Genetic Engineering of Pinus Radiata and Picea Abies, Production of Transgenic Plants and Gene Expression Studies

Molecular Breeding of Woody Plants N. Morohoshi and A. Komamine, editors. 9 2001 Elsevier Science B.V. All rights reserved.

211

GENETIC ENGINEERING OF PINUS RADIATA AND PICEA ABIES, PRODUCTION OF TRANSGENIC PLANTS AND GENE EXPRESSION STUDIES

Christian Walter 1, Sharon Bishop-Hurley 2, Julia Charity 1, Jens Find 3, Lynette Grace ~, Kai Htifig', Lyn Holland ~, Ralf Miiller ~, Judy Moody 1, Armin Wagner' & Adrian Walden 4 tNew Zealand Forest Research Institute Ltd, Rotorua, New Zealand 2 108 Waters Hall, Plant Pathology, University of Columbia, Missouri, M065211, USA ~Tissue Culture Laboratory, Botanic Garden of Copenhagen, O. Farimagsgade 2B, 1353 Kbh.K, Denmark +Vialactia Biosciences, Level 4 Clinical Building University of Auckland School of Medicine, 85 Parl Road, Grafion, Auckland, New Zealand.

ABSTRACT Plantation forestry, based on successful breeding of superior tree genotypes, is becoming more widely used by international forestry companies, since it offers the possibility to grow and manage forests of high economic value and superior quality. However, a number of highly desirable traits are not readily available in the breeding population and may be introduced using desirable genes from other organisms. Forest molecular biology, and in particular tree genetic engineering is now at a stage where the technologies are readily available to transfer specific traits of commercial and scientific interest into forest trees. Our efforts are aimed at the genetic engineering of plantation grown conifer trees such as Pinus radiata, Pinus taeda and Picea abies. Stable transformation technologies have been developed for embryogenic tissue using either Agrobacterium tumefaciens or a Biolistic | particle delivery system. Many genes from other organisms combined with promoters of various origins, were transferred into conifer tissue and transgenic plants recovered. Examples are genes for resistance against herbicides, genes involved in reproductive development, and genes involved in lignin formation. Analysis of transgenic tissue and plants has confirmed successful transfer of genes and their expression. For example, a herbicide resistance gene was introduced into Pinus radiata and Picea abies, and transgenic plants were regenerated and exposed to an operational concentration of the respective herbicide. Spray tests demonstrated the expression of the herbicide resistance gene and the newly acquired resistance of transgenic conifers to the herbicide. Another example is the expression of the endogenous cad gene in sense or antisense orientation in transgenic Pinus radiata tissue and plants. Biochemical analyses indicate that the influence of the inserted gene construct on the expression of the endogenous cad gene can be dependent on the developmental stage of the plant. A further challenge is provided by the growing numbers of potentially useful genes and promoter / gene combinations offering potential for forest trees. Early screening

212 technologies that allow the quick and economic screening of candidate genes need to be developed to enable researchers to make the right choice of a gene for a specific purpose. To this end we are developing tissue culture protocols to produce secondary wall-forming cells and tracheary elements in vitro. These cells may be genetically transformed and may provide an excellent screening and gene expression analysis tool by avoiding the long periods of time otherwise necessary to regenerate and analyse transgenic plants. INTRODUCTION Genetic engineering has contributed to significant improvements in agricultural crops, and plants with engineered resistance against herbicides or insects are used in commercial plantations worldwide [1,2]. This relatively new technology has the potential to improve quality and yield of agricultural products, and newly developed products for human consumption hold the promise to significantly contribute to human health and welfare [3]. The use of agrochemicals can be reduced, leading to a more environmentally acceptable agriculture that is truly sustainable. The development of molecular biology platforms including genetic engineering has somewhat lagged behind in forestry, mainly due to additional challenges related to the long rotation time of these plants, long breeding times and difficulties with tissue culture and genetic transformation protocols [4]. Conventional breeding has been the predominant technique to improve genetic gain in plantation forestry, and many techniques have successfully been applied to improve gain and various growth and performance characteristics [5,6]. Conventional tree

Identification of Superior Traits

~n ~ ~Breedingl

Quality Assurance

~ Introduction of Novel Traits (Genetic Engineering)

_

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|Propagation] Improved planting stock

Figure 1: The production of superior tree germplasm. Major techniques used to provide improved planting stock

213 improvement programs aiming at the production of superior germplasm (Figure 1) have traditionally made use of the identification of superior traits. Also, various breeding techniques and methods of propagation (including both micro and macro propagation) to provide superior planting stock for commercial plantations are used. More recent developments, in particular in the area of molecular biology, have added techniques for quality assurance such as marker-aided selection (MAS) and genetic fingerprinting [7]. Also, over the past 10 years significant progress in developing genetic engineering protocols has been made and they are now available for most major forest tree species of commercial importance. These can provide techniques to transfer traits that are not readily available in the existing breeding population [8,9]. In this paper we review genetic engineering technologies developed at the New Zealand Forest Research Institute and present results from the analysis of transgenic tissue and plants. Further, we discuss results obtained from conifer promoter analysis in a heterologous plant species, and present strategies for more efficient and faster functional analysis of candidate genes.

GENETIC

TRANSFORMATION

OF

EMBRYOGENIC

TISSUE

USING

BIOLISTICS | OR A GROBA CTERIUM TUMEFA CIENS Embryogenic tissue on a maintenance medium [10] is used for transformation with Biolistic | [11] techniques, or via Agrobacterium tumefaciens [4 and Charity, in preparation]. Transformation takes place with embryogenic tissue at an early stage in development and involves a plasmid vector carrying the nptlI gene for resistance against aminoglycoside antibiotics such as kanamycin and geneticin. Geneticin was found to be the better selective agent for the selection of transgenic conifer tissue. The exact concentration ranges from 5 to 35mg / 1, depending on the conifer species and the genotype. Kanamycin was also tested as a selective agent, however it allowed the emergence of escapes ie tissue that grew on selective media but was not transformed. Geneticin selection never allowed such growth. The antibiotic hygromycin was also successfully used to select transgenic radiata pine tissue after transformation with a vector containing the aph4 gene [12]. Tissue resistant to the antibiotic proliferated and became visible usually 4-6 weeks after transformation. At this stage it was transferred to fresh media for further proliferation, analysis and regeneration. For information on protocols, see [12,13,14 and Charity in preparation]. In summary, a range of conifer species were successfully transformed by Biolistic | or Agrobacterium related techniques (Table 1).

Analysis of transgenic material Transgenic tissue and plants were analysed using a variety of techniques to confirm transgenic nature and function of the introduced gene product. This includes histochemical and fluorometric uidA analysis, Southern and Northern hybridisation, RT-PCR, nptlI and Bacillus thuringiensis toxin (crylAc) ELISA, and spray testing with herbicides for functional analysis. Initial molecular analysis with putative transgenic lines was carried out using PCR analysis. DNA isolated from tissue pieces growing on selective media were probed with

214

Species

Transformation Transgenic Molecular technique tissue selected analysis

Regeneration of transgenic plants

Pinus radiata

Biolistic |

+

+

+

Picea abies

Biolistic |

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Pinus taeda

Biolistic |

+

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Abies nordmanniana Pinus radiata

Biolistic |

+

+

+

Agrobacterium

+

+

+

Table 1:

Genetic transformation of various conifer species, analysis and regeneration of plants.

primers initiating the amplification of the nptlI or uidA gene and products were analysed by gel electrophoresis. For example, tissue selected on geneticin after Agrobacterium transformation was tested for the presence of nptlI DNA and confirmed to contain the transgene of interest (Figure 2). Controls including primers for the virD gene from A. tumefaciens did not amplify a corresponding DNA fragment indicating the absence of A. tumefaciens in transgenic tissue. Further analysis of transgenic selected tissue involved histochemical staining for uidA expression and blue colouration was found in most selected lines at varying intensity (data not shown). Southern hybridisation analysis of transgenic P. radiata plants derived from a Biolistic | transformation experiment revealed medium to high copy numbers of the integrated nptlI gene (Figure 3).

Npttl PCR

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Figure 2: PCR analysis of embryogenic tissue of Pinus radiata transformed with Agrobacterium tumefaciens. Abbreviations: M: Molecular marker; W: Water-control; +: Positive (plasmid) control; sp: Space (no DNA); -: Untransformed tissue control; 1-14: Tissue from individually transformed lines.

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Figure 3: Southern hybridisation of transgenic P. radiata plants. Genomic DNA was digested with either the enzyme EcoRI or HindIII. 1-4: Transgenic lines, 5: non-transformed control. This is typical for transclones that are produced using Biolistic | techniques, in contrast to Agrobacterium transformation where single or low copy numbers of the transgene can usually be expected. High copy numbers can have negative effects on gene expression stability and correct long-term gene expression. This is particularly important with trees that have generation times of 30 years and more. Transgenic conifer tissue has also been assayed using ELISA (Enzyme Linked ImmunoSorbent Assays) to confirm the expression of nptlI and the presence of the neomycin-phosphotransferase enzyme in transgenic tissue and plants [ 14]. The assay is very specific and sensitive and was positive for most transgenic tissue lines and also transgenic plants in the field and the greenhouse (Figure 4).

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Figure 4: nptlI ELISA with transgenic Picea abies plants grown in the greenhouse. Abbreviations: C: Non-transformed control; 1 and 2: Positive control plants (assayed positive in earlier experiments); a-h: transformed Picea abies plants.

216 In this assay, most transgenic lines, with exception of line S1 showed levels of the NPTII protein significantly higher than the control. Line S 1 may have lost expression of the nptII gene, or the expression is very low compared to the other lines. The results also show that foreign gene expression is highly variable within the different lines tested. This may reflect a position effect of the introduced gene, copy number effects or silencing of transgenes after integration. The nptII ELISA is now routinely used to confirm transgenic nature of selected lines early in the process. A similar technique was used to confirm the expression of the Bacillus thuringiensis toxin gene (cryIAc) in P. radiata embryogenic tissue (Figure 5). The 11 lines tested were co-transformed with a vector containing the selection gene nptII and a vector containing the Bt gene. Nine of the 11 lines confirmed transgenic by nptII-PCR, showed high levels of Bt toxin present in the cells. A further line, confirmed transgenic by nptII and cryIAc-PCR, did not express the cryIAc gene as evidenced by ELISA. Another line was negative in cryIAc PCR but positive in nptII PCR. The results indicate that the co-transformation frequency for the two unlinked genes was 91%.

FUNCTIONAL ANALYSIS: EXPRESSION OF A HERBICIDE RESISTANCE GENE IN TRANSGENIC CONIFERS:

P. radiata and Picea abies embryogenic tissue was transformed with a construct containing the nptII selection gene, and a second construct containing the bar gene for resistance against the herbicide phosphinothricin [15]. The gene product of the bar gene, a phosphinothricin-acetyl-transferase modifies phosphinothricin by the addition of an acetyl group. Acetylated phosphinothricin is no longer an active herbicide and is rapidly degraded by plants and microorganisms [16, 17]. Transgenic P. radiata and Picea abies plants were regenerated from 35 independent transformation events, and propagated in a greenhouse. Samples were spray tested with operational concentrations of the herbicide to confirm resistance. Six cry 1Ac E L I S A

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217 weeks after spraying, all non-transformed control plants were dead, whereas transgenic plants continued to grow unaffected by the herbicide. This is the first demonstration of a commercially useful gene expressed functionally in a conifer. The experiments also demonstrated that even very low expression levels of bar (as determined by Northern blotting experiments) were sufficient to render transgenic plants resistant against the herbicide [ 15]. F U N C T I O N A L ANALYSIS: EXPRESSION OF E N D O G E N O U S PINUS RADIATA GENES AND THE FUNCTION OF THEIR P R O M O T E R S IN A H E T E R O L O G O U S SYSTEM:

Using a differential screening approach, a series of different cDNAs specifically expressed in male cone tissues of P. radiata were isolated. Northern hybridisation experiments confirmed the expression of the respective genes in male cone tissue, and absence of expression in other tissue types. Analysis of DNA sequences and homologies between the sequences, groups one of the cDNAs isolated (PrCHS 1) with plant chalcone synthase genes, and another sequence (PrLTP2) with a clade of lipid transfer protein genes. In situ expression analysis confirmed the expression of PrCHS 1 mainly in the tapetum of developing male reproductive structures, whereas PrLTP2 was confirmed to express mainly in developing microspores [ 18]. Based on sequence information and using a Clontech Genome Walker kit, the genomic upstream sequences of PrCHS1 and PrLTP2 were amplified and cloned upstream of a uidA reporter gene (H6fig, in preparation). Arabidopsis thaliana plants were transformed with these constructs using a vacuum infiltration technique [19]. Transgenic seed were selected by germination on antibiotic containing media and resulting plants were selfed to produce heterozygous transgenic populations. Plants from the T3 generation were grown to maturity and their uidA expression patterns analysed. Microscopical studies included phase-contrast and a very sensitive ultradarkfield technology. The results indicate that the PrCHS 1 promoter directs expression towards the Arabidopsis tapetum and to microspores at an early stage in development (pollen mother cells). The activity of the PrLTP2 promoter was also observed in these tissues, however the onset of expression was later, at the tetrad stage of microspore development. This study demonstrates that sequences upstream of differentially expressed conifer genes are evolutionary conserved with respect to their function in two such distant species as A. thaliana and P. radiata. Further experiments will be based on these sequences to specifically direct expression of genes to the tapetum cells. As an example, a cytotoxin gene fused to this promoter has the potential to abolish tapetum function and lead to male sterility. F U N C T I O N A L ANALYSIS OF THE CAD GENE IN TRANSGENIC RADIATA PINE

Lignin biosynthesis is essential in terrestrial plants, contributing significantly to the properties of wood forming tissue. In pulp and paper making however, lignin needs to be removed. This is an expensive process and it generates environmentally unfriendly waste products. Therefore, various approaches were made to reduce lignin content or change its composition to improve the process and efficiency of pulp and paper production. We are using both sense and antisense suppression to understand and manipulate lignin biosynthesis related gene expression.

218 Little data is available on the effectiveness of different suppression mechanisms based on sense or antisense constructs in gymnosperms. We compared the effect of both sense and antisense constructs in suppressing the endogenous expression of cad, a lignin related gene encoding for Cinnamyl Alcohol Dehydrogenase, in P. radiata. More than 100 transgenic P. radiata lines containing a cad sense or antisense construct were generated. CAD assays were performed to quantify the activity of intracellular CAD in transgenic and non-transgenic control tissue at different stages in development. Our results indicated that the sense construct used in this study was more efficient in suppressing the endogenous CAD activity compared to the antisense construct. The resulting CAD activity in suppressed lines was also monitored over time [Wagner in prep]. Figure 6 summarises the development of the CAD activity in line cad sense1 in different tissue types over a period of approximately 18 months. This line presented an over-expression phenotype at the embryogenic tissue stage, severe suppression at the seedling stage (approximately 6 weeks old plantlets) and an easing suppression level after 18 month of development. As a general rule, it was found that the suppression level was strongest in tissues with high endogenous CAD activity. Results demonstrate that suppression can be dependent on developmental changes in the plant. The reduced level of suppression in older material may be a function of the promoter activity, or an indication for silencing of sense-overexpression constructs. Long term studies with transgenic trees may be necessary to fully understand this phenomenon.

2000"

control r - - 1 cad sen.,

cad Activity pkat/mg protein

1500"

1000

500

Embryos

Seedlings

Trees

Figure 6: Development of CAD activity in transgenic line cad s e n s e l and wildtype control G95-9 over time

219 SECONDARY CELL WALL FORMING TISSUE CULTURE CELLS AS AN EARLY ASSAY SYSTEM FOR WOOD DEVELOPMENT RELATED GENES: The analysis of foreign gene expression in transgenic trees such as shown above, is time consuming and the long generation times of conifers often make efficient screening of candidate genes impossible. The problem is evident when the large number of potentially useful genes isolated from a variety of organisms, is compared with the numbers of genes and promoter / gene constructs that are analysed in current genetic engineering programs. A possible solution is an approach where the function of genes is tested in tissue culture systems rather than developing and mature trees. Many genes could be tested simultaneously, and the technology should also be much more efficient in terms of time and space needed. Further, field-testing can be avoided and this should reduce cost for compliance with local and national regulations. We have developed an approach where radiata pine cells are grown in tissue culture, and induced to form secondary cell walls. (Figure 7). Current research concentrates on the development of a genetic engineering technology for cells developing secondary walls. These should be useful as an assay system for genes and promoters related to secondary cell wall formation. Protocols for the chemical analysis of very small amounts of cells were also developed. This involves pyrolysis followed by GC/MS, to evaluate lignin content of tissue culture samples (Figure 8).

Figure 7:

Confocal image of a tracheary element induced from P. radiata tissue cultured cells

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Spectrum 1 shows a typical chromatogram of milled wood lignin of P. radiata after pyrolysis. Spectrum 2 was obtained after pyrolysis of parenchymatous radiata pine callus cells grown on maintenance medium. The peaks, typical for lignin were significantly reduced in spectrum 2, indicating a low lignin content. After transfer of the cells to a medium inducing secondary cell wall formation, the corresponding spectrum shows typical lignin peaks (spectrum 3). The peaks found at the retention times of 29.75 and 38.28 are characteristic for guaiacol and 4-vinyl guaiacol respectively, which are breakdown products of softwood lignin.

221 CONCLUSIONS: Protocols for genetic engineering of conifers have been developed and a range of genes have been successfully transferred into embryogenic tissue. Transgenic plants have been regenerated and molecular and functional analysis have confirmed the expression and function of introduced genes. Future challenges include the development of techniques to ensure correct expression of transgenes over the life span of engineered trees, and functional analysis of many candidate genes using quick and efficient functional assays. REFERENCES

1. C. James, A. F. Krattiger, 'Global review of the field-testing and commercialisation of transgenic plants: 1986 to 1995, the first decade of crop biotechnology', International Service for the Acquisition of Agri-Biotech Applications (ISAAA) Brief No. 1. ISAAA, Ithaca, N. Y., 1996. 2. H.W. Kendall, R. Beachy, T. Eisner, F. Gould, R. Herdt, P. H. Raven, J. S. Schell, M. S. Swaminathan, 'Bioengineering of crops: report of the World Bank. Environmentally and Socially Sustainable Development Monographs', 1997. Series No. 23. World Bank, Washington, D. C. 3. M.L. Guerinot, 'the Green Revolution strikes Gold', Science 2000, 287, 7984-89. 4. C. Walter, S. D. Carson, M. I. Menzies, T. Richardson, M. Carson, 'Review: Application of biotechnology to forestry - molecular biology of conifers', World Journal of Microbiology and Biotechnology 1998, 14, 321-330. 5. M. J. Carson, R. D. Burdon, S. D. Carson, A. Firth, C. J. A. Shelbourne, T. G. Vincent, 'Realising genetic gains in production forests', in: Proceedings IUFRO working parties on Douglas fir, Lodgepole pine, Sitka and Abies spp. Breeding Genetic Resources. Session: Genetic gains in production forests. Olympia, Washington, 1989 6. C. J. A. Shelbourne, M. J. Carson, M. D. Wilcox, 'New techniques in the genetic improvement of Radiata pine. Commonwealth Forest Review, 1989, 68: 3. 7. P. H. Wilcox, H. V. Amerson, G. Kuhlman, G. H. Liu, D. M. O'Malley, R. R. Sederoff, 'Detection of a major gene from resistance to fusiform rust disease in loblolly pine by genome mapping', Proceedings of the National Academy of Science, 1996, USA 93, 3859-3864. 8. C. Walter, L. J. Grace, in: Molecular Biology of Woody Plants, Vol 2, S. M. Jain and S. C. Minocha (eds), 1999, Kluwer, pp 79-104. 9. T. Tzfira, A. Zuker, A. Altman, 'Forest-tree biotechnology; genetic transformation and its application to future forests', TIBTECH 1998, 16, 439-446. 10. D. R. Smith, 'Growth medium US patent number: 5,565,355', 1996. 11. T.M. Klein, E. D. Wolf, R. Wu, J. C. Sandford, 'High-velocity microprojectiles for delivering nucleic acids into living cells', Nature 1987, 327, 70-73. 12. A.Wagner, J. Moody, L. J. Grace, C. Walter, 'Stable transformation of Pinus radiata based on selection with Hygromycin B', New Zealand Journal of Forestry Science 1997, 27(3), 280-288. 13. C. Walter, L. J. Grace, A. Wagner, A. R. Walden, D. W. R. White, S. S. Donaldson, H. H. Hinton, R. C. Gardner, D. R. Smith, 'Stable transformation and regeneration of transgenic plants of Pinus radiata D. Don', Plant Cell Reports 1998, 17, 460-468.

222 14. C. Walter, L.J. Grace, S.S. Donaldson, J. Moody, J. E. Gemmell, S. van der Maas, H. Kvaalen, A. Loenneborg, 'An efficient biolistic transformation protocol for Picea abies (L) Karst embryogenic tissue and regeneration of transgenic plants', Canadian Journal of Forest Research 1998, 29, (10), 1539-1546. 15. S. L. Bishop-Hurley, R. J. Zabkievicz, L. J. Grace, R. C. Gardner, C. Walter, 'Conifer genetic engineering: transgenic Pinus radiata (D Don) and Picea abies (Karst) plants are resistant to the herbicide Buster', Plant Cell Reports, 2001, in print. 16. M. De Block, J. Botterman, M. Vandewiele, J. Dockx, C. Thoen, Gossel6, N. Rao Movva, C. Thompson, M. van Montagu, J. Leemans, 'Engineering herbicide resistance in plants by expression of a detoxifying enzyme', The EMBO Journal 1987, 6, 2513-2518. 17. W. Gotz, E. Dorn, E. Ebert, K. H. Leist, H. Kocher, 'HOE 39866, a new nonselective herbicide: Chemical and toxicological properties; Mode of action and metabolism', Proceedings of the Ninth conference of the Asian Pacific Weed Science Society, 1983. 18. A. R. Walden, C. Walter, R. C. Gardner, 'Genes expressed in Pinus radiata male cones include homologous to anther specific and pathogenesis response genes', Plant Physiology, 1999, 121, 1103-116. 19. N. Bechthold, G. Pelletier, 'In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration', Methods Mol B iol 1998, 82, 259-266.