农杆菌介导的海岛棉茎尖再生体系及其遗传转化影响因子的研究

采用根癌农杆菌LBA4404和EHA105介导,对海岛棉的茎尖再生体系及其转化影响因子进行了研究。茎尖培养与棉花体细胞胚胎发生相比,不同基因型的茎尖再生频率差异不明显;在9种不同的激素组合中,以6-BA0.5 mg/L NAA0.1 mg/L与1/2MSB5 IBA 0.1 mg/L对茎尖的再生效果较好;获得较高转化频率的海岛棉培养条件为:海岛苗茎尖在OD600值为0.6~0.8农杆菌菌液中感染20~30 min,共培养3 d后进行转接,在含有不同激素和抗生素的各类培养基中进行抑菌和筛选继代培养,选择压以50~150 mg/L梯度进行;农杆菌以EHA105菌株的转化效果较好;抗性苗用SDS-CTAB法提取其总DNA,进行PCR扩增检测,有7株PCR检测呈阳性反应。由此,初步证明外源抗虫基因已经进入到再生植株的细胞核中。

Study on Regeneration System and Influencing Factors of Genetic Transformation of G. barbadense Shoot Apex Mediated by Agrobacterium

Regeneration system and influencing factors for genetic transformation of G.barbadense shoot apexes mediated by Agrobacterium LBA4404 and EHA105 were studied.In comparison of the culture of G.barbadense shoot apex with the occurrence of cotton cell embryo,the regeneration frequency of different genotype didn't show significance.Among 9 different combinations of hormones,6-BA 0.5 mg/L NAA0.1 mg/L and 1/2MSB5 0.1 mg/L IBA combined were the best for the generation.The higher transformation frequency was detected under the conditions of subsequently infection for about 20-30 minutes in Abarobacteriunz suspension with OD600 value about 0.6-0.8 followed by co-culture for 3 days.The regeneration culture was conducted in different medium with different growth hormones and antibiotics under the selection marker 50-150 mg/L in the medium.The transformation efficiency and frequency of Agrobacterium EHA105 strains were optimized in the protocol.The results of PCR analysis preliminarily showed that the BT gene in the T-DNA had been integrated into G.barbadense cotton genome.