Estimation of overall efficiencies of Agrobacterium-mediated transformation system in tomato (Pusa ruby) based on the expression of universal marker genes at both RNA and protein levels

Tien Van Vu*, Nirupam Roy Choudhury, Vinh Nang Do, Sunil Kumar Mukherjee

Abstract


Tomato (Solanum lycopersicum L.) is a valuable source of nutritional as well as medicinal subtances for human’s healthcare. However, as the cultivation of tomato is profoundly affected by both biotic and abiotic stresses, generation of transgenic tomatoes which can tolerate/resist these stresses assumes great importance. In the present study, we have optimized and for the first time estimated overall efficiencies of Agrobacterium-mediated transformation system for tomato Pusa ruby cultivar based on the expression of universal marker genes (GUS and GFP) at both RNA and protein levels. Two different formulas of plant growth regulators, 1.5 mg/l of 6-benzylaminopurine (BAP) combined with 0.5 mg/l of indole-3-acetic acid (IAA) and 0.5 mg/l of zeatin combined with 0.1 mg/l of IAA, were used for regeneration of transgenic plants from cotyledonous tissues, and our data revealed significant differences of transient expression of marker genes for these two combinations. The overall efficiencies of the transformation system was calculated as a ratio of the number of the intact transformants (counted as 1 intact plant for 1 explant used for transformation) to the total explants used and were observed to be 4.55 ± 1.02% and 3.11 ± 0.71% for GUS gene and GFP, respectively. Molecular analyses of the GFP transgenic plants indicated various levels of post-transcriptional gene silencing. This system can also be used to efficiently transform gene silencing constructs (RNAi, artificial microRNA expression cassettes) or protein expressing constructs into tomato Pusa ruby cultivar.

Keywords


Agrobacterium-mediated transformation; GUS/GFP marker genes; Post-transcriptional gene silencing; Shoot regeneration; Transgenic tomato plants

Full Text:

PDF

Refbacks

  • There are currently no refbacks.




Copyright (c) 2013 Annals of Plant Sciences

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.