Preparation of Sporoplasts for Studies of Pollen Physiology
Sporoplasts (pollen protoplasts) are produced in quantity from pollen of Lilium longiflorum Thunb. by exposure to 4-methylmorpholine N-oxide (MMNO). Treatment for 1 h at 25°C with aqueous MMNO, 15% w/v, in buffered solution, pH 5.3, containing 0.5% cellulase, 0.25% pectinase and 0.1% bovine serum albumin loosens the attachment of the sporoplast to exine but fails to effect release. Displacement from exine is achieved by a series of three treatments. First, MMNO-treated pollen is suspended in a buffer containing 0.5 mM EDTA, 0.6 M mannitol and 10 mM MES, pH 5.5 which effects release of sporoplasts from exines (25–30%). Then it is suspended in a buffer containing 5 mM CaCl2, 0.6 M mannitol and 5 roM MES, pH 5.5. Finally, it is suspended in 5 mM CaCl2, 0.3 M pentaerythritol and 3 mM MES, pH 5.3. Separation of sporoplasts from exines and undigested pollen is accomplished on a discontinuous sucrose density gradient. Purified sporoplasts are readily ruptured by passing the suspension through a 15 um3 nylon mesh. Data is presented on cell-free distribution within selfgenerated Percoll gradients of phytase and a phytate-rich component.
KeywordsSucrose Phosphorus EDTA Germinate Syringe
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