Stable Integration and Expression of Chitinase Gene in Transgenic Tomato

Early blight caused by Alternaria solani is a major production constraint in the cultivation of tomato. Conventional breeding has met with limited success. Therefore the transformation of tomato with gene encoding pathogen cell wall degrading enzyme chitinase isolated from Trichoderma harzianum was carried out for expression of resistance against A. solani. The present study was done to identify homozygous lines with stable and high transgene expression from the progeny of primary transformants (T1) of tomato cultivar 'Arka vikas'. T-1 plants were screened by PCR using gene specific primer. 15 T-1 plants were selected based on percent disease index (PDI) and similarity to parent phenotype for advancing to T-2 generation and two homozygous lines were identified. Gene expression analysis for chitinase gene was carried out in randomly chosen plants from the progeny of two T-1 homozygous lines and one T-1 hemizygous line through quantitative RT PCR. The chitinase gene expression in plants of two homozygous lines was 2.5-4.0x higher than that of hemizygous lines. The seedlings of homozygous plants in T-2 and T-3 generation were also screened for disease expression by in vitro leaf bioassay as well as challenge inoculation with Alternaria solani.