甘蓝型油菜胞质体大量制备技术的研究
STUDIES ON BULK PRODUCTION OF CYTOPLASTS FROM HYPOCOTYL OF BRASSICA NAPUS
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摘要: 用Percoll密度梯度离心法和吖院橙(acridineorange,AO)结合光照处理两种方法,均从甘蓝型油菜下胚轴原生质体制备到胞质体、在2个Percoll梯度、30000g(18000r／min)与12C离心60min的条件下,获得了含胞质体80％以上的群体;以含80、100、120mg／LAO的酶液酶解下胚轴原生质体,纯化后分别给以3h、2h、lh光照(光强度:4000 lX)可使原生质体几乎不能分裂,但保持5d以上的生活力。由于光照导致AO－DNA复合体的DNA降解,造成核不可逆失活,从而可以认为经AO与光照处理的原生质体是胞质体。Abstract: Bulk cytoplasts were produced from hypocotyl protoplasts of B. napus by means of a discontinuous Percoll density gradient centrifugation or treatment with acridine orange (AO)followed by exposure to light irradiation. More than 80% cytoplastswere obtained in the interphase between WSsolution and 5% Percoll in a discontinuoustwo step gradients after 60 min centrafugation at 12 C30 000 g(18000 r/min). The protoplasts treated with 80 mg/L AO (14～18 h during maceration) plus 3 h irradiation(4000 lx), 100 mg/L AOplus 2 h irradiation or 120 mg/L AO plus 1 h irradiation seldom divided in culture,but could survive for more than 5 days. As light induced fragmentation of AO-DNA complex resulting in degradation of DNA,the nucleus inactivated irreversibly,thus the products may by considered as cytoplasts.
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Keywords:
- Brassica napus /
- Cytoplast /
- Percoll /
- Acridine orange
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