根癌农杆菌对甘蓝型油菜的转化及转基因植株的再生

TRANSFORMATION OF BRASSICA NAPUS USINGAGROBAC TERIUM TUMEFACIENS AND REGENERATION OF TRANSGENIC PLANTS

用根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍ ｅｆａｃｉｅｎｓ）共培养法把外源基因导入甘蓝型油菜（Ｂｒａｓｓｉ－ｃａ ｎａｐｕｓＬ．）主要栽培品种“云北２ 号”，获得转基因植株。所用外植体为带有１—２ ｍ ｍ 子叶柄的完整子叶，根癌农杆菌为Ａ２０８ＳＥ（ｐＴｉＴ３７－ＳＥ， ｐＲＯＡ９３）。Ｔｉ质粒ｐＲＯＡ９３ 带有ＮＰＴⅡ及ＧＵＳ嵌合基因。共培养２ 天后转到附加２５ ｍ ｇ／Ｌ卡那霉素的分化培养基（ＭＳ＋ ４．５ ｍ ｇ／ＬＢＡＰ）上。ＡｇＮＯ３ 和羧苄青霉素促进芽的分化，头孢霉素则有抑制作用。最高转化频率为２７％ 。把分化出的茎芽切下，插入含有２５ ｍ ｇ／Ｌ卡那霉素的生根培养基中。羧苄青霉素不利于根的形成。把完整抗性植株移入盛土壤的盆中，生长状况良好。测定β－葡糖苷酸酶活性，８４％ 明显高于对照。以ＮＰＴⅡ基因作探针进行Ｓｏｕｔｈｅｒｎ ｂｌｏｔ分析。

Seeds of Brassica napus L.cv.“Yunbei 2” were surface sterilized and germinated on hormone free MS medium.After 4—5 days the cotyledons were excised in such a way that each has a 1—2 mm petiole was remained at its base.These cotyledons were used as the explants for tissue culture and genetic transformation.This paper first deals with the improvement of the medium for shoot regeneration.Of the elements tested, AgNO 3 and carbenicillin enhanced shoot regeneration.The highest frequency (52%) was obtained on MS medium containing 4 5 mg/L BAP, 20 μmol/L AgNO 3 and 500 mg/L carbenicillin.An efficient gene transfer system based on the regeneration procedure was established.After 2 days of cocultivation with Agrobacterium tumefaciens strain A208SE (pTi T37 SE, pROA93), the explants were transferred onto selection medium containing 25 mg/L kanamycin.After 1 5 months shoots emerged from 27% of the explants inoculated.They were excised and transferred onto rooting medium containing 25 mg/L kanamycin and 200 mg/L cefotaxime which is better than carbenicillin for root induction.Whole plants were transplanted into pots, and grew well in the phytotron.Transformation was confirmed by β glucuronidase assay and Southern blotting analysis.