Studies On Construction Of Regeneration System And Genetic Transformation Of Puccinellia Chinampoensis

Puccinellia chinampoensis is a special gramineous plant like rice and wheat, which can survive well in the saline and alkali environment with severe drought and salt stress. The difference from rice or wheat is Puccinellia chinampoensis has evolved a series of resistance mechanism via years of adaptation in the saline and alkali soil. In this study, transformation system of Puccinellia chinampoensis has been constructed and optimized, following with optimized regeneration system, through plant tissue culture technique. The whole constructed system will be pre-determinative for the further studies as gene interference.In this study, some crucial factors which affected the embryo culturing using Puccinellia chinampoensis as the main plant material were discussed comprehensively and systemically. And the genetic transformation of put-LS2 mediated by Agrobacterium tumefaciens was followingly conducted using the above callus. The main results obtained in this study contains as follows.In the callus induction process of Puccinellia embryo, different sterilant and sterilized time to seed embryo showed different effects and induced situation obviously. In this study, the best combination was 70% ethanol for 3min and 10% sodium hypochlotous for 30min. The rate of callus induction was different in the medium containing different concentrations of 2,4-D and the optimized was 4 mg/L. The altered medium I (N6max+MSmin+2×MSFe+5×MSvitamin (inositol no change)) was chosen to be the basic medium to induce the callus. The time of cultivation in light also have effect on the callus induction. The shorter of the light time, the higher rate of the callus induction would get.In the callus subculture of Puccinellia chinampoensis seeds,different mediums showed different impacts. The result showed that the modified medium S(MSmax+4×MSmin+MSFe+ N6vitamin) was chosen as the basic subculture medium.1mg/Lphytohormone ABA was added to the medium to improve the quality and the quantity of embryo callus.In the differentiation of callus of Puccinellia chinampoensis embryo, MS was the differentiation medium. Different ratio of hormone had different effects on differentiation. The result indicated that the best was 6-BA 0.4mg/L+IAA 0.04mg/L.Sucrose was substituted with sorbital and maltose, which could inhibit callus browning obviously and increase differentiation rate.500mg/L proline was added into induction medium, succeeded medium and differentiation medium to effectively prevent callus from browning and to improve differentiation rate.In the transformation test of Puccinellia chinampoensis callus, using kanamycin (Kana) to screen transformed callus, the optimized concentration was 50mg/L. The beat way of remove of bacterial is filtration. pBl121-GUS was transformed into the callus mediated by Agrobacter-ium tumefaciens, and the transformation rate was 47.14%.