PhD

The present study entitled ‘Identification of graft transmissible resistant factors and development of siRNA mediated resistance in Cassava against Cassava mosaic virus’ was carried out during the period 2012-2017 at the Department of Plant Pathology, College of Agriculture, Vellayani. The study was carried out with the objective of identification of transferability of resistance factor from resistant cassava, Sree Padmanabha to susceptible, Vellayani Hraswa by grafting and to develop siRNA mediated technology for the development of cassava plant resistant to Cassava mosaic geminivirus.
Grafting experiments were conducted using resistant Sree Padmanabha as root stock and susceptible Vellayani Hraswa as scion. Symptoms like leaf mosaic, chlorotic spots, reduction in leaflet size and stunting of plants were noticed in susceptible variety. Virus concentration was found to be less in grafted plants. Grafting experiments showed the expression of an extra protein by SDS-PAGE and Coomassie staining in grafted plants which is around 38 kDa. Molecular weight of the new protein revealed the presence of extracellular protein in grafted samples. The extra proteins found in the grafted plants are assumed to be transferred from Sree Padmanabha to Vellayani Hraswa by the process of grafting.
The study also involved the development of an intron hairpin RNA vector against replicase gene of SriLankan cassava mosaic virus and introduction of this construct into embryogenic cells via Agrobacterium mediated transformation. A protocol for somatic embryogenesis in cassava variety, Vellayani Hraswa was developed by using immature leaf lobes as explants. The young leaf lobes from tissue culture plantlets produced through meristem culture was used for embryogenic callus formation. Cremish white calli was initiated in Murashige and Skooge (MS) medium supplemented with picloram 12 mg L-1 in dark. For embryogenesis, the calli were transferred to MS medium supplemented with BA 2µM and NAA 1µM which resulted in the production of glassy elongated somatic embryos. The germinated cotyledonary embryos were then regenerated into plantlets by culturing in MS medium supplemented with BA 1mg L-1.
Effort was taken to construct an intron hairpin RNA vector and the gene targeted for silencing was the replicase gene of SriLankan cassava mosaic virus (SLCMV). Total DNA was extracted from virus infected plants and the whole replicase gene was isolated using gene specific primers. Sequencing of the whole gene was done. BLAST analysis showed 98% similarity to replicase gene of various isolates of SLCMV. The sequence was then subjected to miRNA target prediction and restriction mapping to select suitable region for the construct. Based on this information, a fragment of 397 bp towards the 5’ end was amplified by designing a set of primers with anchored restriction sites. The primers anchored with Xho and Kpn 1sites were used for the amplification of sense strand and the primers anchored with Xba and Cla 1sites were used for amplification of antisense strand. Selected region was amplified to form sense and anti-sense fragments and cloned to pTZ57R/T cloning vector. Inserts were then released from pTZ57R/T using the corresponding restriction enzymes. The sense and anti-sense fragments were then integrated in the primary vector pHANNIBAL on either side of the pdk intron which facilitated the formation of intron hairpin RNA construct. The intron hairpin RNA construct in pHANNIBAL contained CaMV35S promoter, sense strand, pdk intron, antisense strand and OCS terminator in the order with Not 1 restriction sites. After confirmation of integration by restriction digestion, the Not1 fragment with sense and anti-sense strand were released from pHANNIBAL and ligated to the digested Not1 site in the lacZ gene of binary vector pART27 containing antibiotic resistant marker nptII and spec. the binary vector was confirmed for the presence of insert by transferring to DH5α cells and colony selection by blue white screening. Plasmid DNA isolated from transformed colonies grown on Luria agar medium supplemented with 100 mg L -1 spectinomycin were confirmed for the presence of insert. After confirmation of insert in the binary vector, it was transformed to Agrobacterium tumefaciens strain LBA4404 via freeze thaw method. Transformed colonies were selected on kanamycin selection medium at 100 mg L -1 and confirmed for the presence of binary vector and ihpRNA insert using nptII primers and primer for sense and antisense strands by PCR reaction.
Cotyledons excised from the somatic embryos were transformed with LBA4404 having pART 27 by co-cultivation and the transformed embryos were selected with antibiotic pressure (Kanamycin 100 mg L-1). DNA was isolated from the transformed somatic embryos and confirmed for the presence of insert using forward primer of sense fragment and reverse primer of antisense fragments. Transformed embryos were subjected to regeneration.