United States

Department of

Agriculture

Forest Service

Southern Forest

Experiment Station

New Orleans,

Louisiana

Proceedings of the

Southern Regional Information

Exchange Group Biennial

Symposium on Forest Genetics

General Technical Report

SO-108

June 1994 Huntsville, Alabama

July 8-I 0,1992

“Applications of Vegetative Propagation in Forestry”

Proceedings of the

Southern Regional Information

Exchange Group Biennial

Symposium on Forest Genetics

Edited by

G. Sam Foster

and

Alex M. Diner

Sponsored by

Southern Regional Information Exchange Group

and

Southern Forest Experiment Station

Published by

Southern Forest Experiment Station

New Orleans, Louisiana

1994
PREFACE

This volume is the proceedings of the 1992

Southern Regional Information Exchange Group

(SRIEG) Biennial Symposium on Forest Genetics.

The meeting was held in Huntsville, Alabama, USA

on July 8-10, 1992 and was entitled, “Applications of

Vegetative Propagation in Forestry.” The nine

papers were divided into three sets under the

headings: “Tissue Culture,” “Rooted Cuttings,” and

“Propagule Growth, Development and Applications.”

The authors of each paper were given the task of

synthesizing the current state of knowledge for their

appointed topic. The meeting was co-hosted by

SRIEG, the USDA Forest Service Southern Forest

Experiment Station, and Alabama A&M University.

This proceedings may be referenced as a 1993

publication.

The Southern Regional Information Exchange

Group is sponsored by the University Experiment

Stations in the Southern Region.

The editors, G. Sam Foster and Alex M. Diner,

express their gratitude to Lyn Thornhill, who

compiled and formatted the proceedings into their

final publication form. We also thank the authors for

their major effort in writing the manuscripts and

giving the presentations at the Symposium.

Special thanks go to the moderators for the

symposium: Henry V. Amerson, Tissue Culture;

Hans van Buijtenen, Rooted Cuttings; and Barbara

McCutchan, Propagule Growth, Development and

Application, Their participation greatly enhanced

the symposium.

Members of the Steering Committee are

acknowledged for their tireless and superb help in

the local arrangements for the Symposium:

USDA Forest Service

G. Sam Foster

Jimmy L. Reaves

Alex M. Diner

Jane L. Ford-Logan

Chuck Grimes

Lyn Thornhill

G. Sam Foster

Program Chair

Southern Forest Experiment Station

Normal, AL

ii
Contents

Tissue Culture

TISSUE CULTURE MICROPROPAGATION OF CONIFERS

0 . J . SCHWAFU, S . E . SCHLARBAUM. AND J. A. BURNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

IN VITRO PROPAGATION OF HARDWOODS

S. A. MERKLE AND R. N. TRIGIANO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................. 23

REGENERATION OF THE GENETICALLY ENGINEERED CONIFER - THE IMPORTANCE OF THE

BIOLOGICAL SYSTEM

DAVID D. ELLIS, ALEX M. DINER AND YINGHUA HUANG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Rooted Cuttings

MACROPROPAGATION OF CONIFERS BY STEM CUTTINGS

FARRELL C. WISE AND THOMAS D. CALDWELL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

ROOTED CUTTING MACROPROPAGATION OF HARDWOODS

S. B. LAND, JR. AND MIKE CUNNINGHAM *............................................... .**..*.......................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

THE ROOTING ENVIRONMENT FOR CUTTINGS FROM FOREST TREES

JANE L. FORD-LOGAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...............................

97
Propagule Growth, Development and Application

FIELD PERFORMANCE COMPARISONS BETWEEN VEGETATIVE PROPAGULES AND SEEDLINGS OF

LOBLOLLY AND SLASH PINES

LEWIS J OHN F RAMPTON. JR. ,...................................,....,..,,............,,,,.,..........................,,..,.............,.................................115

APPLIED VEGETATIVE PROPAGATION PROGRAMS IN FORESTRY

CLEMENTS C. LAMBETH. GARY A. RITCHIE, AND BRIAN STANTON . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

RESEARCH APPLICATIONS WITH VEGETATIVE PROPAGULES

DARROLL D. SKILLING. KENNETH F. RAFFA. DANIEL J. R OBISON, AND PAUL BERRANG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

iv
Tissue Culture

Moderator:

Henry V. Amerson

School of Forestry

North Carolina State University

Raleigh, North Carolina

1

TISSUE CULTURE MICROPROPAGATION OF CONIFERS1

0. J. Schwarz, S. E. Schlarbaum, and J. A. Burns2

Abstract. The future of micropropagation technology is

bright with respect to its immediate utility as an important tool in

tree breeding programs and to its use as a mass production

methodology of germplasm produced by traditional and

biotechnological means. In general there are three broad

regenerative pathways that allow the in vitro vegetative

propagation of conifers: (1) regeneration through somatic

embryogenesis, (2) regeneration from adventitious meristems, and

(3) regeneration via proliferation or enhancement of bud break of

existing meristems. This paper outlines the current understanding

of the major in vitro propagation strategies based upon these three

regenerative pathways and discusses some of the key challenges

that remain to be addressed.

INTRODUCTION

Significant progress has been made in exploring

opportunities for the use of in vitro-propagated

conifers in forestry since the first report of plantlet

regeneration in Pinus palustris Mill via

organogenesis from mature zygotic embryos

(Sommer et al. 1975). Since this first report there

has been a proliferation of micropropagation

technologies and applications of those technologies

that offer considerable promise for use in the forests

lPaper presented at the 1992 Southern Regional

Information Exchange Group Biennial Symposium,

Huntsville, AL, July 8-10, 1992.

2Associate Professor, Botany, University of

Tennessee, Knoxville; Associate Professor,

Department of Forestry, Wildlife and Fisheries,

University of Tennessee, Knoxville; Ph.D.

Candidate, Department of Horticulture, University

of Georgia, Athens.

of the world. These technologies encompass an

array of techniques designed for the “true-to-type”

propagation of selected genotypes (Debergh and

Read 199 1).

Assessment of the progress and significance of the

application of micropropagation technologies to the

vegetative regeneration of forest trees has been

the subject of numerous publications (Boulay

1987a,b, Gupta 1988, Thorpe et al. 1991, von Arnold

1991, Zobel 1992). Zobel(1992) concluded that

“Currently, the most promising use for tissue culture

and biotechnology is to supply desired genotypes

developed by the tree breeder, in the proper

physiological stage, so that they can be reproduced

in mass using conventional rooted cutting

methodology.” Similarly, von Arnold (1991)

suggested that the contribution of in vitro vegetative

propagation to the improvement of forest trees

included the “exploitation” of existing “plus”

genotypes and the production of commercially

valuable new genotypes. Boulay (1987a) also

emphasized the importance of clonal forestry, but

discussed problems implementing these new

technologies on an operational scale. He suggested

that the slow incorporation of in vitro methodologies,

for vegetative propagation on a massive scale, was

3
the result of resistance from forest nursery

operations because of past orientation to seedling

production and lack of experience in the production

of rooted cuttings. The low cost of forest tree

seedling production, coupled with the lack of elite

clones and numerous technical difficulties was also

said to contribute to the slow acceptance of these

technologies. Despite known problems and those not

yet encountered, the future of micropropagation

technology appears to be bright with respect to its

immediate utility as an important tool in tree

improvement programs and to the promise for its

use as a mass production methodology of elite

germplasm.

The selection of a specific approach for in vitro

vegetative propagation of conifers is often based

upon objectives such as the development of

totipotent cellular regenerative systems designed as

a vehicle for gene insertion for the eventual

production of transgenic plants, the propagation of

valuable, improved germplasm to allow field testing

across broad environmental gradients, or the mass

production of proven (mature) genotypes for large

operational plantings (Zobel 1992). The attainment

of any of these objectives requires overcoming

numerous biological barriers established through

millennia of evolutionary trial and error. The basic

problem faced by researchers is to overcome and

successfully manipulate the innately programmed

developmental sequencing responsible for the

production of the normal phenotype.

It is generally agreed that there are three

different regenerative pathways (van Arnold 1991,

Gupta 1988) used for the in vitro vegetative

propagation of conifers. The pathways are (1)

regeneration through somatic embryogenesis, both

direct and indirect; (2) regeneration from

adventitious meristems; and (3) regeneration via the

proliferation or enhancement of axillary bud

development of existing meristems. All of these

propagation pathways are currently being

investigated worldwide by academic and industrial

research groups. The literature contains numerous

publications surveying various aspects of these

micropropagation technologies. Recent papers

include reviews by Boulay (1987131, Fowke and

Hakman (1988), Mehra-Palta and Thompson (19881,

Thorpe (1990) and Thorpe et al. (1991). This paper

presents an overview of the current understanding

of the in vitro propagation strategies used in the

three regenerative pathways listed above and

discusses some of the challenges that need to be

addressed in order to allow widespread use of

micropropagation technologies in forestry.

REGENERATION THROUGH SOMATIC

EMBRYOGENESIS

Somatic embryogenesis has been defined as a

“non-sexual developmental process which produces a

bipolar embryo from somatic tissue” (Tulecke 19871.

There is some confusion in the scientific literature as

to which developmental processes are encompassed

in each stage, e.g., maturation. To facilitate accurate

interchange of information among scientists, it

would be beneficial to develop a common definition

of terms for the substance and process of the

morphogenesis of fully “mature” somatic embryos

and for the developmental processes of germination

and early growth of the newly regenerated plantlet.

The generation of too many “new” terms is not a

concern, as long as they are precisely defined and

uniformly applied. The following describes a

sampling of the current nomenclature and some

areas of possible confusion.

Systems for tracking the ontogeny of somatic

embryo maturation and germination have been

described (Dunstan et al. 1988, Hakman and von

Arnold 1988, Webb et al. 1989). The morphogenic

process is usually divided into four stages, each

stage being defined by a more or less clearly

observable set of morphological characteristics. For

example, Tautorus et al. (1991) provides a clear

definition and illustration of each of the four stages

(stage 1 through 4bl for the maturation of Picea

marianasomatic embryos into plants.

Nomenclature used by Webb et al. (1989) was based

upon the classification system for the development of

zygotic embryos by Buchholz and Stiemert (1945).

This nomenclature describes, in four discrete stages,

the maturation of the somatic embryo but, contrary

to Tautorus et al. (19911, excludes “plantlet”

attainment.

The final two steps, maturation and germination,

are reasonably well demarcated through an increase

in morphological complexity to produce an

anatomically complete and physiologically mature

embryo that resembles its zygotic counterpart. The

maturation process should ideally end with the

production of an embryo that, given the appropriate

environmental cues, is capable of immediate

germination, a developmental state not unlike that

exhibited by the embryo in a fully mature

“quiescent” seed (Roberts et al. 1990a). These last

4
two steps in the production of plantlets have been

variously described by different authors in

attempting to detail the various developmental

hurdles presented by the sometimes recalcitrant

cultured embryos. Becwar et al. (1989) suggested

that the process of regeneration via somatic

embryogenesis be divided into three developmental

stages: “(1) ‘maturation’ representing the growth of

immature somatic embryos to the cotyledonary

stage, (2) ‘germination’ as evidenced by growth of

the radical, and (3) ‘conversion’ indicating the

survival and continued growth of in vitro-derived

plantlets in soil.” Gupta et al. (1991) define the term

“conversion” as the ” . ..process of turning somatic

embryos into plants growing autotrophically in soil.”

The term is further described to include the

germination of a “well formed embryo” on a

germination medium to facilitate the development of

true leaves followed by successful outplanting in

soil. It is also stated that to attain a reasonably high

level of conversion one must start with a fully

developed and mature embryo.

Although there is not total agreement about all of

the details of the ontogeny of somatic embryos

(Becwar et al. 1991, Gupta and Durzan 1987a), most

authors agree that somatic embryos are produced by

a recapitulation of development closely paralleling

the genesis of a normal zygotic embryo. Sharp et al.

(1980) describe two distinct routes to somatic embryo

formation, direct and indirect. The direct

developmental route is defined as the formation of a

new somatic embryo either from explant tissue

without an intervening callus or free-cell stage, or

from a previously formed somatic embryo. The

somatic embryo develops directly from the explant

tissue as a product of a single cell or of a small group

of totipotent vegetative cells. The production of

embryos by indirect somatic embryogenesis proceeds

via callus or cell suspension, or via a single cell or

group of cells derived from existing somatic embryos

(Tautorus et al. 1991). However, according to

Williams and Maheswaran (19861, “Once induction

of embryogenic determined cells has been achieved,

there appears to be no fundamental differences

between indirect and direct somatic embryogenesis.”

Production of embryos via direct or indirect

somatic embryogenesis can affect the genetic fidelity

of the resulting clonal regenerants (Berlyn et al.

1986). The use of a direct somatic embryogenic

pathway may present several advantages, as the

presence of a callus stage in a propagation strategy

has a greater probability of introducing genetic,

developmental, and physiological instability into the

cloned germplasm (Chaleff and Keil 1981, Larkin et

al. 19891. Berlyn et al. (1980) suggested that

“Genetic changes caused by culture conditions and

undetected in tissue culture-derived plants could

lead to large investments in forests of defective

trees.” This possibility emphasizes the need for

screening for genetic defects before the germplasm is

released for plantation deployment (Becwar et al.

1991, Berlyn et al. 1986). Somaclonal variations

were found in Picea abies globular somatic

proembryos and mature somatic embryos obtained

from immature embryos and cotyledons of young

seedlings (Lelu 1988). A positive note can be

sounded for the genetic stability of embryogenic

cultures of Picea glauca x engelmannii complex

(Eastman et al. 1991). After extensive isozyme

analysis and tracking of culture morphology, the

authors concluded that “...embryogenic cultures

initiated from interior spruce embryos show a high

degree of genetic stability in that the morphological

behavior and isozyme phenotype were always

consistent with that of the explant genotype.”

The successful establishment of an efficient

somatic embryogenic propagation system for forest

trees is predicted to have many advantages (Cheliak

1991, Durzan and Gupta 1988, Gupta et al. 1991,

Thorpe et al. 1991) for all aspects of forestry.

According to Thorpe et al. (19911, the difficulty and

time required to induce rooting of shoots produced

through the various organogenic-based

methodologies is eliminated, because a mature

somatic embryo comes with complete bipolar

development of apical and root meristems. Somatic

embryogenesis offers an efficient method for the

production of large numbers of “emblings” (Libby

1986) through the development of automated, largebatch, cell suspension cultures (Durzan and Durzan

1991). In addition, there is the opportunity for the

application of genetic engineering technologies to

embryogenic protoplasts derived from embryogenic

cells and tissues (Gupta et al. 1988, Wilson et al.

1989) in order to produce transgenic plants.

Induction

The “process” of in vitro plantlet regeneration via

somatic embryogenesis has been described as the (1)

induction and/or initiation of embryogenic callus on

the explant, (2) proliferation of the resulting

embryogenic tissue, (3) maturation of the somatic

embryos, and (4) germination of the fully mature

somatic embryos (von Arnold 1991). The first step in

the regeneration process, the induction of the

embryogenic state in the explant, requires the

5
redirecting or resetting of the developmental

program controlling the morphogenic fate of the

targeted cells. It is unknown to what extent this

involves a relatively minor developmental

redirection, which may be the case for the

embryogenic callus protruding from the micropylar

end of a female gametophyte (Becwar et al. 1990,

Finer et al. 1989), or a major resetting and

redirecting of the morphological endpoint of the

explant, as might be the case for the induction of

embryogenic callus from developing young seedling

tissue (Attree et al. 1990a, MO and von Arnold 1991).

Even in the same species, newly induced

embryogenic tissue/callus can vary slightly in

cellular morphology with respect to the presence or

absence of well-formed somatic embryos (Becwar et

al. 1990, Gupta and Durzan 1987a,b). In general,

however, the standard description for conifer

embryogenic tissue is a glossy, translucent, white,

mucilaginous cellular mass. This tissue contains a

variable mix of elongated cells, aggregates or clumps

of densely cytoplasmic cells, embryo initials, and

sometimes later stage embryos (Finer et al. 1989,

Gupta and Durzan 1986, Gupta and Durzan 1987a,

Hakman et al. 1985, Laine and David 1990, Lu and

Thorpe 1987).

The initiation of the embryogenic state in explant

tissue is reasonably complex, as it involves an

interplay of variables that include, but are not

restricted to, explant age and physiological

condition, incubation medium, and environment.

The choice of a specific combination of these

variables is increasingly based upon a review of the

literature of related systems and a growing

understanding of the physiology and biochemistry of

the embryogenic process. Comparative studies of

media composition, genotype, and culture conditions

have all contributed to an understanding of the

importance that each plays in the initiation process

(Attree et al. 1990a, Becwar et al. 1990, Hakman

and von Arnold 1985, Jain et al. 1989, Kvaalen and

von Arnold 1991, Roberts et al. 1989, Tautorus et al.

1990, Tremblay 1990, Webb et al. 1989). Continued

research is being focused on discovering and then

optimizing the factors that promote the induction of

somatic embryogenesis. These include the selection

of the optimum developmental stage of the explant

donor, the selection of the culture medium plus

hormone additives, and the provision of appropriate

environmental conditions for culture development

(Tulecke 1987, Webb et al. 1989). Tautorus et al.

(1991) have recently published an excellent review

of the “most recent directions in research of conifer

embryogenesis.” These authors present a tabulation

of conifer species that have produced somatic

embryos. Six genera of conifers are represented

Abies, Lark, Picea, Pinus, Pseudotsuga, and

Sequoia. Twenty species and species complexes are

included, with the genera Picea and Pinus each

having six species represented.

The explant tissues used to initiate somatic

embryogenesis encompass a range of developmental

states, including reproductive generative structures

(megagametophytes containing the developing

zygotic embryo) (Becwar et al. 1990, Finer et al.

1989, Nagmani and Bonga 19851, immature and

mature zygotic embryos (Jain et al. 1989, Wann et

al. 1989, Webb et al. 19891, hypocotyl and

cotyledonary tissue from seedlings (Becwar et al.

1988, Krogstrup 1986, Lelu et aL 19871, and recycled

cotyledonary somatic embryos (Eastman et al. 1991,

MO et al. 1989). The majority of investigators use

immature to mature zygotic embryos as the explant

to study induction of embryogenic callus in conifers

(Durzan and Gupta 1988, Tautorus et al. 1991).

Webb et al. (1989) studied the relationship between

the efficiency of embryogenic callus induction and

the production of shoot-forming callus (i.e.,

caulogenesis) in Picea glauca and P. engelmanii as

related to the degree of zygotic embryo maturation.

They found that the stage of active cotyledonary

development was most competent for embryogenic

callus induction, in agreement with several previous

reports for spruce zygotic embryos (Becwar et al.

1988, Hakman and Fowke 1987, Lu and Thorpe

1987). Interestingly, they also reported that

competence for caulogenesis seemed to be inversely

related to the degree of maturation of the spruce

zygotic embryos. Becwar et al. (19881 compared

several species of Picea and Pinus with respect to

the optimal developmental stage for initiation of

embryogenic tissue. They concluded that precotyledonary embryos were optimal for Pinus species

and that initiation in Picea was most efficacious

when post-cotyledonary embryos were targeted for

explant use. In addition, the origin of the

embryogenic tissue differed between the two genera.

In the three Pinus species, P. serotina, P. strobus,

and P. taeda, embryogenic tissue seemed to

originate from the suspensor region of the

precotyledonary embryo. In contrast, the hypocotyl

and cotyledons of more mature post-cotyledonary

zygotic embryos of Picea produced the embryogenic

tissue.

As knowledge of the basic culture requirements

for induction of somatic embryogenesis from

immature tissues has increased, parallel successes

6
have been achieved in chronologically older and,

consequently, morphologically more advanced

explant tissues. Mature zygotic embryos removed

from seed that had been in long term cold storage

have produced embryogenic callus (Tautorus et al.

1990, Tremblay 1990). Tremblay (1990) succeeded

in regenerating plantlets from embryogenic callus of

Picea glauca produced from mature zygotic embryos

that had been stored for 3 to 11 years.

The induction of embryogenic callus was reported

for young seedling material of Picea (Attree et al.

1990a, Rrogstrup 1986, MO and von Arnold 1991).

Embryogenic callus was induced on 12-day-old

seedling tissues of Picea mariana grown from seed

that had been stored for 10 years (Attree et al.

1990a). These calli were established in liquid

suspension culture and subsequently transferred to

a maturation medium on rigid filter paper supports.

As embryos matured, they were separated from the

main callus mass in order to facilitate continued

development. One hundred and eleven plants were

recovered, transferred to pots, and moved to a

growth chamber.

Proliferation

Embryogenic callus, or perhaps more correctly

embryogenic tissue (sensu Tautorus et al. 1991), is

used to accomplish the second stage of in vitro

plantlet regeneration, “proliferation.” At this stage,

alterations can be made in protocols to foster

different proliferation methods. Several options are

available for multiplication of embryogenic tissue.

Multiplication of the existing tissue can be achieved

in its initial morphological state. Depending upon

the species and genotype, embryogenic tissues can

continue to be grown on the induction medium

(Becwar et al. 1990, Finer et al. 1989, Jain et al.

1989, MO et al. 1989, von Arnold and Woodward

1988, Webb et al. 1989) or they may require a

medium reformulation (Becwar et al. 1990, Laine

and David 1990). Proliferation of the embryogenic

tissue can also take place in liquid suspension

cultures. The advantages of this approach to

somatic embryo production have been amply

discussed (Becwar et al. 1988, Dunstan 1988,

Durzan and Gupta 1988, Gupta et al. 1991). Liquid

suspension culture of embryogenic tissue provides

an opportunity for enhanced growth rates over

growth on solid medium, providing an avenue for

the economical production of trees for reforestation

(Lulsdorf et al. 1992). Culture variables that

influence growth rate and overall culture

productivity in liquid systems are now being

elucidated. Rrogstrup (1990) described the

relationship between suspension culture density and

proliferation rate of stage 1 somatic embryos of

Picea sitchensis initiated from mature zygotic

embryos. Dunstan (1988) maintained embryo

cultures of Picea glauca for extended periods of time

through subculture in liquid medium. These

cultures yielded 150 to 300 stage 1 embryos per

milliliter of culture medium, with a 3-to &fold

multiplication every 7 days. Although the embryo

productivity levels demonstrated were still not at

levels required to fill even a small part of the yearly

seedling requirement for reforestation of any

commercial species, this methodology demonstrates

the potential for the development of large-scale

bioreactor production of somatic embryos. Tautorus

et al. (1991) lists eight species of conifers that have

been established from embryogenic tissue in shakeflask suspension cultures (Abies nordmanniana,

Picea abies, Picea glauca, Picea mariana, Pinus

caribaea, Pinus strobus, Pinus taeda, and

Pseudotsuga menziesii).

Suspension cultures of embryogenic cells have

been used as a source of protoplasts that have been

regenerated into somatic embryos. Regeneration of

somatic embryos from protoplasts was first reported

for Pinus taeda (Gupta and Durzan 1987b) and

Picea glauca (Attree et al. 1987) and later for

Pseudotsuga menziesii (Gupta et al. 1988), Picea

mariana (Tautorus et al. 19901, Pinus caribaea

(Laine and David 1990), and haploid Larix decidua

(von Aderkas 1992). Plantlet regeneration from

protoplast cultures has been achieved in Picea

glauca (Attree et al. 1989) and Larix x eurolepis

(Klimaszewska 1989). The regeneration of somatic

embryos and then plantlets from protoplasts may

provide the necessary vehicle for the application of

genetic engineering to conifers (Tautorus et al.

1991).

Maturation

The process of somatic embryo maturation,

broadly defined as the production of embryos

physiologically and morphologically capable of

germination and early development, is currently

poorly understood. The efficiency of the production

of embryos to this “mature” developmental state has

been relatively low (Dunstan 1988, Gupta and

Durzan 1987a, Hackman and von Arnold 1988, von

Arnold 1987) and varies with plant genotype

(Jalonen and von Arnold 1991, Webster et al. 1990).

Considerable improvement in the efficiency of

bringing somatic embryos to the mature state has

7
resulted through the use of abscisic acid (ABA)

(Becwar et al, 1989, Dunstan et al. 1988, Dunstan et

al. 1991, Durzan and Gupta 1987, Gupta and

Durzan 198713, Roberts et al. 1990a, von Arnold and

Hakman 1988), partial desiccation at high relative

humidity (Roberts 1991, Roberts et al. 1990b), and

manipulation of the gaseous environment of the

culture vessel (Kvaalen and von Arnold 1991).

Somatic embryos are usually proliferated on

medium containing various levels of growth

regulators, after which they are transferred to

medium with a reduced level of hormone and

containing ABA (Attree et al. 1990b, Atree et al.

1992, Dunstan et al. 1988, Roberts et al. 1990a,b). In

general, the combination of the reduced growth

regulator levels, a brief exposure to ABA, and partial

drying at high relative humidity stimulates

development of fully mature embryos. The

importance of using the appropriate level of ABA in

the medium during the maturation process was

emphasized by Roberts et al. (1990a). The

maturation pattern of ontogenetic development

followed by somatic embryos of Picea glauca x

engelmannii hybrids was dependent upon the media

concentration of ABA. Without ABA, little or no

development occurred, and a range of embryo

morphologies were produced as the ABA

concentration of the medium was increased. Low

levels of ABA (1 to 10 PM) produced “shooty

embryos;” intermediate levels (10 to 20 PM)

produced bipolar embryos that often precociously

germinated, whereas 30 to 40 FM of ABA produced

opaque cotyledonary embryos that entered a

quiescent stage of arrested development. When

indolebutyric acid (IBA) was added to the ABAcontaining medium, the number of mature embryos

was significantly increased. Incorporation of ABA

and IBA into the medium resulted in a

developmental synchrony in the somatic embryos

similar to that found in the maturation and

germination of naturally produced zygotic embryos

(Roberts 1991). Somatic embryos generated through

this protocol began to accumulate proteins (Roberts

1991) and lipids (von Arnold and Hakman 1988) in a

manner similar to that of maturing conifer zygotic

embryos (Feirer et al. 1989).

Germination

The final step in plantlet production,

“germination,” followed by successful early growth

(i.e., conversion [Becwar et al. 19891), has

operationally been somewhat better defined and

separated from maturation through the application

of ABA and the partial drying step mentioned above.

Somatic embryo germination has been variously

described as “root and hypocotyl elongation”

occurring in a synchronized manner (Roberts et al

1990b), as ‘I... the transition from mature somatic

embryos to green plantlets and involves clearly

visible root as well as cotyledon and hypocotyl

elongation” (Attree et al. 1990b), or simply as

“radical growth” (Becwar et al. 1989). The methods

used to encourage germination of somatic embryos

usually involved transfer of mature embryos to a

germination medium lacking growth regulators

(Attree et al. 1990b, MO and von Arnold 1991,

Roberts et al. 1990b) with or without a

pregermination desiccation treatment (Roberts et al.

1990b). Becwar et al. (1989) found that the

placement of mature somatic embryos on the

support medium was critical to the efficiency of

germination. The highest percent germination, 56%

for Picea abies, resulted when the cotyledons and

apical meristem were inserted in agar with the

hypocotyl and radical end projecting downward into

the air inside an inverted vessel. The mature

embryos were sometimes singulated (i.e., removed

from the whole calli) and cultured in individual

vessels to enhance germination efficiency (Attree et

al. 1990b, Becwar et al. 1989, Roberts et al. 1990b).

Few studies describe the moderate- to large-scale

soil establishment and subsequent long-term field

performance of emblings. Becwar et al. (1989)

reported a-year survival of six phenotypically

normal Picea abies emblings. Webster et al. (1990)

presented a detailed study of the propagation and

regeneration of 71 genotypes of embryogenic

cultures of Picea glauca x englemanii obtained from

immature embryos. They reported an 80% or greater

survival rate of emblings for most genotypes after

one season’s growth under nursery conditions. Their

results suggested that ”. ..somatic embryogenesis can

be used for production of planting stock for a range

of interior spruce genotypes.”

REGENERATION FROM ADVENTITIOUS

MERISTEMS

Clonal propagation via adventitious meristems

involves the induction of unipolar shoots (Thorpe et

al. 1991) on explants followed by shoot excision and

induction of root meristems. Because of the

similarity in methodologies used to initiate

adventive root production on microshoots produced

via both adventitious meristems and axillary buds

and their subsequent acclimation to the ambient

8
environment, a combined discussion addressing

rooting and plantlet acclimation is presented in this

paper following the section on Regeneration From

Axillary Buds.” In conifers, the process of adventive

meristem production can follow two major

developmental sequences: direct or indirect

organogenesis (Hicks 1980).

Indirect organogenesis occurs when there is an

initial production of callus tissue from the primary

explant, followed by the appearance of meristemoids

(Torrey 1966). It is believed that these meristemoids

are, at first, not determined (i.e., capable of

producing either shoot or root primordial. Progress

toward plantlet regeneration in conifers via this

methodology has been limited. However, initiation of

shoot me&terns via indirect organogenesis from

callus has been reported (Ball 1950, Gladfelter and

Phillips 1987, Kaul and Kochhar 1985, Konar and

Singh 1980, Simola and Honkanen 1983, Winton

and Verhagen 1977). A low frequency of plantlet

regeneration has been achieved in Larix x eurolepis

(Laliberte and Lalonde 1988) and P. eldarica

(Gladfelter and Phillips 1987) from buds initiated

from long-term callus cultures.

The process of direct organogenesis is defined as

organ formation directly from the primary explant

in the absence of an intervening callus stage (Hicks

1980). The predominant morphogenic route

reported for adventitious shoot formation in conifers

has been through direct organogenesis without the

involvement of an intermediate callus stage (Thorpe

and Biondi 1984). In a comparative study of six

species from two genera of conifers, Thorpe and

Pate1 (1986) described organogenesis as the

formation of meristematic centers or meristemoids

which developed into bud primordia and “...finally

adventitious shoots with apical domes and needle

primordia.”

As noted in the introduction, the first complete

plantlet regeneration of a conifer (Pinus palustris)

was accomplished by Sommer and Brown (1974) and

Sommer et al. (1975). The buds were produced on

the cotyledons of mature zygotic embryos,

presumably via a direct organogenic route. The

step-wise procedure used in their studies is typical of

methods used to induce direct organogenesis in

conifers. The procedure involved transfer to several

different media in order to induce, first, apical bud

initiation, followed by induction of root primordia,

and finally, continued plantlet development. Mature

embryos were excised from seed and placed on a

basal medium containing both an auxin and

cytokinin. During the next four to six weeks, areas

on the zygotic embryos’ cotyledons differentiated

shoot buds. These areas were subsequently excised

and transferred to a second medium for further

development into plantlets. The authors concluded

that, in order to facilitate plantlet regeneration,

differentiated shoot apices should be transferred to a

medium capable of inducing root formation. A final

transfer to yet another medium was required to

facilitate root elongation and further plantlet

development.

Shoot apices, needles, hypocotyls, epicotyls,

cotyledons, zygotic embryos, dormant buds, needle

fascicles, and lateral buds have been used as

explants for the induction of adventitious bud

meristems (John 1983, Thorpe and Biondi 1984).

Ideally, explant material should come from

genetically superior trees, proven by or selected from

progeny tests. However, there is a general

recalcitrance of explants derived from older, more

mature trees to form adventitious buds and roots

(Bonga 1987, Durzan 1984, Hackett 1987). The

majority of successful reports of plantlet

regeneration in conifers have resulted through the

use of juvenile explant material.

The procedure typically followed to accomplish

plantlet regeneration by direct organogenesis can be

divided into four steps: (1) initiation of shoot

meristems, (2) development and elongation of shoot

buds, (3) root meristem initiation and development,

and (4) plantlet acclimation to the ambient

environment (Thorpe and Biondi 1984, Thorpe et al.

1986, Thorpe et al. 1991). The details of each of the

procedural steps required to accomplish plantlet

regeneration vary somewhat depending upon the

species of conifer and the type and developmental

stage of the explant. What follows is a brief

summary of the process from shoot initiation to the

production of a shoot ready for rooting. A discussion

of the rooting and acclimation procedures required

to produce plantlets is presented after the section on

“Regeneration From Axillary Buds.”

Shoot initiation

Adventive shoot meristems can be initiated by the

application of a cytokinin (benzylaminopurine [BAI,

isopentenyl adenine, kinetin, etc.) (Webb et al. 1988)

alone, or in combination with an auxin and/or other

plant growth regulators (Chang et al. 1991, Sen et

al. 1989) over a range of concentrations, exposure

times, and delivery methods (Bonga and Durzan

1987, Thorpe and Biondi 1984). Extensive

9
experience with a range of coniferous species has

indicated that maximum bud yields are obtained

through a specific combination of explant and

cytokinin-containing medium (Bornman 1983,

Thorpe and Pate1 1986, von Arnold and Eriksson

1979, von Arnold and Hawes 1989). Typically, a

single application of BA at about 25 PM (Thorpe et

al. 1991) will produce the desired effect. Abscisic

acid (ABA) has been found to enhance shoot

formation on loblolly pine cotyledonary explants via

the organogenic process (Sen et al. 1989). When

ABA was added to an induction medium containing

both BA and naphthaleneacetic acid (NAA), both the

shoot number and average fresh weight of loblolly

pine microshoots increased significantly.

Explant cells responding to the hormone

treatment undergo transformation into meristematic

centers or meristemoids that ultimately produce

buds (see discussion above). Although there are

some exceptions, most plantlet regeneration systems

rely on a timed exposure to the hormone-containing

medium followed by explant transfer to a hormonefree medium designed to promote further

development of the newly induced buds (Bronson

and Dixon 1991, Jang and Tainter 1991).

A notable exception to this approach is a method

described by Aitken-Christie et al. (1988) that relies

on BA for initial induction of meristematic tissue

followed by continued long-term exposure to the

growth regulator. Mature zygotic embryos were

cultured on medium containing BA, with

subculturing every 21 days for 12 weeks. During

this time, the embryos produced “pieces” of

meristematic tissue that were easily dislodged from

the explant. Subsequently, all tissues were

subcultured every 4 weeks to fresh medium

containing BA. The “pieces” of tissue were

meristematic nodules capable of being maintained

and multiplied for extended periods of time. A

single embryo of Pinus radiata was reported to have

produced 5480 pieces of nodular tissue after 13.5

months in culture. Each piece of nodular tissue had

the potential of producing an average of 68

elongated shoots. The authors estimated that

“...260,000 trees could be produced from a single,

good-reacting seed in 2.5 years.” It is clear from the

description of this regeneration system that there is

a need for its application to the propagation of other

coniferous species where limitations exist in the

numbers of clonal propagules producible per

genotype.

Shoot Elongation and Development

The development and elongation of buds,

resulting from the production of cell division centers

or meristemoids, is usually accomplished by

transferring the hormonally treated explant to a

medium without hormones. Activated charcoal may

or may not be added to the hormone-free medium as

required to enhance elongation (Thorpe and Biondi

1984, Thorpe et al. 1991). The time required to

obtain shoots of sufficient size (i.e., usually >5 mm in

length) for excision and rooting was highly variable.

The required development and elongation may be

achieved from a single transfer to basal medium for

6 to 10 weeks as with buds induced on cotyledonary

explants of Pinus oocarpa (Franc0 and Schwarz

1985) and loblolly pine (P. taeda) (Amerson et al.

1988, Mott and Amerson 1981). Sequential transfers

to fresh basal medium, however, may be required to

produce rootable shoots as in Picea glauca and P.

mariana (Rumary and Thorpe 1984). In Pinus

oocarpa, the yield of rootable shoots can be increased

by subculturing the remaining explant tissue, after

removal of the larger shoots, in the same basal

medium to allow elongation of the remaining smaller

shoots and development of new adventitious buds

(Franc0 and Schwarz 1985). In general, larger

shoots tend to root at a higher percentage than

smaller shoots produced on the same explant.

Shoot yield can be significantly increased by

“hedging” or removal of the top portion of the shoot

and reculturing both the severed top and base

(Aitken-Christie and Thorpe 1984). In response to

the removal of the apical meristem, axillary bud

break is stimulated in the basal portion of the shoot

(see discussion in the following section). This

technique was used by Aitken-Christie and Thorpe

(1984) in combination with an adventive

organogenic procedure in Pinus radiata to produce

large numbers of rootable axillary shoots in 3-month

cycles and resulted in at least a four-fold

multiplication for every cycle.

REGENERATION FROM AXILLARY BUDS

Micropropagation utilizing axillary meristems

differs from adventive shoot production only in the

ontogenesis of shoot production. After axillary bud

stimulation and elongation, rooting and acclimation

procedures are essentially the same as those used

with shoots of adventive organogenic origin.

10
Stimulation of axillary and fascicular meristem

development is accomplished by hormonal treatment

of the explant tissue. Explants have been obtained

from juvenile to mature conifers, and regeneration

has been achieved in several Pinus species (Table 1).

Table 1. Selected list of publications highlighting in

vitro microshoot production from axillary and

fascicular bud meristems.

Pinus Species

Explant

Material AuthorsNear

Pinus brutia Ten. Z-month-old Abdullah

Pinu.s eldarica

Medw.

seedling tips

6-month-old

seedlings

seedling shoot

tips

seedlings

et al. (1984)

Abdullah

et al. (1986)

Gladfelter and

Phillips (1987)

Wagley et al.

(1987)

Pinus elliottii 30- to 40-day old Burns et al.

Engelm. seedlings (1991)

Pinus nigra

Arnold

3-week-old seed- Jelaska et al.

ling shoot tips, l- (1982)

and a-year-old

resting vegetative buds

20-day-old

seedlings

KolevskaPletikapic et al.

(1983)

Pinus oocarpa’

Schiede

distal portion Baxter et al.

(1Omm) of (1989)

hypocotyl with

cotyledons attached

(newly germinated

seedling)

Pinus pinaster

Aiton

seedling stem

apices

Pinus mdiata

D. Don

seedling shoot

tips

adventitious

clonal shoots

David and David

(1977)

David et al. (1978)

Horgan and

Aitken (1981)

Aitken-Christie

and Thorpe (1984)

1 1

Pinus strobus

L.

in vitro Aitken-Christie

hedges and Jones (1987)

l- to a-week old Kaul(1987)

seedlings

Pinus sylr~estri.3 a-week-old Bornman and

L. seedling plumules Jansson (1980)

seedling apical Skripachenko

segments (1982)

seedling apices Zel et al. (1988)

2- to 17-week-old Toribio and

seedling apices Pardos (1989)

Pinus taeda 2- to 3-year-old Stomp (1985)

L. plants

11-year-old Amerson et al.

hedges ( 1988)

‘also Pinus caribaea var. hondurensis Morlet and Pinus

tecunumanii Equihu and Perry (Pinus pat&a Schiede

and Deppe ssp. tecunumanii [Equiluz and Perry])

Axillary shoots produced in Pinus species

generally arise from preexisting quiescent

meristems. These meristems are located adaxial to

juvenile needles, in the axils of cotyledons, in the

normally dormant short-shoot surrounded by needle

primordia that make up a fascicular budlet, and

primarily, in older plant material, adaxial to scale

leaves (cataphylls) (David 1982, Lanner 1978, Stomp

1985, Toribio and Pardos 1989). The procedure

required to regenerate plantlets utilizing these

axillary and fascicular meristems parallels that used

in adventive shoot production. The basic steps are

(1) stimulation of me&tern development, (2)

elongation of the developing shoots, (3) induction of

adventitious roots, and (4) acclimation of the

regenerated plantlet to the ambient environment.

Because of the size and vigor of the microshoots

produced by axillary meristems, rooting can often be

accomplished under ambient environmental

conditions directly in commercial soil rooting

mixtures. Where this is accomplished, the third and

fourth steps can be combined, resulting in a savings

of in vitro culture time, growth chamber space, and

labor.3

3Schwarz, 0. J. Unpublished
There are several advantages to shoot

proliferation via axillary meristems over the de nouo

organogenic route. Clonal plantlets derived from

axillary shoots have been reported to be genetically

stable, showing no higher frequency of spontaneous

mutations than plants derived through seed

(Abdullah et al. 1986, Hussey 1986). In addition, the

time required for the production of axillary shoots is

shorter than that needed for clonal shoots of

adventitious origin (Abdullah et al. 1984, Burns et

al. 1991, Kolevska-Pletikapic et al. 1983, Zel et al.

1988).

Stimulation of Bud Meristem Development

Stimulation of axillary bud development has been

approached in several ways, depending upon the

developmental stage of the explant donor. Amerson

et al. (1988) successfully produced multiple

microshoots from 11-year-old loblolly pines that were

severely hedged prior to explant removal. The donor

trees were hedged to stimulate the production of

shoots possessing “juvenile” morphology with

elongating primary needles. Other investigators

have used juvenile explant materials, starting with

seedlings of various ages up to 3-year-old plants

(Table 1).

Explants are disinfested and treated with a

cytokinin to stimulate the development of axillary

meristems present in the explant tissues. The

method of exposure to the cytokinin varies.

Generally, explants are obtained from both juvenile

and mature sources and incubated on a cytokinincontaining medium for varying periods of time

(Abdullah et al. 1986, Horgan 1987, Skripachenko

1982, Toribio and Pardos 1989, Zel et al. 1988).

They are subsequently transferred to a hormone-free

basal medium to allow the axillary buds to elongate

sufficiently for rooting.

Several variations of a pulse exposure method

have been described. Zel et al. (1988) obtained

maximum axillary bud activation in explants from 9-

week-old P. syluestris L. seedlings after a &hour

soak in an aqueous BA-containing solution.

Amerson et al. (1988) pulse-treated explants from

11-year-old clonal loblolly pines for 1 to 2 minutes by

immersion in a 70% ethanol solution containing BA.

A slight modification of this method was successfully

applied to hypocotylary explants of &week-old slash

pine seedlings (Burns et al. 1991). The slash pine

seedling explants were pulse-treated in an aqueous

70% ethanol:O. 1% dimethyl sulfoxide (DMSO)

solution containing BA for 45 seconds. This

shortened exposure time was sufficient to produce

an average of three to seven harvestable axillary

shoots per genotype, depending upon seed source.

The concentration of BA used to treat the various

types of explants varied with maturation state and

method of hormone exposure. In general, the more

juvenile the explant, the lower the cytokinin

concentration required to stimulate axillary bud

development.

An in vitro methodology for the spontaneous

production of axillary shoots without exogenous

hormone application has been described for several

Pinus species by Baxter et al. (1989). Axillary shoot

production depends solely upon the repeated topping

or hedging of elongating seedling shoots and the

repeated harvesting and elongation of the resulting

axillary shoots. A high level of interclonal variation

was found with respect to axillary shoot production.

The interclonal variability with respect to axillary

shoot production also varied between species under

constant cultural conditions. Greater than 1000

explants were produced in 1 year by 11.8% of the

Pinus oocarpa clones compared to Pinus caribaea at

2.5%, and Pinus tecunumanii at 0%.

Elongation of Axillary Shoots

After activation of the quiescent meristems by the

cytokinin treatment, explants were usually

transferred to basal medium with a decreased

cytokinin concentration (Abdullah et al. 1987) or to

basal medium without hormones to promote shoot

growth (Abdullah et al. 1986, Burns et al. 1991,

David 1982, Horgan 1987). This shoot elongation

phase may require only a single transfer to achieve

an appropriate shoot size for subsequent rooting

(Amerson et al. 1988, Burns et al. 1991). The

methodology used to promote axillary shoot

elongation is essentially the same as that described

for shoots of adventive origin (see discussion above).

ADVENTITIOUS ROOTING AND ACCLIMATION

Adventitious Rooting

To complete plantlet regeneration, elongated

shoots obtained from either adventive or axillary

meristems were placed under various regimes

designed to induce adventitious root meristem

formation. Mohammed and Vidaver (1988) reviewed

the literature concerning the procedures for rooting

and acclimation of micropropagated conifers. These

authors provided an extensive tabulation of rooting

12
treatments that have been used with conifer tissue

cultures. Adventitious rooting was found to be

influenced by the method of application and

concentration of the rooting hormone, physiological

state of the shoot (“shoot quality”), age of the

original explant donor, donor genotype, and

temperature. In addition, the morphogenic origin of

the microshoot, i.e., shoots of adventive organogenic

origin as opposed to pre-existing axillary buds, also

affects rooting success4 and the subsequent field

performance of the plantlets (Abdullah et al. 1989).

A review of the literature describing rooting

protocols indicates that a general trend has

developed for in vitro adventitious root induction.

The process involves the exposure of the basal

portion of the microshoot to the inducing hormone.

Root-inducing protocols include a relatively short

duration pulse (Aitken-Christie and Thorpe 1984,

Bornman 1983, Chesick et al. 1991, Pate1 and

Thorpe 19861, an extended hormonal exposure

(Bronson and Dixon 1991, Chesick et al. 1991,

Coleman and Thorpe 1977, Mohammed et al. 19891,

a dip in rooting powder (Pate1 and Thorpe 19861, or

simply maintaining the shoots on basal media

without hormones to allow spontaneous rooting

(Burns et al. 1991, David 1982, Horgan and Aitken

1981, Webb et al. 1988). The efficiency of

adventitious rooting is highly variable and remains

one of the key problems encountered in conifer

plantlet regeneration in vitro (Burkhart and Meyer

1991, Haissig et al. 1992, Mohammed and Vidaver

1988). It has been found that rooting occurs when

tissues are exposed for a specified time to an auxin

(in the form of indole-3-acetic acid [IAAI,

naphthalene acetic acid [NAA], indole-3-butyric acid

[IBA]), alone or in combination with a cytokinin

(e.g., BA). The microshoot is then transferred to a

hormone-free basal medium to promote the further

development and elongation of newly initiated root

meristems. Usually the concentration of mineral

nutrients in the basal medium is reduced to one-half

to one-fourth that used for shoot initiation. The

soluble sugar (sucrose, maltose, etc.) level varies but

is usually set at 1 to 2%. Photoperiods from 12 to 24

hours and both reduced and elevated temperatures,

when compared with those used for shoot induction

and elongation, have been used in rooting protocols.

The parameters of rooting protocols are highly

dependent on an array of culture variables and

environmental conditions that must be adjusted to

the specific genotypes to be rooted. The presence or

4Schwarz, 0. J., unpublished.

absence of charcoal in the agar rooting medium

(Mohammed and Vidaver 19881, the use of

gibberellin synthesis inhibitors in combination with

auxin (Burkhart and Meyer 1991), the substitution

of peat-perlite for agar as the support medium, and

the addition of boron to the induction and elongation

medium (Mohammed et al. 1989) are all examples of

culture variables shown to affect upon rooting

efficiency. For a detailed discussion of these and

other related culture variables, see Mohammed and

Vidaver (1988) and Thorpe et al. (19911.

Plantlet Acclimation

Problems associated with the acclimation of

conifer plantlets to ambient environmental

conditions can be minimized if rooting is carried out

under ex uitro conditions directly to soil in the

greenhouse. Under these circumstances, both

rooting and acclimation are accomplished in a single

step. Experience with several conifer species (e.g.,

Cupressus lusitanica, Pinus oocarpa, Pinus elliottii)

has encouraged further development of this

combined approach.5 Of greatest concern when

transferring in vitro-generated plantlets to the

greenhouse and eventually to field conditions is

excessive water loss. This is usually related to the

absence or greatly reduced levels of epicuticular wax

on the leaf (i.e., needle) surfaces (Sutter 1982).

Early methods of transitioning usually involved

placing the regenerant in a pot of soil covered with a

container or beaker located in the laboratory to

facilitate its close observation. The plantlet was

carefully watered to maintain a condition of high

humidity without saturating the soil. After an initial

period, usually ranging from 10 to 14 days, the

container/beaker was gradually raised to reduce

humidity. At the end of an additional 14 days, the

container/beaker was completely removed, and the

plantlet was ready for transfer to the greenhouse.

This level of attention is not possible when large

numbers of plantlets are generated. The availability

of humidity control systems for greenhouses has

enabled acclimation of in vitro-rooted plantlets and

the single-step, direct-to-soil rooting of microshoots.

By no means has the problem of low rooting

efficiency and plantlet mortality during acclimation

been solved; however, greenhouse environmental

control technology has provided an added tool with

which to help solve the problem.

%chwarz, O.J., unpublished.

13
PERSPECTIVES AND CHALLENGES

Many challenges and limitations need to be

overcome before the mass propagation of forest trees

via in vitro methods can become routine practice. All

of the micropropagation technologies reviewed in

this paper are still in their infancy, in particular,

with reference to cloning efficiency and, more

importantly, with respect to understanding of the

primary biological mechanisms controlling the

processes of differentiation and growth. The

disciplines of genetics, plant physiology,

biochemistry, and molecular biology will be required

to gather basic knowledge on nutrition, hormonal

action, and the control of the expression of

totipotency in cells of both juvenile and mature

tissues (Haissig 1989, Haissig et al. 1987, Haissig et

al. 1992, Hussey 1986). Significant accomplishments

have been made in regeneration in vitro when the

explant is derived from seed or other juvenile

tissues. However, successful application of current

micropropagation technologies to a mature tree that

has been identified as genetically superior is not

routinely accomplished. The reason for limited

success is that selection can only be reliably made at

a time when the individual is morphologically/

developmentally mature and, in comparison to

embryonic tissue or seedlings, has, to a great degree,

lost its morphogenetic plasticity (Bonga 1987, Mason

and Gill 1986). Ununger and Ekberg (1987) believe

that “The most serious constraint associated with

clonal forestry is the physiological changes within

the tree that are related to its aging.” Accordingly,

the use of cuttings for propagation of Picea abies

Wnunger and Ekberg 1987) and other conifer

species (Mason and Gill 1986) in commercial

ventures has been limited to trees of 4 to 6 years of

age.

Difficulties similar to those associated with the

rooting of coniferous species from cuttings of mature

trees are encountered in every stage of

micropropagation technology. Figure 1 illustrates

the developmental progression towards maturation

of a typical coniferous forest tree, together with

representative explant materials that have produced

developing microshoots and, in the case of the

juvenile materials, have routinely regenerated

plantlets and emblings. There have been numerous

approaches to increasing the micropropagation

efficiency of more mature, less juvenile explanted

tissues of both hardwood and coniferous forest trees

(Franclet et al. 1987). In general, the methods

outlined rely on two approaches: (1) the utilization of

those portions of the mature tree thought to be

juvenile by virtue of their placement within the tree

or the timing of their initial morphogenesis

(Ballester et al. 1990, Durzan 1990) and (2)

rejuvenation of selected tissues and organs through

cultural manipulation, chemical treatment, or a

combination of both (Bonga 1987, Thorpe and Harry

1990). In some instances, published research has

relied on an empirical approach to experimental

design, based mainly upon previous successes.

Major advances are likely to emerge when the

existing body of empirical knowledge can be

combined with an increased understanding of the

basic mechanism.s that control the transition from

juvenility to maturity.

In consideration of the three major in vitro

propagation pathways discussed in this review, the

most promising in the long term appears to be

somatic embryogenesis. This propagation route

offers the greatest potential for large scale

commercial application, while providing the ideal

system for genetic engineering. Unfortunately, this

system is the least developed with respect to its

efficacy and species applicability. In the interim, the

opportunity to use clones of selected trees provided

via organogenic or axillary bud propagation in

existing tree improvement programs should be

vigorously pursued (Zobel 1992).

LITERATURE CITED

Abdullah, A.A.; Grace, J.; Yeoman, M.M. 1986.

Rapid micropropagation of Calabrian pine from

primary and secondary buds on shoot explants.

Canadian Journal of Forest Research.

16(3): 637-641.

Abdullah, A.A.; Grace, J.; Yeoman, M.M. 1989.

Rooting and establishment of Calabrian pine

plantlets propagated in vitro: Influence of growth

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eds. Handbook of plant cell culture. Crop species.

New York: Macmillan Publishing Company: 435-

470. Vol. 2.

Thorpe, T.A.; Harry, I.S. 1990. Special problems and

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Tremblay, F.M. 1990. Somatic embryogenesis and

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21
CONIFER MICROPROPAGATION DIFFICULTY INDEX

m-m

TURE

YEARS

II--1-----------1- EXPLANT

a STALKS CF IMMATURE CONES

r

rr,

BUDS

NEEDLE FASCILES

zm BASAL ORTHOTROPIC SHOOTS

s

9

ADOLESCENT e

B

3-7 YEARS Fl BUDS w

5i NEEDLE FASCILES

8

2

2 SEEDLING

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MATURE SEED

*

s

G

s

2 COTYLEDONS

-r HYPOCOlYlARY EXPLANT

s

l

8

: WHOLE EMBRYO

s EXCISED COTYLEDONS

P

WHOLE FEMALE GAMETOPHYTE

p DEVELOPING IMMATURE EMBRYO

tn

Figure 1. The developmental stages in the life of a typical conifer as they relate to examples of increasingly

mature explant tissues that have been used for micropropagation. For most conifer forest trees, the ease of

vegetative propagation, whether by traditional or in vitro methods, decreases with increasing maturation of

the donor tree.

2 2
IN VITRO PROPAGATION OF HARDWOODS’

S.A. Merkle and R.N. Trigiano2

Abstract.--Despite substantial advances in propagation of hardwood forest trees

during the last decade with regard to the number of species successfully regenerated

and frequency of propagules produced, there are still relatively few systems for

southern hardwood species that are useful for operational purposes. Shoot or bud

multiplication systems, while regarded as producing the most true-to-type-propagules,

are labor-intensive and multiplication rates are relatively low. Organogenic systems,

while offering the potential for higher-frequency propagule production, still require

multiple labor-intensive steps for production of plantlets ready for field planting, and

may be subject to higher amounts of within-clone variation due to the origin of the

shoot meristems. Embryogenic systems appear to offer the highest potential for

large-scale propagule production at a low per unit cost, by high multiplication rates

and by eliminating the multiple steps required by the other systems. However, very

few embryogenic systems have been initiated from mature hardwood trees, limiting

propagation of plants of proven genetic value. The combination of different in vitro

regeneration systems (and conventional propagation systems) may provide the best

potential for producing material from superior genotypes on an operational scale.

Examples of current in vitro propagation technology with southern hardwoods

include shoot multiplication and organogenic systems for sweetgum (Liquidambar

styraciflua), organogenic regeneration systems for eastern cottonwood (Populus deltoides)

and embryogenic regeneration systems for yellow-poplar (Liriodendron tulipifera), black

locust (Robinia pseudoacacia) and American chestnut (Castanea dentata).

INTRODUCTION

Historical Perspective and Current Effort

In vitro regeneration systems offer a number of

potential applications for mass propagation of

superior genotypes of hardwood forest tree species.

Hardwood forest trees were among the first plants

‘Paper presented at the 1992 Southern Regional

Information Exchange Group Biennial Symposium,

Huntsville, AL, July 8-10, 1992.

2School of Forest Resources, University of

Georgia, Athens, GA 30602; Department of

Ornamental Horticulture and Landscape Design,

University of Tennessee, Knoxville, TN 37901

cultured in vitro. Gautheret (1940) reported

formation of adventitious buds in cultured cambial

explants of smooth-leaved elm (Ulmus campestris),

demonstrating that an exogenous source of sugar

was required for bud production, while high levels of

indoleacetic acid (IAA) in the medium inhibited bud

formation. Jacquiot (1949) extended the research

with this species, using trees up to 180 years old as

tissue sources, Mathes (1964) reported in vitro

production of both roots and shoots in callus cultures

of quaking aspen (Populus tremuloides), although

apparently no plantlets were produced. It was not

until four years later that Wolter (1968) produced

entire P. tremuloides plantlets in vitro by inducing

shoot formation with benzylaminopurine (BAP) and

subsequently rooting the shoots in vitro. Winton

(19681, working with triploid P. tremuloides callus,

also obtained complete plantlets, demonstrating for

23
the first time in vitro propagation of a forest tree

with a superior genotype.

Since the pioneering work with Populus,

hundreds of hardwood tree species have been

propagated in vitro, although much of this work was

done only on an experimental rather than an

operational scale. In the United States, during the

last decade, research priority was placed on

commercially important coniferous species.

However, a number of laboratories have contributed

to substantial progress in the field of hardwood

tissue culture, resulting in the availability of a few

tissue culture regeneration systems that are ready

to be employed for applied purposes. It would be

unrealistic to attempt to review all of the tissue

culture research reported to date with North

American hardwoods. However, it is also true that,

whereas a number of comprehensive reviews of in

vitro propagation of coniferous trees are available,

the same cannot be said for North American

hardwood trees. Fortunately, there are a number of

books which, between them, contain chapters on the

in vitro propagation of virtually all of the important

hardwood genera represented in North America. We

would refer those looking for information on

manipulation of individual hardwood genera in vitro

to books edited by Bajaj (1986, 1989,1991) or Bonga

and Durzan (1987).

Types of In vitro Propagation Systems

Systems for propagation of plants from sterile

culture, also known as micropropagation, fall into

three broad categories, one of which relies on

multiplication of preformed structures, and two of

which rely on de nouo generation of either plant

organs or embryos (i.e. morphogenesis). These three

systems are axillary shoot or bud multiplication

(a.k.a. axillary enhancement), organogenesis and

somatic embryogenesis.

Production of plantlets from axillary shoots is

similar to vegetative propagation via rooted cuttings,

in that shoots are excised from source tissue and

rooted individually to multiply the original

genotype. The differences are that, with axillary

shoot systems, the procedures are performed in

vitro, under aseptic conditions, and that

manipulations in vitro can be used to greatly

multiply the number of propagules obtained. These

include media supplements and culture conditions

which induce axillary buds in the cultured tissues to

elongate to form shoots. These shoots can then be

24

excised and either rooted in vitro, or placed back in

culture for further enhancement of axillary

branching. Thus, unlike rooted cutting

macropropagation, conditions can be applied in vitro

which promote multiplication of new shoot material.

Organogenesis is the de nouo production of plant

organs (buds, shoots, roots) from tissues or from

callus. In many cases, initial explants are induced

to form callus and subsequently shoots. Then,

similar to axillary shoot methods, the shoots are

induced to elongate, are excised and rooted

individually. Somatic embryogenesis is the de nouo

production of structures resembling zygotic embryos,

either from organized tissues or from callus.

Structures classified as somatic embryos must be

bipolar (possessing both root and shoot poles) and

have no vascular connection to the source tissue.

Somatic embryos may be derived either through

direct or indirect embryogenesis. In direct

embryogenesis, embryos are formed essentially by

multiplication of a zygotic embryo explant, i.e by

embryo cloning (Sharp et al. 1980). Indirect

embryogenic systems involve a dedifferentiation of

nonembryonic tissue to form a callus from which

somatic embryos arise (Sharp et al. 1980). These

two forms of somatic embryogenesis have an impact

on the potential application of embryogenic systems

for propagation and will be discussed further.

In vitro propagation methods covering all three

routes of regeneration have been widely reported for

hardwood forest trees. However, even after years of

experience, only a few methods are being applied or

have a real potential to be applied for operational

production of hardwood forest tree propagules. Our

purposes of this review, therefore, are to (1) present

a view of the goals of in vitro production of

hardwoods, with regard to operational applications,

(2) discuss features which impact the operational

utility of in vitro propagation methods, (3) Compare

different in vitro propagation methods with regard

to their relative advantages and disadvantages for

operational application to hardwood species, (4)

present a number of examples of in vitro

propagation systems for hardwood species that

demonstrate the current state of the art for

hardwood micropropagation, and (5) briefly

speculate about some advances in methodology

which are likely to have some impact on application

of this technology to hardwood forest trees in the

future. We have chosen to limit the scope of the

examples in two ways: We will concentrate mainly

on southern hardwood species, although a few
systems for other hardwoods will be cited, and we

will focus on describing those systems which are

state-of-the-art for in vitro technology for these

species.

GOALS AND DESIRABLE FEATURES OF IN

VITRO PROPAGATION SYSTEMS

Goals of In vitro Propagation of Hardwoods

A primary goal of vegetative propagation is to

capture the total genetic superiority of the parent

material, which includes both additive and

nonadditive genetic components. In addition, clonal

propagation systems allow the application of a very

high selection differential, since whole new

populations of plants can be cloned from just a few

elite individuals. Theoretically, since somatic

tissues are used as the starting material, trees

regenerated from these tissues should be exact

clonal copies of the ortet. However, unlike

macropropagation systems, some in vitro

propagation methods have the potential to introduce

significant new genetic or epigenetic changes

(somaclonal variation) into propagules regenerated

from them, so that the parent material is not truly

replicated. Furthermore, the nature of much in

vitro propagation technology for hardwood species

excludes clonal propagation of proven genotypes,

since many of these systems are limited to

employing explants from juvenile, genetically

unproven material.

A second principal goal of micropropagation that

parallels conventional vegetative propagation is

simply to mass propagate plants, bypassing sexual

reproduction, which for some reason may be

expensive, inefficient or impractical. However, in

vitro propagation can also be used to overcome

problems associated with macropropagation,

including limitations on available parent material or

space. In addition, just as propagation through

seeds may be limited by biological factors, certain

species or even individual genotypes may be

recalcitrant to conventional vegetative propagation

techniques, such as rooting of stem cuttings. In

these cases, in vitro techniques may succeed where

macropropagation fails.

In addition to the advantages shared with

conventional vegetative propagation, in vitro culture

has a number of associated applications that are

unique. For example screening of genotypes for

such traits as disease resistance can be accomplished

in vitro, followed by production of propagules from

the selected material. This technique has already

been applied with some coniferous trees (Amerson

and Mott 1990). In vitro selection has also been

used in conjunction with somaclonal variation to

generate new variants in culture, which can then be

propagated in vitro. This technique has been used

to generate hybrid Populus variants that are

resistant to the herbicide glyphosate (Michler et al.

1992) and eastern cottonwood (Populus deltoides)

variants that show increased or decreased resistance

to leaf rust caused by Melampsora medusae

(Prakash and Thielges 1989).

Another potential application of in vitro culture

which may eventually succeed for hardwood trees is

generation of somatic hybrids via protoplast fusion.

For example, protoplasts of Citrus species from

different genera which were unable to hybridize

sexually, were fused and somatic hybrid plantlets

regenerated (Grosser et al. 1988). Although

regeneration of somatic hybrids has not been

reported for a hardwood forest tree to date,

protoplasts of a number of species have been isolated

and induced to regenerate cell walls and divide to

eventually produce protoplast-derived plantlets (e.g.

Russell and McCown 1986, Sticklen and Lineberger

1985, Merkle and Sommer 1987). Finally, in vitro

culture provides the only route for generation of

genetically engineered genotypes of hardwood trees.

Gene transfer technologies including

Agrobacterium-Ti plasmid mediated gene transfer,

electroporation, microinjection and microprojectile

bombardment all depend on the ability to culture

cells in vitro in order to select the transformed cells,

usually by employing drug-resistance marker genes.

Once transformed cells are obtained, these can be

cultured to regenerate transformed plantlets. It is of

special value to be able to employ cultures capable of

regeneration from single cells, since this ability

raises the probability that transformants derived

from these cultures will be nonchimeric.

Desirable Features for In vitro Hardwood

Propagation Systems

The ability to regenerate a particular hardwood

species from tissue culture does not necessarily

guarantee that the method will be useful for

operational propagation of that species. To be

adapted for production purposes, an in vitro

propagation system must have a number of

attributes that make it an economically feasible

alternative to conventional techniques. A number of

these attributes are determined by the type of in

25
vitro propagation system (i.e. shoot multiplication,

organogenesis or somatic embryogenesis).

A primary consideration for propagation purposes

is the frequency with which plantable propagules

are produced. Due to the high labor inputs for

initiating and maintaining plant tissue cultures and

producing field plantable stock from them, costs are

high compared to conventional systems. Thus, in

order to compete with macropropagation methods on

a per unit cost basis, micropropagation systems must

possess the potential to produce thousands of trees

from a single explant in a limited time. Therefore,

an in vitro propagation system must be amenable to

scale-up, in order to lower the cost per propagule.

Additional savings in labor costs may be obtained if

the system can be automated. A very important

factor with regard to the successful application of an

in vitro propagation system is the ability of the

cultures to generate plantlets that are true-to-type,

i.e. that are faithful to the phenotype of the parent

material. Since one of the theoretical advantages of

clonal propagation is that members of the clone will

be exact copies of the desirable ortet, one of the

primary motivations for clonal propagation is lost if

the regenerants vary significantly from the

phenotype of the ortet. Also, when in vitro cultures

are started from immature, genetically unproven

material, which is the case for many hardwood tree

cultures, clonal uniformity is still a desirable feature

for the regenerants. Thus, in vitro propagation

methods with a high potential for somaclonal

variation are less desirable for clonal production

than those that can guarantee propagules with

uniform performance.

Finally, since even very efficient in vitro

propagation methods are still more expensive on a

per unit cost basis than seedlings for most species,

micropropagation methods must be able to offer

some value-added component to the regenerated

trees in order to make the system economically

feasible (Redenbaugh et al. 1987). A value-added

feature is a phenotypic character that in vitro

regenerated propagules possess that is unavailable

in trees regenerated by other means, which makes

up for the added cost of the culture-derived trees.

One example of such a value-added feature is

genetically engineered genotypes, carrying and

expressing foreign genes for such traits as insect or

disease resistance. Another such feature may be the

production of rare hybrid trees that cannot be

achieved in large numbers in a reasonable time

period through conventional breeding,

2 6

ADVANTAGES AND DISADVANTAGES OF

DIFFERENT IN VITRO PROPAGATION SYSTEMS

After a number of years of experience with the

three in vitro regeneration methods with a variety of

hardwood forest tree species, there is some basis for

comparison of the different methods. Using the

criteria listed in the previous section, these methods

are compared with regard to their relative

advantages and disadvantages for application to

propagation of southern hardwood trees. However,

as the examples will show, the success of a given

method appears to be highly species-specific, with

shoot multiplication systems working well for some

species, while embryogenic systems are clearly

superior for others. We choose to emphasize here

that it is counterproductive to view these different

systems as competing. Indeed, it may ultimately be

more beneficial to combine the strongest features of

the different systems in order to achieve the desired

goal, or even to combine in vitro propagation

systems with conventional vegetative propagation

systems, which has been accomplished in one of our

later examples.

Axillary Shoot Multiplication

Probably the main advantage of axillary shoot

multiplication methods for hardwood trees is that

since all propagules are derived from preformed

buds, this method provides the best guarantee that

propagules will be true-to-type. Since shoots arise

from meristems present in the explant, there is little

chance of introducing somaclonal variation into the

propagules. Currently, axillary shoot multiplication

methods offer the best (and sometimes only) route

for propagating mature genotypes. Mature

genotypes of such species as black locust (Robinia

pseudoacacia) have been propagated via axillary

shoots, while organogenic and embryogenic systems

for these species have relied on immature starting

material.

Given that production of plants from axillary

shoots has some similarities to rooted cutting

propagation, it might be expected that it has a

number of disadvantages in common with this

macropropagation technique. One of these is the

relatively low frequency of propagule production

associated with this technique. Although repeated

cycles of in vitro culture can ultimately produce

thousands of propagules, large input of labor and

facilities are required to produce operationally

useful numbers of plants. Axillary shoot
multiplication methods are also relatively laborintensive, requiring substantial handling, both for

cycling of cultures and for production of plantlets.

Plantlet production can require multiple steps,

including induction, shoot elongation, shoot excision,

rooting and acclimatization of plantlets, each of

which may require significant input of labor.

Finally, because of multiple handling steps, axillary

shoot multiplication methods may not be as

amenable to scale-up or automation as other

methods for hardwood forest tree propagation,

although recent advances in robotics may change

this potential.

Organogenesis

The primary advantage of organogenic

regeneration methods over axillary shoot methods is

that some of these methods may have the potential

for higher frequency plantlet production in a shorter

period of time than axillary shoot systems. Callus

can be grown in large quantities with less demand

for inputs of labor and space, and adventitious shoot

production can be used to achieve high

multiplication rates. However, like axillary shoot

methods, organogenesis-based methods may require

the labor-intensive steps of shoot elongation,

excision, rooting, and plantlet acclimatization to

produce field-plantable stock. In addition, unlike

axillary shoot methods, organogenic methods,

especially those requiring an intermediate callus,

are associated with the production of significant

amounts of somaclonal variation in the regenerated

plantlets. Thus, the resulting plantlets from a given

clone may display unacceptable variation.

Somatic Embryogenesis

Somatic embryogenesis has been cited by many

authors as the in vitro regeneration system of choice

for economical production of clonal populations of

forest trees (e.g. Gupta et al. 1991). Certainly, this

type of system has a number of powerful advantages

over axillary shoot multiplication methods and

organogenesis. However, it should be noted that the

efficiency of embryogenic methods varies widely

with species. One important advantage of

embryogenic methods over both axillary shoot and

organogenic methods is the potential for very high

frequency regeneration. Depending on the species,

virtually unlimited numbers of embryos can be

generated from a single explant. In addition,

embryogenic cultures of many species can be grown

in liquid, allowing production and handling of

thousands of embryos at one time. Thus, in

comparison to axillary shoots and organogenesis,

somatic embryogenesis offers the potential for high

volume, large-scale propagation which can be

translated into significant labor savings. Greater

economies of scale may be possible if bioreactor and

continuous culture technologies can be applied to

embryogenic systems (e.g. Styer 1987, Stuart et al.

1987). The feature of embryogenic methods which

may ultimately have the most impact for mass

propagation of hardwood trees is the fact that the

product is an embryo. The morphological and

physiological similarity of somatic embryos to zygotic

embryos means that they are complete propagules in

themselves, with embryonic roots, shoots and leaves,

and, most importantly, the “program” to make a

complete plant. Thus, unlike other in vitro

propagation methods, no separate shoot elongation

or rooting steps are required for plantlet production.

Furthermore, many embryogenic systems produce

singularized embryos, requiring no excision from

source tissue or other embryos in order to be

handled. These characteristics further lower labor

inputs and give somatic embryos the potential for

direct delivery to the greenhouse or field as

“artificial seeds” (Redenbaugh et al. 1987, Fujii et al.

19921, thereby eliminating the need for laborintensive transplanting.

A major disadvantage of embryogenic

propagation methods for hardwood species is that

the bulk of forest tree embryogenic systems reported

to date have relied on immature tissues (i.e. from

seeds or seedlings) as explanting material. Thus,

the material being propagated is of unproven genetic

value. Most reports of somatic embryogenesis in

tree species are in reality reports of “embryo

cloning,” in which the zygotic embryo is induced to

replicate itself indefinitely. Another drawback of

forest tree embryogenic systems concerns the low

frequency of plantlet production or “conversion” of

somatic embryos to plantlets. Although numerous

embryogenic systems have been reported for forest

trees, some of which produce embryos at high

frequencies, a major bottleneck has been induction

of embryo maturation and subsequent production of

field-plantable stock. Currently only a few species of

North American hardwoods can be propagated via

somatic embryogenesis from multiple clonal lines in

sufficient numbers to enable establishment of useful

field tests.

Finally, the relative advantages or disadvantages

of somatic embryogenesis for propagation of a given

species depend on whether the system developed is

direct or indirect. Direct embryogenic systems,
where there is no intermediate callus stage have

been cited as having a very low potential for the

production of variants (Ozias-Akins and Vasil 1988,

Maheswaran and Williams 1987). Thus plantlets

produced via this route should be very faithful to

their clone. However, embryos produced via direct

systems tend to proliferate as clumps of mixtures of

globular and other stage embryos. Thus, they may

not grow well in suspension cultures and will

require more handling to produce individual

plantlets, raising labor costs. Conversely, indirect

embryogenesis, in which embryos are produced from

callus, has a higher potential for maintenance in

suspension culture and for production of

singularized embryos, which may be synchronized in

development for ease of handling. However, the

callus origin may give indirect embryogenic cultures

a higher potential to introduce unwanted variation

into the clonal population (Maheswaran and

Williams 19871.

ILLUSTRATIONS OF HARDWOOD IN VITRO

PROPAGATION SYSTEMS

The following examples demonstrate that the

three in vitro propagation methods have the

potential to be adapted for operational use with

some hardwood species. Here, we have concentrated

on choosing systems with southern hardwood species

as examples, with the inclusion of a few other

hardwoods that are good illustrations of what can be

accomplished with a particular species or method.

Sweetgum

There are more published reports on in vitro

propagation of sweetgum (Liquidambar styraciflua)

than any other major southern hardwood tree,

either via axillary shoot multiplication @utter and

Barker 19851, adventitious shoot formation (Sommer

1981, Sommer et al. 1985, Brand and Lineberger

1988,1991, Chen and Stomp 1991) or somatic

embryogenesis (Sommer and Brown 1980). Here,

three systems are summarized, one of which is

currently being applied for operational production of

sweetgum clonal material.

Sommer et al. (1985) described an organogenic

regeneration system for sweetgum which was

capable of producing up to 50 plantlets per culture

on approximately a monthly basis, depending on

clonal line. To initiate the cultures, seedlings were

germinated aseptically and hypocotyl sections were

explanted on a semisolid Risser and White’s (1964)

medium with 0.1 mg/l IAA and 0.5 mg/l BAP to

initiate adventitious shoot production. For shoot

multiplication, Sommer et al. (19851 found that a

liquid Blaydes’ (Witham et al. 1971) medium with

0.01 mg/l naphthaleneacetic acid (NAA) and 0.5 rngfl

BAP was superior to an agar-solidified medium

(Sommer 1981). Adventitious shoots were rooted

with up to 90% efficiency on a modified Risser and

White’s (1964) basal medium. Sommer et al. (19851

also reported on nursery bed performance of the

plantlets derived from adventitious shoots, noting

some problems with root girdling.

Brand and Lineberger (1988,1991) also reported

on an organogenic regeneration system for

sweetgum, in which adventitious shoots were

induced to form directly on leaf explants, without

formation of callus. In the first study, leaf and

petiole tissues were harvested from shoot cultures

initiated from lateral buds of 25- to 30-year-old trees

and explanted onto a Woody Plant Medium (WPM;

Lloyd and McCown 1980) with 2.5 mg/l BAP. Shoot

formation on leaf tissues occurred most frequently at

or near breaks in major vasculature. Wounding the

leaves by cutting across the lamina and vasculature

increased the number of shoots formed per explant

up to 18 for one sweetgum variety. Over 88%

rooting of the excised shoots was obtained with a 2-

min basal dip in 200 mg/l IBA prior to transfer to a

peat/vermiculite mixture in covered trays. Later,

Brand and Lineberger (1991) applied the same shoot

induction treatment to obtain up to 57 shoot

meristems per explant when leaves from intact

plants were used instead of leaves from shoot

cultures. Leaf developmental stage exerted an

impact on shoot meristem production, since leaves

that were 50% expanded produced up to 90 shoot

meristems per explant for one sweetgum variety.

Workers at Union Camp Corporation have

applied a sweetgum shoot multiplication method to

propagate selected genotypes to be used in

operational production of sweetgum. The method,

outlined briefly by Feirer and Wann (19891, and

modified from that of Sutter and Barker (19851,

depends on shoot multiplication from apical

me&terns of selected trees up to several years old.

Dormant buds or greenwood cuttings were sterilized

by application of both mercuric chloride and bleach.

Excised shoot tips were placed on WPM with 0.01

mg/l NAA and 2 mg/l BAP. Shoot cultures developed

mainly from axillary buds, and were maintained on

the same medium. Shoots were given a rooting
pretreatment in vitro, by placing excised shoots on

l/3-strength WPM with 0.1 mg/l indolebutyric acid

(IBA) and 1% sucrose for 3 weeks. Pretreated shoots

were moved to peat plugs for root emergence under

fog. Over 100 mature trees selected from genetic

tests and seed orchards have been propagated using

this method (personal communication, S.R. Wann,

Union Camp Corp., Princeton NJ). To date, over

6000 plantlets have been produced from over 60

clones, with no obvious phenotypic variants. The

method is in use in Union Camp’s vegetative

propagation program for “clone capture,” i.e. to make

sufficient copies of a selected tree for planting in a

cutting orchard. Union Camp’s sweetgum program

is an excellent example of a combination of

vegetative propagation methods (i.e.

micropropagation and rooted cuttings) for a specific

application, and, to our knowledge, represents the

only application of in vitro propagation to

operational production of selected genotypes of a

southern hardwood.

Eastern Cottonwood

More cell and tissue culture research has been

reported on Populus than for any other hardwood

genus. For the purposes of this review, we will focus

attention on eastern cottonwood (Populus deltoides),

which is well known for its capacity to be highly

productive in the southeastern United States. For

further information on Populus tissue culture, we

refer the reader to reviews by Ahuja (1987), and

papers on protoplast culture and somatic

embryogenesis by Russell and McCown (1986) and

Michler and Bauer (19911, respectively.

Coleman and Ernst (1989,1990,1991) conducted

the most intensive research program with

organogenic regeneration of eastern cottonwood,

although their research concentrated more on the

control of adventitious shoot regeneration than on

optimizing plantlet production. In the 1989 study

internodal explants from 3 clones collected from a

stool bed were explanted onto modified WPM with

various concentrations of the cytokinins BAP, zeatin

and 2-isopentenyladenine (2iP). Zeatin proved to be

the best cytokinin for adventitious shoot production,

while BAP was apparently phytotoxic. A zeatin

concentration-by-genotype interaction was also

observed. Further investigations (Coleman and

Ernst 1989) revealed that when zeatin concentration

was held at 0.5 mg/l, there were strong genotypic

differences in shoot regeneration among 16

genotypes. Coleman and Ernst (1990) further

defined shoot regeneration competence and callus

determination in eastern cottonwood in terms of

developmental stages, finding some genotypes that

were competent for direct shoot regeneration, other

genotypes requiring initial culture on callusinducing medium with 0.5 mg/l2,4-

dichlorophenoxyacetic acid (2,4-D) to acquire shoot

regeneration competence, and some genotypes which

would never regenerate shoots. Later, Coleman and

Ernst (19911, using SDS-PAGE and 2-D

electrophoresis, showed that certain proteins were

associated with explants determined for either shoot

regeneration versus callus growth.

Adventitious shoot regeneration was also

reported for eastern cottonwood using leaf disk

explants (Prakash and Thielges 1989, Uddin and

Dinus 19901, although regeneration has been limited

to a few genotypes. Prakash and Thielges (1989)

cultured leaf disks on modified WPM with 1 PM

NAA and 1 uM BAP to obtain callus from the cut

edges of the explants. Callus was subsequently

transferred to modified WPM with 0.1 PM

thidiazuron (TDZ) for production of adventitious

shoots. Shoots were elongated on WPM with 0.5 pM

BAP, excised, and grown on WPM with 0.1 pM BAP

prior to rooting on peat plugs. When the resulting

somaclones were screened for resistance to

Melampsora leaf rust, some individuals displayed

increased resistance or susceptibility. Uddin and

Dinus (1990) also reported adventitious shoot

production from eastern cottonwood leaf sections

cultured on WPM supplemented with NAA and

BAP, which were elongated by reducing growth

regulator concentrations to half-strength. Excised

shoots could be rooted on WPM supplemented with

IBA. The authors proposed to use this regeneration

system as the basis for Agrobacterium-Ti plasmidmediated gene transfer.

Another development involving in vitro

technology for eastern cottonwood should be noted.

Bradshaw et al. (1992) are constructing a restriction

fragment length polymorphism (RFLP) map in a

Populus pedigree founded by a black cottonwood (P.

trichocarpu) female and a P. deltoides male. Stem

explants of the P. trichocurpu parent on zeatinsupplemented WPM produce adventitious shoots

with 100% frequency, while those from the P.

deltoides parent fail to produce adventitious shoots.

In the Fz and backcross, continuous segregating

variation from O-100% is found. By correlating

adventitious shoot formation in vitro with RFLP

genotypes, Bradshaw et al. (1992) intend to

elucidate the genetics of organogenesis in the genus.

2 9
Silver Maple

Although silver maple (Acer saccharinurn) is not

generally considered to be a major southern

hardwood, recent progress in micropropagation of

this species may have implications for propagation of

other hardwoods, especially with regard to the

application of the relatively unknown plant growth

regulator, TDZ. Preece et al. (1991a,b) cultured 2.5

cm shoot tips or nodal explants from juvenile and

mature trees on a Driver and Kuniyuki (1984)

medium supplemented with different concentrations

of four amino purine cytokinins (BAP, kinetin,

zeatin and 2iP) and TDZ, a substituted phenylurea

compound. TDZ at 10 nM was found to be optimum

for production of large numbers (over 100 after 4

months) of long shoots by both juvenile and adult

nodal explants. The authors believed that in

addition to long-term exposure to TDZ, the high

frequency shoot production could be attributed to

decapitation of the longest shoots in the mass to

release apical dominance and the development of a

massive callus base, which provided physical

support for the shoots in liquid culture and may

have aided with nutrient uptake from the medium.

Microshoots of juvenile origin which were 1.5 cm or

longer could be rooted at a high frequency in the

greenhouse under intermittent mist, with no auxin

treatment, while rooting of microshoots of adult

origin apparently was improved by a l&second dip

in 1 Mm IBA. Preece et al. (1991b) concluded that

their micropropagation method could be applied

successfully to a wide range of silver maple

genotypes. To date hundreds of micropropagated

trees have planted in the field and have performed

comparably with bare-root seedlings (Preece et al.

199la).

Black Locust and Other Woody Legumes

While there are a number of woody leguminous

species in North America, the one with substantial

commercial potential as a forest species is black

locust (Robinia pseudoacacia). However, results

with in vitro propagation of black locust and other

North American woody legumes may have

considerable value if it can be applied to other

members of the group, since woody legumes are of

great importance as timber species in other parts of

the world, especially in the dry tropics.

Black locust, while not particularly associated

with the southern United States, is of particular

interest in this review because it has been

regenerated in vitro by axillary shoot multiplication,

organogenesis and somatic embryogenesis, and the

relative merits of the three regeneration methods

can be compared for the same species. It should also

be noted that black locust micropropagation has

been accomplished from both immature and mature

tissues. Chalupa (1983) described an axillary shoot

system initiated from nodal segments of one- to

three-year-old seedlings. Explants were cultured on

a Murashige and Skoog’s (1962) medium (MS)

supplemented with 0.4-0.6 mg/l BAP and 0.05 mg/l

IBA. Shoots were multiplied by excising axillary

shoots and culturing nodal segments from them on

fresh medium. Shoots were rooted on a modified

Gresshoff and Doy’s (1972) medium (GD) with 0.3

mg/l NAA and 0.3 mg/l IBA. Similarly, Barghchi

(1987) employed axillary bud explants from a-year

old cutting-derived plants cultured on MS medium

with 0.25-1.0 mg/l BAP to obtain 5-fold shoot

multiplication every month. Shoots were rooted on

half-strength MS medium with 1 mg/l IBA. In both

reports, plantlets were readily established in soil.

Davis and Keathley (1987) induced axillary shoot

production from 20- to 30-year-old black locust trees

by culturing excised dormant winter buds on MS

medium supplemented with 3.2 FM BAP. Shoots

were elongated on MS medium with 0.3 yM BAP

and rooted on l/10 strength MS medium with 0.2

mg/l IBA. Adventitious shoot production was also

induced in black locust using callus derived from

seedling hypocotyl explants cultured on MS medium

with 10 PM BAP (Han and Keathley 1989). Shoots

derived from the callus cultures and transferred to

MS medium with 0.32 pM BAP produced an average

of 5 new shoots per culture in 4 weeks.

Embryogenic cultures of black locust were

initiated at a low frequency by culturing immature

seeds (2-4 weeks postanthesis) on MS medium with

4 mg/l2,4-D and 0.25 mg/l BAP for 1 week prior to

transfer to MS basal medium (Merkle and Wiecko

1989, Merkle 1991). Clumps of somatic embryos

emerged from the immature seeds, apparently after

forming directly from tissues of the zygotic embryo.

New somatic embryos continued to be produced for

some time via direct, repetitive embryogenesis from

the radicles of older embryos. However, repetitive

embryogenesis was terminated when the embryos

germinated precociously, which also resulted in

spindly plantlets. Precocious germination was

limited by subculturing clumps of somatic embryos

to MS medium with the sucrose concentration raised

from 3% to 10% and by interposing a layer of filter

paper between the embryos and the semisolid

3 0
medium, thus changing the osmotic environment of

the embryos. As a result of the changed culture

conditions, proembryogenic masses (PEMs; Halperin

1966) were produced by the somatic embryos. PEMs

may be thought of as very early stage proembryos

that are rapidly proliferating via repetitive

embryogenesis before they develop further. We

consider this form of embryogenesis to offer the best

potential for very high frequency somatic embryo

production and for gene transfer applications (see

section on yellow-poplar). PEMs rapidly proliferated

in either semisolid or liquid lOA40N medium (Finer

and Nagasawa 1988), a medium originally developed

for soybean suspension cultures, supplemented with

3 mg/l2,4-D (Merkle 1991). Hundreds of black

locust somatic embryos were produced by

transferring approximately 0.5 g of PEMs to

hormone-free liquid lOA40N medium (Arrillaga and

Merkle, unpublished). Embryos were germinated

either on l/2-strength MS medium or, at low

frequency, directly on peat/vermiculite potting mix

in GA7 vessels (Magenta Corp.).

Somatic embryogenesis has also been reported in

other North American woody legumes, including

eastern redbud (Cercis canadensis; Trigiano et al.

1988, Trigiano and Beaty 1989, Geneve and Kester

1990) and yellowwood (Cladrustis Mea; Weaver and

Trigiano 1991). In all cases, the explants were

immature embryo tissues. Redbud embryogenesis is

of special interest since somatic embryos were

formed directly from cotyledons (Trigiano et al.

1988), indirectly from callus (Trigiano and Beaty

1989) and even from adventitious roots (Trigiano,

unpublished).

Oaks and Chestnuts

In vitro propagation of members of the Fagaceae

has been difficult, with virtually no reports of any

method with the potential for application for mass

propagation. However, we believe recent progress

with somatic embryogenesis with members of this

family is noteworthy, especially considering the high

value of products derived from these species, in

particular the genus Quercus. In the past few years

a number of European oak species have been

regenerated via somatic embryogenesis, including

English oak (Quercus robur; Chalupa 1990),

Durmast oak (Quercus petrueu; Chalupa 1990) and

cork oak (Quercus suber; Bueno et al. 1992). Among

North American oak species, Gingas and Lineberger

(1989) regenerated northern red oak (Quercus

rubru) plantlets via somatic embryogenesis.

Although the highest frequency somatic embryo

production was achieved from immature (late heart

to early cotyledonary stage) zygotic embryos

cultured on MS medium with 1 mg/l2,4-D and 1

mg/l BAP, the highest number of normal, bipolar

embryos resulted from culturing the explants on

hormone-free MS medium. A few embryos were

induced to convert to plantlets by desiccating them

inside empty, sealed Petri plates for 2-3 weeks,

apparently allowing them to break epicotyl

dormancy. Embryogenic cultures of white oak

(Quercus ulbu) were generated using the same

procedure, although no plantlets were generated.

Gingas (1991) reported production of somatic

embryos from partially expanded male catkins of

swamp white oak (Quercus bicolor) cultured on MS

medium with 1 mg/l2,4-D. Callus which

proliferated from the base of individual male flowers

produced somatic embryos following transfer to MS

medium with 1 mg/l BAP. Normal, bipolar embryos

transferred to l/2-strength MS basal medium

produced plantlets. This report is of particular

interest because it is one of the few demonstrating

that embryogenic cultures can be initiated from

mature tree tissues (see section on advances

expected in the near future).

There is renewed interest in research with the

American chestnut (Custuneu dentutu), which was

virtually eliminated from the eastern forests of the

United States during the first half of the 20th

century by chestnut blight, caused by the fungus

Cryphonectriu parasitica. Part of this effort is the

establishment of in vitro regeneration systems for

future application in clonal propagation of resistant

genotypes and for genetic engineering purposes.

Micropropagation procedures have been reported for

immature tissues by Keys and Cech (1982) and for

mature tissues by Read et al. (1985), who cultured

5 cm apical portions from forced buds on WPM

supplemented with 0.5 mg/l BAP. Recently, Read

(1992) reported improved axillary shoot production

from American chestnut cultures by substituting low

concentrations (0.05 yM) of TDZ for BAP. Merkle et

al. (1991) initiated embryogenic American chestnut

cultures from immature seeds cultured on WPM

with 4 mg/l2,4-D and 0.25 mg/l BAP. PEMs,

apparently derived from the immature zygotic

embryo, transferred to hormone-free WPM

developed into cotyledonary embryos, but no

plantlets were produced.

Yellow-Poplar

Somatic embryogenesis in yellow-poplar

(Liriodendron tulipiferu) tissue cultures was

31
reported by Merkle and Sommer (1986). Immature

zygotic embryos cultured on a Blaydes’ (Witham et

al. 1971) induction medium with 2 mg/l2,4-D and

0.25 mg/l BAP produced a fast-growing, nodular

callus, since reclassified as PEMs. Similar to many

embryogenic systems, PEMs maintained on

induction medium continued to proliferate without

further development, but transfer to hormone-free

(basal) medium allowed somatic embryos to develop

from the PEMs. Embryos converted to plantlets at a

low frequency, following transfer to a modified

Risser and White’s (1964) plantlet development

medium.

Since the first report, the yellow-poplar

embryogenic system was improved in two ways.

First, by initiating cultures from developing zygotic

embryos collected at regular intervals throughout

the summer, it was found that the optimum stage of

development for initiation of an embryogenic culture

was the late globular to early heart stage, which

occurred approximately 8 weeks postpollination in

the southeastern U.S. (Sotak et al. 1991). By

applying this information, embryogenic cultures

were routinely obtained from up to 50% of explants

(Merkle, unpublished). Secondly, the flexibility

inherent in cultures that proliferate as PEMs was

exploited to scale-up production of somatic embryos

with a high potential for conversion to plantlets

(Merkle et al. 1990, 1991). Embryogenic yellowpoplar suspension cultures were initiated by

transferring PEMs to liquid induction medium in

Erlenmeyer flasks and maintaining them on a

gyratory shaker, with transfer to fresh medium

every 3 weeks. Although large numbers of somatic

embryos were obtained by transferring sizefractionated PEMs to liquid basal medium,

conversion of these embryos to plantlets was very

low (Merkle et al. 1990). Therefore, a protocol was

adopted in which PEMs were size-fractionated on

stainless steel screens and the 38 pm - 140 pm

fraction was collected on a disk of filter paper using

a Buchner funnel. Then, the filter paper with PEMs

was plated on semisolid induction medium. Within

2-3 weeks, a roughly synchronous population of

mature somatic embryos developed from the PEMs

on the filter paper. Embryos transferred from the

filter paper to basal medium lacking casein

hydrolysate formed plantlets at frequencies up to

84%, depending on the clonal line (Merkle et al.

1991).

The high-frequency plantlet production of the

fractionation/plating procedure offered the

opportunity to initiate the first large-scale field test

of somatic embryo-derived trees of a southern

hardwood. PEMs representing 9 embryogenic lines

were increased for embryo production by growing

suspension cultures in 250 ml Erlenmeyer flasks.

Suspension cultures were size-fractionated and

plated as described above and over 1000 somatic

embryos of each of the clones were produced and

shipped to cooperators at the University of

Tennessee for germination, plantlet development,

transplanting to soil and acclimatization. As

embryos germinated, they were transferred to

plantlet development medium in GA7 vessels and

grown for 2-3 months in a growth room. Plantlets

were transplanted to a peat/vermiculite potting mix

in planting containers, grown in a misted

greenhouse for 2-3 months and transferred to the

greenhouse. By the end of the growing season, of

approximately 8700 plantlets transferred ex vitro,

about 5500 (63%) were hardened off and transferred

to the shadehouse where buds were set in the fall.

Over 99% of the plantlets broke dormancy and

continued growth the following spring (Merkle et al.

1991). Plantlets were maintained for another season

in the shadehouse and planted in replicated field

tests using both clonal block and clonal mix designs

in March-April, 1992.

Although not directly related to propagation, it

should also be noted that growth of PEMs in

suspension culture facilitated development of a

system for efficient production of transgenic yellowpoplar trees (Wilde et al. 1992). Embryogenic

yellow-poplar suspensions, size-fractionated to

enrich for single cells and small cell clusters less

than 140 ym in diameter, made excellent target

material for gene transfer via microprojectile

bombardment. Antibiotic-resistant callus colonies

were maintained under strict selection in antibioticsupplemented liquid medium to block growth of

nontransformed cells, virtually guaranteeing

production of nonchimeric embryos and plantlets.

Thus, due to the great potential for manipulation

offered by embryogenic cultures (i.e. growth as

suspensions and ability to size-fractionate)

nonchimeric transgenic trees were efficiently

generated.

Earlier, rare hybrid trees were used as an

example of a value-added feature potentially

available via tissue culture propagation. Hybrids

between the North American yellow-poplar (L.

tulipiferu) and the Chinese tuliptree (Lifiodendron

chinense) were reported to be heterotic, displaying

32
growth rates superior to those of either parent

(Parks et al. 1983). Seedling-derived trees of this

hybrid, however, remain rare in the U.S., due to the

availability of very few Chinese tuliptree parents.

To test the applicability of embryogenic cultures for

propagation of rare hybrid trees, cultures were

initiated from immature embryos resulting from

controlled pollinations of L. tulipiferu female parents

with pollen collected from one of the few mature L.

chinense trees in the U.S.. Media and conditions

were the same as described by Merkle and Sommer

(1986). Averaged across the 4 hybrid families, 12%

of the explanted embryos produced embryogenic

cultures. Unlike pure yellow-poplar embryogenic

cultures, however, hybrid cultures tended to

proliferate as clusters of repetitive globular embryos,

rather than as PEMS. Thus, none of the hybrid

lines was amenable to growth in suspension culture,

limiting large-scale propagation. Nevertheless,

approximately 100 embryos from different hybrid

lines were converted to plantlets, some of which

displayed rapid growth following transfer ex vitro

(Merkle et al. 1992).

ADVANCES IN IN VITRO PROPAGATION

EXPECTED IN THE NEAR FUTURE

Although a number of authors have speculated

on the future impact of in vitro propagation on

forestry, this discussion is focused on a few

potentially-important advances which may impact in

vitro propagation of hardwoods in the short term.

First, a number of recent reports have appeared on

the promotive effects of plant growth regulators that

have not been widely used with hardwood species

previously, notably phenylurea compounds with

cytokinin-like activities, such as TDZ and CPPU [Nphenyl-N-(2-chloro-4-pyridyl) urea]. These

compounds have been shown to have strong

developmental effects, especially with regard to

adventitious shoot production, with a number of

hardwood genera, including Custunea (Read et al.

1992), Fruxinus (Bates et al., in press), Jugluns

(Neuman et al., in press) and Populus (McCown et

al. 1991). It is likely that additional work with

application of these growth regulators for in vitro

propagation of hardwoods will be reported in the

near future.

Other major advances concern somatic

embryogenesis, which probably has the highest

probability of ultimately providing propagules on an

economical scale. First, it is likely that success will

be achieved with somatic embryogenesis from

mature tissues (or at least tissues of known genetic

value) of hardwood species in the near future.

Already there are reports of somatic embryogenesis

from male flower parts of Quercus (Gingas 1991) and

Aesculus (Jorgensen 1989). Also, Michler and Bauer

(1991) used leaf tissues of a Populus hybrid of

known genetic value to obtain somatic embryos.

Similarly, as more cultures are initiated from male

flower parts, there will likely be reports of

embryogenesis from haploid tissues (androgenesis),

generating either haploid lines, or homozygotic

diploid lines which would prove useful in tree

improvement.

Another area which is likely to come into

widespread application for operational propagation

of hardwood trees is long-term storage of

embryogenic cultures, primarily using

cryopreservation. Since most of the embryogenic

systems reported to date depend on material of

unknown genetic value, trees derived from these

cultures need to be tested for field performance

before being released for production programs. Even

if researchers are eventually able to clone mature

genotypes of a particular hardwood species via

somatic embryogenesis, there will still be a need for

field evaluation of trees derived from these cultures.

However, under continuous culture, the useful life of

most embryogenic cultures does not exceed a few

years. Therefore, embryogenic cultures must be

held in a suspended state while trees derived from

them are tested. Once field testing is complete,

cultures of those clones with the best field

performance can be scaled-up for production.

Fortunately, embryogenic cultures of angiosperms

seem to be very amenable to cryopreservation, and

this technique has already been applied for longterm storage of embryogenic cultures of Betulu

(Mannonen and Monger 1992).

CONCLUSIONS

In vitro propagation of hardwood trees has a long

history, and has progressed over the past few

decades, despite the relatively small research effort

in this area. With a few exceptions, most in vitro

propagation systems for southern hardwoods are not

adequate for routine clonal propagation of trees, due

to low multiplication rates, high labor costs,

limitation to genetically unproven material, or other

features that make them economically

uncompetitive with seedlings or macropropagation.

A few systems summarized here demonstrate that in

vitro propagation has the potential to be applied for

33
operational propagation programs. As techniques

are improved to raise the frequency of plantlet

production, lower labor costs, guarantee clonal

fidelity, propagate mature genotypes, and integrate

value-added traits into propagules, it is likely that in

vitro propagation systems will be increasingly

applied for operational propagation of hardwood

forest trees.

ACKNOWLEDGEMENTS

The authors wish to thank the following people

for their assistance in providing much of the

information in this review: Dr. John Preece, Dr.

Steven Wann, Dr. Rafique Uddin, Dr. C.S. Prakash,

Dr. Gary Coleman and Dr. Harry Sommer.

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37

REGENERATION OF THE GENETICALLY ENGINEERED CONIFER -

THE IMPORTANCE OF THE BIOLOGICAL SYSTEM’

David D. Ellis, Alex M. Diner and Yinghua Huan2

Abstract.--Genetic engineering of forest trees offers a unique opportunity to

selectively add single important traits to superior trees while still maintaining

the mix of genes responsible for the superior phenotype. Within the conifers,

the only two examples of stable regeneration of transformed plants include

transformation of Larix decidua by Agrobacterium rhizogenes and transformation

of Picea glauca by particle acceleration. Reliance on identifying particular attributes

of the biological system into which the DNA is delivered, has played an important

role in the development of these two transformation systems. While the precise

attributes that are important for allowing gene transfer are unknown, numerous

factors such as the meristematic state of the explant, the rate of cell division and

the age of the explant all play a role3.

INTRODUCTION

Genetic engineering of forest trees offers several

possibilities to complement traditional tree breeding

programs. Genetic engineering technology can

selectively add a single trait to superior trees while

still maintaining the existing combination of genes

responsible for the superior phenotype. Then, with the

use of vegetative propagation methods, genetically

engineered individuals can be clonally multiplied.

Although there are few genes currently available for

‘Paper presented at the 1992 Southern Regional

Information Exchange Group Biennial Symposium,

Huntsville, AL, July 8-10, 1992.

2Department of Horticulture, University of

Wisconsin-Madison, Madison, WI; USDA Forest

Service, Southern Forest Experiment Station, Alabama

A&M University, Huntsville, AL; Forestry Dept.,

Oklahoma State University, Stillwater, OK

3Acknowledgments: The authors would like to

thank Dr. Ken Raffa and Rodney Serres for their

critical reviews of the manuscript.

traits important to forest trees, the outlook is

promising that genes for such traits as lignin

biosynthesis, specific gravity, and pest resistance will

be available in the near future. Unfortunately,

methods to genetically transform forest trees are

available for only a few species, including Populus

(poplar) (Fillatti et al. 1987, Pythoud et al. 1987,

McCown et al. 1991), Lireodendron tulipifera (yellow

poplar) (Wilde et al. 1992), Larix decidua European

larch (Huang et al. 1991) and Picea glauca (white

spruce) (Ellis et al., submitted). This limited success is

not due to the inability to insert foreign DNA into plant

cells; rather it is due to the scarcity of tissue culture

systems for the regeneration of plants containing the

inserted genes.

There are only two examples within the conifers, of

regeneration of genetically engineered trees.

Transformed Larix plants were regenerated using

Agrobacterium, a plant-pathogenic bacterium which

during infection transfers a segment of its DNA into

the plant genome (Huang et al. 1992). In this case, the

apparently high susceptibility of seedling tissues to

inoculation with Agrobacterium and the competence of

those tissues for hairy root or shooty tumorigenesis

proved critical for success. Genetic transformation of

Picea was achieved using microscopic gold particles

which were coated with DNA and propelled into cells

by a process known as particle acceleration (Ellis et al.,

39
submitted). In this case, the identification of a specific

stage of embryo development was important not only

for the regeneration of transformed plants, but also for

the expression of the introduced genes. Clearly, the

reliance on the identification of particular

developmental stages of plants used for transformation

studies is crucial not only for expression of foreign

genes, but also for the ability to regenerate

transformed plants.

Successful transformation of plants depends on

matching several biological determinants with many

physical constraints. With the various gene transfer

methods currently available, simple placement or

transfer of DNA into a plant cell is no longer a limiting

factor. However, targeting of the DNA to a particular

cell within a complex tissue or organ competent for

regeneration is a major limitation. Further,

microculture methods which complement gene transfer

technology do not exist for most plants. Even when a

microculture method does exist for a particular plant,

the identity of those cells which divide to form new

plants is often unknown. In addition, the cell

containing the DNA must be competent to express the

introduced genes. This paper discusses these areas of

gene transfer in relation to coniferous forest trees, and

speculates on the different causes and effects which

cellular biology may have on the success of gene

transfer.

GENE TRANSFER

Genes which are transferred into plants, are placed

into single cells. Simple penetration of the cell wall

and plasma membrane is only a small portion of the

process. The DNA must then pass through the

cytoplasm and the nuclear membrane, before finally

arriving intact in the nucleus. In order for a stable

transformation event to occur, the DNA must then be

incorporated into the plant’s genome. Although it is

known that all of these events must happen, the

mechanisms involved from the entry into the plant cell

to the integration into the plant genome are unknown.

However, the use of inoculation/regenerationcompetent cells is critical; thus: the need to determine

the temporal “window of competence” for such cells.

Once integrated into the genome of a particular cell,

those genes on the inserted sequence must be

expressed in order to establish a functional

transformation event. Expression of the inserted genes

depends on several factors including 1) the site of

insertion and the copy number of the inserts, 2) the

promoter or regulatory element driving the gene, 3) the

gene itself, 4) the specific types of tissues or organs for

inoculation, and 6) the physiological and developmental

state of the transformed cell. Although each of these

factors is discussed briefly, focus is placed on the fourth

variable, the state of the recipient cell.

GENE INSERTION

There is some evidence that introduced genes can be

targeted to a particular site in the genome by

homologous recombination; yet in general, the site of

DNA insertion is assumed to be random. Although

insertion may be random, the effect of (1) those DNA

sequences flanking the introduced DNA, (2) the

number of copies and (3) the sequence of the introduced

DNA will all affect gene expression. The expression

level of the introduced DNA was postulated to be

higher if the DNA were integrated into, or close to an

area in the genome which is being actively transcribed.

Although the precise mechanisms by which the

flanking genomic sequences affect the expression of the

introduced DNA is unknown, it is clear that certain

regions of the genome, such as the condensed regions

containing heterochromatin, are not actively

transcribed. It therefore follows that DNA inserted

into these regions will also not be actively transcribed.

Further, inserted endogenous promoters might

override regulatory elements used to drive the

introduced genes. Although little experimental

evidence exists to explain the influence of endogenous

flanking DNA sequences on the expression of

introduced DNA, the influence of the endogenous DNA

on the expression of inserted DNA is generally referred

to as the “position effect”.

CO-SUPPRESSION

Multiple copies of genes were also been shown to

influence introduced gene expression. Known as “cosuppression”, this phenomenon accounts for the

suppressed expression of introduced DNA when either

multiple copies of a gene are inserted into the genome,

or when introduced DNA encodes for a gene already

present in the genome. The best example of this latter

case is in Petunia where introduction of an exogenous

chalcone synthase (CHS) gene shut off expression of the

endogenous gene (Napoli et al. 1990). In some cases,

methylation of the DNA is associated with cosuppression; yet this has not been shown to be

responsible for the suppression of gene expression in

all cases of co-suppression.

40
PROMOTERS

Each somatic cell within a plant theoretically

contains the same complement of genes. What

differentiates a leaf from a stem or flower is the

expression of which genes are turned on or off in the

cells in these tissues or organs. The regulatory

sequences of DNA responsible for the expression of one

set of genes and not another are known as promoters.

Promoters are sequences of DNA adjacent to the gene

which contain the necessary information to precisely

regulate the expression of the gene so that it is

expressed only in the proper cell type and at the proper

time during development. It has been generally

assumed that a specific regulatory sequence, or

promoter, can be used to express any gene attached to

it in the manner programmed by that promoter.

Therefore, the expression of introduced genes could be

restricted to certain tissues or developmental stages.

Clearly, the promoter used to drive the expression of

the introduced gene plays a major role not only in the

overall level of gene expression, but also in the location

within a regenerated plant in which the genes will be

expressed. While there are no reports on the

differential expression of genes by different promoters

in transformed forest trees, numerous studies have

investigated the expression of various genetic

constructs using transient assays (Bekkaoui et al.

1990, Wilson et al. 1989, Ellis et al. 1991).

Since the mechanisms responsible for expressing

genes are present throughout the nucleus, an

introduced gene can be expressed prior to stable

integration into the genome. Such expression is known

as transient gene expression. Therefore, with transient

gene expression assays, the expression of different

promoters can be tested quickly in various cell types

without the need for stably incorporating the DNA into

the genome. The abovementioned studies on transient

expression in conifers have confirmed the expression of

a number of angiosperm promoters. The ability to use

angiosperm promoters to express genes in conifers

demonstrates the universal nature of the genetic code

and opens the door for differential regulation of

inserted genes in conifers. This is crucial because few

conifer promoters have been isolated.

While these studies have shown that angiosperm

promoters function in conifers, transient assays have

failed to demonstrate tissue-specific expression (Ellis

et al. 1991). The ability to rapidly test for tissue- or

cell-specific expression using transient assays would be

a very powerful tool, as the differential expression of

genes in long-lived perennials may be very useful. It is

important to note, however, that although these

promoters do not function in a tissue-specific manner

in transient assays, they may express a gene in a

predicted inducible or tissue-specific manner once

stably integrated into the plant genome. This has been

the case in herbaceous plants where promoters did not

give the predicted inducible or tissue-specific

expression in transient assays, but showed proper

expression when stably integrated (D. Russell, personal

communication). There is no reason to expect that the

predicted inducible or tissue-specific expression from

these promoters would not occur in forest trees once

genes are stably integrated.

To date, all the examples of stable integration of

introduced DNA in forest trees have utilized promoters

which are not generally expressed in an inducible or

tissue-specific manner. Examples of these are

cauliflower mosaic virus (CaMV) 35s promoter and

nopaline synthase (nos) promoter (McCown et al. 1991,

Ellis et al., submitted). As transformation systems are

developed for forest trees, the testing of numerous

promoters in stably transformed plants will surely

follow.

STABLE GENE TRANSFER USING

AGROBACTERIUM

Several studies have focused on the identification of

Agrobacterium strains which infect conifers (Sederoff et

al. 1986, Clapham et al. 1986, Ellis et al. 1989, Morris

et al. 1989, Stomp et al. 1990, Huang et al. 1991,

Huang and Tauer, personal communication). These

studies demonstrated some important factors for the

application of Agrobacterium for gene transfer in

conifers including, 1) a high degree of variation in

infectivity by different Agrobacterium strains; 2)

infectivity is different for different conifer species or

genotypes; 3) infectivity is dependent on the

developmental stage of the tissue inoculated. The

original approach to Agrobacterium work in conifers

was to confirm that Agrobacterium would infect

conifers similar to other plants and then to identify

Agrobacterium strains which could infect conifers at a

high frequency. As a serendipitous by-product of this

approach, developmental stage-specific interactions

affecting Agrobacterium infection were identified. For

example, all Agrobacterium tumefaciens strains

infectious to white spruce seedlings infected young

tissue at a higher frequency than older tissue (Ellis et

al. 1989). A number of these strains also had a higher

frequency of infection on the younger tissue of both

Picea engelmannii (Engelman spruce) and Picea

sitchensis (Sitka spruce).

41
Infection by Agrobacterium of younger actively

growing tissues is not unique to spruce and was

reported in other conifers (Diner and Karnosky 1987,

Morris et al. 1989, Stomp et al. 1990). Possible

explanations for the higher incidence of infection in the

younger tissue could be a lack of resins in younger

tissue (Diner and Karnosky 1987), differential phenolic

production following wounding in old vs. young tissue

(Ellis et al. 1989), the relative level of stem woodiness

(Stomp et al. 1988) and differences in the rates of cell

division (A. Stomp, personal communication).

Interestingly, the level ofAgrobacterium infection was

the same in both young and old tissue in Pseudotsuga

menziesii (Douglas-fir) (Ellis et al. 1989), suggesting

that older tissue in Douglas-fir does not undergo the

same rapid changes associated with maturation that

occur in the other species. In addition to species and

ontogeny specificity, successful inoculation with

agrobacteria is also genotype-dependent. Recent

results from studies of loblolly pine genotype

transformation potential suggest that host genotype

moderates infection (Huang and Tauer, personal

communication).

LARIXAS A CONIFER MODEL FOR

AGROBACTERIUiW INFECTION

Larix decidua has proven to be very amenable to

transformation, tumorigenesis and the regeneration of

transformants using Agrobacterium vectors.

Preliminary attempts in 1985 to inoculate L. decidua

germlings with A. rhizogenes pRi11325 resulted in the

differentiation of tumorigenesis within 30 days. The

choice of germlings was deliberate (Diner and

Karnosky 1987), although use of older hosts was later

attempted (Huang et al. 1991). The extreme

susceptibility of succulent, rapidly growing tissues of

other conifers to inoculation with agrobacteria has

since been more broadly investigated (Ellis et al. 1989,

Huang and Tauer personal communication, Morris et

al. 1989). In Larix, tumor morphology at inoculated

apical wounds included multiple shooty clusters, later

shown (Huang et al. 1991) to be transformed

regenerants. Tumor morphology was specific to the

site of wound-inoculation. Inoculation of a very shallow

wound through the center of the shoot apex induced a

large, confluent teratoma-like mass of tiny buds, all of

which failed to develop further (A. Diner, unpublished).

Inoculation of the apex wounded more deeply and

through the cotyledonary axis resulted in up to 10

progressively developing shoot buds, while only roots

grew from inoculated wounds made to the lower

hypocotyl. These specific tumor types could form

either individually on an appropriately inoculated

germling, or collectively on a single germling

inoculated with pRi 11325 at the three respective sites.

In contrast, wound-inoculation of L. decidua

germlings with A. rhizogenes A4pARC8 (Simpson et al.

1986) at any of these same three sites elicited only root

tumorigenesis (A. Diner, personal communication).

Frequency of tumorigenesis was virtually lOO%,

similar to that using pRi11325; transformation was

verified by Southern blot analysis (Y. Huang, personal

communication). Only infrequent host death by

unidentified causes, precluded confumation of

successful transformation.

Transformed regenerant plants are not uncommon

consequences of tumorigenesis induced either by A.

tumefuciens or A. rhizogenes (Ondrej et al. 1984, Ondrej

and Biskova 1986, Shahin et al. 1986, Jouanin et al.

1987, Noda et al. 1987, Morris and Robbins 1992),

while spontaneous, well-defined shoot organogenesis

from in situ Agrobacterium-induced tumors is more

rare.

Shoot or root tumorigenesis may be a result of

concentrations and proportions of phytohormones to

which the transformed cells are exposed in their

microenvironment, as well as to the duration of that

exposure. Differential morphogenesis from cell

cultures or organized tissue explants has been

classically described to be under phytohormone control.

It is unlikely that the organogenic role of a

phytohormone is direct; control of the organ-initiating

event is more likely mediated by substances whose

syntheses are promoted or’ inhibited by the respective

phytohormone(s). Indeed, both those transformed

tissues (roots and shoots) which are phytohormoneindependent and those normal tissues to which

phytohormones are exogenously applied elaborate

novel transcription products, several of which appear

to be shared (Burrell et al. 1986). In addition to the

phytohormone independence conferred by

transformation (Klee et al. 1987), there is an

enhancement of tissue sensitivity to phytohormones

(Maurel et al. 1991). Organogenesis may thus be an

almost spontaneous consequence in a population of

cells transformed both to auxin independence and

extreme sensitivity.

Studies suggest that tumorigenesis and tumor

morphology are controlled by 4 factors: (1) the plasmid

(Klee et al. 1987), (2) the host (Delmotte et al. 1991), (3)

their interrelationships (Stachel et al. 1985), and (4)

the site ofAgrobacterium inoculation of the host plant

(Gresshoff et al. 1979, Huang et al. 1991) which may be

merely localized anatomical expressions of factor three.

Certainly, there are physical and temporal gradations

42
in the availability of all pertinent phytohormones in

the shoot. The concentration gradients of auxin and

other phytohormones in the shoot may thus be

responsible for effects on morphology (Burrell et al.

1986) and gene transcription (van Slogteren et al. 1984)

in transformed shoots grafted to normal stems, and

may help to explain the site-specific Larix tumor

morphology developed from inoculation with A.

rhizogenes pRi11326, as described above. Beyond cell

competence for differentiation and regeneration, tumor

morphology in some hosts depends upon the specific

plasmid employed (Huang 19911. In the case of the two

most studied Ti plasmids, the octopine type induces

undifferentiated tumors , while the nopaline type

induces teratomous tumors with some shoot

differentiation. Generally speaking, transformed plant

cells carrying wild-type T-DNA of an armed Ti plasmid

are unlikely to be regenerated to plants. However,

successful examples of plant transformation and

regeneration using oncogenic strains of agrobacteria

have been reported in a variety of angiosperm species

(Tepfer 1990, Yang and Simpson 19811. Further use of

the A. rhizogenes Ri plasmids is relatively

straightforward for regeneration of whole plants

following tissue transformation, as compared to the Ti

plasmid. As we see it, A. rhizogenes strain 11326 is a

special strain, because it carries the nopaline-type Ri

plasmid. The pRi11325 is a unique system for gene

transfer and recovery of transformants via spontaneous

shoot organogenesis in European larch (Huang et al.

1991).

That very young (nonwoody) conifer tissues appear

so susceptible to inoculation and transformation by

Agrobacteria (Diner and Karnosky 1987, Morris et al.

19891, may be less a consequence of the absence of

bactericidal secondary products in those tissues, than

the simple availability of viable, physiologically

dynamic tissues to which a particular strain of

Agrobacterium may be virulent. Cell cycle phases exert

considerable effects on transformation efficiency using

particle bombardment (Iida et al. 1991). Synchronous

cells bombarded at M and G2 phases were transformed

at 4 to 6 times the frequency of those bombarded at S

and Gl phases. In order to develop effective protocols

for integrative transformation and regeneration, some

studies have focused on the use of meristematic cells as

targets (Potrykus 1991). These types of cells have been

shown competent for transformation, but are often not

competent for regeneration in many plant species

generally considered recalcitrant to that process. This

may suggest that competence for transformation is not

pertinent to competence for regeneration. Possibly, a

specific cellular developmental stage is liable to

differentiation, particularly in response to some

43

trigger. In the case of spontaneous organogenesis

following transformation, we speculate that a “window

of competence” exists for a cell, tissue or organ, and is a

specific and short fraction of a developmental stage.

Thus, synchronous cells may be collectively competent

for gene transfer and regeneration (Huang 1991). This

may be one plausible explanation for initiation of

adventitious shoot buds from transformed cells of

European larch.

Certainly, the mere fact that cells may be in

synchrony ought not to be pivotal to their

transformation frequency in Larix apices or hypocotyls

inoculated with Agrobacterium, though the juvenility

and active growth of germlings implies that some

synchrony likely occurs at least adjacent to the

meristem, potentiating the availability of cell

aggregates collectively undergoing common phases of

division. The transformation and early cell

“commitment” to specific tumor morphology in any of

these aggregates may regulate similar commitment by

other transformed cells in that or another aggregate.

There are suggestions that pre-existing primordia

influence the character of primordia formed later

(Poethig 1991).

However, another form of “synchrony” appears to be

maintained in older tissues of woody species, causing

rhythmic and direct effects on cambium division and

differentiation (Stieber 1985). Stieber suggested that

unidentified, mobile “function-determining factors” act

in synergy with auxin to control cell differentiation.

These factors, if real, may also affect tissues of the

germling such that their concentration(s) at any locus

synergistically controls the early differential

morphologic development of organs from a transformed

cell.

STABLE GENE TRANSFER USING PARTICLE

ACCELERATION

To achieve transformation using particle

acceleration, microscopic gold or tungsten particles are

coated with DNA and shot into cells. Although

generally inefficient in yielding stable integrative

transformation, one major advantage of this technique

over other direct gene transfer techniques is that

particle acceleration allows the transfer of genes into

cells intact in tissues. Theoretically, DNA can be

inserted into any cell which is impacted by the particle.

Foreign genes were expressed in all conifer tissues

exposed to particle acceleration including embryos,

seedlings, megagametophytes, xylem, pollen, needles,

buds, cell suspension cultures, embryogenic callus, cell
aggregate cultures and roots. While almost all of this

expression was transient, it yielded valuable

information on factors involved in the expression of

introduced genes in the various tissues competent for

regeneration. For example, freshly excised embryos of

white spruce expressed introduced DNA at a relatively

low level. Pretreating the embryos on bud induction

medium containing cytokinin (Ellis et al. 1991) prior to

particle acceleration resulted in a dramatic increase in

the level of gene expression. This increase continued

for up to seven days of pretreatment, and then

stabilized so that a longer pretreatment period was

ineffective. Clearly, whatever change was induced by

the bud induction treatment which was responsible for

the increase in gene expression, occurred within seven

days.

The cellular changes which occurred on bud

induction medium were well documented for numerous

conifer species. Within this seven day period, changes

in cell division patterns from anticlinal to periclinal

were observed which resulted in the proliferation of

meristemoids. These meristemoids continued to

divide, and within seven days the epidermal and

subepidermal cell layers associated with the

meristemoids contained localized areas of densely

cytoplasmic isodiametric cells characteristic of

meristematic cells. The induction of meristematic

tissue was suggested to be important for the expression

of introduced genes in spruce (Ellis et al. 19921, poplar

(McCown et al. 1991) and Vuccinium (cranberry)

(Serres et al. 1992). The dependence of gene expression

on a specific physiological and developmental stage of

the cell may relate to the above discussion concerning

Agrobucterium, in which both biochemical changes and

cell attributes such as cell division rates were thought

to be related to the ability ofAgrobacterium to infect

tissues.

Whether the expression of foreign genes in woody

plants is directly related to meristematic cells and

whether the cells which are induced with a

pretreatment are truly “meristematic” have been

debated. The important issue, however, involves

identification of those cellular attributes which account

for their higher competence to express introduced

genes. If the analogy to a meristematic state is

continued, it can be assumed that these cells have the

ability to rapidly divide, and that they have a high rate

of metabolic activity. These two factors may be related.

Further, a cell with high metabolic activity would

likely be active in endogenous gene expression with all

the functions for gene transcription and translation

actively expressed. It would only follow then, that

genes introduced into such a cell would also have a

higher probability of being expressed.

How this may be related to the regeneration of

stably transformed plants is that 1) DNA replication

may aid in the incorporation of foreign DNA, and 2)

expression of introduced genes is required for selection

of transformants based on antibiotic selection. There is

indirect evidence that a certain phase (S phase) in the

cell cycle may be a prerequisite for DNA integration

into the genome. Possibly, the importance of this DNA

synthesis phase involves many DNA repair and

synthesis mechanisms which are active during this

phase and which may aid in the incorporation of the

DNA into the genome. Unfortunately, these

experiments are difficult both to perform and interpret.

Therefore this topic remains a black box. In contrast,

the expression of introduced genes is definitely

required for selection. The incorporation of a gene

encoding for resistance to an agent (antibiotic or

herbicide) which is normally lethal to a plant cell is

critical to the identification of a single cell containing

the introduced DNA in a large population of

untransformed cells. If the gene encoding resistance to

the selective agent were not expressed, selection for

that cell containing the inser’ted DNA would not be

possible.

PICEA AS A CONIFER MODEL FOR

PARTICLE ACCELERATION

The time course of gene expression over an eight

week period following DNA insertion by particle

acceleration was investigated in white spruce (Ellis et

al. 1992). The time course of expression with

numerous promoters is characterized by a high initial

level of gene expression which is maintained for seven

days. Fourteen days after particle acceleration,

however, this expression decreased dramatically to a

lower level of expression which was maintained for up

to eight weeks (56 days). This type of decay in gene

expression may allow for the selection of transformed

cells in a rapidly dividing plant system such as in

Nicotiana tabucum (tobacco). But in conifers which

grow considerably slower in culture, it is desirable to

maintain maximal gene expression over a longer period

of time. Interestingly, this characteristic time course

of gene expression remained unaltered with many

different cultural treatments. Some treatments, such

as placing the tissue at cooler temperatures (22’) after

particle bombardment altered the baseline level of gene

expression, yet had no affect on the decline of gene

expression after 7 days.

44
This rapid decline in gene expression was first

thought to represent the degradation of DNA in the cell

prior to its incorporation into the genome. However,

using an inducible heat shock promoter, it was

discovered that maximum gene expression was induced

for up to four weeks, suggesting that the DNA was

remained intact and that expression of the introduced

DNA was somehow shut off. This theory was recently

supported by the use of a heat shock, which resulted in

maximal expression activity of other promoters

maintained beyond the initial 7 day period. The

phenomena which caused the decrease in gene

expression following particle acceleration are presently

unknown. Yet, based on enzyme stability, it is known

that the introduced gene was probably transcribed

during the first few days following introduction into the

plant cell. Further, S-azacytidine has no effect on the

time course of expression, suggesting that methylation

(or azacytidine reversible methylation) is not the cause

of the loss of gene expression.

As discussed above, the developmental and/or

physiological stage of the target explant plays a major

role in gene expression. It is important to keep in mind

that the recovery of stable transformants is “a numbers

game”, since only a small percentage of the cells

containing the introduced DNA will express the genes.

And only a small percentage of those cells expressing

the introduced DNA will stably integrate the DNA into

the genome. Therefore, anything that is done to

enhance the probability of selecting out the cells

containing the integrated DNA will be advantageous.

One clear advantage is to sustain maximum expression

of introduced genes over a longer period. Interestingly,

in contrast to zygotic embryos, the time course of gene

expression in somatic embryos followed a different

pattern. With somatic embryos maximum gene

expression was maintained for over 3 weeks. This is

further support for the reliance on a biological entity

for sustained gene expression.

The difference in gene expression over time between

zygotic and somatic embryos may be explained by

reference to the discussion on the importance of a

“meristematic” state for transformation success.

Somatic embryos are more physiologically juvenile

than mature zygotic embryos. Since the former have

not undergone dehydration, many of the changes

associated with embryo maturation are incomplete.

Although synthesis of storage proteins is initiated in

later stage somatic embryos, these embryos can be

regarded as immature, as evidenced by their ability to

form embryogenic callus at a high frequency. In early

work on the initiation of embryogenic callus from white

spruce, it was found that immature embryos were the

most competent to form embryogenic callus (Becwar et

al. 1987). This supports the inference that somatic

embryos are more juvenile or closer to the

meristematic state, and may help to explain the

maintenance of maximum gene expression in somatic

embryos.

The ability to survive selection was as important as

the expression of the introduced genes in the

development of a transformation system for white

spruce. Since embryogenic callus is not initiated until

3-6 weeks after placement on induction medium,

keeping the embryo alive, while at the same time

suppressing non-transformed cells from dividing was

the strategy. Senescent conifer cells produce several

secondary products such as phenolics, which are

detrimental to the survival of surrounding cells.

Therefore, if selection pressure kills non-transformed

cells, the chances of recovering the few transformants

are greatly reduced due to autotoxicity. Again, the

developmental stage of the explant plays a major role

in developing a selection method. The young

precotyledonary stages of somatic embryos did not

survive for four weeks when exposed to a relatively low

level of kanamycin selection (5 pg/ml). In contrast, this

level of selection was inhibitory, yet not lethal to later

post-cotyledonary developmental stages.

The identification of the factors important for the

expression of foreign genes and selection of

transformants using the appropriate developmental

stage of somatic embryos competent to form

embryogenic callus, has resulted in development of a

transformation system for white spruce (Ellis,

unpublished). The system utilizes particle

acceleration, and during the course of the past year

more than 15 independent transformed embryogenic

cell lines were differentiated. All cell lines tested

expressed the 6glucuronidase (GUS) gene and were

resistant to ordinarily toxic levels of kanamycin,

indicating expression of the neomycin

phosphtransferase (NPT) gene. Transformed embryos

and seedlings were differentiated from most of these

embryogenic lines, and seedlings from two lines were

transferred to the greenhouse.

A third gene which has been inserted into all of the

transformed spruce lines encodes for the proteinaceous

insecticidal cryIA endotoxin from Bacillus thuringiensis

(B.t.). Preliminary evidence from feeding trials with

Choristoneura fumiferana (spruce budworm) indicates

that this gene was expressed at a low level. Parallel

studies by our group in entomology have observed the

feeding behavior of insects and toxicity of this protein

to spruce budworm, and showed that sublethal levels of

45
the toxin inhibit feeding, causing a decrease in larva

weight. This property of B. t. is a good indicator of B.t.

expression in transformed tissues (Ramachandran et

al., in press). When spruce budworms were fed either

transformed embryogenic callus or seedlings, there was

a significant decrease in larval weights after 6 days,

compared to weights of those larvae placed on control,

non-transformed tissue.

CONCLUSIONS

Development of a gene transfer technology for forest

trees, particularly conifers, depends on a complex set of

biological interactions which operate on the frontiers of

modern molecular biology. The precise mechanisms for

gene insertion to the genome of plant cells is in need of

considerable study. Once inserted, gene expression

depends on the interaction of numerous factors. In

attempting to match and overlap these factors to

optimize a transformation method, it is easy to lose

sight of the importance of the biological (plant) aspect

of this process. As more work is done with

“recalcitrant” or underexploited species, it is clear that

the developmental and physiological stage of the tissue

plays a crucial role in transformation success.

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Jouanin, L.; Guerche, P.; Pamboukdjian, N. [and

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McCown, B.H.; McCabe, D.E.; Russell, D.R. [and

others]. 1991. Stable transformation of Pop&us and

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4 7
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48
Rooted Cuttings

Moderator:

Hans van Buijtenen

Texas Forest Service and

Texas A&M University

College Station, Texas

49

MACROPROPAGATION OF CONIFERS BY STEM CU’ITINGS1

Farrell C. Wise and Thomas D. Caldwell

Abstract. Trees, particularly conifers, have not been readily propagated

by stem cuttings. This document groups significant biological barriers to

rooting into major problem areas, and provides examples and possible solutions

from the conifer literature within each area. Barriers discussed are genotypic

variation, maturation, variability within trees, inadequate testing of complex

cultural influences, and poor early form. Although advances have been adequate

to allow large-scale rooting in some species, macropropagation is likely to become

more widely adopted when components of these biological barriers, and especially

their interactions, are investigated more deeply. Four key research issues, of

both biological and logistical natures, must also be resolved before cuttings of

any specific species can be commercially rooted on a large scale. A commercial

example of overcoming biological barriers to rooting of loblolly pine cuttings is

presented.

INTRODUCTION

Growers of coniferous trees have tried to apply

asexual propagation techniques to their crops for

many years. Successes have been difficult, but have

been achieved by identifying key physiological

barriers and then adapting techniques to capitalize

on or enhance natural variability. This document

concentrates on attempts to propagate conifers by

stem cuttings, and demonstrates how major

problems have been attacked.

Specifically, answers will be provided to this question: “Why has mass propagation of coniferous trees

via stem cuttings been so difficult?”

lPaper presented at the 1992 Southern Regional

Information Exchange Group Biennial Symposium,

Huntsville, AL, July 8-10, 1992.

2Research Scientist, Westvaco Corporation, Box

1950, Summerville, SC 19484; Research Biologist,

USDA Forest Service, Southern Forest Experiment

Station, Box 2008, GMF, Gulfport, MS 39503.

Five major barriers or problem areas are

discussed: (1) the weak or variable genetic rooting

capacity of many desirable species, (2) reduced

rooting and growth due to maturation, (3) rooting

variability among cuttings collected within a tree,

(4) insufficient testing and incomplete success of

cultural enhancements, and (5) poor form of rooted

cuttings from some species. Since many

physiological processes are interrelated,

physiological influences on rooting are likewise

interrelated, and these five areas are therefore not

completely discrete. The authors have selected this

format primarily for ease of communication and will

identify interactions between topics as appropriate.

For each area, literature focusing on stem cutting

propagation has been chosen to illustrate specific

problems and solutions. Rather than reviewing all

pertinent works within a problem area,

representative data that make good examples will be

discussed. Examples are world-wide, but many are

from North America. These works appear within

forestry, Christmas tree, and horticulture literature.

A few examples use plant parts other than stem

cuttings and these have been distinguished by

underlining (e.g. hypocotyls) for clarity.

51
INADEQUATE GENETIC ROOTING

CAPACITIES

Problem

Many desirable species can be rooted only on an

experimental scale. In reviewing research

publications that address rooting, there apparently

has been no definitive comparison of rooting

variability among the more important conifers.

However, enough information exists to categorize

the various species as easy, moderate, or difficult-toroot regardless of the phenotypic traits, climatic

conditions, or seasonal changes associated with,

vegetative propagation. This is not to say that any

one species will never achieve acceptable levels of

rooting, but that some species will require more

intensive propagation methods. The following

describes one species chosen from each of the

categories to better illustrate the genetic variability

in rooting.

Vegetative propagation of Norway spruce (Picea

abies L. [Karst.]) is well established in Europe.

Stem cuttings of this species form adventitious roots

at their bases quite easily, and results have been

well documented (Kleinschmit 1977, Bentzer 1981).

Rooting rates of 95% or greater have been

commonplace. Propagation methods that were

described by these authors, although similar in

nature, contain sufficient differences to be labeled

unique, yet all have produced impressive rooting

percentages.

Slash pine (Pinus elliottii Engelm.), a moderate

species, has rooted at satisfactory percentages when

exposed to outdoor nursery beds (Frampton and

Hodges 1989) or controlled greenhouse

environments (Reines and Bamping 1960). Rooting

from hedged trees has ranged from 0% to 73%,

depending on specific techniques employed (Bower

and van Buijtenen 1977, Cunningham 1981). It has

typically rooted within the 65-75% range for some

refined trials (unpublished data, T. D. Caldwell) and

is considered to be easy to root by some ‘researchers3.

There is no full-scale production system for this

species at this time,

3R.H. Smeltzer, Section Leader, Asexual

Propagation, Forest Productivity and Research,

International Paper, Route 1, Box 1, Bainbridge GA

31717.

Loblolly pine (Pinus taeda L.), considered to be

the most economically important of the southern

pines, has the added distinction of being one of the

most difficult conifers to propagate by rooted

cuttings (Gardner 1929, Greenwood and Nussbaum

1981, Foster and Shaw 1987). Most attempts to root

loblolly pine have generally reported successes

within a range of 0%-60%. In direct comparisons

with slash pine, cuttings have consistently rooted

more poorly, with infrequent exceptions (Reines and

Bamping 1960, Reines and Bamping 1962, Frampton

and Hodges 1989). As with slash pine, the

propagation methods are quite varied (Foster 1988,

Foster and Shaw 1987, Greenwood and Nussbaum

1981, Frampton and Hodges 1989).

Solutions

Selection.--Despite poor overall rooting with some

species, there is encouraging information showing

rooting variability among full-sib families within all

species tested, and even greater variability between

members of the same family (Foster 1990, Anderson

et al. 1992, Haines et al. 1992). Recent work

(Haines et al. 1992) attempted to determine

relationships between rooting and morphological

features of shoots from hedges of five full-sib

families of hybrid tropical pines (P. caribaeu Morelet

var. hondurensis Barr. and Golf. X P. tecunumanii

(Schw.) Eguiluz and Perry). Rather than finding

strong morphological markers, the genotype identity

(clone) proved to be the best indicator for both

greenhouse and field responses, followed by the

family identity. Significant associations with

morphological features will be covered later.

This information indicates that screening for

families and individuals that possess strong rooting

capacities can have a significant positive impact on

the overall rooting success of a propagation program.

Obviously, selecting for good rooting could reduce

potential growth gains, but the exact impact is not

known. There is evidence with western hemlock

(Z’suga heterophylla (Raf.) Sarg.) that there may be

a positive genetic correlation between rooting ability

and initial growth of the rooted cutting (Foster et al.

1985). Regardless, this type of screening process can

be conducted readily.

Understanding the Molecular Basis.--The longterm need is to better understand the role of gene

expression in adventitious root initiation. It is fairly

well established that rooting ability has a strong

genetic component. Bower and van Buijtenen (1977)

5 2
demonstrated variation among clones of slash pine.

In screening several hundred loblolly clones, Foster

(1990) found that a wide range of cuttings rooted.

Determining what is different between a hard-toroot genotype and an easily-rooted genotype can

possibly lead to the identification of the gene(s)

involved in the actual rooting process. Haissig et al.

(1992) have recently reviewed strategies for

understanding the genetic control of rooting that

would use recent developments in molecular

genetics to compare rootable and non-rootable plants

at the levels of transcription and translation. Initial

studies most likely would not employ conifers

because the described technologies are more readily

applicable to the herbaceous species and woody

angiosperms used to develop them. However, the

implications of discoveries made in these studies

should be far-reaching, and conifers should be

adaptable to some of the described approaches.

Summary

The capacity to form adventitious roots under

specific conditions varies widely both within and

between conifer species. For desirable species that

root poorly, selecting outlying families or individuals

that root well can greatly improve overall

propagation success, but the impact on other

desirable traits is unknown. When selection can be

coupled with cultural procedures that maintain

juvenility and that otherwise assure strong rooting,

even highly recalcitrant species should be adaptable

to cutting propagation.

PRONOUNCED MATURATION EFFECTS

Problem

Poor Rooting.--Gardner (1929) was one of the first

to conclude that the capacity for stem cuttings to

form roots declined rapidly with tree age. Among

the 21 species he attempted to root were seven

conifers, including four pines. Even at that time

pines were “ordinarily considered very difficult to

propagate by cuttings.” One-year-old wood from

these pines rooted reasonably well at age one (46-

98%), and much poorer two years later (O-12%).

Loblolly pine was the most recalcitrant. Although

data were not presented, the ‘author also noticed

that cuttings from one-year-old trees rooted faster

than those from older plants.

Although Gardner established the principle of

declining rooting with age, he used only limited age

5 3

ranges, and a rooting environment that was only

partially defined and presumably poor. Steele and

others (1990) rooted dormant cuttings of Sitka

spruce (Picea sitchensis (Bong.) Carr.) taken from

the uppermost whorls of trees of various ages

between 1 and 39 years. They were rooted under

fairly uniform greenhouse conditions, and percent

rooting data were fitted to the Gompertz equation.

For this species, rooting declined dramatically

between ages 4 and 11 indicating a pronounced

maturation effect for this character.

Rooting is more precisely connected with phase of

the cutting wood than age of the donor plant. The

capacity to form adventitious roots is one of several

characteristics that have been associated with the

juvenile phase. As a seedling develops can occur on

different parts of the same tree. In general, the

lower- and inner-most areas contain tissues that

remain juvenile. Therefore, rooting capacity should

be expected to vary with the cutting’s original

position in a tree.

Grace (1939) was one of the first to demonstrate

this phenomenon with conifer cuttings. Dormant

cuttings were collected from the upper and lower

thirds of an l&year-old Norway spruce tree and

rooted in a greenhouse propagation frame. Not only

did cuttings from the more juvenile lower crown root

more often than those from the upper crown (75% v.

43%), but they also produced longer roots (15 v. 9

cm). Some control over the decline in rooting with

maturation can therefore be exerted by selecting

cutting wood from the lower sections of trees.

Poor Growth.--Unfortunately, cuttings that have

been rooted from somewhat mature material may

grow slowly or be otherwise affected. Early results

from a slash pine trial revealed a dramatic decline in

height growth as the source for cuttings advanced in

age (Franklin 1969). More recently, cuttings from

seven-year-old radiata pine (Pinus rudiata D. Don)

trees were planted with 1.5/O nursery seedlings and

assessed annually for 15 years. Heights were

statistically similar for both propagule types

throughout the study, but diameter growth was

greater for the seedlings (Klomp and Hong 1985).

Further analyses (Menzies and Klomp 19881

indicated that cutting-derived trees had about 13%

less volume under bark, primarily due to reduced

diameter growth (Table 1). In summarizing

many radiata pine studies, these authors state that

there is virtually no growth reduction for cutting

donors of radiata pine up to age three, but that older
sources induce slower diameter growth and poorer rejuvenate mature material, there are no consistent

rooting. methods for complete rejuvenation.

Solutions

Rather than just accepting the limitation of maturation, tree propagators have attempted to improve

rooting success by managing the juvenile phase of

the donor material. This can be achieved in many

ways, but these approaches generally involve either

rejuvenation of mature material or maintenance of

juvenility,

Rejuvenation.--Hackett (1985) has summarized

various approaches used to attempt rejuvenation in

woody plants. The most germane to mass

Maintenance ofJuvenility.--Until rejuvenation

techniques are perfected, selection and development

of clones must rely on the maintenance of juvenility.

One strategy is serial propagation, which

theoretically uses the rejuvenating influence of

successive rooting of cuttings from previously rooted

cuttings to maintain high rooting potentials.

Rleinschmit’s (1974) early work describes extensive

development of Norway spruce clones where rooting

averaged 90%. Although repropagation from rooted

cuttings for several “phases” did not improve

rooting, it was maintained at these high levels.

Table 1. Growth of radiata pine rooted cuttings from 7-year-old

trees and of 1.6/O nursery seedlings after 15 years. Condensed

from Menzies and Klomp (1988).

Difference Statistical

Response Cuttings Seedlings V . significance of

seedlings’ difference’

Height 19.4 19.8 -2% ns

Cm)

Diameter

at breast

height

kal)

34.5 37.2 -1% * *

Volume

Cm’)

0.71 0.82 -13% **

‘RCuttings - seedlings) ! cuttings] X 100.

2ns=not significant, **=t-test significant at 0.01% level.

propagation of conifers by cuttings involves,

ironically, the “rejuvenation” of plants by the

rooting process itself. As an example, cuttings were

rooted from 11 Douglas-fir (Pseudotsugu nenziesii

(Mirb.) France) trees that were 12-22 years old from

seed, and clonal field plantings were established

from them. Three years after rooting, cuttings were

collected from these trees and another set was

collected from the lower crowns of the original

seedling donors (Black 1972). Rooting was much

greater from cuttings that originated from the

grown rooted cuttings (38% vs. 11% for those from

original donors) even though both donor types were

genetically identical and both were the same age

from seed. In addition, needle and bud morphologies

were reported to have reverted to more juvenile

appearances than those on the original donors.

Although this approach and others may partially

Unfortunately, recent work (Dekker-Robertson

and Kleinschmit 1991) indicates that subtle but

important maturation does occur with serial

propagation. Norway spruce clones were derived

from four-year-old trees and then repropagated from

rooted cuttings at three-year intervals for a total of

seven serial propagation steps (“phases”). New

clones were also started from new four-year-old

seedlings at each three-year interval and similarly

repropagated. Measurements of nursery plants,

made three years after rooting, were averaged for

each phase and compared with those of non-selected

seedlings. Several indicators were significantly

decreased by later propagation phases. For

example, height at age three was reduced from

about 36 cm after one propagation phase to about 32

cm after six phases. Since several measures were

highest after the second propagation phase, it was

postulated that the rooting process may have

reinvigorated the shoot meristems, rather than

rejuvenating them, presumably by bringing them

closer to the root systems. The authors also

demonstrated that the maturation effects could be

reduced by eliminating the slowest-growing (and

perhaps most mature) clones from the last three

phases. The net disadvantage, however, was that it

would take lo-15 years to identify superior clones

and that these clones would be adequately juvenile

for mass production for only another 10 years.

Shearing growth to prevent cuttings from arising

from mature wood is another technique to maintain

juvenile rooting. Copes (1983) began with lo- and

13-year-old Douglas-fir trees of 14 clones, pruned

the lower 2 m annually, and rooted dormant cuttings

from the sheared regions over eight years. Rooting

capacity over all clones was not just maintained but

actually increased about 3% per year to a maximum

5 4
of 67%. Therefore, meristems were again apparently

partially rejuvenated or reinvigorated.

Heavy shearing of young plants to arrest

maturation in cutting wood is currently practiced

with several species. Experiences with radiata pine

in the southern hemisphere provide a good example.

Libby et al. (1972) were among the first to test

earlier observations that cuttings from hedged

plants rooted more like tree-form plants of the same

height rather than of the same age. Plants from 68

clones were either sheared to l-m-tall box-shaped

hedges or grown in modified tree form beginning at

age two. Cuttings were collected from the upper

surfaces of hedges or upper crowns of trees and

rooted annually for five years. By the third year,

cuttings from hedges rooted significantly more often

than cuttings from tree-form plants (31% v. 8%), and

this difference was essentially stable for the next

two years. In the fifth year the cuttings from hedges

fewer male and female flowers. Stem form was

improved for cuttings from tree-form origin. Since

radiata pine is grown intensively for sawtimber, the

authors postulated that partial maturation could be

exploited to improve timber quality. Although

volume would have to be sacrificed somewhat,

trunks would be straighter, and there would be

fewer and smaller basal branches to remove.

Summary

Rooting of most shoots decreases dramatically

with tree age. Propagators have adapted to this

limitation by selecting material from the more

juvenile sections of the tree, and by enhancing or

maintaining juvenile rooting through propagation or

shearing. Although these approaches have been

somewhat successful for sustaining good rooting

capacities, subsequent growth often indicates that

subtle maturation has occurred.

loo--

90--

60-- 0

E

70--

0

B-S-0,

P

60-- .

0

\

: ’

. 4‘ \

\

c SO-- . ,0

\

5

i4

4Q-- 9”‘.

*

k!

30-- ,

I

&

1 I

I I I I I i 1

J F M A M J J A s 0 N D

MONTH

-*- Loblolly - o- Douglawfir -m- Whlto pine

Figure L-Monthly rooting of cuttings from 2- to 3-year-old non-sheared loblolly pine, 8- to lo-year-old sheared Douglas-fir, and 17-

year-old non-sheared eastern white pine averaged over two years. Adapted from Reines and Bamping (1960,1962), Roberts and

Fuchigami (1973), and Kiang et al. (1974).

also produced significantly more roots per rooted

cutting (7.4 v. 5.9).

VARIABLE ROOTING WITHIN A TREE

Cuttings of both origins for 32 of these clones

were planted in pairs for further evaluations

(Bolstad and Libby 1982). After seven growing

seasons total heights were similar for cuttings of the

two origins, but bole volume was 31% greater for

cuttings derived from hedges. In addition, trees of

hedge origin showed more juvenile characteristics

like larger branches, more lower branches, and

Problem

Given a specific set of potential donor trees of a

known age, the rooting capacities of severed cuttings

will change with position and time.

Cutting Position Effects.--As mentioned

previously, cuttings collected from the lower crowns

of Norway spruce (Grace 19391 rooted better than

5 5
those from the upper crown. Although this may be

related to greater maturation in the upper crown, it

is also an example of a gross positional effect. More

localized effects have been found in the strongly

pyramidal-shaped conifers like spruces and firs.

These species have a very regular pattern of

branching from the primary axis of the trunk. Data

from Black (1972) generally indicated that tertiary

axes from nine-year-old Douglas-fir trees rooted

better (64-69%) than secondary (47%) and

quaternary (41%) axes unless the tertiary axes were

small (41%).

Time Effects.--Rooting success with cuttings from

the same trees can also change with time. It has

been generally stated that cuttings from narrowleaved conifers in temperate regions tend to root

best when collected from late fall to late winter

(Hartmann and Kester 1983). For example, cuttings

from eight- to ten-year-old sheared Douglas-fir

rooted best between November and April (Fig. 11,

depending on supplemental treatments (Roberts and

Fuchigami 1973). In this test, rooting of dormant

shoots was poorest in September and October when

buds were most deeply dormant. It was postulated

that exposing dormant shoots to cold brings

endogenous biochemical promoters and inhibitors

into balance for successful rooting. Cuttings from

young loblolly pines also rooted best when collected

in January, but rooting was generally low for this

species (Fig. 11 (Reines and Bamping 1960, 1962).

There have been solid exceptions to the pattern of

good cold-season rooting. Kiang and others (1974)

rooted eastern white pine (Pinus strobus L.) cuttings

from 17-year-old trees at monthly intervals for two

years and found best rooting occurred with June

cuttings (Fig. 1). This was associated with the

presence of rapidly expanding shoots at the distal

ends of one-year-old wood at the time of severance.

The situation can become further complicated when

secondary and tertiary axes are examined

separately. Girouard (1975) tested rooting of

Norway spruce cuttings collected from these two

origins at two- or three-week intervals for two years.

Rooting was generally most successful in mid-spring

(about 53-57% for one year). Secondary axes rooted

best in April while maximum rooting for tertiary

cuttings occurred in May. Smaller rooting peaks for

both origins occurred between September and

November.

Although hedging eliminates mature upper

crowns that would produce cuttings with reduced

rooting capacity, the resulting juvenile shoots may

still be subject to seasonal fluctuations. For

example, cuttings from six slash pine clones that

were four years old from seed were severed monthly

from 1.2 m outdoor hedges (Bower and van

Buijtenen 1977). Rooting was generally greatest for

May cuttings (67%) and poorest (0%) for those

collected in September. These results directly

conflict with those using slightly younger, nonhedged trees where May was an intermediate month

(Reines and Bamping 1960) or the poorest month

(Reines and Bamping 19621, and September was an

intermediate month (Reines and Bamping 1960,

1962). Although physiological changes due to

hedging may be partly responsible for the

discrepancy, other cultural and genetic differences

are likely to have contributed greatly.

There can also be great variation in rooting from

the same trees between years. When Norway spruce

shoots were collected over two years (Girouard

19751, rooting was generally better in the second

year of the study despite the additional age of the

donor plants. This was demonstrated by the rooting

maxima for lateral shoots being approximately lo25% greater in the second year than in the first. In

a study with Scats pine (Pinus syluestris L.)

(Boeijink and Broekhuizen 1974), the year-to-year

component of variation was reported to be greater

than the genetic component for rooting.

“C” Effects.--A special case of rooting variability

can occur when rooted cuttings from a tree have

been established as plants to produce more cuttings.

This practice is critical to the development of true

clones for widespread planting. Although

genetically identical, rooting of cuttings can vary

depending on the environmental preconditioning of

individual donor plants. This effect has frequently

been referred to as a common effect, or “C!” effect, by

extending Lerner’s (1958) definition to asexually

propagated material. It should be noted that the

effect of cutting position, discussed previously, is

another type of common effect.

Some studies have accounted for the variability

among clonal cutting donors. For western hemlock,

variation of rooting success among genetically

identical donor plants was 6%, compared to 31%

variation among genetically different clones selected

randomly from a wild stand (Foster et al. 1984).

Variation of this magnitude could partially obscure

the selection of clones with strong inherent rooting

capacities. Analogous variation in tamarack (Larix

Zaricina Du Roi) was 11% among clonal cutting

donors vs. 31% among random clones for one year,

56
but the ‘KY effect was insignificant for a second year

(Farmer et al. 1992). The authors suggested that

these effects would probably not be problematic, but

could not be ignored.

Solutions

There are several approaches to handling

variability within trees. Positional effects can be

circumvented by selecting cuttings from specific

locations within a tree. Selection for strong rooting

capacity must be tempered with consideration for

positional effects on growth rate and form. The

latter will be discussed further in a subsequent

section.

Heavy shearing to arrest maturation greatly

reduces positional effects by limiting the origins of

cutting production to a small, juvenile region. As

previously discussed, these shoots are generally

uniform in size and possess strong inherent rooting

capacities.

Seasonal variability can also be accounted for or

manipulated to some degree. For dormant cuttings,

Struve (1980, 1982) developed a chill-unit

accumulation model for eastern white pine based on

a daily weighing method used for peaches

(Richardson et al. 1974). Using needle fascicle

cuttings from 54 six-year-old trees, he found that

58% of the variation could be accounted for by the

model, whereas only 47% could be accounted for

using calendar date as a predictor. Unfortunately,

similar modeling of artificially chilled seedling top

cuttings only explained a maximum of 5% of rooting

variability. Since the test population was rather

heterogeneous, the author suggested that accuracy

might be improved if models were developed for

individual clones.

Artificial exposure to constant cold has been used

to simplify chilling of dormant cuttings. Miller et al.

(1982) severed cuttings from five-year-old Fraser firs

(Abies fraseri (Pursh) Pair.) in early October after

shoots had received only 60 total hours below 5°C.

They were assumed to be fully dormant, and were

exposed to O-12 weeks of dark storage at 4°C in

sealed plastic bags before rooting. Rooting increased

dramatically with only four weeks of chilling, but at

least eight weeks of chilling was required to achieve

50% budbreak.

Wise et al. (1985a) used these results to postulate

that mediocre rooting and budbreak observed with

cuttings collected in mid-winter might be related to

competition between the two processes for limited

reserves. By chilling only four weeks to stimulate

good rooting, the authors achieved up to 92% success

(depending on crown position and subsequent

treatments) with dormant Fraser fir cuttings

severed from 14-year-old sheared trees. Cuttings

chilled eight weeks generally rooted about 20% less.

After further chilling so that all rooted cuttings

received a total of 11 weeks at 4”C, new shoot

growth for those initially chilled only four weeks was

about twice as large as for those initially chilled

longer. Thus the separation of rooting from

budbreak in this fixed-growth species seems to have

improved both responses.

To reduce variability among genetically identical

cutting donors, Foster et al. (1984) recommend

treating donor plants of the same clones uniformly.

This would include establishing these plants from

cuttings that were the same size and from the same

position on the original tree. It should also include

growing these plants under similar cultural

conditions.

Summary

Due to the changing physiology of the donor

plant, the physiological predisposition towards

rooting varies strongly with the season in which

cuttings are collected. Although winter cuttings of

most conifers root relatively well, some have been

rooted successfully at other times under certain

conditions. Variability in rooting due to positional

effects in tree-form donors can be reduced by

selecting well-defined cuttings. Choosing tertiary

axes from lower tree crowns may favor cuttings with

a strong rooting capacity, but subsequent growth

from these propagules may be undesirable. When

rooted cuttings are further cultured to produce

clonal cuttings, positional effects and differences in

growing environments can contribute to significant

non-genetic variability unless conditions are

standardized.

INCOMPLETE TESTING AND SUCCESS OF

CULTURAL ENHANCEMENTS

Problem

Propagators have tested a multitude of cultural

treatments to improve rooting of cuttings, and these

can be categorized and subdivided in many ways. In

general, cultural enhancements can boost rooting

success somewhat, but cannot entirely compensate

5 7
for poor rooting due to genetically inferior clones,

maturation of cutting wood, or improper timing of

collection. Despite the broad range of cultural

factors that may influence rooting, relatively few

good studies have been conducted on conifers;

especially with juvenile cuttings. Still, a

comprehensive review of these factors is covered

elsewhere (Davis et al. 1988), and this discussion

will highlight those that have shown promise for

future enhancement of conifer rooting. For the

purpose of this discussion, cultural enhancements

are defined as manipulative inputs attempting to

enhance yields of quality rooted cuttings that are

made before, during, or after shoot severance. They

will be covered in that order.

Pre-severance treatments.--The most important

cultural manipulations conducted on donor plants

are (1) treatments that influence the health and

vigor of the plants, and (2) shearing treatments that

affect yield and quality of juvenile cuttings from

hedges.

Health and vigor.--Reports on procedures that

produce good rooting from healthy, vigorous stock

plants come primarily from horticultural literature,

and contain evidence that is often anecdotal. Few of

these reports deal with conifers important to

commercial forestry. As an example, a symposium

covering the effects of donor plants on propagation

success (Read 1987) described or implied benefits

from soil amendments, mulches, cautious use of

herbicides, insect and pathogen controls, light

exclusion, carbon dioxide enrichment, proper

irradiance levels and photoperiods, and chemical

growth regulators. Of the 137 references cited that

dealt with specific species, only 10 involved

coniferous species. Four of these ten were tissue

culture studies, and most of the rest are covered

somewhere in this document. The underrepresentation is more likely due to absence from

the literature rather than editorial bias. Although

these proceedings are neither an exhaustive review

of donor plant conditioning, nor directly applicable

to coniferous forest species, the above factors that

they identified may be important for future

examination.

There is further descriptive evidence, somewhat

obscure, from successful horticultural nurseries.

Iseli and Van Meter (1983) outlined the following

procedures that produced healthy, economical

cuttings of ornamental Norway spruce cultivars with

enhanced rooting capacities:

“1. The nursery acquired stock plants

which were containerized and

maintained at optimum nutrition

levels.

2 . Weed, pest and disease vigilance was

maintained and good control

procedures established and adhered

to on all containerized stock plants.

3 . Use of herbicides as a weed control

measure was suspended to ensure

the optimum health and rootability

of the plants. This requirement has

been gradually relaxed with the

limited use of Ronstar with no

apparent deleterious effects.

It was felt that if the wood could be taken from

super healthy and vigorous plants where

disease and pest problems were minimal that

good results would accrue; results vindicate

that belief. ”

For forestry applications, Kleinschmit

(1974) generalized that “rooting response

can be increased markedly by systematic

fertilizing” for juvenile Norway spruce.

No data were presented.

There is scant direct evidence of specific benefits

from proper fertilizing, watering, and light

conditions. Enright (1959) applied supplementary 4-

12-4 fertilizer and cottonseed meal to three-year-old,

non-hedged nursery plants of three conifers and

detected large increases in rooting percentages

(Table 2). Although the fertilization effect was

Table 2. Rooting percentages of cuttings severed from three-yearold plants of three species, with and without supplementary

fertilizer. For fertilized trees, 4-12-4 was incorporated into nursery

beds at 560 kg/ha before planting, and top-dressed at 187 kg/ha

during the first growing season. Cottonseed meal of undefined

analysis was also incorporated and then top-dressed at 1121 kg/ha

and 374 kg/ha, respectively. Means were calculated over three

summer collection times from Enright (1969).

Supplementary Norway

fertilizer Spruce

With 67

Without 16

Pitch

pine

64

1

E. white

pine

59

14

5 8
100

9 0

6 0

7 0

6 0

5 0

4 0

3 0

2 0 30 40 5 0 6 0 70 6 0

LENGTH OF ROOTING PERIOD (DAYS)

Figure 2.--Effect of h-radiance on root formation ofPinus syluestris hypocotyl cuttings. Seedlings were grown under 8 watts/m2 (filled

circles) or 40 watts/m2 (open circles) for five weeks prior to rooting. Data from Hansen et al. (1978).

dramatic, extremely low responses from the nonfertilized but very juvenile plants indicates these

donors were probably highly deficient. With

juvenile Sitka spruce, a balanced and “moderate”

nitrogen, phosphorus, and potassium regime applied

in the growing season prior to cutting collection

improved rooting over two other treatments (twice

the moderate rate and a “high nitrogen” regime)

(Mason and Keenleyside 1987). Although rooting

from the moderate N-P-K treatment was 98%,

compared to 90% from the other two treatments, it

was not statistically better than the 95% achieved

with a standard, low-fertility regime.

Light exclusion and water stress treatments have

also influenced rooting in Sitka spruce (van den

Driessche 1985). Containerized donor plants were

propagated from hedges that were 15 years old from

seed, and were 2 years old from cuttings when given

38% shade for 5 or 7 months, or not shaded.

Dormant cuttings subsequently rooted at 53% from

both shade treatments and only 34% from nonshaded controls. Although various forms of light

exclusion have been shown to improve rooting in

many angiosperms, this is one of the few examples

that has shown a benefit to stem cuttings of a

conifer. Two moisture levels were also

superimposed on this study: (1) normal watering to

maintain moist soil and (2) supplemental watering

withheld about eight weeks in July and August.

Only 33% of cuttings from stressed plants rooted

while normal watering induced 53% success.

Although this is good evidence for a water stress

effect, the stress imposed was apparently quite

extreme since 62% of non-shaded donor plants died

before cuttings could be collected.

Donor plant light conditions have been explored

further with a somewhat artificial system using

Scats pine (Hansen et aL 1978, Hansen and

Ernstsen 1982). For these studies seeds were

germinated for one week, seedlings were grown five

weeks under various light levels in growth

chambers, and hypocotyl cuttings were then floated

on aqueous solutions for rooting. Cuttings from

seedlings grown at a relatively low light intensity, 8

watts/m2, rooted more quickly and more frequently

than those grown at 40 watts/m2 (Fig. 2) (Hansen et

al. 1978). This corresponded to lower contents of

various carbohydrates, as seen in cuttings of some

herbaceous species, and suggested that high light

intensities induced supraoptimal carbohydrate

contents for rooting.

Photoperiod was also important, indirectly, in a

similar study (Hansen and Ernstsen 1982).

Seedlings germinated throughout the year were

grown in natural light at either natural

photoperiods or 4-hour days. Constant short days

nearly always boosted rooting, but mid-summer

cuttings generally rooted poorly regardless of

photoperiod. This implied that, in this system, the

total radiant energy received during bright summer

days was enough to stifle rooting even if exposure

were short.

Cutting yields.--Beside improving rooting

potential, cultural treatments can improve cutting

yields. Although there are few reports comparing

such treatments, New Zealand’s Forest Research

Institute has studied many manipulative techniques

to produce good uniform cuttings from radiata pine

5 9
seedlings. One novel, intensive example (Fig. 3)

involved growing seedlings 14 months, and then

rather than hedging, pruning off succulent top

growth and pinning the leaders horizontally to the

ground to remove apical dominance (Menzies et al.

1985). Fascicular buds then expanded into upright

cuttings, and were thinned during the season to

improve individual quality. When dormant cuttings

were collected, the shoot closest to the root system

was retained intact on each plant. After these

shoots grew early the next season, they were pruned

and pinned to repeat the process. This procedure

was developed primarily to multiply scarce

quantities of genetically superior seed, but can

potentially produce juvenile cuttings for several

years and demonstrates the highly manipulable

character of pines. Production systems will probably

need to be tailored to meet the unique requirements

of each of the desired species.

Summary

Cultural treatments that ensure the health and

vigor of coniferous donor plants have been

infrequently described and occasionally tested. A

few studies have indicated benefits from controlled

fertilization and light manipulation, but the full

value of these treatments for enhancing rooting

potential has yet to be realized. Similarly, managing

juvenile plants by hedging or other techniques will

be important for optimizing cutting production, but

there is little public information on potential

methods.

Month: 0

(early spring)

14

Treatments at severance.--The selection and

removal of specific shoots from donors can influence

rooting success. Although evidence conflicts, cutting

length is more likely to influence cutting quality

than to influence percent rooting success of juvenile

shoots. For example, six-year-old Norway spruce

shoots cut 5-20 cm long rooted at about 94%

regardless of length (Radosta and Volna 1989).

However, root collar diameter, the percentage of

cuttings with branched root systems, and plant

height after two years increased directly with

original cutting length. Similarly, Fraser fir

cuttings lo-26 cm long rooted around 78%, but the

number of roots per rooted cutting increased with

length (Miller 1982). Mean root length also

increased with sizes up to 22 cm long. Conversely,

rooting of shoots from hedges of a hybrid tropical

pine (Pinus curibaea Morelet var. hondurensis Barr.

and Golf. X Pinus tecunumtmii (Schw.) Eguiluz and

Perry) decreased as length increased from 10 to 49

cm (Haines et al. 1992). As with other species, roots

per cutting and age two height increased with

original cutting length.

Haines and others (1992) assessed several additional

phenotypic characters to develop selection criteria

for cuttings from hybrid tropical pine hedges.

Shoots were collected in late summer so they were

not dormant. Although genotype was the strongest

predictor of rooting success, significant

improvements in rooting and growth were predicted

by selecting shoots with long primary needles, short

but expanding secondary needles, and bases greater

2 0 25

Figure 3.--Method for producing large cuttings from seedlings of radiata pine. Seeds are sown in early spring and seedlings are grown one

year. In the spring of the second year, leaders are pinned horizontally to remove apical dominance, and emerging fascicular shoots are then

thinned. Dormant cuttings are rooted the following fall. The main shoot is severed (arrow) to leave one fascicular shoot, and the pinning

process is repeated the next spring. Adapted from Menzies et al. (1985).

60
Aug S 0 N D J I= M API

COLLECTION MONTH

Figure 4.-Seasonal rooting averaged over two years for cuttings severed from 3- to lo-year-old sheared Douglas-fir trees and rooted with (+)

or without (open circles) post-severance auxin treatment. Data horn Roberts and Fuchigami (1973).

than 2 mm in diameter. Several other characters

were less important. These criteria not only guide

cutting severance, but indicate improvements can be

made by precise timing of collection and hedge

culture methods.

Post-severance treatments.--h wide array of

manipulations can be imposed before cuttings are

subjected to the rooting environment. Most have

been reviewed extensively elsewhere (chapters lo15, 17,20 in Davis et al. 1988). Certain chemical

treatments and handling procedures are most likely

to be important for rooting conifer species. Due to

the extensive nature of the topic, only major points

Haines and others (1992) assessed several

additional phenotypic characters to develop selection

Au&S.--These hormones have been used to

stimulate rooting for decades, but the practice

involves many variables. Besides variable rates,

there are several common chemical forms, and

options for applying them as powders or liquids. For

liquids, the solvent can be varied (usually an

aqueous alcohol solution or pure water) and the

treatment period can range from less than one

second to several hours. Techniques and benefits

have been summarized by Blazich (Chapter 10 in

Davis et al. 1988).

The general benefit of auxin applications is to

improve the quantity and quality of adventitious

root systems on cuttings already somewhat

predisposed to root. For example, a commercial

100

90

00

7 0

6 0i

1500 3000 4600

IBA RATE (PPM)

Figure 5.--Interaction of indole-3-butyric acid (IBA) concentrations, collection date, and shoot origin on rooting of Fraser fir stem cuttings

collected June 23 (triangles), July 14 (circles), and August 4 (squares), from upper thirds of tree crowns. Redrawn from Wise et al. (1985b).

will be briefly discussed.

Among the many chemicals applied to conifer

cutting bases, the most beneficial are probably

auxins, fungicides, and shoot growth retardants.

auxin preparation improved rooting of cuttings from

sheared 8- to lo-year-old Douglas-fir trees (Fig. 4),

but cuttings collected late summer through mid-fall

still rooted poorly (Roberts and Fuchigami 1973).

Highest percentages were still obtained during the

6 1
four months that had strongest inherent rooting

capacity, but auxin hastened the transition to good

winter rooting, perhaps by substituting for chilled

buds. The major point, therefore, is that auxin

applications have enhanced rooting, but they have

not completely overcome poor physiological

conditions due to maturation, branch order, cultural

preconditioning, or other factors.

New synthetic auxins have shown promise in the

last decade. Using jack pine (Pinus banksiana

Lamb.) seedling tops, Haissig (1983) found that Nphenyl indolyl-3-butyramide (NP-IBA) increased

percent rooted, number of roots, and longest root

length over equimolar concentrations of a standard

auxin, indole-3-butyric acid (IBA), and non-treated

cuttings. Cuttings from non-sheared 7-year-old jack

pine trees that were treated with NP-IBA tended to

root slightly more frequently than those dipped in

an IBA solution that was 37% stronger than the NPIBA solution. Phenyl indole-3-thiobutyrate (P-ITB)

and phenyl indole-3-butyrate (P-IBA) also were more

efficient at stimulating rooting by some measures.

Although these new auxins have improved rooting of

a coniferous species, they have apparently remained

largely untested with potential systems using

juvenile donor plants.

Some studies have found no auxin benefit.

Possible explanations could be that cuttings were

not collected in the proper condition (as seen in the

example in Fig. 41, were not subjected to an

appropriate auxin treatment, were capable of

rooting at high percentages regardless of auxin

treatment, were collected from a biased genetic base,

or combinations of these. Auxin response was

strongly influenced by genotype of lodgepole pine

(Pinus contorta Dougl.) cuttings from nine- and tenyear-old trees (Bowen et al. 1975). For one clone,

rooting was less than 10% without auxin, increased

to a maximum of about 75% near the mid-range of

rates tested, and decreased to about 45% at the

highest level. Two other clones did not respond to

auxin; one rooted above 80% and another below 10%

regardless of concentration. This clearly indicates

that genotypes must be accounted for when refining

auxin treatments, but similar work with juvenile

shoots has not been widely published.

Interactions of timing and auxin rate have also

been noted. For example, Fraser fir cuttings were

severed from upper and lower crowns of sheared

14-year-old trees near the end of the annual growth

flush and treated with relatively low auxin rates

before rooting (Wise et al. 198513). For upper crown

cuttings, those severed in late June (before terminal

buds were visible) rooted relatively well without

auxin, but rooting percentage increased with a weak

concentration and decreased markedly at higher

rates (Fig. 5). Cuttings collected three weeks later

only rooted well with auxin applications, yet rooted

less well at the highest rate. After another three

weeks, shoots were generally unresponsive although

increasing auxin rates increased rooting slightly.

Cuttings from the lower crown generally rooted in

slightly higher percentages, as expected, but were

more responsive to higher auxin concentrations.

These results suggest that the internal complement

of rooting promoters and inhibitors in softwood

cuttings is highly dynamic, and that extensive

testing may be necessary to find auxin treatments

that enhance rooting by supplementing endogenous

auxin.

In summary, auxin treatments have a strong

record of enhancing rooting, but the wide array of

possible treatment combinations makes exhaustive

testing difficult. Testing is further complicated by

interactions of awin treatments with other

important factors such as genotype and the

physiology of shoots at specific times. Research to

refine auxin treatments on shoots from juvenile

conifer hedges is largely absent from the literature,

but past trends indicate that gains of uncertain

magnitude may be possible if other factors are

properly accounted for.

Fungicides.--Applications of these chemicals are

generally recommended for improving adventitious

rooting (Hartmann and Kester 19831, and benefits

have been shown for some conifers. Grigsby (1966)

achieved 40% rooting with 25% captan plus 0.8%

IBA applied as a powder to the bases of cuttings

from six-year-old loblolly pines. This was three

times greater than IBA alone. Cuttings from nonsheared 13-year-old eastern white pines responded

more to fungicides than IBA levels when bases were

dipped in treatment powders (Thielges and Hoitink

1972). No cuttings rooted without fungicide,

regardless of IBA rate. Rooting percentage tended

to increase with IBA rate when 5% benomyl was

added, and results were generally best when 5%

benomyl and 25% captan were both used. The

fungicides were thought to indirectly enhance

rooting by inhibiting pathogen attacks, rather than

by directly stimulating root initiation. Although

fungicide treatments have been beneficial for rooting

somewhat mature conifers in the past, direct

evidence is lacking for benefits in juvenile rooting

systems.

62
Synthetic shoot growth retardants (SSGR).--These

are used to improve the appearance of many

ornamental crops, and have also been found to

stimulate rooting in many cases. Their influences

on rooting, and those of chemical inhibitors, have

been recently reviewed by Davis and Sankhla

(Chapter 13 in Davis et al. 1988). For conifers, one

published report indicated that the SSGR

paclobutrazol increased rooting in hypocotyl cuttings

of loblolly pine (Rutter and Ingram 1988). The

highest concentration (25 ppm) and longest soak

period (24 hours) induced greatest rooting (93%).

Water soak controls and a commercial auxin quick

dip induced 73% rooting. Despite demonstrated

benefits, breadth and magnitude of rooting

stimulation have apparently not been sufficient to

warrant commercial adaptation of SSGR

applications.

Chemical combinations.--Significant work on the

effects of combined chemical treatments on three

pine species was summarized by Hare (1974).

Independent experiments using cuttings from lyear-old slash pine, 12-year-old Japanese black pine

Pinus thunbergiana France), and 13-year-old

shortleaf pine Pinus echinata Mill.) generally

indicated that a specific powdered mixture of two

auxins, the fungicide Captan, one SSGR, and

sucrose applied to cutting bases was most effective.

Juvenile slash pine cuttings collected four times

throughout the year rooted at 100% with the

complete or near-complete mixture.

The above formulation was referred to as “Hare’s

Powder” by later researchers. Unfortunately

subsequent work with the preparation gave mixed

results, and loblolly pine experiments provide an

example. Hare’s Powder improved rooting to 21%,

from 6% with IBA alone, in cuttings from nonsheared 4-year-old loblolly pines (Greenwood et al.

1980). Pousujja (1981) reported increases ranging

from 12% to 79% over IBA alone using one- to fiveyear-old non-sheared trees, but could not repeat

high success rates in later experiments. For shoots

from six-year-old hedges, those treated with the

mixture always rooted similar to those that received

0.4% IBA regardless of season of cutting collection

(Mahalovich et al. 1987). As with simple auxin

treatments, it appears that complex mixtures of

chemical rooting stimulants will have to be

developed for specific production systems that

account for juvenility, season of collection, and other

influences.

Handling.--Handling treatments are the last to be

discussed and are difficult to define and specify. In

general, they consist of collection, storage and

transportation techniques that seek to maintain or

improve a strong rooting capacity in cuttings. Since

cuttings are removed from their source of water,

nutrients, and other growth factors, and can be

temporarily subjected to fairly hostile environments,

procedures should be adapted to maintain cuttings

in the best health. However, references to handling

procedures are infrequent, and those usually contain

anecdotal evidence. For example, handling

procedures have been important for rooting

ornamental cultivars of Norway spruce:

“All cuttings are taken early in the morning

before heat of the day. They are collected in

plastic tubs with a small amount of water in

the base. When returned to the propagation

house, cuttings are placed in a specially

constructed bench which is endowed with a

mist system to ensure the needles always

possess moisture, thereby minimizing the

possibility of shock. We have carried over for

twenty-four hours cuttings in this environment

with no apparent harm to rooting.” (Iseli and

van Meter 1983)

Grigsby (1971) compared two distinct sets of

handling methods for rooting influences using shoots

from non-sheared six-year-old loblolly pines.

Dormant cuttings collected in early morning, always

held vertically, cold stored for two days, and not

recut before sticking rooted at 10%. Those collected

in late morning, kept horizontal, and recut and

stuck a few hours later without cold storage only

rooted at 5%. The experiment was conducted with

fairly mature material and was not designed to

detect the most important handling factor(s), but

gives some evidence that the way cuttings are

processed can impact subsequent rooting. Hard

evidence apparently has not been published for

juvenile conifer rooting systems.

Solution and Summary

Some cultural manipulations made before, during,

or after cutting severance have greatly affected

rooting. Most have not been studied extensively,

and few have been tried in juvenile production

systems. Focused testing of these techniques within

well-defined propagation programs may continue to

enhance rooting, especially with selected families or

individuals of recalcitrant species.

6 3
POOR FORM OF ROOTED CUTTINGS

Problem

Initial growth of rooted cuttings should be good in

order for propagation to be fully successful.

Reductions in shoot growth due to maturation of

donor plants (Menzies and Klomp 1988, DekkerRobertson and Kleinschmit 199 1) and sub-optimal

timing (Wise et al. 1985a) have already been

mentioned. In addition, rooted cuttings of many

conifers exhibit a branchlike, plagiotropic

(pZugios=oblique, tropos=turning) growth habit that

can persist for a period of several years. For space

considerations, this section will only discuss early

plagiotropic growth.

One of the most interesting examples of the

severity of the problem is provided by Black (1973).

Douglas-fir cuttings from two clones, two and three

years old, were planted normally in containers,

planted in containers and staked vertically, or

planted in the bottoms of containers and suspended

in the air. After two years, the orientation of the

leader growth within each clone was essentially the

same for all treatments. The clone with the history

of more severe plagiotropism grew about 35” from

vertical and the other at about 14”. Therefore the

severity of plagiotropic growth seems to be a strong

genetic character that is not easily modified.

Severity of plagiotropism also interacts with

maturation. Roulund (1975) demonstrated that

three-year-old cuttings of Norway spruce grew more

obliquely as the age of the donor plant increased.

He concluded that cuttings should be selected from

four-year-old or younger trees in order to obtain

good rooting and growth for this species.

Plagiotropic growth has a rather curious

physiological basis. It is generally known to be

mediated by the formation of compression wood on

what was originally the upper (adaxial) side of the

branch, which is opposite of where it is usually

formed. Starbuck (1979) also found greater cambial

activity on adaxial sides of Douglas-fir cuttings, and

increased short-term extension growth on adaxial

sides of softwood cuttings. Although he also

associated these processes with a gradient of

apically-applied, radiolabeled auxin towards the

adaxial sides, the gradient did not result from

vertical reorientation. Biochemical mechanisms

have not been elucidated further for conifer cuttings.

Although plagiotropic growth is primarily a

problem with conifers that possess very regular

branch orders, like spruces and firs, mild cases have

been noted in the senior author’s loblolly pine

propagation program. Cuttings that were rooted in

the greenhouse from greenhouse-grown hedges,

transplanted into small tubes, and then returned to

the greenhouse grew obliquely. Those from outdoor

hedges that were transplanted into 4-liter containers

and then returned outdoors grew upright. To

determine whether light intensity, rather than root

restriction, was mediating the response, cuttings

from both origins were rooted in a shaded

greenhouse and exposed to three light levels at wide

spacing for nine months. Growth deviations from

vertical increased linearly with decreasing light

intensity for cuttings from both origins, but bending

of greenhouse cuttings was exaggerated under

denser shade. Adaxial compression wood actually

began forming during the 12-week rooting period in

most cuttings of both origins. This is evidence that

pines are not immune to plagiotropism problems, but

that propagators have some control over the severity

of its expression.

Solutions

Other cultural manipulations to reduce

plagiotropic growth have met with mixed success.

Black (1973) was generally unable to enforce upright

growth by staking rooted Douglas-fir cuttings from

sheared trees, and the active bending actually broke

stakes attached to several cuttings. With X-yearold sheared Fraser fir trees, Wise (1986) reduced

bending of one-year-old rooted cuttings by delaying

original softwood shoot severance by about three

weeks until lignification provided internal vertical

support (33” v. 68”). A simple external wire support

during rooting could be substituted for the delay (35”

v. 6S0).

Results have also been mixed with more juvenile

material. Rooted primary branch tips from coast

redwood (Sequoia semperuirens D. Don (Endl))

hedges were generally more upright than those from

secondary branch tips (Power et al. 1988). However,

seedling branch tips of the same orders grew more

upright than corresponding cuttings from the

hedges, which were seven years from seed. This

suggests that shoot selection from hedges can be

used to reduce plagiotropism in some species, but

that hedge maturation may circumvent the

improvement.

6 4
As a final example, Bentzer (1988) attempted to

establish a “signal” within terminal buds that would

cause shoots to grow upright. The objective was to

restrain upper expanding shoots in an upright

position so that their developing terminal buds

would possess the “signal” for future upright growth.

He used mesh cylinder bags on 3-year-old Norway

spruce trees that had been serially propagated 3

times (14 years old from seed). He also pruned the

leader from similar sets of trees (both with and

without mesh bags), and selected cuttings by branch

order. Unfortunately, restraining new growth

upright actually increased the degree of

plagiotropism, but it also improved rooting success

(Table 31. It was concluded that just removing the

leader improved cutting quality somewhat,

especially if branch tips were collected only from the

upper two whorls.

maintaining juvenility, correctly timing and

choosing cuttings, imposing some beneficial cultural

treatments, and minimizing early plagiotropic

growth, there is great promise for even the most

recalcitrant coniferous species. Enhancements can

still be made in each area, and important

interactions require further exploration. Cultural

manipulations remain largely untested, especially

with juvenile shoots, so additional attention to

production, treatment, and handling of cuttings may

also improve rooting capacity in some species.

Regardless of the approach taken, a strong rooting

capacity must still be captured by the correct rooting

environment (see from these proceedings: FordLogan 1993).

To adapt rooting technology to many forest

conifers, several key research questions still

command attention for each species.

Table 3.--Rooting success and growth form (divergence from vertical) of Norway spruce cuttings from three-yearold plants that had leaders removed or upper growth forced vertically within cylindrical mesh bags prior to

severance. Data from Bentzer (1988).

Treatment Rooting Plagiotropy

None (control)

Leader removed

Vertical bag

Leader removed plus vertical bag

Dercent’ iic!2Le2

90b 2.1 b

92b 2.0 b

98 a 2.8 a

98 a 2.7 a

‘Means compared within columns by Duncan’s multiple range test, 5% level.

21=900-820, 2=82”-72”, 3=72”-58”, 4=68”-46”, 5=<45” from horizontal

Summary

Although plagiotropic growth has frustrated

efforts to produce good-quality cuttings, it is

certainly more problematic with some species than

others. Treatments that retard maturation seem to

also reduce the severity of plagiotropism, but do not

completely arrest it. Correct timing, shoot selection,

and clonal selection can foster a more upright initial

growth habit, but physical restraints have not been

generally successful.

GENERAL SUMhIARY AND INTERPRETATION

OF KEY RESEARCH ISSUES

Great progress has been made towards scaling the

barriers to successful stem cutting propagation with

conifers. By imposing some genetic selection,

How should rooted cuttings be used in tree

improvement?--There are essentially two options: to

deploy clones or to multiply scarce quantities of

high-value seed. Historically, rooted cuttings have

been used to perpetuate individual genotypes that

possessed outstanding combinations of traits,

primarily of horticultural crops. These genotypes

could have been discovered by chance or through

concerted breeding efforts. The population of

genetically identical plants that would result from

propagating one such genotype would be a clone.

The application of clonal techniques to forestry

involves not only developing the methods to produce

and root cuttings, but identifying the superior

individuals to be propagated. In practice, clones are

selected from field tests and then multiplied by

cuttings into usable numbers for afforestation.

Since maturation of selected trees would make them

65
difficult to propagate by the end of the test, a

separate set of plants representing each clone in the

test is maintained in the juvenile phase throughout

the test. This option can consequently be expensive

and time-consuming, but allows the exact genetic

duplication of truly outstanding genotypes. For the

future, it will also allow the perpetuation of

individuals with genetically engineered traits

without requiring the traits to be sexually

transmissible.

The other option, often referred to as vegetative

multiplication, generates juvenile plants from a

limited supply of superior seed, and then roots

cuttings from these seedlings. Methods for

producing and rooting cuttings are required, but no

additional testing or selection is needed.

Consequently, the rooted cuttings are, genetically,

only as good as the seeds that are multiplied by

these methods. On the other hand, maturation is

not a major problem since individual hedges are only

retained to produce cuttings for only one to a few

years. These techniques are often used with species

that do not readily produce adequate quantities of

seed. Alternately, they are often coupled with

breeding programs that identify superior families at

frequent intervals, and that want to deploy large

numbers of trees of these families with a minimal

lag time. In a recent world survey of cutting

propagation in commercial forestry, it was concluded

that “the primary use of rooted cutting technology is

for bulk production of genetically improved half or

full-sib families” (Ritchie 1991).

The choice of use depends primarily on the goals

of each specific tree improvement program and its

breeding component. Complicated or expensive

methods may be necessary to clonally propagate

some highly recalcitrant species. Consequently, the

application of rooted cutting technology to these

species may be limited to the simpler process of

vegetatively multiplying seed.

How should production systems be okveloped?--

Focused research will be needed to develop unique

production systems for each species. Since juvenile

shoots have historically been critical to success, each

system most likely will be based on maintaining

juvenile donor plants through shearing or serial

propagation. In addition, systems must work with

identified sources of variability to generate cuttings

with strong inherent rooting capacities. For

example, cutting production should be timed so that

juvenile shoots will be severed and rooted during

periods of proven success. Promising seedling

populations of known pedigree will doubtless be

chosen as the starting material for vegetative

multiplication, and for the development of clones.

Additional selection can be used to perpetuate the

best-rooting genotypes, or to cull those that root

most poorly. However, this must be balanced

against possible reductions in gains of economic

importance (e.g. volume growth).

Cultural manipulations have often enhanced

rooting, and tested treatments should be integrated

into cutting production systems. Donor plants must

be grown for the clear purpose of generating

maximum quantities of cuttings with high rooting

capacities. Therefore, maintenance of good health

will be essential, and development of cultural

treatments to ensure this through specific

manipulations of water, fertilizer, light, and

competition control may often be warranted.

Cutting wood should be collected using defined size

standards since size will affect the quality of

adventitious root systems, and consequently the

uniformity and repeatability of cutting crops.

Chemical treatments applied after severance have

frequently boosted rates of successful rooting or of

root system quality under certain conditions.

However, variability of response among and within

species, and the wide array of potential chemicals,

concentrations, and application methods will make

exhaustive testing difficult. Regardless, strong

reliance on these post-severance techniques is not

warranted since they historically have not

completely compensated for cutting wood that was

not already somewhat predisposed towards rooting.

It is most likely that general systems developed for

species will eventually be adapted to specific clones

and climates to further refine rooting success.

Rooting of cuttings has historically been laborintensive. Horticultural crops have commanded

relatively high prices to compensate for high labor

costs per plant. Cutting collection, preparation, and

rooting have generally not been automated because

of the complexity of the tasks required, and the

diversity of horticultural crops and producers. For

forestry, there will be a strong economic incentive to

minimize labor costs per plant due to the prevalence

of low-cost seedlings. Fortunately, the large

numbers of cuttings required to satisfy planting

needs, and the relatively few nurseries that have

historically produced forest tree seedlings, will help

justify research expenditures directed towards

automation. Future systems for rooting conifer

cuttings are likely to draw upon existing and

emerging technologies in materials handling,

6 6
mechanical sensing, robotics, and artificial

intelligence.

How can we circumvent maturation?--Maturation

has long been a major barrier to rooting, and

successful propagation systems have been built

around the maintenance of juvenile donor plants. In

addition to enhanced rooting potential, juvenile

phase shoots have generally been more uniform in

size, and have produced plants that are largely

similar to seedlings in form and growth rate.

Unfortunately, there is evidence that procedures to

maintain juvenility are not completely successful.

Advanced research with Norway spruce clones has

indicated that serial rooting of donor plants will not

completely arrest maturation (Dekker-Robertson

and Kleinschmit 1991). If it is assumed that longterm hedging is similarly insufficient, propagation of

specific conifer genotypes as clones will not be

possible for an indefinite period of time. However,

for vegetatively multiplied radiata pine, the slight

maturation that has occurred on hedges may

actually improve the quality of sawtimber harvested

from the rooted cuttings (Menzies and Klomp 1988,

Arnold 1990).

With current technology, it takes many years to

develop clones for afforestation. Genotypes must be

field tested while juvenile material of the same

genotypes is maintained separately. After superior

clones are selected, it often takes one or more years

to multiply these genotypes into large numbers of

donor plants that will produce sufficient quantities

of rooted cuttings for deployment. If maturation of

genotypes proceeds slowly regardless of

repropagation or shearing, testing and

multiplication may leave little time for clones to be

effectively utilized in planting. For Norway spruce,

it was estimated that clones would be adequately

juvenile for only 10 years following lo-15 years of

testing and multiplication (Dekker-Robertson and

Kleinschmit 19911. Burdon (1991) stated that the

feasibility of developing clonal forestry techniques

for radiata pine “is still dominated by maturation.”

Additional research must make the development

of clones more efficient. Ideally, methods to

completely rejuvenate mature genotypes would

eliminate the need for maintaining juvenile plants

during clonal tests and, perhaps more important,

might allow clonal tests to be composed of superior

individuals selected from plantations or seedling

progeny tests. Complete rejuvenation would also

theoretically extend the useful lives of conifer clones

for an indefinite period of time. Although

rejuvenation techniques have been summarized

(Hackett 1985), none have been complete enough to

have been adapted to the testing and multiplication

of clones. New rejuvenation techniques that are

partially successful might be effective if coupled with

hedging or serial propagation. Alternative methods

to store genotypes during clonal tests, such as in

vitro cold storage, may also retard maturation.

Development of procedures to hasten testing and

multiplication of clones will also be important.

Reliable early selection techniques, through

conventional means or molecular biology, could

shorten or eliminate clonal tests. Rapid

multiplication procedures that may not be

economically feasible for direct afforestation, like

micropropagation or somatic embryogenesis, might

instead be employed to multiply the number of donor

plants for the production of field cuttings. In

general, the creative synthesis of new and existing

techniques should ultimately be able to extend the

useful lives of conifer clones.

Is the technology economically feasible?--

Commercial application of rooted cutting technology

must be cost effective. The economic benefits of

asexual propagules must surpass their added costs,

and both of these elements require extensive

investigation. If clones are to be deployed as rooted

cuttings, the superiority and uniformity of selected

genotypes must be quantifiable and repeatable.

Selecting and deploying clones that are substantially

superior to improved seedlings will be difficult

because the process is complicated by maturation

during the testing and multiplication phases, as

discussed above. In recalcitrant species, intense

selection for clones with strong rooting capacities

may restrict potential growth gains, or require

expensive clonal tests with large populations.

Assuming that the superiority and longevity of

clones can be demonstrated, and that integrated

propagation procedures can be developed, will the

costs be justified? The question must obviously be

addressed to specific production systems in specific

climates. One example that compared radiata pine

planting stock of equal genetic quality estimated

that rooted cuttings from juvenile donor plants

would cost 40-130% more to produce than seedlings

(Menzies et aL 1985). To develop superior clones,

costs incurred during clonal testing and

multiplication would have to be added, but these

would have to be weighed against the greater

productivity of the clones. Although the question is

simple to ask, the complex array of specific costs and

benefits will make it difficult to answer.
Despite the immensity of some of the biological

barriers to propagation, the relatively slow rate at

which they have been solved through widely

dispersed studies, and the research questions that

remain, some conifers are being propagated on a

large scale. To close, an example will demonstrate

how solutions to the various problem areas were

assembled into a commercial system for rooting

conifer cuttings in the southeastern United States.

EXAMPLE OF COMBINED SOLUTIONS WITH

LOBLOLLY PINE:

INTERNATIONAL FOREST SEED COMPANYS

PROPAGATION PROGRAM 1988-1991

This program emphasized the production of clonal

loblolly pine cuttings that were resistant to fusiform

rust (Cronurtium quercum (berk.) miyabe ex Shirai

F. dp. fisifirme) and that produced good wood

volumes (Foster and Shaw 1987). Seedlings

resulting from controlled pollinations were exposed

to rust screening methods at the U.S. Forest Service

Rust Resistance Screening Center in Asheville,

North Carolina (Anderson et al. 1983). Rust-free

survivors at least six months old were then planted

and maintained in hedge rows. Shearing techniques

encouraged the development of many succulent

juvenile shoots. Greenhouse rooting and field

growth were assessed for cuttings from each hedge,

and subsequent selections resulted in the

establishment of a cutting orchard. The latter

produced high quality cuttings that were rooted for

planting stock.

One key to this clonal propagation program was

the identification of donor plants that would

repeatedly produce easily rooted cuttings. This

required several propagation trials for each donor

plant over a 12- to 18-month period. Later,

genotypes were retested periodically to identify

reductions in rooting capacity. A 2.8 ha cutting

orchard resulted from these tests, and was composed

of hedges of varying age classes. It provided an

excellent opportunity to observe the effects of

cultural regimes and shearing on several age classes

of donor plants. Drip irrigation was used to provide

accurate fertilization and consequently to ensure

good health of hedges. Best hedge height was about

15-18 cm.

The rooting potential of genotypes identified as

good rooters was enhanced by shearing; however,

clones that rooted poorly were not significantly

influenced by shearing (T. D. Caldwell, unpublished

data). Further attempts were made to arrest

maturation or rejuvenate older stock possessing

desirable traits by serially propagating clones. The

success of this technique has not yet been confirmed

with mature stock. Two- to three-year-old stock

plants were serially propagated to increase the

numbers of desired clones. In most cases, the good

rooters continued to express high rooting

percentages. Unfortunately, genotypes expressing

poor rooting were not retained for comparison since

the commercial objective was to screen for good

rooting clones. Consequently, the efficacy of serial

propagation to enhance rooting ability of poorrooting clones is unknown.

Although the orchard was capable of producing

cuttings at least three times each year, the actual

harvest only occurred in late fall. The decision to

root only one cycle per year was made to circumvent

problems with harsh weather and seasonal rooting

variability. The extreme temperatures in the

summer months adversely affected the vigor of the

cuttings during harvest and propagation, which

resulted in significantly lower rooting percentages

(T. D. Caldwell, unpublished data). Earlier trials

had indicated that the fall and winter collections

were more likely to provide easily-rooted cuttings.

Cuttings were therefore harvested by hand after

becoming dormant. The actual dates of collection

were usually after 15 November and before 10

December. Cuttings with terminal buds were

preferred, but proximal shoot segments without

terminal buds were also collected. These were

pruned such that a lateral bud would eventually

develop apical dominance.

Cuttings were placed upright in cold storage (3”

C) for 24-36 hours. Before setting, the proximal end

of each cutting was dipped in a proprietary version

of Hare’s powder (Hare 1974). Each was then set in

a 97 ml cavity containing a soilless medium

comprised of 60% perlite and 40% sphagnum peat

moss. Planting density was 1000 cuttings per

square meter.

Cuttings were then placed in a greenhouse for

approximately 16 weeks where temperature,

humidity, and light were closely monitored. Day

temperatures were maintained at 25-26” C; night

temperatures at 23-24” C. Winter air temperatures

were increased using a radiant heating system

located near the greenhouse roof. Relative humidity

ranged from 85% to 92%. The variation in humidity

corresponded roughly to day and night periods since

66
elevated day temperatures caused an increase in the

moisture-holding capacity of the greenhouse air.

A traveling gantry boom irrigation system,

positioned approximately 45 cm above the cuttings,

provided intermittent mist. Utilizing normal

waterline pressure of approximately 4 kg/cm2 (60

psi) and nozzles that dispersed fine water droplets at

a rate of 0.23 liter/min (0.06 gpm), a fog-like mist

was deposited over the needle surfaces as the gantry

boom traveled the length of the greenhouse.

Manipulating the speed and frequency of travel

regulated the amount of moisture deposited on the

foliage. Uncallused cuttings required more frequent

misting than cuttings that had callused or that had

initiated root primordia. Operation of the gantry

system was therefore regulated to match moisture

applications to the physiological state of the

cuttings. Two sources of artificial light extended

natural day lengths to 16 hours during twelve of the

sixteen weeks. Stationary 300-watt incandescent

lamps were combined with similar lights attached to

the misting boom to provide sufficient irradiance.

Approximately 10 weeks into propagation,

temperatures and day lengths were gradually

reduced to induce dormancy and to promote more

root development with less shoot elongation. During

weeks 11 and 12, the greenhouse would be returned

to outside ambient temperatures. Day temperatures

would therefore range from 5 to 12” C; night

temperatures from 5 to 7” C. The cuttings were

returned to natural day lengths by the end of week

12.

The rooted cuttings remained within the

greenhouse until the following spring after all

danger of a late freeze had passed. The rooted

cuttings were usually moved outdoors after 15 April.

Cuttings then received soluble 20-20-20 general

purpose fertilizer at ten-day intervals through the

summer. The last application occurred in mid- to

late September when irrigations with clear water

were substituted to lower nitrogen levels in

preparation for dormancy. Fungicides and

pesticides were applied as needed. The rooted stock

was considered to be field ready by late fall,

approximately 12 months from cutting harvest.

This rooting system was designed specifically to

propagate one coniferous species. Many

components, however, were similar to those in

vegetative propagation programs found primarily

within the ornamental nursery industry. It was

critical to control humidification, temperature,

nutrition, medium, and seasonal harvest timing.

Many factors were interrelated and required balance

to stimulate development of adventitious root

systems. Two factors that caused loblolly pine to be

difficult to propagate vegetatively were (1) the

inevitable loss of the strong juvenile rooting

capacity, which often resulted in dramatic

reductions in rooting success, and (2) the resulting

requirement for intensively selecting clones that

maintained strong rooting capacities past the second

or third shearing cycle (about 2 years of age).

Barriers to successful propagation were

successfully scaled to institute this program. Since

loblolly pine was, and will continue to be, an

important species, selection within the species to

provide outstanding growth, survival, and rooting

capacity was important. By shearing the donor

plant, maturation was retarded and many succulent,

juvenile shoots were forced into growth. How long

this technique can arrest maturation will probably

be dictated by the species, and by the individual

donors and their response to continuous hedging

and other treatments. Collecting cuttings in the fall,

when they had a relatively strong rooting capacity,

improved crop success and consistency. Water and

fertilizer applications ‘enhanced productivity and

ensured that cuttings on healthy hedges would have

a strong capacity to form roots. A highly-controlled

greenhouse provided the correct balance of

environmental factors that allowed cuttings to root.

Currently, there are no large scale clonal

propagation programs for loblolly pine. The costs of

such a labor- and equipment-intensive program may

continue to deter many organizations from pursuing

the necessary technology. However, as the biological

barriers to rooting are scaled, and logistical

problems are resolved, clonal propagation should

become a viable forest regeneration option for this

and other coniferous species.

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73

ROOTED CUTTING MACROPROPAGATION OF HARDWOODS

S.B. Land, Jr. and Mike Cunningham2 3

Abstract.--Factors affecting regeneration from cuttings of hardwood tree

species include (a) rejuvenation and conditioning of the stock plant,

(bl environmental conditions and physiological status of the stock plant,

(c) genetic differences in rooting ability, (d) seasonal timing of collection,

(el cutting size, node characteristics, and position on the stock plant,

(fl treatments of cuttings, and (g) planting methods.These factors are

discussed and illustrated with examples for hardwood species. A condensed

table of recommendations for cutting macropropagation of 16 hardwood genera

is included, and the current status of several operational programs is given.

Management of within-clone variability is discussed.

INTRODUCTION

Macropropagation with shoot and/or root

cuttings has been accomplished for many hardwood

tree species. These cuttings can provide an

economically viable alternative for artificial

regeneration if (a) unrooted hardwood shoot cuttings

can be planted directly in the field with high rooting

success or (b) rooted cuttings can be mass produced

in a nursery/greenhouse and outplanted to the field

at an acceptable cost. Situation (a) can be met for

some (but only a few) hardwood timber species, as

are identified later. Situation (bl requires either

cheap labor, highly mechanized nursery methods,

rapid growth with short rotations (energywood,

1 Paper presented at the 1992 Southern Regional

Information Exchange Group Biennial Symposium,

Huntsville, AL, July 8-10, 1992

2 Professor of Forestry, Mississippi State

University, P. 0. Drawer FR, Mississippi State, MS

39762; Research Scientist, Union Camp Corporation,

P. 0. Box 345, Hagan, GA 30429

aApproved for publication as Journal Article

PS8052 of the Mississippi Agricultural & Forestry

Experiment Station, Mississippi State University.

pulpwood), or high-valued products (tine furniture

wood, urban trees, multiproduct agroforestry

species, or horticultural nut and fruit species). Some

species are so difficult to root that they will probably

never be regenerated with cuttings.

We will first define some terms used throughout

the paper and in the rooting literature. General

factors affecting rooting success from shoot and root

cuttings of hardwood tree species will then be

reviewed and illustrated with examples. These

factors include (a) rejuvenation and conditioning of

the stock plant, (b) environmental conditions and

physiological status of the stock plant, (c) genetic

differences in rooting ability, (d) seasonal timing of

collection, (e) cutting size, node characteristics, and

position on the stock plant, (0 treatments of

cuttings, and (g) planting methods. The third

section of the paper provides specific

recommendations for cutting macropropagation of 16

hardwood genera and summarizes the current

status of several operational hardwood programs

using cutting macropropagation in the United

States. The final section discusses the need for

control of within-clone variability in production

programs, once the methods for getting high rooting

success have been developed. Sources of withinclone variability that are discussed include (a)

phenotypic (non-persistent) variation, (b) epigenetic

75
variation, (c) genetic variation within clones, and (d)

variation caused by pathogens. A suggested

strategy of seven steps is given for management of

cutting production programs to control these sources

of within-clone variability.

TERMINOLOGY

The rooting literature is notorious for jargon. To

assist the reader, several terms and concepts used in

this paper are defined below. These definitions come

from Hartmann et al. (1990, Chapters 1,8, and 9)

and from the Society of American Foresters’

“Terminology of Forest Science, Technology,

Practice, and Products” (SAF 1983). Additional

definitions are provided at appropriate places

throughout the paper.

Adventitious.--Adventitious refers to a plant part

that develops outside the usual order of time and/or

position (outside the normal order of plant

development and ontogeny). Adventitious buds

(and shoots) arise from any plant part (stem, leaf,

or root) other than terminal, lateral or latent buds

on stems. The origin of adventitious buds is usually

in callus or lateral meristematic tissue, and these

buds have no vascular connection with the pith. In

contrast the terminal, lateral, and latent buds have

a vascular connection with the pith, indicating that

they originated from terminal meristematic tissue

and were preformed in the normal order of plant

development. The latent buds are dormant buds

lying under the bark, and they begin to grow when

dormancy is broken by wounding or by increased

sunlight on the stem. Adventitious roots arise

from any plant part (stem, leaf, or root) other than

from terminal meristems in root tips. Adventitious

buds and roots are initiated in response to some

wounding and/or chemical stimulus. Adventitious

roots can be either preformed roots (develop on

stems while the stems are still attached to the

parent plant) or wound roots (occur in response to

a wounding effect, and can develop after the plant

part is removed from the parent plant).

Cuttings.--A cutting is a segment or part

(greater than a few cells or piece of tissue) that is cut

from a living stem, limb, root, or leaf. It is used to

produce a whole new plant through the development

of adventitious roots and/or shoots. Stem or limb

cuttings require only the development of

adventitious roots, and these cuttings can be

classified according to the degree of lignification

(hard woodiness) of the tissue. Softwood cuttings

(also called summerwood or greenwood cuttings) are

those cut from the current year’s tissue while the

parent plant is actively growing. These cuttings are

taken early in the growing season from near the

growing tip, have little lignification, and are soft and

fleshy with a high water content. Hardwood

cuttings (also called dormant cuttings) are those

collected during the dormant season of the year.

They are lignified woody cuttings and may come

from the most recent past growing season (usual) or

from earlier growing seasons. Semi-hardwood

cuttings (also called firm softwood cuttings) are

taken late in the growing season from the current

year’s growth and are partially lignified. Root

cuttings are usually taken from lignified woody

roots and must initiate both a new shoot system

(from an adventitious bud) and new adventitious

roots. Sometimes these cuttings have adventitious

shoots (root sprouts) already formed while the root

was still attached to the parent plant. More

commonly, the adventitious roots and shoots are

formed after the root cutting is detached from the

parent plant. In the present paper the word

“cutting” usually means stem, limb, or sprout

cuttings. A root cutting will be specified as “root

cutting”.

Sprouts.--A sprout (also called a shoot) is a

young, slender, aerial outgrowth from a woody plant

part (stem, limb, or root). Sprouts are often used as

sources for cuttings, because they retain some

juvenility and give cuttings with higher rooting

success. Coppice sprouts (also called stump

sprouts or stool shoots) are any shoots arising from

adventitious or latent buds near the base of a woody

plant that has been cut back (detopped). Epicormic

sprouts (also called water sprouts) are shoots

arising from adventitious or latent buds on the stem

or on a limb of a woody plant. The development of

these sprouts is usually in response to wounding or

to an increase in sunlight reaching the stem.

Coppice sprouts are epicormic sprouts, but epicormic

sprouts also include shoots further up the tree.

Root sprouts (also called root suckers) are aerial

shoots arising from adventitious buds on roots or

rhizomes below ground.

Rooting ability.--Rooting ability (also called

rooting capacity or rooting success) refers to the ease

of adventitious root initiation on cuttings (and

adventitious shoot initiation on root cuttings). This

is typically measured by the percentage of cuttings

which establish a root and shoot system and survive

7 6
for some defined period of time (such as to the end of

the first growing season).

Juvenility and maturity.--Any tree in its life

cycle goes through embryonic, juvenile, transitional,

and mature (adult) phases of growth and

development. The juvenile phase has a greater

ability to develop adventitious roots and shoots than

the mature phase, but the mature phase can

produce flowers while the juvenile phase cannot.

These are epigenetic changes, meaning that the

differences result from changes in gene regulation

rather than changes in the genetic makeup of the

plant. Because the juvenile phase exhibits the

higher rooting ability, that phase is preferred for

macropropagation with cuttings. Therefore, an

important topic in macropropagation is how to

control juvenile-mature changes (the aging process).

Possibilities include (a) use of the most juvenile part

of the tree (the base of the stem) and (b) reversions

of the aging process. This aging process involves

both physiological and ontogenetic aging.

Physiological aging is identified by reduced

growth rate, change in type of branching, and

exhaustion of nutrient content as the source plant

gets older and larger in size (Hartmann et al. 1990,

Struve and Lineberger 1988). It is not localized in

the meristem and can be reversed by renewal or

invigoration of shoots through pruning, nutrient

additions, or stand density control. Invigorated

shoots may root better than non-invigorated shoots,

but they can also retain mature traits such as

flowering (Hackett 1988). Ontogenetic aging

refers to the epigenetic change from the juvenile to

the mature phase. It is genetically programmed,

localized in the meristem, and not related to

exhaustion of nutrients. Rejuvenation represents

the reversal of the ontogenetic aging process.

Rejuvenated shoots will root easily, will usually not

flower, and will exhibit morphologically juvenile

traits, such as juvenile leaf forms.

FACTORS AFFECTING ROOTING OF CUTTINGS

FROM HARDWOOD TREE SPECIES

Age and Conditioning of the Source Plant

Rooting ability of shoot cuttings decreases with

increasing tree age for hardwood tree species. For

some of the eucalypts rooting success declines so

rapidly that cuttings collected from one-meter-tall

seedlings will not root (Hartney 19801. Typically,

however, once successfully rooted first-generation

cuttings of mature trees are obtained, they will

produce rejuvenated cuttings of increased rooting

capacity. This has been reported for Eucalyptus

tereticornis Sm. x E. camaldulensis Dehn. by

Franclet (1983). The problem is getting the rooted

first-generation cuttings from the mature source

plant.

Several methods listed by Hackett (1988) for

improving rooting from mature plants include: (a)

severe pruning or hedging, (b) grafting mature

scions on juvenile rootstocks, (cl adventitious bud

initiation, and (d) in vitro propagation. The first

three are discussed here, and the fourth is included

in the paper by Scott Merkle (1993) at this

symposium.

Severe Pruning or Hedging.--Many hardwood

species can produce coppice sprouts from the base of

a tree. These sprouts often have high rooting ability

that is comparable to cuttings from seedlings, since

they originate from a juvenile part of the tree. This

characteristic has been the basis for a number of

operational macropropagation programs throughout

the world, where stool beds or cutting orchards are

established from selected trees and repeatedly

coppiced or severely pruned to produce cutting

material. Franclet (1983) believes that this form of

hedging results in selection of me&terns which

remain juvenile and stabilized at that maturation

level, rather than true rejuvenation, In addition to

maintaining juvenility, pruning or hedging of stock

plants controls plant shape for ease of stock

management and cutting collection, controls the

timing of flushes, and increases cutting production

(Scott 1987).

There may be instances where severe pruning or

coppicing is not an option for the production of

epicormic shoots (e.g. the selected tree is needed for

breeding purposes). One alternative method is to

partially girdle selected trees to temporarily relieve

apical dominance and allow coppice sprouts to

develop. This technique has been successfully

employed to initially propagate Quercus rubra L.

(Harmer 19881, yellow-poplar (Liriodendron

tuliperifera L.1 (M&pine and Kormanik 19721,

sweetgum (Liquidambar styraciflua L.1 (Kormanik

and Brown 1973) and several Eucalyptus L’Her.

species (Hartney 19801. Juvenile sprout material for

macropropagation has also been obtained through

the application of cytokinins to the base of trees to

induce latent bud break. Mazalewski and Hackett

(1979) reported that the application of cytokinins as

a lanolin paste or water-ethanol solution to the base

of Eucalyptus fzcifolia F, Muell. trees was successful

77
in inducing bud break in the lignotubers and in the

upper trunk region. However, only shoots obtained

from the lignotubers possessed high rooting ability.

They concluded that the position on the trunk from

which epicormic shoots were obtained reflected

rooting ability of the original seedling nodes from

which they were derived.

Grafting Mature Scions on Juvenile Rootstocks.--

A second alternative (when the selected tree must be

retained for breeding purposes) is the grafting of

mature scions onto seedlings. This is followed by

either (a) hedging or partially girdling the grafted

tree after a few years to stimulate epicormic sprouts

or (b) repeated grafting in serial fashion at short

intervals on successive seedlings. The procedure

has been an effective method of obtaining cuttings

with increased rooting ability for several species.

Farmer and Besemann (1974) combined grafting and

severe pruning of selected black cherry (Prunus

serotina Ehrh.) to substantially improve the rooting

of softwood cuttings propagated under mist.

However, there were no non-pruned controls to

determine if grafting or pruning effects had

improved rooting. Paton et al. (1981) proposed that

the improved rooting of cuttings from mature scions

of Eucalyptus grandis Hill ex Maiden. grafted onto

juvenile seedlings is a result of a closer association

between the shoot tip and the base of the stem

and/or roots (“root-shoot gradient”). They compared

rooting ability and levels of the growth regulator G

(a compound which has been shown to inhibit

rooting of E. grandis at high concentrations and

promote rooting at lower levels) for (a) cuttings

taken from the distal and basal positions on adult

trees and (b) scions of adult trees grafted onto

seedlings. In each case they found that epicormic

shoots at the basal position had lower G

concentrations and higher rooting percentages.

Franclet (1983) and Hackett (1988) believe that E.

grandis cuttings taken from epicormics at the base

of mature scions grafted on seedlings have been

rejuvenated. Repeated grafting in serial fashion is

also thought to rejuvenate cuttings when used for

Eucalyptus x trabutii (= E. botryoides Sm. x E.

camaldulensis) (Hartmann et al. 1990) and E.

camaldulensis (Franclet 1983).

Adventitious Bud Initiation.--Adventitious

shoots on roots or sphaeroblasts of Populus

tremuloides Michx., UZmus L. species, and apple

cultivars produce rejuvenated cuttings of high

rooting success (Hackett 1988). Aspen tree

improvement programs rely on the production of

these shoots from root segments for

macropropagation, The techniques described by

Benson and Schwalbach (19701 consist of collecting

root segments from selected trees, culturing the

roots under greenhouse conditions to produce

adventitious shoots, and then rooting the softwood

sprouts. Success rates of from 70 to 90 percent are

obtained with most aspens. A similar system has

been used for sweetgum (Farmer 1966). Stimulation

of adventitious buds for coppice and epicormic shoots

by severe pruning/hedging and by grafting have

been discussed above.

Environmental Conditions & Physiological Status of

the Stock Plant

Macropropagation of selected trees under forest

conditions severely restricts the capabilities of stock

plant environmental control. If large numbers of

cuttings are required, it is best to establish stock

plants in cutting production orchards (stool beds).

Doing so provides a number of advantages: (a) tree

health is known, (b) management is easier, since the

propagator controls plant nutrition, weed control,

pruning, pest and disease control, and all aspects of

plant manipulation; and (cl tree growth is more

uniform, since cuttings root more evenly (Scott

1987). Most operational rooted cutting programs

have established cutting production areas for these

reasons.

Nutritional status of the stock plant, and

particularly the carbohydrate-to-nitrogen ratio

(C/N), is frequently discussed as an important factor

for root initiation. High C/N has been shown to

improve rooting for a number of species (Moe and

Anderson 1988). One method of obtaining this high

ratio is to reduce N supply to stock plants

(Hartmann et al. 1990). In a study examining clonal

differences in rooting of sycamore coppice sprouts,

Cunningham (1986) found significant differences in

rooting between clones grouped by the progeny test

block in which the stock plants were located. These

blocks also differed in soil N content. Rooting

percentages for the four blocks were 68,59, 54, and

35, and these corresponded to soil N levels (ppm) of

473, 705,650 and 822, respectively. While no firm

conclusions could be drawn regarding the effects of

soil N levels on rooting due to a lack of common

genotypes among treatments, clones from the

progeny test block with low soil N rooted

significantly better than those from the high soil N

block.

Carbohydrate status of cuttings can be increased

by girdling shoots prior to severance. The technique

7 8
developed by Hare (1976a, 1976b,19771 for a

number of species involves removing a small strip of

bark around the stem to allow for the accumulation

of carbohydrates as well as other translocated

substances at the distal end of the girdle. After six

to eight weeks the girdled shoot is removed from the

tree and used for propagation. In nearly all cases,

Hare has found that cuttings from girdled shoots

root significantly better than their nongirdled

controls.

Cutting macropropagation is also influenced by

the light conditions under which stock plants are

grown. Increasing light irradiance in the

greenhouse may increase the carbohydrate content

of the cuttings. However, light reduction has often

been beneficial for rooting. This may be because

flowering and vegetative growth is inhibited to

release more carbohydrate for rooting (Hartmann et

al. 1990). Reducing stock plant irradiance through

shading and reduction in photoperiod is a common

practice to enhance rooting (Bachelard and Stowe

1963, Hansen 1987). Total exclusion of light from

newly emerging shoots for a period of time prior to

propagation (etiolation) has proven effective in

promoting rooting for a number of species

(Hartmann et al. 19901. A slight modification of this

procedure, where light is excluded from the cutting

base prior to propagation (banding), may also

improve rooting. The girdling procedures of Hare

(1976a, 197613, 1977) would be considered banding,

since the girdled portion of the stem is covered with

aluminum foil. Mixed results have been obtained in

etiolation trials with forest species. Kormanik and

Brown (1973) found that etiolated shoots of

sweetgum and yellow-poplar rooted poorly while

Farmer and Hall (1973) found that black walnut

(Juglans nigru L.) etiolated shoots rooted well.

Additional explanations for the effects of etiolation

and banding on rooting include changes in stem

tissue anatomy (less lignification, decreased cell wall

thickness and reduced cell differentiation) and

increased tissue sensitivity to exogenously applied

auxin (Bassuk and Maynard 1987).

Genetic Differences in Rooting Ability

There is a wide range in rooting ability for

species, geographic origin, and genotype just as

there is for other more commonly measured traits of

forest trees. One of the first reports of tree-to-tree

variation in rooting was for red maple (Snow 1939).

Since then genetic variation has been reported for a

large number of species (Haissig and

Riemenschnieder 1988). The selection of good

genotypes can dramatically improve overall rooting

percentages, provided rooting ability is under

genetic control. For example, eucalypt programs are

finding that clonal means for rooting percentages of

genotypes selected for growth characteristics can

range from 0 to 100 percent (Campinhos and

Ikemori 1980).

Studies evaluating the level of genetic control

have found broad-sense heritabilities on a clone

mean basis to be fairly high, but few estimates of

additive and non-additive genetic variance are

available. Wilcox and Farmer (1968) estimated

broad-sense heritabilities for eastern cottonwood

(Populus deltoides Bartr. ex Marsh.) to be from 0.32

to 0.89 for a number of rooting traits. With a

different population of eastern cottonwood, Ying and

Bagley (1977) estimated broad-sense he&abilities

for numbers of roots initiated to be 0.8 and 0.86, and

they detected significant levels of additive variance.

Rooting abilities for both sycamore and sweetgum

have comparable broad-sense heritability estimates

to those for eastern cottonwood, but estimates for

additive variance were nearly zero (Cunningham

1986, Cunningham 1989). One common limitation

to each of these studies is the use of open-pollinated

families, where no direct estimates of non-additive

variances can be obtained. In addition, the work

with sycamore and sweetgum used only a limited

number of families and clones within families, so the

precision of estimates was low.

Seasonal Timing of Collection

The time of year in which cuttings are collected

can have a dramatic effect on rooting and may be

the key to success for many species. Easily rooted

species, such as those possessing preformed roots,

can generally be rooted all year and are

operationally rooted using hardwood cuttings. In

contrast, many species can only be rooted using

softwood cuttings. Sweetgum is a good example of

such a species. Cuttings from either tree crowns or

coppice shoots root fairly well when collected during

the growing season; however, dormant material will

not root (Cunningham 1989) (Table 1). Hardwood

cuttings can generally be collected any time during

dormancy provided they are outplanted at the

proper time, and proper storage procedures have

been followed (Fege 1983, M&night 1970).

However, if cuttings are set immediately following

collection, some differences may be seen. Kefeli and

Turetskaya (1965) found that willow cuttings

collected in the fall rooted poorly in comparison to

spring collections. But, chilling or removal of buds

7 9
Table l.--Influence of cutting type and location on sweetgum

rooting success

Cutting Type

Cutting position Softwood Hardwood

from early collections of sweetgum coppice sprouts,

while not having higher rooting percentages, were

significantly larger at time of planting than later

collections (Table 2). Increasing lignification and

concentrations of root-inhibiting growth regulators

in later collections is a problem for eucalypt species

(McComb and Wroth 1986, Paton et al. 19811.

% rooted Type of Cutting Material Collected

Coppice sprout 8 4 0

Tree crown 5 2 1

from the fall-collected cuttings improved rooting

success to levels equal to those of spring-collected

cuttings. They attributed the poor rooting to the

accumulation of flavonoid compounds in the buds

during the fall which neutralize the stimulating

effects of auxin. Over the winter months the

concentrations of these inhibitors decline to levels

where root initiation can proceed in the spring.

Cutting Position on Tree or Sprout.--Hardwood

cuttings from the basal portion of sprouts tend to

root and grow better than cuttings from more distal

portions of the stem. This has been reported by

several authors for sycamore (Briscoe 1963, Land

1983, Nelson and Martindale 1957). Nelson and

Martindale (1957) and Land (1983) separated

potentially confounding effects of cutting diameter

in their analyses and demonstrated that cutting

position affected first-year height and seventh-year

dbh and volume, respectively.

The time of collection during the growing season

is very important to rooting success of softwood

cuttings. For some species rooting success is best

when cuttings are collected early in the season,

whereas for others a semi-hardwood cutting

collected in late summer is best (Dirr and Heuser

1987, Hartmann et al. 1990). For deciduous species

collection time during the growing season will also

impact the length of time for root and shoot growth

to occur prior to the onset of dormancy.

Cunningham (1989) found that cuttings derived

Findings similar to those for sycamore have also

been reported with Populus L. species (Hansen and

Tolsted 1981, Schroeder and Walker 1991).

Differences in performance based on cutting position

have been attributed to the number of preformed

root primordia and carbohydrate levels. Distal

positions on poplar shoots tend to have fewer root

primordia than basal cuttings (Smith and Wareing

1972al. Although they have higher concentrations of

carbohydrates, the total amount of carbohydrate in

the stem is less for shoot tip cuttings than for

cuttings from the sprout base (Fege and Brown

19841.

Table 2.--Influence of growing season collection date on rooting and growth of sweetgum cuttings from coppice sprouts

Collection

Date

Rooting

success

Cutting

Height

Root-collar

Diameter

Root

Growth1

June 21

June I

91 a

July 7

July 19

22 Gm HM 32

88a2 159a 4.2a 65a

14.0 b 3.9 b 51 b

91 a 11.9 c 3.5 c 37 c

89 a 11.8 c 3.1 d 10 d

1 Root growth was measured as the percentage of rooted cuttings with suffkient root development to form a complete plug when removed

from the container.

2 Collection date means followed by the same letter within a column are not significantly different at the 5 percent level.

8 0
For softwood cuttings the situation is reversed.

Cuttings taken from the shoot tip generally root

better than those from farther down the shoot

(Leakey and Coutts 1989, Cunningham 1989). The

better response of apical softwood cuttings has been

attributed to higher concentrations of root

promoting chemicals at the shoot apex and a lower

degree of stem lignification (Hartmann et aL 1990).

Cutting Size and Node Characteristics.--

Softwood cuttings are typically short (8 to 15 cm

long), and consist of the current year’s growth after

some degree of lignification has occurred. Leaves

are always attached, as rooting percentages will

drop dramatically if these leaves are removed (Bilan

1974, Bachelard and Stowe 1963, Gear-y and

Harding 1984). Macropropagation programs with

For Populus deltoides in the South, the average

cutting length used for field planting is 50 cm.

Longer cuttings might be used on drier sites

(McKnight 19701, and shorter cuttings might be

used for nursery propagation (Cunningham 1986).

In other regions of the United States, hardwood

cuttings are usually shorter, and average 20 cm

length (Fege 1983). Diameters of hardwood cuttings

typically range from 13 to 25 mm. Cuttings larger

than this will root, but they are more difficult to

handle (Fege 1983, McKnight 19701. The survival

and growth of field-planted Salix alba L. cuttings

increase markedly as cuttings increase in size up to

a diameter of 19 mm and length of 20 cm (Burgess et

al. 1990). Rooting success declines for sycamore

cuttings with less than a 15mm diameter, but is

equally good for 15- to 22-mm-diameter cuttings

Table 3.--Influence of cutting diameter, number of nodes, and proximity of bottom node to base of cutting on rooting success of

25-mm hardwood sycamore cuttings

Basal Diameter No. Nodes Below Ground

Length from Cutting

Base to Lowest Node

Diameter Rooted Number Rooted Length Rooted

mm 5 % Ha

<=12 10 0

13-14 17 1

16-16 4 2 2

17-18 5 6 3

19-20 5 2

21-22 5 0

0 o-2 5 3

2 7 3-4 3 2

3 8 5-6 3 4

3 6 7-a 2 5

9-10 3 3

>lO 14

eucalypt species generally consist of cuttings with

two to four nodes, depending on internode length.

Eucalypt cuttings for the Aracruz program in Brazil

contain two nodes and four leaves to allow for some

leaf senescence (Campinhos and Ikemori 1980).

However, Geary and Harding (1984) reported that

while Eucalyptus camaldulensis cutting yields from

stock plants were doubled by using shorter cuttings

with two nodes, rooting success of the two-node

cuttings was significantly lower and not sufficient to

produce more rooted cuttings than the four-node

propagules.

Hardwood cuttings usually are collected from

one-year-old sprouts during the dormant season.

(Land et al. 1991) (Table 3). Improvements in

survival and growth with increasing cutting

diameter may differ by clone. In a study comparing

the effects of cutting diameter on growth of three

Populus clones, Dickmann et al. (1980) found that

cuttings from 6- to 19-mm diameter survived and

grew equally well for 2 of the 3 clones, but declined

for cuttings below lo-mm diameter for the third

clone.

Hardwood cuttings should consist of several

nodes, and location of the nodes is important. Land

et al. (1991) found for sycamore that having at least

two nodes below ground and a node within two cm of

the cut base resulted in best rooting (Table 3).

Having a node near the top of the cutting, and

having the correct type of buds present at the nodes,

81
may be critical for rooting of Pop&s species.

Radwan et al. (1987) found that the best-growing

cottonwood cuttings were those possessing axillary

buds, rather than dead buds or spent buds (nodes

where a branch had elongated). The authors

hypothesized that the slower growth exhibited by

cuttings with spent or dead buds was a result of the

time lag involved in the development of suppressed

or adventitious buds at the nodes.

Treatment of Cuttings

Hardwood cuttings are often stored prior to

planting as a convenience to nursery managers.

This storage may or may not affect rooting success.

In some cases cold storage is required to improve

rooting ability. Fege (1983) recommends storage of

Populus cuttings at temperatures near freezing for

short term handling and at slightly cooler

temperatures (-loo C) for longer-term storage (five

months or more). The cuttings stored at these lower

temperatures had higher concentrations of sugars,

lower concentrations of starch, and slightly higher

total carbohydrates (Fege and Brown 1984).

However, storage temperature had little effect on

performance of these same clones in subsequent field

trials (Fege and Phipps 1984).

Sometimes, chilling can significantly improve

rooting success when hardwood cuttings are to be

rooted under greenhouse conditions. Sycamore

cuttings collected in the fall from mature ortets

rooted at 58 percent when treated with auxin and

stored at 4’ C for one month compared with 35

percent rooting for cuttings treated with auxin and

immediately set in the greenhouse (Hare and Land

1982). Smith and Wareing (1972b) found that fallcollected Populus cuttings given 10 weeks of chilling

at 3’ C produced significantly more roots than

unchilled seedlings. The improved rooting may have

been a response of increased endogenous auxin

levels that were found in chilled compared with

unchilled cuttings.

Other storage treatments for hardwoods include

warm temperature callusing and bottom heat

callusing. These two procedures involve treating

dormant fall-collected cuttings with root-promoting

chemicals and then either (a) storing them under

warm, moist conditions for several weeks followed by

planting in the nursery (warm temperature

callusing) or (b) storing them under normal winter

or cool conditions with the base of the cutting placed

in damp packing material over bottom heat

(Hartmann et al. 1990). Warm temperature

callusing significantly improved rooting success of

Pyrus communis L. hardwood cuttings. Noncallused

controls rooted at 0 and 1 percent in 2 studies, and

the best auxin treatments for the callused cuttings

in those studies rooted at 45-72 percent (Hartmann

et al. 1963).

Rooting of both hardwood and softwood cuttings

can be improved by treatment with various

chemicals. The most commonly used root-promoting

substance is some form of auxin. The literature

abounds with reports of successful rooting of species

treated with auxins that otherwise would not root or

rooted very poorly. Blazich (1988) provides a good

summary of the use of auxins to promote rooting and

lists four advantages of their use: (a) increased

percentage of cuttings that initiate roots, (bl

increased rate of root initiation, (c) increased

number of roots initiated and quality of roots, and

(d) improved uniformity of rooting.

Other plant growth regulators evaluated for

their effects on adventitious rooting have provided

mixed results. For most of the classes of growth

regulators there have been both promotive and

inhibitory effects reported: A good summary of

research with each class of compounds is provided in

the book “Adventitious Root Formation in Cuttings”

(Davis et al. 1988).

In addition to treatment with auxins, the leaves

on softwood cuttings from species with large leaves

are often trimmed to improve water relations.

Geary and Harding (1984) compared the rooting of

Eucalyptus camaldulensis cuttings with trimmed

and entire leaves and found that a significantly

greater percentage of cuttings with trimmed leaves

rooted and flushed. Leakey and Coutts (1989)

trimmed the leaves of Triplochiton scleroxylon

Schumann cuttings to three different sizes (lo,50 or

100 cm2). After 42 days of propagation, the largerleafed cuttings had greater dry weights and

carbohydrate levels. However, fewer of these largeleafed cuttings rooted, possibly because they had a

large leaf area that reduced leaf water potential

prior to root emergence. The authors hypothesized

that rooting ability is partially determined by a

balance between transpiration and photosynthesis.

Environmental Conditions During Rooting

Rooting soil medium, fog or mist systems,

temperature, and light in the greenhouse or nursery

can affect rooting success. Since the topic is covered

82
by another paper at this symposium (Ford-Logan

19931, it will not be discussed here.

Planting Methods

Once greenhouse- and nursery-grown cuttings

have been rooted, they can be handled similarly to

seedlings. Softwood cuttings are typically grown in

containers and subsequently field planted when they

reach an appropriate size and when soil conditions

are suitable (Campinhos and Ikemori 1980,

Cunningham 1989). Hardening-off treatments

involving reductions in watering and shade are

important for these softwood rooted cuttings before

planting in the field.

Field-planted unrooted cuttings require good

hardwood site preparation prior to planting

(Huppuch 19601. In addition, weed control through

cultivation or chemicals is essential until crown

closure (Kennedy 1975, Huppuch 1960, McKnight

1970). Cuttings have traditionally been planted

vertically leaving 2.5-5 cm of the top of the cutting

above ground. However, Hansen et al. (1991) have

recently reported that planting the cuttings flush

with the soil surface gives fewer multiple-stem trees

for hybrid poplars, and there are no effects on

growth or mortality. There has been some research

evaluating the potential of horizontally planted

cuttings in furrows. Good success has been obtained

with (a) green ash V’ruxinus pennsyluanica Marsh.)

cuttings derived from seedling sources (Kennedy

1972) and (b) sycamore sprouts (Briscoe 1969). One

disadvantage to horizontally planted cuttings is that

multiple stemmed trees are usually produced.

In the South, hardwood cutting plantations have

been successfully established throughout the

dormant season (Briscoe 1963, Briscoe 1973, Nelson

and Martindale 1957). Mild winters usually keep

the ground from freezing, and root initiation can

take place while shoots are still dormant (Nelson

and Martindale 1957). In more northern locations

where soil freezing and thawing occur, fall plantings

can meet with poor success due to frost heaving.

Hansen (1986) recommends that spring planting be

delayed until soil temperatures are above 10’ C.

When soil temperatures are below this value

cuttings will not grow roots, will expend storage

reserves, and will experience water stress.

CUTTING MACROPROPAGATION OF SOME

HARDWOOD TREE SPECIES

Recommendations for 16 Genera

Summary comments on difficulty of rooting

cuttings and on the best methods for maximizing

rooting success are presented in Appendix Table 1

for 16 hardwood genera that have been used for

wood production in the United States. The difficulty

of rooting has been subjectively divided into four

classes in that table. Class “A” species are those that

are very easily rooted (they usually have preformed

root initials on stem cuttings) and can be planted

directly in operational field plantations as dormant,

unrooted hardwood cuttings. Either juvenile or

mature cuttings can usually be used, although

cuttings from one-year-old shoots on top-pruned

stock plants in stool beds are preferred. Class “B”

species are slightly more difficult to root. However,

hardwood stem cuttings from juvenile material will

root easily without treatment and can be planted as

dormant, unrooted cuttings in operational field

plantations. Mature material must be either

rejuvenated (or invigorated), or the cuttings must be

rooted with customized treatments in the nursery or

greenhouse, before outplanting. Class “C” species

are somewhat difficult to root, but rooting can be

accomplished with customized treatments in

greenhouses or nurseries (as summarized under

“Comments” in Appendix Table 1). Only rooted

cuttings of these species could be outplanted

operationally. Class “D” species are very difficult to

root. In some cases, no reported successes in rooting

are available in the literature. Cutting

macropropagation on an operational scale is not an

option for these species, at least with current

technology.

Some Operational Programs

Cutting macropropagation is used operationally

for forestry in the United States for Populus

CAigeiros Duby and Tucumuhucu Spach sections),

Eucalyptus (species shown in Appendix Table l),

and S&ix L. species and hybrids.

Populus deltoides (eastern cottonwood) has long

been planted as unrooted hardwood cuttings in the

Mississippi River valley. These plantations

represent approximately three-fourths of all

commercial poplar plantings in the United States

(DeBell 1992). One example is the Fitler Managed

Forest owned by James River Corporation in

western Mississippi, where 45-cm cuttings from oneyear-old shoots on stools in a cutting production

nursery are collected and planted during the winter.

P. trichocurpu Torr. and Gray x P. deltoides hybrids

0 3
are being planted as unrooted 30-40 cm hardwood

cuttings in Washington and Oregon. Many of the

clones have been developed by the University of

Washington/Washington State University Poplar

Research Program. The plantations are being grown

on a six-year rotation for pulpwood. James River

Corporation already has plantings of 3480 ha in the

lower Columbia River Valley (some of which are now

being harvested), and Boise Cascade Corporation

expects to plant 8 100 ha in eastern Washington by

1996 (DeBell 1992). Other hybrid poplars (such as

P. deltoides x P. nigra L., P.candicans Ait. x P.

berolinensis Dippel, and P. charkowiensis x P.

caudina [= P. nigra var Caudina Tenorel) (Hansen

et al. 1991) are being planted as unrooted hardwood

cuttings in many parts of northeastern and northcentral United States and southeastern and southcentral Canada. An example of the magnitude of

these plantings is the Hybrid Poplar Plantation

Network, which is a cooperative effort between the

U.S. Forest Service, the Department of Energy,

Energy Performances Systems Inc., and the

Electrical Power Research Institute. Twenty-three

plantations in Minnesota, Wisconsin, North Dakota,

and South Dakota were being managed in the

Network in 1991, and a 20,200 ha plantation to fuel.

a 100 megawatt tree-burning power plant was

planned for 19924

Rooted cuttings of Eucalyptus species are being

used by members of the Eucalyptus Improvement

Association Inc. in California to establish shortrotation (lo-year) plantations for energywood and

pulpwood. The primary species for the clonal

forestry program are Eucalyptus camaldulensis, E.

grandis, E. viminalis Labill, and E. gunnii Hook. x

dalrympleana Maiden (Sachs et al. 1988). There

were 4000 growers of Eucalypts in California in 1991

with a total area of approximately 35,600 ha in

plantations. In Brazil, Aracruz Florestal company is

planting 15 million rooted cuttings per year ofE.

grandis on a six-year rotation for high-quality,

bleached sulfate pulp (Ikemori 1984, Zobel 1992,

Zobel et al. 1983).

Unrooted hardwood cuttings of a hybrid willow

(Salix matsudana Koidz. x alba) from New Zealand

are being produced by Austree, Inc. in Pescadero,

California (four nurseries in California and private

4Reported in Tresearch Newsletter (Volume 2 No.2,

Spring 1991) from the Department of Soil Science,

University of Wisconsin, 1525 Observatory Drive,

Madison, Wisconsin.

nurseries in Indiana, Montana, Nebraska, and

Saskatchewan). The cuttings are used to rapidly

establish windbreak, energywood, and pulpwood

plantations and for land reclamation plantings. A

single male clone (sterile), trademarked as

“Austree”, has been planted on a limited scale

throughout the United States and Canada. Tree and

shrub willows (Salix alba, S. purpurea L., S. x

rubens S&rank, S. viminalis L., S. amygdaloides

Anderss., S. bebbiana Sarg., S. discolor Muhl., S.

eriocephala Michx., S. exigua Nutt., S. lucida Muhl.,

S. pellita Anderss. ex Schneid., and S. petiolaris

J.E.Sm.) and their hybrids are being planted as

unrooted hardwood cuttings at very close spacings

(15-45 cm) by the College of Environmental Science

and Forestry at the State University of New York,

the Forest Genetics Laboratory at the University of

Toronto, and the Ontario Ministry of Natural

Resources to test the ultrashort-rotation

(“woodgrass”) concept of bioenergy plantations5 .

These are small scale, experimental plantings.

There are currently no commercial Salix bioenergy

plantings in the northeastern United States or

southern Canada, although Domtar Corporation in

Ontario, Canada, established some small

experimental plantings of S. alba for pulpwood in

1990.

MANAGING WITHIN-CLONE VARIABILITY

All cuttings from the same clone do not perform

identically. Within-clone variability can be due to

phenotypic (non-persistent developmental and

environmental) effects, epigenetic (persistent phase

change or gene regulation) effects, somatic genetic

changes and chimeras, and systemic or non-systemic

pathogenic effects. The objective of vegetative plant

propagation is to reproduce, year after year, a

specific cultivar that is true-to-type with a minimum

of within-clone variability.

Phenotypic (Non-persistent) Variation

Periphysis is the carryover effect of the ortet’s

(or rootstock’s) environment on cutting performance,

but without causing a permanent genetic or phase

5Personal letter and progress report provided by Mr.

Richard F. Kopp, Senior Research Support

Specialist, State University of New York, College of

Environmental Science and Forestry, Syracuse, New

York. Principal investigators are Drs. Edwin H.

White and Lawrence P. Abrahamson.

84
change (Barnes and Burley 1987, Hartmann et al.

1990). Rootstocks of the same clone that are grown

under different environmental and cultural

conditions can produce cuttings that perform

differently in early growth rates and hardiness.

Mixtures of these cuttings can give large withinclone variability. The management questions are (a)

what are the best cultural conditions for rootstocks

to provide cuttings of uniformly high production

potential, and (b) how long do the periphysis effects

last? Plantation spacing might be adjusted to reduce

the impact of these effects on competition-caused

variability. Forestry cutting macropropagation

programs should expect that different nurseries, or

even in different portions of the same nursery, may

have periphysis effects on cutting performance after

outplanting. Standardized greenhouse procedures

for mass production of rooted cuttings in containers

will reduce this effect.

Epigenetic Variation

Epigenetic effects include cyclophysis

(variation in phase [age] of cuttings from different

parts of a tree) and topophysis (variation in growth

form of cuttings from different parts of a tree)

(Barnes and Burley 1987, Hartmann et al. 1990).

These persistent, long-term effects are not caused by

genetic differences, but can be transferred to later

crops of cuttings by serial vegetative propagation.

Thus, they are more serious sources of within-clone

variation than periphysis effects, and are more

difficult to control.

As discussed earlier in this paper, cuttings from

different parts of large, mature trees can differ in

ontogenetic age and in physiological age. Cuttings

from near the base of the stem are more juvenile

than those from further up the stem and in the

crown (Briscoe 1963, Preece et al. 1991). These are

effects of ontogenetic age of the meristem. In

addition, there can be differences in physiological

age and vigor of cutting sources within the crown.

Hare and Land (1982) found that annual growth

increment of limbs was positively correlated with

rooting success of hardwood cuttings from mature

sycamore trees, so that selection of limbs with

greater growth increments within the crown gave

greater rooting. The correlation was not improved

by including tree age (lo-60 years old) in the

regression equation. As a result of within-tree

variation in ontogenetic and physiological ages of

cutting sources, the population of ramets produced

from the first vegetative cycle of expansion of a

mature tree may be highly variable in rooting

ability, growth, and morphology. This heterogeneity

is transmitted to the cuttings of successive

vegetative generations (Franclet 1983). Withinclone selection must be applied (usually for

juvenility) to obtain a stool bed population of similar

phase for production of uniform cuttings, and this

homogeneity in phase must be maintained over

successive vegetative cycles by methods of

rejuvenation and invigoration.

The other epigenetic effect on variation within

clones is topophysis, which occurs extensively in

conifers and sometimes in hardwood tree species

(Hartmann et al. 1990). Ramets produced from

cuttings of upright shoots will grow vertically

(orthotropic), while cuttings taken from lateral

limbs will grow more-or-less horizontally

(plagiotropic). Careful selection of source of

cuttings is required to reduce this type of withinclone variation for those species that exhibit

topophysis. Plagiotropic growth in hardwoods has

been reported for coffee (Coffea arubicu L.)

(Hartmann et al. 1990), several Eucalyptus species

(Durrand-Cresswell et al. 19821, black locust6 , and

(potentially) the tropical hardwood genus

Dipterocurpuceue Blume. (Barnes and Burley 1987).

Genetic Variation Within Clones

Genetic variation within clones originates from

somatic cell mutations that occur during expansion

and production of cuttings from the selected clone.

These are bud-sports or bud-mutations, since the

mutation occurs in one of the mitotic cells in the

apical meristem of a bud. The rate of such

mutations is low, but when operational programs

produce literally millions of cuttings for propagation

the probability for some mutations to occur

spontaneously somewhere in the clone is high.

Latent mutations, such as those affecting a mature

trait, may remain undetected for many years and

could be rapidly expanded in a serial propagation

system with stool beds.

Because of the arrangement of cells in an apical

meristem, mutation of a single cell invariably results

in a bud and shoot containing a mixture of mutated

and nonmutated tissue (a chimera). Most

gymnosperms and the roots and young embryos of

angiosperms have unstructured apical meristems

6Personal communication from Dr. Bruce

Bongarten, School of Forest Resources, The

University of Georgia, Athens, Georgia.
that result in sectorial chimeras (part of the internal

and external tissues are mutated) (Hartmann et al.

1990). However, most angiosperms have structured

apical meristems that result in periclinal and

mericlinal chimeras (external layer of tissue is

mutated and internal core is not, or vice versa).

This is important, as the cells used to produce the

cutting will determine whether it is a mutant type

or nonmutated type. For example, most cuttings

involve perpetuation of the apical meristem and

would contain the periclinal chimera of mutated

epidermis and non-mutated core. If propagation

uses adventitious shoots that arise internally,

however (such as softwood cuttings from root

sprouts), there would be a reversion to the nonmutated genotype of the inner tissue. With sectorial

chimeras this would not happen. Thus, in hardwood

tree species genetic differences can arise among

individual ramets as a result of bud mutations and,

subsequently, the source and type of cuttings used.

Variation Caused By Pathogens

Pathogens such as fungi, bacteria, viruses,

mycoplasma-like organisms (MLOs), rickettsia-like

organisms (RLOs), viroids, nematodes, and insects

and mites can infect and be transmitted in cuttings

(Hartmann et al. 1990). The pathogens can affect

growth, form, or leaf characteristics and thereby

contribute to within-clone variability. Some of the

pathogens are easily detected from visual

examination of cuttings, such as infection of Populus

deltoides cuttings by wood-boring insects and fungicaused canker diseases (Morris et al. 1975). Infected

cuttings can be discarded during grading at the

nursery. However, many pathogens cannot be

detected by simple visual examination, and the

incidence of these pathogens can rapidly increase in

cutting-production systems. Most operational

forestry cutting programs rely on what Hartmann et

al. (19901 call the “Pedigree Selection” system --

regenerated stool beds are established with cuttings

from the previous stool beds, and the only selection

is a visual inspection to avoid “off-type” plants or

insect/disease-infected cuttings. According to the

authors, that system has historically resulted in ‘I...

eventual contamination by viruses and other

pathogens, which in some instances have threatened

the viability of the entire industry.”

Management to Control Within-Clone Variability

Operational forestry programs relying on rooted

cutting macropropagation should recognize that

within-clone variability exists or will occur. They

should develop management plans to minimize these

effects. Some suggested strategies are given below.

I. Develop stoolbed procedures to produce uniform

cuttings, and grade all cuttings at time of collection

and packaging.

(a) Use uniform nursery site, uniform spacing

among stools, and uniform fertilization and

irrigation practices.

(b) For rootstocks in the nursery, plant

pathogen-free cuttings that are uniform in size

and bud characteristics.

(4 Grade harvested unrooted cuttings by

diameter, length, bud characteristics, and

absence of visible infection at time of packaging,

and discard those that don’t meet standards.

(d) Before outplanting rooted cuttings, grade

by sprout size, number and distribution of roots

(if bare-root cuttings), and absence of visible

infection. Discard those that don’t meet

standards.

II. Develop a “foundation stock” of a limited number

of pathogen-free, genetically true-to-type ramets of

each clone.

(a) Molecular markers might be used to

determine if each ramet of a clone is genetically

identical and if each cell layer in the apical bud

has the same genotype.

(b) Shoot-apex culture or heat treatments

(Hartmann et al. 1990) could be used to obtain

pathogen-free material from infected material.

(c) For species with recurrent apomixis (such

as apple), apomictic seeds having the same

genotype as the mother tree can be used to

produce pathogen-free, juvenile individuals.

III. Maintain the foundation stock in one or more

locations under protected conditions to prevent

infection by pathogens. The foundation stock should

be located at least l/2 mile from the production

nursery and plantations, in order to separate the

plants from infective agents (particularly for insect-,

nematode-, or pollen-vectored diseases and viruses).

IV. Change field position within the nursery each

time stool beds are regenerated. The old stoolbed

site should be cleared of all rootstock, fumigated,

8 6
and used for other crops for several years before

being used again as a stoolbed.

V . If a decline in growth or an increase in withinclone variability of cuttings occurs after several

cycles of stoolbed regeneration with cuttings from

the previous stool beds, start a new stool bed with

cuttings from the foundation stock. Use good

sanitary procedures (such as repeated alcohol dips of

saws and use of sterilized rooting containers and

rooting media) in collecting and expanding cuttings

from the foundation stock.

VI. Maintain juvenility in the foundation stock by

cutting back tops to produce stump sprouts each

year, and re-establish rootstocks every 2-3 years

from the stump sprouts or from root suckers. Use

good sanitation procedures to prevent spread of

pathogens.

VII. Continually monitor foundation stock for

infection or bud-mutation.

(a) Remove any “off-type” plants observed in

visual inspections, check for non-visible

mutations with molecular-marker analyses, and

develop culture-indexing or virus-indexing

methods (Hartmann et al. 1990) for detecting the

presence of latent pathogens.

(b) If any pathogen contamination is detected,

then develop a new, pathogen-free foundation

stock by shoot-apex culture and/or heat

treatments (Hartmann et al. 1990).

SUMMARY

Rooted cuttings can be obtained from most

hardwood tree species, but with widely varying

degrees of rooting success. Literature cited and data

presented in this paper indicate that chances of

success are increased by the following:

(a) use juvenile shoot or root cuttings, either

from young plants, from sprouts arising near the

base of older trees, or from rejuvenated (or

invigorated) tissue;

(b) produce shoot or root cuttings in stool beds

where high C/N ratios in stock plants and

reduction in stock plant irradiance may be

accomplished;

(c) select individual clones for high rooting

ability;

(d) collect the shoot or root cuttings in the

most appropriate season for that particular

species (hardwood versus softwood for shoot

cuttings);

(e) select hardwood shoot cuttings from the

base of sprouts, while softwood cuttings should

come from shoot tips;

(0 retain trimmed leaves on softwood shoot

cuttings, and for hardwood cuttings use large

cuttings (13-25 mm diameter) that have multiple

(>2) nodes per cutting (with one near the base

and one near the top);

(g) for some species, provide a cold storage or

warm temperature/bottom heat callusing

interval (with or without an auxin chemical dip,

depending on species) between collection and

planting of hardwood cuttings; and

(h) plant rooted or unrooted cuttings on

intensively prepared sites in the field after the

danger of frost heaving is past, followed by

thorough weed control until crown closure.

Some species in 16 hardwood genera have been

classified into four classes based on the relative ease

of obtaining rooting success. Class “A” species are

those that root very easily (usually have preformed

root initials on stem cuttings) and can be planted

directly in the field as unrooted hardwood cuttings.

The genera Platanus L., Populus, and Salk contain

these species. Not surprisingly, species and hybrids

of Populus and Salix are involved in operational

cutting macropropagation programs in the United

States. At the other end of the range in ease of

rooting are the class “D” species. These species are

very difficult to root, and cutting macropropagation

on an operational scale is not an option with the

current level of technology. The genera Carya

Nutt., Fraxinus L., and Juglans L. contain many of

these species.

Managing within-clone variability will be a

concern in cutting macropropagation programs, once

the methods for obtaining and maintaining high

rooting success have been developed. The objective

of these programs is to consistently reproduce a

specific cultivar that is genetically true-to-type and

has a minimum of within-clone variability. Sources

of variability include (a) phenotypic (non-persistent,

87
non-genetic) variation caused by environmental

differences among different rootstocks of the same

clone, (b) epigenetic (persistent, non-genetic)

variation caused by differences in degree of

juvenility or growth habit of cuttings from different

parts of a tree, (c) genetic differences arising from

mutations within a clone, and (d) differences caused

by pathogen infections of some of the cuttings or

rootstocks from a clone. A management strategy to

control within-clone variability is suggested that

would include (a) development of stoolbed

procedures to provide uniform environmental

conditions for all rootstocks, (b) grading all cuttings

for uniformity at time of collection and packaging,

(c) development and maintenance of a pathogen-free,

genetically true-to-type, juvenile foundation stock of

all clones at a different location from the production

stoolbed nursery, (d) rotating field positions in the

nursery each time the stool beds are regenerated,

and (e) starting a new production nursery with

cuttings from the foundation stock, using good

sanitary procedures to prevent pathogen infection, if

the old production nursery begins to decline in

growth or increase in within-clone variability.

LITERATURE CITED

Bachelard, E.P.; Stowe, B.B. 1963. Rooting of

cuttings ofAcer rubrum L. and Eucalyptus

camaldulensis Dehn. Australian Journal of

Biological Science. 16: 751-767.(l)’

Barnes, R.D.; Burley, J. 1987. Vegetative

propagation for improved tropical forest trees. In:

Abbott, A.J.; Atkin, R.K, eds. Improving

vegetatively propagated crops. New York: Academic

Press: 211-227.(2)

Bassuk, N.; Maynard, B. 1987. Stock plant

etiolation. HortScience. 22: 749-750.(3)

Benson, M.K.; Schwalbach, D.E. 1970. Techniques

for rooting aspen root sprouts. Tree Planters’ Notes.

21: 12-14.(4)

Bilan, M.V. 1974. Rooting of Liquidambar

styraciflua cuttings. New Zealand Journal of

Forestry Science. 4: 177-180.(5)

7Reference number is for use with Appendix Table 1.

Blazich, F.A. 1988. Chemicals and formulations used

to promote adventitious rooting. In: Davis, T.D.;

Haissig, H.B.; Sankhla, N., eds. Adventitious root

formation in cuttings. Advances in plant science

series. Portland, OR: Dioscorides Press: 132-149.

Vol. 2.(6)

Briscoe, C.B. 1963. Rooting cuttings of cottonwood,

willow, and sycamore. Journal of Forestry. 61:51-

53.(7)

Briscoe, C.B. 1969. Establishment and early care of

sycamore plantations. Res. Paper SO-50. New

Orleans, LA: U.S. Department of Agriculture, Forest

Service, Southern Forest Experiment Station.

18 p.(8)

Briscoe, C.B. 1973. Extended planting seasons for

sycamore and cottonwood. Res. Note SO-160. New

Orleans, LA: U.S. Department of Agriculture, Forest

Service, Southern Forest Experiment Station.

3 p.(9)

Burgess, D.; Hendrickson, O.Q.; Roy, L. 1990. The

importance of initial cutting size for improving the

growth performance of Salk alba L. Scandinavian

Journal of Forest Research. 5: 215-224.(10)

Campinhos, E., Jr.; Ikemori, Y.K. 1980. Mass

production of Eucalyptus spp. by rooting cuttings. In

Symposium on genetic improvement and production

of fast-growing trees; Sao Pedro, Sao Paulo, Brazil:

l-16.(11)

Cunningham, M.W. 1986. Evaluation of the

potential of clonal forestry for a population of

American sycamore. Raleigh, NC: North Carolina

State University. 89 p. Ph.D. dissertation.(l2)

Cunningham, M.W. 1989. Evaluation of the

feasibility of clonal forestry for sweetgum--final

report. Unpublished report of the Hardwood

Research Program. On file with: North Carolina

State University, Raleigh, NC. 72 p.(13)

Davis, T.D.; Haissig, B.E.; Sankhla, N., eds. 1988.

Adventitious root formation in cuttings. Advances in

plant science series. Portland, OR: Dioscorides

Press: 132-149. Vol. 2.(14)

DeBell, D.S. 1992. Activities related to poplar and

willow cultivation, exploitation, and utilization.

National Poplar Commission of the USA. Period:

1988-1991. Unpublished paper on file with: U.S.

Department of Agriculture, Forest Service, Forestry

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Sciences Laboratory, 3625 93rd Ave. SW, Olympia,

WA. 41 p.(15)

Dickmann, D.; Phipps, H.; Netzer, D. 1980. Cutting

diameter influences early survival and growth of

several Populus clones. Res. Note NC-261. St. Paul,

MN: U.S. Department of Agriculture, Forest Service,

North Central Forest Experiment Station. 4 p.( 16)

Dirr, M.A. 1990. Manual of woody landscape plants:

their identification, ornamental characteristics,

culture, propagation and uses. 4th ed. Champaign,

IL: Stipes Publishing Company. 1,007 P.(17)

Dirr, M.A.; Heuser, C.W. 1987. The reference

manual of woody plant propagation: from seed to

tissue culture. Athens, GA: Varsity Press, Inc. 23

PW)

Durrand-Cresswell, R.; Boulay, M.; Franclet, A.

1982. Vegetative propagation of Eucalyptus. In:

Bonga, J.M.; Durzan, D.J., eds. Tissue culture in

forestry. Boston: Martinus NijhoeDr. W. Junk

Publishers: 150-181.(19)

Farmer, R.E., Jr. 1966. Propagation of sweetgum by

softwood stem cuttings. In: Proceedings of the 8th

conference on southern forest tree improvement;

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9 0
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9 2
Appendix Table l.--Summary comments and references for rooted cutting macropropagation of 16 hardwood genera.

SDecies

Acer

(maples)

negundo L.

rubrum L.

saccharinum L.

saccharum Marsh.

Alnus incana Moench.

(alders) glutinosa Gaertn.

Bet&a nigm L.

(birches) papyrifera March.

Carya

(pecans,

hickories)

illinoensis K.Koch

(many others)

Diffrcu1ty

of Rootid

B

B

B

C

C

C

C

C

D

D

Rooting of juvenile and mature material is successful for negundo,

rubrum, and saccharinum, but saccharum is more difficult and must

use juvenile cuttings. For rubrum and saccharinurn, juvenile cuttings

may not need growth hormones, but treat single-node cuttings of

mature trees with either 8000ppm NAA quick-dip, or with IBA or K-IBA

at 3000 to 6000ppm quick-dip, or treat 23 cm cuttings with 2-4 nodes

with 1OOOppm IBA and SOOppm NAA solution, and place under mist

over bottom heat. Timing is critical, for cuttings collected in August or

later have poorer rooting. Semihardwood cuttings collected in

September and treated with 8OOOppm IBA-talc rooted readily for

negundo. Softwood juvenile cuttings collected in June gave some

rooting success for saccharum, wounding did not help, and hormones

did not improve success in some reports and did (O%-4% quick-dip IBA

solution) in others. Much tree-to-tree variation in rooting ability of

rubrum.

Use 8000 ppm IBA-talc for stem cuttings (incana) or June-July softwood

cuttings (glutinosa); untreated cuttings don’t root.

Timing is very important, and any period of dryness on the leaves’

surfaces insures failure; use 1OOOppm IBA in 60% alcohol (or K-IBA) on

June-July softwood cuttings (nigra), and either 2000ppm IBA-alcohol

quick-dip or 8000ppm IBA-talc on wounded semihardwood cuttings

taken in August-September (papyrifera). One study of papyrifera

indicated low rooting success for softwood cuttings (1500ppm K-IBA

quick-dip) from grafted scionwood of mature trees, that hedging and

defoliation of grafts were not effective, but that serial propagation

(using previously rooted cuttings as stock plants) more than doubled

rooting success (from 20 to 44%).

No reports of successfully rooted cuttings of hickories; minimal success

for pecan -- best for juvenile wood and root cuttings, using 1% IBA-talc

and/or 1OOOppm IBA-solution.

Ref. (by

a!?J

1,17,18,

41,62,70

17,18,41

17,18,41,

71

17,18,41

IA= Hardwood cuttings from juvenile and mature material will root without rooting hormones and can be planted directly in the field;

B= Cuttings from juvenile material may root readily without hormones, but cuttings from mature material require specific

treatments/conditions to get high (950% rooting);

c = Cuttings from juvenile material require specific treatments/conditions to give high (>EiO%) rooting success, but cuttings from

mature material provide only low to moderate (lo%-50%) rooting success even under the best treatments/conditions; and

D= Cuttings from juvenile and mature material are dif8cult to root (~10%) under any treatments/conditions.

93
Eucalyptus

(eucalypts)

grandis Hill ex Maiden.

camaldulensis Dehn.

tereticornis Sm.

gunnii Hook.

cladocalyx F.Muell.

deglupta Sm.

robusta Sm.

(many others)

Fraxinus

(ashes).

Juglans

(walnuts)

americana L.

pennsylvanica Marsh.

nigm L.

regia L.

Liquidambar formosana Hance

(sweetgums) styraciflua L.

Liriodendron

(yellow poplar)

tulipfera L.

D

C

D

D

C

C

C

Eucalypts are considered difficult to start from cuttings as a genus;

there is large variation in rooting ability among species and within

species; 78 species reported to form roots on stem cuttings taken from

seedlings or basal epicormic shoots; in general, it is relatively easy to

propagate by cuttings provided leafy (firm softwood) cuttings are taken

from very young seedlings or epicormic shoots at the base of the tree

(sometimes produced by partial girdling); timing is important -- best in

spring or fall when growth rate high (California) or when cuttings not

too woody or too succulent during growing season (Brazil); 4-node

softwood cuttings with trimmed leaves best; dip in 6000 ppm IBA-talc or

quick-dip in 3000-8000ppm K-IBA solution and stick in

vermiculite:perlite (not peat) under mist (must keep leaves moist); may

use bottom heat (California); rooting in 2-5 wks. and planted to field in

lo-12 wks.; problems are plagiotrophic growth (cladocalyx) and

unbalanced distribution of roots (wind throw).

Cuttings of americana have been nearly impossible to root, even from

young trees; horizontal planting of seedling cuttings has been successful

for pennsylvanica.

Cuttings are very difficult to root; some success for cuttings from

seedling plants, but poor from older trees (unless rejuvenated by serial

grafting on young seedlings [nigra x regial); etiolated shoots from

juvenile plants girdled and 1% IBA-lanolin put above girdle in AprilJune, covered in foil, and subsequently severed from plant to give some

rooting (nigra); softwood cuttings rooted for regia, hut rooted cuttings

can’t be disturbed and survival is poor.

Softwood cuttings from adventitious shoots on root segments can be

rooted; softwood cuttings collected from shoot tips on crowns and stump

sprouts of mature trees in early June, treated with 4000ppm IBAsolution or 8000ppm IBA-talc, and placed in peat:perlite under mist root

well; overwintering may be a problem. Hardwood cuttings will not root

unless girdled, treated at the girdle with IBA + PPZ [l-phenyl-&methyl5pyrazolonel + sucrose + captan, and subsequently severed at the

girdle before planting; etiolated shoots don’t improve rooting.

Considered difficult to root, particularly from mature plants (juvenility

is important if species is to be rooted in high percentages). Leafy stem

cuttings (tree age unknown) collected in July with the basal cut 1 cm

below a node rooted 52%. Boot cuttings and cuttings from stump sprouts

have been rooted. Partial girdling tree and using basal sprouts has

increased rooting, but etiolated shoots don’t root.

1,11,19,

27,28,41,

42,44,53,

66,61,64,

74.75

18,46

18,22,27,

41

5,13,17,

18,20,36,

41,49

17,18,41,

49,64

94
Platanus

(sycamores)

x acerifolia Miller

ex Munchh.

occidentalis L.

Populus Aigeiros Duby:

(pop1ars, deltoides Bartr.

cottonwoods, and ex Marsh.

aspens) nigm L.

Tacamahaca Spach:

balsamifera L.

trichocarpa Torr.

and Gray

Leucoides Spach:

heterophylla L.

Deuce Duby:

alba L.

x canescens (Ait.) Sm.

gmndidentata Michx.

tremu,loides Michx.

Prunus serotina Ehrh. C

(cherries) avium L. C

A

B

Cuttings of x acerifolia provide high rooting success, even from trees as

old as 45 yrs (96% rooting with no hormone trt) and from unrooted

hardwood cuttings planted directly in the field (70% rooting and

survival); NAA is valueless, and IBA is no better than the untreated

control; leafy June softwood cuttings collected after the shoots have

begun to mature can be rooted, and success is highest with cuttings

taken from young vigorous stock plants cut back annually during the

dormant season (“invigoration”); hardwood cuttings should be taken

after leaf fall in the autumn from the current season’s growth and be

15-23 cm long with 4 buds (top cut just above a bud and lower cut just

below a bud). Cuttings of mature occidentalis are more difficult to root

than x acerifolia, but juvenile cuttings (l-yr-old seedlings and coppice

stump sprouts on young stock plants) root easily and can be planted

directly in the field as unrooted hardwood cuttings; 58% of hardwood

cuttings collected from mature trees were rooted when treated with a

quick-dip of 0.5% IBA + 0.5% PPZ [l-phenyl-3-methyl&pyrazolonel in

50% ethanol, then dipped in 20% powdered sucrose and 5% captan in

talc, and put in cold storage for one month before planting; rooting

success for hardwood cuttings from 1-yr-old sprouts on mature, pruned

trees was highest for 2&m cuttings having a basal diameter of 16-20

mm, 2-3 nodes, and the lowest node within 2 cm above the basal cut.

Reduced N fertilization of stock plants and growing them in full sun

may increase rooting of cuttings; horizontal planting of cuttings from

sprouts has provided successful rooting.

7,w,12,

17,18,27,

35,38,41,

43,50,51,

6 0

Hardwood cuttings of most poplars Wgeiros, Tacamahaca, and

Leucoides sections, and the species alba) root readily, but not the gray

poplars and aspens (see below); cuttings are usually 25-45 cm pieces (6-

19 mm diameter) of dormant 1-yr-old shoots collected from coppiced

stock plants in stool beds (not collected before October in Minnesota)

that may be stored in a cooler (-10 C) for up to six months and are then

planted in the field as unrooted cuttings during the early spring (soil

temperature >lO C) in the northern U.S. (all winter in the South,

without cold storage); cuttings from the base of stoolbed sprouts root

and grow better than cuttings from distal portions, because they have

more preformed root initials; roots form from these root initials to give

90% to 100% rooting success; however, greater uniformity in growth and

fewer multiple stems are obtained when cuttings have healthy axillary

buds (not from branched portion of stem and not from stem tips) and are

planted so that the top of the cutting is flush with the soil surface; weed

control is essential after planting.

The gray poplars and aspens (x canescens, grandidentata, and

tremuloides) can be propagated from root cuttings and from softwood

cuttings from root suckers (5-10 cm root pieces collected and placed in

moist peat moss in February, and soft shoots that develop on these root

pieces by late March can be rooted under mist with bottom heat and

IBA-talc). Aspens and Leucoides poplars root least readily from softwood

cuttings, while Aigeiros and Tacamahaca poplars are easiest to root.

4,7,15,

16,17,18,

23,24,25,

27,31,32,

33,41,47,

56,63,66,

68,69,72,

73.76

Softwood cuttings collected in spring and early summer from (a) root l&21,27

suckers on root cuttings, (b) seedlings, and (c) mature trees (which were

grafted in pots and pruned to promote shoot formation) have been

rooted with 8000ppm IBA-talc under mist in sand:peat. Wide tree-totree variation was observed in rooting success (O-100%).

95
Quercus

(oaks)

laurifdia Micbx.

lyrata Walt.

nigra L.

paluetris Muenchh.

phellos L.

robur L.

shumardii Buck].

virginiana Mill.

(other oaks)

Robinia

(black locust)

pseudoacacia L. B

Salix

(willows)

Ulmus

(elms)

nigro Marsh.

alba L.

(many others)

alata Michx.

americana L.

C

C

C

C

C

C

C

C

D?

A

A

C

C

Attempts to propagate oaks by cuttings have usually been failures,but

the species listed here (except nigm) have been successfully rooted as

juvenile leafy, semihardwood cuttings. Juvenility is most important.

Semihardwood cuttings collected in July (firm cuttings after first

growth flush hardens), quick-dipped in 10000-20000 K-IBA solution, and

placed in 2 perlite:l peat under mist and 50% shade gave 60-80% rooting

in 7-10 wks. Girdling stock-plant shoots and treating the girdle with

IBA + PPZ + sucrose + captan before severing the shoot increased

rcoting (76%) of 19 to 57-yr-old nigra trees. Basal sprouts from partially

girdled robur have been rooted.

Root cuttings of 7-13 mm diameter and 10 cm length have given greater

than 90% rooting success in Hungary when dug in the early spring and

planted directly into nursery rows with the distal end down. Softwood

cuttings from coppice sprouts can be rooted under mist with near 100%

success. Rooting media containing high levels of peat should be avoided,

as cuttings are sensitive to excessive moisture. Hormones are not

required, but quick-dip in 10000-16000 IBA in ETOH can improve

rooting slightly. Plagiotropism is a problem, unless softwood cuttings

are taken from coppice sprouts or root suckers.

Willows root readily by either hardwood or softwood stem cuttings (have

preformed root initials) or by root cuttings. Hardwood stem cuttings can

be collected anytime after leaf fall, but fall-collected cuttings may root

more poorly than spring-collected cuttings and require chilling or

removal of buds to improve rooting success. Spring cuttings (and chilled

fall cuttings) can be stuck directly in the field. Rooting success is 90-

lOO%, even for very large cuttings (survival and growth of alba

increases with increasing diameter up to 19 mm, and not affected by

diameter above that size). Rooting hormones don’t help. Softwood

cuttings can be rooted in high percentages in peat:perlite under mist. A

“willow rooting cofactor” is present in willow stems and has sometimes

been used to promote rooting of hard-to-root plants.

Elms can be propagated from root cuttings, softwood cuttings from root

cutting sprouts, and softwood leafy stem cuttings. Root cuttings are

collected in December-January, cut into 5-8 cm lengths, dusted with

captan, and placed in rooting medium over 2O’C bottom heat. When root

sprouts become woody, they are treated with 8000ppm IBA-talc and put

under mist. Semi-lignilled softwood leafy stem cuttings that were

collected in early June, cut into 5-10 cm lengths with l+ leaves, treated

with 8OOOppm IBA-talc, and stuck in peat:perlite under mist rooted

94%. Moisture during the period immediately after insertion in the

rooting medium is critical. Also, the juvenility factor is very important

in elm rooting success, as stump sprouts from 30 cm above ground gave

83% rooting, stump sprouts from 1.8-2.3 meters above ground gave 64%,

and cuttings from mature trees gave 38%. Roots on rooted cuttings are

very brittle and can be easily damaged, so rooting in individual pots is

recommended.

17,18,37,

39,41

17,!8,41,

48 L

7,10,17,

l&41,45,

76

17,18,29,

41

2 Personal communication from Dr. Bruce Bongarten, School of Forest Resources, The University of Georgia

96
THE ROOTING ENVIRONMENT FOR CU’ITINGS FROM FOREST TREES1

Jane L. Ford-Logan2

Abstract.--This paper reviews the requirements of the rooting environment as they

affect the induction of adventitious roots on cuttings, as reported from studies on various

forest tree species. Evidence for the involvement of growth regulators in root induction

is discussed. The environmental conditions which promote rooting include a porous

rooting medium, intermittent mist, bottom heat, extended photoperiods, and relatively

low light. High humidity is a critical factor in maintaining tissue turgidity until new

roots have developed. Both total irradiance and light quality are important components

of the stock plant environment, and these factors interact with nutrient availability to

influence subsequent rates of net photosynthesis and rooting of cuttings. Rooting

medium combinations of sphagnum peat, perlite, and sand appear to give fairly high

rooting success for cuttings of forest tree species. Optimal rooting environments enhance

the efficiency of rooted cutting production systems, which subsequently encourages the

use of vegetative propagules in forest tree improvement programs.

INTRODUCTION

Clonal reforestation requires the development of

economically efficient production systems for

vegetative propagules. The potential benefits of

clonal propagation of forest trees, particularly

conifers, to forestry have been discussed by many

authors (Rediske 1978, Libby and Rauter 1984,

Boulay 1987, Greenwood et al. 1991). The

realization of these benefits is hampered by the lack

of suitable technology for vegetative propagation of

lPaper presented at the 1992 Southern Regional

Information Exchange Group Biennial Symposium,

Huntsville, AL, July 8-10, 1992.

2Plant Physiologist, USDA Forest Service, Southern

Forest Experiment Station, Alabama A&M

University, Normal, AL 35762

The use of trade or firm names in this publication is

for reader information and does not imply

endorsements by the U.S. Department of

Agriculture of any product or service.

many coniferous species. Progress is continuing in

the search for practical, efficient methods to clonally

propagate by means of rooted stem cuttings (Rauter

1983, Foster and Shaw 1987, Williams 19871, needle

fascicles (Rudolph and Nienstaedt 1964, Cohen 1975,

Struve and Blazich 1984, Koh et al. 19901, and by

cell, tissue, and organ culture (Mott 1981, David

1982, Bonga 1983, Amerson et al. 1984, Amerson et

al. 1988). Rooting cuttings and micropropagation

are the two most important methods for cloning that

can be employed in the operational production of

forest tree propagules.

Propagation of most conifers and hardwoods by

rooted cuttings is difficult and variable in success.

The ability of plants to form adventitious roots is

controlled by a complex array of interacting factors,

including nutrition, environment, genetics and other

factors relative to the plants, and numerous

endogenous and exogenous compounds (Hartmann

et al. 1990). This array of factors becomes

ineffective as a trigger for adventitious root

formation if any one of them is limiting.

One of the most fundamentally important and

least experimentally studied aspects of adventitious

9 7
root formation is the rooting environment. Very

little literature exists on the environmental

conditions needed to induce roots on cuttings of

conifers and hardwoods. What is reported on this

aspect of vegetative propagation generally concerns

herbaceous or softwood cuttings from horticultural

species.

The purpose of this paper is to review what is

presently known about the requirements of the

rooting environment for the propagation of forest

trees. This paper is written to document specific

points, rather than generalities from past research,

and to further improve the understanding of the

rooting environment.

Background Studies

Comprehensive studies of environmental factors

which promote rooting of cuttings of forest tree

species have included Pinus spp. (Hare 19741,

Douglas-fir (Pseudotsugu menziesii rMirb.1 Franc01

(Brix and Barker 19731, western hemlock (Tsuga

heterophylla lRaf.1 Sarg.) (Brix and Barker 19751,

loblolly (Pinus taedu L.1 and slash pine (Pinus

elliottii Engelm.) (van Buijtenen et al. 19751, and

southern pines (Marino 1982).

WATER RELATIONS

Water Quality

Water quality is an important factor in rooting

cuttings (Hartmann et al. 1990). For good results

the available water should not contain total soluble

salts in excess of 1400 ppm -- ocean water averages

about 35,000 ppm. The salts are combinations of

such cations as sodium, calcium, and magnesium

with such anions as sulfate, chloride, and

bicarbonate. Water containing a high proportion of

sodium to calcium and magnesium can adversely

affect the physical properties and water-absorption

rates of rooting media and should not be used for

irrigation purposes.

Problems can arise in maintaining the proper

calcium/magnesium levels, both being important

elements for plant growth, when soilless media are

used. Purified water may not contain adequate

amounts of these elements, requiring supplementary

fertilizers be added when calcium is 25 ppm or less

and the magnesium level is 15 ppm or less.

Hard water containing relatively high amounts of

calcium and magnesium (as bicarbonates and

sulfates) can be a problem in mist-propagating units.

Hard water softened by replacement of the calcium

and magnesium in the water by sodium ions is toxic

to plant tissue and should never be used for

irrigation or misting.

Better, but more expensive methods of improving

water quality include deionization or reverse

osmosis. Deionization removes calcium, magnesium,

and sodium by substituting hydrogen ions for these

elements. Reverse osmosis changes a more

concentrated solution to a less concentrated solution

eliminating unwanted salts from an otherwise good

water source (Barnstead 1971).

Humidity

The environmental requirements for adventitious

root initiation in stem cuttings are those that

minimize physiological stress in the cutting.

Providing a high relative humidity to reduce

transpiration losses is one means of reducing stress.

By doing so, the vapor pressure of the atmosphere

surrounding the cutting is maintained close to that

in the intercellular spaces of its leaves. Relative

humidity is defined as the ratio between the actual

weight of moisture and the total amount of water

that can be held by a unit volume of air at a specific

temperature and pressure expressed as a percentage

(Mastalerz 1977).

There are numerous propagation systems used in

commercial horticulture to maintain high humidity,

which are usually based either on spraying mist,

fogging, or enclosing the cuttings in polyethylene.

The advantages of polyethylene systems have been

known for many years (Loach 1977). Low-pressure

misting systems for maintaining a film of moisture

on cutting surfaces without saturating the rooting

medium have been greatly enhanced and simplified.

Mist vs. No-mist Propagation

There are many cases where mist has been found

to be beneficial or essential for rooting forest tree

species. Examples include loblolly pine and slash

pine (van Buijtenen et al. 19751, and eucalyptus

(Reuveni et al. 1990). In contrast to the norm, the

use of intermittent mist or fog generated high

humidity growth chambers, there are cases where

these can be disasterous to a rooted cutting

production operation.

98
Leakey et al. (1990) experimented with a wide

range of timber and multi-purpose tree species from

both tropical moist forests and semi-arid areas.

They compared rooting under mist with rooting in

an improved, low-technology, no-mist propagator,

and demonstrated the advantages of conditions

without mist. A third treatment in which a mist

propagator was enclosed in polyethylene, resulted in

even less rooting and higher mortalities due to

rotting. No-mist propagators used in Kenya to

produce clonal material of Prosopis juliflora have

achieved success rates of greater than 75%.

Similarly, cuttings ofAcacia tortilis and Terminalia

spinosa were rooted more easily in the high

humidity conditions of a no-mist propagator. When

the rooting of cuttings of Ricinodendron heudelotii

was tested both under mist and in no-mist

propagators with ‘Seradix 2’ commercial rooting

powder, rooting by day 21 was best without mist

(75% vs 50% under mist). In a parallel study on

rooting media, a comparison between juvenile

Gmelina arborea cuttings set in fine sand under

mist and the no-mist propagator, showed better

rooting in the no-mist propagator.

There are certain positives ascribed to the use of

“low-tech,” no-mist propagators (Leakey et al. 1990).

These high humidity polyethylene propagators are

inexpensive to construct, are very effective and have

no essential requirements for either piped water or

electricity. The advantage seems to be particularly

great for the dry-zone species which can be very

susceptible to rotting under mist. By being enclosed

and continuously moist, the cuttings are not

subjected to the extremes of saturation and water

stress that can occur when misting frequency is not

timed to match changes in the weather.

Brix and Barker (1975) conducted humidity

control studies comparing rooting in western

hemlock using three humidity control systems: an

intermittent misting system with a Geiger Mist-AMatic control (HCl), flats with cuttings enclosed in

clear plastic sheeting (4 mil) and placed under

intermittent mist (HC2), and a plastic enclosed box

with no mist and shaded from direct sunlight (HC3).

Neither heated air or bottom heat was provided to

the cuttings, other than that required to keep the

greenhouse frost-free. They evaluated six

temperature conditions using the three humidity

control systems (Table 1). Rooting averaged 33% for

Regime 1,42% for Regime 2, and 45% for Regime 3.

Since there were no statistically significant

differences among regimes, Regime 3 using plastic

covered boxes with no mist was eventually adopted

as the standard system.

Table l.--Temperature conditions and humidity controls for

cuttings of western hemlock (adapted from Brix and Barker 1975)

Regime Temperature Condition Humidity Control

No.

1 unheated greenhouse, intermittent mist (HCl)

unheated soil1

2 unheated greenhouse, flats covered under mist

unheated soil’ W-32)

3 unheated greenhouse or box plastic covered, no

outside box, unheated mist (HC3)

soil1

4 warm greenhouse intermittent mist (HCl)

f20°C), warm soil (2OOC)

5 unheated greenhouse or box plastic covered, no

outside box, warm soil mist fHC3)

(2OOC)

6 unheated greenhouse, intermittent mist (HCl)

warm soil (2OOC)

‘Thermostat set at 2OC to keep soil frost-free.

Quantity of mist fall is an important influence on

the rooting of loblolly pine cuttings. Greenwood et

al. (1980) found that too much mist inhibited rooting

of loblolly pine cuttings. Sixty percent rooting was

achieved in 4-year old loblolly pine cuttings with a

misting regime of 0.05 to 0.1 mm/h. Variation in

mist fall accounted for 75% of the variation in

rooting observed, with 64% of cuttings rooted in a

portion of the bench receiving 0.05 mm/h of mist and

16% in an area receiving 0.77 mm/h, demonstrating

that low quantities of mist resulted in the best

rooting.

LIGHT

Light is necessary for photosynthesis, and for this

reason it is usually also necessary for a good rooting

response. Light requirements with respect to

rooting cuttings of forest trees are difficult to

ascertain due to little work being carried out with

these species. In order to understand its effect on
rooting, light must be interpreted with respect to

photoperiod (daylength/duration), intensity

(irradiance/photon flux), and quality (wavelength).

Photoperiod

The German plant physiologist, Sachs (1882)

postulated the existence of a specific root-forming

substance manufactured in the leaves, which moves

downward to the base of the stem, where it promotes

root formation. Later, it was shown (van der Lek

1925) that vigorous bud growth promotes the

development of roots just below the buds in cuttings

of some plants. Bouillenne and Went (19331 and

Went (1934) found substances in cotyledons, leaves,

and buds which stimulated rooting of cuttings and

called this material “rhizocaline”. Removal of the

buds from cuttings in certain plants or leafless

cuttings almost completely stopped root formation

(van der Lek 1925, Leakey et al. 19821, especially in

species lacking preformed root initials. This

response was shown in hardwood cuttings (Fad1 and

Hartmann 1967). If hardwood cuttings are taken in

midwinter when the buds are in the rest period,

they have no stimulating effect on rooting, but if the

cuttings are made in early fall or in the spring,

when the buds are active, they show a strong rootpromoting effect.

Photoperiod influences cambial and bud activity

as well as rooting of cuttings of woody plants (Nitsch

195713). Wareing and Smith (1963) concluded that

photoperiod affects rooting either by influencing the

activity of the shoot apex or the hormone production

in leaves. Leaves and buds are known to be

powerful auxin producers, and the effects were

observed directly below them, showing that polar

transport was involved. The fact that auxin is

involved in the process of adventitious root initiation

is well established (Blakesley et al. 1991).

Baker and Link (1963) studied the effect of

natural daylength, l&hr, and 24-hr photoperiods on

the rooting of cuttings of 26 woody species, and

observed an earlier breaking of rest with extended

photoperiods in the propagating bench. They found

photoperiod had little influence on rooting; however,

when dormancy was a factor, extended photoperiods

exerted some root-promoting effect.

Similar results were seen in a study conducted by

Miller et al. (1982) examining rooting capacity and

visible terminal bud activity of Fraser fir (Abies

fraseri [Purshl Poir.) stem cuttings as influenced by

duration of postseverance chilling and photoperiod

during rooting. Cuttings received SD (short-day) or

LD (long-day) treatment during a lo-week

propagation period. There was no significant

relationship between photoperiod during rooting and

the number and length of roots per rooted cutting.

Rooting was primarily contingent upon IBA

(indolebutyric acid) treatment and chilling, although

long-days had a strong promotive effect when

cuttings were chilled less than 6-weeks. This study,

and the one by Baker and Link (1963) showed that

long-days may compensate for a shortened chilling

or rest period in the rooting of cuttings of some

woody species.

Snyder (19551, working with cuttings of Japanese

yew (Taxus cuspidatu) taken in December, found

that an 18-hr photoperiod stimulated shoot growth

of cuttings while in the bench, whereas no bud

activity occurred with 8-hr photoperiods. He found

no difference in rooting between the two

photoperiods. Waxman (1957) reported summer

cuttings of flowering dogwood (Corms florida)

produced twice as many roots in an 18-hr

photoperiod as in a 9-hr photoperiod, and 1 l/2 times

as many roots as cuttings under normal daylengths.

Lanphear and Meahl(1961) observed that juniper

(Juniperus horizontalis “Plumosa”) cuttings rooted

significantly better under 24- (77.8%) and 18-hr

(79.2%) photoperiods than under normal daylengths

(40.3%) when rooted during the autumn. They

reported that cuttings under these three

photoperiods showed 68.8,31.2, and 0.7% bud break,

respectively. According to Nitsch (1957a), cutting

growth of staghorn sumac (Rhus typhinu) and

sycamore Wutunus occidentalis L.) was stopped

completely under SD (lo-hr), whereas under LD (14-

hr or longer) growth was vigorous. Wareing and

Roberts (1956), working with black locust (Robiniu

pseudoucubiu L.), concluded that long days maintain

cambial activity, whereas short days tended to

decrease the activity.

A study by Bhella and Roberts (1974) determined

the effects of photoperiod in combination with

rooting temperature on bud and cambial activity and

subsequent rooting in stem cuttings periodically

sampled from field-grown Douglas-fir. An 18-hr

photoperiod significantly increased cambial activity,

rooting (percentage and quality), bud respiration,

and also hastened bud break of stem cuttings as

compared with cuttings propagated under 9-hr

photoperiod. They concluded that cambial stimulus

from leaves under LD may be due to high levels of

auxin resulting from such photoperiods, and

100
conversely, the cessation of cambial activity under

SD may result from insufficient auxin production

under such conditions. Rooting response was

modified by sampling date and temperature of the

rooting medium with rooting being the least from

August-November (O-4%) and greatest in December

and January (1526%). Their results with Douglasfir are in agreement with those of Lanphear and

Meahl(1961) for juniper, and with Wareing and

Smith (19631 for poplar (Populus robusta). Van

Buijtenen et al. (1975) also reported significantly

better rooting of loblolly and slash pine cuttings

under long rather than short photoperiods.

Haugh (19781 elucidated that the LD (18hr)

enhancement of rooting reported by Bhella and

Roberts (1974) was not a photoperiodic response, but

a beneficial effect of greater accumulations of

photosynthate, the results of which were higher

levels of starch and enhanced root regeneration. He

attributed the absence of rooting in September, even

in the presence of increased starch content, to the

lack of root initiation.

Root initiation can proceed if a correct balance of

nutrients (carbohydrates - particularly starch and

proteins) and rooting co-factors are available (Stoltz

and Hess 1966a, 1966b, Hess 1969, Jain and Nanda

1972). An increase in root initiation with LD was

correlated with increased carbohydrates (Hess 1969)

and a higher auxin content (Wareing and Smith

1963, Heide 1964).

Intensity

Light may also have negative effects on rooting as

shown in earlier investigations (Kawase 1965,

Hansen and Eriksen 1974). With leafy aspen

(Populus spp.) cuttings, a low (8 W mm21 light

intensity was found to give the best rooting

(Eliasson and Brunes 1976). High (40 W mm21 light

intensity decreased rooting when applied to the

stock plants and to the cuttings during the rooting

period.

Eliasson et al. (19771 illustrated this same effect

with experiments using cuttings from 6-week old

seedlings of Scats pine (Pinus syluestris L.).

Seedlings were grown and the cuttings rooted at the

same two light intensities (8 and 40 W rnm21. The

light treatment during seedling growth had the

strongest influence on the subsequent rooting,

rather than light treatment during the rooting

period. Similarly, cuttings from seedlings grown at

low light intensity rooted faster and formed more

roots than cuttings from the seedlings grown at high

light intensity (Hansen et al. 19781. Photosynthesis

decreased in all treatments during the rooting

period. There was an increase in dry weight and

starch content in cuttings rooted at the low light

intensity. This showed that the low light intensity

used in this experiment was more than sufficient for

production of assimilate for growth and rooting of

pine cuttings.

Eliasson et al. (1977) found higher levels of the

auxin IAA (indole-3-acetic acid) in cuttings grown at

low light intensities than in cuttings grown at high

light intensity. The cuttings also showed a positive

rooting response to an external supply of the auxin

IBA. These results indicate that light influences the

hormonal relationships of the cuttings. At least

some of the inhibitory effect of high light intensity

may be due to a changed hormonal balance.

There is no generally accepted hypothesis for the

adverse effect of high light intensities on rooting.

This inhibitory effect of light has been found to be

strong in some other species, including Norway

spruce (Picea abies IL.1 Karst.), especially if the stem

base of the cutting is illuminated during the rooting

period (Eliasson et al. 1977). Light influences many

biochemical, physiological, and anatomical

properties of plants. The best compromise may be to

optimize light intensity for photosynthesis and

growth, but endure the small negative effects on

rooting.

Quality

Stoutemyer and Close (1946) reported radiation

in the orange-red region of the spectrum seems to

favor rooting of cuttings more than that in the blue

region, but there are conflicting reports on the

influence of light quality on both stock plants and on

the rooting of cuttings (Hartmann et al. 1990). In

contrast, Shapiro (1958) reported red light (above

680 nml to be more inhibitory to rooting than blue,

green, or far-red light on stem cuttings of Lombardy

poplar (Populus nigru “italica”).

TEMPERATURE

Air Temperature

Air temperature in combination with bottom heat

is important for rooting. Ambient air temperatures

can influence the length of time required for rooting.

Ideal temperatures appear to be in the range of 20°C

101
to 22oC for many forest tree species (Rauter 1983)

even though Rauter (1971) noted no detrimental

effects on rooting spruce (Picea spp.) cuttings

exposed to temperatures ranging from 10°C to 32OC.

Low temperatures increase the length of time

required to root cuttings. Generally cuttings root

well in cool, moist air surrounding the tops, and

warm conditions around the base. This temperature

gradient allows greater activity at the base, while

minimizing respiration and moisture stress at the

top of the cutting.

Brix and Barker’s (1975) rooting studies of

western hemlock cuttings evaluated the effect of

temperature of the environment during rooting.

They evaluated six temperature conditions using

three humidity control systems (Table 1). The most

consistently good results were obtained when

neither the air nor the soil was heated. Regime 4

(warm air and warm soil combination) was generally

the worst. The unheated air and warm soil

condition, Regimes 5 and 6, was consistently good,

but was inferior to unheated air and soil in two out

of three trials. Based on favorable results obtained

under the plastic cover (HC31, they subsequently

rooted cuttings achieving humidity control combined

with no heating of air or soil in an outside

propagation box (Regime 3).

High rooting percentage is achieved with cuttings

of loblolly and slash pine at a temperature of 27OC

during cooler months and 32OC during the summer

(van Buijtenen et al. 1975). The rooting medium is

similarly maintained at 27OC.

Soil Temperature

It is well known that adding auxins to the base of

stem cuttings generally increases the percentages of

rooted cuttings, root counts, and total root weight

per cutting. Wells (1985) reported that the efficacy

of auxins in promoting the rooting of cuttings

depended on the temperature of the medium. This

was observed by Hinesley and Blazich (1981) when

rooting Fraser fir stem cuttings maintained at two

different soil temperatures. IBA alone and

wounding + IBA significantly increased the rooting

percentage for both rooting medium temperatures,

but the improvement was more pronounced at the

higher temperature. At the lower temperature, IBA

treatments had a negligible effect on the number

and length of roots, but these were significantly

increased at the higher temperature.

Heating the medium to enhance the rooting of

cuttings is a general practice during vegetative plant

propagation. Numerous authorities recommend

warming the medium to gain a higher percentage of

rooted cuttings, faster rooting, and more roots per

cutting (M&one 1962, Gislerod 1983). Hartmann et

al. (1990) recommended keeping the medium

temperature higher than the air temperature to

speed cutting root development relative to shoot

development. The objective is to induce root activity

before shoot growth occurs in the cuttings.

Elevating the temperature of the rooting medium

above the air temperature is particularly important

when low-pressure mist systems are used for

propagation. The relatively low water temperature

of the mist combined with the cooling effects of

evaporation often result in suboptimal temperatures

in the rooting medium. Rapid desiccation of the

cuttings or rapid drying of the rooting medium may

occur with excessively high temperatures.

Therefore, control of the temperature in the rooting

medium is essential. Loach (19881 reviewed the

methods by which bottom heat can be controlled.

Hinesley and Blazich (1981) observed that visible

bud activity was greatest for IBA treated Fraser fir

cuttings in the absence of bottom heat, but was

considerably less at a higher rooting medium

temperature, 20 vs. 24OC, respectively. This

decrease in visible terminal bud activity

accompanying increased rooting response was

unexpected.

Nelson (1966) summarized information on the

role of bottom heat in the rooting of cuttings. H e

concluded bottom heat was more important in the

forcing of growing plants than in the rooting of

cuttings. Poole and Waters (1971) observed

reductions of as much as 50% in time required for

propagation of tropical foliage plants during the cool

months of the year, when rooting temperature was

maintained between 24 and 29OC. Halma (1931)

concluded that a rooting temperature of 24 to 26OC

was necessary for propagation of softwood cuttings

of trees and shrubs. Hamada (1959) emphasized the

importance of bottom heat in propagation of

mulberry Worus alba L.). Schwartze and Myhre

(1947) observed that bottom heat at 21XoC

increased rooting of blueberry (Vaccinium spp.)

cuttings in March, but was not needed in June.

The study by Bhella and Roberts (1974) showed

that rooting temperature had a pronounced effect on

102
the extent of callus formation on the base of

Douglas-fir cuttings. In auxin-treated cuttings,

callus formation was fair at 10, good at 18, and

excellent at the 26OC rooting temperature,

irrespective of SD or LD phoboperiod. Rooting

temperature did not influence cambial activity in the

first year of their two-year study, but it did in year

two. While cuttings rooted significantly better

under 26 and 18OC temperatures, these

temperatures enhanced rooting without affecting

bud activity. They concluded that 10°C was too low

for optimum metabolic activity and callus formation.

Leakey et al. (1982) conducted two experiments

testing the effects of different propagating bed

temperatures on root initiation in cuttings of

Triplochiton scleroxylon K. Schum., an important

timber tree of West Africa. The first experiment

tested temperatures between 20 and 31°C without

auxins, and the second tested temperatures ranging

between 23-38OC with auxins. In both experiments,

temperatures markedly affected rooting. In

experiment I, the percentages of rooted cuttings

after week eight increased from zero at 20°C, to 21%

and about 36% at 24.5 and 29-31°C, respectively.

The rooting percentages were generally greater in

experiment II, where the auxin-treated cuttings

rooted more rapidly and reached a higher final

percentage at 28OC than at 23OC. At higher

temperatures (33OC and 38OC) rooting was initially

similar to that at 28OC!, but cuttings which had not

rooted within four weeks started to deteriorate.

While approximately 80% of the surviving cuttings

rooted in the 28-38OC range, the numbers of

survivors at 23OC was significantly greater than at

33OC and 38OC. More roots developed per cutting at

33OC (2.03 f 0.26) than at 23OC (1.60 f 0.16).

Raising the temperature from 25 to 30°C

increased and accelerated rooting of Eucalyptus

camaldensis Dehn. cuttings having a relatively

intermediate to high rooting capability (Reuveni et

al. 1990). Bottom heat did not improve rooting of

clones which had low rooting capability. Carville

(1979) also observed the rooting of conifer cuttings

was promoted by slightly higher temperaures in the

base zone.

ROOTING MEDIUM

Cuttings of woody plants are propagated in a

variety of media. Reisch (1967) concluded that the

particular medium components were of secondary

importance in the rooting response, but the

resultant physical properties and the management

of the medium were of primary concern.

Physical properties of a medium include such

parameters as total porosity, bulk density, particle

size distribution, air space, water holding capacity,

and saturated and unsaturated hydraulic

conductivity (Reisch 1967, Hartmann et al. 19901.

Of these, aeration and moisture content appear to be

the most important properties of a propagation

medium. The choice of rooting medium used to

propagate conifers and hardwoods is usually by

availability, cost, etc. Only a few tests have been

specifically designed to evaluate rooting media.

Tilt and Bilderback (1987) conducted a very

elaborate study looking at the physical properties of

propagation media and their effects on rooting three

woody species. Cuttings of a conifer, Leyland

cypress (x Cupressocyparis leylandii “Haggerston

Grey”), a broad-leaved evergreen, holly, (IZex x

“Nellie R. Stevens”), and a deciduous tree, crape

myrtle (Lugerstroemia indica) were inserted in 11

media. The physical properties of seven media were

engineered by manipulating particle size

distribution of a 1:l (v/v) aged pine bark:composted

hardwood bark medium. Four other propagation

media were tested for comparison. With container

capacity air space ranging from 12%-40%, and water

held after drainage in the root zone ranging from

35%~55%, variation in rooting response of cuttings

occurred between species and media, but differences

could not be attributed to the physical properties of

the various media. Also, no relationship was

detected between rooting response and engineered

combinations of hardwood bark and pine bark. Only

one medium (1 sphagnum peat:1 coarse sand)

showed any consistency (within 10%) in rooting

between species, and this medium contained the

least air space of those compared.

The medium not only influences the percentage of

cuttings which form roots, but also the type of root

system developed. This observation was tested by

Copes (1977) with cuttings from 2- to 4-year old

Douglas-fir trees. Twenty-one combinations of

sphagnum peat, perlite, vermiculite, and sand in l:O,

2:1, l:l, and 1:2 ratios were evaluated. The rooting

media yielding the highest percentage of rooted

cuttings were: perlite-sand (1:l) - 78%; vermiculitesand (1:l) - 71%; and sphagnum peat-vermiculite

(2:l) - 68%. Large differences in root thickness,

flexibility, and branching were noted between

cuttings grown in different rooting media.

103
For Douglas-fir cuttings, Brix and Barker (1973)

determined that a mixture of equal volumes of fine

peat moss, washed coarse sand, and coarse grade

perlite provided favorable aeration, drainage and

moisture retention for rooting with the water regime

used. The same medium was adopted with favorable

results for western hemlock cuttings (Brix and

Barker 1975). This is a three component mixture of

the same ingredients that Copes (19771 found

successful for rooting Douglas fir.

Eliasson et al. (1977) carried out experiments

under greenhouse conditions with intermittent mist

and supplementary light. Several rooting media

were evaluated, but most experiments used

sphagnum peat or perlite. Cuttings taken from twoyear-old seedlings of Norway spruce throughout the

year, rooted well regardless of the rooting media

used. The average rooting obtained with this

material was 80% .

Cuttings taken monthly (October 1975-September

1976) from 1 l-year-old Norway spruce rooted poorly

in perlite (15% average rooting) and somewhat

better in peat (42%). Good rooting was obtained

with this material in July in both these media (79%

in perlite, 73% in peat) (Eliasson et al. 1977). van

Buijtenen et al. (1975) recommended a 50-50

mixture of perlite and vermiculite for rooting

cuttings of loblolly pine and slash pine, this was also

recommended by Hare (1971).

Leakey et al. (1990) conducted studies using five

tree species and investigated the effects of four

different rooting media: (ii) gravel, (ii) 50:50 gravel

with sawdust, (iii) fine sand, and (iv) 50:50 fine sand

and sawdust. Each medium was tested with

cuttings dipped in a range of IBA concentrations (O0.8%). There were substantial differences between

species with regard to rooting success on the

different media. Single-node juvenile cuttings of all

five species rooted well (70-95%) on their best

medium. Cordia alliodora rooted best in fine sand,

with or without sawdust, while rooting of Vochysia

hondurensis cuttings was detrimentally affected by

the incorporation of sawdust into both gravel and

fine sand. In contrast, sawdust enhanced rooting of

Eucalyptus deglupta cuttings in both gravel and

sand, while Gmelina arborea and Albizia guachapele

rooted well in all media. The mature cuttings of G.

arborea did not root as well as the the juvenile

cuttings, especially in pure gravel.

Reuveni et al. (1990) successfully rooted cuttings

of Eucalyptus camaldulensis Dehn. in a wide range

of media under intermittent mist. Rooting

percentage did not differ significantly among the

media used. They concluded that various media

could be used for rooting eucalyptus cuttings, as

long as the medium contained components that

improve aeration. Similar results were obtained in a

number of other species when peat, perlite, and

vermiculite in various combinations, were

components of media producing the higher

percentages of rooted cuttings.

Other studies show no significant difference in

rooting among media, including Carter (1984) with

tamarack (Larix Zaricina [Du Roil K. Koch) cuttings

and Williams (19871 with western white pine (Pinus

monticola Dougl.) cuttings.

More information is needed about media effects,

especially regarding variations in gas-filled pore

spaces of substrate without including measurements

on other parameters such as mineral nutrients or

PH.

MYCORRHIZA

Mycorrhizae formed on the roots of plants by

naturally occurring or artificially introduced fungal

symbionts are known to enhance the growth and

development of their host plants. There is evidence

that the fungi increase nutrient uptake in the host

plant and produce growth promoting or regulatory

compounds (Slankis 1973). Indirect evidence that

the fungi release these materials in the host root

comes from anatomical and physiological changes in

mycorrhizae that can be induced or mimicked by

pure preparations of these compounds and/or culture

filtrates from the fungi. Therefore, the question is

whether ectomycorrhizal fungi produce growth

promoting substances that could enhance the

rooting of cuttings of vegetatively propagated forest

trees.

It is now well established that ectomycorrhizal

fungi release IAA (Moser 1959, Ek et al. 1983, Gay

and Debaud 19871, which enhances the rhizogenic

activity of the host plant and is responsible, at least

in part, for the typical morphology of

ectomycorrhizae. In different pine species,

ectomycorrhizal fungi induce prolific initiation of

short roots that are dichotomously branched

(Slankis 1973). Long roots are generally

nonmycorrhizal but they can be invaded by the

symbiotic fungi, which suggests that they are under

direct influence of the fungi (Gay 1990).

104
Linderman and Call (1977) were the first to show

that the growth-promoting substances produced by

mycorrhizal fungi could promote rooting of woody

plant cuttings. Experimenting with 13 different

ectomycorrhizal fungi, they demonstrated an

increase in the rooting percentage and root volumes

of cuttings of bearberry (Arctostuphylos uua-ursi L.

Spreng.) and huckleberry (Vuccinium ouatum

Parsh). This stimulation occurred before or in the

absence of any mycorrhizal association. Similar

results were reported by Navratil and Rochon (1981)

on poplar cuttings, and by Branzanti et al. (1985)

and Cristoferi et al. (1986) on hardwood cuttings of

fruit trees, which are nonectomycorrhizal plants.

Stein et al. (1990) also observed stimulated

rhizogenesis of black spruce (Picea marianu (Mill.)

cuttings by addition of Luccuria bicolor (Maire)

Orton and Suillus cuvipes (Opat.1 Smith & Thiers to

the rooting medium over controls, although the best

rooting percentage and highest number of roots was

formed on auxin-treated cuttings. The results

observed with European larch (Lurk decidua)

cuttings were different, in that the two fungal

symbionts had no significant effects on rooting. No

ectomycorrhizal formation occurred in S. cuvipes

inoculated medium with either tree species.

Gay (1990) studied the effect of the

ectomycorrhizal fungus Hebelomu hiemale Bres. and

its culture filltrate on in vitro rooting of Aleppo pine

(Pinus hulepensis Mill.1 isolated shoot hypocotyls to

determine if ectomycorrhizal fungi could enhance

adventitious root formation. P.‘haZepensis

hypocotyls did not root in the absence of hormonal

treatment, whereas the rooting percentage was

87.3% in the presence of 5 PM IAA. In the presence

of Trp (tryptophan), which is a precursor of IAA, H.

hiemale strongly enhanced rooting of hypocotyls

cultivated in the absence of any hormonal

treatment. In the presence of 0.1 mM Trp, the

rooting percentage of the inoculated hypocotyls was

96.6%, whereas it was only 7.6% in the absence of

the fungus. H. hiemule culture filtrate obtained in

the absence of Trp did not contair 1AA and did not

stimulate rooting of the hypocr s. In contrast, a

culture filtrate obtained in the presence of Trp

contained IAA; an ethyl acetate extract from this

filtrate resulted in 100% rooting. Different fractions

were isolated by thin-layer chromatography from the

IAA-containing filtrate and studied for their effect

on rooting. It was demonstrated that IAA was

responsible for the rhizogenic activity of H. hiemale.

These results suggest that ectomycorrhizal fungi

which rapidly metabolize exogenously supplied Trp

to IAA could be a suitable tool to enhance rooting of

gymnosperms. They also confirm previous reports

(Libby and Conkle 1966, Bowen et al., 1975, Smith

and Thorpe 1975) that rooting of gymnosperm

cuttings (e.g. radiata pine [Pinus rudiutu D. Don1 or

lodgepole pine [Pinus contortal) can be enhanced by

auxins. It must also be emphasized that

ectomycorrhizal fungi also release cytokinins,

gibberellin-like compounds, and ethylene (Gay

1988).

Mechanism of Effect

Several mechanisms could be operating with this

enhanced rooting response. The rooting effect of

these fungi is probably due to a nonspecific

mehanism and could be ascribed to growth

regulators (e.g. auxins, cytokinins, ethylene or

gibberellins), vitamins, or other metabolites released

by the fungi (Slankis 1973, Gay 1990). It can also be

postulated that inoculation of cuttings with these

fungi could modify their hormonal balance and

subsequently their rooting ability. Navratil and

Rochon (19811 suspected hormonal exudates

liberated by the mycelium of Pisolithus as a cause of

the enhanced shoot and root development in poplar

cuttings.

Other possible mechanisms for the rooting

phenomenon include the mycorrhizal fungi

producing substances which may act protectively or

synergistically with the growth substances already

in cuttings (Linderman and Call 1977). Levisohn

(1956) suggested that these fungi may be altering

the nutrient status of the medium, or may be

releasing substances that are inhibitory to other

microbes that cause cuttings to deteriorate.

Endomycorrhizal Inhibition

Endomycorrhizal colonization has also been

shown to stimulate growth of greenhouse-grown

seedlings of hardwood species (Kormanik and

McGraw 1982, Melichar et al. 19861. However,

there are results which show that the potential

benefits associated with endomycorrhizal inoculation

were not expressed in plant growth measurements

of cuttings during the root production phase.

Sword et al. (1991) conducted an experiment to

identify the effect of inoculation using a mix of three

Glomus spp. isolates on the root growth rate of

eastern cottonwood (Populus deltoides Bartr. ex

Marsh.) cuttings. Results indicated that

endomycorrhizal inoculation appeared to have a

negative effect on growth during greenhouse
production. There was no significant effect on stem

length, stem diameter, or on stem, root, or foliar dry

weights of the cuttings. The leaf surface area of

inoculated cuttings was significantly less than that

of uninoculated cuttings. Shoot and root growth

were either unaffected or inhibited due to

endomycorrhizal inoculation.

Sword et al. (1991) attributed negative root

growth rate and leaf surface area responses to a

combination of factors. They suggested that

endomycorrhizal inoculation may have affected root

system morphology. Also, the physiology of

hardwood cuttings may provide some explanation for

the growth inhibition observed. Nanda et al. (1971)

reported the importance of having adequate

exogenous glucose, in addition to IAA, for rooting of

etiolated Populus cuttings. It has been reported

(Tschaplinski and Blake 1990) that a proper balance

of nutritional and regulatory compounds determines

the rooting ability of this genus. The rapid rate of

shoot growth of cuttings as compared to seedlings

suggests that starch availability for initial root

growth may be more limiting in cuttings than in

seedlings. Furthermore, the shoot growth rate of

cuttings compared to seedlings suggests that

production of growth regulators in shoot

meristematic tissues may be greater in cuttings. As

a result, growth responses of cuttings and seedlings

inoculated with endomycorrhizal fungi may differ.

Another explanation for the negative effect on

growth is that a limited availability of photosynthate

for root growth, due to higher metabolic

requirements of plants with endomycorrhizal fungal

associates than without, may have contributed to

reduced growth of the host. Other factors which

may help to explain the unexpected decrease in

growth of inoculated cuttings in this particular

study, may involve competition between the three

endomycorrhizal isolates, in combination with low

greenhouse light conditions.

THE ROOTING FACILITY

Container (Enclosed) vs. Open Mist Beds

The appropriate choice of propagation facility is

based on the level of humidity and the amount of

temperature control that is required to root a

particular species, with enclosed beds offering more

control over these factors. Sensitivity of the plant’s

tissues to varying moisture levels, is another

consideration. Both systems are usually shaded

which is essential in most systems to keep water

stress to a minimum (Loach 1977). It has been

shown that mist inside a closed polyethylene tent

reduced the leaf-to-air vapor pressure gradient more

effectively than conventional open mist beds

(Grange and Loach 1983).

Grange and Loach (1984) compared rooting of 81

species of leafy cuttings in open and polyethyleneenclosed mist systems. Enclosing a mist system in a

polyethylene tent affected the environment and

cuttings in several different ways. Because the leaf

surfaces were wetter and the humidity greater

under enclosed mist, water loss from the cuttings

was reduced and the water potential of the foliage

was significantly greater (less negative) than under

open mist. Light levels were 20% lower and air

temperatures averaged 5OC higher in enclosed mist

than in open mist.

A comparison of rooting performance in the

enclosed mist and open mist systems suggests that

the enclosed mist system would be preferable for the

propagation of cuttings susceptible to water stress,

though physiological principles show open systems

are likely to be superior for net photosynthetic gain,

but inferior in terms of water stress (Grange and

Loach 1984). Other factors such as leaf wetness,

which can cause rotting, must also be considered.

Greenhouse vs. Nursery vs. Field Rooting

In propagation procedures, cuttings are

sometimes planted directly in the nursery bed or

field, but most often they are started in a blended

mix in some type of container. Rooting in these

three environments is segregated by the degree of

environmental control available within each system.

l Conventional greenhouse rooting can usually be

designed so that humidity, temperature, light,

and other parameters (e.g. CO2 enrichment) can

be either precisely controlled or relatively

controlled. The cuttings are stuck into

individual rooting containers or into rooting

beds. Humidity is controlled with fog or ultralow volume mist. Temperature inside the

greenhouse can be controlled by heaters,

evaporative coolers, exhaust fans, and movable

side walls or vents.

l A covered nursery bed is usually a plastic-covered

metal frame installed over several nursery beds.

Humidity and temperature, based on whether

106
beds are located indoors or outdoors, can be

controlled with this system.

l Field rooting allows for no environmental control.

Water/fertilizer is the only element of the

environment that can be provided to cuttings,

generally through some type of drip irrigation

system.

The high cost of plantation establishment dictates

that the very best planting stock be used. Methods

to produce rooted stock have met with variable

success. Many times the decision on which

propagation facility to use has been a purely

economic rather than a practical decision based on

efficiency of the method. Most often, foresters have

met less success by adopting methods for

establishing and managing large plantations than

those accomplished with cottonwood cuttings

(M&night 1970, Randall and Miller 1971). Under

ideal circumstances, cottonwood performs as no

other tree species can.

The lack of success has prompted the

investigation of potential methods of improving

rooting ability and ease of propagation to acceptable

levels for commercial production. Cunningham

(1986) evaluated the rooting ability of cuttings from

one-year-old coppice of American sycamore for 80

ortets (4 trees from each of 20 half-sib families) in a

greenhouse and a nursery environment. Rooting

percentages averaged 97.26 for the greenhouse and

56.29 for the nursery study. Greenhouse rooting

success was excellent for all but a few clones, and

nursery rooting, while not as high as greenhouse,

was encouraging. The quality of the nursery-rooted

cuttings and the number of clones with good rooting

success (20% of the clones rooted at 80% or better)

indicated that it was feasible to produce American

sycamore from nursery-rooted planting stock.

Findings were inconclusive in a study conducted

by Foster (19781, in which he compared the rooting

behavior of loblolly pine stem cuttings in an open

medium, and in 164-ml plastic Ray Leach Supercell

containers, both under mist. Rooting success was 39

and 24%, respectively, showing no significant

differences. Although there was a large degree of

variability in this experiment, there were some

advantages to using containerized cuttings,

including easier and quicker rooting observation,

and less damage during lifting of cuttings rooted in

the open medium.

While the cost of rooting and growing large

numbers of planting stock in the greenhouse may be

prohibitive, particular clones of high value for

growth and wood properties “may” justify the added

expense of nursery rooting. Clonal selection for

nursery rooting ability would probably place most

emphasis on improving percent rooting, as opposed

to other selection traits (Cunningham 1986).

Higher rooting percentages would save money with

less area needed for stock plants and smaller

nursery requirements to produce the same quantity

of planting stock. Greenhouse rooting, with its high

rate of success, could be used in the initial stages of

multiplying the number of stock plants for mass

production of cutting material.

CONCLUSIONS

Based on the potential impact in forestry that

vegetative propagation by rooted cuttings can have,

the search for more efficient methods to produce

operational planting stock must continue.

Production of quality propagules, similar to those

which are routinely achieved for Japanese cedar

(Cyptomeria juponica IL.F.1 Don), Norway spruce,

radiata pine, and eastern cottonwood, by rooted

cuttings is anticipated in the future. Many of these

trees have been growing under plantation conditions

sufficiently long to demonstrate the success of clonal

stock and provide impetus to solve the problems of

difficult-to-root genera and species. Other important

commercial forest species, such as loblolly pine,

slash pine, and Douglas-fir are in various stages of

development to support a production program for

forest plantations.

This review shows that there is incomplete

understanding of the various interactive factors

involved in providing optimal environmental

conditions for the rooting of cuttings from forest

trees, and the results from some prior studies have

often raised difficult questions. The purpose of

studies cited in this paper was to better understand

the processes underlying improved rooting by

following what is documented about humidity, water

relations, light, air and soil temperature, the rooting

medium, mycorrhiza, and the rooting facility.

Optimal conditions encourage further

development towards fully operational clonal

forestry. Rigorous control of the rooting

107
environment promises to improve the success of

rooting larger numbers of cuttings. More studies

are required to understand exactly how

ectomycorrhizal fungi affect rhizogensis. However,

the ability of the fungi to enhance rooting of

coniferous cuttings in absence of any auxin supply

has been demonstrated, and may represent an

alternative to the use of auxins for the vegetative

propagation of conifers from cuttings.

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Propagule Growth, Development

and Application

Moderator:

Barbara McCutchan

Westvaco Company

Forest Research

Summerville, South Carolina

113

FIELD PERFORMANCE COMPARISONS BETWEEN VEGETATIVE PROPAGULES

AND SEEDLINGS OF LOBLOLLY AND SLASH PINES1

Lewis John Frampton, Jr.2

Abstract.--Current knowledge of the field growth of loblolly and slash pine

tissue culture propagules and rooted cuttings relative to seedlings is reviewed.

Generally, tissue culture propagules produced via axillary- fascicular

micropropagation from epicotyls and rooted cuttings from juvenile ortets

perform similarly to seedlings under field conditions. Tissue culture

propagules produced via the Cotyledon System and rooted cuttings from older

ortets generally express mature characteristics including reduced growth

relative to seedlings. The knowledge and ability to manipulate the degree of

planting stock maturation will be required in order to effectively develop

operational vegetative propagation programs for these species.

INTRODUCTION

Currently, numerous forest products companies

in the Southern U.S. are interested in developing

commercial vegetative propagation systems for

loblolly pine (Pinus taeda L.), slash pine (Pinus

elliottii var. elliottii Engelm.) or both. Their

incentive is to capture the benefits associated with

deploying reforestation planting stock either of elite

control-pollinated crosses and/or clones.

Often, it has been assumed that vegetative

propagules will perform similarly to seedlings in the

field. However, the process of vegetative

propagation, or even factors unique to a specific

method of vegetative propagation, may cause the

resulting propagules to behave dissimilarly to

seedlings. In order to fully capture the benefits of

this technology, an understanding of and ability to

lPaper presented at the 1992 Southern Regional

Information Exchange Group Biennial Symposium,

Huntsville, AL, July 8-10, 1992.

2Propagation Scientist, Weyerhaeuser Company,

Southern Forestry Research, Box 1060, Hot Springs,

AR 71902

manipulate any differences between vegetative

propagules produced via various methods and

seedlings is necessary.

The objective of this manuscript is to review

the current knowledge of field performance of

loblolly and slash pine vegetative propagules

relative to that of seedlings.

TISSUE CULTURE PROPAGULES

Loblolly Pine

Three methods of tissue culture propagation of

loblolly pine have been summarized: 1)

organogenesis of adventitious shoots and roots from

cotyledon explants (Cotyledon System), 2)

micropropagation via fascicular and axillary shoots

obtained from juvenile, adolescent and mature

explants and 3) somatic embryogenesis using

immature zygotic embryos as a starting source

(Amerson et al. 1988, Greenwood et al. 1991).

The current knowledge of the field performance of

propagules produced from each of these tissue

culture methods is reviewed below.

115
Cotyledon System.--Establishment of field trials

comparing loblolly pine Cotyledon System

propagules and seedlings began in the late 1970’s

and early 1980’s so that field performance of

propagules produced in this manner has been

studied intensively.

Growth and Survival--While Cotyledon System

propagules generally survived field planting as well

as seedlings, they displayed reduced early height

and diameter growth relative to seedlings (Amerson

et al. 1985). For example, in a series of eight field

trials established during 1981, the mean fourth year

height of these propagules was significantly less

than that of seedlings from the same open-pollinated

families (4.40 versus 5.06 m, respectively) (Amerson

et al. 1988, Frampton 1986). Large differences in

early diameter growth between these Cotyledon

System propagules and seedlings were also found

(Mott et al. 1984). Measurements through year eight

in one of the 1981 trials located near Monroeville,

Alabama, verified that this trend continued into

later years (Mott et al. 1990). However, propagule

types in these studies were established in paired

row-plots so that measurements subsequent to

crown closure were confounded with competition

effects. Nevertheless, it is clear from these and

other studies that Cotyledon System propagules

grow considerably slower than seedlings.

Shoot Morphology.--Differences in shoot

morphology between Cotyledon System propagules

and seedlings were often casually observed. A

detailed assessment of various characteristics which

change with maturation in loblolly pine (Greenwood,

1984) (bud size, number of growth cycles, number of

branches, etc.) after two growing seasons at one field

trial in Jesup, Georgia, verified quantitatively that

Cotyledon System propagules produce more maturelike morphology relative to seedlings of the same

open-pollinated families (McKeand 1985). A later

set of measurements in the same trial after four

growing seasons and in a Raleigh, North Carolina,

trial after six growing seasons revealed that these

morphological differences between propagule types

lessen at older ages (Mott et al. 1987).

Fusiform Rust Resistance.--Generally, Cotyledon

System propagules have shown less fusiform rust

(caused by Cronartium quercuum (Berk.) Miyabe ex

Shirai f. sp. fusiforme) infection than have seedlings.

In five of the eight field trials established in 1981,

rust infection levels exceeded 60%. The Cotyledon

System propagules in these five trials had a lower

incidence of gall formation at age four than did

seedlings from the same open-pollinated families (46

versus 71%, respectively) (Frampton 1986).

Wood Specific Gravity. -- Juvenile wood specific

gravity of Cotyledon System propagules and

seedlings were compared at three field trial locations

each of a different age: Lewiston, North Carolina

(age two), Jesup, Georgia (age three) and Raleigh,

North Carolina (age six) (Frampton and Jett 1989).

A significant difference in the mean specific gravity

between propagule types was found only in the

youngest material (0.387 for the seedlings versus

0.356 for the cotyledon system propagules.

Root Systems.--Cotyledon System propagule root

systems were generally less developed than those of

seedlings. Under greenhouse culture they typically

produced less root dry weight and fewer lateral roots

in the upper portion of their root systems than did

seedlings (McKeand and Allen 1984, Frampton and

McKeand 1987). The main differences between

Cotyledon System propagule and seedling root

systems appeared to be morphology rather than

physiology since their ability to uptake nutrients

was similar based on root surface area (McKeand

and Allen 1984). Root excavations of Cotyledon

System propagules and seedlings confirmed that

these morphological root system differences persist

after 2.5 years in the field (Frampton and McKeand

1987).

Cause of Differences. --Differences between

Cotyledon System propagules and seedlings were

recognized early so that in 1982, two field trials were

established to better understand these differences

(Frampton and Isik 1987). Both of these field trials

contained Cotyledon System propagules and

seedlings from the same open-pollinated families as

well as two additional in vitro-produced propagule

types: excised embryos and rooted hypocotyls. As in

other field trials, after 3.5 growing seasons, the

Cotyledon System propagules were shorter in

height, had less fusiform rust incidence and

displayed more mature morphology relative to the

seedlings. The two additional propagule types

performed similarly to the seedlings for all traits

assessed. Designed comparisons among these four

types of propagules suggest that the origin of

differences between Cotyledon System propagules

and seedlings lies with in vitro shoot production

methodology rather than with the tissue culture

environment or rooting procedures.

116
Another field trial designed to understand

differences between Cotyledon System propagules

and seedlings was established in late 1983

(Anderson et al. 1992). After one growing season,

scion of Cotyledon System propagules and seedlings

were grafted onto rootstock of Cotyledon System

propagules and seedlings of the same openpollinated families so that four grafted combinations

were obtained. Three growing seasons after

grafting, Cotyledon System roots accounted for 0.3 m

of height growth loss and 1.0 cm of basal diameter

growth loss while the Cotyledon System shoots

accounted for 0.6 m of height growth loss and 1.4 cm

basal diameter growth loss. The roots of Cotyledon

System propagules used in this study were initiated

and developed for 18 months prior to grafting under

the control of Cotyledon System shoots. This may

account for the contribution of the Cotyledon System

roots to growth reduction in this trial that was not

apparent in the earlier pair of trials (Frampton and

Isik 1987). As in the earlier pair of trials, three

years’ data in this trial indicated that the expression

of mature shoot characteristics of Cotyledon System

propagules is controlled almost entirely by their

shoots rather than roots.

One additional field trial was established in 1988

to understand differences between Cotyledon System

propagules and seedlings (Frampton et al. 1992). In

this trial, seedlings, Cotyledon System propagules,

axillary-fascicular micropropagules originating from

epicotyls (see below) and axillary-fascicular

micropropagules originating from Cotyledon System

shoots were established. Based on measurements

assessed during the fifth growing season, the height

and diameter at breast height (DBH) of the

Cotyledon System propagules and the

micropropagules derived from Cotyledon System

shoots were significantly less than that of the other

propagule types which were similar to each other.

Again, these results suggest that the method of

adventitious shoot production from cotyledons

results in dissimilarities from seedlings in the

propagules produced. Further, these differences are

maintained through up to three cycles of

micropropagation.

Axillary-Fascicular Micropropagation Juvenile

Sources.--One field trial containing axillaryfascicular micropropagules of loblolly pine was

established near Cantonment, Florida, in 1988

(Frampton et al. 1992). As mentioned above, this

field trial contained axillary-fascicular

micropropagules originating from epicotyls,

seedlings, Cotyledon System propagules, and

axillary-fascicular micropropagules originating from

Cotyledon System shoots. The micropropagules

originating from epicotyls were also produced using

three levels (0,25 and 50 mg/l) of benzylaminopurine (BAP) in the shoot initiation procedure.

Unlike the cotyledon-origin adventitious shoots, the

micropropagules in this field trial grew similarly to

the seedlings based on first and fifth year height and

diameter assessments. Their first year shoot

morphology was also similar to seedlings. Further,

the three levels of BAP used for shoot initiation did

not have any significant effect on the subsequent

growth and development of these micropropagules.

Adolescent and Mature Sources.--Although

techniques exist to propagate adolescent and mature

loblolly pine via axillary-fascicular micropropagation

(Amerson et al. 1988), no field performance data

have been reported. Such propagules would be

expected to express some degree of rejuvenation.

Their rate of maturation relative to seedlings would

be of great interest. Data from such trials could

contribute greatly to the current understanding of

maturation and rejuvenation.

Somatic Embryogenesis.--Currently, intensive

research efforts are focused on developing this

technology as a potentially more productive tissue

culture system for loblolly pine. Although a small

number of loblolly pine somatic seedlings have been

field-planted to date, no reports of their field

performance currently exist in the literature.

Slash Pine

Although the same methods of tissue culture

propagation reported for loblolly pine could be

applied to slash pine, considerably less research has

been conducted on this species. In one field trial

established near Yulee, Florida, slash pine

Cotyledon System propagules were compared to

seedlings. Three-year-results showed a similar but

smaller difference in height growth between these

propagule types than was observed for loblolly pine.

No reports of the growth of slash pine propagules

produced via other tissue culture methods currently

exist.

Rooted Cuttings Loblolly Pine

Growth.--Growth of loblolly pine rooted cuttings

was compared with that of seedlings in two field

117
trials established in central Arkansas during 1978

and 1980 (Foster et al. 1987). In the older trial,

rooted cuttings from four-year-old ortets were

significantly smaller in height (P~0.05) and DBH

(P<O.Ol) at age six than were seedlings from the

same open-pollinated families. In the younger trial,

cuttings from one-year-old ortets were significantly

(PsO.01) larger in height and DBH than rooted

cuttings from five-year-old ortets and seedlings from

the same open-pollinated families. The mean height

and DBH of the rooted cuttings from five-year-old

ortets and seedlings were not significantly different

from each other. However, the seedlings in this trial

were one growing season younger and considerably

smaller in size than the rooted cuttings when the

trial was initially established. The seedling height

growth curve was parallel to that of the rooted

cuttings from one-year-old ortets and steeper than

that of cuttings from the five-year-old ortets.

Together, the results from these trials

demonstrate the negative effect of increased ortet

age on the growth of loblolly pine rooted cuttings

and suggest that the early field growth of rooted

cuttings of one-year-old ortets and seedlings is

similar. Results from a smaller field trial comparing

rooted cuttings from young ortets with seedlings of

the same five control-pollinated families

corroborates this conclusion (Foster 1988). Height

and DBH after three years in the field were not

significantly different for the rooted cuttings and

seedlings in this trial.

Shoot Morphology--Shoot morphology

characteristics were measured in the three studies

discussed above (Foster et al. 1987, Foster 1988).

Differences between rooted cuttings and seedlings

were not consistently found. However, when

differences were detected, the rooted cuttings

displayed less stem taper, fewer growth cycles and

fewer branches than did the seedlings.

Fusiform Rust Resistance--No loblolly pine rooted

cutting field trials with fusiform rust infection have

yet been reported. However, two studies utilizing

artificial inoculation and disease development of

greenhouse-grown containerized seedlings and

rooted cuttings have been conducted.

In the first study (Foster and Anderson 1989),

full-sib families of seedlings were inoculated and

symptoms were assessed using standard protocol

(Anderson et al. 1983). After symptom assessments,

those seedlings without visible gall formation were

developed into hedges. Cuttings from these hedges

were rooted, inoculated and assessed using the same

protocol as for the seedlings.

In this study, the mean percentages galled for the

rooted cuttings and seedlings were approximately 4

and 53%, respectively. The range in seedling and

rooted cutting family means were 41-64% and O-11%

galled, respectively. Further, a resistant and

susceptible seedling checklot included with the

rooted cutting inoculations averaged 50 and 80%

galled, respectively.

While the results from this study showed a

dramatic difference in fusiform rust resistance

between seedlings and rooted cuttings, the following

confounding factors make it impossible to conclude

to what degree, if any, that this difference is due to

inherent differences between propagule types: 1)

the cuttings had been screened for resistance while

the seedlings had not; and 2) succulent epicotyl

tissue was inoculated on the seedlings while second

year growth was inoculated on the rooted cuttings.

In the second study (Frampton and Walkinshaw

1988, Unpublished Data), clones of cuttings along

with seedlings from the same 14 open-pollinated

families were inoculated together using standard

protocol (Anderson et al. 1983). In this study, the

rooted cuttings were sheared and new fascicular

shoots were allowed to develop so that the inoculated

tissue was in a similar stage of development.

Additionally, there was no differential selection for

resistance between the rooted cuttings and

seedlings.

Results of this study also revealed a marked

difference in resistance between the rooted cuttings

and seedlings: 19 versus 43% galled, respectively.

These results suggest that only a portion of the

difference in propagule type resistance reported in

the first study was a result of screening for

resistance. While phenological differences between

rooted cuttings and seedlings cannot be ruled out in

the later study since there are differences between

growth and development of fascicular shoots versus

epicotyls, it appears that there may be a large

degree of resistance associated with using rooted

cuttings rather than seedlings as planting stock.

Ultimately, rooted cutting and seedling comparisons

must be made under field conditions on high rust

hazard sites to corroborate this conclusion.

118
Slash Pine

Growth.--An early report (Hoeskstra and

Johansen 1957) suggested that ortet age influenced

subsequent growth of vegetative propagules of slash

pine. Although seedling comparisons were not

included, air-layers from six-year-old ortets were

substantially taller than those from 23-year-old

ortets after 1 year in a nursery. In another study

without seedling comparisons, slash pine air-layered

from the main stem and first-order branches of fouryear-old ortets were reported to grow similarly

(Greene 1962).

In a later reported study established from 1944 to

1947 (Franklin 1969), the effect of ortet age on

growth was quantified. Height and DBH

measurements of 21- to 24-year-old rooted cuttings

were regressed on ortet age (ranging from 12 to 28

years). The regression equations suggested that

each ortet year reduced height and DBH of the

rooted cuttings by about 12 cm and 39 mm,

respectively. In another field trial involving airlayered and grafted material; similar relationships

were also found (Franklin 1969).

A planting of slash pine air-layers from ll-yearold ortets was established in 1957 and 1958 in

northeast Florida near Olustee (Schultz 1972a and

b). This trial was designed as a split-plot factorial to

study the effect of various cultural treatments of

oleoresin production. The plots in the study were

separated by one or two rows of commercial

seedlings.

While the fastest growing air-layered clone had

three times the merchantable stem volume as the

slowest growing clone at age 12, the overall mean

merchantable stem volume of the air-layers was

almost identical to and not statistically different

from that of the seedlings (Schultz 1972a). The

mean height and DBH of the air-layers were slightly

greater that those of the seedlings after 30 years:

21.8 versus 22.8 m and 32.6 versus 34.7 cm,

respectively. Inspection of DBH (inside bark) growth

curves reconstructed from increment core

measurements revealed that during certain periods

of the rotation, the air-layer mean diameter equaled

or exceeded that of the seedlings. These differences

in growth between the air-layers and seedlings were

slight, however, when compared to the variation

among the clones of air-layers: 18.9-24.3 m in height

and 22.9-41.2 cm in DBH at age 30.

119

The similar growth of the propagule types in this

study, despite the relatively old air-layer ortet age,

may be attributed to two factors: 1) air-layers are

likely to be of much better vigor with more profuse

root systems than rooted cuttings due to the benefits

of rooting attached to the parent tree and 2) the airlayers were likely taller at establishment since slash

pine air-layers were typically up to 60 cm in length

(Mergen 1955). However, the results from this field

trial suggest that it may be possible to reverse the

negative effect of ortet age on growth by providing

special care to improve the planting size, vigor and

root system of vegetative propagules from older

ortets.

Root Systems.--Root development of ll- to 12-year

old air-layers (from ll-year-old ortetsl and seedlings

were compared in the field trial mentioned above

which was established on a moderately deep sandy

soil (Schultz 197213). The air-layers and seedlings

did not differ significantly in total root surface area

or in shoot growth (see study discussed above,

Schultz 1972a). However, all seedlings developed

well-defined tap roots with primary lateral roots at

depths of 0 to 1.5 m, while most air-layers had one to

three heavy sinkers .arching from the root collar to

the maximum root depth which sometimes

developed lateral roots. The average depth to which

roots developed were 2.3 and 2.6 m for air-layers and

seedlings, respectively. Although not statistically

significant, seedlings averaged 44% more primary

lateral roots and 79% more sinker roots than did airlayers. The average root diameter differed only

slightly between the two propagule types. The root

systems of individual trees varied greatly except

that members of air-layer clones were remarkably

similar to each other.

GENERAL DISCUSSION

Generally, vegetative propagules of loblolly and

slash pine survive and grow normally under typical

field conditions and do not exhibit gross aberrations.

No reports of plagiotropic field growth of slash and

loblolly pine vegetative propagules exist. While both

tissue culture propagules and rooted cuttings of

these species commonly display plagiotropic growth

in the greenhouse, apparently, under field

conditions, this response is corrected by the

production of compression wood.

When differences between vegetative propagules

and seedlings have been reported, the vegetative
propagules displayed reduced growth, mature shoot

morphology or both. While reduced growth is

undesirable for commercial forestry applications,

many of the mature characteristics such as reduced

fusiform rust infection, less stem taper and fewer

branches are highly desirable characteristics. The

ortet age at which the tradeoff between fast growth

and desirable shoot characteristics occurs is not

known for loblolly and slash pines. More data

regarding the influence of ortet age on the growth of

rooted cuttings are available for Monterey pine

(Pinus rudiatu D. Don). In this species, rooted

cuttings from young ortets (one- to three-years-old)

perform at least as well as seedlings. Rooted

cuttings from slightly older ortets (four- and fiveyear-old) display some loss of early diameter growth

but have better form with less malformation

(Menzies et al. 1991).

Commercial rooted cutting programs designed to

bulk-up elite control-pollinated families or clones

will require the use of hedges and/or serial

propagation. These techniques are reported to slow

down the maturation process in other species.

However, for loblolly and slash pine, little is known

about this process, especially with relation to

subsequent field performance.

Obviously, further field trials are needed to

provide the knowledge necessary to develop effective

vegetative propagation systems for loblolly and slash

pine. While not emphasized in this review, many of

the past field trials comparing vegetative propagules

and seedlings have various shortcomings. The

following recommendations are provided to help

circumvent some of these shortcomings in future

field trials of this type (see Frampton and Foster

1992 for further details): 1) compare propagule types

of similar genetic quality such as from paired

open-pollinated families, 2) make every effort to

culture the propagule types in a manner so that they

can be planted at the same time and be of

comparable size and physiological condition, 3) use

block plots for propagule types rather than row or

single tree plots in order not to confound competition

with propagule type effects after crown closure and

4) measure the trials at early ages and frequent

intervals.

CONCLUSION

Vegetative propagules of loblolly and slash pines

often perform similarly to seedlings under field

conditions. When this is not the case, vegetative

propagules generally display reduced growth and

mature-like morphological characteristics. Better

understanding how to control these differences will

be necessary in order to effectively utilize vegetative

propagation technology in the future and may

become a key advantage of using this technology.

LITERATURE CITED

Amerson, H.V.; Frampton, L.J., Jr.; McKeand, SE.

[and others]. 1985. Loblolly pine tissue culture:

laboratory, greenhouse and field studies. In: Henke,

R.R.; Hughes, K.W.; Constantin, M.J.; Hollander,

A., eds. Tissue culture in forestry and agriculture.

New York: Plenum Press: 271-287.

Amerson, H.V.; Frampton, L.J., Jr.; Mott, R.L.;

Spaine, P.C. 1988. Tissue culture of conifers using

loblolly pine as a model. In: Genetic manipulation of

woody plants. Hanover, J.W.; Keathley, D.E., eds.

New York: Plenum Press: 117-137.

Anderson, A.B.; Frampton, L. J., Jr.; McKeand, S.E.;

Hodges, J.F. 1992. Tissue-culture shoot and root

system effects on field performance of loblolly pine.

Canadian Journal of Forest Research. 22: 56-61.

Anderson, R.L., C.H. Young and J.D. Triplett. 1983.

Resistance Screening Center Procedures Manual.

USDA Forest Service State and Private Forest

Report. No. 83-1-18.

Foster, G.S. 1988. Growth and morphology of rooted

cuttings and seedlings of loblolly pine and their

genetic analysis. In: Proceedings of the 10th North

American forest biology workshop; 1988 July 20-22;

University of British Columbia, Vancouver, BC: 67-

78.

Foster, G.S.; Anderson, R.L. 1989. Indirect selection

and clonal propagation of loblolly pine seedlings

enhance resistance to fusiform rust. Canadian

Journal of Forest Research. 19: 534537.

Foster, G.S.; Lambeth, CC.; Greenwood, M.S. 1987.

Growth of loblolly pine rooted cuttings compared

with seedlings. Canadian Journal of Forest

Research. 17: 157-164.

Frampton, L.J., Jr. 1986. Field performance of

loblolly pine tissue culture plantlets. In: Breeding

theory, testing and seed orchards: Proceedings of the

joint meeting of IUFRO (International Union of

120
Forest Research Organizations) working parties

conference; Williamsburg, VA. Raleigh, NC: North

Carolina State University-Industry Tree

Improvement Program: 547-553.

Frampton, L.J., Jr.; Amerson, H.V.; Leach, G.N.

1992. [In review]. Influence of tissue-culture method

on ex vitro growth and development of loblolly pine.

Canadian Journal of Forest Research.

Frampton, L.J., Jr.; Foster, G.S. 1992. Field testing

vegetative propagules. In: Ahuja, R.; Libby, W.J.,

eds. Clonal forestry: genetics, biotechnology and

application. Berlin: Springer Verlag: 110-134.

Frampton, L.J.; Isik, K. 1987. Comparison of field

growth among loblolly pine seedlings and three

plant types produced in vitro. Tappi Journal. 70:

119-123.

Frampton, L.J.; Jett, J.B. 1989. Juvenile wood

specific gravity of loblolly pine tissue culture

plantlets and seedlings. Canadian Journal of Forest

Research. 19: 1,347-1,350.

Frampton, L.J., Jr.; McKeand, S.E. 1987.

Characterization of the root and shoot systems of

young loblolly pine propagules. In: Annual progress

report supplement: special project on tissue culture.

Raleigh, NC: North Carolina State University.

[Number of pages unknown]

Franklin, E.C. 1969. Ortet age has strong influence

on growth of vegetative propagules of Pinus elliottii

Engelm. In: 2nd world consultation of FAO (Food

and Agriculture Association) and IUFRO

(International Union of Forest Research

Organizations) on forest tree breeding; Washington,

DC: 815-827.

Greene, J.T. 1962. Air-layered branches of slash

pine will develop into straight trees. Journal of

Forestry. 60: 135.

Greenwood, M.S. 1984. Phase change in loblolly;

pine: shoot development as a function of age.

Physiologia Plantarum. 61: 518-522.

Greenwood, M.S.; Foster, G.S.; Amerson, H.V. 1991.

Vegetative propagation of southern pine. In: Duryea,

M.L.; Dougherty, P.M., eds. Forest regeneration

manual. Dordrecht; Boston; London: Kluwer

Academic Publishers: 75-86.

Hoekstra, P.E.; Johansen, R.W. 1957. Growth of

planted slash pine air-layers. Journal of Forestry.

55: 146.

McKeand, S.E. 1985. Expression of mature

characteristics of tissue culture plantlets derived

from embryos of loblolly pine. Journal of the Society

for American Horticultural Science. 110: 619-623.

McKeand, S.E.; Allen, H.L. 1984. Nutritional and

root development factors affecting growth of tissue

culture plantlets of loblolly pine. Physiologica

Plantarum. 61: 523-528

Menzies, MI.; Klomp, B.K.; Holden, D.G. 1991.

Optimal physiological age of propagules for use in

clonal forestry. In: Proceedings of the Forest

Research Institute/New Zealand Forest Products,

Ltd. clonal forestry workshop; 1989 May l-2:

Rotorua, New Zealand. FRI Bull. 160: 142-146.

Mergen, F. 1955. Air-layering of slash pines. Journal

of Forestry. 53: 265-270.

Mott, R.L.; Amerson, H.V.; Frampton, L.J., Jr. 1984.

Annual progress report: special project on tissue

culture. Raleigh, NC: North Carolina State

University. [Number of pages unknown.]

Mott, R.L.; Amerson, H.V.; Frampton, L.J., Jr.

1987. Annual progress report: special project on

tissue culture. Raleigh, NC: North Carolina State

University. [Number of pages unknown.]

Mott, R.L.; Amerson, H.V.; Somers, P.W. 1990.

Annual progress report: special project on tissue

culture. Raleigh, NC: North Carolina State

University. [Number of pages unknown]

Schultz, R.P. 1972a. Inherent vigor influences

growth of slash pine more than intensive cultural

treatments. Silvae Genetica. 21: 26-28.

Schultz, R.P. 197213. Root development of intensively

cultivated slash pine. Soil Science Society of America

Proceedings. 36: 158-162.

121

APPLIED VEGETATIVE PROPAGATION PROGRAMS IN FORESTRY1

Clements C. Lambeth, Gary A. Ritchie, and Brian Stanton2

Abstract.--Since the 1970’s there has been a surge in the use of vegetative

propagation and reforestation on a commercial scale. The genetic

improvement strategies and methods of vegetative propagation are reviewed

with particular emphasis on the two largest programs in the United States,

one at James River Corporation and the other at Weyerhaeuser Company.

The benefits, problems and limitations in the implementation of vegetative

propagation on a commercial scale are discussed.

INTRODUCTION

Before the 1970’s very few commercial

applications of vegetative propagation existed in

forestry. A notable exception is the centuries old

program of clonal plantations of sugi (Cryptomeria

juponica D. Don) in Japan (Ohba 1983). Eucalypts,

radiata pine (Pinus rczdiutu D. Don) and Norway

spruce (Piceu &es [L.] Rarst) have been

regenerated by rooted cuttings since the 1970’s, and

during the 1980’s efforts have expanded to several

other species. In the past 20 years there has been a

surge in the number of organizations worldwide that

use vegetative propagation on a commercial scale.

Survey results indicate that at least 70 million

conifer cuttings are produced annually in 20

countries (Ritchie 1992a). We are unaware of

similar survey results for hardwoods but worldwide

production of hardwood rooted cuttings is probably

lPaper presented at the 1992 Southern Regional

Information Exchange Group Biennial Symposium,

Huntsville, AL, July 8-10, 1992.

21nternational Forestry R&D Manager, Smurfit

Group, P. 0. Box 626 Callahan, FL 32011; Senior

Scientist, Weyerhaeuser Company, G. R. Staebler

Forest Resources Research Center, 505 North Pearl

Street, Centralia, WA 98531; Research Associate,

James River Corporation, 349 N.W. 7th Avenue,

Camas, WA 98607.

more than twice that of conifers. The largest

programs are in Brazil, The Congo and South Africa

where eucalypts are planted in monoclonal

plantations (Brandao 1984, Delwaulle 1985, Denison

1989). Vegetative propagation as a proportion of

total regeneration is expected to grow considerably

in the coming decades and may become the norm for

many species which are now being regenerated by

seedlings.

There are a number of publications on

operational vegetative propagation programs,

besides those already mentioned to which the reader

is referred (Armson et al. 1980, Arnold and Gleed

1985, Bentzer 1981, Foster and Shaw 1987, Gill

1982, Johnsen 1985, Kleinschmidt 1974, Lambeth et

al. 1992, Ritchie 1992b, Zobel 1992). A complete

review of operational programs worldwide will not

be attempted here. For a synopsis of international

regeneration activity with conifer rooted cuttings

the reader is referred to a recent article by Ritchie

(1992a). Emphasis is given here to: 1) benefits of

rooted cuttings which make them so attractive to

many organizations, 2) tree improvement and

propagation systems being employed, and 31

problems and limitations of rooted cutting

production. Two large-scale, vegetative propagation

programs in the United States are used as case

studies.

123
POTENTIAL BENEFITS OF VEGETATIVE

PROPAGATION

At this time all applied vegetative propagation

programs in forestry use rooted cuttings though

some promising research continues on

micropropagation systems, the most promising of

which is somatic embryogenesis. These programs

began for various reasons depending on the species,

final product and ease of propagation. Some of the

more commonly cited reasons and benefits follow.

Gains in Growth Rate

Most programs are aimed at the production of

clonal plantations or multiplication of high value

full- or half-sib families from seed orchards or

hybrids. Potential gains in yield vary greatly from

species to species but can be substantial when

compared to the best available seedlings. Most

conifer programs are aimed at bulking up seed of

seed orchard genotypes. Notable exceptions are sugi

in Japan and Norway spruce in southern Sweden

where emphasis is placed on the testing and

deployment of clones. By contrast, all hardwood

rooted cutting programs are aimed at the production

of clones. This is due to their ease of propagation

and absence of serious stock plant maturation

problems. Detailed systems of propagation and tree

improvement options are covered in a later section.

Maturation of stock plants can negate or reduce

potential genetic gains in growth. Cuttings from

mature stock plants often exhibit poorer rooting,

slower growth and morphological abnormalities such

as plagiotropism as compared with their

counterparts from juvenile stock plants or as

compared to seedlings of equal genetic value.

Improved Quality

Some rooted cutting programs have been justified

primarily on improved quality of the final product,

especially in radiata pine (Arnold 1985, Spencer

1987). Apparently, cuttings maintain some of the

mature qualities of older stock plants resulting in

fewer and smaller branches and straighter stems,

In some programs, expectations in growth rate gains

may be nominal and, in other cases, there is an

acceptance of reduced growth of rooted cuttings in

favor of their higher quality. The clonal eucalypt

program at ARACRUZ has made impressive volumeper-hectare gains of over 50% over nonimproved

seedlings, but testing for specific gravity and

pulping qualities is also a crucial part of the

program (Brandao 1984).

Speed of Propagation of High Value Genotypes

One of the most commonly cited reasons for the

use of rooted cuttings is very slow seed production in

orchards, especially in species such as Norway

spruce in northern Scandinavia and black spruce

(Picea mariana [Mill.] B.S.P.) in Canada (Armson

1980, Gill 1982, Johnsen 1985, Ritchie 1992a,

1992b). Small quantities of seed from the best openpollinated or full-sib families are multiplied to

commercial quantities. In many areas of the tropics,

especially near the equator, exotic conifers may

produce little to no seed (Gallegos 1983, Dvorak and

Lambeth 1992) and vegetative propagation may be

necessary, or at least a financially attractive

alternative to producing seed.

Reduced System Cost

Rooted cuttings of conifers typically cost two to

three times that of seedlings when one looks at only

the nursery phase. However, it is invalid to look at

only one phase of propagation. One option now

being explored is the possibility having only small

seed orchards produce small quantities of seed

which can be multiplied by rooted cuttings. The cost

of such a system may be less than operating a fullscale orchard and conventional nursery for seedling

production. Some of the economic gains from

planting rooted cuttings will be offset by higher

propagation costs using current rooting techniques,

but more inventive propagation systems and more

cost-effective rooting procedures will certainly

change this situation.

James River Corporation roots cuttings of poplars

directly in commercial plantations with no need for a

nursery, thereby achieving lower regeneration costs

than those possible with conventional seed orchards

and nurseries. ARACRUZ has found rooted cuttings

to be less expensive than seedlings of Eucalyptus

grandis (Hill, ex Maiden) and its hybrids because

the time cuttings spend in the nursery is less than

that for seedlings which reduces the necessary

nursery space (Dr. Bruce Zobel, Zobel Forestry

Associates, pers. comm.). The savings are

substantial because ARACRUZ plants year-round.

In the beginning of rooted cutting research and

early operations, the system savings were largely

overlooked but, in the future, more rooted cutting

programs will be started when the decision of

124
whether or not to establish a new orchard becomes

necessary.

Uniformity and Predictability of Improved Stock

In established programs which produce

monoclonal plantations of eucalypts and sugi, crop

uniformity is cited as an advantage (Brandao 1984,

Lambeth 1992, Ohba 1985). In the case of eucalypts,

the predominant uniformity advantage is uniform

wood which can be processed more efficiently and

which produces a more uniform pulp. In some

programs only about 20 tested clones, out of

thousands tested, can be used for regeneration;

while in seed orchard programs it is common to

regenerate with only a few families selected from a

few hundred tested. In the case of a clonal program,

it is much easier to find 20 clones of similar wood

qualities due to the high selection intensity than it

would be in the seed orchard program where

selection is lower and wood quality varies among

individuals within a family.

Hypothetically, uniform clonal plantations should

also have less inter-tree competition and, therefore,

less competition-induced mortality than seedling

populations. Detailed studies have not yet proven

this hypothesis, but one can observe more late

mortality in stands from seed than in clonal

plantations.

Seed orchard families, especially open-pollinated

families from the first generation, are highly

variable, and their superiority over unimproved seed

lots is often difficult to observe and may be

unpredictable under field conditions. As tree

improvement programs mature, geneticists will find

it more and more important to provide commercial

genotypes which are visually superior in a

commercial setting in order to justify continued

existence of decades-old programs. More uniform

genotypes will help make this possible.

Improved Survival?

Although there has been great concern about the

quality of rooted cutting root systems and their field

adaptability, most studies indicate that, when

overall rooting percent is acceptable, cuttings

survive as well or, in some cases, better than their

seedling counterparts (Lambeth et al. 1992, Ritchie

1992a, Struve and McKeand 1990). Lambeth et al.

(19921 observed statistically significant higher

mortality in seedlings than in rooted cuttings of

Eucalyptus grandis in the first year with continued

higher mortality in the seedlings in the second and

third years. Seedling check lots consistently ranked

low for survival in 16 clonal trials. By the third year

the difference was 11% (83% survival for seedlings

versus 94% for clones), a statistically and

operationally significant difference. Ritchie et al.

(1992c) found that Douglas-fir (Pseudotsuga

menziesii [Mirb.] France) rooted cuttings survived as

well as seedlings and transplants when matched for

genetics, stem caliper and root system quality.

International Forest Seed Company (Tom Caldwell,

pers. comm.) has also found that rooted cuttings of

loblolly pine (Pinus taeda L.) have higher survival

than seedlings of comparable genetic quality.

Cuttings of radiata pine have been found to exhibit

better survival than seedlings in a harsh

Mediterranean climate (Ritchie et al. 1992a).

All of the reasons for the better survival of

cuttings are not well understood. Douglas-fir rooted

cuttings tended to be more cold-hardy and had a

deeper dormancy than did seedlings in one study

(Ritchie et al. 19928. In some cases the cuttings

also had larger root collar diameter than seedlings

which may have improved their survival. Libby and

Rauter (1984) point out that seed from openpollinated seed lots from orchards may have a

greater number of dominant lethal alleles due to

natural self-pollination, whereas select trees which

are vegetatively propagated should have few to none

depending on the age of selection. Competitive

dominance may result in higher mortality in very

small trees found in seed-derived populations as

mentioned above. In some cases, rooted cuttings

may have greater root biomass than seedlings.

While rooted cuttings cannot always be counted

on to have better survival than seedlings, neither

should one assume that cuttings will have poorer

root systems or poorer adaptation to field conditions.

Other Advantages

The benefits mentioned above are the primary

ones cited by organizations involved in applied

vegetative propagation programs. Other more

subtle but important advantages, especially for

clonal forestry, are discussed in detail by Libby and

Rauter (1984). They include capturing gains due to
genotype-environment interaction (matching clones select tree, and rooting environment requirements.

to site), avoidance of pollen contamination, ease of Examples will be used to describe the options in

hybrid production, and ease of eliminating and Figures 1 and 2 where possible. In some cases, both

adding clones to hedge orchards versus seed rooting and nursery facilities may not be necessary

orchards. Another advantage is that vegetative but both are shown because it is the most common

propagation provides a method of producing system for vegetative propagation. “Propagation lab”

valuable species such as Alaskan yellow cedar indicates that a laboratory or other highly controlled

E 80

:

70

;

80

50

I 40

; 30

R 20

HT. DBH VOL/TR. SURV. VOL/HA. STRAIT. BRANCH. CROWN

TRAIT

-AQE 1 YEAR AGE 2 YEARS 0 AGE 3 YEARS

Figure 1. Average percentile ranking of seedling check lots versus clones of !&&yg&~Q!Xl&i in

18 trials in Colombia

(From Lambeth et al. 1992)

(Chamaecyparis nootkatensis D. Don.) which cannot

be propagated easily from seed (Ritchie et al. 1992a).

TREE IMPROVEMENT STRATEGIES AND

PROPAGATION SYSTEMS

As already discussed, there are a number of

reasons for using vegetative versus sexual

propagation. There are a number of strategies for

vegetatively multiplying a select tree or clone as

well. Figures 1 and 2 illustrate several of the

commonly used propagation systems in applied

programs as well as some systems that are at the

research stage. Some of these options (referred to as

“clonal options”) produce clones as the final

genotypes (Figure 1) while others (referred to as

“seed-vegetative propagation options”) simply

multiply desirable seed lots (Figure 2). The

appropriate system depends greatly on the ease of

rejuvenation (or preservation of juvenility) of a

and/or aseptic conditions may be required, especially

for tissue culture and embryogenesis.

Clonal Options

Clonal options are usually preferred because they

provide greater genetic gain and produce a uniform

crop for commercial use. These options entail the

vegetative multiplication of individual trees to

commercial quantities, and there are a number of

approaches being used or at least attempted.

Option I.--In this option, cuttings are taken

directly from trees and planted directly into a

commercial plantation. This is the approach of

James River Corporation in the floodplains of the

Colombia River in Oregon and Washington.

Cuttings are taken from second and third order

branches from the best clones in plantations (Table

1). This, the simplest and cheapest of options, works

when the species roots well under field conditions

126
HEIGHT (m) DBH (cm)

3.5

3

AGE - YEARS

m HT. - SEEDLINGS HT. - CLONES

0 DSH - SEEDLINQS DBH - CLONES

Figure 2. Mean heights and diameters of seedling check lots versus clones of Eucalvotus grand@

in 16 trials in Colombia

(From Lambeth et al. 1992)

and when tree branches are sufficiently “juvenile”

for good growth after rooting. Most species require

more elaborate nursery conditions for rooting or a

cutting orchard for rejuvenation or maintenance of

juvenility using sprouts from stumps or hedges.

Option 2.--In Japan, branches are collected from

“mature” trees and either rooted directly in the

nursery and, from there, go directly to a plantation,

or they are rooted for purposes of establishing a

cutting orchard from which commercial levels of

cuttings are collected. This option is more akin to

the system used for eucalypts with the exception

that the operation begins with branches from a

mature tree, an option which is unworkable in

eucalypts due to maturation.

Option 3.--This system is used throughout the

tropics for eucalypt species. After identifying the

select tree it is necessary to produce stump (or basal)

sprouts which are more “juvenile” than the branches

of a mature tree. Branches of large trees typically

do not root well and have mature morphological

characteristics shortly after rooting. Another

difference between this option and options 1 and 2 is

that it is usually necessary to have a special (high

humidity) facility for rooting as well as an outdoor

nursery area for acclimatization.

Option 4.--For most species, it is necessary to

somehow rejuvenate or maintain juvenility of a

select tree for ease of propagation and desirable

growth. It appears to be easier to maintain

juvenility than to rejuvenate. Many programs are

aimed at maintaining juvenility of a genotype which

has been cloned for purposes of testing under field

conditions.

One promising avenue now under intensive

research involves the clonal multiplication of

individuals through somatic embryogenesis. For a

genotype, some somatic embryos (or sprouted tissue)

can be placed in cryogenic storage while others are

used in the establishment of clonal trials. Once the

clone has been tested, the stored material is then

retrieved and again multiplied in a propagation lab

and further propagated in a rooting facility and/or

nursery setting. Commercially, the simplest option

would be to further multiply stored embryos to

commercial level, encapsulate them and sow them

directly into a nursery much as is done with seed.

However, a great deal more research is needed to

make artificial seed a viable option. Furthermore, it

remains to be seen whether laboratory cultures can

retain their juvenility indefinitely. Do they mature

with time, as do trees and hedges?

127
Option B--When juvenile material can not be

cryogenically stored it may be possible to maintain

clones in hedges while the clonal test is being

conducted. This is the approach used in clonal

programs of Norway spruce and was being used by

International Forest Seed Company for loblolly pine

(Foster and Shaw 1987). The difficulties in this

option are the cost of maintaining hedges for all

clones tested and the fact that it may not be possible

to maintain juvenility long enough. Hedges

typically mature to some extent when compared to

seed-derived plants (Bolstad and Libby 1982). In

some cases, a certain degree of maturation can be

tolerated, i.e., the effects of maturation are offset by

the high genetic value of the clones produced.

Options 6 and 7.--For several years, it was

thought that propagules from callus tissue or

embryogenesis of mature tissue from a select tree

would result in rejuvenation because they would be

formed from undifferentiated tissue. However,

embryogenesis of mature tissue has proven to be

quite difficult and tissue culture plants from mature

tissue tend to exhibit mature characteristics

(Greenwood et al. 1991). Although this was the most

common approach in the beginning of this area of

research, it is receiving less and less emphasis.

Seed - Vegetative Propagation Options.--When

rejuvenation or maintenance of sufficient juvenility

is not possible, then other options must be used;

however they typically serve only for bulking up

high value seed. These options begin with seed or

scions from a select tree but depend on the

production of seed somewhere in the propagation

system. For purposes of demonstration,

conventional systems which produce seedlings as

well as vegetative propagation options will be

shown. The objective is to show that these

vegetative propagation systems have competing

technologies of similar genetic gain which rely on

seed production for commercial plantings.

Option &--Seed can be taken directly from a

select tree in a forest or genetic test. Such seed may

not be found in commercial quantity, in which case

it may be desirable to multiply it vegetatively. An

example is the approach being used in Colombia for

both Pinus tecunumanii (Schwerd.) and P.

maximinoi (H.E. Moore). These are two highly

promising species which are only beginning to be

used for commercial plantations, The price of

unimproved P. tecunumanii seed from Central

America placed in Colombia is about $5OO/kilo and,

as yet, good seed production sites have not been

found in Colombia. As stated earlier, some tropical

pines produce little to no seed in areas near the

equator. Seed from select mother trees in Central

America have been tested in Colombia and Smurfit

Carton de Colombia is returning to the original

select mother trees and collecting seed which is

being multiplied by creating hedges and rooting

cuttings. This same approach is even more critical

for reducing foxtailing in P. maximinoi. This species

normally foxtails at very high percentages but

studies have shown that the progeny of some mother

trees produce very low percentages of foxtailing

while others foxtail at 100% (Urrego and Lambeth

1989).

Option 9.--The seed alternative to option 8 is to

collect directly from the select tree for commercial

purposes. Examples would be seed production areas

(where plantations or natural stands are thinned to

leave the best trees) or seedling seed orchards

(where genetic trials are genetically thinned).

Option IO.--This is the clonal seed orchard

favored by many organizations around the world.

Open-pollinated, supplementally-pollinated, controlpollinated and hybrid seed can be produced in an

orchard setting. Considerable labor may be required

for making controlled or supplementally pollinated

seed, but it is still an option that is favored in many

regions of the world.

Option Il.--In this option, high value (or scarce)

seed from the clonal seed orchard is used to establish

a cutting orchard (usually hedges) from which

sprouts are used for rooted cutting production on a

commercial scale. This approach is being used by a

number of organizations including the Douglas-fir

program at Weyerhaeuser Company (Table 2).

Hedges are retained only a short period of time in

order to assure the production of juvenile cuttings.

This approach is typically used when plants from

hedges which are maintained for too many years,

lose desirable characteristics such as rootability or

growth rate. This approach is also used when there

is insufficient evidence to determine maturation

effects in long-term hedges.

Option 12.~-Again, there is hope that embryos

from somatic embryogenesis may be encapsulated

and their sowing mechanized in such a way that

option 12 may be cheaper than option 11. Somatic

embryogenesis in this system would be easier than it

128
would be in options 6 and 7 since it would be

conducted on seed embryos.

COMMERCIAL SCALE APPLICATIONS IN THE

UNITED STATES

There are two commercial scale vegetative

propagation programs which offer contrasting

species (hardwood versus conifer) and different

approaches to vegetative propagation (clonal versus

bulking up of seed) and age of the programs (32

versus 5 years).

James River Corporation

James River Corporation (Table 1) has planted

poplar cuttings in the South (the approach is similar

to option 3 in Figure 1) since 1960 and in the

Northwest (a system similar to option 1 in Table 1)

since 1982. The program is justified primarily by

the fact that planting of cuttings directly in the field

is considerably cheaper than production of seedlings

in a nursery for commercial plantings, and by the

substantial gains that can be made in the

commercial deployment of superior clonal

plantations. Program features can be seen in Table

I.

Tree Improvement Strategies.--The basic tree

improvement strategy in the Northwest is

interspecific hybridization (eastern X black

cottonwood1 followed by combined family and within

family selection. The same strategy is used in the

South though species hybridization is replaced with

intraspecific matings of eastern cottonwood (Populus

deltoides Bartr. ex Marsh.). Selected eastern

cottonwood genotypes serve in the breeding

programs of both regions, having been chosen on the

basis of performance in clonal trials in the South.

Currently, parental selection of black cottonwood

(Populus trichocarpa Torr. and Gray) takes place in

natural stands. Future breeding stock will come

from a 1993 trial of 1,700 black cottonwood clones.

Implicit in this strategy is the assumption that

clonal values are suitable indicators of breeding

values in both inter- and intraspecific matings.

Currently there are 58 and 102 full-sib families of

eastern cottonwood and hybrid cottonwood,

respectively, in various stages of clonal evaluation.

Factorial mating designs with parental

assortative pairings are used in both regions.

Progeny are evaluated for one year in a nursery

after which time approximately 50% are massselected for clonal screening. Initial selections are

made at age two in the Northwest and at age four in

the South. Clonal multiplication of these selections

commences with greenhouse propagation of 1,000

rooted cuttings per clone. The multiplication

operation continues another three years through

stoolbed culture in the South and through field

plantings in the Northwest. At this point, a

rotation-age evaluation of the screening trial is

conducted to either confirm or refute the superiority

of the earlier selections. Replicated monoclonal plots

are also established to provide realistic growth and

yield data.

In the long term, the southern improvement

program will focus on recurrent breeding. Long

term breeding in the Northwest will not focus on

advanced generations as there does not appear to be

much promise in the F2 hybrid generation.

Consequently, a reciprocal recurrent selection

program designed to improve both parental species

for hybridization is being developed.

On the operational side, both programs will likely

increase their acreage under cultivation in future

years. With continued clonal development, it is

anticipated that yields will increase or rotations will

shorten. Of equal concern is the desire to broaden

the genetic diversity of the operational clone pools,

especially in the Northwest where other hybrids

may be pursued.

Coppicing in second rotation stands is the only

competing technology to the development of new

clones. While refined in the South, questions

remain about effective ways to use coppice in the

Northwest.

Weyerhaeuser Company

Main program features can be found in Table 2.

The program began in 1987 with the objective of

bulking up elite full-sib seed of Douglas-fir (option

11). Even if control crossing were possible, orchard

capacity would be sufficient to produce only eight

percent of regeneration demands. Vegetative

propagation easily moves this figure up to 100%.

Only one-year-old stock plants are used for

cutting production in order to avoid negative effects

on growth and rooting due to maturation. Growth of

stock plants is accelerated. By the end of the year,

stock plants produce 50 cuttings which are rooted in

129
the greenhouse and then transplanted to a

conventional bareroot nursery (essentially a 1 + 1).

The cost of cuttings is two to three times higher than

that of seedlings. The target is 1.5 times that of

seedlings.

Rooted cuttings are now being planted on the

highest site land near the mill in order to get the

highest financial return for high value families. In

addition, they are not planted on sites which have a

low probability of remaining in company ownership

through rotation such as suburban growth areas and

scenic corridors.

Tree Improvement Strategies.--Select full-sib

families for bulking up are identified from about

4,000 parents across six breeding zones in 8 to 12-

year-old first generation tests. These selections

come either from tested crosses or from elite parents

which have not been tested as specific crosses.

Second generation seed will be available for testing

within one or two years. It is anticipated that the

best second generation families will be bulked up

with cuttings in a similar manner.

There is a clearly articulated strategy and rules for

allocation of families to sites, maximum contiguous

acres of a family, maximum percentage contribution

of a single family to any year’s total regeneration

and the number of families to be planted.

The program is expected to grow considerably in

the next few years if cost reduction efforts continue

to be successful and if field tests continue to

demonstrate high gain potential. Ritchie (199213)

provides more detail about the Weyerhaeuser

program.

PRINCIPAL PROBLEMS IN APPLIED

VEGETATIVE PROPAGATION PROGRAMS

Maturation of Stock Plants

Perhaps the most commonly cited problem which

limits the development of applied rooted cutting

programs is reduced growth and rooting potential

resulting from maturation of stock plants (Armson et

al. 1980, Arnold and Gleed 1985, Bolstad and Libby

1982, Gill 1982, Greenwood et al. 1991, Johnsen

1985, Libby 1974, Rauter 1983, Shelbourne 1987,

Spencer 1987, Struve and McKeand 1990).

Nonetheless, some are exploiting the mature

characteristics (Armson et al. 1980, Arnold and

Gleed 1985, Bolstad and Libby 1982, Gill 1982,

Libby 1974, Libby and Rauter 1984, Spencer 19871,

especially those programs aimed at high quality,

solid wood products. However, many programs

either do not rely on solid wood products or do not

find that the improved quality of more mature

cuttings is sufficient to offset the losses in yield or

increased nursery costs due to poorer rooting.

Maturation is a problem in many species, perhaps

even in stump sprouts of eucalypts (Lambeth et al.

1992) which are commonly rooted on a commercial

scale and are thought to be without maturation

problems.

One difficulty that tree breeders may have in the

assessment of rooted cuttings is evaluation time.

Clones from plus trees of Eucalyptus grandis had

more mature characteristics than did nonselect

seedlings with less branching, larger leaves and

slower growth in the first year (Lambeth et al.

1992). By the second year (l/3 rotation) the clones

had surpassed seedlings in growth and their

superiority increased through the third year (l/2

rotation) (Figures 3 and 4). This delayed expression

of superiority of clones over nonselect seed lots has

also been observed in clonal programs in Brazil (Dr.

Bruce Zobel, pers. comm.) and South Africa (Dr.

Neville Denison, pers. comm.). It is suspected that

the clones are more mature than seedlings and,

therefore, have slower relative growth rate in the

early establishment year(s). However, as seedlings

pass out of the exponential growth phase associated

with juvenile stock, the clones begin to express their

true genetic superiority in the mature phase of

growth.

It will become more important to understand the

growth curve of rooted cuttings (especially those

from older hedges) and their state of maturation

versus seedlings as operational programs develop.

This will be especially important for long-rotation

conifers. The evaluation period for comparison

seedlings versus cuttings may depend on a

difference in shape of the growth curve as has been

observed in eucalypts. In the compressed time

frame for eucalypts, clones surpassed seedling

checks between l/3 and l/2 rotation age. If the same

occurs in conifers then many trials may be too young

to evaluate the difference between cuttings versus

seedlings for the long term. On the other hand, if

cuttings are considerably older physiologically than

seedling counterparts, they may never catch up in

growth rate.

130
cost

In most conifer programs, cost of cuttings is still

considerably higher than that of seedlings primarily

because of the special conditions needed for rooting

and the labor-intensive nature of rooted cuttings

(Arnold and Gleed 1985, Gill 1982, Greenwood et al.,

Ritchie 199213). Three things will make the costs of

vegetative propagation systems more favorable: 1)

Mechanization of harvest and sticking of cuttings or

transplanting of rooted cuttings will lower cost

considerably. Mechanization has not received much

emphasis in the past because of the uncertainty of

the applicability of rooted cutting systems. 2)

Simpler rooting systems will be developed and costs

of facilities will drop. 3) System costs will be

examined more closely and, in many cases, it will be

concluded that a rooted cutting system will be

competitive with seedlings, especially when larger

genetic gains are possible with cuttings. In some

programs, seed orchards will be obsolete or at least

much smaller orchards will be established with the

sole objective of feeding rooted cutting programs.

The system advantage of rooted cuttings will not

only be in the form of cost savings, but also through

faster implementation of genetic gains (Arnold 1985,

Gill 1982, Matheson and Lindgren 1985, Libby 1974,

Libby and Rauter 1974, Rauter 1983, Ritchie 1992b).

Environmental Restrictions

Given the current worldwide concern about

biodiversity and monocultures, many researchers

and plant propagators are concerned that vegetative

propagules may be regulated at some future date to

the point that full potential gains may not be

exploited. Furthermore, it is possible that

investments in biotechnology and vegetative

propagation may be lost if the planting of genetically

engineered genotypes or clones is too restrictive.

Such concerns may discourage the development of

vegetative propagation programs. For example, the

Swedish government places restrictions on how

clones should be tested and specifies the number of

clones that must be planted in mixtures (Anon.

1982). In Sweden, the potential benefits of uniform

clonal plantings can not be realized because

monoclonal plantings are illegal.

It would behoove forest geneticists to begin

studies on the genetic variation and risks of

different deployment strategies of not only clones

but seed orchard families as well whether they be

open-pollinated or full-sib families. For example, it

has been suggested that genetic diversity of clonal

mixtures may be greater than that of openpollinated families from seed orchards when

appropriate mixtures are used (Libby and Rauter

1984). What about a mosaic of monoclonal plantings

versus clonal mixtures or open-pollinated family

blocks versus plantations of unimproved seed? From

a legal standpoint, what are the private landowner’s

rights in deciding which genotypes to plant and in

deciding how to deploy them? These issues must be

dealt with quickly in order to avoid problems that

may arise when significant quantities of plant

material emerge from biotechnology and vegetative

propagation programs.

Competing Technologies

Those vegetative propagation options which bulk

up high value seed lots such as full-sib families

(Figure 2) must compete with seed orchard options

which, perhaps, have not been investigated

thoroughly enough. These include supplemental

mass pollination, control crossing, two-clone

orchards and relocation of orchards to other areas of

faster and more abundant production of cones,

higher seed set, no pollen contamination and low

labor costs for making crosses. Slash pine (Pinus

elliottii Engelm.) is a good example of the latter. In

its natural range, slash pine produces commercial

quantities of seed in orchards at 10 years of age or

later and suffers from considerable pollen

contamination. In Zimbabwe or southern Brazil,

seed production occurs at five years of age, is more

abundant than in the U. S., areas of low pollen

contamination can be found, and labor costs for

making controlled crosses is lower than in the U.S..

Scion export for seed orchard establishment overseas

and seed import costs would be nominal but import

and quarantine restrictions would have to be studied

very carefully.

Tree breeders must take some risk in deciding

whether or not to put research into vegetative

propagation or seek alternative seed production

systems for the production of full-sib families. This

decision is difficult when there is insufficient

research in either area. Furthermore, somatic

embryogenesis is a competing technology for rooted

cuttings and seed orchard crosses in the production

of full-sib families since all three techniques would

produce the same level of genetic gain, assuming one

system has no more maturation problem than the

others.

131
CONCLUSIONS

Worldwide commercial applications of vegetative

propagation are increasing at a dramatic rate.

Exploited advantages are increased growth rate

from high value genotypes in the form of clones or

bulked up full-sib families, increased crop uniformity

and predictability of wood quality and other

characteristics, greater quality of final products

associated with more mature characters such as

reduced branching and knot size, faster

multiplication of high value families due to slow seed

orchard production, and reduced system cost by

elimination or downsizing of seed orchards. There

are several genetic strategies and propagation

systems in use. The appropriate strategy depends

on the ease of propagation and rejuvenation (or

maintenance of juvenility) and potential genetic

gains. Some produce clones for commercial planting

while others simply multiply high value seed lots.

The cost of the latter must be competitive with

alternative breeding technologies such as

supplemental pollination, two-clone orchards or

control crossing on a commercial scale.

Primary limitations in the implementation of

vegetative propagation and reforestation are cost

due to the labor-intensive nature of rooted cuttings

or tissue culture, maturation of stock plants (usually

hedges) which reduces rooting percent and growth

rate in the field, and the fear of environmental

restriction on the use of new materials from

expensive research programs. Some of these

limitations are being overcome by research into cost

reduction methods through mechanization and

studies of methods to maintain juvenility (or

rejuvenation) of select trees or clones.

The United States lags behind other countries

such as Brazil, The Congo, Japan, South Africa and

several western European countries in the level of

use of vegetative propagation on a commercial scale.

Two notable exceptions are the programs at James

River Corporation, which plants clonal plantations

of poplars on floodplains of the Mississippi and

Columbia rivers, and Weyerhaeuser Company,

which has a rapidly growing program of vegetative

multiplication of high value full-sib families for

regeneration in Oregon and Washington.

LITERATURE CITED

Anon. 1982. Regulations on clonal forestry with

Picea dies. In: Statute book SKSFS 1982: Swedish

National Board of Forestry. [Number of pages

unknown].

Armson, K.A.; Fung, M.; Bunting, W.R. 1980.

Operational rooting of black spruce cuttings. Journal

of Forestry. 78: 341-343.

Arnold, R.; Gleed, J.A. 1985. Raising and managing

radiata pine cuttings for production forests.

Australian Forestry. 48: 199-206.

Bentzer, B. 1981. Large scale propagation of Norway

spruce (Picea dies) by cuttings. In: Symposium on

clonal forestry. Res. Note 32. Uppsala, Sweden:

Swedish University of Agricultural Science,

Department of Forest Genetics: 33-42.

Bolstad, P.V.; Libby, W.J. 1982. Comparisons of

radiata pine cuttings of hedge and tree-form origin

after seven growing seasons. Silvae Genetica. 31(l):

9-13.

Brandao, L.G. 1984. The new eucalypt forest:

lectures given by the 1984 Marcus Wallenberg prize

winners at the symposium in Falun, Sweden on

September 14, 1984. In: Proceedings of the 1st

Marcus Wallenberg symposium; 1984 September 14;

Falun, Sweden: Marcus Wallenberg Foundation: 3-

15.

Delwaulle, J.C. 1985. Clonal plantations of hybrid

Eucalyptus in the Congo. Bois et For&s des

Tropiques. 208(2): 37-42.

Denison, N.P. 1989. Practical problems encountered

in developing an applied programme for eucalypt

species. In: Breeding tropical trees: Proceedings of

IUFRO (International Union of Forest Research

Organizations) conference; 1988 November 28 -

December 3; Pattaya, Thailand. Oxford, United

Kingdom: Oxford Forestry Institute: 435436.

Dvorak, W.S.; Lambeth, C.C. 1992. Results of a

survey to determine the cone and seed production of

Pinus tecunumanii in the tropics and subtropics. In:

Breeding tropical trees: Proceedings of IUFRO

(International Union of Forest Research

Organizations) conference; Cali, Colombia.

[Publisher and number of pages unknown]

132
Foster, G.S. 1986. Making clonal forestry pay:

breeding and selection for extreme genotypes.In:

Breeding theory, testing and seed orchards:

Proceedings of the joint meeting of IUFRO

(International Union of Forest Research

Organizations) working parties conference; 1986

October 13-17; Williamsburg, VA. Raleigh, NC:

North Carolina State University-Industry Tree

Improvement Program: 582590.

Foster, G.S.; Shaw, D.V. 1987. A tree improvement

program to develop clones of loblolly pine for

reforestation. In: Proceedings of the 19th southern

forest tree improvement conference; 1987 June 16-

18; College Station, TX. Springfield, VA:

Department of Commerce, National Information

Service: 17-21.

Gallegos, CM. 1983. Flowering and seed production

of Pinus caribaea var. hondurensis (results of a

worldwide survey)--fast growing trees. Silvicultura.

29: 84-87.

Gill, J.G.S. 1982. Comparisons of production costs

and genetic benefits of transplants and rooted

cuttings of Picea sitchensis. Forestry. 56: 61-73.

Greenwood, S.G.; Foster, G.S.; Amerson, H.V. 1991.

Vegetative propagation of southern pines. In: Forest

regeneration manual. Dordrecht, the Netherlands:

Kluwer Academic Publishers: 76-86. Chapter 5.

Johnsen, 0. 1985. Successive bulk propagation of

juvenile plants from full-sib families of Norway

spruce. Forest Ecology and Management. 11: 27 l282.

Lambeth, C.C.; Endo, M.; Wright, J. 1992. [In press].

Genetic analysis of 16 clonal trials of Eucalyptus

grandis and comparisons with seedling check lots.

Forest Science.

Libby, W.J. 1974. The use of vegetative propagules

in forest genetics and tree improvement. New

Zealand Journal of Forestry Science. 4(2): 440-447.

Libby, W.J.; Rauter, R.M. 1984. Advantages of clonal

forestry. Forestry Chronicle. 4: 145-149.

Kleinschmidt, J. 1974. A programme for large-scale

cutting propagation of Norway spruce. New Zealand

Journal of Forest Science. 4: 359-366.

Matheson, A.C.; Lindgren, D. 1985. Gains from

clonal and the clonal seed-orchard options compared

for tree breeding programs. Theoretical and Applied

Genetics. 71: 242-249.

Ohba, K. 1985. Cryptomeria in Japan. In: Zsuffa,L.;

Rauter, R.M.; Yeatman, C.W., eds. Clonal forestry:

its impact on tree improvement and our future

forests: Proceedings of the 19th meeting of the

Canadian Tree Improvement Association--part 2;

1983 August 22-26; Toronto, Canada. Chalk River,

Ontario: Canadian Forestry Service: 145-162.

Rauter, R.M. 1985. Current status of

macropropagation. In: Zsuffa, L.; Rauter, R.M.;

Yeatman, C.W., eds. Clonal forestry: its impact on

tree improvement and our future forests:

Proceedings of the 19th meeting of the Canadian

Tree Improvement Association--part 2; 1983 August

22-26; Toronto, Canada. Chalk River, Ontario:

Canadian Forestry Service: 58-74.

Ritchie, G.A. 1992a. The commercial use of conifer

rooted cuttings in forestry: a world overview. New

Forests. 5: 247-275.

Ritchie, G.A. 1992b. [In press]. Commercial

production of Douglas-fir rooted cuttings at

Weyerhaeuser Company. Proceedings of IUFRO

(International Union of Forest Research

Organizations) conference on vegetative

propagation; Bordeaux, France.

Ritchie, G.A.; Tanaka, Y.; Meade, R.; Duke, S.D.

1992c. Establishment and early field performance of

Douglas-fir rooted cuttings compared to seedlings

and transplants. Tech. Rep. 050-3423/15.

Weyerhaeuser Company. 18 p.

Ritchie, G.A.; Tanaka, Y.; Duke, S.D. 1992d.

Physiology and morphology of Douglas-fir cuttings

compared to seedlings and transplants. Tree

Physiology. 10: 179-194.

Shelbourne, C.J.A.; Thulin, I.J. 1974. Early results

from a clonal selection and testing programme with

radiata pine. New Zealand Journal of Forestry

Science. 4(2): 387-398.

Spencer, D.J. 1987. Increased yields of high quality

veneer and sawn timber from cuttings of radiata

pine. Australian Forests. 50(2): 112-117.

133
Struve, D.K.; McKeand, SE. 1990. Growth and

development of eastern white pine rooted cuttings

compared with seedlings through 8 years of age.

Canadian Journal of Forest Research. 20: 365-368.

Urrego, J.B.; Lambeth, C.C. 1989. Provenances and

family variation in foxtailing of Pinus maxciminoi

H.E. Moore in the Andean mountains of Colombia.

In: Breeding tropical trees: Proceedings of IUFRO

(International Union of Forest Research

Organizations) conference; 1989 November 18 -

December 3: Pattaya, Thailand: IUFRO Working

Parties: 503 p.

Zobel, B. 1992. Vegetative propagation in production

forestry. Journal of Forestry. 90(4): 29-33.

134
Table l.--Features of a James River Corporation rooting cutting program

SPECIES: South - Eastern Cottonwood (Populus deltoides)

Northwest - Black Cottonwood (Populus trichocarpa)

X Eastern Cottonwood Hybrid

REGION: Mississippi (Mississippi and Louisiana) and Colombia (Oregon and Washington)

Rivers Floodplains

OBJECTIVE:

REASON:

Produce Fast-Growing Clonal Plantations

Best Clones From Full-Sib Families Exceed the Family Mean by 10% in

Height and 20% in DBH

REGENERATION ACTIWN:

SOUTH

- Since 1960 - 7,000 ha Planted

- Clones 67% of 690 ha Annual Planting

NORTHWEST

- Since 1982 - 4,500 ha Total

- Clones 100% of 400 ha Annual Planting

FINAL PRODUCT:

SOUTH

- High Quality Coated papers

NORTHWEST

- Groundwood Communications Grade Paper

ROTATION:

SOUTH

- 10 Years

NORTHWEST

- 6Years

PROPAGATION SYSTEM:

SOUTH

- Cuttings from l-year-old Whips in Stool Beds

- 4-Year Rotation in Stool Beds

- Cuttings 20-50 cm Long and .6-2.5 cm Diameter

NORTHWEST

- Cuttings from 1st and 2nd Order Branches in 2-year-old Commercial Stands

- Cuttings 30-35 cm Long and .6-2.0 cm Diameter

BOTH REGIONS

- Rooting Directly in the Field (96100% Success)

LIMITATIONS AND CONCERNS: Genetic Diversity in Breeding Programs

FUTURE ACTIVITY:

SOUTH

- Recurrent Breeding and Testing

NORTHWEST

- Reciprocal Recurrent Selection for Good Hybrid Performance

BOTH REGIONS:

- Emphasis on Better Clones from Breeding Programs

- Increased Clonal Planting

135
Table 2.--Features of a Weyerhaeuser Company rooted cutting program.

SPECIES: Coastal Douglas-Fir (Pseudotsugamentiesii)

REGION:

SITE TYPES:

ORJECTIVJZ:

Western Washington and Oregon

High Site Index, Low Elevation

Multiply Limited Quantity of High Value Orchard Seed such as Full-Sib Families

REASON: 11525% Gains in Site Index

REGENERATION ACTIVITY:

1987 - First Crop

1991- 36 ha

1992 - 40 ha

1993 - 700 ha Planned

1994 - 800 ha Planned

Of 10,000 ha Total Annual Regeneration

FJNAL PRODUCT:

ROTATION:

High Value Solid-Wood Products, Pulp

40-60 Years

PROPAGATION SYSTEM:

- l-Year-Old Potted Stock Plants in a Greenhouse

- Winter Dormant Cuttings - approx. 50 per Stock Plant

- Greenhouse Rooting

- Transplant to a Bareroot Nursery in the Fall

- One Additional Year in the Nursery

GUIDELINES:

- Limited Contiguous Area of a Single Full-Sib Family

- Limited % of Area of Annual Regeneration to a Single Full-Sib Family

LIMJ!I’ATIONS AND CONCERNS:

- Cost (Target 1.5 x Seedlings)

- Future Environmental Restrictions on Deployment of Select Material

FUTURE ACTJS’ITYz

- Increased % Regeneration with Rooted Cuttings

- Perhaps Smaller Seed Orchards Which Will Produce Control Cross Seed for Vegetative Propagation

136
RESEARCH APPLICATIONS WITH VEGETATIVE PROPAGULESl

Darroll D. Skilling, Kenneth F. Raffa, Daniel J. Robison, and Paul Berrang2

Abstract.--Vegetative propagules have recently received more attention

in forest tree improvement due to more efficient techniques for plant

multiplication including tissue culture. Vegetative propagules are being used in

research on air pollution, entomology, and pathology to develop more sensitive

tests for screening putative resistant materials. These techniques offer both

speed, and the ability to produce large numbers of new genotypes.

INTRODUCTION

The use of vegetative propagules probably dates

back prior to written history when the first

humanoid poked a willow branch in a sandbar and

came back later to see a young tree growing in place.

So what we are discussing today is not exactly the

cutting edge of science. What has changed,

however, is the ability to take ancient techniques

and modify them to provide us with tools that can be

used to help us in today’s tree improvement work.

In this paper we discuss the value of vegetation

propagules in research. We describe some systems

that have important research applications.

Although these research techniques may help us

develop trees with genetically improved traits, they

are just another approach to tree improvement.

Information from these techniques will supplement

lPaper presented at the 1992 Southern Regional

Information Exchange Group Biennial Symposium,

Huntsville, AL, July 8-10, 1992.

2Chief Plant Pathologist, USDA Forest Service,

North Central Forest Experiment Station, St. Paul,

MN 55 108; Professor of Forest Entomology,

Department of Entomology and Forestry, University

of Wisconsin-Madison, Madison, WI 53711; Research

Assistant, Department of Entomology and Forestry,

University of Wisconsin-Madison, Madison, WI

53711; Center Manager, USDA Forest Service,

Center for Forest Environmental Studies, Dry

Branch, GA 3 1020.

more classical breeding programs, but they will not

replace them.

The plant material developed in the research

laboratory must still be subjected to field testing and

may become stock material for future tree breeding.

Having made this qualifier, let’s look at some of

these techniques and see how vegetative propagules

can be used to study interactions between insects,

air pollution, and diseases as stress components in

forest trees.

In research, we try to hold variables constant

except for the independent variable we are studying.

With forest trees, this can often be difficult as each

individual tree is a variable and in most cases is

somewhat different genetically from every other

tree. To study uniform populations we devise

systems to combine members of various families so

that each treatment has approximately the same

representative genotypes present. Through

statistical methods it is then possible to separate out

the variation due to treatment from that variation

due to genetic variability in the plant material. A

more sensitive test, however, is to deal with

identical genotypes in each treatment. This can be

achieved by the use of vegetative propagation

systems in research studies.

In forest pathology we have a system called the

“disease triangle”. In this triangle the development

of a forest disease is the result of three variables.

These are the genetics of the host, the genetics of the

pathogen, and the variation within the environment.

A change in any one or more of these three parts of

137
the triangle may have a major impact on disease

development.

If we want to study the genetics of the fungus we

must hold the genetics of the host and the

environmental factors constant and vary only the

races of the pathogen. If we wish to study the

genetics of the host, in this case a tree, we maintain

the genetics of the pathogen constant as well as the

environmental factors and vary only the genetics of

the tree. This is why pathologists working with

identical vegetative propagules in tissue culture can

detect smaller differences in genetic resistance than

would be possible in field studies. How important

these small differences are depends on the purpose

of the study.

The idea of working with a single tree genotype

under controlled environmental conditions is of

course not limited to forest disease research. In this

paper, we will discuss insects, air pollution, and

diseases, but the general principal will remain the

same.

APPLICATIONS TO AIR POLLUTION RESEARCH

General Comments

Traditionally, air pollution research has been

done by plant pathologists and, if one accepts some

pathologists’ definition of disease, air pollutants are

just a type of disease. In air pollution, however, the

disease triangle is severely truncated because

pathogen genetics is not a factor. Within the forest

air pollution field, the triangle is often even more

reduced because most researchers have only a

passing interest in the genetics of the host. The

reasons air pollution researchers would use

vegetative propagules are similar to the reasons

most forest pathologists would use them. First, the

use of genetically identical plants makes it possible

to obtain a more uniform plant response to specific

types of air pollution stress. Second, the ability to

repeatedly replicate a specific genotype means that

vegetative propagules can be used to increase the

continuity between experiments or over space and

time. Third, the ability to produce multiple copies of

individuals makes it possible to investigate aspects

of genetic variation that are not available with plant

material grown from seed. In many cases,

researchers obtain several of these benefits

simultaneously.

138

Air pollution researchers in agronomy and

horticulture have made extensive use of inbred lines

and hybrid varieties in their work. They have made

tremendous advances in their research due, in part,

to their ability to quickly and economically produce

large numbers of genetically similar plants.

Research on ozone injury on tobacco is just one

example of their successes. Starting in the early

1950’s tobacco growers throughout eastern United

States began reporting crop injury that seemed to be

related to the weather. It was not until the late

1950s that ozone was recognized as the cause of this

injury. By 1960 tobacco growers were starting to

plant resistant varieties (Heggestad 1966) and today

ozone injury is rare on this crop. Breeders of a wide

variety of crops continually make improvements in

the air pollution tolerance of a wide variety of

species. Inbred lines and hybrid varieties like those

used in agronomy and horticulture are practically

non-existent in forestry, but clones produced

through vegetative propagation have the potential to

provide forest air pollution researchers with many of

the same benefits. However, to date, there have been

very few applications of this technology to forest air

pollution research.

When forestry research lags behind agricultural

or horticultural research, it is often due to the fact

that foresters enter the field at a later date. That is

not the case at least with ozone. Researchers

working with trees were some of the first people to

study the effects of ozone on plants.

Examples of Work

Verification of Injury Observed in the Field.--

Some of the earliest ozone work done with forest

trees involved the use of vegetative propagation. In

the late 1950’s the USDA Forest Service began to

investigate a decline of eastern white pine in the

southern Appalachians that was called post

emergence chronic tipburn. There was a great deal

of apparent variation in susceptibility to this disease

with some individuals showing extensive damage

while others showed no damage. Early work

indicated that the disease was not associated with

pathogenic fungi (Berry and Hepting 1964) or

nutrition (Dochinger 1964). Reciprocal grafting

experiments established that the disease was not

graft-transmissible and that differences in

susceptibility between seedlings seemed to be

genetically controlled (Berry and Hepting 1964). In

the early 196Os, Berry and Ripperton (1963)

established ozone as the probable cause of this
disease by producing clones of susceptible

individuals and showing that ramets maintained in

ozone-free air did not develop the disease while

plants fumigated with ozone did develop the disease.

One might expect that the success of these early

researchers might encourage later forest air

pollution workers to make frequent use of vegetative

propagation to minimize genetic variation in

susceptibility. However, this has not been the case.

Traditionally, foresters have used genetically diverse

groups of trees propagated from seed to reforest

areas, and most forest air pollution workers have

chosen to work with tree seedlings.

Develop Better Bioindicators of Ozone.--

Pioneering ozone researchers developed clones of

plants that were useful as indicators of the presence

of phytotoxic levels of ozone (Berry 1973). Interest

in this work declined as accurate mechanical

monitors became more widespread. Interest in this

technique has been revived recently because it is

viewed as a way of monitoring the effects of ozone

rather than its mere presence.

The first bioindicators consisted of a single clone

or inbred line which was selected for sensitivity to

the pollutant of interest. Many factors other than

ozone can affect the response of these plants to ozone

including soil moisture, nutrient availability, and

temperature. Heagle (1991) has developed a pair of

white clover (Trifolium repens L.) clones, one

sensitive to ozone and one resistant, for use as

bioindicators. When the growth of just one of these

clover clones is compared to ambient ozone

concentration, the correlation is, at best, weak.

However, these two clones were selected from the

same clover cultivar and presumably react similarly

in response to most other environmental factors

other than ozone. When the relative growth rates of

these two clones were compared in ambient ozone

there was a strong correlation (Heagle, USDA

Agricultural Research Service, Raleigh, NC,

personal communication).

The USDA Forest Service has the responsibility

of preventing air pollutants from affecting resources

in Class 1 Wilderness Areas. The agency is also

prohibited from installing conventional mechanical

monitors in these areas by wilderness legislation,

One option being used is collecting data on

symptoms of injury on sensitive plants in the field.

Field surveys indicate that phytotoxic levels of ozone

exist in many wilderness areas, but natural

variation in ozone tolerance within species limits the

conclusions that can be drawn from data collected in

natural populations. The Center for Forest

Environmental Studies plans to develop paired

clones of native bioindicators that would function

much like the clover clones already described.

Study Patterns of Genetic Variation.--Relatively

little work has been done with the genetics of air

pollution tolerance in trees, but there tends to be a

substantial amount of variation for these traits

within species of forest trees. For example,

Karnosky and Steiner (1981) reported that some

green ash Wraxinus pennsylvanica Marsh.)

provenances showed 10 times as much sulfur dioxide

injury and 60 times as much ozone injury as other

provenances of the same species. They also found

significant differences among families within

provenances, but like most of the researchers who

have studied genetic variation in air pollution

tolerance, they were unable to evaluate differences

among individuals within families because they

were working with seedlings.

In the few cases where researchers have

vegetatively propagated individuals within clones

they have found that differences among individuals

can be a major component of variation. While

studying the effect of sulfur dioxide on height

growth of Norway spruce (Picea abies CL.1 Karst.),

Scholze and Venne (1989) found that in clean air,

differences among provenances accounted for most

(54-63%) of the genetic variation, but that in the

presence of the pollutant differences among clones

within families accounted for most (30-32%) of the

variation. Variation due to differences among

families was relatively small regardless of sulfur

dioxide level. Berrang and Karnosky (1986, 1991)

did not evaluate differences among families within

provenances, but they did find that differences in

ozone tolerance among clones within provenances of

quaking aspen (Populus tremuloides Michx.) were

substantially larger than differences among

provenances.

If the amount of variation among individuals in

these two species is typical of other trees, geneticists

working with air pollution tolerance would have

considerably more variation to work with if they

included vegetatively propagated clones in their

studies.

Another advantage of using clonal propagation to

study patterns of genetic variation is that the actual

139
plants in the population are being sampled rather

than their offspring. When one propagates

population samples from seed, the samples can

include seedlings that are extremely sensitive to air

pollution and would not survive long in ambient air.

Berrang et al. (1986, 1991) showed, using

vegetatively propagated aspen, that populations

from polluted areas do not include the sensitive

clones found in pristine areas suggesting that

sensitive individuals are eliminated by ambient

levels of air pollution in polluted areas.

Breeding for Air Pollution Tolerance.--In

reference to this topic, Zobel and Talbert (1984)

stated “there is no area of forest tree breeding in

which greater emphasis is needed.” However, major

programs specifically designed to develop air

pollution resistant trees do not exist in the United

States. This is probably due, in part, to the fact that

the most common pollutants in this country are

regional. Because the effects are spread uniformly

over broad areas, it is difficult to convincingly

demonstrate that the problem is severe enough to

justify a major breeding program. Forest air

pollution researchers have instead concentrated on

determining the magnitude of air pollution effects.

There are only a few estimates of narrow sense

heritability of air pollution tolerance in trees but

they suggest heritability is low to moderate for

height growth (Gerhold 1977, Scholz and Venne

1989). Given the fact that there are tremendous

differences in tolerance to air pollutants among

individuals, specific combining ability and/or nonadditive components of variation may be important

components of genetic control. If this turns out to be

the case, the ability to vegetatively propagate large

numbers of ramets from selected individuals should

be an important component of breeding programs.

Study Mechanisms of Resistances--There is a

growing interest in discovering the various

mechanisms by which trees and other plants can

resist the effects of air pollutants. Some of these

mechanisms allow plants to avoid exposure to air

pollutants while others enable plants to avoid

reductions in growth when they are exposed to air

pollutants.

Air pollutants must enter the plant leaf for

exposure to occur. Stomates are the main barrier to

the uptake of gases, and several agricultural crops

appear to avoid air pollution exposure by closing

their stomates. There is a possibility that those

characteristics that affect the transport of pollutants

within the leaf may affect ozone sensitivity.

Examples of such characteristics could include the

amount of intercellular space in the leaf mesophyll

and the reactivity or solubility of pollutants with cell

membranes. Little evidence exists to show that

these characteristics are related to air pollution

tolerance in trees.

Some plants may tolerate exposure to pollutants

by scavenging ozone or the free radicals formed from

ozone. One example of such a mechanism would be

actively increasing levels of ascorbic acid in response

to exposure to ozone. This is true in several

agronomic crops and Chevone et al. (19891 reported

greater concentrations of ascorbate in ozoneresistant genotypes of white pine than in ozonesensitive genotypes of the same species. Very little

work has been done with trees in this area, but

extensive work with other plants suggests that

several enzyme systems may play a role in

protecting plants from the effects of air pollutants

including superoxide dismutase (Bennett et al. 1984)

and peroxidase (Jager et al. 1985).

There is limited evidence that suggests plants

may tolerate exposure to pollutants by developing

repair mechanisms. There is very little evidence of

this sort for trees, but work with agricultural crops

has shown that exposure to ozone can increase

concentrations of ATP (Pell 1974) or that proportion

of respiration presumably required for maintenance

(Amthor and Cumming 1987).

Mechanisms that depend on stomata1 closure

would presumably require that intact plants be used

when genotypes are screened for tolerance to

pollutants. However, it may be possible to develop in

vitro tests for screening genotypes in cases where

mechanisms depend on scavenging ozone or repair of

injury. Tree breeders may be able to use somaclonal

variation to obtain more resistant genotypes in these

cases.

There can be differences among resistant

individuals within a single population of a single

species in the mechanisms by which they avoid the

effects of air pollutants. For example, Karnosky

(Michigan Technological University, Houghton, MI,

personal communication) has found that some clones

from a single population of quaking aspen in

Wisconsin avoid injury by closing stomates during

ozone exposures. Because there can be such variety

between individuals, it may be difficult for

140
researchers working with differences between

families or between provenances to identify

physiological mechanisms that explain the

differences in sensitivity they have absorbed.

Working with single clones would probably increase

the likelihood that these researchers could identify

specific mechanisms by which plants resist the

effects of air pollutants.

Comparing Response of Seedlings and Mature

Trees.--Much of the forest tree work has

concentrated on seedlings, and an important issue is

how closely seedling responses represent the

responses of older trees. Two approaches have been

used to evaluate differences. One technique involves

exposing individual branches of mature trees to air

pollutants at the same site that whole seedlings are

being fumigated. A second technique assumes some

degree of cyclophysis or persistent age effects and

involves comparing the responses of clonal material

grafted from mature trees with seedlings grown

from seed collected on the same trees. Using this

technique, Sue Kossuth showed that the response of

tissues of different ages to ozone was similar for

diameter growth, but not for height growth (SCFRC

1990).

APPLICATIONS OF VEGETATIVE

PROPAGATION TO BASIC AND APPLIED

RESEARCH ON INSECT RESISTANCE IN TREES

INTRODUCTION

The current interest in tree defense mechanisms

arises from both basic ecological and applied forestry

perspectives. Basic ecologists are concerned with

the selective pressures exerted on plants by insects,

and conversely, on insect counter adaptations for

overcoming plant resistance traits. Trees are of

particular interest because they raise the

evolutionary paradox of long-lived hosts co-existing

with rapidly reassorting parasites. The forestry

interest arises out of the need for improved trees.

Tree breeders attempt to identify, select, and breed

for heritable traits, including those that may

improve overall yield by conferring protection

against pests. Both approaches have produced

major advances during the last two decades. The

ecological perspective has fostered synthesis and

development of robust theories. For example, the

levels and types of plant commitment to defense can

be at least partially predicted based on the relative

availability of carbon and nitrogen, insect feeding

guild, fitness values of various plant tissues, and

plant life histories (Bryant et al. 1985, Coley et al.

1985, Bazzaz et al. 1987, Raffa and Berryman et al.

1987, Mattson et al. 1988, Price et al. 1989).

Resistance breeders, on the other hand, have

contributed most of our genetic knowledge on plant

defense mechanisms (e.g., Hanover 1975, 1980). A

number of agricultural, and a few tree, varieties

have been successfully developed for insect, or

combined insect and disease, resistance.

Progress toward both objectives has been

enhanced by the recent integration of these

previously isolated perspectives. Basic evolutionary

and ecological theories can sometimes provide

guidance to breeding and pest management

programs. Conversely, genetically well-defined

selections and/or hybrids can greatly facilitate

analysis and understanding of basic evolutionary

processes.

Despite this progress, both approaches have

major limitations. Many ecological theories remain

largely untested, and are difficult to approach by

traditional approaches. Rigorous hypothesis testing

requires experimental control of environmental and

genetic factors. Natural ecosystems are often highly

variable and complex, and while they foster

comparative studies, are less amenable to

experimentally controlled analysis. Likewise,

resistance breeding is operationally very difficult

with trees, and has advanced less rapidly than in

agriculture. The long generation times and large

size of trees can seriously impede the breeding and

rapid evaluation needed for improvement programs.

The difficulties facing both basic ecology and

applied breeding can be partially overcome by using

research models that employ vegetatively

propagated materials. Sexually propagated crosses

are required to characterize the extent and potential

of heritable variation in resistance, but subsequent

use of vegetative clones offers the uniform genetic

base and control needed to address more specific

questions. Full integration of propagation by seed

and vegetative means provides the highest

likelihood of success (Libby 1973, Dickman and

Stuart 1983, Mordiek 1983). Vegetative propagation

can also help breeders reduce some of the delays in

tree evaluation (Libby 1973). Site-specific

requirements, effects of silvicultural treatments, and

site-by-treatment interactions can be rapidly and

reliably quantified by planting clones in split-plot

141
designs. Vegetative propagation becomes

particularly valuable in contending with the broad

mosaic of traits that are required of plantation trees,

because these vegetative clones allow for multiple

traits to be more rapidly assayed and deployed.

Rapidly Growing Hardwoods as a Model System

Fast growing hardwood species, such as Populus

and Sulix, provide an example of trees that are

amenable to several methods of vegetative

propagation. They also contain a range of heritable

traits needed both for testing ecological theories

(Price et al. 1989, Whitham 1989) and improving

production systems (Dickman and Stuart 1983,

Mohrdiek 1983). Within-clone variation is low,

which allows environmental effects to be more

accurately measured. The high between-clone

variation provides a broad genetic background for

testing ecological theories and guiding tree

improvement programs. Cuttings from rapidly

growing hardwoods, for example, can be rapidly

produced for glasshouse studies, and thereby

expedite research assay and design. Glasshouse and

growth chamber experiments must always be

verified under more realistic conditions, however

(e.g., Robison and Raffa 1992 a,b).

Our work has concentrated on rapidly growing

hybrid poplars. The extent to which the potential of

these trees will be realized, both for traditional fiber

uses and as a biomass alternative to fossil fuels,

depends on inter-related biological and economic

considerations. The major insect defoliator of

poplars is currently the cottonwood leaf beetle

(Chrysomela scripta F.); two lepidopterans, the

forest tent caterpillar (Mulucosomu disstriu Hubner)

and the gypsy moth (Lymuntriu dispur L.), also

pose significant threats (Caldbeck et al. 1978,

Dickman and Stuart 1980, Ostry et al. 1989). Our

research team includes specialists with both

traditional and molecular expertise. In the following

sections, we will not attempt to exhaustively list all

of the benefits of vegetative propagules, as a number

of excellent reviews are already available (Libby

1974, Toda 1974, Hartney 1980, Copes 1981, Mott

1981, Zobel 1981, Burdon 1982). Rather, we will

provide a few illustrations of how these models

facilitate both basic ecological understanding and

resistance breeding in insect research.

Contributions of Vegetative Propagation Research to

a General Understanding of Tree-Insect Interactions

When ecologists evaluate plant reproductive

“strategies”, there is an implicit assumption that

photosynthate allocation to one process, such as

growth, is at the expense of other processes such as

defense (Lorio 1986, Bazzaz et al. 19871. This view

has intuitive appeal, but is difficult to test because of

the operational problems described above. These

physiological alternatives, if valid, have implications

both for evolutionary theory and tree improvement

strategies. Vegetatively propagated Populus has

enabled us to test some of these assumptions

(Robison and RafTa 1992 a,b,c). Among 15 hybrid

poplar clones assayed for susceptibility to

Lepidoptera, Coleoptera, rodents, and Septoriu

fungi, there were no significant relationships

between primary productivity and susceptibility to

any pest. At least within Populus, these artificially

developed lines do not support the assumption of

“tradeoffs” between growth and defense (Robison

and Raffa 1992 a,b,c). Each system is likely to

differ, but these results indicate that the assumption

of tradeoffs should be rigorously tested despite its

intuitive logic. This research also lends caution to

the widely accepted and appealing view that plant

defense theory can provide strong guidance to

breeding programs (Robison and Raffa 199213). From

a positive standpoint, however, this research also

suggests that clones having both rapid growth and

high resistance properties can be developed.

A second area of interest concerns the

relationship between insect behavior and plant

defense. Behavioral aversion has long been

recognized as a key element of plant resistance to

insects (Painter 1941). From a basic evolutionary

perspective, behavioral aversion also raises the

question of whether some plants have evolved

chemicals that innately repel insects, or conversely,

whether insects have evolved the ability to orient

away from harmful chemicals (Miller and Strickler

1984). The above clones were tested for a variety of

forest tent caterpillar performance factors (Robison

and Raffa 1992). Early instar larvae preferred

clones that best supported larval growth, which

lends support to the view that insects select the most

beneficial plants. However, there were no

relationships between insect choice, either by larvae

or adults, and larval survival, and late instar larvae

sometimes preferred clones that delayed their

development times. These results do not support the

hypothesis that insect preferences always reflect

optimal choices. This example illustrates how

vegetatively propagated clonal Populus can provide

the strictly controlled experimental conditions

142
needed to dissect specific components from complex

plant-insect interactions, and thereby refine overly

generalized hypotheses. It also adds to our

understanding of the multifaceted components of

plant defense systems, even against a single

herbivore.

A third area of plant-insect interaction theory

concerns differences between specialists and

generalists (Bernays and Graham 1988). This issue

also impacts applied forest entomologists and

silviculturalists, because the feasibility of employing

multiple-species plantings to reduce pest

reproductive success depends on each insect’s host

range. Clonal hybrid poplar cuttings have helped

provide some insights into the relationship between

host plant chemistry and insect feeding breadth.

The principal secondary compounds present in

poplar, phenolic glycosides, have been shown to

reduce Lepidoptera performance (Lindroth and

Hemming 1990). Ecological theory predicts that

feeding by generalists is usually incited by

ubiquitous plant chemicals, such as sucrose, and

deterred by specific allelochemicals that can inhibit

growth and survival (Dethier 1982). Conversely,

specialists can metabolize the toxins produced by a

particular plant species, and are often attracted to

these allelochemicals. For example, feeding by the

cottonwood leaf beetle, a specialist on Populus, is

incited by tremulacin (Bingaman and Hart 1992

a,b). Thus, ecological theory predicts an inverse

order of preference among clones by the forest tent

caterpillar, a moderate generalist, and the

cottonwood leaf beetle. Assays conducted by Robison

and Rtia (1992a) confirmed this relationship.

Although most work on plant-insect interactions

has concentrated on chemical defenses, recent

attention has included plant architecture (Floate

and DeClerk-Floate 1992). Evidence from natural

systems suggests that certain plant forms can either

support or restrain insect performance. Again, it is

difficult to rigorously test such theories under

controlled conditions. However, hybrid poplar clones

provide a wide diversity of plant morphology, which

could facilitate testing of these ideas. Among our 15

clones, for example, the ratio of tree height to

diameter varies by a factor of over 2X, the ratio of

bole to branch dry weight varies by nearly 2X, and

the length of secondary stems can vary from 3.2 m to

0 m after 2 years (Robison and Raffa 1992a). The

value of this system as an experimental model is

heightened by the fact that both insect resistant and

insect susceptible clones occur among each category,

which facilitates comparative studies.

A final illustration of how vegetatively

propagated trees can contribute to our

understanding of plant-insect interactions concerns

the multiple functionality of allelochemicals. Earlier

models of plant-insect coevolution assumed a

relatively simplistic interaction, in which single

insect species were selected for by patterns of plant

chemistry. Recent studies have attempted to

develop more inclusive models that consider the

relative impa.cts of multiple selective pressures

(Bernays and Graham 1986, Price 19901.

Evaluations of hybrid poplars (Robison and Raffa

1992d) revealed clonally based allelopathic weed

suppression. Interestingly, one such clone also

exhibits high resistance to Lepidoptera, Septoria,

and rodents. Preliminary evidence suggests that

one chemical group, phenolic glycosides are at least

partially involved in protection against this broad

array of biotic stress agents (Robison and Rtia 1992

a,b,d). This interpretation seems compatible with

models of evolutionary parsimony (Bazzaz et al.

1987 ), whereby organisms produce the fewest

necessary amounts of metabolically costly materials.

Future research is needed on this question, but the

clonal Populus model will continue to be extremely

useful.

Contributions of Vegetative Propagation to Insect

Resistance Breeding Programs

Breeding for insect resistance is confounded by

the need for an optimal combination of traits, not

just the ability to avoid damage by one particular

species (Hanover 1975, 1980). Insect resistance

must be kept in perspective. For a particular species

or clone to be practical, it must first have good

rooting properties, adapt to the particular soil

conditions under which it will be grown, be droughtand temperature-tolerant, grow rapidly, possess

desirable form and fiber qualities, and show overall

high survival (Libby 1973, Wilkinson 1973, Cannel1

1978). Likewise, within the general area of pest

resistance, there is little value to planting trees that

are simply immune to one target species. Under

field conditions, trees are exposed to a broad range

of microbial, insect, vertebrate, and weed pests.

Even “resistance” to a particular pest species needs

to be further delineated according to toxicity, growth

inhibition, tolerance, and (with animals) behavioral

deterrence.

143
The need for a complex of desirable traits is a

formidable obstacle for tree improvement. The time,

size, and variability constraints described earlier are

magnified when a specified group of properties is

required. Vegetative propagation methods such as

grafting, rooting, cuttings, and tissue culture can

expedite this process, particularly if integrated with

an ongoing sexual crossing program. Research

operations still require a large amount of time and

effort, but an otherwise insurmountable task can be

made feasible.

Burdon (1982) listed the major uses of vegetative

propagation in breeding programs as preserving

genomes, providing clonal seed orchards for mass

propagation, directing mass propagation of trees

with full use of non-additive genetic variation,

providing genetic information, and providing

material for physiological research. Insect

resistance studies fall mostly within the last

application. We will refer to our work with hybrid

poplar clones to illustrate how a large number of

traits can be screened fairly rapidly.

Hybrid poplar clones in our study showed a high

range of performance in the field, with survival

ranging from 97% to 0%. Height growth varied by a

factor of approximately 2X, but tolerance rankings,

measured as relative growth rates following a

controlled level of defoliation, are distinct from

primary growth rates. In general, there is an

inverse relationship, but two clones deviate

from this pattern (Robison and RafTa 1990). These

two clones offer the combined features of rapid

growth, high survival, and defoliation tolerance.

No such compatibility appears available for

combining resistance to the forest tent caterpillar

and cottonwood leaf beetle. There is generally an

inverse relationship between resistance to these two

groups, with the useless exception of two clones that

are susceptible to both (Fig. 1A). No clones fall in

the region, marked by a triangle, that represents

resistance to both defoliators. The best combination

occurs with a clone ranking fourth most resistant to

the cottonwood leaf beetle, and fifth most resistant

to the forest tent caterpillar. Thus, there appears

little chance of obtaining combined resistance,

especially considering the opposing responses to

phenolic glycosides described above. Conversely,

there does appear to be potential for combined

resistance to Lepidoptera and rodents (Fig. 1 B).

Although there is no statistically significant trend

overall, three clones fall within the top four groups

most resistant to both pests. These results can be

further expanded by considering other pest

combinations. There is generally an inverse

relationship between cottonwood leaf beetle and

Septoria canker resistance (Fig. 10, and no clones

have a combined ranking better than fourth and

fifth, respectively. A second opportunity for

combined resistance appears to arise with Septoria

canker and Lepidoptera. Despite the lack of overall

relationship, three clones ranked at least in the top

five for resistance to both pests (Fig. 1D).

Thus, the most promising clones, along with their

shortcomings, can be rapidly identified. This can

provide strong guidance for tree improvement

programs. For example, we can plan ahead for the

likelihood that any clone with resistance to

Coleoptera will require additional protection from

Lepidoptera, rodents, and Septoria. This protection

could be provided through tolerance, transgenic

resistance, or an Integrated Pest Management

program targeted to these pests (Oliveria and

Cooper 1977, Caldbeck et al. 1978, McNabb et al.

1982). Conversely, clones with Lepidoptera

resistance will probably require special efforts

against Coleoptera, but there is good potential that

they can be resistant to Septoria and rodents.

Fortunately, at least one highly productive clone is

associated with resistance to each pest (Robison &

Raffa 1992 a,bl.

A second major area in which vegetative

propagation can facilitate insect resistance research

is in the application of biotechnology, or more

specifically, the integration of biotechnology with

traditional breeding methods. Direct transfer of

insect resistant traits can provide protection to

normally susceptible clones (McCown et al. 1991a,

Robison et al. 1992). For example, expression of Bt

endotoxin in clone NC5339, protected seedlings

against gypsy moths and forest tent caterpillars

(Fig. 2). Related Bt genes are effective against

cottonwood leaf beetles (Bauer 1990, Ramachandran

et al., in press), but these have not yet been inserted

into trees. Benefits from genetically engineered

resistance could include reduced pesticide

applications, and the increased economic

competitiveness of biomass alternatives to fossil

fuels.

However, a number of ecological dangers could

accompany widespread deployment of such toxins

(Gould 1988, Raffa 1989). As with pesticides,

resistant cultivars, or clones (McNabb et al. 1982),

144
n

0

0

0

.-

0

0

0

0

0

0

0

0

0

0

0

/

.

0

0

145

vYaldoa103 01 A.llllol.ua38n*
TRANSGENIC RESISTANCE TO LEPIDOPTERA

*

Bt *

* P < 0.05

Control

(Treated vs Control)

.Ol .l 1 10 100

Leaf Consumption

(Log sq. cm)

Figure 2. Transgenic resistance against Lymantria dispar and Malacosoma disstria in poplar seedlings expressing Bt endotoxin (Data from

McCown et al. 1991a).

any widespread deployment can favor resistant

biotypes, reduce natural enemy populations, and

indirectly cause secondary pest outbreaks. Very

carefully conceived deployment strategies are

needed to manage these potentially adverse effects.

The sheer number of variables that must be

considered to accurately assess the environmental

safety of genetically engineered trees could delay

development of this potentially beneficial

technology. Vegetative propagation can greatly

expedite this process. For example, our group’s

current efforts are aimed at developing planting

mosaics, that involve mixtures of clonal and

transgenic resistance (McCown et al. 1991, Ratfa et

al. 1992, Ramachandran et al. 1992 a,b; Robison et

al. 1992 e). Within this framework, only the Bt gene

active against Lepidoptera would be inserted into

beetle-resistant clones, and only the Coleopteraactive Bt would be placed into Lepidoptera-resistant

clones. Some less resistant, but highly tolerant,

clones might be included in these plantings to

provide reservoirs for the susceptible insect gene

pool. We have also proposed that within-plant

spatial, temporal, and wound-inducible expression

might reduce biotype evolution and secondary pest

eruptions (Rtia 1989).

Vegetatively propagated plants are helping to

transfer this theoretical framework into reality. For

example, we can simulate the patterns of withinplant expression described above, by artificially

applying appropriate Bt extracts to foliage. Once we

evaluate the relative value of each tactic, molecular

biologists can prioritize their efforts accordingly.

For example, application of Bt to only the young

foliage causes beetles to feed only on older foliage

(Ramachandran et al., in prep). This does not

provide complete plant protection, but can reduce

damage substantially, and allows some Btsusceptible beetles to survive.

Vegetatively propagated research material can

also increase the efficacy of transgenic plants.

Interactions between Bt and host allelochemicals

can greatly affect toxicity, for example (Krischik et

146
al. 1988). Thus, a judicious choice of clones can

synergize interactions between leaf chemistry and

transformed properties.

CONCLUSION

Vegetative propagation can greatly expedite

research on insect resistance in trees. In some

cases, the relative ease of propagation could

determine whether or not a particular objective is

achievable, given practical constraints on time and

resources. Research fostered by vegetative

propagation has far reaching implications to basic

ecology and evolution, the development of highyielding insect-resistant trees, and the

environmentally safe implementation of genetic

engineering.

TREE PROPAGULE/MICROBE GENETIC

INTERACTIONS

In our forest pathology project we use vegetative

propagules for a variety of purposes. Our mission is

to develop systems for rapidly imparting resistance

to specific diseases of forest trees. This program,

which started about seven years ago uses the

phenomenon of somaclonal variation to obtain

genetic change without going through the long

process of traditional sexual breeding.

Somaclonal variation is the genetic variation

exhibited by plants grown in aseptic culture.

Although the causes of somaclonal variation are not

completely understood, the phenotypic and genetic

variation found in various plant crops indicates that

many factors are involved. These include minor

point mutations, polyploidy, transposable elements,

aneuploidy, and epigenetic changes. Somaclonal

variation can result from preexisting genetic

variation that is expressed in regenerated plants, or

it can be induced during the tissue culture process

itself (Scowcroft and Larkin 1983).

I would like to give a few examples of how

vegetative propagules have helped our research

program at the North Central Forest Experiment

Station.

Hybrid poplar-Septoria canker-.--Our original

research with the process of somaclonal variation

was with hybrid poplar. The study was conducted to

demonstrate the potential application of somaclonal

variation for increasing disease resistance in a tree

species. Populus was chosen because of its

widespread importance as a source of fiber and

energy. This genus is also conducive to whole plant

regeneration by a number of vegetative propagation

systems including tissue culture. Yields from hybrid

poplar plantations are seriously limited by the foliar

and canker diseases caused by the fungus Septoria

musiua Peck. Our objective was to take highyielding hybrid poplar clones and increase their

resistance to Septoria canker. Callus cultures of

susceptible clones were initiated from stem

internode explants taken from greenhouse plants.

These callus cultures were maintained for 5 to 13

months before shoot proliferation was induced.

Elongated shoots were excised and rooted before

they were placed in the greenhouse. New, fully

expanded leaves were collected from these plants

and inoculated with conidia of S. musiua on the

abaxial surface. Plants with increased resistance to

Septoria were recovered from two of the three

previously susceptible clones. Many regenerates

were significantly more resistant to Septoria canker

than were the parent plants (Ostry and Skilling

1988). These putative resistant plants have now

been field-tested for resistance for 6 years. Many of

these plants continue to show resistance to Septoria

canker. The best selections have been asexually

propagated for more extensive field evaluation.

Although the field testing of this plant material

requires several years, we were able to produce a

number of Septoria-resistant plant lines within a

year after starting this study. In this situation,

using vegetative propagules has been very

productive both in the final results and in the

shortened time needed to produce these results.

European larch - Gremmeniella canker.--The first

attempt to produce somatic variants in a conifer

species was with European larch (Larix decidua

Mill.). Our objective was to produce resistance to

Gremmeniella canker caused by the fungus

pathogen Gremmeniella abietina (Lagerb.) Morelet.

In this model system the cotyledon culture technique

was used to produce somatic variants with

resistance to G. abietina (Diner et al. 1986). The

resulting plantlets were inoculated in vitro with

conidia of G. abietina. Plantlets that survived three

inoculations were then multiplied both in vitro and

ex vitro from axillary buds to produce clonal lines

with putative resistance. At present we have 50

clonal lines that are being field-tested in Wisconsin

and New York for resistance to G. abietina.

147
American chestnut - Chestnut blight.--One of our

cooperators, Dr. Dennis Fulbright, at Michigan

State University, has identified American chestnut

(Castanea dentata (Marsh.) Borkh.) germplasm with

high levels of resistance to chestnut blight caused by

the fungus Cryphonectria parasitica (Murr.) Barr.

Dr. Fulbright is developing in vitro propagation

techniques for multiplication of these disease

resistant trees. This research has progressed to the

point where the plantlets from resistant trees are

rooting very well, although survival is reduced when

the plants are placed on a peat-perlite mixture. He

is also developing in vitro inoculation techniques for

screening germplasm prior to field planting. This

technique saves both time and money. In this study

the use of vegetative propagules will insure that our

germplasm orchards will maintain plants with the

original genes for resistance. Whether this

propagation system can produce asexual plant

material at a reasonable price per unit is not known.

Butternut - butternut canker.--The last example I

want to present is our research on the development

of butternut (Juglans cinerea L.) with resistance to

butternut canker caused by the fungus Sirococcus

clavigignenti-juglandacearum Nair, Kostichka, and

Kuntz.

Forest populations of butternut trees are rapidly

being lost as a result of this fungus. Because of this

disease, butternut is being considered for the

endangered species list. Our program uses a three

part approach to produce resistant butternuts. The

first part is the establishment of grafted clonal lines

of butternut from putative resistant trees, These

are inoculated with the butternut canker fungus to

determine resistance. We now have 20 clonal lines

that are being evaluated. The second part of the

program is to develop techniques to rapidly

propagate resistant lines from these grafted trees.

The objective here is to maintain the resistant genes

in the asexually produced progeny. Rooting of

butternut plantlets from axillary buds has been

accomplished with juvenile material. Our next step

will be rooting of shoots from the grafted plants.

The third part of this project is to look for somatic

variants that may have canker resistance. In this

work we are collecting immature butternut during

the month of July. Our results indicate that

immature cotyledonary tissue of J. cinerea is

amenable to successful induction of somatic

embryogensis. Work is continuing on development

of entire plants from this embryogenic material. If

this is successful, it would provide us with an

additional source of butternuts that may have

resistance to this serious canker disease.

From the examples that we have included in this

paper, it is obvious that vegetative propagules have

an important role in the research arena. In many

cases they supply us with additional opportunities to

develop new plant material or to conduct more

sensitive tests on existing plant material. They have

limitations and must be used with caution, but the

system offers both speed and the ability to produce

large numbers of resistant clones. Tree

improvement is a difficult process under the best of

conditions. Anything that will increase the success

of this work has real value. We feel that vegetative

propagules can contribute to this success.

ACKNOWLEDGMENT

The work on insects and vegetative propagation

was supported by the University of Wisconsin -

Madison College of Agricultural and Life Sciences,

and the UW-Madison Graduate School. The critical

reviews by D. Ellis and R. Guries are greatly

appreciated.

.

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. 1

Foster, G. Sam; Diner, Alex M., eds. 1993. Proceedings of the 1992 Southern

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Nine papers address a range of issues in applications of vegetative propagation

in forestry. The three subtropics were tissue culture, rooted cutting, and

propagule growth, development, and application. This proceedings may be

referenced as a 1993 publication.
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