AN EFFICIENT METHOD OF ISOLATION AND TRANSFORMATION OF PROTOPLASTS FROM TOMATO LEAF MESOPHYLL TISSUE USING THE BINARY VECTOR PCAMBIAA 1302

Abstract: This is a new method of isolation and transformation of protoplasts from tomato leaf mesophyll tissue using polyethylene glycol. The binary vector pCambia 1302 carrying green fluorescence protein sequence as reporter was used and the transformed protoplasts were selected using hygromycin. Important parameters which are critical for the quality of protoplast and efficiency of transformation, such as molecular weight of polyethylene glycol, kinds of osmoticum used and period of incubation, were standardized. The transformed protoplasts were screened by the selectable marker and further confirmed with PCR. The protoplasts were analyzed with propidium iodide and fluorescence microscopy. The transformation efficiency was studied with the assay of the green fluorescence protein. This is the first protoplast isolation protocol for the Pusa Ruby variety of tomato and also the first protocol to use hygromycin as the selectable marker for tomato.

Keywords: Tomato, protoplast, mesophyll, pCambia, PEG, fluorescence, GFP, hygromycin.