Evaluation Of Growth Performance And Forage Quality Of Transgenic Alfalfa(Medicago Sativa) Under Field Condition

Drought,salinity,high temperature and other abiotic stresses have posed a serious threat to global crop cultivation and ecological environment.Alfalfa（Medicago sativa）is an excellent leguminous forage contained rich nutrition and good palatability,and is known as"the queen of forage".However,alfalfa is not suitable for large-scale cultivation in harsh environment of northwest of china because of its limited tolerance,which is an important limiting factor of alfalfa planting.Zygophyllum xanthoxylum is a xerophyte lived in desert t and developed abundant mechanisms of stress tolerance over the course of its evolution,and it possesses a variety of stress tolerance genes.In previours experiments of our laboratory,the genes ZxNHX1 and ZxVP1-1encoding vacuolar membrane Na⁺/H⁺antiporter（NHX1）and H⁺-pyrophosphatase（VP）,which involved in Na⁺accumulation for cell osmotic regulation,were co-transformed into alfalfa,and the T₁ transgenic alfalfa lines show enhanced salt and drought tolerance.In addition,the cuticle synthesis related gene ZxABCG11 of Z.xanthoxylum was fused with the epidermal-specific promoter MtML1 from Medicago truncatula and AtCER6 from Arabidopsis,respectively,for genetic transformation of alfalfa,which significantly improved the drought and heat tolerance of transgenic lines.However,the growth performance and forage quality of these transgenic materials in the field condition are still unclear.Therefore,on the basis of previous studies,the present work systematically analyzed the growth performance of the above transgenic alfalfa lines under field conditions and evaluated its forage quality,which laid a foundation for further breeding new alfalfa cultivars with higher yield,stronger resistance and better quality.The main results are as follows:（1）Under field condition,leaf size,forage yield,dry and fresh weight of ZxNHX1-ZxVP1-1transgenic alfalfa were 56.4%,74.5%,31.7%and 45.1%higher than those of wild type,respectively.The stem thicknes was also significantly higher than for wild type.In addition,the net photosynthetic rate and water use efficiency were 19.3%and 45.1%higher compared to the wild type respectively during initial flowering stage.These results indicated that ZxNHX1-ZxVP1-1transgenic alfalfa had better growth performance.（2）Under field condition,leaf size of AtCER6-ZxABCG11 and MtML1-ZxABCG11 transgenic alfalfa increased 49.8%and 54.1%respectively.Compared with the wild type,the plant height of the two transgenic lines were decreased,and there were no significant difference in shoot dry weight,shoot fresh weight and forage yield.Interestingly,the branch number of AtCER6-ZxABCG11transgenic alfalfa increased whereas stem thickness of MtML1-ZxABCG11 transgenic alfalfa increased.In terms of photosynthetic performance,the net photosynthetic rate and water use efficiency of both transgenic lines were significantly higher than that of the wild type.These results suggested that the yield of AtCER6-ZxABCG11 and MtML1-ZxABCG11 transgenic alfalfa had no obvious advantage compared with the wild type,but their photosynthetic performances were better than wild type.（3）The flowering period of the three transgenic alfalfa was different from that of the wild type.ZxNHX1-ZxVP1-1 transgenic alfalfa bloomed at the same time as wild type,but its flowering period was longer than wild type for 19 days.AtCER6-ZxABCG11 and MtML1-ZxABCG11 transgenic alfalfa bloomed later than wild type for 17 days and its flowering period was longer than wild-type for 38 days.（4）Under field conditions,the contents of crude protein and crude fat in ZxNHX1-ZxVP1-1transgenic alfalfa were significantly higher than those in wild type,while its acid detergent fiber,neutral detergent fiber and lignin were significantly lower than those of the wild type.Correspondingly,the relative feed value of ZxNHX1-ZxVP1-1 transgenic alfalfa was significantly higher than the wild type for 35.4%.The results showed that the forage quality of ZxNHX1-ZxVP1-1 transgenic alfalfa was better compared to wild type and it is suitable for popularization and application as the fine forage.（5）Under field conditions,the crude protein and ash contents of AtCER6-ZxABCG11transgenic alfalfa were significantly lower than those of wild type.There was no significant difference in crude fat,neutral detergent fiber,acid detergent fiber,lignin and relative feed value between AtCER6-ZxABCG11 line and wild type.In MtML1-ZxABCG11 transgenic alfalfa,the crude fat content was significantly lower than that of the wild type,while there was no significant difference in crude protein and ash contents compared with wild type.The neutral detergent fiber and lignin were significantly higher than those of the wild type,while the acid detergent fiber had no significant difference from the wild type.Its relative feed value was significantly lower than wild type for 12.9%.The results showed that the overexpression of ZxABCG11 did not improve the forage quality of transgenic alfalfa,but it has the potential to be used for ecological control of grass species because of its strong stress tolerance.