高山红景天毛状根培养系统的建立

【作者】 徐洪伟；

【导师】 陆静梅；

【作者基本信息】 东北师范大学 ， 植物学， 2004， 硕士

【摘要】 本文利用发根农杆菌A4，R1601菌株和 ATCC15834菌株侵染高山红景天，通过对外植体的选择、外植体最适生长条件的优化、农杆菌侵染条件的优化、除菌条件的优化、毛状根生长条件的优化，建立了高山红景天的毛状根培养系统。研究表明，农杆菌A4；R1601；ATCC15834侵染高山红景天能产生毛状根，其中，ATCC15834和A4菌株侵染率高；外植体以叶片最好，发根农杆菌菌液浓度在OD600=0.4~0.5；乙酰丁香酮浓度为50μmol/L；侵染时间为25分钟；共培养时间为2天；共培养的培养基的pH值为5.0；抑菌培养基添加“羧苄青霉素150㎎/l +头孢霉素100㎎/l”的培养条件最佳。PCR检测结果表明：所获得的高山红景天毛状根在500bp处有与rolC基因相同大小条带存在。PCR-Southern分子杂交检测进一步证明了该条带是rolC基因， 高山红景天基因组中整合了来源于Ri 质粒的rolC基因。转化的毛状根在不添加外源激素的培养基上自主地快速生长并表现出了典型的毛状根特征。研究表明：在1/2 MS0液体培养基、散射光、温度为20℃左右、pH4.5~4.8等条件下，毛状根生长迅速。更多还原

【Abstract】 To establishment of hairy roots cultures of Rhodiola sachalinensis A.Bor with Agrobacterium rhizogenes strains (A4，R1601, and ATCC15834), the following experiment parameters and procedures were optimized, such as selecting some suitable explants, the growth condition for explants, the period of Agroinfiltration, and the growth condition for hairy roots. The research results showed that hairy roots of Rhodiola sachalinensis A.Bor were induced with A. rhizogenes strains (A4，R1601, and ATCC15834), the strains (A4 and ATCC15834) of which had the better effect. It has also been found that the leaf as explants was superior to these organisms. The bacterium concentration OD600=0.4-0.5 and AS (50μmol/L) to the medium for 25 minutes increased the infiltration frequency. The infection frequency was generally higher when the co-cultivation durations of 2 days and co-cultivation medium at pH 5.0 was applied. The addition of Cb (150㎎/l) and Cef (100㎎/l) to the medium showed the optimum resistant-bacterium efficiency. PCR and PCR-Southern blot revealed the integration of rolC gene from Ri T-DNA into the plant genome. The transformed roots grew rapidly on hormone-free medium and showed typical hairy root phenotype. The optimum growth conditions of hairy roots were identified as follows: 1/2 MS0 liquid medium, light free, temperature 20oC, pH 4.5-4.8.更多还原