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猛仔仔铁杆木虫 (正式写手)
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[求助]
关于水稻原生质体培养的问题
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最近在做水稻原生质体分离实验，但是感觉自己的细胞壁酶解液不能降解细胞壁，实验方法根据已经发表的文献作参考，具体如下： Dehulled seeds of rice (Oryza sativa L.) cultivar Nipponbare were sterilized with 75% ethanol for 1 min. These seeds were further sterilized with 2.5% sodium hypochlorite for 20 min, washed at least five times with sterile water and then incubated on 1/2 MS medium with a photoperiod of 12 h light (about 150 μmol m-2 s-1) and 12 h dark at 26°C for 7-10 days. Green tissues from the stem and sheath of 40-60 rice seedlings were used. A bundle of rice plants (about 30 seedlings) were cut together into approximately 0.5 mm strips with propulsive force using sharp razors. The strips were immediately transferred into 0.6 M mannitol for 10 min in the dark. After discarding the mannitol, the strips were incubated in an enzyme solution (1.5% Cellulase RS, 0.75% Macerozyme R-10, 0.6 M mannitol, 10 mM MES at pH 5.7, 10 mM CaCl2 and 0.1% BSA) for 4-5 h in the dark with gentle shaking (60-80 rpm). After the enzymatic digestion, an equal volume of W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl and 2 mM MES at pH 5.7) was added, followed by vigorous shaking by hand for 10 sec. Protoplasts were released by filtering through 40 μm nylon meshes into round bottom tubes with 3-5 washes of the strips using W5 solution. The pellets were collected by centrifugation at 1,500 rpm for 3 min with a swinging bucket. After washing once with W5 solution, the pellets were then resuspended in MMG solution (0.4 M mannitol, 15 mM MgCl2 and 4 mM MES at pH 5.7) 望有经验的网友给与指点，万分感谢！ |
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