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Chapter34
Biotechnology, Biodiversity, and Bioethics 1
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 m ethods 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 w ill 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
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
'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 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
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.
Biotechnology, Biodiversity, and Bioethics
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.
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 transgenes 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.
Biotechnology, Biodiversity, and Bioethics
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.
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: 13951396.
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.
Biotechnology, Biodiversity, and Bioethics
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: 686691.
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.
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