A simple and efficient Agrobacterium-mediated transformation of tomato

Abstract

A reproducible transformation system for the tomato genotype IPA-3 using Agrobacterium tumefaciens and cotyledon explants was developed. Disarmed Agrobacterium tumefaciens - GV 3101 carrying binary vector p35S-2-SFR was used in the genetic transformation study. The binary vector contained GUS under the control of cauliflower mosaic 35S (CaMV35S) promoter. Factors governing the efficiency of Agrobacterium-mediated transformation include age of explants, bacterial concentration, preculture period and co-cultivation time were optimized. The frequency of GUS expression varied from 2.66 to 23.16 % under different treatments. The Agrobacterium (OD 600=1) inoculum when diluted to 1:20 recorded maximum GUS expression on treatment duration of 30 minutes. The 10 day old seedling explants i.e. cotyledons also proved superior to transformation over 7 and 12 day old. The effect of 1 day pre-culture was also significant over 2 and 3-day. Similarly, co-cultivation period also showed significant effect on percent GUS expression and thereby transformation efficiency. The present protocol can be considered as a simple and reproducible protocol for obtaining high frequency of transformation in Tomato.