Protoplasts from Phytolacca dodecandra L'Herit (endod) and P-americana L (pokeweed)

被引:0
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作者
Koch, PE [1 ]
Bonness, MS [1 ]
Lu, H [1 ]
Mabry, TJ [1 ]
机构
[1] UNIV TEXAS,DEPT BOT,AUSTIN,TX 78713
关键词
Phytolacca; protoplast; ribosome-inactivating protein; pokeweed antiviral protein; tissue culture;
D O I
暂无
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
Pokeweed (Phytolacca americana L.) and ended (P. dodecandra L'Herit) produce ribosome-inactivating proteins which are sequestered in leaf cell walls. These proteins display strong antiviral activity. To aid in studying the antiviral mechanism, we developed protocols to isolate protoplasts from suspension culture cells and leaves. Ninety-five percent of pokeweed or ended culture cells were converted to protoplasts using 2% cellulase, 0.25% pectinase, 0.2 M mannitol, 2% sucrose, 15 mM CaCl2, Murashige and Skoog salts, pH 5.7. Viability was >85% after 24 h. Culture-derived protoplasts were purified by centrifugation through a 15% sucrose pad. Protoplasts collected from the supernatant were then pelleted in 0.3 M mannitol. Pokeweed leaves provided respectable yields (4 x 10(6) protoplasts/g f w) of partially-purified viable protoplasts when digested in solution containing 1% cellulase, 0.2% Pectolyase, 0.4 M mannitol, CPW salts, 0.5 mM MES, pH 5.6. We were unable to completely separate cell debris from mesophyll protoplasts, which were small and easily damaged by centrifugation. Ended leaves were found to be resilient to several digestion enzymes tested.
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页码:824 / 828
页数:5
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