Agrobacterium Tumefaciens-mediated Transformation With Salt-tolerant SOS Genes In Alfalfa

Medicago sativa L.is one of the most widely distrubted perennial leguminous forage in the world,which is also an important forage feed for animal husbandry in China.It has high yield,enriched nourishment,balanced amino acid composition,and livestock and poultry are all fond of food.With the development of ecological civilization and the development of modern livestock and grass industries,there is an urgent need for water-saving,drought-resistant and salt-tolerant new varieties.The use of molecular breeding meathod is an effective way to accelerate the development of new salt-tolerant varieties of alfalfa.Salt overlay sensitive(SOS)family genes regulate ion balance and have been shown to enhance salt tolerance in plants such as Arabidopsis haliana,rice,tobacco,and turfgrass,but the application of these family genes in alfalfa to improve salt tolerance is less reported.The present study is based on the establishment of a high-frequency regeneration system of alfalfa,optimization of the Agrobacterium-mediaied genetic transformation system with SOS family genes(SOS2 and SOS3)to obtain the salt tolerant transgenic lines,and to further identify and evaluate of salt resistance in the green house and field trial.The main results are as follows:I.Through callus and cotyledonary nodes culture in vitro,a high-frequency callus regeneration andin vitro organ regeneration system were established.Ten alfalfa varieties including,'Jacklin','Algonquin','Golden Empress I','Golden Empress 2','Queen 2000','Plato','Suntory','Gannong No.3','Aohan,and 'Longdong' were used as experimental materials.Callus induction and differentiation,adventitious bud induction and elongation and radication were systematically studied.The results showed that the callus induction rate of hypocotyls of 'Jacklin' was up to 100%,and the differentiation rate was as high as 69.75%.The induction rate of adventitious buds for 'Golden Empress 2' was 76%,and the differentiation rate was found 71%,while the rooting rate was as high as 96%.2.The factors influencing genetic transformation of alfalfa embryonic callus mediated by Agrobacterium tumefaciens were discussed,and the genetic transformation system was optimized.In Agrobacterium-mediated multiple genes transformation system,100 ?mol·L-1 acetosyringone can increase the transformation rate by 2-3 times;the optimal infection density OD600 value was 0.6;the optimal infection time was 20 min.The optimum concentration of cefotaxime as the best sterilizing agent was 500 mg L-1,and the optimum selection pressure of Glu was 5.0 mg·L-1.In the later period,the concentrations of antibacterial agent and the screening agent could be appropriately reduced.3.Twelve herbicide-resistant plants obtained were subjected to PCR,Southern hybridization and RT-PCR molecular detection.Five positive plants were confirmed.It was proved that the exogenous multi-genes have been integrated into the genome of alfalfa and the SOS3 and Bar genes have been transcribed.The obtained transgenic strain T2-1 was propagated in a potted way using the shoot cutting technique,and 60 transgenic T2-1 strain group materials were obtained.4.The obtained transgenic strain T2-1 was identified for indoor salt tolerance in pots,and it was proved that the transgenic lines had significant salt tolerance compared to the wild type.After treatment with 0,100,200 and 300 mM NaCl for 8 days,the phenotypes,plant height,chlorophyll content,leaf area,fresh weight,stem diameter and branches number and other agronomic traits of transgenic line T2-1 were better than that of wild type controls plants;In the wild-type control,the contents of K+,Ca2+,and Pro in leaves and the activities of SOD,POD and CAT enzymes were increased and higher than those in the wild-type controls.The accumulation of Na+,MDA content,and relative conductivity were lower in the leaves than in the wild-type controls.The results showed that the antioxidant activity of the transgenic T2-1 line increased,and the active oxygen was eliminated;the accumulation of osmotic adjustment substances increased and the osmotic adjustment ability was improved;the membrane lipid peroxidation was low and the membrane structure was maintained.It may be due to the regulation of the expression of the SOS pathway gene to excrete Na+ out of the cell,selectively absorb more K+,maintain the dynamic balance of intracellular Na+ and K+,reduce the Na+ toxicity,thus increasing the salt tolerance of the transgenic alfalfa.5.The salt tolerance of the transgenic strain T2-1 was further confirmed in the field,which was proved that the transgenic lines had higher salt tolerance than the wild type.The growth of transgenic line T2-1 and wild-type alfalfa were inhibited to varying degrees,but the plant height,leaf area,yield,chlorophyll content and net photosynthetic rate of transgenic line T2-1 were better than those of wild type.The transpiration rate of transgenic line T2-1 were lower than the wild-type.The antioxidant enzymes activity,proline,soluble protein and soluble sugar content,and the content of K+ and Ca2+ and K+/Na+ratio of transgenic line T2-1 were higher than wild type controls,whereas the MDA content and relative conductivity of transgenic line T2-1 were lower than those in the wild type.The results indicated that the overexpression of Arabidopsis SOS genes in alfalfa enhanced the photosynthetic property of transgenic alfalfa and increased yield,meanwhile improved the antioxidant enzymes activity and maintained membrane stability,enhanced the translocation activity of Na+/K+ antiporter and reduced or avoided salt injury under salt adversity stress,then improved the salt tolerance of transgenic line T2-1.