Studies On Transformation Of AtNDPK2Gene Into Medicago Sativa L.

Alfalfa （Medicago sativa L.） was a rich nutritional forage which was widely grown all over the wrold.However, Low temperature, salinization, drought and other adversities environment which seriously restrictedthe plant growth, reduced productivity and threated the survival of crop. It leaded to the continuousdegradation of the vegetation and the ecological environment. Therefore, by means of genetic engineering, weselected excellent stress-related genes to cultivate new alfalfa varieties, which not only established a solidtheoretical basis for the study of genetic breeding, but also promoted the development problems of animalhusbandry.This experiment established “Ao han” and “long mu801” hypocotyls’ regeneration system in theHeilongjiang Province.“Ao han” as the research object made its hypocotyls’ regeneration system asnucleoside diphosphate kinase gene2（AtNDPK2） transformed receptor. The mediated transformation Methodfor Agrobacterium EHA105to transform and obtain transgenic alfalfa plants. The transgenic plants weremolecular biological detected and salt stress analyzed the results as follow:1. The hypocotyl of “Ao han”and “Long mu801” could be induced callus on the improved SH basicmedium.0.15mg·L^-1KT and2mg·L^-12,4-D induced into embryogenic callus, the hormones of0.1mg·L^-1KTwas in favour of embryogenic callus differentiation.“Ao han”callus induction rate was100%, embryogeniccallus differentiation rate was53.3%;“Long mu801” callus induction rate was98.3%, embryogenic callusdifferentiation rate was37.5%; Rooting rate of both varieties was100%on1/2MS.2.350mg·L^-1Cefotaxime （Cef） could effectively inhibit the growth of agrobacterium EHA105,50mg·L^-1kanamycin （Kan） did not affect the hypocotyl embryogenic in transformation system. The results ofantibiotic selection pressure were: the hypocotyls of embryogenic callus induction rate was not affected byconcentration of Cef; the hypocotyls were sensitivesed to Kan, the high concentrations of Kan suppressedembryogenic callus formation. The result of study on preculture time, co-culture time and selecting methodsfound: obtaining resistance embryogenic callus of5d preculture,2d co-culture time and delay selecting wasthe highest, the transferred rate was23.3%.3. The23transgenic plants were detected by PCR,20transgenic plants were obtained. Leaves oftransgenic plants and control plants of SOD、POD activity and MDA content were measured under differentconcentrations of NaCl stress, observing SOD、POD activity of transgenic plants that was higher than thecontrol, but MDA was lower at0.4%～1.0%NaCl. It fully explained that the genetic transformation ofAtNDPK2gene enhanced alfalfa plants to salt stress.