An attempt to standardize VIGS based knockdown tool for floral Organ Identity Genes in Coccinia grandis

Abstract

Various forms of sexual systems are found among angiosperms and evolution of dioecy is one of the most extreme changes in the course of plant development. Coccinia grandis, a dioecious species belonging to Cucurbitaceae family was characterized by our lab as a good model system to study factors involved in sex expression in dioecious flowering plants. To elucidate the function of Organ Identity Genes (OIGs) or any other candidate genes in sex expression, we need a genetic transformation system for C. grandis which is currently unavailable. In this study a frequently used transient silencing technique named Virus Induced Gene Silencing (VIGS) was standardized for C. grandis. Viral vectors derived from Tobacco Rattle Virus (TRV), a bipartite, positive-strand RNA virus were used to silence phytoene desaturase (PDS) whose disruption shows a photobleaching phenotype. At the beginning of the experiments, the available vector containing Nicotiana tabacum PDS (NtPDS) was used for silencing which showed a very low silencing efficiency. To improve on this, a 224 base pair (bp) native C. grandis PDS sequence was isolated and cloned into the MCS region of TRV vector. Photobleaching phenotype was seen as yellowing of leaves starting 2 weeks post infiltration. Molecular analysis using semi-quantitative RT-PCR showed lower levels of PDS in infected plants compared to that in uninfected plants. Hence, VIGS technique could be used as a fast and an efficient gene knockdown tool in C. grandis. Concurrently, three fragments of C. grandis PISTILLATA (CgPI), a B class gene were chosen for knockdown studies. These fragments were amplified from RNA extracted from the flowers and cloned individually into the TRV vector and infiltrated into the plants. Further work is under progress to study the effects of CgPI silencing.