Plant Physiol. Journal of Pharmacology and Experimental Therapeutics
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Plant Physiology 93:857-863 (1990)
© 1990 American Society of Plant Biologists

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Molecular Biology and Gene Regulation

A Polyethylene Glycol-Mediated Protoplast Transformation System for Production of Fertile Transgenic Rice Plants 1

Akio Hayashimoto2, Zhijian Li and Norimoto Murai

Department of Plant Pathology and Crop Physiology, College of Agriculture, Louisiana State University, Baton Rouge, Louisiana 70803-1720

We have established an efficient procedure for protoplast transformation and regeneration of fertile transgenic plants of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Protoplasts were mixed with a plant-expressible hygromycin resistance gene and treated with 25% (w/v) polyethylene glycol. Stringent selection of transformed colonies was applied to 14-day-old regenerated protoplasts in the presence of 95 micromolar of hygromycin B for 12 days. After selection, 450 and 200 resistant colonies were recovered per million treated Taipei 309 and Nipponbare protoplasts, respectively. Southern hybridization analysis of hygromycin-resistant cell lines and regenerated plants indicated that 1 to 10 copies of transferred DNA were integrated at 1 to 4 loci of the rice genome. Southern DNA analysis suggests that the introduced plasmid DNA may form concatemers by intermolecular recombination prior to integration. Four Taipei 309 and 39 Nipponbare transgenic rice plants were regenerated and grown to maturity in the greenhouse. Two Taipei 309 and 35 Nipponbare plants set viable seeds. Agronomic traits of Taipei 309 transgenic plants and inheritance of the hygromycin resistance trait by progeny of the selfed transgenic plants were analyzed.

2 Present address: Department of Biotechnology, Ishihara Sangyo Kaisha, Ltd., Kusatsu, Shiga 525, Japan.

1 This work was supported in part by grants from the Louisiana Education Quality Support Fund (1987-90)-RD-A-6 and from Ishihara Sangyo Kaisha, Ltd. to N. M.




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