建立青菜(Brassica　chinensis)农杆菌介导法基因转化体系

Establishment of an Agrobacterium mediated Genetic Transformation System for Common Chinese Cabbage( Brassica chinensis )

以青菜地方品种矮脚黄、苏州青的子叶、子叶柄、真叶为外植体，在改良ＭＳ培养基上离体培养获得再生植株。在不定芽诱导培养基中添加２～２０ｍｇ／ＬＡｇＮＯ３可显著提高子叶不定芽诱导频率；添加０．１ｍｇ／ＬＮＡＡ和１～２ｍｇ／ＬＣＰＰＵ既可提高不定芽诱导频率，又可获得较多数量的不定芽。用含ＣｐＴＩ基因和ＮＰＴＩＩ基因的农杆菌感染子叶，获得了具有卡那霉素抗性的再生植株。培养基中添加羧苄青霉素促进子叶再生不定芽，头孢霉素和卡那霉素则抑制子叶再生。卡那霉素敏感性测定结果表明，当培养基中含卡那霉素１０ｍｇ／Ｌ时。

The cotyledons,cotyledon petioles and leaves of Brassica chinensis cv.Aijiaohuang and Suzhouqin were used as transformation recipients.The regenerated plants were obtained on the modified MS medium with growth regulators.The presence of 2-20 mg/L AgNO 3 on the shoot induction medium markedly enhanced shoot regeneration frequencies from cotyledons.The percentage of shoot regeneration and the number of shoots per cotyledon were increased on the medium supplemented with 0 1 mg/L NAA and 1 to 2 mg/L CPPU. By means of Agrobacterium mediated transformation,plantlets resistant to kanamycin were obtained from cotyledons of Brassica chinensis. The Agrobacterium tumefaciens strain LBA4404 harbouring mini Ti plasmid pRCL27 carrying the CpTI gene and NPTII gene was used for transformation.Carbenicillin could increase percent shoot regeneration, while cefotaxime reduced shoot regeneration.The test also showed that shoot regeneration from cotyledons was inhibited by kanamycin.All of shoots turned white on the medium with 10 mg/L kanamycin.Therefore,10 mg/L kanamycin was applied to the selection of transformed shoots.