根癌农杆菌介导的骏枣愈伤组织遗传转化体系的研究

采用农杆菌介导法,对骏枣愈伤组织遗传转化体系进行研究.结果表明：进行遗传转化的合适条件为：愈伤诱导培养基MS＋6-BA 0.4 mg/L＋2,4-D 1.2 mg/L;浸染之前对愈伤组织进行6d的避光培养可提高愈伤组织在共培养过程中的成活率;愈伤组织在OD600=0.4～0.6的农杆菌菌液中浸染10 min,农杆菌LBA4404的生长情况最好;在28℃黑暗条件下共培养放置16 d对转化最适宜;头孢霉素的抑菌浓度为250 mg/L;卡那霉素的筛选浓度为125 mg/L.经PCR检测,初步证明外源目的基因已整合到骏枣基因组中,初步实现了农杆菌介导的骏枣愈伤组织遗传转化,与以骏枣茎叶外植体建立的遗传转化体系相比,以骏枣愈伤组织为外植体共培养时间长,有利于转化.

Study on Agrobacterium Tumefaciens-mediated Genetic Transformation System for Callus of Ziziphus jujube cv.Junzao

The transformation system of Ziziphus jujuba cv. Junzao callus was studied by Agrobacterium mediated method. The results showed that：the appropriate conditions for genetic transformation： callus induction medium is MS ＋ 6 - BA of 0.4 mg of/L ＋ 2, 4 - D of 1.2 mg of/L; It could be improved the survival rate of callus in the process of co culture that callus of Ziziphus jujuba cv. Junzao were dark culture before contaminated ; The Agrobac- terium tumefaciens LBA4404 was grew best when callus was contaminated about 10 min in bacteria liquid（ OD600 = 0.4 ~0.6） ;It was suitable for conversion that Callus and bacteria liquid were culture together about 16 d at 28 ℃ under dark condition ; Cephalosporium inhibitory concentration was 250 mg/L; the screening concentration of kana- mycin was 125 mg/L; It was indicated that foreign gene had been integrated into the genome of Jun jujube by PCR detection. Compared with the stem and leaf of Ziziphus jujube cv. Junzao, the explants as callus were cultured for long time, conducive to the transformation.