Study On Regeneration System Of Medicago Falcata L. Of Xinjiang And Transformation Of AK-APR Gene By Agrobacterium Tumefaciens

Medicago falcata L. is perennial leguminous forage, which is essential valuable germplasm for the alfalfa breeding because of excellent quality, rich nutrition, strong resistance and adaptability. By using seed of germplasm of Medicago falcata L.as material, this picked from Qitai, Xinjiang. Regeneration plants were obtained by callus induction and differentiation. This study aimed at hard seed, explants, hormones (2,4-D and KT), and the media, discussing the influence factors of M. falcata L. tissue culture. With the method of agrobacterium-mediated technique, the gene with sulfur amino acid AK-APR was transferred to M. falcata L., and then the primary genetic transformation method of M. falcata L. was founded. Results were as follows:(1) The method of hard broken seed of M. falcata L.:placed24hours in the4℃low temperature, and soaked15～20minutes by sulfuric acid(95%), finally, reflushed30minutes with water; with this method the germination rate reached to90%.(2) Regeneration system of M. falcata L. in Xinjiang:hypocotyl was the most suitable explant for callus induction, and the callus induction rate was reached to85%; the MS medium was more effective than the improved SH medium for the callus induction, which contained that the MS+0.5mg/L2,4-D+0.8mg/L KT+30g/L sucrose+7g/L agar, and the callus induction was80%; light green and light yellow callus were differentiation very well in the culture medium, which contained MS+0.2mg/L2,4-D+0.4mg/L KT+1g/L CH+20g/L sucrose+7g/L agar, and the differentiation rate reached to30%; rooting medium contained1/2MS+20g/L sucrose+8g/L agar.(3) The differentiation medium,which was added reagent study showed that:(a)the addition of glutathione(25m g/L)could prevent the callus from browning, which is more effective than ascorbic acid and activated carbon;(b)the addition of CH (1g/L) can promote the differentiation of callus.(4) The AK-APR gene transformation system,which receptor was M. falcata L. callus:the Light green or light yellow callus were immersed in the Agrobacterium-mediated bacterium fluid (OD600=0.3)15minutes, which were cultured in MS medium for36hours, and then transfered to the MS medium (including0.2mg/L2,4-D,0.4mg/L KT,1g/L CH,25mg/L GSH,50mg/L Kan,500mg/L Carb) about60d,finally, the differentiated seedlings were obtained.(5) After three strains of transgenic plants were obtained, we extracted the total DNA of transgenic and non-transgenic plants, and then do PCR according to the specific primers, which designed by AK-APR gene sequences. The AK gene fragment was founded at500bp, but the APR gene fragment at700bp, so we can concluded that the sulfur amino acid AK-APR gene had been transferred into the M. falcata L.,...