United States 

Department of Transient Expression of GUS in 

Agriculture 

Forest Service 

Southern 

Research Station 

Research Note 

SRS-7 

Bombarded Embryogenic Long 

and Eastern White Pine 

Alex M. Diner, Allan Zipf, Rufma Ward, 

Yhghua Huang, and George Brorm, Jr. 

May 1999 

Abstract 

Embryogdc tissue cultures derived from immature zygotic embryos of 

longleaf, loblolly, and eastem white pine were maintained in culture for up 

to 2 years, then bombarded with gold particles coated with a gene 

construct containing the GUS reporter gene fused to an adenine 

feme promoter from an algal vh. Physiological expression 

of GUS was observed in cultures of all three pine species within 24 horn 

but not at 7 days. Expression of GUS activity was recorded Fn somatic 

embryonal heads of varied stages of development, suspensor cells and 

other cells of mixed ontogeny. Collective expression of GUS in small 

clusters of cells suggested inheritance of the reporter gene through early 

mitotic events. The presence of multiple discrete sites of GUS expression 

common in individual somatic embryos was indicative of densely 

associated multiple transformation events, and was enhanced by reducing 

the sample distance. This is a first report of transgene expression in 

longleaf and white pine. 

and others 1992). In such trmformation attempts, 

expression of the newly introduced genetic loci has been 

most commonly transient for several possible reasons 

(Clapham and others 1999, although temporal expression 

has been lengthened (Clapham and others 1995), and even 

stabilized in some cases (Ellis and others 1993). 

Our laboratories at Alabama A&M and Oklahoma State 

Universities cooperate on the development of methods for 

both vegetative popagation of southern pines and genetic 

manipulation for genotype improvement. The work reported 

here describes the results of microprojectile bombardment of 

embryogenic cultures of three southern pines, using an algal 

virus gene promoter as yet unreported for application with 

tree genetic manipulation. This is a frrst report of transgene 

expression in longleaf and white pine embryogenic tissues. Keywords: Biolistics, gene gun, gene promoter, GUS, Pinus palustris 

Mill., P. strobus L, P. taeda L. 

Materials and Methods 

Introduction 

Microprojectile-mediated DNA transfer has been shown 

effective for a variety of woody angiosperms and 

gymnospems. In the latter, the target tissues have included 

cotyledons (Stomp and others 199 1, Sul and Korban 1994), 

mature pollen (Hay afld others 1994, Li and others 19941, 

somatic embryogenic cultures (Clapham and others 1995, 

Duchesne and Chmest 199 1, Bomineni and others 1994, 

UiBlter and others 1994), mature somatic embryos 

(Robertson and others 1992), vegetative buds and budderived calli (konen and others 1994), and xylem (Loopstra 

Diner is a Research Physiologist with the U.S. D 

Forest Service, Southern Researeh Station, Department of Plant and Soil 

Science, Alabama A&M University, Normal, AL 35762; Zipf and Ward 

are Assistant Professors and Brown is an Associate Professor with the 

Department of Plant and Soil Science, Alabama A&M University, Normal, 

AL 35762; and Huang is an Associate Professor with the Department of 

Forestry, OMahoma State University, Stillwater, OK 74078. 

Preparation of Embryogenic Tissues 

for Bombardment 

Embryogenic cultures of white and longleaf pines were 

initiated and maintained for 2 years as described by Kaul 

(1 995) and PJagmani and others (1 993). Embryogenic 

cultures of loblolly pine were initiated &om immature 

embryos collected in Idabel, OK, during mid-July 1995. 

Seeds excised &om the cones were surface sterilized in 10 

percent household bleach for 10 minutes, then ~sed. 

Megagametophytes removed fiom their developing seed 

coats were hersed for 5 minutes in 5 percent bleach, then 

rinsed and cultured on DCR medium (F3ecwar and others 

1990) containing 2,4-dicfiloropheno;l;yacetic acid (2,4-D) 

( 12 pM [molaq ) and benqlaminopurine PAP) (4 pM) 

solidified using 0.2 percent Gelrite. After 8 to 10 weeks, 

embryogenic calli were isolated and subcultured bi-weekly 

or tri-weekly for 4 months onto a fieshly prepared medium 

of the same composition. At the end of that period 
(December 10,1995), embryogenic cultures of all three 

species were transferred to a pH 5.8,6.5 percent agar 

(Sigma Chem. Co., St. Louis, M0)-solidified Brown and 

Lawrence medium (Brow and Lawrence 1968) containing 

2.5 percent sucrose, and modified to contain glutamine (10 

) as the sole source of amino nitrogen. The medium was 

sulh~plemented with 2,4-D (8 pM) and BAP (4 pM). All 

cultures were dark-incubated at 20-22 OC and subcultured 

every 3 weeks to freshly prepared medium. 

Five to 7 days prior to bombardment, the embryogenic 

culture of each species was transferred to a fieshly plated 

medium, to occupy an approximate circular area of 2- to 3- 

cm diameter in the center. Six replicate cultures were 

prepared for each species, of which one would be an 

unbombarded control. 

Microprojectile Bombardment 

Procedures used for microprojectile preparation and coating 

with DNA have been described by Heiser (1992). Each 

culture was bombarded once with 1 pg gold particles onto 

which had been precipitated "psah/lTGUS25" (provided by 

A. Mitra, University of Nebraska, Lincoln, NE), containing 

the Chlorella vinrs adenine methyltransferase gene promoter. 

(Mitra and Higgins 1994) linked to the GUS reporter gene 

(Jefferson and others 1987). Twenty-five pg DNA was 

precipitated onto 3.6 mg gold particles. Initial bombardment 

employed a sample distance of approximately 6.3 cm. To 

increase particle pattern density, and thus greater potential 

transformation frequency, this was later reduced to 5 cm. 

All bombardments were carried out using a rupture-disc 

pressure of 1,100 psi. The apparatus employed for 

bombardment was the Biolisticm particle delivery system 

PDS- 1000 (DuPont, Wilmington, DE). Following 

bombardment, petri dishes containing the cultures were 

incubated in the dark at 20 to 22 OC. Cultures were sampled 

at 24 hours, 48 hours, and 7 days. Sample size was 

approximately 0.5 cc. GUS activity was assayed as 

described (Jefferson and others 1987). 

Results and Discussion 

All bombarded embryogenic cultures expressed GUS 

activity at 24 hours, while the controls did not. 

Bombardment at a target distance of 6.3 cm produced 

approximately eight impactsiGUS-expression sites per 

0.5 cc tissue, irrespective of species. Bombardment at 5 cm 

approximately doubled that effect, as measured at the 

maximum (40X) magnification provided by the binocular 

dissecting photomicroscope that was used to examine the 

microcentrihge tube-content of bombarded tissue. 

However, greater resolution that was later provided by the 

compound microscope employed for photomicroscopy of 

sformant cells in thin layer, showed both multiple GUSexpressing sites on unit structures such as single embryos 

(fig. 1) and nmerous isolated individual cells or small 

clumps (fig. 2) showing the same characteristic blue color. 

This observation was especially true of tissue bombarded at 

5 cm. 

Twenty- four and 48-hour assays showed GUS activity in 

single cells, as well as in small clusters of cells (fig. 2), 

apparently resulting from mitotic events following 

bombardment. Cells in both somatic embryo heads and 

suspensors showed GUS expression (fig. 1). Where cells 

were densely packed in the former, stain appeared either to 

have diffused into adjacent cells or to have reacted with 

substrate that had diflksed from the specifically bombarded/ 

GUS-expressing cell. This phenomenon was also reported 

for bombarded pine cotyledons (Stomp and others 199 1). 

The promoter employed here is a 85 1-base-pair upstream 

region frorn an algal virus methyltransferase gene, heretofore 

shown effective for expression in transformants of a small 

number of both monocots and dicots (Mitra and others 

1994) No applications of this promoter to tree 

transformation have been reported. And, although our 

assays showed no GUS activity after 7 days, such a 

temporal loss of expression might be reduced through the 

use of certain medium osmotica or of different promoters 

(Martinussen and others 1994, Clapham and others 1995). 

Our work with somatic embryogenesis and genetic 

transformation of embryogenic tissues is continuing. 

Acknowledgments 

This work was supported by the U.S. Department of 

Agriculture, Historically Black Colleges and Universities 

Cooperative Research Program grants 19-92-0 14, 19-92- 

049, and 19-95-057. 

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