Department of Energy
August 12, 2013
August 12, 2013
SBIR / 2014
October 15, 2013
NOTE: The Solicitations and topics listed on this site are copies from the various SBIR agency solicitations and are not necessarily the latest and most up-to-date. For this reason, you should use the agency link listed below which will take you directly to the appropriate agency server where you can read the official version of this solicitation and download the appropriate forms and rules.
The official link for this solicitation is: http:--science.doe.gov-grants-pdf-SC_FOA_0000969.pdf
DOE's Office of Biological and Environmental Research (BER) Genomic Science Program supports DOE mission-driven fundamental research to identify the foundational principles that drive biological systems. Development of innovative approaches for sustainable bioenergy production will be accelerated by a systems biology understanding of non-food plants that can serve as dedicated cellulosic biomass feedstocks and microbes capable of deconstructing biomass into their sugar subunits and synthesizing next generation biofuels from cellulosic biomass. Genomic Science Program research also brings the -omics driven tools of modern systems biology to bear for analyzing interactions among organisms that form biological communities and between organisms and their surrounding environments. BER established three Bioenergy Research Centers (BRCs) in 2007 to pursue the basic research underlying a range of high-risk, high-return biological solutions for bioenergy applications. Advances resulting from the BRCs are providing the knowledge needed to develop new biobased products, methods, and tools that the emerging biofuel industry can use. The three Centers are based in the Southeast, the Midwest, and the West Coast, with partners across the nation. DOEs Lawrence Berkeley National Laboratory leads the DOE Joint BioEnergy Institute (JBEI) in California, DOEs Oak Ridge National Laboratory leads the BioEnergy Science Center (BESC) in Tennessee, and the University of Wisconsin-Madison leads the Great Lakes Bioenergy Research Center (GLBRC). The goal for the three BRCs is to understand better the biological mechanisms underlying biofuel production so that these mechanisms can be redesigned, improved, and used to develop novel, efficient bioenergy strategies that can be replicated on a mass scale. Many of these mechanisms form the foundation for the BRCs inventions and tech-transfer opportunities, which enable the development of technologies that are critical to the growth of a biofuels sector. Successful applicants will propose R&D that will lead to biofuel commercialization utilizing one of the TTOs listed below. Applications that propose technologies related to a TTO but that do not directly utilize a TTO will not be funded. Applications should include sufficient preliminary data and scientific detail so that expert reviewers will understand both the potential benefits and the challenges that may be encountered in carrying out the proposed research. Challenges should be identified, and solutions should be proposed that will explain how the PIs team will overcome the challenges. Applications should address potential risks such as biocontainment challenges as well as strategies to mitigate those risks.
The JBEI has developed a method for providing industrial host microbes with resistance to valuable but potentially toxic molecules, such as solvents and fuel-like compounds. Providing such tolerance is a crucial step in engineering organisms to produce desirable substances. The scientists used efflux pumps to confer resistance on E. coli and developed a library of the most effective pumps for protection against several compounds, such as geraniol, limonene, pinene, and farnesyl hexanoate. These compounds represent biogasoline, biodiesel, and biojetfuel candidates. Moreover, the method for deriving this library is applicable to determining the most effective pumps for any given host and target compound. As metabolic engineering increases the biological production titers of compounds, there is a growing need to overcome limitations posed by each compounds toxicity, inhibition of cell growth, and intracellular feedback inhibition (i.e., the slowing of production by accumulated product). Until now, these problems have been addressed primarily through combinatorial approaches, such as adaptation, genome shuffling, and random mutatgenesis. These techniques may work under certain settings but are often not transferrable to other hosts or target compounds, because they do not identify the mechanism of the resistance. On the other hand, the JBEI technology uses a known, transferrable mechanisman efflux pumpto optimize the tolerance of various hosts to any compound of interest. In several cases where the target compound is highly water immiscible, successful export of the compound from the cell can also improve product extraction from the culture.
The JBEI has developed a technology that overcomes lignin recalcitrance without negatively affecting plant growth and development. The JBEI researchers produced novel monolignols called stoppers that, when incorporated in lignin chains, reduce the incorporation of additional monolignols in the chain. (Monolignols are the building blocks of lignin). As a result, the size and degree of polymerization of the lignin polymer is reduced. This technology is designed to produce the stoppers only in lignified woody tissues, i.e., vessels and fibers, to avoid any interference with plant defense mechanisms against pathogens and UV stress.
The JBEI has developed a technology that can be used to fine-tune desirable biomass traits in plants. A key feature of the invention is the design of an artificial positive feedback loop whereby a transcription factor induces increased transcription of itself. Gene promoters are selected according to the desired outcome, for example, to improve saccharification efficiency or to raise the level of desirable hexose sugars in relation to hard-to-ferment pentoses. Some promoters can boost secondary cell wall deposition of cellulose; others can decrease deposition of lignin or hemicellulose (xylan). With similar promoter engineering, increased wax production can be directed to the epidermal layers of a plant, improving drought tolerance and efficient water use while preserving energy for increased production of biomass. This versatile technology can be used to improve crops used for biofuels and paper production; provide livestock with more digestible forage; extend the range of crops to marginal land; or produce stronger timber for construction, among other applications. Unlike other genetic engineering methods, when applied to increasing secondary cell wall deposition, the JBEI technologies alter biosynthesis in plant fibers but not in vascular tissue or leaves. Thus they do not adversely affect growth, fertility, or the fruit- or grain-bearing capacity of the plants. Because this new method involves dominant traits and uses genetic promoters that are part of conserved pathways, it will be applicable across many species, including polyploids and sterile plants. Moreover, its application does not require sequencing of the entire genome of the target plant or the presence of a particular variety or cultivar. To date, the technology has been applied to three applications: 1. Controlling Lignin Deposition: To fine-tune lignin deposition, the scientists started with a mutant of Arabidopsis that under-produces lignin in all tissues. The JBEI scientists then selectively restored lignin biosynthesis to vascular tissue but not fiber cells by expressing a wild-type allele under the regulation of a promoter that is expressed only in vascular tissue. The engineered plants were morphologically and developmentally identical to the wild type, but they had a total lignin level that was approximately 33% less. When tested with several different pretreatment methods, biomass from the engineered plants had a saccharification efficiency 1.5-2.3 times greater than that of wild types. 2. Controlling Xylan Deposition: Using a method similar to that described above, the scientists started with a mutant with a defective allele for a key gene in xylan biosynthesis. They then selectively restored expression of a normal allele to vascular tissue only. The resulting plants have a reduced amount of hemicellulose relative to cellulose. Thus, compared to wild types, these plants can be pretreated more easily for biofuel production, yield more glucose per unit of biomass, and produce fewer low-value byproducts such as pentose from biofuels production or black liquor from pulping. 3. Increasing Wood Density and Drought Tolerance: In this application, promoters are used in a positive feedback loop to increase traits such as wood density or drought tolerance. To boost wood density, JBEI scientists upregulated a transcription factor that induces the expression of genes involved in secondary cell wall synthesis in native tissues. This upregulation occurs only in fiber cells and in a manner that does not interfere with growth, cell expansion, or nutrient transport. When this technology was combined with the fine-tuning of lignin deposition, stem density was increased by almost 20% and the saccharification efficiency was two- to three-times greater than that of wild types. While boosting yields, the technology can also decrease the cost of transporting biomass from the field to the biorefinery.
Researchers at the JBEI have developed a suite of technologies to engineer feedstock plants with increased galactan content. Galactans are composed of hexoses that are easily fermented, in contrast to the hemicellulose xylan, the most abundant non-cellulosic component of biomass, which is composed of pentoses, which are difficult to ferment. By increasing galactan content, the JBEI technology has the potential to increase digestibility and yield of fermentable sugars of feedstocks. Galactan also has the potential to substitute for pentosans, polysaccharides made of pentoses, in lignocellulosic biomass. Specifically, the technology involves overexpressing Arabidopsis beta-1,4-galactan synthase genes. These can be expressed alone or in conjunction with genes involved in UDP-Galactose biosynthesis, thus increasing the availability of UDP-galactose to increase both beta-1,4-galactan in the cell walls and, more generally, the galactose component of cell wall matrix polysaccharides. Furthermore, the genes may be expressed in conjunction with genes encoding specific UDP-galactose transporters with a preference for pectin-related transport. The technology is applicable to a large number of feedstock plants including Arabidopsis, poplar, eucalyptus, rice, switchgrass, pine and others.
The JBEI has developed a technology that employs in yeast cells a trait-changing strategy that has been applied to fine tune desirable biomass deposition in plants. Crucial to this strategy is the design of a genetic switch, or transcription factor, containing an artificial positive feedback loop (APFL) within its DNA sequence. Once inserted in yeast, the switch regulates expression of desired new traits, while the embedded feedback loop induces increased transcription of the switch itself, sustaining the production of those traits. The APFL strategy was first employed successfully to fine tune secondary cell wall synthesis in the model plant Arabidopsis. The new JBEI technology extends this strategy with a feedback loop that works in yeast, which is a model organism for many types of fungal cells. By identifying key genes in yeast that can be controlled in this manner, the researchers have demonstrated that this technology for plants can be adapted to entirely different organisms. In yeast, it confers traits that can potentially transform fungal cell cultures into efficient factories for the production of chemicals ranging from biofuels to pharmaceuticals.
Researchers at the JBEI have generated a new synthetic pathway in cells to 1-deoxyxylulose-5-phosphate (DXP). Both routes allow more direct conversion of carbon to terpenoid compounds circumventing the typical, but inherently inefficient, route to DXP. The JBEI process results in the conservation of 17% of carbon being converted to terpenoid products. The novel pathways to DXP entail conversion of xylulose-5-phosphate to DXP, which circumvents the loss of CO2 and provides a higher theoretical yield, particularly if xylose is included as a carbon source. It also provides a second metabolite pool (the essential pentose phosphate pathway) for isoprenoid biosynthesis. In the case of having a mixed carbon source (for example, xylose and glucose from a hemicellulose feedstock), it is envisioned that a large fraction of the xylose component could be primarily converted to the isoprenoid product since the carbon is diverted at the entry point into metabolism (xylulose-5-P). The novel routes into the DXP pathway could also be used in conjunction with the normal DXP-mediated route to maximize flux.
JBEI has identified a rice acyltransferase gene, LOC_Os06g39390, for which increased gene expression reduces ferulic acid composition. JBEI has shown that incubation with cellulases releases more sugars from plant wall leaf material with over expression of the acyltransferase compared with wild type wall material. The plants with increased acyltransferase expression exhibit little or no significant changes in vegetative morphology and seed mass and no change in biomass compared with wildtype plants with normal levels of the acyltransferase. This is the first demonstration that increased expression of a native plant gene modifies ferulic acid cell wall content and affects cell wall digestibility. Unlike dicots, grasses incorporate the phenyl propanoid ferulic acid into the cell wall matrix polysaccharide arabinoxylan. Ferulic acid can then undergo reactive oxygen species mediated reactions to form covalent crosslinks between neighboring phenylpropanoid residues of arabinoxylan and lignin, making the biomass difficult to saccharify.
Researchers at the JBEI have developed a genetically modified host cell that increases production of fatty acids and their derivatives. Specifically, the JBEI team found that increased concentration of cellular fadR, a transcriptional factor protein that regulates genes responsible for fatty acid activation and several genes in the fatty acid degradation pathway, lowers fatty acid degradation rate and enhances unsaturated fatty acid biosynthesis, resulting in an increase in total fatty acid production. The current approach to increasing fatty acid yield is engineering thioesterase enzymes, which are responsible for converting fatty acyl-CoA into fatty acids. But this method has limited success. JBEIs regulation of fadR expression overcomes these shortcomings. Researchers introduced a plasmid that contained the fadR gene under the control of an inducible promoter and measured its effect on fatty acid production. Total fatty acid yield reached 5.2 g-l, six times more than the yield using a previous fatty acid production strain. Results correspond to approximately 75% conversion of the carbon source. Additional testing to understand fadRs mechanism indicated that fadR increases fatty acid production by changing cells overall metabolism rather than acting on one specific gene. This technology also includes a dynamic sensor-regulator system (DSRS), developed by the researchers to detect metabolic changes in microbes during the production of fatty acid-based fuels or chemicals and control the expression of the specific genes at work to improve production.
Scientists at the JBEI are developing a device that can be used to efficiently assemble DNA parts, such as genes encoding enzymes, into multiple combinations, and then screen the resulting combinatorial library to identify combinations with the most desirable properties. The device combines into one microfluidic chip all of the steps necessary for this process: assembly of DNA parts; transformation and expression of these assemblies in whole-cells or cell-free platforms; cell culture; and functional assays using techniques such as colorimetric reporters, cell labeling-sorting, fluorescence imaging, and-or spectroscopy. The JBEI invention could be used in engineering plants and enzymes for better biofuel production, or developing crops that are more resistant to pathogens or drought. The device places each component in the processDNA parts, reagents, cells, assayable markersinto discrete droplets that flow through microfluidic channels on a chip. Specific droplets are fused at designated times and locations in the channels to precisely control every reaction and incubation step. Throughout the process, each combination of biological parts is kept spatially separated from the other combinations. Thus, each droplet comes off the chip with its function assessed and its combination of parts known. In addition, droplets can be removed from the chip at different points throughout the process to obtain various intermediate products such as recombinant DNA, transformed cells, labeled cells, or protein cocktails.
Researchers at the JBEI have developed a technology to preferentially produce and extract sugars produced by the direct acid hydrolysis of biomass from an aqueous solution of ionic liquids such as 1-ethyl-3-methylimidaolium chloride. JBEI researchers have extracted over 80% of hexose and pentose sugars, indicating that the JBEI approach is a significant improvement in the field of biomass saccharification using ionic liquid. The JBEI invention uses solvent extraction technology, which is based on the chemical affinity of boronates or other organic acids to complex sugars, to extract sugars from the aqueous phase. Solvent extraction technology has been shown to successfully remove sugars from aqueous solutions in the paper pulping industry. JBEI researchers have optimized this proven technology for the recovery of sugars from biomass pretreatment processes utilizing ionic liquid pretreatment techniques also developed at JBEI.
Researchers at the JBEI have developed a pretreatment technology using ionic liquids that efficiently extracts sugars from a combination of mixed feedstocks. Any ionic liquid used for biomass pretreatment or cellulose hydrolysis by thermostable cellulase may be used. Until now, no known technology could efficiently pretreat and liberate sugars from mixed feedstock streams. The JBEI technology has been successfully demonstrated in a mixture of softwood (pine), hardwood (eucalyptus), grass (switchgrass), and agricultural (corn stover) feedstocks. In tests, sugar yield reached 0.8 mg-ml within 6 hours and 1 mg-ml after a 24-hour period. The ability to recover a higher tonnage of biomass per acre where a variety of crops are present due to intercropping, row cropping, relay cropping and similar cultivation methods has the potential to significantly lower the cost of lignocellulosic biofuel and biomaterials production. A pretreatment that is effective on a wide range of lignocellulosic feedstocks will further lower overall biorefinery costs.
Researchers at the JBEI have identified a glyco-syltransferase encoded by a rice gene that is critical for xylose biosynthesis in plant cell walls. Inhibiting the expression of the gene, Os02g22380, in bioenergy plants reduces the plants lignin content, thus reducing recalcitrance of their cell walls and increasing the amount of soluble sugar that can be extracted from them. The technology is applicable to wheat, rice, corn, switchgrass, sorghum, millet, miscanthus, sugarcane, barley, turfgrass, hemp, bamboo and Bracypodium. Mutant rice plants based on this finding demonstrated reduced height with leaves deficient in xylose as well as ferulic acid and coumaric acid, acids linked with the inhibition of microbes ability to covert sugars to fuels. Using a promoter to limit the action of this gene to non-vascular tissue could improve plant height to compare favorably with wild type plants.
Researchers at the JBEI have developed a technology to create a more efficient workflow for hydrolytic enzyme discovery and enzyme cocktail optimization by providing fast, efficient analysis of native glycans using high specificity mass spectrometry-based enzyme assays. In the JBEI technology, native substrates are used for enzyme activity screening and then tagged for efficient mass spectrometry analysis. The tagged mixture is assayed using mass spectrometry-based arrays that enable high throughput screening from microwell plates. Integrating this technology with acoustic printing has yielded extremely high throughput (three minute-384 well plate) mass spectrometry arrays. In the case of hydrolytic enzyme library screening, if incomplete hydrolysis is observed, the mixture can be screened for additional enzymes that would complete the hydrolysis. As enzymes are added, the mixture can be used to screen for other enzymes to add to the cocktail until the desired conversion of biomass is achieved.
UW-Madison GLBRC researchers have developed compositions and methods that expand the ability to make, express and identify target polypeptides, including enzymes capable of enhancing the deconstruction of biomass into fermentable sugars. This approach uses a cell-free system to express enzymes and other polypeptides in a combinatorial manner. Because the system is cell-free, the enzymes can be assayed without intermediate cloning steps or purification of the protein products. This system also is more reliable than conventional methods for analyzing biomass transformation because it does not utilize living systems, which could rapidly consume soluble sugars. This system could be used to efficiently screen enzyme combinations for effective deconstruction of biomass from different feedstocks and under different conditions.
UWMadison GLBRC researchers have developed a method of using translation coupling to quickly and reliably determine whether a given host is capable of expressing the gene product of any given gene. This method could be used to monitor protein translation efficiency in bacterial cells which can be very important in the discovery and screening work around producing microbes to ferment biomass-derived sugars to biofuels and biorenewable chemicals. This method utilizes antibiotic resistant in a way that confers resistance only if the transgene is translated into protein, allowing for more real time monitoring of recombinant protein production.
UWMadison GLBRC researchers have developed genetically modified E. coli that are capable of overproducing fatty acid precursors for medium- to long-chain hydrocarbons. The modified bacteria can be used to ferment biomass-derived sugars to fatty acids. These fatty acids can be separated from the fermentation media and subsequently used as feedstock for biofuels and biorenewable chemicals based on medium- and long-chain hydrocarbons. The modified bacteria were transformed with exogenous nucleic acids to increase the production of acyl-ACP or acyl-CoA, reduce the catabolism of fatty acid products and intermediates, and-or reduce feedback inhibition at specific points in the biosynthetic pathway.
UWMadison GLBRC researchers have developed a method to impart ethanol tolerance to yeast. The toxicity of alcohol to microbes such as yeast is a bottleneck in the production of ethanol from biomass-derived sugars through fermentation. The Elongase 1 gene encodes ELO1, an enzyme involved in the biosynthesis of unsaturated fatty acids in yeast. This gene could be incorporated into an industrial yeast strain to increase the amount of ethanol produced from biomass. An industrial fermentation yeast strain with increased ethanol tolerance could be widely applicable in reducing costs and energy consumption.
UWMadison GLBRC researchers have identified 10 genes in yeast that are involved in xylose fermentation. Efficient fermentation of biofuels and biorenewable chemicals from biomass-derived sugars would benefit from microbes that can utilize both glucose and xylose. These genes could be used to create an organism by modifying one that normally utilizes glucose to one that can ferment both xylose and glucose for enhanced biofuel production. These genes may be used in various combinations to produce useful industrial strains.
UW-Madison GLBRC researchers have identified Streptomyces sp. ActE, isolated from wood wasps, as an excellent source on enzymes capable of efficiently degrading cellulose from both pretreated and nontreated biomass. The secretome of ActE can be utilized to digest a lignocellulosic materials, resulting in feedstock that can be further used to produce biofuels or biorenewable chemicals. Specific genes have also been identified that encode enzymes capable of digesting different substrates such as xylan, chitin, cellulose, or biomass. The secretome or enzyme combinations could be developed into mixtures for efficiently accessing useful subunits of lignocellulosic biomass.
Biodiesel can substitute for conventional petroleum diesel in almost all applications. Oftentimes, use of biodiesel requires engine modification since biodiesel has different solvent properties and often degrades natural rubber. Since use of biodiesel is increasing rapidly, alternative biofuel supplies are needed to accommodate the growing demand. Michigan State Universitys GLBRC inventions provide a source and production method for novel plant oils, acetyl-triacylglycerols (ac-TAGs), with possible uses as biodiesel-like biofuel and-or as low-fat food ingredients. By combining an ac-TAG-related enzyme with a method for catalyzing large-scale synthesis of ac-TAGs, in a single crop, many benefits can be obtained. The inventions have lower viscosity and fewer calories per mole than TAGs. Pilot experiments by the inventors have achieved approximately a 60 mole percent accumulation of ac-TAGs in seed oil.
Production of alternative fuels such as biodiesel is on the rise around the world and in the U.S. due to a strong and growing desire to reduce dependency on petroleum-derived diesel fuel. The acceptance of biodiesel has been slowed due to its higher cost relative to petroleum-derived diesel. The higher cost of biodiesel is directly related to the cost of feedstock used for biodiesel production, which is often derived from crops also used for food. The displacement of food crops by energy crops causes higher food prices and is fueling a rapidly growing social, environmental, economical and political push to move away from food crops for alternative fuel production. Michigan State Universitys GLBRC technology increases the oil storage capacity in plants and could help lower biofuel feedstock costs by enabling higher oil yields per acre of feedstock crops. The invention causes plant oil to accumulate in the leaf and stem structures of the plant. Plant oil normally accumulates in seeds. By altering the function of the rigalactosyldiacylglycerol (TGD) proteins, oil accumulates in the leaf and stem structures, which have greater potential oil storage capacity. This allows for more oil to be produced per acre.
Currently, there is a great interest in using plant biomass, instead of grain, to produce ethanol. Starch can easily be used to make ethanol and would improve ethanol production from cellulose. In most plants, though, starch accumulated during the day is usually broken down each night, resulting in very little starch accumulation in the leaves. The quantity of starch present in the leaves of a plant will affect the gross yield and processing efficiency. Since currently existing high starch plants cannot degrade their starch early in their life, they do not grow as fast as plants that can degrade their starch, thus resulting in reduced yields. Michigan State Universitys GLBRC technology relates to the creation of a genetically modified crop that might be used for the production of bio-ethanol or directly as an animal feed. Specifically, this invention increases the yield of easily degraded polymers, such as starch, in plants by blocking starch degradation at a developmental point late in the life cycle of the plant. The accumulation of starch in plant leaves is controlled through transgenic expression of an RNAi construct that inhibits expression of normal starch turnover.
Production of fuels and value-added chemicals from plant biomass often requires pretreatment of the biomass. Pretreatment increases the capital equipment needs and costs of the final product. Additionally, the use of seeds as a feedstock has been controversial, with some claiming that use of seeds for chemical production is increasing the cost of food. What is needed is non-food plant material such as stems or leaves that requires less pretreatment. This GLBRC technology is a method and composition for improving the digestibility of plant biomasses by increasing the methylesterification of homogalacturonan (HG) in the plant cell wall. Methylesterification is increased via the overexpression of certain methyltransferases. The methyltransferases act on HG molecules before they are delivered to the apoplast, thus not interfering with the amount of de-esterified HG in the cell wall. Presence of highly demethylesterified pectin improves digestibility of plant biomasses while maintaining normal amounts of esterified HG prevents negative effects on the plants mechanical strength and growth. Ultimately, this technology reduces cost of pretreatment in terms of money and time, leading to more efficient biofuel or green chemical production, improved forage crops, and more easily pulped trees.
Production of alternative fuels such as biodiesel is on the rise around the world and in the U.S. due to a strong and growing desire to reduce dependency on petroleum-derived diesel fuel. The acceptance of biodiesel has been slowed due to its higher cost relative to petroleum-derived diesel. The higher cost of biodiesel is directly related to the cost of feedstock used for biodiesel production, which is often derived from crops also used for food. The displacement of food crops by energy crops causes higher food prices and is fueling a rapidly growing social, environmental, economic, and political push to move away from food crops for alternative fuel production. This GLBRC technology is plants modified to divert metabolic activity from carbohydrate storage to oil storage in vegetative tissues. Enhancement of TAG synthesis in Arabidopsis is achieved via up-regulation of the TAG biosynthesis pathway and acyltransferase over-expression. This results in enhanced energy content of plant biomass by up to 6% without any detrimental effects in Arabidopsis. Model experiments indicate that the plant material is excellent forage, with animals fed the high-TAG material showing increased weight gain. The increased energy density makes the biomass particularly suited for pyrolysis.
The unwanted dissemination of transgenic genotypes from one plant cultivar to another via pollen dispersal is a significant problem that often prevents field testing and consumer use of commercially-valuable genetically modified plants. Researchers at the University of Tennessee have developed a novel genetic system for rendering male plant pollen sterile without the concomitant cytotoxic effects of the only other pollen sterilization system currently in use. This advance is a watershed for anyone working with transgenic plants where containment of hybrid genotypes to specific plant cultivars or species is essential. This system is functional in dicots (e.g. tobacco) and is currently being tested in monocots, (e.g. switchgrass). This technology represents a major step forward in enabling innovation in fields as diverse as horticulture, agriculture, and biofuel production, permitting economically valuable greenhouse-to-field application in that it renders male plant pollen sterile, thus preventing unwanted fertilization and unwanted spread of transgenic plant genes; works in both monocots and dicots; and has no cytotoxic effects