甘蓝子叶作为外植体转Hyg^r－Km^r基因的技术研究

Studies on Techniques of Tranfering Hyg￣r and Km￣r Genes to Cabbage with Its Cotyledon Explants

本文报告了以甘蓝（ｂｒｅｓｓｉｃａｏｌｅｒａｃｅａ）子叶为外植体，成功地将抗潮霉素（Ｈｙｇ~ｒ）基因和抗卡那霉素（Ｋｍ~ｒ）基因导入甘蓝品种，培育出转基因植物的技术研究成果．以ＭＳ为基本培养基，添加ＩＡＡ２ｍｇ／１，ＢＡ４ｍｇ／１，蔗糖２０ｍｇ／１和琼脂８．０ｍｇ／１，进行高再生率基因型选择，结果１９个品种的再生芽发生率在０％～９０％之间。对高再生率的品种进一步试验，进行不同激素浓度的适宜培养基的选择。结果表明：以ＩＡＡ３ｍｇ／１和ＢＡ４ｍｇ／１的ＭＳ固体培养基诱导甘蓝子叶，其再生芽发生率高达９５％～１００％；每个外植体平均可诱导出２．４～３．９个再生芽。抗生素耐性试验结果指出，培养基中含有５００ｍｇ／１羧苄青霉素几乎时再生芽的发生率和再生芽个数无影响，只是诱导的再生芽相对发育缓慢，体态弱小。用卡那霉素和潮霉素所进行的致死试验结果是：虽然致死率因基因型不同而有所不同，但培养基中含有Ｋｍ２０ｍｇ／１和Ｈｙｇ２０ｍｇ／１以上基本可以使所有的外植体死亡而无幼芽的再生。选择时期的试验结果显示，在外植体与ＥＨＡ１０１农杆菌共培养３～ｌ０ｄ范围内，共培养６ｄ和７ｄ进行选择分别获得３％～５％的转化率。

This paper reported the results of tranfering the genes of resistance to Hygromycinand Karamycin into Cabbage using its cotyledon explomts. The rates of regeneration shootsfrom 19 varieties of cabbage were 0% ￣ 90% in the basic MS medium added IAA2mg/l,BA4mg/1, sucross 20mg/1 and agrose 8.0mg/1. The selective experiment of hormonal concertratiorsand proportions showed that there were 95% ￣ 100% shoot regeneration rate and 2.4-3.9regeneration shoots per explant in the MS solid medium containe IAA3mg/1 and BA4mg/1. Wealso found that carbenicill in (500mg/1) in the medium had nearly no influence on shootregeneration rate and shoot reeeneration .number except that the regeneration shoots developpedslowly. The lethal test with Kanamycin and hygromycin in the mrdium could lead to dead of allthe explants. During 3 days-10days of co-culture of the explants with agrobacteriumtumefaciens EHA101 3%￣ 5% rate of the transgenic plants were obtained on the 6th dayand 7th day