208 

chlorocatechol 208 

chlorophenol hydroxylase 208 

chlorophyll content 19, 35, 92 

chloroplast 21, 103, 116, 144, 145, 147-

149, 187-191, 297, 299, 305 

chloroplast DNA (see: cpDNA) 

chloroplast rONA spacer region 145 

chloroplastic transit peptide 188 

chlorosis (chlorotic) 45, 189, 297 

chlorsulfuron 30, 52, 56, 61, 68, 170, 

207, 211, 270 

Chondrostereum purpureum 269, 270, 293 

Choristoneura 178 

Choristoneura fumiferana (C. fumiferana) 

(spruce budworm) 178, 293 

Choristoneura occidentalis(C. occidentalis) 

(western spruce budworm) 293 

Choristoneura pinus (C. pinus) (jack pine 

budworm) 294 

chroma tin 279, 297, 299, 301 

chromosome 34,36,49,60,92, 101,151, 

221-223, 229, 274, 279, 295, 297 

· chromosome damage 47 

chromosome doubling 221 

chromosome landing 109, 235, 

297 

chromosome rearrangement 229,297 

chromosome transplantation 220 

chromosome walking 297 

Chrysanthemum spp. 254, 292 

Chrysomela 

populi (poplar leaf beetle) 175, 294 

scripta (cottonwood leaf beetle) 174, 

175,178,241,255,256,259,294 

tremulae (aspen leaf beetle) 58, 86, 

89,175,176,179,294 

CHS (chalcone synthase) 65, 96 

chymotrypsin 174 

chymotrypsin inhibitor (see also: 

proteinase inhibitor) 52 

cinnamaldehyde 197, 200, 204 

cinnamate-4-hydroxylase (C4H) 194 

cinnamic acid 194, 197 

cinnamoyl-CoA reductase (CCR) 194, 

195, 203, 205 

cinnamyl alcohol 66, 68, 86, 194, 195, 

200, 202, 205 

dehydrogenase (CAD)(POPCAD) 

(EuCAD) 65-67, 86-88, 194-197, 200-203 

cis-acting DNA sequence 297, 309 

Citrus 33, 34, 292, 294 

sinensis 216, 218, 292 

cladistic 106, 297 

classical breeding 155, 220, 222, 224-232 

classical genetic 220, 224, 228, 229 

climate change 44, 138, 268 

clonal deployment 224 

clonal identification 105 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 
clonal planting 236 

clonal propagation 8, 15, 48, 97, 116, 

273,274,277,278,297 

clonal trial 101, 109, 224, 228 

eM (centiMorgan) 107, 222, 297 

co-cultivated explant 75 

co-cultivated leaf disc 70 

co-cultivation 54, 56, 175, 297 

co-integrate vector (co-integration 

vector) 297, 310 

co-suppression 94, 96, 97, 214, 279, 298 

CO (CONSTANS) 212, 216 

C02 sequestration 251 

cobalt 207 

coding region 76, 126, 144-146, 148, 151, 

170, 297 

coding sequence 70, 71, 86-88, 102, 151, 

198,297 

codling moth 185, 186 

codominant 102-105, 108, 143, 222, 297 

codon 167, 168, 170, 179, 296, 297, 302, 

309 

codon preference 165, 170 

Coffea 

arabica 34, 292 

cold storage 7, 36, 44-46, 49 

Coleoptera 3, 58, 87, 89, 174, 178, 179, 

254, 256, 257, 259-261 

collar shoot 7, 297 

Colletotrichum 264, 294 

gloeosporioides 267, 271, 294 

lindemuthianum 174, 294 

commercial field yield 247 

commercial plant production 236 

commercial production 19, 44, 179, 236 

commercial-scale 14 

commercial-sized plantation 242 

compatibility 106, 214, 254, 259, 296, 

297,302,308 

competence 61-63, 79, 141, 298 

competition 2, 86, 103, 114, 260, 261, 

263,268 

complementary DNA (see: eDNA) 

COMT (caffeic acid/5-hydroxyferulic 

acid-0-methyltransf~rase)(OMT) 67, 

194-203 

confirmation 52, 113, 215, 263 

conidiophore 298 

conidium (conidia) 157, 167, 298 

coniferaldehyde 202 

coniferin 196 

coniferyl alcohol 194-196, 200, 303 

coniferyl alcohol oxidase 195 

consensussequence 168 

conservation 3, 44, 47, 48, 67, 76, 106, 

132, 170, 208, 209, 233, 236-243, 273, 276, 

277,309 

conserved 67,76,80, 110,130,132,134, 

143-148, 151, 296, 302, 309 

CONSTANS (CO) 212, 216 

constitutive expression 86, 168, 198, 

216,298 

constitutive promoter 86, 168, 170, 208 

conventional breeding 51, 165, 166, 275 

copper 173, 215 

coppice (coppicing) 13, 193, 206, 247, 

298 

copy number 52, 57, 85, 90, 93, 94, 96, 

161,175,198,298,305 

corolla 298, 304 

cortex 94 

Corynebacterium 

diphtheriae 214, 294 

Cotesia 

melanoscela 257, 294 

cottonwood leaf beetle (Chrysomela 

scripta) (C. scripta) 174, 175, 178, 241, 

255,256,259,294 

cotyledon 132, 134, 274, 298, 301 

4-coumarate-3-hydroxylase (C3H) 194 

p-coumaric acid 194, 200 

p-coumaroyl-CoA-3-hydroxylase 

(CCoA3H) 195 

p-coumaryl alcohol 194, 196, 303 

cpDNA (chloroplast DNA) 103, 107, 

116, 119, 143-148, 297, 304 

CRE (cAMP response element) 126 

crop protection 251 

cross-hybridization 276 

cross-pollination 220, 298 

cross-resistance 254, 298 

crossing over 222, 298, 307 

crown gall 1, 51, 52, 150, 161-163, 293, 

309 

crsl-1 52, 55, 56, 207 

cry (Bt endotoxin gene) 2, 55, 252, 253 

cryiA 9, 87, 178, 179, 181-183 

255,256,261 

crylB 178, 180-182, 253, 254 

cryiC 181 

cryll 178 

cryllA 181 

crylll 178 

crylllA 87, 179, 184, 254, 256 

crylV 178 

cryopreserve (cryopreservation) 41, 44-

47,298 

cryoprotection 45 

Cryphonectria parasitica (C. parasitica) 

(chestnut blight) 166, 167, 233, 294 

CT AB (hexadecyltrimethylammonium 

bromide) 117 

cultivar 53, 157, 253, 254, 258, 298 

culture condition 11, 15, 24, 36, 75, 92, 

138, 229 

culture filtrate 158 

Cupressaceae 134, 292 

cysteine 167, 170, 173, 174, 176 

cysteine protease inhibitor (OCI) 52, 

55, 85-87, 174-176 

cysteine proteinase 173-175 

cytokinin (see also: BA; kinetin; and 

zeatin) 7, 8, 11, 14, 19, 30, 38, 51, 53, 62, 

75, 78, 79, 83, 128, 161, 298, 305 

cytology 298 

cytoplasm 63, 71, 96, 103, 106, 188, 207, 

221,229,298,299,303,305,307 

cytoplasmic DNA (see also: cpDNA, 

mtDNA) 103, 106 

cytosol 70, 71, 188, 190 

cytotoxin 213-215, 252, 298 

cytotoxic gene 252 

D 

2,4-D (2,4-dichlorophenoxyacetic acid) 

10-12, 15, 19, 29, 30, 38, 46, 52, 56, 92, 

208, 274, 296 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 

daminozide 215 

de novo 133, 225, 298 

deamination 194, 298 

defense 3, 131, 156, 158, 173, 187, 254-

256, 302, 305 

defensin 167, 298 

DEFICIENS (PTD) 213 

deglycosylation 125 

degree of branching 35 

dehydrogenate 195,298 

deletion 102, 103, 105, 143, 305 

delivered cost 243, 245 

demethylation 96, 298 

denaturation 117, 152, 298, 305 

deoxyribonucleic acid (DNA) 298 

deployment 176, 181, 223, 224, 234, 249, 

251-254,258,261,275 

depolymerize 193, 202, 298 

detoxification 187, 188, 206-208 

detritivore 257, 258 

developmental influence 52, 84 

developmental process 10, 11, 77 

developmental stage 10, 11, 85, 110, 

141,155,178,214,239,306 

dextran 45 

DHDPS (dihydrodipicolinate synthase) 

66 

diamondback moth (Plutella xylostella) 

180, 294 

diazoacetyl-compound 173 

2,4-dichlorophenol 208 

2,4-dichlorophenoxyacetic acid (see: 

2,4-D) 

dicotyledon (dicot) 33, 65-67, 76, 142, 

145, 168, 298 

DIFP (diisopropylphosphofluoridate) 

174 

dihaploid 221, 229, 298 

diisopropylphosphofluoridate (DIFP) 

174 

dimer 125, 128, 135, 174, 196, 298 

dimethyl sulfoxide (DMSO) 45 

dioecious 220, 229, 298 

diphtheria toxin subunit A (DTA) 214 

diploid 36-38, 65, 97, 221, 298, 305 

Diptera 178, 257 

direct gene transfer (see also: biolistics, 

electroporation, DNA microinjection) 

12, 60-63, 165 

discounted cash-flow 241 

disease 1, 3, 23, 42, 44, 51, 55, 78, 96, 

108, 150, 155, 157-158, 161, 163, 164, 174, 

209,268,269,273,276,293,294,304 

disease rating 157 

disease resistance 3, 113, 150, 155-

158, 161-164, 168, 174, 193, 223, 224, 

275 

disulfide bond 132, 133, 135, 137, 167 

dithiothreitol (OTT) 31, 136, 175 

DMSO (dimethyl sulfoxide) 45 

DNA-banding pattern 119 

DNA extraction 7, 117 

DNA fingerprint 103, 104, 112, 115, 116, 

118-121,123,222,228-231,295,298 

DNA hybridization 103 

DNA marker (genetic marker) 2, 79, 

101-111, 115-121, 143, 145-148, 220-223, 

228-231, 266, 267, 297, 300, 302, 306 

DNA methylation 38, 92, 95, 98, 99, 274 

315 
DNA microinjection 24, 299, 303 

DNA polymerase 101, 114, 116, 117, 

123, 222, 299 

dominant 77, 93, 95, 102, 104, 110, 130, 

134,143,180,183,184,222,275,296,299 

dormant (dormancy) 5, 9, 11, 58, 78, 82, 

83, 94, 100, 116, 125, 127, 129, 130, 179, 

221, 236, 299, 306 

Douglas-fir (Pseudotsuga menziesii) (P. 

menziesii) 76, 82, 134, 183, 254, 293 

down-regulated (down-regulation) 88, 

196, 197, 200-202, 213, 299 

Driselase 27 

Drosophila 113 

DTA (diphtheria toxin subunit A) 214 

DIT (dithiothreitol) 31: 136, 175 

durability 168, 169, 266 

durable resistance 109, 168, 169 

Dutch elm disease (Ophiostoma ulmi) (0. 

ulmi) 41, 166, 294 

E 

early flowering 74, 76, 78, 81, 215-217 

early selection 1, 105, 158, 221, 231, 302 

earlywood 79-81,299,302 

ecological interaction 150, 176 

economic 1-3, 10, 13, 33, 34, 44, 69, 74, 84, 

86-88, 104, 108, 109, 116, 150, 161, 165, 176, 

180, 181, 193, 203, 205, 206, 209, 223, 224, 

229, 231, 232, 241, 245, 247-249, 251, 254, 

255,258,272,278,279 

economic evaluation 245,247,248 

economic injury level 254, 255 

ecophysiological 251 

ectomycorrhiza 155, 160, 208, 210, 211, 

294,299,303 

ectopic 77, 96, 215, 217, 276, 299 

ectopic expression 77, 215, 217 

ectopic gene expression 77 

EDTA (ethylenediaminetetraacetic acid) 

117,174 

Elaeis 

guineensis 292 

electric discharge method 61,63 

electron transport 187, 190 

electrophoresis (electrophorese) (electrophoretic) 64, 102-105, 113, 114, 117, 120, 

133, 135-137, 139, 140, 148, 152, 235, 298, 

299,306,308 

electroporation 24, 30, 41, 51, 60, 61, 63, 

64, 74, 91, 98, 210, 299 

ELISA (enzyme-linked immunosorbent 

assay) 52,.175, 299 

elongation factor 214, 299 

embryo 11,13-15,17, 19,23,41,64,132-

135, 139-141, 217, 220, 221, 229, 231, 232, 

235,299-301,306,308 

embryo culture 14, 15, 23, 217, 235 

embryorescue 220,221,229,231,232 

embryogenesis 2, 10-12, 19, 21, 23, 40, 

48, 49, 64, 130-133, 138-141, 221, 222, 

230, 231 273, 274, 277, 299, 308 

embryogenic 10-12, 19, 23, 24, 49, 133, 

141,274 

embryogenic callus 11, 23, 49 

embryogenic cell culture 24 

embryoid 11, 12, 299 

316 

endogene (endogenous gene) 78, 90,93-

96,276 

endomycorrhiza (VA mycorrhiza) 208, 

299,303 

endopeptidase 173 

endoplasmic reticulum (ER) 126, 132, 

133, 299, 301 

endoprotease 128 

endosperm 132, 139, 141, 142, 299 

endotoxin (-endotoxin) 2, 9, 55, 62, 86, 

87, 89, 177-186, 252-254, 256, 257, 260, 

262, 296, 299 

endpoint 266, 267, 269, 271 

energy cost 241-245 

energy crop 44, 186, 193, 299 

enhancer 63,67, 176,177,198,299,302 

5-enol pyru ylshikimate-3-phosphate 

synthase (EPSP) (see: EPSP) 

ent-kaurene acid 215 

entomopathogen 253,270 

environmental consequence 251 

environmental factor 77, 94, 95, 100, 

109, 127-129, 155, 215, 249, 267, 306 

environmental impact 86, 87, 209, 212, 

217, 259, 260, 263-265, 267, 270 

environmental risk 249, 250, 258, 261, 

264, 266, 268-270 

environmental safety 212, 249, 251, 258 

environmental stress 113, 187-189, 191 

enzyme 26, 27, 30, 34, 52, 66, 67, 69, 72, 

73, 81, 82, 87, 102-106, 113, 126, 131, 

142-147, 151, 152, 173-175, 177, 187, 188, 

194, 197, 198, 200-205, 208, 222, 260, 297, 

299, 302, 304-308 

enzyme activity assay 52 

enzyme assay 197, 198 

enzyme-linked immunosorbent assay 

(ELISA) (see: ELISA) 

epicuticular wax 19, 23 

epidemiology (epidemiological) 150, 

264, 269, 299 

epigenetic 97, 100, 273, 274, 299 

epigenetic change 36, 92, 93, 95, 96, 

99,223,273 

epigenetic effect 98, 229 

epimutation 97 

epistasis (epistatic) 96, 99, 299 

epoxy propane 173 

EPSP 52, 70-72 

EPSP synthase 70-72, 207 

Eranthis hyemalis 137, 140, 292 

Erwinia amylovora 167, 294 

escape of engineered germplasm 258 

escape of transgene 251, 277 

Escherichia coli (E. coli) 91, 113, 188-191, 

207, 218, 308 

ESF12 (synthetic analogue of PGLa) 

167-169 

EST markers (expressed sequence tags) 

105 

establishment cost 206, 242, 243 

ester 173, 299 

ethanol 152, 199, 201, 202, 241, 245-247, 

259 

ethylene 21, 79 

ethylenediaminetetraacetic acid (EDT A) 

117, 174 

etiology 150 

EuCAD (Eucalyptus cinnamyl alcohol 

dehydrogenase) 67,86,87 

Eucalyptus 33, 34, 65-67, 74, 82, 86, 101, 

103, 104, 108, 110-113, 203, 215-218, 235, 

292 

Eucalyptus globulus (E. globulus) 215, 

292 

Eucalyptus grandis (E. grandis) 82, 104, 

112,235,292 

Eucalyptus lansdowneana (E. 

lansdowneana) 216, 218, 292 

Eucalyptus nitens (E. nitens) 103, 111, 

215, 218, 292 

Eucalyptus occidentalis (E. occidentalis) 

215, 217, 292 

Eucalyptus regnans (E. regnans) 103, 292 

Eucalyptus urophylla (E. urophylla) 112, 

292 

euchromatin 299 

Euglena gracilis 144 

eukaryote 99, 100, 168, 297, 299, 300, 

302, 304, 307 

evolution 3, 86, 92, 96, 107, 110, 112, 

114, 121, 123, 140, 143, 149, 179-186, 250, 

252-254, 256-263, 266, 267, 270, 272, 273, 

275,276,278,297,305 

evolutionary consequences 266 

ex situ 43, 44, 48, 300 

ex vitro 6, 7, 70, 156, 237, 300 

ex vitro growth 7 

ex vitro rooting 237 

exogenous 15,92,156,253,300 

exon 253,260,300,302 

exopeptidase 173 

exotic introduction 251 

exotoxin 214, 218, 300 

explant 5-7, 10-16, 19, 21, 22, 31, 37, 41, 

45, 52-54, 56, 60, 62, 63, 92, 156-159, 197, 

222, 236-238, 274, 300, 304, 307 

explant source 7, 15, 37, 92 

expression pattern 67, 78, 80, 85, 86, 

138, 181, 217, 300 

extension 104, 105, 117, 152, 185, 232, 

286 

F 

F1 10, 32, 34, 95, 107-111, 144, 164, 227, 

275, 300 

F2 74, 108-110, 222 

Fabaceae 133,134,140,145,149,292 

fasciated stem 78 

feedback inhibition 77 

feeding assay 175, 180, 183 

feeding test 176 

female catkin 19, 21 

feral population 251 

fertile (fertility) 54, 64, 76, 78, 129, 166, 

208,213,214,252,279 

fertilizer 183, 206, 207, 209, 216, 237, 

243 

ferulate-5-hydroxylase (F5H) 194 

ferulic acid 67-69, 83, 194, 197-200 

fiber 2, 77-79, 87, 116, 155, 183, 193, 

206,220,241,247,264,297,310 

fiber length 247 

field-grown 7, 11, 24, 39, 46, 84-88, 130, 

179,236 

field resistance 157, 180 

field screening 157 

USDA Forest Service Gen. Tech. Rep. RM~TA-297. 1997 
field test 155, 157, 158, 166, 179, 182, 

231, 265, 267-269 

field trial 3, 39, 84-88, 97, 111, 166, 176, 

202, 209, 224, 229, 231, 232, 267 

financial cost 242-244, 246 

financial impact 245 

fingerprint analysis 104 

fitness 90, 97, 153, 180, 267, 268, 270, 

275,276 

flanking DNA 300 

FLO (FLORICAULA) 212, 213, 217 

FLO/LFY (PTFL) 213, 214 

floral bud 216 

floral development 82, 212, 216 

floral differentiation 213 

floralgene 212,213,252 

floral homeotic gene 82, 212-214 

floral meristem 212-214 

floral meristem identity gene 212, 214 

floral organ 212-214 

floral organ identity gene 212, 214 

floral promoter-cytotoxin 252 

FLORICAULA (FLO) 212, 213, 217 

flower (flowering) 1, 2, 5, 19, 22, 55, 58-

60, 74-76, 78, 79, 81-83, 87, 95, 96, 98-

100, 125, 133, 141, 142, 161, 162, 165, 

168, 172, 175, 179, 188, 197, 198, 202, 

208, 212-216, 252, 275, 279, 292, 295, 296, 

298, 303-305, 308 

flower bud 19, 22, 216 

flower color 95, 96, 99, 100 

flower development 212, 213 

flower initiation 59, 75, 83, 216 

flower primordium 75 

flower production 212 

fluorescence 112, 266, 300 

fluorometer (fluorometry) 85, 152, 300 

folding of mRNA 168 

folivore 254 

force 40, 87, 236, 247, 261, 302 

foreign DNA 54, 58, 60, 64, 100, 296, 

300,309 

foreign gene 3, 49, 52, 84, 90, 92-94, 273, 

277, 300, 309 

forest tent caterpillar (Malacosoma 

disstria) (M. disstria) 9, 62, 89, 178-183, 

253-256, 294 

frost hardiness 77 

frost tolerance 108 

frosty spot (Frs) 95 

fruit 33, 40-43, 95, 110, 124, 165, 215, 

217, 269, 293, 295 

fuel 1,193,243,245,247 

full-sib progeny (full-sib offspring) 229, 

300 

fungicide 174, 241, 242, 300 

fungistatic 156, 174, 300 

fungus (fungi) 24, 115, 151-153, 155-

158, 167, 170, 173, 174, 208, 257, 266, 

267, 293-296, 300-303, 305, 309, 310 

Fusarium 41, 167, 174, 294 

G 

oxysporum 167, 294 

solani 174, 294 

G-box 126,300 

GA (Gibberellic acid) (Gibberellin) 78, 

79, 128,215 

galactose-(1-3)-N-acety !galactosamine 

125 

galactose-(1-4 )-N-acetylglucosamine 

125 

gallic acid 184 

gamete 213, 275, 299, 300, 308, 310 

gametoclonal (gametoclone) 33, 34, 36, 

300 

gametoclonal variation 34, 36, 92, 98 

gametophyte (gametophytic) 104, 134, 

214, 300, 303, 305 

gas exchange 34, 39, 40 

gas chromatography 105, 198, 300 

Gaussian model 269 

gelling agent 19 

GEM (genetically engineered microbe) 

266 

gene construct 62, 93, 94, 168-170, 197, 

198 

gene containment 212, 214 

gene escape 250-252, 276, 277 

gene expression 11, 13, 56, 60-63, 65-68, 

70-72, 75, 77, 79-81, 85-87, 93-95, 127, 

132, 134, 138, 175, 179, 180, 196, 197, 

200, 213, 223, 230, 252, 276, 298-300, 307, 

309 

gene flow 251,266 

gene frequency 253, 300 

gene introgression program 110 

gene mapping 2 

gene mutation 274 

gene regulation 52, 67, 127, 131, 132, 

252,297 

gene silencing 85, 87, 90, 94-97, 214, 

277, 300 

gene transfer 24, 30, 51, 52, 60-62, 90-

93, 97, 165, 169, 223, 228, 230, 264, 265, 

273,275,276,300,305 

generation time 78, 79, 105, 155, 254, 

273, 300 

genetic code 168, 266, 297 

genetic contamination 230 

genetic diversity 13, 105, 106, 224, 230, 

273-277 

genetic engineering 2, 5, 7, 24, 30, 51, 

71, 74, 77-81, 84, 90-94, 116, 138, 161-

164, 165-170, 173-176, 178-184, .187-191, 

193-203, 206-209, 212-216, 223, 228, 249-

259, 264-269, 273-278, 310 

genetic fidelity 223 

genetic fingerprint (see also: DNA 

fingerprint) 105, 221 

genetic gain 220, 221, 229-232, 300 

genetic improvement 116, 119, 273, 275, 

277 

genetic instability 90-92, 95, 97, 273, 

274, 276, 277 

genetic loci 109 

genetic manipulation 21, 74, 92, 151, 

277 

genetic map 74, 79, 91, 101, 103-109, 

116, 206, 222, 223, 228, 229, 231, 232, 

273,300 

genetic marker (see: DNA marker) 

genetic pollution 212 

genetic resource 48, 231, 233 

genetic selection 208, 209 

genetic stability 33, 48, 92, 93, 274 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 

genetic transformation 13, 34, 48, 53, 

63, 67, 84, 87, 90, 91, 93, 94, 161-163, 

220-223, 228, 230-232, 253, 275, 277, 295, 

300 

genetic variability 221, 228, 229, 231, 

232,267,275 

genetic variance 110, 301 

genetic variation 33, 44, 47, 92, 101, 

143, 148, 220, 222, 224, 228, 230, 231, 

273-277,295,303,307 

genetically engineered microbe (GEM) 

266 

genetically engineered organism (GEO) 

250-252,257,266,267 

genetically modified tree (GMT) 87 

genome map (see: genetic map) 

genome size 51, 65, 67 

genome structure 110 

genomic DNA 65, 104, 144, 152, 161, 

222, 295 

genomic library (libraries) 65, 103, 104, 

297, 300 

genotype 7, 8, 10, 13, 24, 27, 29, 30, 37, 

44, 52-54, 60, 62, 63, 65, 68, 92, 94, 95, 

101, 105, 108-110, 132, 155, 156, 165, 166, 

180, 206, 207, 216, 220-223, 228-232, 236-

239, 253, 254, 274, 275, 277, 299, 300, 

302,305,308 

genotype x environment interaction 

110, 216, 305 

genotypic fidelity 47, 221, 222, 231 

Geographic Information Systems (see: 

GIS) 

germination 11, 131-133, 167, 237, 274 

germplasm 1, 2, 12, 44, 45, 47, 48, 103-

105, 113, 220, 230-232, 250, 258, 273, 277, 

301 

germplasm analysis 104, 105 

germplasm preservation 2, 12, 44, 45, 

273, 277 

GFP (Green Fluorescent Protein) 86 

Gibberellic acid (Gibberellin) (see: GA) 

Ginkgo biloba 134, 292 

Ginkgoaceae 134,292 

girdling 215 

GIS (Geographic Information System) 

269, 300 

globulin 132-134 

Glomerella 294 

cingulata 294 

B-1,3-glucanase 96 

p-glucocoumaryl alcohol 196 

glucose 28, 29, 297, 301 

glucoside 196, 301 

a-glucuronidase (glucuronidase) (GUS) 

7, 55, 56, 61-63, 67, 85, 86, 88, 91, 94, 95, 

137, 162, 182, .198, 208, 307 

glutamine 11, 30, 128 

glutathione 55, 187-191, 207 

glutathione reductase (GR) 55, 187-191, 

207 

glutelin 132, 133. 

glycerol 45, 198, 200 

Glycine max 70, 292 

glycoside 183, 254, 255, 295, 301 

glycosylation (glycosylated) 125, 126, 

135, 137, 138 

glyphosate 52, 70-72, 93, 207 

glyphosate tolerance 52, 70-72 

317 
gn1 (see also: 8-1,3-glucanase} 96 

Golgi apparatus (dictyosome} 133, 301 

GR (glutathione reductase} 55, 187-191, 

207 

Gram-negative bacteria 167, 297, 301 

Gram-positive bacteria 167, 294, 297, 

301 

Gram's staining reaction 301 

gravity 79, 80 

Green Fluorescent Protein (GFP) 86 

growth and development 74, 75, 77-80, 

92, 175,201 

growth and yield 40, 74, 228, 247 

growth medium 26 

growth rate 34, 36, 51, 62, 63, 85, 247, 

256, 257, 266 

growth regulator (see: plant growth 

regulator} 

guaiacyl 194, 196, 199, 201 

guideline 166, 209, 254, 258, 265 

Guinier 1, 290, 291 

gunpowder method 61, 63 

GUS (8-glucuronidase) 7, 55, 56, 61-63, 

67, 85, 86, 88, 91, 94, 95, 137, 162, 182, 

198, 208, 307 

gymnosperm 33,51,65, 125,126,132-

134, 139-142, 194, 196, 298, 301 

gypsy moth (Lymantria dispar) (L. dispar} 

62,178,179,253,257,294 

H 

hairpin loop 168 

hairy root disease (see also: 

Agrobacterium rhizogenes) 51, 55, 78, 

293,307 

halogenated phenolics 206, 208 

haploid 15, 33, 36, 38, 92, 221, 222, 229, 

273, 296, 298, 300, 301, 303, 305 

haploid culture 33 

haploid plantlet 15 

haploid tissue culture 221 

heat stress 165 

heavy metal 174, 206-209 

hedging 236-238 

height and diameter growth 223 

height/ diameter 110 

height growth 2, 45, 110, 183 

Heimburger 1, 291 

Heliothis 174, 181, 294 

virescens 181, 294 

zea 174,294 

helium gas 61 

helium propulsion method 63 

hemicellulase 27 

herbicide 2, 34, 36, 37, 52, 70, 72, 84, 91-

93, 96, 146, 187, 189, 207-209, 222, 223, 

241-243,247,252,267,269,275,305,308 

herbicide-binding protein (Qb) 146 

herbicide resistance (herbicideresistant} 52, 84, 212, 267 

herbicide tolerance 2, 34, 36, 37, 70, 

84,91, 93, 96, 207, 223 

herbivore 251, 253, 256, 257 

heritability (heritabilities) (heritable) 

38, 80, 92, 93, 109, 207, 221, 223, 229-231, 

253, 256, 301, 303 

heterochromatin (heterochromatized) 

96, 301 

318 

heterodimer 125, 301 

heterogeneous (heterogeneity) 60, 125, 

254-256, 301 

heterokaryon 30 

heterologous 65, 67, 71, 106, 181, 200, 

222 

heteroploid 37 

heteropolymer 193, 195, 301 

heterosis 10, 110, 161, 224, 228, 301 

heterotrophic 131, 301 

heterozygote 183,299,307 

heterozygous (heterozygosity) 95, 96, 

101, 102, 104, 105, 220, 221, 273, 275, 

301, 302 

heterozygous loci 102, 104 

hexadecyltrimethylammonium bromide 

(CTAB) 117 

hexanucleotide 145 

Hibiscus sabdariffa 26, 30, 292 

high-density linkage map 105 

histochemistry 85, 301 

homeostasis 79,301 

homeotic gene 212-214, 301 

homologous 65, 93, 94, 96, 97, 103-105, 

134, 135, 143, 152, 167, 212, 214, 222, 298, 

301, 307, 309 

homology 65, 67, 76, 92, 94-96, 135, 137, 

homozygote 183 

homozygous 95,102,104,105,221,229, 

301,302 

homt1 (OMT) 66 

homt2 (OMT) 66 

horizontal gene transfer 264 

horizontal resistance 109, 306 

hormone (see: plant growth regulator) 

host-parasite interaction 155, 156, 175 

host-pathogen interaction 158 

hostrange 265-267,294 

hostres~tance 1, 150,158,176,255 

host-specific toxin 155, 156, 158 

HPLC (High Pressure Liquid Chromatography) 202, 203 

HPT (hygromycin phosphotransferase) 

55, 56, 208 

human serum response factor (SRF) 213 

Hyalophora cecropia 168, 294 

hybridization 10, 30, 52, 67, 90, 94-96, 

102, 104, 106, 144, 197, 220-222, 230, 231, 

251, 254, 258, 267, 275-277, 295, 301, 302, 

306, 308 

hybrids spontanes 1 

hydrogen bond 45, 135 

hydrogen peroxide 187 

hydroxamic acid derivative 174 

3'-hydroxy (3'-0H) 306, 309 

hydroxy-acetosyringone (OH-AS} 56 

4-hydroxycinnamate-CoA ligase (4CL) 

194 

hydroxycinnamyl alcohol 194 

5-hydroxyconiferyl alcohol 200 

5-hydroxyferulic acid 194, 197-200 

hydroxylation 194-196, 301 

p-hydroxyphenyl 194 

hygromycin 55, 56, 63, 96, 208 

hygromycin phosphotransferase (HPT) 

55, 56, 208 

hypermethylation 95 

hypertonic 24 

hypha(hyphae) 298,301,303 

hypocotyl 158, 301 

Hypoxylon canker 157, 158, 294 

Hypoxylon mammatum (H. mammatum) 

156-158, 294 

IAA (indole-3-acetic acid) 21, 77, 296 

IAA-biosynthesis gene 76-79 

iaaH (indoleacetamide hydrolase} 

51, 55, 77, 161-163 

iaaM (tryptophan monooxygenase} 

51, 55, 77, 161-163 

IBA (indole-3-butyric acid) 19, 46, 163, 

296 

ice-nucleating bacteria 264, 266, 301 

IGS (intergenic spacer} 144-146, 151, 

302 

illumination 45, 189 

immunochemical 125, 188 

immunochemical analysis 188 

immunochemical localization 125 

immunogenic analysis 137 

immunology 266 

impact assessment 264-270 

impact prediction 269 

imported willow leaf beetle (Plagiodera 

versicolora) (P. versicolora) 174, 175, 294 

in planta 175, 301 

in situ 44, 48, 80, 236, 238, 301 

in vitro culture 2, 5, 11, 13, 15, 19, 21, 

44, 45, 47, 53, 85, 94, 221, 230, 231, 236-

239,278 

in vitro feeding assay 175 

in vitro propagation 13, 44, 53, 221, 222, 

230, 231, 303 

in vitro roots 237-239 

in vitro screening 155, 158, 167 

in vitro selection 85, 155, 156, 207, 209, 

222,229 

in vivo 14, 34, 56, 131, 166, 170, 174, 199, 

298,301 

inbreeding (inbred) 151, 224, 230, 301 

incompatibility 106, 214, 296, 302, 308 

indirect selection 108, 223 

indole-3-acetic acid (IAA) 21, 77, 296 

indole-3-butyric acid (IBA) 19, 46, 163, 

296 

infect (infection) 51-54, 108, 155, 157, 

158, 161, 163, 165, 167, 174, 276, 294, 

298, 306, 309 

inflorescence meristem 212 

initiation codon 168, 296, 302 

inoculum 56,155,157,310 

inoculum titer 56 

insect 1, 62, 84, 86, 87, 155, 165, 168, 

174, 175, 178-184, 209, 212, 241, 244, 249, 

252-258, 266, 302, 310 

insect attack 165, 182, 183 

insect biotype 86, 180, 183, 253 

insect-eating bird 266 

insect resistance 86, 181-184 

insect-resistant 249 

insect tolerance 84, 182 

insecticide 180, 181, 183, 241, 242, 253, 

254,258 

insertion 97, 223, 310 

instability 38, 56, 87, 90-92, 94-97, 179, 

214,273,274,276,277 

USDA Forest SeNice Gen. Tech. Rep. RM-GTR-297. 1997 
instar 175, 176, 178, 256, 302 

Institute of Poplar Culture 1 

Integrated Pest Management (IPM) 254, 

255 

integration 30, 54, 56, 60, 63, 79, 90, 92-

94,96,197,198,208,209,220,228,231-

233,277,297,308,310 

intercellular space 19, 20, 156, 157, 302 

intergenic spacer (IGS) 144-146, 151, 

302 

internal transcribed spacer (ITS) 151, 

152, 302 

internode (internodal) 13-16, 53, 54, 75, 

77, 156, 163 

interspecific 10, 15, 101, 106-109, 152, 

161,220,221,224,229-231,275 

intracellular 45, 70 

intraspecies differentiation 116, 253 

intraspecific variation 103-107, 253 

introgression (introgressive) 41, 43, 94, 

103, 105, 106, 109, 110, 112-114, 123, 276, 

275, 302 

intron 65, 145, 297, 302 

inventory control 245, 246 

inverted repeat (inverted-repeated) 

146,302,304 

inverted-repeat region 144 

investment 232, 233, 241, 243, 254 

iodoacetamide 17 4 

iodoaceta te 17 4 

IPM (integrated pest management) 254, 

255 

IPT (isopentenyltransferase) 51, 55, 161 

lsatis 269, 292 

tinctoria 269, 292 

isoenzyme (see: isozyme) 

isogenic 151, 296, 302 

isolate 11, 24, 25, 30-32, 46, 52, 65, 66, 

71, 74, 75, 80, 81, 95, 126, 137, 138, 148, 

151, 155, 157, 302 

isolation medium 27, 30 

isomer (isomerization) 133, 135, 302 

isopentenyltransferase (IPn 51, 55, 161 

isotonic 24 

isozyme 34, 36, 102, 103, 105, 108, 116, 

222,228,302 

ITS (internal transcribed spacer) 151, 

152, 302 

J 

Juglans (walnut) 125, 292 

juvenile characteristic 80, 238 

juvenile-mature correlation 155, 158 

juvenile-mature trait relationship 220 

juvenile phase 5, 138 

juvenile tissue 34 

juvenilewood 80 

K 

kanamycin 54, 56, 62, 63, 67, 91, 93, 96, 

163, 175, 275 

kinetin 19, 29, 298 

KM8P (Kao and Michayluk media) 28, 

30 

Kunitz inhibitors (see also: proteinase 

inhibitor) 131 

L 

l'Union Allumettiere, S.A. (UNAL) 1 

Laccaria bicolor (L. bicolor) 155, 294 

laccase 195, 203 

Larix 103, 126, 134, 216, 292, 294 

Larix decidua (L. decidua) 126, 292 

Larix laricina (L. laricina) 103, 292 

Larix leptolepis (L. leptolepis) 216, 292 

Larix occidentalis (L. occidentalis) 132, 

292 

larva (larvae) (larval) 62, 175, 176, 178-

180, 182, 183, 253, 255-257, 293, 302, 306 

latewood 79-81, 299, 302 

leader sequence 70, 71, 168, 302 

leaf area 110, 175, 179 

leaf disc 14, 70, 92, 162, 183, 18~, 189, 

190 

leaf disk 11, 54, 157 

leaf enlargement 77 

leaf explant 11, 12, 15, 54, 62, 237 

leaf phenology 108, 110 

leaf plastochron index (LPI) 85, 302 

leaf primordia (leaf primordium) 75 

leaf micro-cross section technology 

(MCS) 236-240 

leaf rust 150-152,157,294 

leaf spatial position 85 

leafspot 108,150,157,166,167,294 

leaf thickness 39 

leaf-tissue explants 11 

LEAFY (LFY) 74-76, 78, 79, 81, 138, 212, 

214,216 

least-cost basis 241, 243 

lectin 131, 137 

legumin 132 

Leguminosae 173 

lenticel 16, 302 

Lepidoptera 256, 257, 293 

leucine zipper 126, 296, 302 

library screening 74 

ligand 302, 307 

light 11, 29, 46, 56, 75, 77, 87, 118, 156, 

157, 187-191, 295, 299, 305 

lignification 65, 67, 68, 79, 193-203, 302 

lignin 67, 79, 81, 84, 86-87, 185, 193-203, 

223,230,252,302,303 

lignin biosynthesis 81, 87, 193-203, 

223 

lignin metabolism 84, 86, 87, 193-203 

lime 241 

linkage 101, 103-105, 107-110, 116, 196, 

202,222,223,245,302 

linkage analysis 103, 222 

lipoxygenase 131 

liquid culture 21, 28 

liquid (nonpersistent) endosperm 132 

Liquidambar styraciflua (L. styraciflua) 

208,292 

Liriodendron tulipifera (L. tulipifera) 135, 

292 

Litchi chinensis (L. chinensis) 216, 292 

loblolly pine (Pinus taeda) (P. taeda) 103, 

104, 215, 266, 293 

locus (loci) 79, 102-105, 109-110, 116, 

126, 165, 212, 214, 222, 223, 295, 297, 

300,302,306,307 

LOD scores 110 

long-day condition 76, 127 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 

long-lived species 84, 251, 254 

long-term strategies (long-term 

stratagy) 224, 228, 231 

long-term stability 38, 87, 252, 254 

low durability resistance 266 

low-temperature storage 45, 46 

luciferase (luxF2) 55, 56, 275, 276, 307 

LUMIDEPENDENS 212 

luxF2 (luciferase) 55, 56, 275, 276, 307 

Lycopersicon 93, 95, 167, 292 

chilense 95, 292 

esculentum (tomato) 167, 292 

Lymantria dispar (L. dispar) (gypsy moth) 

62,178,179,253,257,294 

lytic peptides 167 

M 

M13 universal primer 118 

macerozyme 26, 27 

macro-propagation 

Macrozamia 134, 292 

communis 134, 292 

MADS box 212, 213, 216 

magainins 167,298,302 

maintenance cost 242, 245, 247 

maize (Zea mays) 53, 55, 66, 95, 97, 144-

146,196-198,200,252 

Malacosoma 178, 253, 254, 256, 294 

disstria (M. disstria) (forest tent 

caterpillar) 9, 62, 89, 178-183, 253-

256, 294 

malt extract 11 

Malus 33, 34, 167, 215, 216, 292 

domestica 167, 292 

hupehensis 216, 292 

Manduca sexta (M. sexta) (tobacco 

hornworm) 174, 294 

mannitol 28, 29, 304 

mannopine synthetase (MAS) 70-72, 77, 

91 

mannose 125 

map-based gene cloning 223 

MAR (matrix attachment region) 

(nuclear scaffold attachment region) 

(SAR) 97, 303, 304, 308 

marker 56, 67, 79, 91, 93, 101-110, 116, 

119, 138, 143-148, 150, 175, 207, 208, 220, 

222, 223, 229-231, 252, 266, 275, 296, 297, 

300,302,303,308 

marker-assisted selection (markeraided selection) 110, 223, 229, 302, 

308 

marker gene 56, 91, 252, 266, 296, 

297, 303 

Marssonina 108, 157, 294 

brunnea 108, 157, 294 

Marssonina leaf spot 157 

MAS (mannopine synthase) 70-72, 77, 91 

mass clonal propagation 48 

mass production 10, 12 

material input 243 

maternal inheritance 144 

mating 108, 109, 220, 224, 228-231, 301, 303, 

306 

matrix attachment region (MAR) (matrixattachment region) (nuclear scaffold 

attachment region) (SAR) 97, 303, 304, 308 

319 
maturation (mature) 10, 11, 38, 39, 91, 93, 

132-134,202,274,304 

mature tissue 156,274 

MCM1 213 

measurement endpoint 266 

Medicago 144, 292 

megagametophyte 134, 303 

megaspore 303 

meiosis 10, 297, 299, 301, 303, 306, 308 

Melampsora leaf rust 157 

Melampsora 38, 108, 109, 150, 152, 157, 

294 

Melampsora larici-populina (M. larici-

. populina) 109, 152, 157, 294 

Melampsora medusae (M. medusae) 152, 

157,294 

Melampsora occidentalis (M. occidentalis) 

152, 294 

membrane 24, 61, 103, 166, 189, 190, 

298, 299, 304-308 

membrane-active peptide 166 

membrane disruption 189, 190 

Mendelian inheritance 103, 104, 229 

mercaptoacetyldipeptide 174 

2-mercaptoethanol (B-mercaptoethanol) 

117, 175 

meristem 5, 7, 13, 55, 62, 74, 76, 78, 80, 

212-214, 221, 222, 231, 239, 295, 303 

mesophyll cell 19, 21, 80, 168 

messenger RNA (see: mRNA) 

metabolic pathway 68, 81, 206, 305 

metal 174, 191, 207, 208 

metal-chelating agent 174 

metallo-proteinase 173, 174 

Metasequoia 126, 292 

glyptostroboides 292 

methoxylation 194, 196 

methyl 66, 173, 189, 207, 208, 298, 303 

0-methyl transferase (OMT) (COMT) 

(see also: caffeic acid/5-hydroxyferulic 

acid-0-methyltransferase) 65-67, 87 

methylation 92, 94-97, 195, 196, 198, 

274,303 

micro-cross section 236-239 

micro-propagation 2, 7,13, 30, 33, 37, 

44, 230, 236-239, 273, 274, 277, 303 

microbe 254, 257, 264-269, 303 

microbial biotype 252 

microcalli (microcallus) 62 

microcosm 267-268, 303 

microculture 5 

microcutting 6, 7, 238 

microinjection 24, 299, 303 

microorganism 47, 155, 169, 207-209, 

251,293,297,302,303 

microparticle bombardment (see: 

biolistic) 

microprojectile (see: biolistic) 

microprojectile-bombardment (see: 

biolistic) 

microprojectile-mediated DNA 

delivery (see: biolistic) 

microsatellite 104, 118, 122 

microsatellite repeat 118 

microscopy 12, 15, 21 

microshoot 37, 237-239, 274, 303 

microspore 213, 303, 305 

mineral 30, 45, 75, 80, 310 

mini-cutting 238 

320 

minisatellite marker 222 

mitochondria 103, 106, 116, 143, 147, 

148, 299, 303, 309 

mitochondrial DNA (mtDNA) (see: 

mtDNA) 

mitosis 297, 299, 300, 303, 306 

mode of toxicity 254 

model species 5, 48, 80, 84, 220, 233 

model system 1, 13, 44, 51, 67, 74, 75, 

80, 81, 91, 93, 96, 101, 138, 166, 277 

modeling 168, 183, 268 

moisture stress 158, 216 

molecular biology 51, 101, 116, 128, 

165, 273, 295 

molecular breeding 105 

molecular chaperone 297,303 

molecular mapping 74,251 

molecular marker(see also: DNA marker) 

78, 79, 101, 102, 105, 106, 108-110, 119, 

120,150,273 

molecular polymorphism 150, 151 

Monch 1 

Monet 1 

monitoring 56, 81, 87, 92-94, 138, 157, 

183, 188, 230, 258, 265, 266 

monocot (monocotyledon) 33, 65, 66, 

132,145,295,298,303 

monogenic 105, 109, 110 

monolignol 196, 200, 203, 303 

monomer 67, 174,303 

morphogenesis (morphogenetic) 10, 13, 

15, 19, 90, 274, 303 

morphogenetic response 274 

morphological marker 108 

morphology 11, 12, 36, 38, 61, 77, 85, 

106, 156, 179, 199, 290, 301 

mortality 45, 60, 87, 175, 178-180, 183, 

184, 249, 257 

motif 126, 300 

mRNA (messenger RNA) 66-69, 71, 72, 

86, 96-98, 127-129, 132, 138-142, 165, 

168, 171, 176, 179, 182, 183, 197, 198, 

201, 205, 223, 300-303, 305-309 

mRNA secondary structure 66, 71, 

86, 127, 132, 165, 168, 179, 223, 303 

mRNP (ribonucleoparticle) 132, 307, 308 

MS (Murashige and Skoog) medium 7, 

8, 11, 14, 18, 19, 28, 30, 37, 46, 47, 54, 75, 

163, 237, 238 

mtDNA (mitochondrial DNA) 103, 106, 

116, 119, 120, 143, 148, 303, 304 

Muhle Larsen 1 

multi-trophic process 250, 257, 258 

multigene family (multigene families) 

126, 127 

multiple resistances 181 

Murashige and Skoog (MS) (see: MS 

medium) 

Musa 33,34,37,292 

mutant 12, 52, 55, 56, 70, 72, 80, 81, 91, 

95, 196, 197, 200-202, 207, 276, 303, 305 

mutant acetolactate synthase (crsl-1) 

52, 55, 56, 207 

mutation 7, 34, 36, 45, 75, 80, 92, 94-97, 

105, 143, 163, 169, 178, 180, 181, 196, 

212, 213, 229, 274, 276, 303-305, 307, 308 

mycelium (mycelia) 301, 303 

mycoherbicide 269 

mycorrhiza (mycorrhizae) 155, 208, 294, 

299,303 

Mycosphaerella (Septoria) 150, 151, 157, 

166, 167, 174, 294 

N 

populicola 150, 294 

populorum 150, 157, 166, 167, 174, 

222,294 

NAA (a-napthaleneacetic acid) 

(napthaleneacetic acid) 11, 15, 19, 29, 38, 

56, 62, 92, 157, 238, 296 

N-ethyl maleimide 174 

N-glycoslyation 133 

NADPH 188, 305 

naphthaleneacetic acid (see: NAA) 

necrosis 8, 157, 189, 190, 303, 310 

nematode 251, 310 

neomycin phosphotransferase (NPTII) 

54-56, 63, 67, 91, 94-96, 162, 175, 188, 

197, 198, 275 

NHz:·terminal amino acid 126 

Nicotiana 93, 95, 96, 144-147, 197, 292 

debneyi-tabacum 95 

glauca-langsdorffii 95 

longiflora 95 

plumbaginifolia 96 

tabacum 144-147, 197 

nicotine 183 

nitrate 15, 28, 30, 163, 207, 209, 216 

nitrogen 15, 30, 45, 47, 71, 117, 124, 125, 

127, 128, 138, 163, 209, 215, 216, 237, 

241, 242 

nitrogen availability 124, 127 

nitrogen cycling 124, 125 

nitrogen deposition 124 

nitrogen metabolism 138 

nitrogen redistribution 124 

nitrogen translocation 124 

node 5, 11, 14-16 

nodule 7, 19, 62, 303 

nonallelic 299 

nonallergenic 165 

nonadditive genetic variation 224, 303 

nondenaturing polyacrylamide gel 

(ndPAGE) 105 

nonhomologous 94, 96, 309 

nontarget effect 266, 267 

nontarget organism 174, 249, 266 

nontarget species 251, 264 

nopaline 54, 67, 91, 162, 175, 188, 198, 

275,304 

nopaline synthase (NOS) 54, 67, 91, 94, 

162,175,188,198,275 

normalwood 81 

northern analysis (see: northern blot) 

(see also: RNA blot) 

northern blot (northern analysis) 

(northern hybridization) (see also: RNA 

blot) 52, 67, 71, 72, 161, 175, 304, 306, 

308 

northern hybridization (see: northern 

blot) 

NOS (see: nopaline synthase) 

NPTII (neomycin phosphotransferase) 

54-56, 63, 67, 91, 94-96, 162, 175, 188, 

197, 198, 275 

nuclear DNA 51, 103, 107, 144, 303 

nuclear genome 101, 144, 307, 309'· 

nuclear genomic DNA 144 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 
nuclear scaffold attachment region 

(SAR) (matrix attachment region) 

(MAR) 97,303,304,308 

nucleotide 102-105, 126, 143, 296-298, 

301, 302, 304-308 

nutrient cycling 258 

nutritional status 216 

0 

0-methyltransferase (OMT) (COMT) 

(see also: caffeic acid/5-hydroxyferulic 

acid-0-methyltransferase) 65-67, 87 

oat 76, 77 

OCI (oryzacystatin) (see also: proteinase 

inhibitor) 52, 55, 85-87, 174-176 

octapine synthase (OCS) 91, 275 

oil palm 33, 34, 216, 292 

Olea europaea 33, 34, 216, 292 

oligogenic trait 110 

oligomer 132, 148, 298, 304 

oligonucleotide (10-mer) 104, 148, 304-

306 

omt (see: COMT) 

oncogene 51,56,161,297,304 

open reading frame (ORF) 126, 168, 

170,304 

operating demand 245 

operator 297, 304, 306 

operon 108,144,151,304,306 

Ophiostoma ulmi (0. ulmi) (Dutch elm 

disease) 41, 166, 294 

opine 304 

opportunity cost 242, 243, 304 

optimum rotation. 241, 243 

ORF (see: open reading frame) 

organ 33, 47, 85, 212-214, 298, 305 

organelle 36, 72, 143, 297, 301, 303, 304 

organelle DNA (see also: cpDNA, 

mtDNA) 36,297,303,304 

organelle genome 143, 304 

organic 28, 30, 193, 298, 301, 305, 306 

organic acid 28 

organochlorine (DDT) 254 

organogenesis 10-12, 15, 19, 21, 26, 30, 

38, 70, 221, 222, 230, 273, 274, 277, 304 

orientation 20, 67, 75, 87, 103, 161-163, 

197-200, 214, 295, 299 

ortet 297, 304, 306 

Oryza sativa (rice) 146, 174, 216, 253, 

265,292 

oryzacystatin (OCI) (see also: proteinase 

inhibitor) 52, 55, 85-87, 174-176 

OsMADS1 216 

osmotic 134, 156, 302, 304 

osmotic potential 156, 302, 304 

osmotic pressure 304 

osmoticum (osmotica) 11, 27-30, 45, 304 

outcrossing 220 

over-expression 67, 179, 187, 189, 190, 

216 

oviposit 182, 257, 304 

ovule 15, 304 

Oxford Paper Co. 1 

ozone 70-72, 157, 187, 190, 191 

p 

pA2 (see also: anionic peroxidase) 66 

pA3 (see also: anionic peroxidase) 66 

paclobutrazol 215 

PAGE (see: polyacrylamide gel electrophoresis) 

PAL (phenylalanine ammonia-lyase) 65, 

66, 194, 195, 197 

palindrome (palindromic) 126, 304 

palisade parenchyma 20, 21, 304 

paramutation 94, 96, 97, 304 

paraquat 189, 208 

parasite 150, 155, 156, 176, 257, 301, 

304,305 

parasitoid 257, 294 

parenchyma 20, 21, 79, 125, 127, 132, 

304,308,310 

parental 33, 36-39, 76, 109, 118, 144, 

226,228,300 

parental origin 118 

paromomycin 30 

particle acceleration (see: biolistic) 

particle acceleration DNA delivery 

system (see: biolistic) 

particle bombardment (see: biolistic) 

PAT (phosphinotricin acetyl transferase) 52, 61, 208 

pAtALS (Arabidopsis thaliana acetolactate 

synthase promoter) 67 

paternity analysis 105 

pathogen 78, 84, 108, 150-152, 155-158, 

165-170, 173, 174, 176, 265-267, 293, 294, 

296-298, 302, 304, 306, 310 

pathogen pest 173, 174, 176 

pathogen resistance 84, 165-167, 

170 

pathogen variation 150-152, 157 

pathogenesis 150, 174, 304 

pathogenic fungi 167, 173, 174 

pathology 150, 165, 264, 266, 267, 269, 

299 

pathosystem 158, 304 

Pauley 1, 128 

Paulownia taiwaniana (P. taiwaniana) 30 

Paulownia tomentosa 33, 34, 292 

Paxillus 208,294 

involutus 208, 294 

PCR (polymerase chain reaction) 52, 65, 

67, 81, 93, 101, 103-105, 116-119, 143, 

144, 150-152, 175, 179, 197, 222, 223, 295, 

304,306 

PCR-RFLP 143, 144, 147-149 

pectinase 24, 304 

pectolyase 27 

pedigree 108, 110 

PEG (see: polyethylene glycol) 

pepstatin (see also: proteinase inhibitor) 

173 

peptide 70-72, 126, 133, 165-170, 188, 

298, 304, 306, 309 

peptide bond 173, 306 

peptide stability 165, 168, 170 

peptide target 165 

peroxidase (PRX) 21, 23, 55, 58, 65-69, 

187, 188, 195, 197, 203 

pest-biotype evolution 249-259 

pest resistance 2, 51, 52, 96, 209, 228, 

251,253 

pest-resistance gene 2 

pesticide 2, 167, 178-180, 206, 209, 247, 

249, 250, 253-255, 257, 258 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 

petiole 15, 62, 304 

Petunia hybrida (P. hybrida) (Petunia x 

hybrida) (P. x hybrida) 95, 96 

PGLa 167 

Phaeolus 156, 294 

schweinitzii 156, 294 

Phaeolus vulgaris 133, 292 

pharmaceutical 21 

phase change 5, 84, 299, 304 

phenolic glycoside 181-184, 254, 255 

phenolic 7, 183, 184, 200-202, 206, 208, 

254-256, 305 

phenotype 33, 36, 74-79, 101, 168, 200, 

212, 222, 224, 229, 250, 275, 276, 299, 

300, 305, 307 

phenotypic 21, 33, 36, 37, 45, 47, 74, 77, 

78, 87, 91-93, 95, 96, 101, 109, 166, 197, 

201, 221, 223, 231, 252, 274, 296, 299, 

301,306 

phenotypic fidelity 36, 231 

phenotypic variance 101, 301 

phenotypic variation 33, 45, 47 

phenylalanine ammonia-lyase (PAL) 65, 

66, 194, 195, 197 

phenylmethylsulfonyl fluoride (PMSF) 

173 

phenylpropanoid pathway 66, 194, 305 

phloem 74,94, 125,198,215,296,305,309 

5'-phosphate (5'-P) 300 

phosphatase 126, 129 

phosphinotricin (glufosinate) 52, 55, 56, 

61,208 

phosphinotricin acetyl transferase 

(PAT) 52, 61, 208 

phosphoramidon 174 

phosphorus 241 

photo labile 179,305 

photoinhibition 189, 190 

photoperiod 11, 47, 77, 79, 80, 81, 127, 

215, 216, 236, 237, 305 

photosynthesis (photosynthetic) 24, 40, 

46, 70, 131, 182, 187, 189, 190, 256, 297, 

304,305 

Photosystem I 189, 305 

phyllotaxy (phyllotaxis) 305 

phylogenetic 101, 131-133, 138, 151, 

152, 266, 275, 276, 297, 305 

phylogeny 106, 305 

physiology (physiological) 7, 11, 21, 24, 

48, 57, 70, 85, 90, 124, 128, 135, 138, 156, 

180,181,252,254,256,277 

phytoalexin 305 

phytochemical 254-256 

phytochrome 2, 55, 77 

phytochrome A (phyA) 76-78 

phytochrome B (phyB) 77 

phytohemaglutinin 131 

phytohormone (see: plant growth 

regulator) 

phytoremediation 206-209, 305 

phytotoxic 8, 167, 305 

PIC (Polymorphism Information 

Content) 101, 104 

Piccarolo 1 

Picea (spruce) 33, 34, 85, 94, 134, 144, 

215, 254, 292, 293 

engelmanii 293 

glauca 85, 94, 134, 215, 293 

glauca x engelmanii 134 

321 
Picea abies (P. abies) 134, 293 

PIN2 (proteinase inhibitor II) 2, 52, 54, 

55, 67, 91, 92, 173-175, 182, 275 

Pinaceae 134, 293 

Pinus (pine) 33, 34, 103, 254, 293 

lambertiana 293 

monticola 134, 293 

palustris 293 

pinaster 134, 293 

radiata 103, 293 

rigida 103, 293 

strobus 103, 126, 293 

sylvestris 126, 293 

taeda 103, 104, 215, 266, 293 

pistil 213, 214, 296, 305, 308 

Pisum sativum (P. sativum) 70, 293 

pith cells 94 

Plagiodera versicolora (P. versicolora) 

(imported willow leaf beetle) 174, 175, 

294 

plant breeding 101, 103, 105, 108, 250, 

254 

plant defense 131, 173, 254, 256 

plant development 78, 95, 97, 193 

plant growth 11, 12, 15, 19, 29, 30, 44, 

45, 47, 56, 75, 77, 79, 80, 202 

plant growth regulator (hormone) 

(phytohormone) (plant growth substance) (see also: abscisic acid, auxin, 

cytokinin, ethylene, gibberellic acid, 

thidiazuron) 11, 12, 15, 19, 21, 27, 29, 

30, 37, 38, 44-47, 56, 61, 63, 75, 77-79, 81, 

92, 96, 128, 134, 156, 157, 161, 274, 296, 

298,305 

plant-herbivore interaction 251 

plant/parasite interaction 150 

plant pathology 150, 170, 264, 266, 267, 

269 

plant regeneration 10, 15, 19, 21, 24, 25, 

27, 30, 197, 274 

plant tissue culture 48, 92, 165, 274, 

296, 298, 307 

plant transformation 252, 305 

plantation cost 243 

plantation site 241, 246, 247 

plantlet 11, 13, 15, 19, 21, 31, 39, 54, 62, 

63, 75, 158, 163, 236-238, 267, 274, 307 

plasma membrane 24, 298, 305, 307 

plasmid 51, 54, 56, 70, 91, 94, 161-163, 

175, 188, 198, 275, 296-298, 305, 307, 309, 

310 

plating density 29, 30 

pleiotropy (pleiotropic) 93, 110, 277, 

305 

Plodia interpunctella (P. interpunctella) 

294 

ploidy 36, 156, 166, 295, 305 

Plutella rylostella (P. rylostella) 180, 294 

PMSF (phenylmethylsulfonyl fluoride) 

173 

point mutation 36, 45, 92, 105, 305, 307 

polar transport 77, 305 

pollen (pollination) 1, 2, 13, 19, 60, 105, 

167, 170, 212-214, 220, 230, 251, 252, 258, 

265, 267, 276; 277, 295, 297, 298, 302, 

303,305,308 

pollen dispersal 265 

pollution (pollutant) 2, 44, 187, 189, 

190, 206-209, 212, 257, 258 

322 

polyacrylamide gel electrophoresis 

(PAGE) (SDS-PAGE) 30, 34, 134-136,308 

polyadenylation 162, 179, 305 

polycistronic 151, 305 

polyethylene glycol (PEG) 24, 156 

polyethylene-glycol-mediated gene 

transfer 305 

polygenic 105, 109, 110, 306 

polygenic trait 105, 109, 110, 306 

polymer 193-197, 201, 202, 297, 298, 

301, 304-306 

polymerase 52, 65, 67, 81, 93, 101, 103, 

116, 145, 150-152, 175, 179, 197, 222, 295, 

299, 304-306, 308 

polymerase chain reaction (see: PCR) 

polymorphic DNA 101-110, 116-120, 

143-148, 150, 151, 222, 306 

polymorphism 34, 35, 101-105, 107, 119, 

132, 143, 144, 150-152, 222, 295, 305, 307 

Polymorphism Information Content 

(PIC) 101, 104 

polypeptide 128, 132-135, 137, 173, 293, 

299, 300, 303, 304, 306, 308, 309 

polyploid 36, 295, 305 

polysaccharide 193, 202, 297 

polysome 132,306 

polyvinylpyrrolidone (PVP) 45 

POPCAD 66 

poplar mosaic virus 108 

Populus - see: Index of Populus 

population genetic 101, 150, 273 

position effect 90, 93, 306 

post-transcriptional 94, 96, 97, 127, 133 

post-transcriptional regulation 127, 

133 

post-translational 127, 132, 137, 138, 

168,309 

post-translational processing 132, 

138 

potassium 178, 241 

potato 55, 65, 67, 86, 91, 97, 137, 174, 

182, 252, 265, 275, 293 

PR-1a (tobacco leader sequence) 168, 

182 

precocious flowering 75, 212, 215, 216 

preculture 56 

predator 2,183,253,257,266,296,306 

prediction 266, 268, 269 

preformed meristem 5, 7, 239 

prey 257, 306 

primer 65, 103-105, 108, 117-122, 143-

145, 147, 148, 151, 152, 222, 305, 306 

primordium (primordia) 75, 306 

probe 65, 66, 71, 103-106, 108, 143, 144, 

197, 222, 266, 297, 298, 300, 304, 306, 

307, 308, 310 

production cost 13, 158, 241, 246, 247 

progeny 1, 34, 88, 95, 108-110, 144, 158, 

169, 224, 226-228, 267, 275, 296, 300, 306 

progeny test 224, 228 

prokaryote 306 

prolamin 132 

promoter 62, 63, 67, 68, 70-72, 74, 76, 

77-79, 85-88, 91, 93-97, 132, 137, 138, 

161-163, 168-170, 175, 179, 182, 183, 188, 

197, 198, 200, 208, 213, 214, 216, 252, 

275, 277, 295, 299, 306, 307 

promoter-based suppression 252 

propagation 11, 33, 34, 36-38, 40, 44, 51, 

101, 109, 128, 169, 206, 220, 221, 224, 

228-232, 236, 238, 239, 273, 274, 277, 297, 

303,310 

propeptide 132 

protease 87, 128, 133, 170, 173, 178, 306 

protein bodies (protein body) 133, 134 

protein hydrolysis 124, 128, 133 

protein storage vacuole 125 

proteinase 173-175, 306 

aspartic proteinase 173 

cysteine proteinase 173-175 

metallo-proteinase 173, 174 

serine proteinase 173-175 

proteinase inhibitor 2, 52, 55, 66, 67, 91, 

173-176, 181, 257, 275 

Bowman-Birk Inhibitor (BBI) 175 

Kunitz inhibitor 131 

pepstatin 173 

proteinase inhibitor I (PINl) 173 

proteinase inhibitor II (PIN2) 2, 52, 

54, 55, 67, 91, 92, 173-175, 182, 275 

oryzacystatin (OCI) 52, 55, 85-87, 

174-176 

protoclonal variation 33 

protoclone 33, 35, 38, 92, 306 

protoplast 12, 24, 29, 38, 60-62, 91, 92, 

221, 229, 305, 306 

protoplast culture 2, 7, 24, 27, 28, 30, 

33, 44, 60, 61 

protoplast fusion 24, 30, 221, 230, 

306,308 

protoplast isolation 24-26, 30, 304 

provenance test 228 

Prunus 33,34,126,293 

cerasus 215, 293 

japonica 216, 293 

persica 126, 215, 293 

serotina 269, 293 

PRX (peroxidase) 21, 23, 55, 58, 65-69, 

187,188,195,197,203 

prxA1 55,66 

prxA3a 66 

prxA4a 66 

psbA 144, 146-148 

pseudo-testcross 108 

Pseudomonas 

aeruginosa 214, 294 

fluorescens 179, 294 

solanacearum 167, 294 

syringae 167, 267, 294, 301 

Pseudotsuga menziesii (P. menziesii) 

(Douglas-fir) 76, 82, 134, 183, 254, 293 

· PT AG 213, 214 

PTD 213 

PTFL 213, 214 

PTOMT 66 

pupa (pupae) 175, 176, 183, 306 

purine 304, 306 

PVP (polyinylpyrrolidone) 45 

pyrethroid 254 

pyrimidine 304, 306 

Pyrus nivalis 34, 215, 293 

Q 

QTL (see: quantitative trait loci) 

quantitative resistance (horizontal 

resistance) 109, 181, 306 ·. 

qualitative resistance (vertical resistance) 109, 306 

USDA Forest Service Gen. Tech. Rep. RM-GTR~297. 1997 

\jj;;;'J 
quantitative trait 79, 105, 108, 109, 223, 

302,306 

quantitative trait loci (QTL) 79, 105, 110 

223, 229, 302, 306 

Quercus 105, 133, 144, 147, 293 

accutissima 144, 147, 293 

ilex 133, 293 

petraea 105, 293 

robur 105, 133, 293 

R 

R1 94 

R2 94 

race 157, 306 

race-specific resistance 157 

radical 2, 11, 189, 190, 195 

radicle 301, 306 

radioactive (radioactivity) 63, 102, 196 

ramet 304, 306 

random amplified polymorphic DNA 

(RAPD) 103, 116, 143, 148, 150, 151, 222, 

306 

RAPD analysis (RAPD analyses) 104, 

118 

RAPD banding pattern (RAPD band) 

116, 143 

RAPD fingerprint 105, 148 

RAPD marker 103-105, 108, 109, 116, 

118, 119, 151, 222 

Raphanus sativus 267, 293 

rate of return 242, 243 

raw material 193, 241, 245-247 

ray cell 66, 78, 125, 127, 306 

rbcL 144 · 

rbcS 91, 93, 94, 277 

rONA (ribosomal DNA) 106, 116, 119, 

143-145, 148, 302, 306, 307 . 

rONA spacers 144 

reactionwood 79-81, 306, 307 

recalcitrant 19, 33, 60, 231, 307 

receptor 12,178,180,307 

recessive 180, 183, 184, 229, 275, 307 

reciprocal recurrent selection 224, 227 

recognition site 168 

recombinant DNA (recDNA) 222, 266, 

300,307 

recombinant gene 90, 94, 96, 275, 307 

recombinase 214,307 

recombination 10, 56, 109, 222, 223, 229, 

276,297,298,300,302,307 

recombination frequency 109, 222, 

223, 307 

recombination value 222 

redundant (redundancy) 167, 214 

refugia 180-184, 253, 307 

regenerant 33,34,36-39,274,306,307 

regeneration 10, 13-15, 19, 21-26, 27, 29, 

30, 37-39, 44-47, 52, 54, 56-60, 70, 73, 75, 

88, 91, 94, 97-100, 155, 162, 163, 197, 221, 

231, 273, 274, 277, 307 

regression model 268 

regulations 87, 230, 232, 258, 268, 276, 

277 

regulatory gene 97, 212, 213, 215, 216, 

307 

regulatory sequence motif 126 

rejuvenation 7, 34, 36, 38, 231, 274, 307 

remediate (remediation) (remediatory) 

206-209, 305, 307 

repeat-induced point mutation (RIP) 

92,307 

repellency 254 

reporter gene (see also: CAT, GFP, GUS, 

LUXF2) 7, 56, 67, 85-87, 91, 175, 208, 

275,307 

repressor 304,307 

reproductive development 216, 252 

reproductive potential 224, 229 

reproductive-specific promoter 252 

reproductive sterility 212, 214, 216, 252, 

277 

reproductive tissue 212, 213 

rescue cloning 74 

resistance (resistant) 1, 2, 34, 36-38, 52, 

56, 62, 67, 84, 86, 87, 91, 96, 101, 108-110, 

150, 155-158, 161-163, 165, 166, 168, 170, 

173-176, 178-184, 186-190, 193, 196, 207-

209, 222-224, 228, 249-254, 258, 265-267, 

275, 276, 296, 298, 306-308 

resistance management program 254 

resistance to abiotic stress 221, 223 

resistant allele 182-184 

resistant clone 108, 1'55, 163, 254 

resistant (R) gene 2 

resistant insect biotype 86, 180, 253 

resorb 124, 125, 128 

resorcinol 183 

restriction digestion 145-148, 152 

restriction endonuclease (restriction 

enzyme) 101, 103-106, 143, 144, 152, 168, 

222,307 

restriction fragment length polymorphism (see: RFLP) 

reverse transcriptase 307 

reverse transcriptase-polymerase chain 

reaction (RT-PCR) 67, 179 

reverse transcription 298, 307 

RFLP (restriction fragment length 

polymorphism) 101-108, 111, 116, 119, 

143, 144, 147, 148, 152, 222, 223, 295, 307 

rhizosphere 208, 209, 307 

ribosomal DNA (rONA) 106, 116, 119, 

143-145, 148, 302, 306, 307 

Ri (root-inducing) 51, 307 

Ri-plasmid 51, 307 

Ribes nigrum 216, 293 

ribonuclease (RNase) 214, 307, 308 

ribonucleic acid (RNA) (see: RNA, 

mRNA, rRNA, tRNA) 

ribonucleoprotein particle (RNP) 132, 

307,308 

ribosomal 106, 146, 308, 309 

ribosomal DNA (rONA) 106, 143, 

144, 306, 307 

ribosomal DNA polymorphism 

(rONA polymorphism) 116 

ribosomal protein 307 

ribosomal RNA (rRNA) 143, 144, 

151, 152, 214, 307, 308 

ribosome 299, 303, 306, 307, 309 

ribosome binding site 308 

ribosome inactivating protein (RIP) 

214 

ribulose 

ribulose bisphosphate carboxylase 

131 

ribulose 1,5-bisphosphate carboxylase/ 

oxygenase 124 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 

ribulose-1,5-bisphosphate carboxy 

lase (Rubisco) small subunit pro 

moter 182 

rice (Oryza sativa) 146, 174, 216, 253, 

265, 292, 

ricin 214 

Ricinus communis 214, 293 

RIP (see: repeat-induced point mutation, 

ribsome inactivating protein) 

riparian (area, buffer, zone) 206, 209 

risk assessment 87, 180, 249-258, 264-

269, 277 

risk determination 265, 266, 267, 269 

risk estimation 265, 269 

risk evaluation 250, 269 

risk magnitude 268 

RNA 66, 67, 71, 72, 94, 96, 127, 132, 143, 

144, 151, 165, 168, 175, 179, 197, 198, 

200, 201, 214, 223, 252, 294-296, 298, 300-

304, 306-309 

RNA blot (see also: northern blot) 77, 

201, 308 

RNA polymerase 145, 306, 308, 309 

RNase 214, 307, 308 

RNase gene 86 

RNP (ribonucleoprotein particle) 132, 

307,308 

Robinia pseudoacacia (R. pseudoacacia) 

126, 208, 293 

rol gene (rol) 

rolB 96 

role 55, 76, 78, 79, 91-94, 277 

root (rooted) 10, 13-15, 19, 34, 46, 51-53, 

77, 79, 87, 127, 155, 165, 174, 206-209, 

215, 236-239, 251, 252, 274, 293-298, 303, 

307 

root cutting 13 

root form 35, 51 

root radical 11 

root segment 14, 15, 92, 238 

rootsprout 251,252 

rootsucker 10,236,238,298 

root-inducing (Ri) 51, 307 

Ri plasmid (Ri-plasmid) 51, 307 

rooting 25, 54, 163, 168, 221, 224, 231, 

237,238 

rotation 34, 51, 84, 91, 116, 181, 206, 

229,231,241,243-247,251,252,254 

rpl2 gene (rpl2) 146, 147 

rpoC1 144-146, 148 

rpoC2 144-146 

rrn operon 144 

rRNA (ribosomal RNA) 143, 144, 151, 

152, 214, 307, 308 

Rubisco small subunit (atslA) 71, 182 

RUBISCO 124, 182 

RUBISCO small subunit transit 

peptide 70 

RUBISCO transit peptide 70, 72 

Rubus 33, 34, 161, 293 

rust 108,109,150-152,157,269,294 

rust fungi (rust fungus) 150-152, 

157, 294, 309 

s 

S (Svedburg unit) 132 

Sl nuclease protection assay 127 

s, 38, 127 

323 
52 stage 38 

S-adenosyl-L-methionine synthase 

(SAM) 66 

safety 178, 212, 230, 249, 251, 258, 265, 

267, 276, 277 . 

Salicaceae 1, 13, 33, 34, 133-135, 138, 

293 

Salix 33, 34, 146, 147, 293 

babylonica 34, 293 

caprea 293 

Iucida 167, 293 

pseudolasiogyne 144-147 

smithiana (x smithiana) 126, 293 

viminalis 223, 293 

Salmonella 93, 294 

salt 8, 27, 28, 125, 132, 156, 304, 310 

SAM (S-adenosyl-L-methionine synthase) 66 

Sambucus nigra (S. nigra) 126, 293 

Santalum album 33, 34, 293 

sarcin loop 214 

scaffold attachment region (SAR) 

(matrix attachment region) (MAR) 97, 

303,304,308 

SCAR (sequence characterized amplified region) 103, 104 

sCEC 168 

Schreiner 1 

scolytid 254 

score (scoring) 117, 215 

screen (screening) 25, 28, 38, 54, 74, 81, 86, 

105,108,155-158,200,207,221,224,297,300 

SDS-PAGE (sodium dodecyl sulfatepolyacryl amide gel electrophoresis) 

(PAGE) 30, 34, 135-137,308 

seasonal growth cycle 5 

seasonal nitrogen cycling (seasonal 

nitrogen cycle) 124, 125, 128 

secondary metabolite 21 

secondary structure 165, 168, 170 

seed 2, 131-138, 167, 220, 221, 230, 237, 

251, 252, 254 

seed dispersal 252 

seed production 212 

seed storage protein (SSP) 124, 130-

138 

segregation 96, 101, 104, 157, 222, 308 

selectable marker 56, 67, 91, 96, 175, 

208,275,296,297,308 

selection pressure 2, 132, 180-184, 258 

selective pressure 180, 253, 254 

self-cleaving protease 170 

self-incompatibility 214 

sense 75, 98, 161-163, 197, 198, 200, 201, 

223, 230, 294 

sense strand 145, 147 

sense suppression 252 

sepal 213, 296, 308 

Septoria 150, 151, 157, 167 

Septoria canker 150, 167, 241 

Septoria leaf spot 150, 157, 167 

Septoria (Mycosphaerella) 150, 151, 157, 

166,167,174,222,241,294 

musiva 150, 157, 166, 167, 174, 222, 

294 

populicola 150, 294 

sequence characterized amplified region 

(SCAR) 103, 104 

sequence divergence 145, 308 

324 

sequence tagged site (STS) 105, 107, 

108, 116 

serine 173 

serine proteinase 173-175 

shikimate 52, 55, 72, 91, 194, 207, 308 

shikimate pathway 194, 308 

shoot culture 5-8, 24-30, 44-48, 70, 156, 

158, 236-239 

shoot multiplication 25, 46 

shoot primordia (shoot primordium) 13 

shoot regeneration 13, 15, 19, 29, 33, 47, 

162, 163 

shoot tip 7, 156, 237, 239, 306, 310 

shoot training 215 

short rotation 51, 116, 241, 251 

short-term strategies (short-term 

strategy) 224, 225, 228 

sialic acid 125 

sieve tube (companion cells) 305 

signal polypeptide 132 

silviculture (silvicultural) 2, 34, 39, 206, 

230, 232, 245, 265 

simple recurrent selection 224, 226 

simple sequence 'repeat (SSR) 102, 104, 

108, 118, 222 

sinapyl alcohol 194, 196, 303 

single strand conformation polymorphism (SSCP) 105 

site-directed PCR 150, 151 

site preparation 241, 243, 247 

site-specific amplication 143 

SLG (S-locus glycoprotein) 214 

SLR (S-locus-related) 214 

SOD (superoxide dismutase) 187, 188, 

190 

Sodium dodecyl sulfate-polyacrylamide 

gel electrophoresis (SDS-PAGE) 30, 34, 

135-137, 308 

soil pH 208, 257 

Solanaceae 173 

Solanum 144, 293 

solute leakage 189,190 

somaclonal 34, 35, 37-40 

somaclonal selection 36 

somaclonal variation 2, 33, 34, 36-38, 

40,92, 93, 157, 207, 221, 222, 228-230, 

232, 30'8 

somaclone 34, 36-40 

somatic 10, 30, 34, 60, 92, 97, 273, 274, 

299,308 

somatic embryo 11, 48, 133, 274 

somatic embryogenesis 2, 10-12, 19, 

48, 138, 221, 222, 230, 231, 274, 277, 

308 

somatic hybrid 24-30, 221, 308 

somatic mutation 34 

Sophora japonica 126, 293 

sorghum 196-198, 200 

Southern blot (Southern hybridization) 

(Southern analysis) 52, 94, 103, 104, 144, 

. 295, 304, 306, 308 

soybean 126, 127, 132, 137, 173, 175, 

252, 267, 269, 292 

spacer.143-148, 151,302,308 

spacer sequence 308 

spatial 80, 180, 181, 250, 269 

spatial refugia 181-184 

spatial regulator 212, 213 

SPL-3 216 

Spodoptera exiqua 174, 294 

spongy mesophyll 19, 20,308 

spontaneous mutation 92, 181 308 

spore 152, 157, 167, 178, 179, 182, 236, 

296,298,299,303,305,309 

spore dispersal 269 

spring flush 5 

spruce (see: Picea) 

spruce budworm (see: Choristoneura) 

spur leaf 125 

SQUAMOSA 216 

SSCP (single strand conformation 

polymorphism) 105 

SSP (seed storage protein) 124, 130-138 

SSR (simple sequence repeat) 102, 104, 

108,118,222 

stability 1, 5, 7, 33, 34, 38-40, 84, 86, 90, 

93, 95-97, 110, 132, 138, 165, 168, 170, 

176, 223, 229, 230, 254, 257, 258, 264, 

267, 274, 276, 277, 309 

stability of transgene expression 70, 

84, 85, 87, 97, 252 

stabilization 7, 208 

stabilization phase 7 

stable integration 198, 208 

stalk 19, 304, 308 

stamen 213, 214, 295,.308 

startpoint 296, 308, 309 

Steenackers 1, 176 

stem cutting 37, 38, 236 

stem diameter 39, 110 

stem girth 35 

stem height 35, 40, 110 

stem segment 75, 92, 162, 163 

stem volume 110 

sterile 75, 87, 166, 212-216, 232, 275 

sterile triploid 252 

sterility 86, 166, 184, 212-216, 230, 232, 

250, 252, 258, 275, 277, 308 

sterility-inducing gene 166 

steroidal glyco-alkaloid 183 

stigma 214, 252, 305, 308 

stigma-specific promoter (STIG1)214 

stock plant 46, 236, 238, 239 

stomate (stomata) 18, 308 

stop codon 168 

storage protein 65, 66, 124-128, 131-138, 

308 

Stout 1 

stress 21, 70-72, 86, 87, 95, 155, 156, 158, 

165, 183, 187-191, 208, 216, 220, 221, 223, 

224,228,252,254,274,303 

structural gene 175, 300, 308 

STS (sequence tagged site) 105, 107, 

108, 116 

stylar transmitting tissue-specific gene 

213 

style 305, 308 

subculture (subculturing) 5-7, 10, 11, 

34, 37, 38, 45-47, 61, 63, 163, 308 

subline 224,230 

sucker 309 

sucrose 28, 29, 163, 237-239 

sugar 28, 125, 247, 300, 301, 304, 307, 

309 

sugar alcohol 28 

sulfometuron methyl 207 

sulfur dioxide (502) 189, 190, 208 

sulfur trioxide radical 190 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 
superoxide dismutase (SOD) 187, 188, 

190 

suppression 75, 95, 96, 201, 213, 214, 

223, 252, 296, 298 

survival 19, 45, 47, 62, 90, 97, 155, 175, 

179, 182,.224, 247, 255, 256, 266, 267, 

269,273,276 

susceptible (susceptibility) 1, 2, 51-57, 

108, 156-158, 165, 168, 174, 178, 180-183, 

189,222,253,254,297,306 

suspension culture 10-12, 37, 38, 61, 156, 

303 

suspensor 132 

sustainability (sustainable) 2, 250 

Svedburg unit (S) 132 

sylleptic branch 110 

symbiont 249 

symbiotic 207, 208,303 

synchronization 45, 61, 62 

syntenic map 105, 108 

synthetic peptide sequence 165 

syringaldehyde 197, 202 

syringin 196 

syringyl 194, 196, 199-201 

systematics 106, 116, 131 

Syzygium aromaticum (clove tree) 215, 293 

T 

T-DNA 51, 52, 54-56, 70, 74, 76-78, 94-

97, 161, 175, 188, 197, 198, 201, 275, 296, 

297, 307, 309 

T-DNA border 51, 60, 198, 275 

TA29 (male reproductive tissue-specific 

promoter) 213 

tag (tagging) 109, 116 

tandem array 151, 309 

tandem repeat 104 

tannin 7, 183 

tapetum 213 

tapetum-specific promoter (tapetalspecific promoter) 86, 214 

Taq (Taq DNA polymerase) (Taq 

polymerase) 117, 152 

target insect 87, 178, 184, 257, 258 

targeting 71, 79, 126, 131, 138, 165, 168, 

181, 214, 309 

TATA box 126,309 

Taxodium 

distichum 126, 293 

taxonomy (taxonomic) 35-37, 104-106, 

116,134,143,150,222,257,305 

TDZ (see: thidiazuron) 

telomere 301, 309 

temperature 45-48, 75, 77, 80, 87, 94, 

127, 128, 148, 157, 187, 189, 190, 214, 

216,269,295,298,299,301 

template DNA 116, 151, 152 

temporal 86, 134, 180, 181, 250, 258 

temporal refugia 180, 182, 184 

temporally specific expression 256, 258 

teratoma (teratomatous) 162, 163, 309 

terminal (terminus) 76, 105, 126, 133, 

167,173,179,202,236,239,296,299,309 

terminal amino residue 173 

terminal carboxyl residue 173 

termination 168, 270 

termination codon 168, 304, 309 

termination sequence 165, 170 

tetranucleotide 144, 145, 168 

tetraploid 36-38 

tfdB 208 

tfdC 208 

thidiazuron (TDZ) 15, 29, 30, 56, 62, 

203,238,298 

thioacidolysis 198-203 

thioglycolic acid 202 

a-thionin (thionin) 166 

thymidine 61, 62 

Ti (tumor-inducing) plasmid 51, 56, 

161, 175, 275, 297, 309 

timber 33, 86, 87, 166, 251, 292 

time series analysis 266 

tissue ablation 213 

tissue culture 10, 13, 24, 33, 47, 48, 51, 

56, 61, 62, 76, 80, 84, 85, 92, 138, 155-157, 

163, 165, 206, 220, 221, 223, 228, 229, 

236, 238, 239, 251, 273, 274, 295, 296, 

298, 300, 301, 307, 310 

tissue specific expression 66, 127, 138 

tissue-specific promoter 79, 87, 213 

TMV (see: tobacco mosaic virus) 

tobacco 13, 24, 65-68, 70, 86, 93, 94, 96, 

137, 138, 144-146, 161, 162, 164-168, 179, 

182, 188, 190, 196-198, 200-202, 213, 214, 

216,276,277,292,294 

tobacco hornworm (Manduca sexta) 

174,294 

tobacco mosaic virus (TMV) 154, 

265,294 

tobacco rbcL gene (rbcL) 144 

totipotent (totipotency) 48, 239 

toxA 214 

toxic (toxicity) 27-30, 167, 168, 178-181, 

183, 187, 206-209, 214, 250, 257, 293, 296, 

298,300 

toxin 155, 158, 178-184, 214, 218, 253, 

277 

toxin resistance 158 

tracheid 79, 309, 310 

traditional breeding 78, 90, 101, 108, 

253,275 

trans-acting factor 309 

trans-inactivation 94, 96, 97, 276 

transcription 70, 71, 75, 86, 127, 132, 

147, 295, 298, 303, 305-307, 309 

transcription factor 203, 212, 213, 

216, 297, 309 

transcription unit 151, 296, 309 

transcriptional 67, 71, 72, 103, 127, 132, 

133, 137, 161, 214, 302, 309 

transcriptional start site 126 

transfer DNA (see: T-DNA) 

transfer RNA (tRNA) (see: tRNA) 

transformant 7, 54, 56, 60, 74-79, 85-87, 

90, 93, 94, 96, 97, 138, 168, 182, 197-199, 

201, 230, 252, 309 

transformation 12, 13, 34, 48, 51-57, 67, 

70, 74, 75, 78, 84, 86-88, 90-95, 97, 161-

163, 166, 169, 170, 174, 175, 179, 188, 

197, 198, 201, 207-209, 214, 220-223, 231, 

232,253,257,264, 275,277,295,297, 

300, 305 

transformation frequency 

(transformation frequencies) 54, 56, 

61,75 

transgene escape 251 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 

transgene expression 2, 7, 56, 61, 70, 72, 

75, 84-88, 90, 93, 95-97, 179, 180, 214, 

252, 276, 309 

transgene inactivation 90, 93-97 

transgene instability 87, 94, 97, 277 

transgene regulation 52, 252 

transgenic organism 166, 250, 276, 277 

transgenic plant 24, 30, 52, 60, 63, 67, 

70, 74, 75, 77, 90-97, 161, 163, 165-168, 

170, 175, 181, 187-190, 197, 199, 207, 214, 

216, 223, 230, 231, 253, 254, 257, 258, 

265, 266, 273, 275, 276, 300, 309 

transgenic pollen 251, 267, 276, 277 

transgenic Populus 60, 74, 75, 207, 252-

254, 257, 264, 266, 

transient expression (transient gene 

expression) 60-63 

transit 

transit peptide 70-72, 182, 188, 309 

translation 70-72, 126, 132, 168, 170, 

302, 308, 309 

translation initiation 165, 168, 170, 

309 

translational enhancer 63 

translocate carbon 127, 256 

translocation 70-72, 124, 127, 207, 297 

transport 63, 77, 79-81, 91, 124-126, 128, 

132,138,206,208,209,298,301,305 

transposable element 36, 302, 309 

Ac (Activator) 53, 55 

transposon 94, 153, 309 

tree breeder 101 

tree breeding 1, 74, 78, 79, 101, 220, 221, 

230 

tree improvement 34, 51, 78, 220, 232, 

233 

triazole growth retardant 215 

trichloroethylene (TCE) 208 

triploid 19, 36, 252, 274, 309 

tRNA (transfer RNA) 144, 145, 168, 309 

trnH 147 

trophic level 249, 264 

tropism 77 

trypsin (see also: proteinase) 52, 131, 

173, 174, 293 

trypsin inhibitor (see also: proteinase 

inhibitor) 131, 173 

tumor 51, 54, 93, 95, 161-163, 175, 275, 

296, 304, 309 

tumor-inducing (Ti) plasmid (see: Tiplasmid) 

u 

ubiquitin (UBQ-1) 168, 182, 183, 309 

uidA 85, 86, 94, 208 

Ulmus (elm) 33, 34, 293 

uniconazole 215 

unit cost 243-247 

untranslated leader 182 

urease 131 

urediospore 152,309 

uredium (uredia) 309 

v 

vacuole 45,125,133,138,141,309 

vanillic acid 197 

vanillin 197, 202 

325 
variability 33, 34, 36, 37, 79-81, 93, 96, 

106, 110, 147, 150, 163, 221, 228-232, 241, 

252,253,266,267,274-276,296 

variegation 35, 95 

vascular cambium 15, 16, 74, 77, 79, 94, 

309 

vascular system 10, 193 

vascular tissue 94, 310 

vector 7, 52, 54, 56, 60, 63, 66, 67, 70, 74, 

91, 161, 163, 168, 169, 174-176, 188, 203, 

214, 257, 265, 296, 297, 310 

vegetative propagation 221, 297, 310 

vegetative storage protein (VSP) 124, 

125, 131, 135, 137, 138 

vertebrate 206, 251, 302 

vesicular arbuscular mycorrhiza (VA 

mycorrhiza) (endomycorrhiza) 208, 303 

viability 24, 27, 45, 135, 138 

vicilin type 132 

virulence (vir) 51 

vir gene 51 

vir inducer 56 

virus 37, 63, 70, 76, 85, 86, 91-93, 108, 

197, 198, 208, 216, 265, 266, 275, 276, 

294, 298, 303, 310 

virus X-resistant potato 265 

vitamin 19, 28 

Vitis vinifra 215, 293 

vitrification (vitrified) 18, 19, 21, 310 

VSP (see: vegetative storage protein) 

VSP promoter 137 

w 

walnut Uuglans) 125, 292 

wasp 266 

water status 80 

water stress 216 

watershed 251 

weed 86, 96, 207, 241, 252, 264, 266, 268, 

269 

weediness 166 

western blot (western blotting) 52, 71, 

72, 175,310 

wild flora 265 

WIN (wound inducible) 65 

WIN3 66,67 

WIN3.12 168 

WIN4 126 

WIN6 (chitinase) 66, 67 

WIN6.39b 168 

WINS (chitinase) 66 

wood chip 193, 202, 246 

wood density 79-81, 108 

wood propertiy (wood properties) 2, 

77, "79-81, 223, 224 

wood quality 86, 108, 110, 116, 220 

wood structure 74, 79-81 

Woody Plant Media (WPM) 8, 14, 15, 62 

wound (wounded) (wounding) 12, 27, 

51, 52, 56, 66, 67, 86, 95, 127, 156, 157, 

163,165,167,168,170,175,182,255,258 

wound-inducible (WIN) 2, 55, 65, 66, 

67, 86, 126, 168, 170, 175, 182, 255 

wound-inducible promoter 168, 170, 

255 

wounding response 65 

W51 (xylem ray storage protein) 66 

326 

X 

Xanthomonas populi (X. populi) 108, 157, 

294 

xenobiotic 257, 310 

Xenopus laevis 167 

xylem 74, 77-81,86, 87, 94, 95, 128, 193, 

197-202, 296, 309, 310 

z 

xylem ray 66, 125, 127 

xylem sap 125, 128 

Zea mays (maize) 53, 55, 66, 95, 97, 144-

146, 196-198, 200, 252 

zeatin 8, 14-16, 19, 29, 30, 47, 163, 298 

zeatin riboside ([9R]Z) 78 

zinc 174 

zinc finger 216 

Zinnia 80, 81, 195, 200, 293 

Zygote 183,300,308,310 

zygotic embryo 10, 11, 48, 60, 135, 

274,300,308 

zymogram 103 

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997 
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