This file was created by scanning the printed publication. Errors identified by the software have been corrected; however, some errors may remain. Chapter 13 Transgenes and Genetic lnstability1 M. Raj Ahuja 1985; Prols and Meyer 1992). This suggests that it is diffi­ Introduction cult to predict if a transgene would be stably integrated or expressed in plants, whether herbaceous annuals or woody perennials. Does a chimeric gene integrate either at a spe­ Genetic improvement of forest trees by traditional breed­ cific site or at any available slot in the genomic landscape? ing and selection methods is a slow process. Because of Are these locations assigned by specific endogenous ge­ long life cycles, these approaches may have limited appli­ netic sequences, or does the alien gene choose any loca­ cation in forest trees. Even in annual plant species, tradi­ tion in the genome? Although the answers to these tional approaches involving hybridization and backcrosses questions are currently unknown, research in genetic en­ may take several generations for transfer of desirable gineering is progressing so rapidly that these questions genes. Genetic engineering, on the other hand, offers pros­ may soon be resolved. pects for genetic improvement at an accelerated pace in Forest trees have long life cycles, with an extended veg­ herbaceous and woody plants. etative phase ranging from 1 to several decades. Because A major problem regarding gene transfer is how to regu­ trees are firmly anchored in 1location, they are exposed to late integration and expression of foreign genes in plant changing environments over long periods that may influ­ and animal genomes. Although many promoters of viral, ence their physiology and morphogenetic processes. For bacterial, and plant origins have been tested in herbaceous long-term survival, trees must adapt to the new challenges plants, littlE: is known about their regulatory control in for­ posed by the changing environment. Under such condi­ est trees. Another problem presented by gene transfer is tions, genes conferring low fitness in trees, for example stability of the integrated transgenes in forest tree genomes. some transgenes, may be silenced or eliminated. There­ Although originally transgenes were considered stably fore, genetic transformation in woody plants must be in­ integrated and expressed, recent reports on herbaceous vestigated to understand genetic instability on a short-and crop plants suggest that transgene expression is not always long-term basis. Questions on the genetic instability of stable in the transgenic plants. Gene silencing, partial or transgenes in long-lived forest trees (Ahuja 1988a, 1988b, complete inactivation of recombinant genes or endogenous 1988c) include: Will the foreign genes be stably integrated genes, has been observed in several herbaceous transgenic and expressed immediately in a specific tissue or in all plants (Finnegan and McElroy 1994;Jorgensen 1992, 1995; tissues? Will foreign genes be expressed immediately but Kooter and Mol 1993; Matzke and Matzke 1993, 1995; remain inactive for a long time, then be re-expressed? Will Meyer 1995; Paszkowski 1994). Furthermore, transgene foreign genes undergo rearrangement or be lost during the expression levels vary among independent transformants, long vegetative phase of forest trees? Will foreign genes which may be determined by copy number and/or inte­ cause genetic changes in the host genome via a position gration transgene site (Hobbs et al. 1990, 1993; Jones et al. effect involving 1 or multiple copies of a transgene in the host genome? In this review, I will discuss published experimental data and present theoretical arguments on the stability and in­ stability of transgenes in woody plants using published reports on transgene inactivation in annual herbaceous plants. Recent investigations are also presented on 1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. transgene stability and expression in Populus and other Micropropagation, genetic engineering, and molecular biology woody plants. Because the techniques and methods for of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: genetic transformation by Agrobacterium-mediated gene U.S. Department of Agriculture, Forest Service, Rocky Mountain transfer or DNA delivery through a microprojectile-bom­ Research Station. 326 p. bardment system have been adequately described in ear- 90 Transgenes and Genetic Instability lier reviews (Binns and Thomashow 1988; Charest and Excellent regeneration systems are available in aspens Michel 1991; Chupeau et al. 1994; H ooykaas and (Ahuja 1983, 1986, 1987a, 1993) and poplars (Chun 1993; Schilperoort 1992; Jouanin et al. 1993; Kung and Wu 1993; Ernst 1993), which lend them to genetic transformation van Wordragen and Don 1992) and in other chapters in and regeneration of transgenic plants (Chupeau et al. 1994; this volume, they are not discussed here. Confalonieri et al. 1994; Devillard 1992; Donahue et al. 1994; Fillatti et al. 1987; Fladung et al. 1996; Howe et al. 1994; Klopfenstein et al. 1991, 1993; 1 ilsson et al. 1992; Tsai et a l. 1994). Genetic Transformation Chimeric Gene and Vector System The following conditions must be present before gene Several chimeric genes have been transferred to Populus transfer is possible in plants: 1) an efficient in vitro regen­ by the Agrobacterium-mediated gene transfer system, par­ eration system; 2) a vector system for the transport of the ticle acceleration Dt A delivery system (McCown et al. chimeric gene(s); and 3) an efficient gene transfer system. 1991), and electroporation of plasmids into poplar proto­ Once the transgenic plants are regenerated, there is a risk plasts (Chupeau et al. 1994). In most studies, at least 2 chi­ from genetic instability (internal), and a risk to humans, meric genes were transferred simultaneously, 1 as a animals, and the ecosystem (external). I will address the selectable marker and the other as a reporter gene. The genetic risk, whether caused by gene interaction or exter­ selectable marker genes normally used are antibiotic-re­ nal factors. sistance genes that confer kanamycin or hygromycin re­ sistance to the transformed tissue. Kanamycin resistance is conferred by a bacterial neomycin phosphotransferase Regeneration System (NPTII) gene and 2 promoters, NOS (nopaline synthase) and OCS (octapine synthase), which are used to control its Many excellent in vitro regeneration systems were de­ expression. Alternatively, several alien genes have been veloped in multiple plant species, including woody plants. used as reporter genes. These may include those that are Some of the reasons that Populus is a model system in for­ biochemically detectable, and others that have a charac­ est biote.chnology and genetic engineering include: teristic phenotypic expression. One biochemically detect­ • fast growth, able gene, the GUS (!}-glucuronidase) gene from E. coli bacteria, has been extensively used in many plant genetic • short-rotation cycles, transformation studies. In most studies, GUS expression is driven by a 35S promoter from the cauliflower mosaic • growth on marginal sites, virus (35S-GUS). GUS expression can be histochemica lly detected in transgenic plant tissues. Several different chi­ • vegetative phase normally lasts 7 to 10 years, meric gene constructs were used to confer pest and herbi­ cide tolerance in poplars. A proteinase inhibitor II (PIN2) • in vitro technology offers prospects for early matura­ gene from potato conferring pest tolerance under the con­ tion that may reduce the vegetative phase to3 to 4years, trol of a 35S or NOS promoter (35S-PIN2 or NOS-PIN2) was expressed in a transgenic hybrid poplar clone (P. alba • unisexual flowers, x P. grandidentata cv. ' Hansen' ) (Klopfenstein et al. 1991, 1993). Another bacterial mutant gene, aroA coding for 5- • in vitro regeneration systems are developed for many enolpyruvyl-shikimate 3-phosphate (EPSP), driven by a species, 35S promoter, conferred herbicide tolerance in another hybrid poplar clone (P. alba x P. grandidentata cl.' C5339') • rejuvenation from mature trees is achievable by tis­ (Donahue eta!. 1994). In an earlier study using a chimeric sue culture, gene fusion carrying the aroA gene with a MAS (mannopine synthase) promoter (pMAS-aroA) (Fi llatti et al. 1987), her­ • relatively small genome (1 / 10th the size of a pine bicide tolerance was not well expressed in hybrid poplar genome), leaves (Riemenschneider and Haissig 1991 ). As with N PTII or GUS genes, the morphological effects of PIN2 or aroA • molecular genome maps are available, and expression were undetected, except for the action of the introduced gene product. • genetic trans forma tion and regeneration of In a recent investigation, we used the rotC gene from A. transgenic plants are achievable in certain aspens and rhizogenes, under the expressive control of 35S and rbcS poplars. (from potato) promoters (355-ro/C and rbcS-ro/C), for ge- USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 91 Section II Transformation and Foreign Gene Expression netic transformation of European aspen (P. tremula) and The value of somaclonal variation in horticultural and hybrid aspen (P tremula x P. tremuloides ) clones (Ahuja and agricultural crops has been adequately described (Bajaj Fladung 1996; Fladung et al. 1996). Expression of the ro/C 1990; Evans and Sharp 1983; Evans et al. 1984; Larkin and gene is de tected at the phenotypic level by reduced leaf Scowcroft 1981; Larkin et a l. 1984). I w ill focus on the pos­ size and reduced chlorophyll content that produces a pale­ sible role of the media components, in particular hormones, green colored leaf in comparison to the dark green leaves in the induction of genetic variation during cell culture. I of untransformed aspens. Since expression of ro/C is de­ w ill also argue that using cultured cells for genetic engi­ tected at the morphological level, it offers prospects for neering adds a risk of genetic instability in transgenic continuously monitoring expression during growth and plants. Somaclonal variation apparently occurs at a higher development of transgenic plants.