| In this study, genetic transformation technology system were established with shoot tips of brown cotton Xincaimian No.5 and No.13, the variety cultured in Xinjiang of Gossypium hirsutum L. by the agrobacterium-mediated transformation method and gene gun mediated transformation method. Through the above two kinds of genetic transformation methods, ANR gene into shoot apical meristem of brown cotton, after the kanamycin resistant shoot tips screening, resistant germs inducted to take root, PCR and qPCR molecular detection, transgenic plants were obtained from two brown cotton varieties. and T2 generation seeds of transgenic positive plants were obtained. The establishment of the genetic transformation technology system lays a foundation for genetic transformation of brown cotton in Xinjiang. The main results are as follows: 1.The establishment of agrobacterium-mediated genetic transformation system with shoot tips of brown cottonThe optimal genetic transformation technology system is as follows, under other constant conditions. Shoot tips were isolated from 4 days dark and 2 days light culture old seedlings, activation of agrobacterium suspension to OD600 value is between0.6-0.8, infected 10 min, co-cultivated 48 h. After shoot tips were moved to MSB screening medium containing 8g·L-1agar powder and 125mg·L-1or100mg·L-1kanamycin, then screened for 25 days to obtain the resistant germ. Resistant germs were induced to take roots in the induction rooting medium adding 1g·L-1 activated charcoal to obtain the resistant regenerating plants. After PCR and qPCR detections, five transgenic positive plants were obtained from 1250 Xincaimian No.5 shoot tips, and transformation rate was 0.31%;and three transgenic positive plants were obtained from 1300 Xincaimian No.13 shoot tips, and transformation rate was0.25%. 2. The establishment of biolistics genetic transformation system with shoot tips of brown cottonThe optimal biolistics genetic transformation technology system is as follows, under other constant conditions. Shoot tips were isolated from 4 days dark and 1 day light culture old seedlings, and were cultured for 4h before being bombarded in the osmotic medium. Then shoot tips were bombarded with the microcarriers diameter of 1.0μm, the DNA concentration of 1.0μg·μL-1, the helium pressure of 7.586MPa（1100 psi）, the bombardment distance of 9cm, the vacuum pressure of 0.0946MPa（28inch Hg）. After culture for 4 days in the recovery medium, shoot tips were moved to MSB screening medium containing 8g·L-1 agar powder and 125mg·L-1 kanamycin, then screened for 25 days to obtain the resistant germ. Resistant germs were induced to take roots in the induction rooting medium adding 1g·L-1 activated charcoal to obtain the resistant regenerating plants. After PCR and qPCR detections, sixteen transgenic positive plants were obtained from 5200 Xincaimian No.5 shoot tips, and transformation rate was 0.31%. 3. Preliminary function analysis of GhANR geneTwelve transgenic lines of Xincaimian No.5 and two transgenic lines of Xincaimian No.13 were obtained by the agrobacterium-mediated transformation method and gene gun mediated transformation method. After qPCR quantitative analysis, compared with the control, the relative expression of transgenic positive plants ANR gene were declined by 47.5%85.48% and 65.3175.51, respectively. Compared with wild type,fiber color of the T1 generation transgenic positive plants obviously diluted. |
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