Establishment Of Efficient Regeneration System Of Potato And Transformation Of HAL1 Gene

Saline soil is very important agricultural production source.Greened saline soil is a key way for enlange agricultural areas,develop production,improve ecological environment.Using gene engineering to culture salt tolance plants is a efficient method for saline soil.HAL1 gene is a key factor of salt tolerance, it's expression can regulate Na+/K+ balance.Thought HAL1 gene is not a kind of transfererd protein,it can keep lower Na+/K+ with other gene and transfererd sistem.So HAL1 gene have great potentiality in the gene engineering of salt tolance.HAL1 gene was transformed into potato by transfection of Agrobacterium tumefaciens,the factors effecting on frequency of gene transfer were examined and salt tolerance character of transgenic potato was analysised.The main results are as follows:1. For establishing the regeneration of four potato cultivars,Kexin No.12,Kexin No.13,Dongnong303 and Zaodabai, stem explants were cultivated in vitro from stem explants.The results show that the perfectable medium of callus for Kexin No.12 and Kexin No.13 were MS+6-BA2mg/L+2,4-D1mg/L,Dongnong303 were MS + 6-BA2mg/L + 2,4-D 0.5mg/L ,Zaodabai were MS+6-BA2mg/L+2,4-D0.5mg/L;The adventitious callus induction rate were 100%,100%,100% and 90%.The perfetcable medium for adventitious bud differentiation from stem callus were MS + 6-BA4.0mg/L + NAA 0.1mg/L+GA31.0mg/L for Kexin13, MS + 6-BA2.0mg/L + NAA 0.5mg/L+GA31.0mg/L for Kexin12, MS + 6-BA2.0mg/L + NAA0.1mg/L +GA31.5mg/L for Dongnong303. MS + 6-BA2.0mg/L + NAA0.5mg/L + GA31.0mg/L for Zaodabai.The rate of adventitious bud differentiation were 80%, 90%, 100%, 76.7%.As the result of this experiments, Dongnong303 was chosen to be the receptor of transgene.2. Established the optimal tissue culture system by using Dongnong303 stem explants as experiment material. Kanamycin were used to be selector for Kan-resistant shoots of "Qinxuan NO.1"and"Qinyan 95",20 mg/L Kanamycin was used to be selector for inducing roots.The perfectable medium of callus were MS + 6-BA 2mg/L + 2,4-D 0.5mg/L ,and the medium for adventitious bud differentiation from stem callus were MS + 6-BA2.0mg/L + NAA0.1mg/L +GA31.5mg/L. 60㎎/L and 75 mg/L Kanamycin were used to be selector for Kan-resistant calluses and calluses differentiation,and 60 mg/L Kanamycin was used to be selector for inducing roots.3. Factors were examined and an efficient gene transfer was established.3w Dongnong303 stem explants were pro-cultured for 2d and transferred with OD600=0.6 LBA4404 for 5min.After 2d co-culture,the stem explants were washed by sterile water 4～5 times,and were incubated in callus-induced medium with 250㎎/L Cb.The Kan-resistant plants were obtained.4. The results of PCR amplification showed that four transformants obtained the special bands at 900bp. HAL1 gene had bene integrated into the genome of part of potato plants.Salt tolerant tests implied that 90.9% transgenic potato can rhizogenesis in the concentration of 0.80%-0.9%NaCl,45.6% transgenic potato can get the high tolance of 1.0%NaCl.In contrast,the control can only rhizogenesis in the culture medium with NaCl lower than 0.70%.