DEVELOPMENT OF GENE CONSTRUCTS TARGETING CONSERVED SUPPRESSOR GENE OF CITRUS TRISTEZA VIRUS FOR TRANSFORMATION OF CITRUS PLANT

Developments of gene constructs targeting conserved suppressor gene of Citrus tristeza virus for transformation of citrus plant Citrus is one of the most important commercial and nutritional fruit crops grown in all the geographical zones of India. Citrus tristeza virus (CTV), virus under genus Closterovirus is a destructive viral pathogen causing significant economic losses of citrus worldwide including India. CTV genome contains positive sense ssRNA of 19.3 kb with 12 ORFs encoding 19 putative proteins. It is a transmitted by Toxoptera citricida in semi persistent manner. It infects most of the cultivated citrus species producing various kinds of symptom. In the present study, 12 CTV isolates of different citrus species from different citrus regions of India maintained in insect-proof greenhouse at Advanced Centre of Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi were characterized based on complete ORF 11 (Suppressor gene, 630 nt) of CTV genome. Using a pair of specific primers, the complete ORF11 gene sequences of all the present 12 isolates were amplified, The PCR products were cloned, sequenced, compared and phylogenetic relationships with other Indian and international CTV isolates were determined. The ORF 11 sequences of all the present Indian isolates showed 88-100% nt identity, and all the isolates showed 87-100% among them. Phylogenetic analysis showed that Indian isolates fell into five genogroups, whereas, all the isolates into eight genogroups. Recombination detecting program, RDP3 identified the recombination events in the sequences of majority of the Indian isolates. The isolate G28 was found to be a strong putative recombinant isolate originated by exchange of genetic material from diverse CTV isolates; D1 and HA16-5 as major donors. The Indian isolate TK1 originating in citrus growing areas of Northeast India was found to be a representative of a major group. Thus the suppressor gene of this isolate was selected for development of suppressor gene construct. The suppresser gene of the isolate TK1 was cloned in antisense orientation in pBinAR binary vector using site directed primers forward KK 707 flanked with Sal I and reverse KK 708 flanked with Bam HI. The antisense gene construct, pASTK1-S was made. The construct was transferred to E.coli cell DH5α and further mobilized to Agrobacterium cell EHA105. The regeneration protocol of Kagzilime was standardized using epicotyl segments, 0.75 to 1.0 cm long of in vitro raised 21 days old seedlings and maximum regeneration of 70% was obtained with 4-5 shoots/ explant when media was supplemented with BAP @ 2 mg/l.Overall, 2944 number of explants were cocultivated and transferred to selective medium (MS, BAP @2mg/l, Cefotaxime @ 400mg/l and Kanamycin @ 90mg/l). After that, 30 plants were initially regenerated in selective medium. Finally, four putative transgenic plants, survived, was maintained in selective elongation medium (MS, BAP @2mg/l, Cefotaxime @ 400mg/l, Kanamycin @ 90mg/l and GA3 @ 0.1 mg/l). As the growth rate of regenerated plant is slow and the size is very small, thus the transformed citrus plants were not confirmed by molecular techniques. uhacw ds ikS/ks ds :ikarj.k gsrq flVal fVaLVstk fo"kk.kq dh lajf{kr laned thu dks yf{kr djus okys thu daLVaDV dk fodkl