Polyethylene-Glycol-Mediated Direct Gene Transfer to Indica Rice Protoplasts and Regeneration of Transgenic Plants

Abstract

This is a simple protocol describing a standard procedure of protoplast isolation (Datta et al. 1990a, 1992b), Polyethylene-glycol (PEG)-mediated stable transformation (Datta et al. 1990b, 1992a; Peterhans et al. 1990) and regeneration of transgenic Indica rice plants. Embryogenic suspension cells provide the key source for regenerable competent rice protoplasts. Microspore culture, immature or mature embryos may be used to obtain embryogenic calli, and eventually embryogenic suspension cells can be established. Electroporation does work for rice protoplast transformation. However, PEG-mediated gene transfer to rice protoplasts appears to be the most efficient, reliable, inexpensive, and simplest method when it does work. In this system, large populations of protoplasts can be obtained readily for the transformation and further selection (for a review on gene transfer to plants, see Potrykus 1991). Regeneration of transgenic plants is possible under suitable in vitro conditions through selection. However, the tissue culture response may vary depending on the plant material (genotype) used and handling of suspension cells. The procedure described below has been successfully used for a number of Indica genotypes, e.g., IR43, IR58, IR72 (IRRI breeding lines), Tepi Boro (Bangladesh), Chinsurah Boro II, Basmati 370 (India), and Makalioka 34 (Madagascar), which may provide a suitable system for general Indica rice transformation and regeneration of transgenic plants.