Polyethylene Glycol-Mediated Direct Gene Transfer to Tobacco Protoplasts and Regeneration of Transgenic Plants
The protocol detailed below describes the procedure for the transient and stable transformation of leaf protoplasts obtained from axenic shoot cultures of Nicotiana tabacum L. cv. Petit Havana SR1, a widely used tobacco genotype with model character (Maliga et al. 1973). The protoplasts are isolated following a modified protocol of Nagy and Maliga (1976). The transformation procedure mediated by polyethylene glycol (PEG) is a simplified version of the protocol by Negrutiu et al. (1987). For recovery of stable transformants, treated protoplasts are grown and selected in bead-type culture (Shillito et al. 1983), and plant regeneration from transformed calli is according to Potrykus and Shillito (1986) with modifications.
KeywordsStarch Mercury Agar Pyruvate Folic Acid
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- Nagy JI, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of N. sylvestris. Z Pflanzenphysiol 78: 453–455Google Scholar
- Negrutiu I, Shillito RD, Potrykus I, Biasini G, Sala F (1987) Hybrid genes in the analysis of transformation conditions. I. Setting up a simple method for direct gene transfer in plant protoplasts. Plant Mol Biol 8: 363–373Google Scholar