Feasibility analysis of leaf disc samples produced via agroinfiltration for promoter trapping studies

Promoter trapping is a method used to isolate and characterize regulatory regions from genomes by elucidating the expression of a promoterless reporter gene flanked by two transposable elements. The conventional method of promoter trapping requires the generation of stable transformed tissue culture derived plant lines. Nevertheless,this method can be laborious, time consuming and expensive. As an alternative method, leaf disc samples produced via leaf agroinfiltration was employed in this work. A promoter trapping construct named pCAMDIN was created which contains a promoterless GUS (β-glucuronidase) gene flanked by the left and right T-DNA border using pCAMBIA 1301 and pBI 121. Following that,the protocol for agroinfiltration of tomato plants using both direct agroinfiltration and vacuum agroinfiltration was optimized. Non-destructive protocols for detection of GUS genes were tested and optimised. Following that, GUS gene expression was studied and areas which showed expression were isolated by making leaf punches. Analysis was carried out by Southern blotting and T-DNA fingerprinting to determine the gene copy number.