Division of Biotechnology, Indian Institute of Horticultural Research, Hessaraghatta, Bangalore-560089.
Peanut bud necrosis virus (PBNV) (or Groundnut bud necrosis virus) is one of the most economically important tospovirus infecting tomatoes in India. We have generated transgenic tomato plants cv Arka Vikas resistant to PBNV by incorporating its nucleocapisd gene (n-gene). The n-gene was isolated by RT-PCR from total RNA extracted from infected fruits collected from farmer's fields. The sequence had 98% homology with the n-gene of PBNV, peanut (groundnut) isolate of India. The n-gene was sub cloned into plant expression vector containing plant promoter and a terminator. The construct was electroporated into tomato plants cv Arka Vikas. A total of 48 T1 plants were raised and the genomic DNA was isolated from 25 plants and was checked by PCR with primers specific to n-gene. The PCR amplified product was evaluated by dot blot with an internal fragment of the n-gene. Nine plants had the transgene integrated. One hundred and sixty T2 progenies of four positive T1 plants were raised. These were challenged by mechanical inoculation with purified virions. Forty-four plants were completely free from the disease. Genomic DNA was isolated from these resistant plants and was evaluated by PCR and dot blot. Forty-two plants were positive for the presence of the transgene. This is a first time report of transgenic tomato resistant to tospo virus in the country.
