Progress 06/24/08 to 06/23/12
Outputs
OUTPUTS: Objective I. We analyzed several genes from rice, switchgrass and sorghum to identify those that would serve as suitable targets for manipulation in order to generate high-energy biomass. We also succeeded in generating hundreds of transgenic rice and switchgrass lines, each representing an independent transformation event with our plant transformation vectors. We have developed and validated a method for quantifying mRNA accumulation and starch content in tissues derived from these plants. We have applied these methods to determine which of the transgenic lines have altered levels of expression of the target genes and which accumulate the highest levels of vegetative starch. Objective II. Using the starch quantitation assay that we developed, we were able to identify several transgenic plant lines that accumulate higher levels of starch than do control lines. We were also able to monitor changes in starch accumulation in individual plants at multiple timepoints over a period of several weeks, including young tissue, early inflorescence and senescence. Objective III. We evaluated 4 different pretreatment and hydrolysis strategies for converting high-starch biomass into fermentable sugars. We have also used the resulting biomass in simultaneous saccharification and fermentation processes to produce ethanol from the resulting sugars. Objective IV. We have generated and tested numerous transgenic switchgrass lines. In each case, only plants for which we were able to confirm the presence of the transgene were carried forward, grown to maturity and assayed for starch accumulation. We have developed sorghum transformation vectors as well as the transformation and regeneration protocols that are required for this crop. We have evaluated four different sorghum cultivars for ease of transformation. We also evaluated the influence of growth conditions, age of the recipient tissue, culture conditions, and selectable markers in our transformation/regeneration protocols. We have generated transgenic sorghum plants using our transformation vectors. PARTICIPANTS: Several scientists, engineers, and technicians contributed to this research at Agrivida. Among these were Dr. Oleg Bougri, Dr. Alvar Carlson, Sarah Dohle, Cairn Ely, Jonas Emery, Christine Feulner, Valerie Graves, Dr. Michael Lanahan, Dr. Philip Lessard, Keith McKinney, Meghan Moriarty, Dr. R. Michael Raab, Marjory Rockwell, Aneliya Sakaeva, Dr. Vladimir Samoylov, Dr. Jeremy Schley Johnson, Dr. Binzhang Shen, Jeff Smith, Ada Vaill, and Katie White. Over the course of this research, several of the staff had to be trained in new techniques to accommodate the requirements of this project. A number of junior members of the staff have since left the company, for example to begin graduate school, where they have employed these newly developed skills. At the University of Illinois, Prof. Vijay Singh of the Center for Advanced Bioenergy Research at the University of Illinois, Urbana-Champaign, trained his students in carrying out detailed compositional and fermentation analyses of the biomass that was generated during this project. TARGET AUDIENCES: The outcomes of this research are of value to organizations that are developing cellulosic ethanol production strategies. Producing ethanol from cellulosic biomass requires that fermentable sugars be extracted from biomass, and that these sugars then be converted into ethanol. High starch biomass makes it possible to extract more fermentable sugars from the cellulosic material, which enables higher ethanol yields and lower costs. Organizations that are developing high-energy feed based on forage crops such as sorghum, rice straw, or corn stover would also be able to recognize the value of crops with high vegetative starch content. Higher levels of starch in forage provide more easily-digested carbohydrate in the feed, providing a cheap source of energy for ruminant and non-ruminant animals. PROJECT MODIFICATIONS: Early successes with transgenic rice made it unnecessary for us to continue the strategy of identifying mutant rice lines among publicly available collections to pursue this work. Also, developing chemically-inducible promoters to drive expression of the transgenes in this project proved to be unnecessary, as plants that expressed the transgenes from existing promoters showed strong starch accumulation characteristics with no noticeable effect on the growth, fertility or health of the monocot plants.
Impacts
Objective I. We were able to identify new DNA sequences for target genes in rice, in some cases correcting errors or incomplete records that were available in public databases. Objective II. We were able to identify transgenic rice lines that accumulate up to 10 times as much vegetative starch as the average among control rice lines. To determine whether the changes in starch accumulation can be attributed to suppression of mRNA levels in transgenic tissues, we developed real-time RT-PCR assays for the mRNAs from each of the target genes. We were able to show an inverse correlation between mRNA levels and starch accumulation, which supports the original hypothesis that suppressing expression of specific genes would lead to higher starch phenotypes. We have generated seed from all of the rice lines generated. We have found that the high-starch trait is stably inherited among progeny, and we have generated hundreds of high-starch rice plants for analysis. Objective III. We have found that more fermentable sugars could be recovered from the straw of high starch rice lines than from the straw of control lines, regardless of the pretreatment and hydrolysis strategy that is employed. In fermentation studies, we determined that the higher recovery of fermentable sugars supported higher ethanol production. These findings were corroborated by external collaborators. Objective IV. We were able to generate transgenic switchgrass plants that, on average, accumulate 2 to 3-times as much starch as do control lines.
Publications
- Lessard, P. "Altering Sorghum for Improved Biofuels Production" Proceedings from the 7th Annual 2010 World Congress on Industrial Biotechnology and Bioprocessing (June 27-30, 2010).
- US Patent Application "Plants with altered Levels of Vegetative Starch". (Submitted 2010)
- US Patent Application "Methods And Compositions for Processing Biomass with Elevated Levels of Starch". (In preparation, 2012)
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Progress 06/24/10 to 06/23/11
Outputs
OUTPUTS: We identified several candidate genes that are implicated in mobilizing starch in vegetative tissues during the light periods. Through quantitative real-time PCR analyses (qRT-PCR), we showed that some of these genes displayed diurnal oscillations in the levels of their corresponding mRNAs, further bolstering the inference that these genes are involved in regulating starch accumulation and turnover. We then assembled a series of transgenes to express interfering RNAs targeted at multiple genes in the starch mobilization pathway. Transgenic rice plants carrying each of these transgenes were created. By qRT-PCR, we were able to find that many of these transgenic plants accumulated less mRNA from the targeted genes than control plants. Post-harvest measurements of starch content in the dried, vegetative tissues showed that a subset of these plants carried 3 to 5 times as much starch as did control plants that had been grown side-by-side with the transgenics. In one of these lines, we also observed that high starch content correlated with an abnormally stunted growth phenotype. This phenotype persisted in the second generation, when seed from the selfed T0 plants were grown to maturity, suggesting that plants may be especially sensitive to changes in the expression of at least some of the genes examined in this study. Conversely, a few of the plant lines in which transgenes targeted a gene for different starch-mobilizing enzyme showed especially robust growth, producing taller, bushier plants than were seen among the wild type controls. This phenotype, too, persisted in subsequent generations when seed from selfed plants were grown. We presented a summary of our findings at the BIO 2010 Congress in Washington, DC in June of 2010. We have sent dried, milled biomass from the highest starch-accumulating lines to our collaborator, the University of Illinois, where they are conducting the assays to determine the impact higher starch content has on the production of fermentable sugars during hydrolysis of the cellulosic material. Our work with transgenic switchgrass has so far produced more modest effects, which may be a function of our finding that switchgrass possesses multiple copies of the primary gene target. Thus, suppressing expression of one copy of the gene may be insufficient to induce a strong starch-accumulation phenotype. We have initiated work with sweet and forage sorghum and constructed transgenes that are specific to sorghum homologs. We are currently evaluating our first transgenic sorghum events. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have expanded our strategy to incorporate transgenes encoding new RNAs, reflecting more recent developments in the literature on RNAi-based gene suppression in plants. We have also included a fourth target gene in the starch mobilization pathway, which we hope will increase the likelihood of identifying the most critical regulatory steps in starch utilization. We have developed transgenes that incorporate micro-RNAs, in which endogenous micro-RNA sequences from rice have been modified to target starch mobilization genes, taking advantage of the endogenous gene regulation mechanisms in rice. In addition to the strategies above, we have taken two approaches toward knocking down the expression of two target genes simultaneously. First, we have developed transformation vectors that encode RNAs corresponding to combinations of two different target genes. We have generated at least 20 transgenic plants carrying each of the new vectors, and these plants are currently growing in our greenhouse. Second, we have carried out reciprocal crosses among the best-performing plant lines from the earlier work to determine whether increased starch accumulation can be achieved by simultaneously targeting more than one gene.
Publications
- No publications reported this period
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Progress 06/24/09 to 06/23/10
Outputs
OUTPUTS: Objective I. Identify genes involved in transitory starch hydrolysis in rice. During the most recent period, we completed our analysis of target genes from rice, switchgrass and sorghum. We also succeeded in generating over 100 transgenic rice lines, each representing an independent transformation event with one of our plant transformation vectors. We have developed and validated a method for quantifying the starch content of tissues derived from these plants and applied the method to determine which of the transgenic lines accumulate the highest levels of vegetative starch. Objective II. Characterize the biochemical and physiological effects of transitory starch accumulation in rice and determine the relative potential of targeted genes, or discovered genes from objective I. Using the assay for quantifying starch accumulation that we developed, we were able to identify several transgenic rice lines that accumulate higher levels of starch than do control lines. We were also able to monitor changes in starch accumulation in individual plants at multiple timepoints over a period of several weeks, including young tissue, early inflorescence and senescence. No clear physiological differences have been established between the transgenic rice lines and non-transgenic rice, other than starch accumulation. Objective IV. Generate transgenic sorghum and switchgrass lines carrying the most effective transgenes and assess effects on plant physiology and starch accumulation. During the most recent period, we have completed our evaluation of the transgenic switchgrass lines. In each case, only plants for which we were able to confirm the presence of the transgene were carried forward, grown to maturity and assayed for starch accumulation. We have developed sorghum transformation vectors and are currently expanding our transformation and regeneration efforts for this crop. We have hired an additional team member explicitly to expand our sorghum transformation efforts and are employing a number of different sorghum cultivars in this work. We are investigating the influences of growth conditions, age of the recipient tissue, culture conditions, and selectable agents in our transformation/regeneration protocols as we begin transformation. PARTICIPANTS: R. Michael Raab, Jeremy Schley Johnson, Phil Lessard, Jonas Emery, Ben Gray, Oleg Bougri, Vladimir Samoylov, Valerie Graves, Meghan Moriarty, and Cairn Ely each contributed more than one person month to the second year of this project. Dr. Raab was project director and provided project management and technical oversight. Dr. Schley Johnson provided additional project management. Dr. Lessard led the technical development related to identification and characterization of the genes responsible for the compositional modification and led the analysis of transgenic rice and switchgrass. Mr. Emery provided technical support to Dr. Lessard. Dr. Gray assisted with plant characterization. Dr. Bougri developed the vectors for plant transformation and led the transcriptional analysis work. Dr. Samoylov led the technical development and implementation of rice and switchgrass transformation. Ms. Graves provided technical support to Dr. Samoylov. Ms. Moriarty led the greenhouse work consisting of growing rice and switchgrass. Mr Ely provided technical support to Ms. Moriarty. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Objective I. Our analysis of one of the primary target genes in rice revealed that a region from the 5-prime end of the gene is misassembled/misannotated in the public sequence databases (e.g. Genbank). By sequencing de novo cDNA clones generated in our laboratory, we were able to correct the available sequence information, which has allowed us to design RNAi vectors that should better match the native mRNAs that are produced in rice plants. Objective II. Through these analyses, we were able to identify transgenic rice lines that accumulate 2-3 times as much vegetative starch as the average among control lines. To determine whether the changes in starch accumulation can be attributed to suppression of mRNA levels in transgenic tissues, we developed real-time RT-PCR assays for the mRNAs from each of the target genes. We were able to show an inverse correlation between mRNA levels and starch accumulation, which supports the original hypothesis that suppressing expression of specific genes would lead to higher starch phenotypes. To advance these outcomes further, we have designed a new series of vectors that will simultaneously target new genes, new regions in the mRNA for silencing, and multiple genes simultaneously to further boost starch accumulation in rice tissues. Objective IV. We were able to identify a small number of plants that, on average, accumulate 2 to 3-times as much starch as accumulates in control lines.
Publications
- Lessard, P. "Altering Sorghum for Improved Biofuels Production" Proceedings from the 7th Annual 2010 World Congress on Industrial Biotechnology and Bioprocessing (June 27-30, 2010).
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Progress 06/24/08 to 06/23/09
Outputs
OUTPUTS: Objectives I and II: We have searched public collections of rice lines for mutants that lacked one or more of the targeted genes. The Plant Functional Genomics (PFG) Laboratory in South Korea has catalogued collections of rice lines containing Agrobacterium T-DNA insertions. In many of these lines, the insertion site for the T-DNA element has been partially characterized, which allows the lab to infer that specific genes within the rice genome may be disrupted in a given line. Through the RiceGE: Rice Functional Genomic Express Database (http://signal.salk.edu/cgi-bin/RiceGE), hosted by the Salk Institute, we were able to identify individual rice lines in the PFG collection that would suggest how knocking out individual target genes might affect the compositional modification. We contacted the PFG Laboratory and requested seeds from several of the more promising rice lines based on our analysis. After obtaining USDA APHIS import permits for these materials, we ordered and received the desired seeds. Seeds obtained from PFG were allowed to germinate. We developed an assay to measure the compositional modification, which has been tested on wild type plants and control samples spiked with chemicals to modify their composition. With the assay in place, we have analyzed all of the mutant rice lines. We have also constructed a number of vectors to silence the desired genes in rice. We made these vectors from synthetic genes that were assembled from the published predictions of the corresponding coding sequences in rice. These vectors are now ready for rice transformation and may be tested for transformation of switchgrass as well. Objective IV: Switchgrass transformation in our laboratories has proven to be very reliable, and we have made several transgenic switchgrass lines that silence targeted genes. Additionally, we have completed sequencing of a set of switchgrass gene homologues. Sorghum silencing gene vectors have been constructed using sequence information from the Joint Genome Institute (JGI). Thus far we have focused on the sorghum-specific vectors that target the native genes. However, because we have found very high levels of sequence identity between rice, sorghum, and switchgrass genes, we may test the rice- and switchgrass-specific vectors in sorghum, and vice versa, to see whether the effect of various silencing constructs is maintained across species. We have begun sorghum transformation development and currently have tissue culture and selection working, but are still optimizing Agrobacterium inoculation and the selection of transformation tissue target. PARTICIPANTS: R. Michael Raab, Phil Lessard, Jonas Emery, Vladimir Samoylov, Valerie Graves, Alvar Carlson, Marjory Rockwell, Ana Fidantsef, and Ada Vaill each contributed more than one person month to the first year of this project. Dr. Raab was project director and provided project management and technical oversight. Dr. Lessard led the technical development related to identification and characterization of the genes responsible for the compositional modification. Mr. Emery provided technical support to Dr. Lessard. Dr. Samoylov led the technical development and implementation of rice and switchgrass transformation. Ms. Graves provided technical support to Dr. Samoylov. Dr. Carlson let the greenhouse work consisting of growing rice and switchgrass and contributed to the implantation of rice transformation. Ms. Rockwell provided technical support to Dr. Carlson. Dr. Fidantsef let the characterization of transgenic rice and switchgrass. Ms. Vaill provided technical support to Dr. Fidantsef. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Objectives I and II: The rice T-DNA knock-out lines received from the PFG Laboratory have not shown any abnormal phenotypes and have not resulted in altered composition. It may be that the T-DNA insertion in these lines does not interrupt the gene effectively, did not target the intended gene (PFG has not confirmed whether the T-DNA insertions do interrupt the purported genes in each line, and they estimate that 60% of their T-DNA lines are accurate to the gene they disrupt), or gene interruption does not lead to compositional modification in rice. It is also likely that any phenotype associated with disruption of the target genes would only appear in homozygous plants. This would make it necessary to test segregating T1 populations for each T-DNA insertion line. We are still evaluating these plants and are proceeding with the rice transformation work that aims to silence the individual genes. We intend to self-pollinate the lines and plant the progeny to see whether homozygous progeny have the desired compositional modification. Our early rice transformation efforts allowed us to determine variables and procedures that influence transformation efficiency, and we have established protocols that should allow us to generate sufficient transgenic material to meet the objectives of the study. The first rounds of rice transformation with the vectors described above produced relatively few plants that survived to maturity. We are currently restarting rice transformation in our new facility, where we have tighter control of conditions and staff who are more experienced with transforming rice. From this effort we will soon be able to generate transgenic lines silencing each of the targeted genes. Objective IV: The transgenic switchgrass lines appear phenotypically normal, and while preliminary results on very young plants suggest that some lines have altered composition that produces significantly more fermentable sugars, the experimental analysis needs to be repeated, verified, and altered composition quantified definitively. Initial Southern analysis suggests that there could be as many as five gene homologues in switchgrass, and we have not yet sorted out which cDNA sequence belongs to a specific homologue.
Publications
- No publications reported this period
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