Research Scientist Geneshifters Pullman, Washington
Body of Abstract: Chickpea is the second largest legume crop with a draft reference genome sequence available. Additional characterization of the chickpea genome using gene expression studies has helped us identify candidate gene(s) involved in different plant development stages, such as early growth vigor, podding, and nitrogen fixation. Additionally, classical genetics approaches coupled with next-generation sequencing approaches have helped in the genetic mapping of multiple traits of interest, such as Ascochyta blight resistance. Still, a handful of genes have been functionally validated in chickpeas due to the lack of functional genomics approaches. As a part of the NIFA-USDA-funded project to develop Ascochyta blight-resistant chickpeas using natural variation present in the wild relatives of crop plants, we are optimizing a novel method of alien gene transfer using virus-induced gene silencing. Using a TRV-based virus system, we are optimizing a transient gene silencing system in chickpeas with the phytoene desaturase gene as a positive marker. TRV is a bipartite virus with two genomic components. Equal volumes of the two genomic components, i.e., TRV1 and TRV2 (Carrying gene of interest), were transferred to Agrobacterium Strain (GV3101), and cultures were grown till OD600=1.5. A 1 mL syringe barrel (no needle) was used to deliver the inoculum to the abaxial side of the cotyledon and infiltrate it until the entire cotyledon appeared saturated. Similarly, plants were directly inoculated with virus-plasmid after missing them with FES (abrasive agent). Our first optimization experiment resulted in successful photobleaching in gene-silenced plants. We are currently repeating experiments with additional candidate gene(s) (Magnesium Chelatase (MgChl)) for virus-induced gene silencing (VIGS)and gene expression studies to validate the findings.