Study On Regeneration System Of Alfalfa And Transformation Of AK-APR Gene By Agrobacterium Tumefaciens

Alfalfa is high-quality forage which has excellent quality, rich nutrition, strong resistance, and highyield. Sulfur amino acid plays an important role in animal production, but low sulfur amino acid content inalfalfa protein. Therefore, it is significant for Xinjiang main cultivars of Medivago varia cv. Xinmu No.1.toimprove sulfur amino acid through transforming special gene into alfalfa.By using leaves of the regenerated plants of Xinmu No.1.as material, which are high-frequencegenotypes of callus induction and differentiation, the influence of high-frequence genotype regeneration isstudied in different hormone and concentration combinations. studying the gene with sulfur amino acidAK-APR was transferred to Xinmu No.1With the method of agrobacterium-mediated technique, and thenthe genetic transformation method of Xinmu No.1. was founded. The results are as follows:(1) By using leaves of regenerated plants of Xinmu No.1as explant, washing15minutes by runningwater, then disinfect30.0seconds with75%alcohol and5min with0.1%mercuric chloride, then flushed3-5times with distilled water.(2) The regeneration system of high frequency genotype of Xinmu No.1: the medium of callusinduction was MS+2,4-D2.0mgï¹'L-1+KT0.5mg·L-1，and the callus induction rate was93.3%; thedifferentiation culture medium was MS+2,4-D1.0mg·L-1+KT0.8mg·L-1, and the differentiation ratereached67.0%; The regeneration time was75±3d.(3) The infection receptor was the callus form leaves of plant regeneration: the callus were immersedin the bacterium fluid (OD600=0.6) for10-15minutes, which were under the condition of co-culture onMS medium for3days, and then transfer to the selective medium which contained50.0mgï¹'L-1Kan,700.0mg·L-1Carb, one time15days for subculture, removed Kan with the increasing in the number ofsubculture, and decreased Carb concentration gradually, finally, two regeneration seedlings were obtained;(4) The target gene was detected by PCR; and also by PCR-Southern blot hybridization detectedAK-APR gene, so that the sulfur amino acid AK-APR gene had been transferred into the alfalfa of XinmuNo.1.