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Chapter34 Biotechnology, Biodiversity, and Bioethics1

M. Raj Ahuja

in natural populations, and later become maladapted? Introduction Would transgenic plants generate new diseases and pose a threat to the host ecosystem? These and related ques­ tions are discussed in this chapter. Recent advances in plant biotechnology have enabled genetic improvement of forest trees. Basic techniques in tissue culture, genetic engineering, and molecular biology, developed in herbaceous crops, have been applied to for­ est tree species with varying successes. Although this sug­ Forest Biotechnology gests that biotechnology methods need further investigation and adaptation for application to long-lived Forest biotechnology development, although similar to and highly heterozygous forest trees, several recent ad­ that of herbaceous plants, has subtle differences including vances are promising. These include rapid clonal propa­ the longevity, heterozygous nature, life cycle, and envi­ gation, germplasm preservation, gene transfer, molecular ronment of forest trees. Forest trees have long generation markers, genome mapping, and the isolation, cloning, and cycles, with the vegetative phase ranging from 1 to sev­ expression of genes (Ahuja 1991a, 1993a; Ahuja et al. 1996; eral decades. Once trees are germinated in nature or trans­ Bonga and Durzan 1987). Although considerable progress planted to plantations, they generally remain anchored in was achieved in some of these areas during the past de­ I location where they are exposed to changing environ­ cade, others are in the embryonic stage. ments and other vagaries of nature. Some of these factors Most of the advances mentioned above are discussed may influence their physiology and alter complex mor­ elsewhere in this book. I will address clonal propagation phogenetic processes. During the long life cycle of (in vitro regeneration) and gene transfer (genetic engineer­ trees, many genetic and epigenetic changes are probably ing) in relation to biodiversity and bioethics. Although important to maintain within-tree and within-population somewhat philosophical, the pros and cons of new tech­ genetic diversity, and to assure long-term survival of indi­ nologies should be examined in the framework of their viduals and populations. Therefore, application of biotech­ impact on biodiversity, long-term adaptation, and evolu­ nology to woody perennials should be considered tionary survival of forest trees. Pertinent questions include: long-term goals, as opposed to short-term annual benefits What rules and ethics should people follow for long-term appropriate to crop plants that are harvested and replaced biodiversity conservation? Is the application of biotech­ each year. This suggests a strategy shift for genetic im­ nology, involving clonal propagation, likely to substantially provement and modification, and for maintenance of for­ reduce genetic variation in trees? Would the transfer of est biodiversity. Two promising methods in biotechnology, chimeric genes into forest trees increase the risk of unde­ involving in vitro regeneration and genetic engineering, sirable genetic instability? Would new or foreign genes are discussed in relation to forest tree biodiversity. escape from commercial plantations, become established

In Vitro Regeneration and Genetic ~iversity In vitro regeneration of plants may be accomplished by 'Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. exploiting one of the differentiation pathways, either from Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. haploid or somatic tissues. The pathway from haploid tis­ Micropropagation, genetic engineering, and molecular biology sues involves regeneration by organogenesis, which is the of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: sequential differentiation (usually not simultaneously) of U.S. Department of Agriculture, Forest Service, Rocky Mountain shoots and roots on tissues. The pathway from somatic Research Station. 326 p. tissues may proceed via embryogenesis, which is when

273 Section V Biotechnological Applications

both shoot and root poles differentiate more or less si­ genotypes (Becwar 1993; Gupta et al. 1993; Gupta and Grob multaneously on the embryogenically competent cells. 1995). For an efficient delivery system to plant somatic Somatic embryos are structurally similar to zygotic em­ embryos in soil, somatic embryos were encapsulated to bryos and, following germination, produce somatic produce "artificial seeds" (Redenbaugh and Ruzin 1989). seedlings. This technology should be available for commercial for­ In vitro regeneration by organogenesis has been achieved estry by the year 2,000 (Gupta et al. 1993). with many woody plant species. With most, juvenile ex­ In spite of optimism for somatic embryogenesis-medi­ plants (e.g., embryos, cotyledons, or shoots from young ated clonal forestry, several basic problems must be re­ plants) were used for clonal propagation (Ahuja 1988, solved (Chen and Ahuja 1993). Inducing somatic embryos 1991a, 1993a; Bonga and Durzan 1987; Pardos et al. 1994). is difficult for many commercially important forest tree Rejuvenation by tissue culture from mature tissues remains species and genotypes, or the frequency of induced so­ challenging in many forest tree species, although a degree matic embryos may be too low for practical applications. of in vitro-conditioned rejuvenation was demonstrated in In several forest tree species, there are also problems with some species, including Populus (Ahuja 1983, 1986, 1987, maturation and germination of somatic embryos and de­ 1993b; Ahuja et al. 1988; Chun 1993; Ernst 1993). velopment of encapsulation protocols leading to somatic Since the first report by Winton (1968) on plantlet re­ seedlings. Since somatic embryos usually go thorough a generation from callus cultures of triploid quaking aspen callus development phase, the relative genetic stability of (P. tremuloides), several other species and hybrids of Populus somatic embryos, somatic seedlings, and somatic plants were clonally propagated by tissue culture technology (Ahuja 1991b; Ahuja and Libby 1993) should be investi­ (Ahuja 1988, 1991a, 1993a; Bonga and Durzan 1987). By gated at the morphologic and molecular levels before in­ employing bud explants from 23~ to 40-year-old trees of troduction into clonal forestry programs. European aspen (P. tremula) and hybrid aspen(P. tremula x There are 2 main genetic concerns with tissue culture­ P. tremuloides), clonal propagules were regenerated for derived plants, whether regenerated by organogenesis or multiple genotypes (Ahuja 1983, 1986, 1993b). Micro­ somatic embryogenesis. The first relates to genetic insta­ propagation varied among the aspen trees. Bud explants bility in plant tissue cultures. Plant cells grown in an arti­ from some mature aspens were unresponsive to tissue ficial environment of tissue culture are often under stress, culture milieu, while in others they exhibited successful and are likely to generate genetic and epigenetic mistakes; differentiation of microshoots for plant regeneration. rapid mitotic divisions in the callus phase may contribute Among other factors, regeneration potential of tissue de­ to this instability. Such genetic changes are manifested as pends on the explant source, tissue physiological state, time single gene mutations, chromosome aberrations, and modi­ of year when tissue collection occurs, and age and geno­ fications of DNA methylation patterns (Kaeppler and type of the donor tree. In forest trees, maturation state and Phillips 1993; Phillips et al. 1994). Callus cells are espe­ genotype largely determine the regeneration potential. In cially vulnerable to tissue-culture induced mutations. One aspens, tissues from mature-tree buds apparently do not possible cause of genetic variation in cultured tissues is require special rejuvenation treatments because bud ex­ the use of high concentrations of phytohormones. In par­ plants can grow and differentiate on relatively simple cul­ ticular, the auxin 2,4-dichlorophenoxyacetic acid (2,4-D) ture media. The genotype apparently is predominant in induces a high frequency of changes in chromosome struc­ determining the in vitro morphogenetic response in aspens. ture (Pavlica et al. 1991) and variation in DNA methyla­ In many other hardwoods, such as beech, oak, and most tion patterns (Phillips et al. 1994). New genetic variation conifers, growth and differentiation from tissues of ma­ in micropropagated plants may be reduced by using only ture trees are strongly influenced by the maturation state. low concentrations of hormones and excluding 2,4-D. Our Theoretically, maturation state may also indirectly affect two-step micropropagation method (Ahuja 1984), using genetic diversity by modifying propagation qualities. In an aspen culture medium (ACM) (Ahuja 1983), produced this situation, elite and mature genotypes may not con­ over 10,000 regenerants from bud explants of more than tribute to the overall genetic diversity of the population. 100 European aspen and hybrid aspen mature trees. In our Regeneration by somatic embryogenesis is another cultures, minimal callus formation occurred, and promising technique for large-scale, clonal multiplication microshoots differentiated directly on the bud explants. of woody plant species. In most studies, somatic embryos Very few gross phenotypic variants were observed in these were differentiated from the embryonal axes of zygotic micropropagated aspens; however, undetectable gene embryos. Clonal propagation by somatic embryogenesis mutations may have been present. Likewise, new genetic in several conifer and hardwood species was reported variation could be minimized in other woody perennials (Becwar 1993; Gupta et al. 1993; Gupta and Grob 1995; by using organogenesis or somatic embryogenesis for re­ Redenbaugh and Ruzin 1989). Since somatic embryos can generation. Although variability will always occur, it de­ be grown in liquid media, it should be possible to increase pends on how much and what types are acceptable for their production in bioreactors for mass cloning of elite clonal forestry programs.

274 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. Biotechnology, Biodiversity, and Bioethics

A second concern relates to the potential loss of genetic larger variation that accompanies transfer of genes by the diversity by clonally propagating forests. A common be­ interspecific or intergeneric hybrid route. lief is that clonal forestry leads to genetically uniform for­ Gene transfer by genetic engineering is a one-step pro­ ests, which is a possibility but not a certainty (Libby 1982). cess that bypasses the time-consuming hybridization pro­ Through use of sufficient numbers of pedigreed clones, cedure. However, to establish the mode of inheritance and genetic diversity can be effectively maintained, better con­ transmission of transgenes from male and female gametes, trolled, and even enhanced in clonal forests, as compared hybridization between transgenic and control plants is with management options for zygotic or seedling forestry necessary. Genetic engineering uses chimeric or recombi­ (Libby and Ahuja 1993). Through effective education and/ nant genes constructed by DNA recombinant technology. or regulation, common management errors in testing and The chimeric genes are placed between the transferred deployment can be avoided in a clonal forestry program. DNA (T-DNA) borders of a disarmed tumor inducing (Ti) plasmid from Agrobacterium and transferred to plants mainly by an Agrobacterium-mediated gene transfer Genetic Engineering and Biodiversity method or a biolistic DNA delivery system. Because a few genes are usually located between the T-DNA borders, at Although genetic engineering and hybridization by con­ least 2 or more chimeric genes are transferred to plants ventional breeding can augment genetic variation in during genetic transformation. In most studies, 1 select­ plants, there are important differences between these 2 able marker and 1 reporter gene are included. Other DNA processes. Not only are different techniques used, but the sequences are usually included in the cassette but are typi­ type of genes transferred and the time required also dif­ cally ignored for the analysis. The reporter genes may be fer substantially. In terms of quick returns, the time needed attached to promoters from viruses, bacteria, or plants. For to produce a new genotype can be a critical factor for its example, the gene encoding neomycin phosphotransferase commercial exploitation. Producing and using genetically (NPTII), which confers kanamycin resistance from bacte­ stable woody plants may require a long time span; there­ ria, is put under the regulatory control of a NOS (nopaline fore, a conflict exists between the basic research required synthase) or OCS (octapine synthase) promoter from to generate and test reliable new genotypes, and the com­ Agrobacterium. A proteinase inhibitor II (PIN2) gene from mercial desire for quick returns. potato conferring pest tolerance under control of 35S, a A breeder uses a backcross program to transfer desir­ promoter region derived from the cauliflower mosaic vi­ able genes from 1 species to another. Following hybrid­ rus, or NOS was expressed in transgenic hybrid poplar ization, the hybrid and _its progeny are backcrossed (Klopfenstein et al. 1993). As mentioned, besides the hy­ repeatedly for several generations to 1 parent species so brid chimeric genes, other genetic sequences are often that a single dominant gene or a small set of desirable genes present between the T-DNA borders; these usually remain is transferred. Essentially, this introgressive hybridization cryptic or uninvestigated. Therefore, an array of other procedure selects for a single or a few gene(s) from 1 par­ "hitchhiking" DNA sequences in the T-DNA are trans­ ent in the genetic background of the recurrent backcross ferred to the host plant along with the marker and reporter parent. By using this approach, several useful genes for genes. disease resistance or other traits were transferred in plants Transfer of genes by hybridization is a slow process that (Ahuja 1962; Ahuja and Hagen 1967; Knott 1961; Sears can require years for crop plants and perhaps decades for 1956). Transfer of such genes may involve intergeneric, forest trees. Introgressive hybridization involving transfer interspecific, or intraspecific hybridizations. The F1 inter­ of genes from 1 species to another has been important in generic and interspecific hybrids are usually sterile; how­ plant speciation and evolution. Genes that displayed adap­ ever, male sterility is more common than female. Some tive fitness were retained, while those with low fitness were viable eggs may be formed in these hybrids between gradually eliminated.· Traditional breeding has been em­ widely divergent species or genera; these can be used for ployed for genetic improvement between individuals in a backcrosses to the recurrent male parent. The hybrid and species or between related species, where the genetic differ­ first backcross generations are highly heterozygous and ences are minimal. Alternatively, genetic engineering can exhibit considerable morphological and chromosomal move functional genetic traits between widely divergent variation. The backcross program is accompanied by se­ organisms in a relatively short time. Transgenic plants, in­ lection of phenotypes with the desired trait(s) from the vast cluding Populus, were produced with functional genes from array of genetic variants in the backcross generations. In­ insects (e.g., luciferase gene from the firefly) and bacteria traspecific hybridizations involving the transfer of a domi­ (e.g., herbicide-resistance genes), and transgenic pigs and nant or a recessive allele from the donor to the recurrent rodents were produced with functional human genes. This parent may similarly involve backcross or selfing cycles phylogenetic leapfrogging offers many unique opportu­ to achieve the desired goal. However, released genetic nities to create populations with novel combinations of variability would be relatively less compared with the adaptive (Regal1994) and nonadaptive genes. In ecologi-

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 275 Section V Biotechnological Applications

cally competent organisms, such as forest tree species, in­ short- and long-term affects on the global ecosystem. Bio­ troduced genes of low fitness may be eliminated by natu­ ethical concerns in forestry originated with conservation­ ral selection over the course of several generations. Most ally minded foresters and environmentalists who had an new trait combinations in transgenic plants do not seem interest in maintaining biodiversity and ecosystem stabil­ ecologically adaptive, as with the firefly luciferase gene ity (Coufal and Spuches 1995). Disturbances in 1 compo­ in tobacco or Populus. However, genes conferring resis­ nent of the forest ecosystem can cause devastating affects tance against diseases, pests, frost, or salinity might be on other ecosystem elements. Certain ethical rules and more adaptive in selected ecosystems. biosafety regulations should be followed during the ap­ Although genetic engineering offers options for generat­ plication of biotechnological research. Conservation of the ing new gene combinations that may be adaptive, an inher­ genetic diversity of tree populations and the biodiversity ent risk of genetic instability associated with trans genes exists of the ecosystems in which they grow is imperative. Be­ in herbaceous annual plants and forest trees (see chapter cause of differences in their value judgements, those in­ titled "Transgenes and Genetic Instability" in this volume). terested in quick returns of biotechnological applications Not only is transgene expression unpredictable, but are often in conflict with "go-slow" traditionalists. transinactivation of ectopic or endogenous genes are more Forest trees are long-lived, usually well adapted, and frequent in transgenic plants than was anticipated. This im­ have relatively long generation cycles with vegetative plies that transgenic plants may be genetically unstable when phases that can extend from 1 to several decades. Most they possess certain transgenes. Long-term testing of genetic changes in their genome structure, whether by clas­ transgenic trees is recommended before widespread use. sical mutation or phylogenetic leapfrogging, may gener­ .Biodiversity is defined as variety and variability among ate instability. However, some genetic changes may have living organisms and the ecosystems in which they exist adaptive value over time. According to Tiedje et al. (1989), (Woodruff and Gall1992). Biodiversity may be considered evaluation and regulation of transgenic organisms should at 3 hierarchial levels: 1) genetic diversity; 2) species di­ be based on their biological properties, including pheno­ versity; and 3) ecosystem diversity (Smitinand 1995). In types, rather than on the genetic techniques used to pro­ this chapter, biodiversity is the genetic variation within duce them. Transgenic trees should be evaluated at populations of genetically engineered or aggressively bred different stages of their development by morphological, species in a pl~tation-generated ecosystem. In a chang­ biochemical, and molecular methods to monitor transgene ing environment, populations with diverse pools of ge­ expression and other unexpected genetic changes. Since netic variation should be more ecologically competent, the potential risks of releasing transgenic poplars are dis­ with increased response and survival during large and/ cussed in chapters by Raffa et al. (this volume) and Yang or rapid environmental change. Tree species belong to this (this volume), they are only briefly discussed here. category and have survived the vagaries of nature over The release of transgenic plants into natural ecosystems millions of years. Cross-hybridization between individu­ raises several questions: 1) Will transgenic crops generate als within populations or between species and genera has new viruses and new diseases? (Falk and Bruening 1994); enabled natural gene transfer, which has enriched gene 2) What are the potential risks of cross-fertilization between pools and contributed to survival and evolution. transgenic crops and their wild relatives? (Baranger et al. Transfer of genes between species and genera by .intro­ 1995; Kareiva et al. 1994; Paul et al. 1995); and 3) Will gressive hybridization is a slow process, but natural selec­ transgenic trees be genetically stable on a long-term basis tion allows adequate time to incorporate adaptive gene under field conditions? Whether transgenic crops could combinations and reject those of inferior fitness. Phyloge­ create new viruses and diseases is a small concern for netic leapfrogging by genetic engineering, which bypasses woody plants. Some viruses may infect forest trees, while the traditional genetic tradeoffs (Regal 1994), presents a others may exist as systemic entities. Unless trees are en­ new dimension in biodiversity for long-lived forest trees. gineered for resistance against a specific virus, the risk of Whether genetic engineering-mediated gene transfers con­ creating a virulent new virus by genetic recombination tribute to adaptive biodiversity in forest tree species or seems minimal. Alternatively, interactions between a generate more genetic instability should be examined on transgene or its product with an endogenous tree virus a long-term global basis. might produce a mutant virus that may be harmful to the host. Therefore, it is difficult to predict if transgenic plants could become a source of new viruses. The second concern, escape of transgenic pollen into the environment and cross-fertilization of wild relatives, poses Bioethics in Biotechnology serious biosafety and regulatory problems. In the worst scenario, the engineered plants or their hybrid derivatives Advances in biotechnology, and especially genetic en­ may become invasive and eliminate original untrans­ gineering of plants and animals, inspire reflection on their formed populations. Pollen can travel long distances or

276 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. Biotechnology, Biodiversity, and Bioethics

can be spread by pollinating insects. Forest trees are gen­ in large-scale field facilities, extrapolations from small plots erally cross pollinated; therefore, the spread of transgenic may not provide adequate risk assessment information pollen to related species is considered a genuine biologi­ (Seidler and Levin 1994). cal concern. A recent study shows that containment of re­ combinant pollen or transgenes is unlikely if physical isolation is the only strategy (Kareiva et al. 1994). Genetic engineering of reproductive sterility in trees is another Value Judgement option (Strauss et al. 1995) for containing transgenes. Pre­ viously, it was argued that engineering of male sterility could stimulate faster tree growth and wood production, Recent advances in biotechnology have ushered in a new reduce production of allergic pollen in the atmosphere, and era for the genetic improvement of forest tree species. offer new options for hybrid breeding (Strauss et al. 1995). Micropropagation through organogenesis and somatic Currently, it is thought that some of these suggestions are embryogenesis has been achieved in many woody plants. based on speculation. Studies are needed to determine Biotechnological procedures have also been effectively whether wood production can be increased by decreasing used to preserve woody plant germplasm (Ahuja 1989, floral tissues through manipulating floral-specific genes, 1994; Chun 1993). Various approaches have been used to toxin encoding genes controlled by floral-specific promot­ transfer foreign genes to forest trees. Genetic engineering ers, or other approaches. However, manipulating floral­ can provide exceptional genotypes for integration into specific genes, or introducing the rolC gene from clonal forestry and zygotic forestry programs. However, Agrobacterium rhizogenes with an appropriate promoter, concerns have been raised regarding decreased may produce growth abnormalities in trees. The rolC gene biodiversity by clonal propagation, increased risk of ge­ under control of a 355 promoter causes a degree of male netic instability in genetically engineered plants, and the sterility in tobacco and a multitude of morphological and potential escape of transgenes into natural populations. physiological changes (Schmiilling et al. 1993). The rolC Genetic variation can be maintained and better managed gene under control of 2 different promoters, 355 and rbcS, by employing appropriate numbers of genetically diverse has also been transferred to aspen (Fladung et al. 1996). In pedigreed clones in clonal forestry programs (Ahuja and aspen, 355-rolC transgenics exhibited much smaller, Lil:?by 1993; Libby 1982). Multiclonal blocks of clones are nonlanceolate, pale-green leaves than rbcS-rolC transgenic recommended for conservation of ecosystem biodiversity. aspens, which produced pale-green leaves that were only The highest risk regarding transgenic crops is the es­ slightly smaller than the controls. In tobacco, the 355-rolC cape of transgenic pollen into nature and hybridization of transgene induces lanceolate and pale-green leaves. A wild relatives (Abbot 1994;Angle 1994; Kareiva et al. 1994; transgene may act differently in an annual tobacco plant Regal1994; Seidler and Levin 1994; Williamson 1994 ). Since and a perennial Populus tree. The effects of rolC in tobacco transgenic pollen would be difficult to contain under natu­ or Populus have only been studied under greenhouse con­ ral conditions, especially when transgenic crops are grown ditions. Therefore, whether this pleiotropic gene will be­ commercially, other strategies, including genetic manipu­ have similarly under the 2 sets of environments cannot be lation of reproductive sterility (Strauss et al. 1995), should predicted. Nevertheless, all strategies toward containment be explored for forest trees. Presently, more information is of transgenic pollen or lessening of floral tissues in needed about dispersal, and the ability of transgenic pol­ transgenic trees should be encouraged. len to preferentially pollinate wild relatives (Baranger et Gene silencing has developed into a challenging field al. 1995; Lefol et al. 1995; Paul et al. 1995); or possibly of study that will hopefully contribute toward understand­ transgenic pollen may pose no more risk than normal pol­ ing trans genes and native gene stability. Recently, instances len from untransformed trees. Because of the relative ease of transgene instability were reported (Finnegan and of in vitro regeneration and genetic transformation, Populus McElroy 1994; Jorgensen 1995; Matzke and Matzke 1995). can serve as a valuable model system to evaluate ques­ The bioethic principle requires that all research in genetic tions regarding the ecological risks of transgenic trees. engineering of plants or animals be reported for adminis­ Nevertheless, environmentally compatible applications of trative authorities to conduct proper risk assessment for biotechnology must be developed (Frederick and Egan biosafety regulation of transgenic organisms (Giampierto 1994), and biosafety regulations and directives must be 1994; Miller et al. 1995). More than 800 applications have followed until the risk factors associated with genetically been filed for testing genetically modified organisms in engineered woody plants are assessed and the adequate the environment, most of them in the United States (The means to address them are found. Gene Exchange 1993). Nearly all of these tests were con­ The relevance of bioethics in forestry is emphasized in ducted on small plots, which may not reflect the entire the following quote from a recent paper by Coufal and spectrum of transgene-environment interactions. Since Spuches (1995). "The value of ethics in forestry is unlim­ commercial application of transgenic crops would occur ited, although not always plain to see. At best, ethics com-

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 277 Section V Biotechnological Applications

mands the power of moral authority, an authority that can Ahuja, M.R.; Hagen, G.L. 1967. Cytogenetics of tumor-bear­ often be flouted with few visible consequences, unlike the ing interspecific triploid Nicotiana glauca-langsdorffii and authprity of the laws of nature. If we can believe that eth­ its hybrid derivatives. J. Heredity. 58: 103-108. ics allows us to view the world more clearly and not be Ahuja, M.R.; Libby, W.J ., eds. 1993. Clonal forestry I and II. limited by our scientific perceptions of it; that good for­ Berlin: Springer Verlag. estry management will not be possible unless ethical di­ Ahuja, M.R.; Krushe, D.; Melchior, G.H. 1988. Determina­ mensions are given the same consideration as scientific tion of plantlet regeneration capacity of selected aspen and economic factors; and thus that ethics should be an clones in vitro. In: Ahuja, M.R., ed. Somatic cell genet­ integral part of all forestry curricula ...." ics of woody ·plants. Dordrecht, The Netherlands: Kluwer Academic Publishers: 127-135. Ahuja, M.R.; Boerjan, W.; Neale, D.B., eds. 1996. Somatic cell genetics and molecular genetics of trees. Dordrecht, The Netherlands: Kluwer Academic Publishers. 287 p. Literature Cited Angle, J.S. 1994. Release of transgenic plants: Biodiversity and population-level considerations. Mol. Ecol. 3: 45-50. Abbot, R.J. 1994. Ecological risks of transgenic crops. Trends Baranger, A.; Chevre, A.M.; Eber, F.; Renard, M. 1995. Ef­ in Ecology & Evolution. 9: 280-282. fect of oilseed rape genotype on the spontaneous hy­ Ahuja, M. R. 1962. A cytogenetic study of heritable tumors bridization rate with a weedy species: an assessment of in Nicotiana species hybrids. Gen~tics. 47: 865-880. transgenic dispersal. Theor. Appl. Genet. 91: 956-963. Ahuja, M. R. 1983. Somatic cell genetics and rapid clonal Becwar, M.R. 1993. Conifer somatic embryogenesis and propagation in aspen. Silvae Genetica. 32: 131-135. clonal forestry. In: Ahuja, M.R.; Libby, W.J., eds. Clonal Ahuja, M.R. 1984. A commercially feasible forestry I. Genetics and biotechnology. Berlin: Springer micropropagation method for aspen. Silvae Genetica. Verlag: 200-223. 33: 174-176. Bonga, J.M.; Durzan, D.J., eds. 1987. Cell and tissue cul­ Ahuja, M.R. 1986. Aspen. In: Evans, D.E.; Sharp, W.R.; ture in forestry. Vols. 1-3. Dordrecht, The Netherlands: Ammirato, P. J., eds. Handbook of plant cell culture. New Martinus Nijhoff Publishers. York: Macmillan Publishing Company: 626-651. Chen, Z.;Ahuja, M.R. 1993. Regeneration and genetic varia­ Ahuja, M.R. 1987. In vitro propagation of poplar and as­ tion in plant tissue cultures. In: Ahuja, M.R.; Libby, W.J ., pen. In: Bonga, J.M.; Durzan, D.J., eds. Cell and tissue eds. Clonal forestry I. Genetics and biotechnology. Ber­ culture in forestry. Dordrecht, The Netherlands: lin: Springer Verlag: 87-100. Martinus Nijhoff, Publishers: 207-223. Chun, Y.W. 1993. 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In: ate new viruses and new diseases? Science. 263: 1395- Ahuja, M.R., ed. Woody plant biotechnology. New York: 1396. Plenum Press: 1-10. Finnegan, M.; McElroy, D. 1994. Transgene inactivation: Ahuja, M.R., ed. 1993a. Micropropagation of woody plants. plants fight back. Biotechnology. 12: 883-888. Dordrecht, The Netherlands: Kluwer Academic Publish­ Fladung, M.; Kumar, S.; Ahuja, M.R. 1996. Genetic trans­ ers. 507 p. formation of Populus genotypes with different chimeric Ahuja, M.R. 1993b. Regeneration and germplasm preser­ gene constructs: transformation efficiency and molecu­ vation in aspen-Populus. In: Ahuja, M.R., ed. lar analysis. Transgenic Research. 5: 1-11. Micropropagation of woody plants. Dordrecht, The Frederick, R.J.; Egan, M. 1994. Environmentally compatible Netherlands: Kluwer Academic Publishers: 187-194. applications of biotechnology. Bioscience. 44: 529-535. Ahuja, M.R. 1994. Reflections in germplasm of trees. In: Giampierto, M. 1994. 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