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本文采用萌发六天的甘蓝(Brassica oleracea)下胚轴材料游离原生质体,经纯化后的原生质体产量为1.45×10~6g~(-1)FW,于含有1.5mg/L BA,0.5mg/L NAA和0.5mg/L 2,4-D的KM 8p的培养基中进行液体浅层培养。原生质体培养3—4天后出现一次分裂,七天时统计分裂频率为50.3%,培养15天左右可形成细胞团,3—4周后即可形成肉眼可见的小愈伤组织,统计形成愈伤组织的植板率为1.25%。将愈伤组织转至含有1.5mg/L BA和0.2mg/L 2,4-D的MS固体扩增培养基上进行扩增,其后可在含有2mg/L BA和0.5mg/L ZT的MS分化培养基上分化出芽,其分化率为54.17%,分化芽可于生根培养基中生根形成完整植株,移栽后,在人工气候室中生长良好。在试验过程中,对取材的不同时间,酶解液的不同配比对原生质体产量的影响,以及不同培养基、不同培养密度、不同的激素配比和不同培养方法等方面对原生质体的再生和持续分裂的影响进行了讨论。
In this paper, the free protoplasts of Brassica oleracea hypocotyls were germinated for six days. The yield of the purified protoplasts was 1.45 × 10 ~ 6g ~ (-1) FW. The protoplasts contained 1.5mg / L BA, 0.5mg / L NAA and 0.5 mg / L 2,4-D KM 8p medium for shallow liquid culture. Protoplasts cultured for 3-4 days after a split, the statistical division frequency of seven days was 50.3%, cultured for 15 days to form cell clusters, 3-4 weeks to form a macroscopic callus, the statistics of the formation of callus Planted rate of 1.25%. The callus was transferred to MS solid amplification medium containing 1.5 mg / L BA and 0.2 mg / L 2,4-D for amplification prior to culturing in medium containing 2 mg / L BA and 0.5 mg / L ZT MS differentiation medium, the differentiation rate was 54.17%. Differentiated shoots could be rooted in rooting medium to form intact plants. After transplanting, the shoots grew well in the artificial climate chamber. In the course of the experiment, the protoplast regeneration was studied in different time, the different proportion of the hydrolyzate on the protoplast yield, and different media, different culture density, different hormone ratio and different culture methods And the effects of sustained division were discussed.