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)
 

Executive Summary

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.

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.

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.

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.

Recommendations for Research, Development, and Application

Ethanol Conversion Processes

• 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

Feedstock Quality Measurement

• 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.

Classical Selection and Breeding

• 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
• 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.
• 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.
• 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.
• 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.

Genetic Markers and Maps

• 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.
• 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
• 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.
• 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.
• 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.

Genetic Transformation

• 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.
• 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.
• 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.
• 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.

Coproducts

• 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.
• 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.
• 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.
• 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.