Acquirement Of Transgenic Alfalfa Lines With PeDREB2a And KcERF Intergrated And Analysis Of Their Resistance To Drought Resistance And Salt Stresses

Alfalfa(Medicago sativa L.)is an extremely important herbage in the development of animal husbandry.Its rich protein content,good palatability and so on.And is the highest nutritional value of forage legumes.The supply of barnyard grass in China is in short supply,which seriously lags behind the development of animal husbandry.Drought and salt stress have seriously affected the vegetative growth and reproductive growth of plants.The proportion of farmland occupied by dry land and saline-alkali soil in Northwest China and North China is relatively high.In order to improve land utilization and reduce the competition between pasture and food crops,the use of molecular breeding methods to cultivate new varieties of drought-resistant salt-resistant alfalfa has farreaching significance for the development of China’s grass industry and animal husbandry.Based on the optimized high-frequency regeneration system of alfalfa,this study cloned the gene PeDREB2a::KcERF cloned from the eutrophic plant Populus euphratica and the salty plant Kandelia candida through the Agrobacterium tumefaciens-mediated method and transferred it into the recipient material alfalfa.The results are as follows:(1)Zhongmu No.1,Zhongmu No.3,and Gannong No.4 were selected as plant materials;the leaves were used as explants,and the optimized high-frequency regeneration system was obtained by screening and synthesis of the three plant materials: The wound tissue induction medium was MS+0.2mg/L 6-BA+2mg/L 2,4-D;the optimal differentiation medium was 1/2MS(+20g sucrose);the optimal rooting medium was 1/2MS.Rooting medium + 0.05 mg/L IBA + 0.05 mg/L NAA.(2)Zhongmu No.1 and Gannong No.4 were selected as the acceptor materials and their leaves were used as explants.According to the optimized regeneration system,the target gene was transferred into the receptor material mediated by Agrobacterium tumefaciens LBA4404.Tests were carried out for the transgenic resistance(Kan)lines: 66 Zhongmu No.1 and 27 Gannong No.4.The positive rate of PCR detection was 93.94% and 88.89%,respectively.(3)RT-PCR showed that the transgene positive plants could be stably expressed at the reverse transcription level;under drought stress and salt stress 24 h,the relative expression levels of PeDREB2 a and KcERF genes in the transgenic lines were highest at 12 h;with 20% PEG-3350 and 250mmol/L NaCl stress treatment and physiological indicators,the results show that: compared with the wild-type transgenic plants drought resistance and salt tolerance was significantly enhanced.