| In this research, the potato variety ‘Qingshu9’ in vitro plants were used asexplant materials. Through the study of callus induction and plant regenerating, thesystematic screenings of callus induction mediums, explant forms and callusregenerating mediums were optimized. And the main factors affecting plantregenerating and the genetic transformation via LBA4404Agrobacteriumtumefaciens were studied.The callus induction and regeneration system and the initially genetic transformation system via Agrobacterium tumefaciens of ‘Qingshu9’potato were established. At the same time, the genetic transformating work of several ERFs transcription factors was carried out.1.The stem cuttings were explant materials in the experiment, and the better callus induction medium was MS medium without organic composition+sucrose25g·L-1+agar6g·L-1+myo-insitol100mg·L-1+nicotinic acid2mg·L-1+pyridoxine-HCl（VB6）0.5mg·L-1+thiamine-HCl（VB1）0.4mg·L-1+folic acid0.25mg·L-1+d-biotin0.05mg·L-1+NAA3.0mg·L-1+6-BA6.0mg·L-1. However the bestregeneration medium was MS+sucrose25g·L-1+agar8g·L-1+Casein enzymatic hydrolysate1g·L-1+NAA0.1mg·L-1+6-BA3.0mg·L-1+GA33.0mg·L-1+trans-zeatin riboside1.0mg·L-1.2.In the genetic transformation via Agrobacterium tumefaciens, the optimum pre-incubation duration was3d, and optimum infection duration was between8to10min. The co-culture period was3d, and optimum bacterial concentration was OD600=0.81.0.3.Cefotaxime sodium was used as bactericide, which can restrain bacterium infection without any negative influence on regeneration of explants when the concentration was between200to300mg·L-1.4.The best concentration of kanamycine used as selective agent was75mg·L-1for callus formation and radication. And the best concentration of chloromycetin used as selective agent was30mg·L-1for callus formation and50mg·L-1for radication.5.In the experiment,481stem cuttings and190foliage pieces were infected by Agrobacterium tumefaciens.At last we obtained10transformed plants that carried P1200-TERF1gene. |
PDF Full Text Request