Field Identification Of Saline-alkaline Tolerance Of Alfalfa (Medicago Sativa L.) Cultivars And Its Physiological Mechanism Of Stress Tolerance

Soil salinization and alkalization are an important ecological and environmental problem worldwide.It not only inhibits plant growth but also limits the improvement of crop yield.The soda saline-alkaline(SA)land,characterized by high Na⁺and high p H,is widely distributed in Northeast China and SA stress has become a major obstacle to the local crop yield and economic development.Alfalfa(Medicago sativa L.),as a high yield and high quality forage,has strong SA tolerance.Therefore,screening for SA tolerance and developing SA tolerant cultivars of alfalfa is an important way for effective utilization of the SA land resources,is of great significance to promote the construction of ecological agriculture and the sustainable development of grass industry.In this study,50 alfalfa cultivars were planted in SA land and non-SA land,respectively.and the field identification test for SA tolerance was carried out and screened out several alfalfa cultivars suitable for cultivation in SA land.In addition,the statistics-aided study of growth and physiological indices related to SA tolerance identified Ca²⁺/Na⁺ratio as a critical marker for evaluating SA tolerance in field conditions.Furthermore,to gain some new insight into the molecular mechanism underlying the SA tolerance in alfalfa,this study also conducted a transcriptomic comparison between SA tolerant and sensitive alfalfa cultivars under alkaline conditions simulated by 0 m M and 25 m M of Na2CO3.Additionally,a potential priming effect of the plant stress hormone abscisic acid(ABA)on alkaline tolerance of alfalfa seedlings was studied in laboratory conditions.The main findings obtained in this study are as follows:1.The growth and physiological indices of each cultivar were determined in the evaluation experiment of SA tolerance under the field condition,and the SA tolerance coefficient(SATC)of these indices were calculated.The 50 alfalfa cultivars were classified into 3 categories by the comprehensive evaluation methods of principal component analysis,membership function analysis,and cluster analysis:Category I consisted of strong SA-tolerant cultivars(WL319HQ,WL903HQ,Polarbear,etc.)with D-values of 0.54–0.78,corresponding to 24%of the 50 cultivars.Category II comprised moderate SA-tolerant cultivars(Gannong NO.6,Magnum NO.7,Bingchi,etc.)with D-values of 0.43–0.52,accounting for 32%of the 50 cultivars.Category III represented weak SA-tolerant cultivars(Zhonglan NO.1,Lnstict,WL525HQ,etc.)with D-values of 0.24–0.42;these accounted for almost half(44%)of the 50 cultivars.2.Taking the commonly used saline-tolerance index as the traditional method,the new method was based on the traditional method,and further included the inclusion of SATCs of the indices Ca²⁺,Mg²⁺and their ratio to Na⁺which were affected by alkaline-saline stress.Stepwise forward regression was used to quantify the explanatory power of the different investigated plant growth and physiological variables for predicting the SA tolerance of alfalfa and identify the key variables predicting SA tolerance.The D value of reflecting SA tolerance of the 50 alfalfa cultivars was the dependent variable,with the SATCs of the plant growth and physiological variables set as the independent variables.The results showed:under the traditional method,the SATC of SL explained the most variation(67.90%)in the D response,and the SATC of PRO was the second most important index with an explanatory power of 13.60%for D,followed by the SATCs of K⁺/Na⁺ratio,SDM,SS,K⁺,and Na⁺.However,under the new method,it was the SATC of the Ca²⁺/Na⁺ratio that was the best predictor,explaining 62.50%of the variation in D.The SATC of SL was the second variable to be included,followed by the SATCs of Mg²⁺,Ca²⁺,SDM,PRO,Na⁺,SS and K⁺.3.The laboratory alkaline stress simulation experiment found that chlorophyll content and shoot fresh weight were drastically declined in the alkaline-sensitive cultivar Algonguin(AG)following alkaline treatment simulated by 0-25 mMNa2CO3solutions,while alkaline-tolerant cultivar Gongnong NO.1(GN)maintained a relatively stable shoot fresh weight and chlorophyll content.Compared with AG,GN had higher contents of Ca²⁺and Mg²⁺,the ratios of Ca²⁺and Mg²⁺to Na⁺,proline and soluble sugar,and enzyme activities of peroxidase(POD)and catalase(CAT),under the alkaline conditions.4.Transcriptomes analyses identified three categories of alkaline-responsive differentially expressed genes(DEGs)between the two cultivars:48 genes commonly induced in both the cultivars(CAR),574 genes from the tolerant cultivar(TAR),and493 genes from the sensitive cultivar(SAR).Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses showed that CAR genes were mostly involved in phenylpropanoid biosynthesis,lipid metabolism,and DNA replication and repair;TAR genes were significantly enriched in metabolic pathways,biosynthesis of secondary metabolites,MAPK signaling pathway,flavonoid and amino acid biosynthesis;and the SAR genes were specifically enriched in vitamin B6metabolism.5.Alfalfa seedlings were pretreated with ABA(10?M)for 16 h and then subjected to alkaline stress simulated by 15 mMNa2CO3 solution.Compared with the control,ABA pretreatment significantly alleviated leaf damage and improved the fresh weight,water content and survival rate of alfalfa seedlings under alkaline conditions.Abscisic acid pretreatment reduced accumulation of reactive oxygen species(ROS),increased activities of the antioxidant enzymes superoxide dismutase(SOD)and peroxidase(POD),maintained higher ratios of K⁺/Na⁺,Ca²⁺/Na⁺,and Mg²⁺/Na⁺,and increased accumulation of proline.In addition,ABA upregulated the expression of genes involved in proline biosynthesis(P5CS)and the sequestration of Na⁺in vacuoles(NHX1 and AVP)under alkaline conditions.Abscisic acid priming increased tolerance to alkaline stress by maintaining homeostasis of ROS and metal ions and upregulating osmoprotection and the expression of stress tolerance-related genes.