Functional validation of plant transformation vector with stacked ech42 and bgn from Trichoderma in tomato for fungal disease resistance

Ugale Sharad; Gaurav Sharma; Narayan Moger; Sumangala Bhat; P. U. Krishnaraj

Ugale Sharad Department of Biotechnology, College of Agriculture, University of Agricultural Sciences, Dharwad 580 005
Gaurav Sharma Department of Biotechnology, College of Agriculture, University of Agricultural Sciences, Dharwad 580 005
Narayan Moger Department of Biotechnology, College of Agriculture, University of Agricultural Sciences, Dharwad 580 005
Sumangala Bhat Department of Biotechnology, College of Agriculture, University of Agricultural Sciences, Dharwad 580 005
P. U. Krishnaraj Department of Biotechnology, College of Agriculture, University of Agricultural Sciences, Dharwad 580 005

Keywords: Endochitinase, endoglucanase, southern hybridization, tomato, transgenics

Abstract

Transgenic tomato lines (cv. Pusa Ruby) were generated by using Agrobacterium tumefaciens strain LBA4404 harboring endochitinase (ech42) and endoglucanase (bgn) genes stacked in binary vector (pRAGS121). Ten putative transformants in T0 generation were confirmed by PCR. Progenies of two transgenic tomato lines, CG2 and CG7 showed the presence of transgenes in the T1 generation. Transgene integration and copy number of transgene was assessed using PCR, Dot blot, Southern hybridization in T2 generation. Southern hybridization using DIG-labelled ech42 specific probe revealed the presence of two copies of transgenes. RT-PCR showed expression of ech42 and bgn at transcript level. Chitinase and glucanase assay revealed 4.09 and 3.93 fold higher expression of ech42 and bgn respectively in transgenic plant compared to nontransgenic plant. Bioassay of transgenic plant against Alternaria solani showed 2.97 times reduction in the leaf area infection and against Sclerotium rolfsii showed significant growth inhibition compared to non-transgenic control.