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Reprinted from - Biological Monitoring of Genetically Engineered Plants and Microbes. ' 1991. Proc. of the International Symposium on The Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms. November 27-30, 1990, Kiawah Island, S.C., USA. USDA/CSRS and Clemson University, Washington, D.C. and Clemson, S.C.

A FIELD TRIAL OF TRANSGENIC HYBRID POPLAR TREES: ESTABLISHEMENT AND GROWTH THROUGH THE SECOND SEASON Harold S. McNabb, Jr., ,W~ B;~ Klopfenstein 1 · Roger D. Hanna, Richard B.Hall, Elwood R. Hart, Scott A. Heuchelin and Robert W. Thornburg Iowa State University 2 Ames, Iowa 50011 USA

RM Reprint

SUMMARY In July 1989, 20 transgenic hybrid aspen poplars (Populus alba x P. grandiden.tata cv. Hansen) were field planted with 20 nontransformed trees of the same clone in Ames, Iowa. This planting, under USDA( APHIS permit number 89-109-03, was the first approved field release of a transgenic woody plant in the United States. The trial is designed for. four complete growing seasons, during which foreign gene expression within different tree tissues, and the effect of foreign genes within a genome (in this case, both the nos/NPT II and pin2/CAT!pin2 genes) on tree growth and development will be studied. Before any poplars were planted, local government officials and the press were appraised of the study and planting plans. At the end of the 1990 growing season, transgenic trees had significantly less total basal diameter growth (p ~ 0.05). INTRODUCTION The parent materials for our genetic engineering studies have been three Populus hybrids, two clones of Populus alba L. x P. grandidentq.ta Michx. cv. Hansen and Crandon (Section Leuce) originating in southeastern Iowa, and the Belgian hybrid clone P. deltoides Marsh. x P. nigra L. cv. Ogy (Section Aigeros) having an Iowa cottonwood as grandmother. Agrobacteriunt-mediated transformation ~tudies began in the Summer of 1987 (Chun et al., 1988a; 1988b) with four con~tructs containing the selectable marker gene nos/NPT II and chimeric genes witfi compo~erits of the potato pin2 ge.ne (Thornburg et al., 1987a). Two of the chimeric genes contain the . w9~Qd~induc{bie·;·p'ln..2/CAT construction (Thornburg .et al., 1987b) having .different terminators· (binary. vector plasmids pRT45 and pRT50). The other two contain the constitutive and terminator.pin2 components with either the nos or 35S promoter (plasmids pRT102 and pRTI04). ..

1

Present Address: Forest Sciences Laboratory, Rocky Mountain Range and Forest Experiment Station, USDA Forest Service, East Campus, University of Nebraska, Lincoln, NE 68583-0822 2

Authors McNabb, Klopfenstein, Hanna, Hall, Hart, and Heuchelin are of the Department of Forestry. Thornburg is of the Department of Biochemistry and Biophysics. McNabb, Klopfenstein, Heuchelin, and Thornburg also hold academic appointments in the Department of Plant Pathology. Hart is also of the Departtnent of Entomology. 155

Approximately 200 transformants from this research are available f~_)r study under greenhouse conditions (Heuchelin et al., 1990; Klopfenstein et al., l9oi.J). On July ~8, l S>8lJ. 20 ramets of the transgenic line Tr15 of the clone Hansen were field-planted with 20 nontransformed trees of the same clone in Ames, Iowa. This planting, und~r USDA/ APHIS permit number 89-109-03, was the first approved transformed woody-plant field planting in the USA. The Environmental Assessment and Finding of No Significant Impact prepared by the USDA Biotech~ol~~yqPermit Unit, and sent with the permit, is considered to be an excellent reference~.

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U~der study in this field trial are both the gene expression within different tree tissues over time and space, and the effect of foreign genes within a genome (in this case, both the nos/NPT II and pin2/CAT/pin2 genes) on tree growth and development. PLANTING PREPARATION AND MAINTENANCE Tree Materials: The transgenic (Trl5) and check lines were clone,lby nodal propagation (Faltonson et al., 1983), and were grown in the greenhouse for 6 weeks. Another 10 days were required for hardening the young trees before out-pianting. All plant materials were transported to the field site within closed containers, and in a closed vehicle. Plan.ting of all 40 trees was completed during the afternoon and early evening of July 28, 1990. The trees will remain on site for four complete growing seasons; a time period within which no flowering is expected to occur. The trees are monitored and measured for height weekly during the growing season. Basal diameter measurements and bud-set observations are made at the end of each growing season. During the second growing season (1990), leaf wounding was done weekly. This wounding was performed with hemostat forceps on one-half of the Tr15 and check trees on the main-stem leaf having a leaf plasticron index (LPI) of 3. This wound treatment is used to activate the wound-inducible pin2/CAT chimeric gene (Thornburg et al., 1987b). ·

Planting Site: The test site is located at the northern edge of Ames, Iowa, within the fenced, locked gate, Hind's Experimental Farm of Iowa State University. This test site· is 30 m x 15 m, surrounded by a plowed 3.5 m buffer zone and a 2.44 m chain-link fence. This fence is always locked when work is not being done within the site. The trees are planted in 4-tree plots, 5 plots of transgenic and 5 of nontransformed poplars. Each plot is surrounded by a 1 m buffer zone which is plowed weekly during the growing season to prevent root grafts between trees from different plots and to confine the .root sprouting habit of the hybrid aspens. Public Awareness: Two weeks before the planned introduction of the transgenic poplars into the field, the research project personnel met with city and county officials to appraise them of the planting plans, the nature of the transgenic trees and theJield ~xperiment, and any risks involved during the 4 years of the trial. The press was invited to_ this meeting and excellent coverage resulted. Iowa State University prepared a press kit, and the principal investigators were available in their 156

laboratories during one afternoon for conferences with the press. Special mention should be made of the coverage by the Omaha World-Herald, Omaha, Nebraska. The article by Julie Anderson that ran on July 30, 1989 is an excellent example of high quality reporting of biotechnology issues. Iowa State University and the researchers received positive reactions from the contacts made during these public information sessions. The fact that the trial would be completed before the trees would flower illustrated to the public that the planned research considered possible risks. The only question, raised by a county official, was that of the possible impact of beavers feeding on the trans formants (who are known to damage cottonwoods in the area). Our response was that the chain link fence effectively negates this possibility.

OBSERVATIONS Studies on poplars transformed with the chimeric gene pin2/CAT/pin2 are concerned with the induction of wound-inducible chloramphenicol acetyl transferase (CAT) gene activity. The field trial presents an opportunity to st1,1dy both 1) gen~ expression within different tree tissues over time and space, and 2) the effect of two foreign genes within a tree genome on tree growth and development. Lack of funding during 1990 has so far prevented ·gene expression studies, but growth and development observations continue. Tree height was nearly 20 em at the time of planting. At the end of the 1990 growing season, the mean height of all trees was about 190 em (see Table 1). At the end of the second growing season, only the mean total basal diameter was significantly different (p ~ 0.05 using ANOVA analysis) with the check trees greater in diameter than were the Trl5 trees (Table 1). Total volume (d 2h) also was significantly different for the two groups (p ~ 0.05). No significant differences appeared in bud-set between transgenic and check trees in the fall of 1990. All trees had set terminal buds between 21-28 September. It was hypothesized early in the research that the addition of foreign genes to the genome of a tree might reduce growth. The observed reduction in diameter growth of Tr15, relative to the checks by the end of the second growing season, may not, in the end, be biologically significant. Also, the wounding done weekly to one..;half of the trees (both treatments) did not cause significant growth differences. Only further growth periods will verify the observations made to date. ACKNOWLEDG:MENT Journal Paper No. J-14337 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011 USA. Projects No. 2210, 2731, 2748, 2796. Sponsored in part by funds from the Agricultural Biotechnology Council of Iowa State University, Mcintire-Stennis Forest Research Program, and the ISU Foundation Project 008R, Tree Research Institute. REFERENCES Chun, Y.W., N.B. Klopfenstein, H.S. McNabb, Jr., and R.B. Hall. 1988a. Biotechnical applications in Populus species. J. Korean For. Soc. 77:467-483.

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Chun, Y.W., N.B. Klopfenstein, H.S. McNabb, Jr., and R.B. Hall. 1988b. Transformation of . Populus species by an Agrobacterium binary vector system. 1. Korean For. Soc. 77: 199-207. Faltonson, R., D. Thompson, and J.C. Gordon. 1983. Propagation of poplar clones for controlled-environment studies. In: Methods of Rapid, Early Selection of Poplar Clones for Maximum Yield Potential. A Manual of Procedures. USDA For. Serv. North Cent. For. Exp. Stn. Tech. Rep. NC-18, pp. 1-11. Heuchelin, S.A., H.S. McNabb, Jr., N.B. Klopfenstein, and R.W. Thornburg. 1990. Agrobacterium mediated transformation of Populus x euramericana 'Ogy' using the proteinase inhibitor II gene (pin2). (Abstr.) Phytopathology 8010:976. Klopfenstein, N.B., S.A. Heuchelin, H.S. McNabb, Jr., R.W. Thornburg, R.B. Hall, and.E.R. Hart. 1989. Transformation of hybrid Populus with the proteinase inhibitor II gene. (Abstr.) Phytopathology 799:1004. Thornburg, R.W., G. An, T.E. Cleveland, and C.A. Ryan. 1987a. Characterization and expression of a wound-inducible protei~ase inhibitor II gene from potato. NATO ASI (Adv. Sci. Inst.) Ser., Ser. A Life Sci. 140:121-129. Thornburg, R.W., G. An, T.E. Cleveland, and C.A. Ryan. 1987b. Wound-inducible expression of a potato inhibitor II-chloramphenicol acetyltransferase gene fusion in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA 84:744-748.

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Table I. Tree measurement means (em) for the first two years of field growth, 20 transgenic (Tr15) and 20 check poplars

.YL_l

Height Yr. 2

Total

.YL_l

27.70 21.55 p=0.048*

161.55 168.70 p=0.343

189.70 190.20 p=0.951

0.142 0.105 p=O.l11

Treatment

Check Trl5

Basal Diameter Total Yr. 2

*Means comparisons that are significantly different at p~0.05, ANOVA analysis ........ Ul \!)

1.962 1.774 p=0.051

2.193 1.975 p=0.044*

..,, Biological Monitoring of

Genetically Engineered Plants and Microbes

Edited by

D. R. MacKenzie

and Suzanne C. Henry

Proceedings of the Kiawah Island Conference November 27-30, 1990

Published by Agricultural Research Institute Bethesda, Maryland USA