Journal of Virological Methods ELSEIUER Journal of Virological Methods 51(1995) 297-304 Molecular cloning of Indian tomato leaf curl virus genolme following a simple method of concentrating the supercoiled replicative form of viral DNA K.M. Srivastava *, Vipin Hallan, R.K. Raizada, Govind Chandra, B.P. Singh, P.V. Sane zyxwvutsrqponmlkjihgfedcbaZYXW Plant Virus Laboratory, National Botanical Research Institute, Lucknow- 001, India Accepted 26 July 1994 Abstract DNA-A and DNA-B components of the genome of a whitefly transmitted virus causing yellowing and leaf curl in tomato (ITLCV) were cloned following a simple procedure for isolation of the double stranded replicative form of viral DNA from infected tomato plants. The method is based on extraction of total DNA from infected plants followed by concentration of the double stranded replicative form of viral DNA by an alkaline denaturation procedure identical to that used for isolation of plasmid DNA from Escherichiu coli. The attempted cloning of DNA showed that 95% of the transformants contained plasmids with an insert of either DNA-A (2.75 kb) or DNA-B (2.55 kb). Cloned DNA-A and DNA-B when used as probes could detect DNA-A/DNA-B in total nucleic acid obtained from fresh diseased tissue. Both DNA-A and DNA-B are needed for infection and they have a common region of 166 bases with about 94% nucleotide sequence homology, a characteristic of all bipartite geminiviruses. Comparison of the amino acid sequence of the putative coat protein product of ITLCV with some other mono- and bipartite geminiviruses revealed a maximum of 86% homology with Indian cassava mosaic virus. Keywords: Indian tomato leaf curl virus; Geminivims; Bipartite genome; Replicative form isolation Tomato leaf curl is a devastating disease of tomato crop in India. The symptoms include yellow mosaic, curling and cupping of leaf lamina and stunting of infected plants. The disease has been known for many years (Vasudeva and Sam Raj, 1948; Verma et al., 1975) but proper characterization of the causal agent as a geminivirus has * Corresponding author. Fax: +91 522 282849. 0166-0934/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-0934(94)00122-7