Optimization of Factors Influencing Microinjection Method for Agrobacterium tumefaciens-Mediated Transformation of Tomato.

Vinoth, S.; Gurusaravanan, P.; Jayabalan, N.

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD = 0.2-1.0). The germinated seeds were cocultivated in the MS medium fortified with (0-200 mM) acetosyringone and minimal concentrations of (0-20 mg L) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg L thidiazuron, 1.5 mg L indole-3-butyric acid, 30 mg L kanamycin, and 0-1.5 mg L adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.

PLANT micropropagation; TOMATOES; TISSUE culture; SEEDS; AGROBACTERIUM

Applied Biochemistry & Biotechnology, 2013, Vol 169, Issue 4, p1173

0273-2289

Academic Journal

10.1007/s12010-012-0006-0