甘蓝下胚轴原生质体高效成株系统的建立

ESTABLISHMENT OF HIGH EFFICIENT REGENERATION SYSTEM FROM HYPOCOTYL PROTOPLAST OF BRASSICA OLERACEA

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。

The yield of purified protoplasts isolated from hypocotyl of 6 d old Brassica oleracea seeding was 1.45Z×106g-1 FW. The protoplasts were cultured in KM 8 p liquid medium supplemented with 1.5mg/L 6-BA, 0.5mg/L NAA and 0.5mg/L 2,4-D. The protoplasts started to divide after 3-4 days of culture. The frequency determined at 7 days was as high as 50.3%. Cell colonies were formed after about 15 days of culture. Actually microcalli could be observed at 3-4 weeks. The plate frequency was about 1.25%, The microcalli were transferred onto the proliferation medium (MS+1.5 mg/L 6-BA+0.2mg/L 2,4-D). And until when they were about 3-5 mm in size, then they were transfer- red onto the differentiation medium (MS + 2.0 mg/L BA +0.5 mg/L ZT) to differentiate shoots. The frequency of shoot formation was 54.17%, The shoots rooted in the rooting medium (1/2 MS+0.5 mg/L IBA), and the whole plants were transplanted into pots and grew well in the phy-totron. In our experiments, the effects of hypocotyls from different time and the different enzyme solution on the yield or protoplasts were examined. At the same time, the effects of different culture medium, protoplast density, hormones and culture methods on the regeneration and sustained division of protoplasts were also discussed.