Chapter34

Biotechnology, Biodiversity, and Bioethics1

M. Raj Ahuja

Introduction

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 for­est tree species with varying successes. Although this sug­gests that biotechnology methods need furthe r investigation and adaptation for application to long-lived and highly heterozygous forest trees, several recent ad­vances are promising. These include rapid clonal propa­gation, germplasm preservation, gene transfer, molecular markers, genome mapping, and the isolation, cloning, and expression of genes (Ahuja 1991a, 1993a; Ahuja et al. 1996; Bonga and Durzan 1987). Although considerable progress was achieved in some of these areas during the past de­cade, others are in the embryonic stage.

Most of the advances mentioned above are discussed elsewhere in this book. I will address clonal propagation (in vitro regeneration) and gene transfer (genetic engineer­ing) in relation to biodiversity and bioethics. Although somewhat philosophical, the pros and cons of new tech­nologies should be examined in the framework of their impact on biodiversity, long-term adaptation, and evolu­tionary survival of forest trees. Pertinent questions include: What rules and ethics should people follow for long-term biodiversity conservation? Is the application of biotech­nology, involving clonal propagation, likely to substantially reduce genetic variation in trees? Would the transfer of chimeric genes into forest trees increase the risk of unde­sirable genetic instability? Would new or foreign genes escape from commercial plantations, become established

'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. Micropropagation, genetic engineering, and molecular biology of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station. 326 p.

in natural populations, and later become maladapted? Would transgenic plants generate new diseases and pose a threat to the host ecosystem? These and related ques­tions are discussed in this chapter.

Forest Biotechnology

Forest biotechnology development, although similar to that of herbaceous plants, has subtle differences including the longevity, heterozygous nature, life cycle, and envi­ronment of forest trees. Forest trees have long generation cycles, with the vegetative phase ranging from 1 to sev­eral decades. Once trees are germinated in nature or trans­planted to plantations, they generally remain anchored in I location where they are exposed to changing environ­ments and other vagaries of nature. Some of these factors may influence their physiology and alter complex mor­phogenetic processes. During the long life cycle of trees, many genetic and epigenetic changes are probably important to maintain within-tree and within-population genetic diversity, and to assure long-term survival of indi­viduals and populations. Therefore, application of biotech­nology to woody perennials should be considered long-term goals, as opposed to short-term annual benefits appropriate to crop plants that are harvested and replaced each year. This suggests a strategy shift for genetic im­provement and modification, and for maintenance of for­est biodiversity. Two promising methods in biotechnology, involving in vitro regeneration and genetic engineering, are discussed in relation to forest tree biodiversity.

In Vitro Regeneration and Genetic ~iversity

In vitro regeneration of plants may be accomplished by exploiting one of the differentiation pathways, either from haploid or somatic tissues. The pathway from haploid tis­sues involves regeneration by organogenesis, which is the sequential differentiation (usually not simultaneously) of shoots and roots on tissues. The pathway from somatic tissues may proceed via embryogenesis, which is when

273

This file was created by scanning the printed publication.Errors identified by the software have been corrected;

however, some errors may remain.

Section V Biotechnological Applications

both shoot and root poles differentiate more or less si­multaneously on the embryogenically competent cells. Somatic embryos are structurally similar to zygotic em­bryos and, following germination, produce somatic seedlings.

In vitro regeneration by organogenesis has been achieved with many woody plant species. With most, juvenile ex­plants (e.g., embryos, cotyledons, or shoots from young plants) were used for clonal propagation (Ahuja 1988, 1991a, 1993a; Bonga and Durzan 1987; Pardos et al. 1994). Rejuvenation by tissue culture from mature tissues remains challenging in many forest tree species, although a degree of in vitro-conditioned rejuvenation was demonstrated in some species, including Populus (Ahuja 1983, 1986, 1987, 1993b; Ahuja et al. 1988; Chun 1993; Ernst 1993).

Since the first report by Winton (1968) on plantlet re­generation from callus cultures of triploid quaking aspen (P. tremuloides), several other species and hybrids of Populus were clonally propagated by tissue culture technology (Ahuja 1988, 1991a, 1993a; Bonga and Durzan 1987). By employing bud explants from 23~ to 40-year-old trees of European aspen (P. tremula) and hybrid aspen(P. tremula x P. tremuloides), clonal propagules were regenerated for multiple genotypes (Ahuja 1983, 1986, 1993b). Micro­propagation varied among the aspen trees. Bud explants from some mature aspens were unresponsive to tissue culture milieu, while in others they exhibited successful differentiation of microshoots for plant regeneration. Among other factors, regeneration potential of tissue de­pends on the explant source, tissue physiological state, time of year when tissue collection occurs, and age and geno­type of the donor tree. In forest trees, maturation state and genotype largely determine the regeneration potential. In aspens, tissues from mature-tree buds apparently do not require special rejuvenation treatments because bud ex­plants can grow and differentiate on relatively simple cul­ture media. The genotype apparently is predominant in determining the in vitro morphogenetic response in aspens. In many other hardwoods, such as beech, oak, and most conifers, growth and differentiation from tissues of ma­ture trees are strongly influenced by the maturation state. Theoretically, maturation state may also indirectly affect genetic diversity by modifying propagation qualities. In this situation, elite and mature genotypes may not con­tribute to the overall genetic diversity of the population.

Regeneration by somatic embryogenesis is another promising technique for large-scale, clonal multiplication of woody plant species. In most studies, somatic embryos were differentiated from the embryonal axes of zygotic embryos. Clonal propagation by somatic embryogenesis in several conifer and hardwood species was reported (Becwar 1993; Gupta et al. 1993; Gupta and Grob 1995; Redenbaugh and Ruzin 1989). Since somatic embryos can be grown in liquid media, it should be possible to increase their production in bioreactors for mass cloning of elite

274

genotypes (Becwar 1993; Gupta et al. 1993; Gupta and Grob 1995). For an efficient delivery system to plant somatic embryos in soil, somatic embryos were encapsulated to produce "artificial seeds" (Redenbaugh and Ruzin 1989). This technology should be available for commercial for­estry by the year 2,000 (Gupta et al. 1993).

In spite of optimism for somatic embryogenesis-medi­ated clonal forestry, several basic problems must be re­solved (Chen and Ahuja 1993). Inducing somatic embryos is difficult for many commercially important forest tree species and genotypes, or the frequency of induced so­matic embryos may be too low for practical applications. In several forest tree species, there are also problems with maturation and germination of somatic embryos and de­velopment of encapsulation protocols leading to somatic seedlings. Since somatic embryos usually go thorough a callus development phase, the relative genetic stability of somatic embryos, somatic seedlings, and somatic plants (Ahuja 1991b; Ahuja and Libby 1993) should be investi­gated at the morphologic and molecular levels before in­troduction into clonal forestry programs.

There are 2 main genetic concerns with tissue culture­derived plants, whether regenerated by organogenesis or somatic embryogenesis. The first relates to genetic insta­bility in plant tissue cultures. Plant cells grown in an arti­ficial environment of tissue culture are often under stress, and are likely to generate genetic and epigenetic mistakes; rapid mitotic divisions in the callus phase may contribute to this instability. Such genetic changes are manifested as single gene mutations, chromosome aberrations, and modi­fications of DNA methylation patterns (Kaeppler and Phillips 1993; Phillips et al. 1994). Callus cells are espe­cially vulnerable to tissue-culture induced mutations. One possible cause of genetic variation in cultured tissues is the use of high concentrations of phytohormones. In par­ticular, the auxin 2,4-dichlorophenoxyacetic acid (2,4-D) induces a high frequency of changes in chromosome struc­ture (Pavlica et al. 1991) and variation in DNA methyla­tion patterns (Phillips et al. 1994). New genetic variation in micropropagated plants may be reduced by using only low concentrations of hormones and excluding 2,4-D. Our two-step micropropagation method (Ahuja 1984), using an aspen culture medium (ACM) (Ahuja 1983), produced over 10,000 regenerants from bud explants of more than 100 European aspen and hybrid aspen mature trees. In our cultures, minimal callus formation occurred, and microshoots differentiated directly on the bud explants. Very few gross phenotypic variants were observed in these micropropagated aspens; however, undetectable gene mutations may have been present. Likewise, new genetic variation could be minimized in other woody perennials by using organogenesis or somatic embryogenesis for re­generation. Although variability will always occur, it de­pends on how much and what types are acceptable for clonal forestry programs.

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.

A second concern relates to the potential loss of genetic diversity by clonally propagating forests. A common be­lief is that clonal forestry leads to genetically uniform for­ests, which is a possibility but not a certainty (Libby 1982). Through use of sufficient numbers of pedigreed clones, genetic diversity can be effectively maintained, better con­trolled, and even enhanced in clonal forests, as compared with management options for zygotic or seedling forestry (Libby and Ahuja 1993). Through effective education and/ or regulation, common management errors in testing and deployment can be avoided in a clonal forestry program.

Genetic Engineering and Biodiversity

Although genetic engineering and hybridization by con­ventional breeding can augment genetic variation in plants, there are important differences between these 2 processes. Not only are different techniques used, but the type of genes transferred and the time required also dif­fer substantially. In terms of quick returns, the time needed to produce a new genotype can be a critical factor for its commercial exploitation. Producing and using genetically stable woody plants may require a long time span; there­fore, a conflict exists between the basic research required to generate and test reliable new genotypes, and the com­mercial desire for quick returns.

A breeder uses a backcross program to transfer desir­able genes from 1 species to another. Following hybrid­ization, the hybrid and _its progeny are backcrossed repeatedly for several generations to 1 parent species so that a single dominant gene or a small set of desirable genes is transferred. Essentially, this introgressive hybridization procedure selects for a single or a few gene(s) from 1 par­ent in the genetic background of the recurrent backcross parent. By using this approach, several useful genes for disease resistance or other traits were transferred in plants (Ahuja 1962; Ahuja and Hagen 1967; Knott 1961; Sears 1956). Transfer of such genes may involve intergeneric, interspecific, or intraspecific hybridizations. The F1 inter­generic and interspecific hybrids are usually sterile; how­ever, male sterility is more common than female. Some viable eggs may be formed in these hybrids between widely divergent species or genera; these can be used for backcrosses to the recurrent male parent. The hybrid and first backcross generations are highly heterozygous and exhibit considerable morphological and chromosomal variation. The backcross program is accompanied by se­lection of phenotypes with the desired trait(s) from the vast array of genetic variants in the backcross generations. In­traspecific hybridizations involving the transfer of a domi­nant or a recessive allele from the donor to the recurrent parent may similarly involve backcross or selfing cycles to achieve the desired goal. However, released genetic variability would be relatively less compared with the

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.

Biotechnology, Biodiversity, and Bioethics

larger variation that accompanies transfer of genes by the interspecific or intergeneric hybrid route.

Gene transfer by genetic engineering is a one-step pro­cess that bypasses the time-consuming hybridization pro­cedure. However, to establish the mode of inheritance and transmission of transgenes from male and female gametes, hybridization between transgenic and control plants is necessary. Genetic engineering uses chimeric or recombi­nant genes constructed by DNA recombinant technology. The chimeric genes are placed between the transferred DNA (T-DNA) borders of a disarmed tumor inducing (Ti) plasmid from Agrobacterium and transferred to plants mainly by an Agrobacterium-mediated gene transfer method or a biolistic DNA delivery system. Because a few genes are usually located between the T-DNA borders, at least 2 or more chimeric genes are transferred to plants during genetic transformation. In most studies, 1 select­able marker and 1 reporter gene are included. Other DNA sequences are usually included in the cassette but are typi­cally ignored for the analysis. The reporter genes may be attached to promoters from viruses, bacteria, or plants. For example, the gene encoding neomycin phosphotransferase (NPTII), which confers kanamycin resistance from bacte­ria, is put under the regulatory control of a NOS (nopaline synthase) or OCS (octapine synthase) promoter from Agrobacterium. A proteinase inhibitor II (PIN2) gene from potato conferring pest tolerance under control of 35S, a promoter region derived from the cauliflower mosaic vi­rus, or NOS was expressed in transgenic hybrid poplar (Klopfenstein et al. 1993). As mentioned, besides the hy­brid chimeric genes, other genetic sequences are often present between the T-DNA borders; these usually remain cryptic or uninvestigated. Therefore, an array of other "hitchhiking" DNA sequences in the T-DNA are trans­ferred to the host plant along with the marker and reporter genes.

Transfer of genes by hybridization is a slow process that can require years for crop plants and perhaps decades for forest trees. Introgressive hybridization involving transfer of genes from 1 species to another has been important in plant speciation and evolution. Genes that displayed adap­tive fitness were retained, while those with low fitness were gradually eliminated.· Traditional breeding has been em­ployed for genetic improvement between individuals in a species or between related species, where the genetic differ­ences are minimal. Alternatively, genetic engineering can move functional genetic traits between widely divergent organisms in a relatively short time. Transgenic plants, in­cluding Populus, were produced with functional genes from insects (e.g., luciferase gene from the firefly) and bacteria (e.g., herbicide-resistance genes), and transgenic pigs and rodents were produced with functional human genes. This phylogenetic leapfrogging offers many unique opportu­nities to create populations with novel combinations of adaptive (Regal1994) and nonadaptive genes. In ecologi-

275

Section V Biotechnological Applications

cally competent organisms, such as forest tree species, in­troduced genes of low fitness may be eliminated by natu­ral selection over the course of several generations. Most new trait combinations in transgenic plants do not seem ecologically adaptive, as with the firefly luciferase gene in tobacco or Populus. However, genes conferring resis­tance against diseases, pests, frost, or salinity might be more adaptive in selected ecosystems.

Although genetic engineering offers options for generat­ing new gene combinations that may be adaptive, an inher­ent risk of genetic instability associated with trans genes exists in herbaceous annual plants and forest trees (see chapter titled "Transgenes and Genetic Instability" in this volume). Not only is transgene expression unpredictable, but transinactivation of ectopic or endogenous genes are more frequent in transgenic plants than was anticipated. This im­plies that transgenic plants may be genetically unstable when they possess certain transgenes. Long-term testing of transgenic trees is recommended before widespread use.

.Biodiversity is defined as variety and variability among living organisms and the ecosystems in which they exist (Woodruff and Gall1992). Biodiversity may be considered at 3 hierarchial levels: 1) genetic diversity; 2) species di­versity; and 3) ecosystem diversity (Smitinand 1995). In this chapter, biodiversity is the genetic variation within populations of genetically engineered or aggressively bred species in a pl~tation-generated ecosystem. In a chang­ing environment, populations with diverse pools of ge­netic variation should be more ecologically competent, with increased response and survival during large and/ or rapid environmental change. Tree species belong to this category and have survived the vagaries of nature over millions of years. Cross-hybridization between individu­als within populations or between species and genera has enabled natural gene transfer, which has enriched gene pools and contributed to survival and evolution.

Transfer of genes between species and genera by .intro­gressive hybridization is a slow process, but natural selec­tion allows adequate time to incorporate adaptive gene combinations and reject those of inferior fitness. Phyloge­netic leapfrogging by genetic engineering, which bypasses the traditional genetic tradeoffs (Regal 1994), presents a new dimension in biodiversity for long-lived forest trees. Whether genetic engineering-mediated gene transfers con­tribute to adaptive biodiversity in forest tree species or generate more genetic instability should be examined on a long-term global basis.

Bioethics in Biotechnology

Advances in biotechnology, and especially genetic en­gineering of plants and animals, inspire reflection on their

276

short- and long-term affects on the global ecosystem. Bio­ethical concerns in forestry originated with conservation­ally minded foresters and environmentalists who had an interest in maintaining biodiversity and ecosystem stabil­ity (Coufal and Spuches 1995). Disturbances in 1 compo­nent of the forest ecosystem can cause devastating affects on other ecosystem elements. Certain ethical rules and biosafety regulations should be followed during the ap­plication of biotechnological research. Conservation of the genetic diversity of tree populations and the biodiversity of the ecosystems in which they grow is imperative. Be­cause of differences in their value judgements, those in­terested in quick returns of biotechnological applications are often in conflict with "go-slow" traditionalists.

Forest trees are long-lived, usually well adapted, and have relatively long generation cycles with vegetative phases that can extend from 1 to several decades. Most genetic changes in their genome structure, whether by clas­sical mutation or phylogenetic leapfrogging, may gener­ate instability. However, some genetic changes may have adaptive value over time. According to Tiedje et al. (1989), evaluation and regulation of transgenic organisms should be based on their biological properties, including pheno­types, rather than on the genetic techniques used to pro­duce them. Transgenic trees should be evaluated at different stages of their development by morphological, biochemical, and molecular methods to monitor transgene expression and other unexpected genetic changes. Since the potential risks of releasing transgenic poplars are dis­cussed in chapters by Raffa et al. (this volume) and Yang (this volume), they are only briefly discussed here.

The release of transgenic plants into natural ecosystems raises several questions: 1) Will transgenic crops generate new viruses and new diseases? (Falk and Bruening 1994); 2) What are the potential risks of cross-fertilization between transgenic crops and their wild relatives? (Baranger et al. 1995; Kareiva et al. 1994; Paul et al. 1995); and 3) Will transgenic trees be genetically stable on a long-term basis under field conditions? Whether transgenic crops could create new viruses and diseases is a small concern for woody plants. Some viruses may infect forest trees, while others may exist as systemic entities. Unless trees are en­gineered for resistance against a specific virus, the risk of creating a virulent new virus by genetic recombination seems minimal. Alternatively, interactions between a transgene or its product with an endogenous tree virus 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 serious biosafety and regulatory problems. In the worst scenario, the engineered plants or their hybrid derivatives may become invasive and eliminate original untrans­formed populations. Pollen can travel long distances or

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.

can be spread by pollinating insects. Forest trees are gen­erally cross pollinated; therefore, the spread of transgenic pollen to related species is considered a genuine biologi­cal concern. A recent study shows that containment of re­combinant 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 option (Strauss et al. 1995) for containing transgenes. Pre­viously, it was argued that engineering of male sterility could stimulate faster tree growth and wood production, reduce production of allergic pollen in the atmosphere, and offer new options for hybrid breeding (Strauss et al. 1995). Currently, it is thought that some of these suggestions are based on speculation. Studies are needed to determine whether wood production can be increased by decreasing floral tissues through manipulating floral-specific genes, toxin encoding genes controlled by floral-specific promot­ers, or other approaches. However, manipulating floral­specific genes, or introducing the rolC gene from Agrobacterium rhizogenes with an appropriate promoter, may produce growth abnormalities in trees. The rolC gene under control of a 355 promoter causes a degree of male sterility in tobacco and a multitude of morphological and physiological changes (Schmiilling et al. 1993). The rolC gene under control of 2 different promoters, 355 and rbcS, has also been transferred to aspen (Fladung et al. 1996). In aspen, 355-rolC transgenics exhibited much smaller, nonlanceolate, pale-green leaves than rbcS-rolC transgenic aspens, which produced pale-green leaves that were only slightly smaller than the controls. In tobacco, the 355-rolC transgene induces lanceolate and pale-green leaves. A transgene may act differently in an annual tobacco plant and a perennial Populus tree. The effects of rolC in tobacco or Populus have only been studied under greenhouse con­ditions. Therefore, whether this pleiotropic gene will be­have similarly under the 2 sets of environments cannot be predicted. Nevertheless, all strategies toward containment of transgenic pollen or lessening of floral tissues in transgenic trees should be encouraged.

Gene silencing has developed into a challenging field of study that will hopefully contribute toward understand­ing trans genes and native gene stability. Recently, instances of transgene instability were reported (Finnegan and McElroy 1994; Jorgensen 1995; Matzke and Matzke 1995). The bioethic principle requires that all research in genetic engineering of plants or animals be reported for adminis­trative authorities to conduct proper risk assessment for biosafety regulation of transgenic organisms (Giampierto 1994; Miller et al. 1995). More than 800 applications have been filed for testing genetically modified organisms in the environment, most of them in the United States (The Gene Exchange 1993). Nearly all of these tests were con­ducted on small plots, which may not reflect the entire spectrum of transgene-environment interactions. Since commercial application of transgenic crops would occur

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.

Biotechnology, Biodiversity, and Bioethics

in large-scale field facilities, extrapolations from small plots may not provide adequate risk assessment information (Seidler and Levin 1994).

Value Judgement

Recent advances in biotechnology have ushered in a new era for the genetic improvement of forest tree species. Micropropagation through organogenesis and somatic embryogenesis has been achieved in many woody plants. Biotechnological procedures have also been effectively used to preserve woody plant germplasm (Ahuja 1989, 1994; Chun 1993). Various approaches have been used to transfer foreign genes to forest trees. Genetic engineering can provide exceptional genotypes for integration into clonal forestry and zygotic forestry programs. However, concerns have been raised regarding decreased biodiversity by clonal propagation, increased risk of ge­netic instability in genetically engineered plants, and the potential escape of transgenes into natural populations. Genetic variation can be maintained and better managed by employing appropriate numbers of genetically diverse pedigreed clones in clonal forestry programs (Ahuja and Lil:?by 1993; Libby 1982). Multiclonal blocks of clones are recommended for conservation of ecosystem biodiversity.

The highest risk regarding transgenic crops is the es­cape of transgenic pollen into nature and hybridization of wild relatives (Abbot 1994;Angle 1994; Kareiva et al. 1994; Regal1994; Seidler and Levin 1994; Williamson 1994 ). Since transgenic pollen would be difficult to contain under natu­ral conditions, especially when transgenic crops are grown commercially, other strategies, including genetic manipu­lation of reproductive sterility (Strauss et al. 1995), should be explored for forest trees. Presently, more information is needed about dispersal, and the ability of transgenic pol­len to preferentially pollinate wild relatives (Baranger et al. 1995; Lefol et al. 1995; Paul et al. 1995); or possibly transgenic pollen may pose no more risk than normal pol­len from untransformed trees. Because of the relative ease of in vitro regeneration and genetic transformation, Populus can serve as a valuable model system to evaluate ques­tions regarding the ecological risks of transgenic trees. Nevertheless, environmentally compatible applications of biotechnology must be developed (Frederick and Egan 1994), and biosafety regulations and directives must be followed until the risk factors associated with genetically engineered woody plants are assessed and the adequate means to address them are found.

The relevance of bioethics in forestry is emphasized in the following quote from a recent paper by Coufal and Spuches (1995). "The value of ethics in forestry is unlim­ited, although not always plain to see. At best, ethics com-

277

Section V Biotechnological Applications

mands the power of moral authority, an authority that can often be flouted with few visible consequences, unlike the authprity of the laws of nature. If we can believe that eth­ics allows us to view the world more clearly and not be limited by our scientific perceptions of it; that good for­estry management will not be possible unless ethical di­mensions are given the same consideration as scientific and economic factors; and thus that ethics should be an integral part of all forestry curricula .... "

Literature Cited

Abbot, R.J. 1994. Ecological risks of transgenic crops. Trends in Ecology & Evolution. 9: 280-282.

Ahuja, M. R. 1962. A cytogenetic study of heritable tumors in Nicotiana species hybrids. Gen~tics. 47: 865-880.

Ahuja, M. R. 1983. Somatic cell genetics and rapid clonal propagation in aspen. Silvae Genetica. 32: 131-135.

Ahuja, M.R. 1984. A commercially feasible micropropagation method for aspen. Silvae Genetica. 33: 174-176.

Ahuja, M.R. 1986. Aspen. In: Evans, D.E.; Sharp, W.R.; Ammirato, P. J ., eds. Handbook of plant cell culture. New York: Macmillan Publishing Company: 626-651.

Ahuja, M.R. 1987. In vitro propagation of poplar and as­pen. In: Bonga, J.M.; Durzan, D.J., eds. Cell and tissue culture in forestry. Dordrecht, The Netherlands: Martinus Nijhoff, Publishers: 207-223.

Ahuja, M.R., ed. 1988. Somatic cell genetics of woody plants. Dordrecht, The Netherlands: Kluwer Academic Publishers. 225 p.

Ahuja, M.R. 1989. Storage of forest tree germ plasm at sub­zero temperatures. In: Dhawan. V., ed. Application of biotechnology in forestry and horticulture. New York: Plenum Press: 215-228.

Ahuja, M.R., ed. 1991a. Woody plant biotechnology. New York: Plenum Press. 373 p.

Ahuja, M.R. 1991b. Woody plant biotechnology: perspec­tives and limitations. In:

Ahuja, M.R., ed. Woody plant biotechnology. New York: Plenum Press: 1-10.

Ahuja, M.R., ed. 1993a. Micropropagation of woody plants. Dordrecht, The Netherlands: Kluwer Academic Publish­ers. 507 p.

Ahuja, M.R. 1993b. Regeneration and germplasm preser­vation in aspen-Populus. In: Ahuja, M.R., ed. Micropropagation of woody plants. Dordrecht, The Netherlands: Kluwer Academic Publishers: 187-194.

Ahuja, M.R. 1994. Reflections in germplasm of trees. In: Pardos, J.A.; Ahuja, M.R.; Rosello, R.E., eds. Biotech­nology of trees. Investigacion Agraria Sistemas Y Resursos Forestales, INIA, Madrid, Spain. pp. 227-233.

278

Ahuja, M.R.; Hagen, G.L. 1967. Cytogenetics of tumor-bear­ing interspecific triploid Nicotiana glauca-langsdorffii and its hybrid derivatives. J. Heredity. 58: 103-108.

Ahuja, M.R.; Libby, W.J ., eds. 1993. Clonal forestry I and II. Berlin: Springer Verlag.

Ahuja, M.R.; Krushe, D.; Melchior, G.H. 1988. Determina­tion of plantlet regeneration capacity of selected aspen clones in vitro. In: Ahuja, M.R., ed. Somatic cell genet­ics 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.

Angle, J.S. 1994. Release of transgenic plants: Biodiversity and population-level considerations. Mol. Ecol. 3: 45-50.

Baranger, A.; Chevre, A.M.; Eber, F.; Renard, M. 1995. Ef­fect of oilseed rape genotype on the spontaneous hy­bridization rate with a weedy species: an assessment of transgenic dispersal. Theor. Appl. Genet. 91: 956-963.

Becwar, M.R. 1993. Conifer somatic embryogenesis and clonal forestry. In: Ahuja, M.R.; Libby, W.J., eds. Clonal forestry I. Genetics and biotechnology. Berlin: Springer Verlag: 200-223.

Bonga, J.M.; Durzan, D.J., eds. 1987. Cell and tissue cul­ture in forestry. Vols. 1-3. Dordrecht, The Netherlands: Martinus Nijhoff Publishers.

Chen, Z.;Ahuja, M.R. 1993. Regeneration and genetic varia­tion in plant tissue cultures. In: Ahuja, M.R.; Libby, W.J ., eds. Clonal forestry I. Genetics and biotechnology. Ber­lin: Springer Verlag: 87-100.

Chun, Y.W. 1993. Clonal propagation in non-aspen poplar hybrids. In: Ahuja, M.R., ed. Micropropagation of woody plants. Dordrecht, The Netherlands: Kluwer Academic Publishers: 209-222.

Coufal, J.E.; Spuches, C.M. 1995. Ethics in forestry curricu­lum. J. Forestry. 93: 30-34.

Ernst, S.G. 1993. In vitro culture of pure species non-aspen poplars. In: Ahuja, M.R., ed. Micropropagation of woody plants. Dordrecht, The Netherlands: Kluwer Academic Publishers:195-207. ·

Falk, B.W.; Bruening, G.1994. Will transgenic crops gener­ate new viruses and new diseases? Science. 263: 1395-1396.

Finnegan, M.; McElroy, D. 1994. Transgene inactivation: plants fight back. Biotechnology. 12: 883-888.

Fladung, M.; Kumar, S.; Ahuja, M.R. 1996. Genetic trans­formation of Populus genotypes with different chimeric gene constructs: transformation efficiency and molecu­lar analysis. Transgenic Research. 5: 1-11.

Frederick, R.J.; Egan, M. 1994. Environmentally compatible applications of biotechnology. Bioscience. 44: 529-535.

Giampierto, M. 1994. Sustainability and technological de­velopment in agriculture. Bioscie~ce. 44: 677-689.

Gupta, P.K.; Grob, J.A. 1995. Somatic embryogenesis in conifers. In: Jain, S.M.; Gupta, P.K.; Newton, R.J., eds.

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.

Somatic embryogenesis in woody plants. Vol. 1. Dordrecht, The Netherlands: Kluwer Academic Publish­ers: 81-98.

Gupta, P.K.; Pullman, G.; Timmis, R.; Kreitinger, M.; Carlson, W.C.; Wetty, D. E. 1993. Forestry in the 21st cen­tury: The biotechnology of somatic embryogenesis. Bio­technology. 11: 454-459.

Jorgensen, R 1995. Cosuppression, flower color, patterns, and metastable gene expression states. Science. 268: 686-691.

Kaeppler, S.M.; Phillips, R.L. 1993. DNA methylation and tissue culture-induced variation in plants. In Vitro Cell Dev. Bioi. 29P: 125-130.

Kareiva, P.; Morris, W.; Jacobi, C.M. 1994. Studying and managing the risk of cross-fertilization between transgenic crops and wild relatives. Mol. Ecol. 3: 15-21.

Klopfenstein, N.B.; McNabb, H.S.; Hart, E.R.; Hanna, R.D.; Heuchelin, S.A.;Allen, K.K.; Shi, N.Q.; Thornburg, R.W. 1993. Transformation of Populus hybrids to study and improve pest resistance. Silvae Genetica. 42: 86-90.

Knott, D.R. 1961. The inheritance of rust resistance from Agropyron elongatum to common wheat. Can. J. Plant Sci. 41: 109-123.

Lefol, E.; Danielou, V.; Darmency, H.; Boucher, F.; Maillet, J.; Renard, M. 1995. Gene dispersal from transgenic crops. I. Growth of interspecific hybrids between oil­seed rape and the wild hoary mustards. J. Appl. Ecol­ogy. 32: 803-808.

Libby, W.J. 1982. What is a safe number of clones per plan­tation? In: Heybroek, H.M.; Stephan, B.R.; von Weissenberg, K., eds. Resistance to diseases and pests in forest trees. Wageningen, The Netherlands: Pudoc: 342-360.

Libby, W.J.;Ahuja, M.R. 1993. Micropropagation and clonal options in forestry. In: Ahuja, M.R., ed. Micropropagation of woody plants. Dordrecht, The Netherlands: Kluwer Academic Publishers: 425-442.

Matzke, M.A.; Matzke,A.J.M. 1995. How and why do plant inactivate homologous (trans)genes? Plant Physiol. 107: 679-685.

Miller, H.I.; Altman, D.W.; Barton, J.H.; Huttner, S.L. 1995. An algorithm for the oversight of field trials in economi­cally developing countries. Bio/Technology. 13:955-959.

Pavlica, M.; Nagy, B.; Papes, D. 1991. 2,4-D causes chro­mosome and chromatin abnormalities in plant cells and mutation in cultured mammalian cells. Mutat. Res. 263: 77-82.

Pardos, J.A.; Ahuja, M.R.; Rossello, E.R., eds. 1994. Bio­technology of trees. Proc. IUFRO WP Somatic Cell Ge-

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.

Biotechnology, Biodiversity, and Bioethics

netics, Valsain, Spain. lnvestigacionAgraria Sistemas Y Recursos Forestales, INIA, Madrid, Spain.

Paul, E.M.; Capiau, K.; Jacobs, M.; Dunwell, J.M. 1995. A study of gene dispersal via pollen in Nicotiana tabacum using introduced genetic markers. J. Appl. Ecology. 32: 875-882.

Phillips, R.L.; Kaeppler, S.M.; Olhoft, P. 1994. Genetic in­stability of plant tissue cultures: breakdown of normal controls. Proc. Natl. Acad. Sci. USA. 91: 5222-5226.

Redenbaugh, K.; Ruzin, S.E. 1989. Artificial seed produc­tion in forestry. In: Dhawan, V., ed. Application of Bio­technology in Forestry and Agriculture. New York: Plenum Press: 55-71.

Regal, P.J. 1994. Scientific principles for ecologically based risk assessment of transgenic organisms. Mol. Ecol. 3: 5-13.

Schmiilling, T.; Rohrig, H.; Pilz, S.; Walden, R.; Schell, J. 1993. Restoration of fertility by antisense RNA in ge­netically engineered male sterile tobacco plants. Mol. Gen. Genet. 237: 385-394.

Sears, E.R. 1956. The transfer of leaf-rust resistance from Aegiolops umbellulata to wheat. Brookhaven Symp. Bioi. 9: 1-22.

Seidler, R.J.; Levin, M. 1994. Potential ecological and non­target effects of transgenic plant gene products on agri­culture, silviculture, and natural ecosystems: general considerations. Mol. Ecol. 3: 1-3.

Smitinand, T. 1995. Overview of the status of biodiversity in tropical and temperate forest. In: Boyle, T.J.B.; Boontawee, B., eds. Measuring and monitoring biodiversity in tropi­cal and temperate forests. Center for International For­estry Research (CIFOR), Bogor, Indonesia, pp. 1-4.

Strauss, S.H.; Rottmann, W.H.; Brunner, A.M.; Sheppard, L.A. 1995. Genetic engineering of reproductive sterility in forest trees. Mol. Breed. 1: 5-26.

The Gene Exchange. 1993. Releases of genetically engi­neered organisms. The Gene Exchange 3: 12.

Tiedje, J.M.; Colwell, R.K.; Grossman, Y.L.; Hodson, R.E.; Lenski, R.E.; Mack, R.N.; Regal, P.J. 1989. The planned introduction of genetically engineered organisms: eco­logical considerations and recommendations. Ecology. 70: 298-315.

Williamson, M. 1994. Community response to transgenic plant release: predictions from British experience of in­vasive plants and feral crop plants. Mol. Ecol. 3: 75-79.

Winton, L.L. 1968. Plantlet formation in aspen tissue cul­ture. Science. 160: 1234-1235.

Woodruff, D.S.; Gall, G.A.E. 1992. Genetics and conservation. Agriculture Ecosystems and Environment 42: 53-73.

279