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KMID : 0903519940370050343
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Journal of the Korean Society of Agricultural Chemistry and Biotechnology
1994 Volume.37 No. 5 p.343 ~ p.348
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Delivery of Ti Plasmid into Nicotiana sanderae Protoplasts via Liposomes
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Abstract
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Ti plasmid of A. tumefaciens was labeled with ³H-thymidine, purified and encapsulated into phosphatidylserine (PS) and PS-cholesterol (Chol; 1 : 1 molar ratio) liposomes by lyophilization-rehydration method. PS was supplemented with 1 mole percent octadecyl rhodamine B for fluorometric measurement of PS. Liposomes entrapping ³H-Ti plasmid were fused with Nicotiana sanderae protoplasts by treating with 5 mM CaCl₂ and 10% PEG. The fusion was evidenced by fluorescence microscopic technique. The amounts of Ti plasmid and PS associated with protoplasts were assayed by the radioactivity of ³H-Ti plasmid and by the fluorescence of rhodamine B. About 7.9% of the PS liposome and 7.2% of PS-Chol liposome were fused with protoplasts. During the fusion process, about 30% of the liposomal contents of PS-Chol liposome was leaked. in contrast to about 60% leakage of its contents in PS liposome. Accounting the number of liposomes fused with protoplasts together with the encapsulation efficiency and the leakage of liposomal contents, it was calculated that ca. 1,700 Ti plasmid was transfered into one protoplast by the present method. This result may indicates that the present method transfers enough Ti plasmid into plant protoplast to elicit genetic transformation of plants.
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