MSc

The study entitled “Evaluation of siRNA mediated Banana bract mosaic virus (BBrMV) resistance in banana plants with ihpRNA construct for replicase gene,” was carried out in the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2015-2018. The objectives of the present study were to evaluate siRNA mediated resistance against Banana bract mosaic virus (BBrMV) in banana (Musa spp. Var. Nendran) plants harboring ihpRNA construct for replicase gene by artificial infection and to confirm the expression of siRNA products in infected plants.
Somatic embryos transformed with Agrobacterium tumifaciens strain LBA 4404 carrying the ihpRNA construct (developed as part of Ph.D. thesis work at the Department of Plant Biotechnology) against the replicase gene of BBrMV was used for the study. The ihpRNA construct (1300bp) consisted of sense and antisense fragment of BBrMV replicase gene, connected through a cre intron under the control of ubi promoter and tmL terminator.
The transformed embryos were selected on semisolid MS medium supplemented with BA 2 mg L-1, IAA 0.5 mg L-1and kanamycin 100 mgL-1. The embryos showed germination in 3 weeks of inoculation in half strength semisolid MS supplemented with the same combination of growth regulators in dark. Germinated embryos when transferred to MS medium with BA 2 mgL-1, NAA 1 mgL-1 and kanamycin 100 mgL-1and incubated in light developed green shoots with proper root development.
The transformation of the regenerated plants were confirmed by PCR analysis for the presence of ihpRNA construct, nptII gene (selectable marker) and cre intron using the corresponding primers already designed for this purpose. The DNA isolated from the leaves of transformed Nendran shoots showed amplicons of size 475, 450 bp and 1300bp corresponding to nptII gene, cre intron and ihpRNA construct respectively. The results indicated successful insertion of the ihpRNA construct in the transformed banana plantlets developed.
The transformed plants were hardened under protected condition in the glass house. Three months old hardened plants were challenged with 50 infectious viruliferous aphids for 24hrs immediately after they were allowed acquisition-access to BBrMV-infected banana plant for 30min. Untransformed control plants of the same developmental stage were also similarly inoculated with viruliferous aphids. After infection period aphids were removed from the plants and new aphid were released in two days interval for 2 weeks. After 2 weeks of infection, the plants were tested for the presence of viral RNA in young leaf by RT-PCR using BBrMV replicase gene specific primers. An amplicon of size 733bp corresponding to replicase gene detected in inoculated untransformed control plants, but none of the ihpRNA transformed plants showed infection.
Northern hybridization was carried out to confirm the production of siRNA transcripts in the transgenic plants. The siRNAs isolated from untransformed control and transformed plants were separated on 1.2% formaldehyde agarose gel, electroblotted on a positively charged nylon membrane and hybridized with probes specific to siRNA targeting replicase gene. Strong signals corresponding to 21nt long siRNAs were obtained in samples from transformed plants, clearly indicating the successful siRNA mediated silencing of replicase gene of BBrMV as the cause of virus resistance in transformed plants.
The RNAi technology was successful in conferring resistance against BBrMV in banana (Musa spp. Var. Nendran). The siRNA detected in the transgenics clearly shows that multiplication of the virus in the host was suppressed due to the silencing of replicase gene. This is the first report on siRNA mediated resistance against BBMV in banana. The study suggests the possible use of RNAi technology for developing resistance against other viruses also in banana.