Plant Regeneration And Molecule Detection For The Transgenic Xinjiang Big Leaf Alfalfa Of Cg-NHX1 Gene From Halophyte Chenopodium Glaucum

Following the development of plants resistance and transgenic technique, breed of new resistant alfalfa by transforming heterogenous gene has become more and more remarkable to pepole.So, the establishment of a high-frequence system of regeneration plants is the base of high efficiency transformation for heterogenous gene. Cotyledon, hypocotyl and root of 5-7 day-old seedlings of Xinjiang big leaf alfalfa were selected as explants. MS and 8 groups of 2,4-D with different concentrations from 0.1mg/L to 3mg/L were used to induce callus, then MS and 2,4-D2mg/L and 10 groups of KT with different concertrations from 0.01mg/L to 0.5mg/L were used to induce callus; the shoots were induced in the medium of MS with additional 6-BA 0.5mg/L and NAA 0.05mg/L; MS was used to produce roots. Results showed that: callus tissues had much better growing status and could be used to produce differentiation shoots in medium of MS with 2,4-D 2.0mg/L and KT 0.2mg/L, the average calli frequences were 93.1%, 100% and 100% for cotyledon, hypocotyl and root respectivel. After growing 40-80 days in the medium of inducing regeneration shoot, callus tissues which originated from cotyledon, hypocotyl and root produce regeneration shoots and its regeneration shoots frequence was 50%, 78% and 50% respectively; when shoots grew to about 2cm high, it was transferred into medium of MS to root; afer 14 days, regeneration plants were transferred into flower-soil and its survivable rate was more than 90%. All of cotyledon, hypocotyl and root can obtain regeneration plants in this culturing system. Moreover root can act as a good material of explant to obtain regeneration plant while researches in this field are still poor.Kanamysin is an important factor in the work of selecting positive plant in the experiment of plant transformation. Cotyledon, hypocotyl and root of 5-7 day-old seedling of Xinjiang big leaf alfalfa were selected as explants. Callus tissues were induced in medium of MS with 2,4-D 2.0mg/L and KT 0.2mg/L with addtion of different concertrations of kanamysin(from 30mg/L-70mg/L). Results showed that: the average calli frequence was 14.82% for cotyledon in medium of kanamysin concertrations of 40mg/L in the time of the 10ds of calli induced, and was 0% for cotyledon in the medium of kanamysin concertrations of 50mg/L in the time of the 10ds of calli induced. The average calli frequence was 90%-100% for hypocotyl and root in medium of all different concertrations of kanamysin. Cotyledon has much higher sensitive to kanamysin. So, in our experiment, cotyledon was selected as explant to being plant transformation experiment, and kanamysin concertration was decided to 40mg/L-50mg/L in screening medium.The new variety of salt tolerance alfalfa can be quickly and effectively obtained by transgenic technology. But, the ability of regeneration and transformation of alfalfa is greatly affected by it's different varieties. So, establishing transgenic system of Xinjiang big leaf alfalfa by Agrobacterium mediated gene transfer has importantly promoting effect to breed new variety. Cg-NHX1 gene was cloned from the Chenopodium glaucum. Chenopodium glaucum is the kind of halophyte that has the salt tolerance. The Cg-NHX1 gene was ligated with pBIN438 plant expression vector and transformed into Agrobacterium tumefaciens EHA105. Transgenic Big Leaf alfalfa was gotten by co-cultivating 5-7d old cotyledon with recombinant Agrobacterium tumefaciens. The genomic DNA of transgenic Big Leaf alfalfa was extracted and analyzed by PCR and Southern blot techniques. The results indicated that the Cg-NHX1 gene had been transformed into Xinjiang Big Leaf Alfalfa. The study of optimizing transformation showed that Agrobacterium tumefaciens's concentration of OD600 in 0.2~0.3, and time of cotyledons immersed in 20min are favorable to inducing calli and obtaining the transgenic big Leaf alfalfa successfully.The concertration of salt will inhibit the germination of seed and plant growth. Seeds of Xinjiang big leaf alfalfa were germinated in medium of MS with seven different concertration of NaCl from 0mmol/L to 400 mmol/L. Results showed that: the averaging seeds germination frequence was 23.3% and 0% in medium of MS with 200mmol/L NaCl and 300mmol/L NaCl in the time of the 15ds of culture respectively. The growth of the plants of seeds germinated were obviously inhibited in the medium of adding 100mmol /LNaCl.