Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021.
Virus induced gene silencing or VIGS is now thought to have immense potential in plant reverse genetics. Unlike the other methods available for reverse genetics, such as T-DNA insertion or transposon tagging, VIGS is methodologically simple and would yield faster results. The system is quite well established in N. benthamiana and efforts are now being made to extend the use of VIGS vector for monocots as well. Rice tungro bacilliform virus (RTBV), a pararetrovirus, causes tungro disease in rice. To develop RTBV as a VIGS vector for rice, its derivatives are being developed for self-replication in the rice plant. A full-length infectious clone (pRTBV-Inf) and a minimal infectious clone of RTBV (pRTBV del-Inf) have been constructed. Preliminary investigations using PCR show that both the clones are infectious by Agrobacterium-mediated transfer to rice. Further, a gus fragment has been cloned into pRTBV-Inf (pRTBV-Inf-gus) to check for its ability to silence GUS activity in gus-transgenic PB1 variety of rice. Flourimetric analysis of transgenic plants inoculated with RTBV-Inf-gus suggests a fall in GUS expression. To optimize the conditions under which the two self-replicating clones show maximum infectivity, different strains of Agrobacterium and different inoculation conditions have been tried. Efforts are now being made to improve the efficiency of the infection of pRTBV-Inf and pRTBV del-Inf. Also, efforts are being made to insert a multiple cloning site into pRTBV-Inf to allow easy cloning of the “gene of interest”.
