The Establishment Of Regeneration Systems And BADH Gene Transformation Of Alfalfa

Abstract: Alfalfa (Medicago sativa) is the biggest for the growth area and the highest quality forage grass. Development and application of genetic engineering opened up a new way for the alfalfa in resisting salt breeding. Tissue culture technique of ten alfalfa breeds(Hetian,A'ergangjin,Gannong NO.1,Gannong NO.3,Gannong NO.4,Longzhong,Muge,Defu,Runbule,Sandeli) was researched and the BADH gene was transferred to alfalfa by Agrobacterium mediated transformation in this paper. The main achievements of our rearch are following:1) The establishment of regeneration systems. The test indecated that to different breeds,the regeneration ability isn't same.From stronge to low,the order is:GannongNO.4>Muge>Longzhong>Sandeli>A'ergangjin>Defu>Runbule>Gannong NO.3>Gannong NO.1>Hetian.To different explant,hypocotyl has the best ability to induce callus. The effective media for inducing callus was on MS supplemented with 2,4-D 2.0 mg·L-1 and KT 0.25mg·L-1 and sucrose 30g·L-1, the best breeds for inducing callus was Gannong NO.4 . The effective media for bud inducing was MS containing KT 0.5mg·L-1and NAA 0.1mg/L and sucrose 20g·L-1.The effective root inducing meida was on 1/2MS supplementedwith NAA0.5 mg·L-1 and sucrose 15 g·L-1.Because of the high or low of the regeneration ability,the test conform Gannong NO.4 Alfalfa as the gene transform ingredients.2) Transformation system of BADH gene establishment: 500 mg·L-1 Carb could restrained growing of agrobacterium effectively.According to we established transformation system:Cultivated 7 days hypocotyls as explants as mediator, preculture 2 days, 10 minutes infection time and OD6000.3～0.5, 3 days coculture. After more than 90 days selection 3 regenerated Kan-resistant plantlets were abtained. The result of PCR analysis stated that the detected Kanamycin resistant plants transgenic BADH gene were all positive.