GM Sorghum: Applications to Agriculture in Developing Nations

1. Cindy Lee, Linguistics and Microbial Biology Katrina Linden, Molecular and Cell Biology Stephanie Wu, Molecular and Cell Biology Lab of Peggy G. Lemaux GM Sorghum: Applications to Agriculture in Developing Nations Agrobacterium-Mediated Transformation of Short-Season and Genome-Sequenced Sorghum Why Sorghum? Abstract Why Agrobacterium? Using Agrobacterium tumefaciens as a vector for stably inserting genes, we are introducing the GFP marker and the selection gene, phosphomannose isomerase (PMI), into the genome of two sorghum varieties. The reduced generation time in N247 speeds transformation success; the genome sequence of BTx623 allows for functional analysis studies. Successful transformation requires a means to culture healthy tissue in vitro from immature embryos, to perform selection for transformed tissues, and finally, to regenerate transformed plants. From a dietary standpoint, developing nations have the most to gain from productive genetic engineering of a more nutritious, drought-resistant sorghum. From a biofuels standpoint, nations lacking a sustainable source of energy have the most to gain from improving starch digestibility in sorghum. Developing an efficient, reproducible transformation protocol for these two sorghum lines is critical because of the impact it will have on engineering improved varieties of sorghum. • Competent cells have plasmids engineered with gene of interest and selection factor • Can inject its plasmid into eukaryotic (plant) tissue and insert gene of interest and markers into plant genome • Inserts genes into genome in a much more controlled manner than the gene bombardment gun • Biofuel application in the United States • Staple food crop in much of Africa and the Indian subcontinent • Despite current use as food, sorghum provides inadequate nutritional value and is difficult to digest • Related to barley, from which we can use the barley-high-lysine gene (BHL9) to increase nutritional value of sorghum • Innate ability to grow under harsh conditions Methods Results • Choice of cultivars: N247’s shortened growing season accelerates transformation; BTx 623’s fully sequenced genome allows for functional analysis. • Totipotency: Immature embryos contain undifferentiated cells that can be transformed and later taken back up the developmental ladder. • Treatment optimization: We manipulate variables such as heat treatment, embryo size, and media composition to maximize transformation frequency and viability. • Selection markers: GFP allows us to visualize transgenic tissue; phosphomannose isomerase allows us to grow only these tissues. • Digestion analysis: SDS-PAGE gels, Western blots, and conventional combustion methods characterize protein digestibility; BLAH for starch. Optimization A. Media and Embryo Size B. Heat treatment Protocol Overview 3. Isolate immature embryos and infect with Agrobacterium after various treatments to optimize transformation. After 3 days co-cultivating embryos and bacteria, plate on antibiotic. 8. Harvest mature panicle of seeds for planting and protein/starch analysis. 2. Collect seeds when embryos are a maximum of 2 mm long. 4. Induce callus formation. Choose only white calli, which will give rise to regenerable plant tissue. 6. Plate transgenic calli on regeneration media to induce root and shoot growth. 7. Transplant regenerated material to soil and cultivate to maturity. 9. Use premiere gel-based methods to analyze protein digestibility and BLAH to determine starch digestibility. 1. Plant seeds and grow at 28°C and 12 hours of light per day. 5. Identify transgenic tissue by visualizing GFP marker via fluorescent microscopy. Acknowledgments Many thanks to Peggy G. Lemaux, Joshua Wong, and the Lemaux Lab References Howe, A., Sato, S., Dweikat I., Fromm M., Clemente T.; (2006) Rapid and reproducible Agrobacterium-mediated transformation of sorghum; Plant Cell Reports: 25-8: 784-791 The Amazing Joshua C Wong Zhao, Zuo-yu; Cai, Tishu; Tagliani, Laura.; (2000) Agrobacterium-mediated sorghum transformation; Plant Molecular Biology: 44-6: 789-798