PhD

The present study entitled “Development of small interfering RNA (siRNA) mediated resistance in banana against Banana bract mosaic virus (BBrMV)” was carried out during 2012-2016 in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The study was carried out with an objective to develop siRNA mediated technology for the development of banana plants resistant to Banana Bract Mosaic Virus (BBrMV). The study was conducted in banana cv. Nendran.
A protocol for somatic embryogenesis in banana cv. Nendran was standardized by using immature male flowers as explants. Pale white friable callus with rich cytoplasm was initiated in Murashige and Skooge (MS) medium supplemented with BA (0.1 – 0.5 mgL-1) and picloram (0.5 – 2 mgL-1) incubated in dark with a maximum explant response of 30 per cent. For embryogenesis, the developed embryogenic calli were transferred to semisolid MS medium supplemented with BA 2 mgL-1 and IAA 0.5 mgL-1 which resulted in a maximum of 10 per cent embryogenesis. The glassy elongated monocot embryos were germinated in half strength semisolid MS medium (0.3 per cent Gelrite) supplemented with BA 2 mg L-1 and IAA 0.5 mg L-1 and incubated in dark. A maximum germination rate of 80 per cent was obtained in this medium. The germinated embryos were transferred to MS medium with BA 2 mg L-1 and NAA 1 mg L-1 resulted in 100 per cent Plant regeneration. The plantlets were transferred to coirpith compost in pot trays in mist chamber for one month for hardening and then transferred to polybags with soil and cowdung (1:1) mixture.
To develop siRNA technology to silence the replicase gene of BBrMV, an intron hairpin RNA (ihpRNA) construct was developed. For this a partial mRNA sequence of replicase gene was isolated from BBrMV banana plants. Gene specific primers designed based on the whole genome sequence information retrieved from the GenBank, NCBI. Total RNA from infected banana leaves was isolated and cDNA was prepared using RT-PCR. The partial gene fragment isolated was sequenced and analysed using the bioinformatics tool BLAST. The sequence was subjected to miRNA target prediction and restriction mapping to select suitable region for the construct and further processing. Based on this information a fragment of 400 bp towards the 5’ end was amplified by designing a set of primers with anchored restriction sites. The primers anchored with BamHI and PacI sites were used for the amplification of sense strand and primers anchored with KpnI and SpeI sites were used for antisense strand amplification. The sense and antisense fragments amplified were cloned to pTZ57R/T cloning vector.
In the next step the inserts were released from pTZ57R/T using the corresponding restriction enzymes and were integrated in pSTARLING (primary vector), on either side of the cre intron which facilitated the formation of the hairpin (ihpRNA) construct. Presence of the inserts was confirmed by restriction digestion and electrophoresis. The ihpRNA construct in pSTARLING now contained ubiquitin promoter, ubiquitin intron, sense strand of replicase gene, cre intron, antisense strand of replicase and termination sequence in the order with the NotI restriction sites. This construct was released from pSTARLING and ligated to the digested NotI site in the lacZ gene of the binary vector pART27 containing antibiotic resistance marker nptII and spec. The binary vector was confirmed for the insert by transferring to DH5α and colony selection by blue-white screening. The binary vector with the insert isolated from the white colony, was transferred to Agrobacterium tumefaciens strain LBA 4404 via freeze-thaw method. Transformed colonies were picked up and confirmed the presence of the vector and the ihpRNA insert by PCR.
Somatic embryos were transformed with LBA 4404 carrying the ihpRNA construct the ihpRNA construct and the transformed embryos were selected with antibiotic pressure (Kanamycin 100 mg L-1). Transformed embryos were subjected to regeneration. A maximum regeneration of 25 per cent was obtained after transformation. The regenerants were confirmed for the presence of ihpRNA construct using PCR with specific primers for sense-intron-antisense fragment, npt II and cre intron. The study was successful in developing a siRNA construct for resistance against BBrMV and obtaining transformed Nendran banana plantlets.