Plant Molecular Virology, National Botanical research Institute, Lucknow-226001.
Abstracts of Research Papers Presented during the National Symposium of Indian Virological Society at Unit of Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi-110 012, October 14–1.
Cucumber mosaic virus (CMV), the type member of genus Cucumovirus of the family Bromoviridae, is one of the most economically important plant viruses known and studied till date. CMV is a single-stranded, plus sense RNA virus with a functionally divided genome. It is worldwide in distribution, infects over 1000 species of hosts, including members of 85 plant families, making it the broadest host range virus known and attacks a great variety of vegetable, ornamental and other plants causing severe losses in yield and quality.
Coat protein mediated resistance has been demonstrated as the resistance caused by the expression of a virus coat protein (CP) gene in transgenic plants. The CP gene is the most widely and commonly used transgene for which virus resistant transgenic plants have been developed and there are instances where the resistance spectrum conferred by a coat protein gene was quite broad, as showed for cucumoviruses and potyviruses. Therefore, with the aim to develop Cucumber mosaic virus resistant transgenic tomato plants, the chimeric construct of the coat protein gene of CMV was generated; mobilization of construct to Agrobacterium through triparental matting and transformation of tomato was attempted.
The complete coding region of CP gene was amplified through PCR using CP specific primers and the template cDNA from a CMV-CP clone. The amplified product (650 bp) was eluted by LMP agarose electrophoresis and ligated into the expression vector between 35 promoter and NOS terminator. The E. coli cells were transformed and one selected clone was used for conjugation of Agrobacterium tumefaciens by triparental mating with the help of a helper plasmid. The conjugants were analysed by PCR using CMV coat protein specific primer, which showed positive amplification of 650 pb, the expected size DNA fragment in several conjugants. One such conjugant containing chimeric cassette was chosen for the transformation of tomato explants.
Transformation of Pusa Ruby tomato leaf explants was attempted by the standard protocol described earlier by McCormick et al, 1986. Co-cultivated leaf explants were transferred on selection plate containing appropriate antibiotics (50 mg/L Kanamycin and 250 mg/L Cefotaxim). The regeneration of the selected transformants is in progress. The preliminary results of the co-cultivated tomato leaf explants and their subsequent regeneration will be discussed.
