PhD

Abstract
The study entitled “ihpRNA mediated resistance for Banana bract mosaic virus in Musa spp. by targeting replicase and movement protein genes” was carried out during 2016-2020 in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective of the study was to develop ihpRNA constructs targeting replicase and movement protein genes of Banana bract mosaic virus, and to generate transformants of banana cultivar Grand Naine carrying the ihpRNA cassette and confirm the transformation.
Embryogenic calli of banana var. Grand Naine were developed using the protocol standardised for Nendran in the Department of Plant Biotechnology, College of Agriculture, Vellayani.
Intron hairpin RNA (ihpRNA) vectors were constructed in pSTARLING to produce small interfering RNA (siRNA) against the replicase and movement protein genes of Banana bract mosaic virus (BBrMV). Movement protein (Mvp) and replicase (Rep) gene fragments were amplified from the RNA isolated from the BBrMV infected plants by reverse transcriptase (RT) PCR. The partially amplified gene fragments were sequenced and analysed using BLASTn tool. Replicase gene sequence showed 96.37 per cent similarity with BBrMV infecting banana (Trichy isolate), while movement protein gene showed 100 per cent similarity to BBrMV infecting banana (Philippine isolate). These sequences were analysed using Dicer substrate siRNA prediction tool for identifying Dicer substrates for finding any virus suppressing sequences. Restriction mapping was carried out to avoid recognition sequences present in the cloning site. Based on the results of these analyses replicase gene fragment of 357 bp and movement protein gene fragment of 224 bp were selected for preparing the construct.
Primers were designed to amplify the fragment of 357 bp of Rep and 224 bp of Mvp towards the 5’ end. NEBuilder tool was used to design the primers for amplifying the fragment containing a combination of Mvp and Rep (Mvp-Rep) with a fragment size of 647 bp. Sense fragments were amplified with the primers anchored with KpnI and SpeI sites so as to ligate them to the corresponding cloning site of siRNA vector pSTARLING. The antisense amplified fragments of Rep (357 bp), Mvp (224 bp) and Mvp-Rep (647 bp) were amplified using primers anchored with AscI and PacI sites. The amplified sense and antisense fragments were eluted from agarose gel and cloned in pJET1.2 cloning vector. Later, the cloned fragments with sticky ends were released from pJET1.2 using the corresponding restriction enzymes and integrated into the NotI cloning site of pSTARLING vector flanking the cre intron to favour the formation of the hairpin structure. Presence of the inserts was confirmed by restriction digestion and PCR.
For Agrobacterium mediated transformation, all the three ihpRNA cassettes containing ubiquitin promoter, ubiquitin intron, sense strand, cre intron, antisense strand and termination sequence in the order within the NotI restriction site were released from pSTARLING by digesting with NotI enzyme. Then ligated at NotI site within the lacZ gene of the binary vector pART27 having antibiotic resistance markers nptII and Spec. Integration of cassette within lacZ gene facilitated the selection of transformed colonies by blue-white screening. The white positive colonies were confirmed for the integration of ihpRNA cassette using PCR. The binary vector with the insert was transferred to Agrobacterium tumefaciens strain GV3103 and ihpRNA insert was confirmed by PCR and restriction digestion.
Embryogenic calli were transformed with Agrobacterium strain GV3103 containing three different ihpRNA cassettes and transformed embryos were selected on kanamycin (200 mgL-1) media. Transformed embryogenic calli carrying Rep, Mvp and Rep-Mvp ihpRNA cassettes when transferred to fresh MS medium with 2 mgL-1 BA yielded a regeneration of 9, 8 and 12 percent respectively. The regenerants were confirmed for the presence of ihpRNA construct using PCR with the primers for nptII gene. Integration of each ihpRNA cassette was also confirmed by Real-Time PCR using sense strand and cre intron specific primers. Syntheses of the respective siRNAs in the regenerated plants were also confirmed through PCR using siRNA specific stem loop primers.
The study was successful in developing transgenic Grand Naine plants carrying ihpRNA constructs targeting the replicase and movement protein genes of BBrMV. Synthesis of siRNA by the transgenics indicate that the constructs integrated are functional. The silencing potential of the constructs need to be validated by artificially inoculating the plants with BBrMV.