DEVELOPMENT OF GENOMIC AND GENETIC TOOLS FOR FOXTAIL MILLET, AND USE OF THESE TOOLS IN THE IMPROVEMENT OF BIOMASS PRODUCTION FOR BIOENERGY CROPS

Abstract

Foxtail millet (Setaria italica L.) is a warm-season, C4 annual crop commonly grown for grain and forage worldwide. It has a relatively short generation time, yet produces hundreds of seeds per inflorescence. The crop is inbred and it has a small-size genome (~500 Mb). These features make foxtail millet an attractive grass model, especially for bioenergy crops. While a number of genomic tools have been established for foxtail millet, including a fully sequenced genome and molecular markers, the objectives of this project were to develop a tissue culture system, determine the best explant(s) for tissue culture, optimize transient gene expression, and establish a stable transformation system for foxtail millet cultivar Yugu1. In optimizing a tissue culture medium for the induction of calli and somatic embryos from immature inflorescences and mature seed explants, Murashige and Skoog medium containing 2.5 mg l-1 2,4-dichlorophenoxyacetic acid and 0.6 mg l-1 6- benzylaminopurine was determined to be optimal for callus induction of foxtail millet. The efficiency of callus induction from explants of immature inflorescences was significantly higher at 76% compared to that of callus induction from mature seed explants at 68%. The calli induced from this medium were regenerated into plants at high frequency (~100%) using 0.2 mg l-1 kinetin in the regeneration media. For performing transient gene expression, immature embryos were first isolated from inflorescences. Transient expression of the GUS reporter gene in immature embryos was significantly increased after sonication, a vacuum treatment, centrifugation and the addition of L-cysteine and dithiothreitol, which led to the efficiency of transient expression at levels greater than 70% after Agrobacterium inoculation. Inoculation with Agrobacterium was also tested with germinated seeds. The radicals of germinated seeds were pierced with needles and dipped into Agrobacterium solution. This method achieved a 10% transient expression efficiency. Throughout these analyses, using plasmids with the hygromycin selectable marker, it was determined that 1.5 mg l-1 hygromycin was the optimal dose for genetic transformation of foxtail millet. In contrast, the nptII selectable marker appeared to yield many escapes. Three methods of transformation were employed in an attempt to produce stable transformants. An in planta transformation experiment, similar to the floral dip method used in Arabidopsis, which utilized a red fluorescent protein pporRFP from coral Porites porites and the hygromycin selectable marker, was tested using immature inflorescences. Although several plants were PCR positive using endpoint and Real-Time PCR and there was transient expression using pporRFP and GUS reporters, no plants were positive on Southern blot. Dipping in Agrobacterium may damage the anther or the pistil because seed production was significantly reduced. Agrobacterium transformation using embryogenic calli was also tested. Although hundreds of plants were regenerated from selection, none were positive using PCR. The third method was to wound germinated seeds with an Agrobacterium coated needle, but none of the plants were PCR positive. Although the Yugu1 genotype was recalcitrant to genetic transformation, several avenues of future research should be considered for foxtail millet. Calli from different foxtail millet genotypes should be screened and selected for regeneration potential, and some genotypes may be more amenable to transformation. Additional selectable markers should also be tested as hygromycin appears to be too stringent and there are too many escapes with nptII. This project has provided training for the following personnel: Dr. Xinlu Chen (postdoc), Xiaomei Liu (postdoc), Jayashree Desai (postdoc) and Kyle Berk (Undergraduate researcher). Conference presentations and peer-reviewed journal articles partly supported by this grant includes the following: 1. Baxter H., Equi R., Chen X, Berk K. and Zale J. Establishing Efficient in vitro Protocols For Foxtail Millet (Setaria italica L. cv. Yugi 1). Plant & Animal Genomes XVIII Conference XVIII, San Diego, California, January 2010 2. Chen X, Zale J and Chen F. The Regeneration and Transformation of Foxtail Millet (Setaria italica), A Model Biofuel Crop. Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, Crystal City, Virginia, April 2011 3. Chen, F., Tholl, D., Bohlmann, J., and Pichersky, E. (2011) The family of terpene synthases in plants: A mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. 66: 212-229.