ORNL/Sub/99-4500007253/1
Genetic Modification of Short Rotation Poplar Biomass Feedstock for Efficient
Conversion to Ethanol
Report Prepared by
Dr. Ronald J. Dinus, Consultant
2490 Goshen Road, Bellingham, WA 98226-9556
Telephone/FAX: 360-966-4027 email: dinus@telcomplus.net
Date published: June 2000
Research supported by U.S. Department of Energy, Office of Fuels
Development, Activity No. EB 52 03 00 0
Prepared for Bioenergy Feedstock Development Program, Environmental
Sciences Division, Oak Ridge National Laboratory managed by University of
Tennessee-Battelle, LLC for the U.S. Department of Energy under contract
DE-AC05-00OR22725
Click here for
complete report in PDF format (309k)
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Executive Summary
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Poplar breeders in the United States have focused on raising
adaptability, growth rates, and pest/stress resistance. Significant increases
have been achieved via classical selection and breeding along with intensified
cultural practices. Efficiencies have also been gained in harvesting and
handling. Considering the aforementioned improvements in productivity, the time
seems optimal for choosing feedstock qualities to modify and for undertaking
the necessary research and development. Indeed, shortened rotations resulting
from past research mean that opportunities for modifying feedstock qualities
can be exploited more easily. Many poplars are propagated clonally, thereby
permitting rapid, inexpensive propagation of valuable variants. Thus, poplars
seem an ideal venue for testing and applying techniques to improve feedstock
quality, e.g., reduced lignin content.
Opportunities for manipulating feedstock quality have long been
recognized but have largely gone unrealized because of uncertainties over which
traits to modify for what process. Quality changes can affect process
efficiency in numerous ways, but reliable information concerning impacts is
sparse. Process specialists must cooperate with breeders and growers to
identify leverage points and quantify impacts such that economic weights can be
combined with genetic information to plan and execute effective modification
strategies. The outcome could be poplar varieties better suited to process
requirements, and more likely to reduce ethanol production costs than varieties
with only increased growth.
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Measurement methods that are accurate, rapid, and inexpensive, and
that rely on small, nondestructively collected samples are needed for genetic
modification of feedstock quality. Recent advances in instrumentation, methods,
and economy have been significant and are opening the way to efficient
modification of wood chemical and physical properties and, perhaps, even to
specifying processing conditions for different feedstocks.
Classical selection and breeding can be used to reduce lignin
content, albeit slowly. Raising cellulose content is more difficult, given its
weak genetic control and negative relationship to growth. Both might be altered
more efficiently via interspecific hybridization to expand ranges of genetic
variation and clonal propagation of outstanding offspring. As an alternative,
breeding might better focus on specific gravity or dry wood production, a
highly heritable trait likely to have indirect but favorable effects on lignin
and cellulose contents. Positive outcomes would accrue in production (e.g.,
harvesting and transportation) as well as in processing (e.g., more cellulose
and less lignin). This tack seems particularly useful in that products would be
attractive to a variety of customers.
Developments in molecular biology have led to detection of genetic
markers associated with useful traits. The pace of development has been rapid,
and further improvements in utility and economy can be expected, if only from
undertakings such as the Human Genome Project. In theory, markers can be used
to screen breeding materials and/or offspring from crosses among selected
parents at early ages. Only those with desired markers would be tested in the
field or moved into production, and years of testing large numbers of
individuals with uncertain value could be avoided.
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Markers are available for identifying poplar genotypes, a
considerable aid to managing breeding programs, and for use in research on
poplar taxonomy, hybridization, and genetic variation. Such markers may soon be
extended to early determinations of gender and monitoring potential for gene
flow from transgenic trees. As concerns selection, markers available to date
have proven useful only for qualitative traits, e.g., disease resistance, or
that are highly heritable, difficult and/or expensive to measure, and of high
value. Most traits affecting feedstock quality, however, are quantitatively,
rather than qualitatively, inherited, and useful markers have proven difficult
to identify. Also of concern is unreliability over the diversity of populations
and families used in breeding, tree ages used even in short-rotation forestry,
and environments encountered in commercial programs. In consequence, most
investigators argue that marker-aided selection is not ready for application,
and that investigations should focus on identifying and dissecting quantitative
trait loci. Results could one day allow describing quantitative traits in terms
of gene numbers, individual gene effects, and modes of action as well as
discerning the basis of heterosis. In addition, candidate genes thus identified
might be used to investigate gene function and perhaps transform individuals
with other desirable traits.
Genetic transformation has been advocated as a desirable and
feasible means of improving wood physical and chemical properties. A host of
U.S. and offshore investigators are pursuing research on this front to good
effect, particularly as concerns lignin and cellulose contents. Transgenic
poplar trees with significantly reduced lignin and increased cellulose contents
have been produced and are being evaluated under controlled field conditions.
Transformants are growing faster than nonmodified controls, and are
morphologically and anatomically normal. Commercialization is only a matter of
time, perhaps 5–10 years.
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Several concerns could impede commercialization. Perhaps the major
obstacle is public concern that transgenic trees will spread so-called foreign
genes to natural populations. One means of minimizing, if not avoiding, this
risk is to render transformants sexually sterile. Research has progressed
rapidly, and genetic constructs should be ready for use in poplars within 5–10
years. Other benefits may also accrue; eliminating reproductive structures
could channel more energy, water, and nutrients into wood production. Concern
also exists about mechanisms for commercialization. Developing and applying
transformation technology is expensive. Licensing fees are also a worrisome
issue, since organizations engaged in transformation research generally patent
their creations. Timely technology transfer may require fostering growth of
independent laboratories that license genetic constructs, effect
transformation, and propagate transformants for pools of breeders, nurseries,
and/or growers.
Coproducts clearly could increase feedstock values, and trees could
be modified to produce a variety of useful materials. Even so, genetically
modifying poplars to yield valuable products, e.g., pharmaceuticals, while also
striving to increase growth and feedstock quality would complicate matters and
raise costs. The more that coproduct characteristics diverge from those
optimized for ethanol conversion, the greater the difficulty and expense. Some
coproducts, however, are compatible with ethanol production, e.g., high value
solid wood products such as molding and trim, veneers, and furniture parts.
Accordingly, this seems the best approach to development of poplar coproducts
over the near term.
Balanced support of additional research, development, and technology
transfer is essential to ensure that dividends from the aforementioned advances
are realized in a reasonable time frame. Poplar feedstock bred for rapid growth
and high dry wood substance production and genetically transformed to have
lower lignin and heightened cellulose contents stand to have tremendous impact
on ethanol production efficiency.
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Recommendations for Research, Development, and Application
Ethanol Conversion Processes
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The impacts of changes in poplar feedstock quality on process
efficiency must be clarified. Analyses to date have focused largely on costs of
growing, harvesting, handling, transportation, and storage. Few have addressed
changes in chemical and/or physical properties, which can affect process
efficiency in numerous ways and at a variety of points in the process. Breeders
and growers must know which traits to modify and how to modify them for which
process, as well as how they will be compensated before iThe impacts of changes
in poplar feedstock quality on process efficiency must be clarified. Analyses
to date have focused largely on costs of growing, harvesting, handling,
transportation, and storage. Few have addressed changes in chemical and/or
physical properties, which can affect process efficiency in numerou
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Feedstock Quality Measurement
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Efficient genetic modification requires that feedstock
characteristics be measured accurately, inexpensively, and nondestructively.
Sponsoring further research on and development of efficient instrumentation and
methods should bring yet new capabilities, heightened availability, and further
cost reductions. Fostering establishment and growth of independent testing
laboratories also seems a worthy goal.
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Classical Selection and Breeding
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Poplar breeding and growing programs seem sufficiently mature to
add objectives targeted at improving feedstock quality. Breeding efforts in
future should be designed to reduce costs and raise efficiencies at all
significant leverage points in the production and processing system.
Accomplishing this requires selection indices that combine genetic data with
economic weights associated with impacts on process leverage points.
Stimulating collaborative research, development, and application activities
amongPoplar breeding and growing programs seem sufficiently mature to add
objectives targeted
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Genetic information about important quality traits, as inferred
above, is adequate for the short term. For the long term, however, more and
better information is needed. More reliable data on extent of genetic variation
and control would be useful. Of particular concern, however, is data on
relationships among quality traits and growth, correlations across ages, and
genotype X environment interactions. Results are prerequisite to development of
the selection indices described above, and the associated research is therefore
of high priority.
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Interspecific hybridization offers one means for increasing the
generally narrow ranges of genetic variation in most quality traits, especially
lignin and cellulose contents. Future breeding efficacy demands that such
techniques be evaluated. Poplar species are quite numerous, and many can be
cross bred with relative ease. Research on expanding variability and acquiring
heterosis, therefore, holds considerable promise and commands at least moderate
priority.
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Data on genetic variation and control of hemicellulose content in
poplars are especially scarce. Recent reports on Eucalyptus species
suggest significant variation and strong control. Research on genetic
parameters therefore seems warranted, given the potential for increasing
carbohydrate content and lessening hemicellulose-lignin linkages.
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Modifying quality traits in addition to lignin, cellulose, and
specific gravity could have merit. Reduced moisture content could have effects
similar to or complementary to high specific gravity. Reducing extractives,
ash, and bark amounts, naturally low in poplars, could have favorable, though
incremental, impacts. Information is limited on the extent to and means by
which such traits can be modified. Research to acquire needed genetic data
seems warranted, but is best pursued within investigations of investigations of
the most influential traits.
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Genetic Markers and Maps
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Genetic marker technologies, even now, are being used to identify
poplar genotypes in classical selection and breeding programs. Continued
development of gender determination markers would do much to reduce costs and
increase efficiency. Extension to monitoring potential gene flow from
transgenic trees to natural populations would constitute a valuable
contribution to public acceptance of genetic transformation and, therefore,
deserves high priority.
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The fact that genetic markers currently seem useful only within
families and for a limited suite of traits greatly restricts application. Costs
of both development and application are high and could drain resources from
classical selection and breeding. Meaningful use awaits development of markers
that are generalizable across species; populations and families; environments;
ages; and generations. To best achieve these ends, future research should be
aimed at identifying and analyzing quantitative trait The fact that genetic
markers currently seem useful only within families and for a limited suite of
traits greatly restricts application. Costs of both development and application
are high and could drain resources from classical selection and breeding.
Meaningful use awaits development of markers that are generalizable across
species; populations and families; environments; a
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Attention should also be given to developing consensus genetic maps
such that markers and other genetic information can be transferred freely
across genetic backgrounds, including both poplars and other well-known model
species.
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Research to verify and validate marker applications should be given
high priority and should involve paired comparison trials, i.e., trees chosen
on the basis of marker technologies versus trees obtained via classical
selection and breeding, spanning all or most of a rotation and involving
several environments. Such trials should give particular attention to
establishing the economic feasibility of marker applications.
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Advances in marker technology are likely to be accompanied by
formation of not only more but also more sophisticated university/industrial
cooperatives and private service laboratories. Fostering growth of such
enterprises would facilitate application of any marker technologies that prove
useful to poplar breeding.
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Genetic Transformation
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Methods for poplar transformation are available, and a number of
genes have been inserted and expressed in various species and hybrids. Poplars
actually are considered models for such research, given the ease with which
many can be manipulated and regenerated from cell and tissue cultures.
Improvements in transformation efficiency and extension to wider arrays of
genotypes, however, remain important research needs.
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Genetic transformation has been used successfully to reduce lignin
and increase cellulose contents. To date, most genetic interventions in lignin
biosynthesis have been done at the level of precursor synthesis. Support of
this largely successful research should be amplified and extended to include
that on the physiology, biochemistry, and genetics of precursor transport,
storage, deposition, and polymerization. Intervention at late points in lignin
biosynthesis could have less effect on other metabolic processes than precursor
manipulation. Given the utility of low lignin content, accelerated research on
such topics seems essential.
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Information on cellulose biosynthesis is less abundant than that on
lignin. Accordingly, arguments given above for continued support of lignin
research also apply to that on cellulose. Modifying both lignin and cellulose
contents should have significantly beneficial effects on ethanol production
efficiency.
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Expression of transgenes in appropriate tissues and developmental
stages is critical to successful genetic transformation. Much transformation
research done to date has been conducted with constitutive promoters. Results
have been spectacular, but xylem specific promoters are needed if outcomes are
to be practical and stable. Research on identifying, isolating, and applying
native promoters warrants acceleration.
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Coproducts
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Profitability of ethanol conversion could be enhanced by modifying
trees to yield high value coproducts, but the modifications required to produce
many such materials do not seem feasible. Changes compatible with ethanol
processing, e.g., solid wood products, may be workable, especially over the
near-term. Stimulating production of enzymes used in ethanol processing also
seems lucrative, and research on this front deserves expansion.
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Despite initial successes, the possibility remains that
modification of lignin/cellulose biosynthesis via genetic transformation will
have detrimental effects on survival, growth, pest resistance, and/or other
tree functions over the length of a rotation. Provision for research on
stability and delayed responses is therefore important.
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Potential spread of so-called foreign genes from transgenic trees
into natural populations is a major worry to the public. Minimizing this risk
seems best accomplished by inducing sexual sterility in transformed trees.
Research is this area has been productive, but continued support is critical
for eventual realization of the substantial benefits resulting from genetic
transformation.
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Mechanisms for commercialization of transformed trees also merit
attention. Few breeders; nurseries; and growers, including some large but
research-shy companies, have the financial wherewithal to license genetic
constructs or plant materials, transform their own breeding materials, and/or
engage in joint ventures with transformation research organizations.
Accordingly, developing and stimulating means for timely technology transfer
may be necessary.
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