Location: Wheat, Sorghum and Forage Research2015 Annual Report
1a. Objectives (from AD-416):
The long-term objectives of this project are to develop improved sorghum (Sorghum bicolor) germplasm (Figure 2) for biofuels production by 1) determining genetic, biochemical, and physiological mechanisms controlling traits beneficial to bioenergy conversion technologies (saccharification & fermentation, pyrolysis and combustion), 2) develop and release improved germplasm with these modified traits, and 3) determine the impact of fungal pathogens on sorghum with these modified traits. Over the next five years, the following specific objectives will be addressed: Objective 1: Determine and manipulate the genetic, biochemical and physiological mechanisms controlling the biological pathways involved in non-grain energy sorghum germplasm. Sub-objective 1.A: Determine the effects of six newly identified brown midrib (bmr) mutants on lignin synthesis and the monolignol biosynthetic pathway. Sub-objective 1.B: Develop transgenic lines over-expressing genes in monolignol biosynthesis to determine their impact on lignin content and composition. Sub-objective 1.C: Identify strategies for increasing the sugar content of sweet sorghum juice and improving its biomass composition for thermal conversion. Objective 2: Determine the impact of fungal pathogens on non-grain energy sorghum germplasm, and determine mechanisms of resistance to sorghum pathogens. Sub-objective 2.A: Determine the response of sorghum with modified lignin biosynthesis pathways to stalk pathogens. Sub-objective 2.B: Determine whether modifications in the lignin biosynthetic pathway affect growth of the pathogen Colletotrichum sublineolum within sorghum leaves. Sub-objective 2.C: Identify sweet sorghum parental lines with resistance to stalk pathogens. Objective 3: Develop and evaluate germplasm to improve sorghum for non-grain energy uses. Sub-objective 3.A: Determine the effects of brown midrib (low lignin) mutations alone or in combination on bioenergy conversion via saccharification and fermentation. Sub-objective 3.B: Develop sorghum lines with novel lignin composition and determine the effects on bioenergy conversion. Sub-objective 3.C: Develop sweet sorghum germplasm incorporating bmr6 and bmr12 to reduce lignin and determine the effects of these alleles on end-use quality.
1b. Approach (from AD-416):
The overall objective of this project is to improve sorghum (Sorghum biocolor) as an energy crop with the focus on the biomass or non-grain components of the plant. The project will conduct basic studies on the genetic, biochemical, and physiological mechanisms affecting the composition of sorghum biomass and its conversion to liquid fuels. The focus will be on decreasing or increasing lignin content and/or modifying its composition and on increasing sugar content and yield for juice extraction. Low lignin is desirable for the saccharification and fermentation conversion process while high lignin concentration is desirable for conversion via pyrolysis. The impacts of fungal pathogens on sorghum with compositionally modified biomass will be determined. Germplasm with desirable genes affecting the conversion of sorghum biomass to energy will be developed, fully characterized, and released and deposited in Germplasm Resources Information Network (GRIN) for use by public and private sector plant breeders for developing improved hybrids and cultivars. The project consists of three integrated components: germplasm development, molecular biology, and plant pathology (Figure 1). Molecular and conventional methodologies will be utilized, and the project scale will range from field-level to gene-level. The project also has extensive formal and informal collaborations enhancing our ability to conduct this research. Anticipated products include improved sorghum germplasm for the sorghum seed industry with enhanced energy traits and biotic stress tolerance, and tools to assess the biological pathways that impact bioenergy traits and fungal pathogen responses of sorghum.
3. Progress Report:
Lignin, a key component of plant cell walls, can affect the efficiency of bioenergy conversion. In specific objective 1, the development of transgenic sorghum over-expressing ten genes in lignin synthesis was completed. These plants will be used to determine whether these approaches can alter lignin or affect bioenergy conversion. Over-expression of a transcription factor, SbMyb60 was discovered to activate many of the genes involved in lignin synthesis and increase the amount of lignin within cell walls. SbMyb60 represents a tool to modify biomass from sorghum and other grasses for bioenergy or other renewable purposes. The brown midrib (bmr) mutants of sorghum reduce lignin. The characterization of several new mutations in the Bmr6 gene was completed. These plants represent new materials that can be used for breeding sorghum with reduced lignin content for bioenergy and grazing purposes. Transgenic lines designed to over-express the sucrose synthase gene failed to increase its gene expression above normal levels in sorghum. Therefore, alternative approaches need to be designed to increase sugar contents in sweet sorghum. To determine how drought affects the susceptibility of sorghum to stalk rot diseases, a greenhouse assay under water-deficit conditions was developed in specific objective 2. This assay will be used to determine how bmr and lignin-modified sorghum lines respond to stalk diseases under water stress. Understanding the interactions between lignin, water stress and disease are critical for the development of this climate resilient bioenergy crop. A technique was developed to inoculate sorghum plants with anthracnose, a major sorghum disease. To enhance inoculation, several surfactants are being tested for their compatibility with the pathogen. Together these assays will help address whether changes to lignin in sorghum affect how plants respond to the major sorghum diseases, anthracnose and stalk rots. To effect further reductions in lignin content than observed in single bmr lines, bmr2, bmr6 and bmr12 were combined in the same genetic backgrounds in specific objective 3. Plants containing the combinations bmr2/bmr12 and bmr2/bmr6 were identified, and are currently being screened in the field. Effects on lignin content of these combinations will be determined in field trials during the summer of 2016. To develop sorghum plants with novel lignin composition for bioenergy conversion evaluation, transgenic lines over-expressing ferulate 5-hydroxylase (F5H) and cinnamoyl-CoA reductase (CCR), two lignin biosynthetic genes, were crossed with two bmr lines. Each of four classes (normal, transgenic, bmr, and bmr/transgenic) from this cross was identified, and these plants are growing this summer in the greenhouse. The stalks from these plants will be analyzed to determine how these combining these traits affects lignin and bioenergy conversion. Sweet sorghum lines containing bmr6 and bmr12, which should reduce lignin concentrations, are being developed as future bioenergy feedstocks. Challenges crossing the plants occurred due to different flowering times. Previously developed forage bmr lines will be grown in the 2015 fall greenhouse as a contingency to complete this study.
1. Discovery of New brown midrib (bmr) Mutants in Sorghum In the U.S., sorghum biomass (stalks and leaves) serves as an important forage crop for livestock. In addition, sorghum is being developed as a bioenergy crop for cellulosic biofuels. Cellulosic biofuels are made from breakdown of the cell wall components (cellulose and hemicellulose) of the biomass into sugars and converting these sugars to fuels. A third cell wall component, lignin, makes cell walls resistant to breakdown into sugars. In sorghum, brown midrib (bmr) mutants are a class of mutants, which have reduced lignin content, improved bioenergy conversion and increased livestock digestibility. ARS scientists at Lincoln, NE discovered six novel bmr mutants. The lignin content and the ability to breakdown cell walls into sugars of these new mutants was analyzed. These mutants provide scientists with new tools to improve sorghum biomass for bioenergy and forage uses, and will potentially aid the identification of genes controlling lignin synthesis in sorghum and other bioenergy grasses.
2. Susceptibility of waxy Sorghum Grain to Gain Mold Pathogens. Starch is normally composed of two types of molecules, amylose and amylopectin. Waxy sorghum starch is almost entirely composed of amylopectin, which changes the physical properties of the starch making it more useful for the food and ethanol industries. The waxy grain is also more digestible than normal grain. Therefore, there was a concern that waxy grain would be more susceptible to grain molds than normal grain. ARS scientists at Lincoln, NE screened field grown normal and waxy sorghum lines for grain mold infections, and waxy lines were not more susceptible to these diseases compared to normal sorghum lines. In a second experiment, grain samples from near identical lines that only differed for the waxy trait (normal or waxy) were infected with different grain molds. Disease responses of the seeds and seedlings were measured. Similarly, the waxy lines were not more susceptible than normal lines. These studies demonstrated that waxy sorghum grain is not more susceptible to pathogens then normal grain, which is valuable information for developing waxy sorghum hybrids with resistance to grain molds.
3. Evaluation of waxy Hybrid Sorghum for Use in the Food and Ethanol Industries. Waxy sorghum alters the starch composition of grain, which is useful for the food and ethanol industries. Previously, waxy lines performed poorly compared to normal lines, which has prevented commercialization of the trait. Typically, sorghum hybrids are grown for grain production in the U.S. A set of waxy hybrids were bred and tested by ARS scientists at Lincoln, NE, and the grain yield of waxy hybrids was not reduced compared to the normal hybrids. These lines are some of the first publically-available germplasm that can produce high-yielding waxy hybrids.
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