Study On Alfalfa Tansformation With HAL1 Gene Mediated By Agrobacterium Tumefaciens

Saline soil is a global issue and a very important non-biological force factor which influence agricultural production and ecological environment. How to improve the tolerance of salt and made plants growth is one of the primary research subjects in bioscience.The alfalfa is an important pasturage crop in west of China, which has a high level of economy. It is significant for us to transform special gene into alfalfa by using saline soil efficiently.This experiment was mainly in the aim of setting up the high-frequency regeneration system, and transformed the HAL1 gene into the alfalfa through Agrobacterium-mediated. The main results are the follows:1.We studied the effects of differentiation characters of hypocotyls according to different pretreatments; the results were that the different pretreatments had influences on the differentiation frequency of four kinds of Alfalfa. The correlation analysis between different pretreatments showed that there existed a remarkably significant correlation between pretreatmentⅡa nd pretreatmentⅠa ndⅢ, (P<0.05) and no significant correlation between different kinds of alfalfa. So low temperature pretreatment such as 4℃could improve the rate of calluses inducement and differentiation frequency.2.The best two induction mediums were MS+2,4-D1.0mg/L+6-BA2.0mg/L+0.55%Ager +30g/L Sucrose and MS+2,4-D 2.0mg/L+6-BA 2.0mg/L+0.55%Agar+30g/L Sucrose. The best one differentiation medium was MS+6-BA 0.3mg/L+ KT3.0mg/L +NAA0.1mg/L+0.55% Agar+50g/L Sucrose.3.The study showed that though there was significant correlation between differentiation frequency(P<0.05), the rates of callus inducement of different gene types of alfalfa weren't different , from high to low was North Pole Star(40.2%), Algonquin(37.5%), Empress 2000(35.7%) and Alfalfa of China(21.1%). The sucrose affected the differentiation frequency of the hypocotyls, which was an important factor. The quality concentration of sucrose at 50g/L was better for differentiation of alfalfa.4.Established the optimal tissue culture system by using the hypocotyls of North Pole Star. The concentration of kanamycin at 60mg/L was used to be selector for Kan-resistant calluses and calluses differentiation, and 30mg/L kanamycin was used to be selector for inducing roots.5.Established Agrobacterium-mediated genetic transformation system according to HAL1 gene in a better way. The North Pole Star, which was immersed under 4℃in 2h,were cultivated and achieved explants, and then pro-cultured for 2d and transferred with OD600=0.6 LBA4404 with HAL1 gene which was diluted 200×for 1min.After 2d co-culture with AS, the explants were washed by sterile water then immersed in 2% NaClO for 20min,and washed by sterile water 4~5 times, and immersed in 800mg/L Amp with 0.1% Mannitol for 12h again ,and were incubated in callus-induced medium with 60mg/LKan.The Kan-resistant plants were obtained.6.The results of PCR amplification showed that three transformants obtained the special bands at 900bp.HAL1 gene had been integrated into the genome of alfalfa of North Pole Star.