Screening Of Mutants With Enhanced Cold Resistance Induced By EMS Mutagenesis In Seashore Paspalum

Seashore paspalum(Paspalum vaginatum Swartz)is an important warm season turfgrass which has strong salt tolerance.Seashore paspalum also has strong tolerance to drought,waterlogging,barren,shade and traffic,but low cold tolerance.Therefore,it is important to select new germplasms with cold tolerance for breeding of seashore paspalum.Sea Spray is a variety of seashore paspalum that can be propagated by seeds.Cold tolerance of seashore paspalum and other three warm season turfgrasses species were analyzed in this study.We found that seashore paspalum had the lowest cold tolerance among the tested turfgrass.On this basis,a mutant population of seashore paspalum using ethylmethylsulfone mutation by treating callus of seashore paspalum with EMS,and lines with cold tolerance were identified by selecting regenerated plants.Followed are the main results.1.Comparison of cold tolerance of four warm season turfgrass speciesCold tolerance of four warm season turfgrass species incuding manilagrass,hybrid bermudagrass,centipedegrass and seashore paspalum was evaluated by measuring the temperature that 50%lethal(LT50).Each species of plants were divided into two groups;one group was treated by low temperature(5?)for one week for cold acclimation(CA),and the other group was not treated by low temperature as non-acclimation(NA).The results showed that all CA-treated plants had lower LT50 than the NA plants,indicating that cold acclimation improved cold tolerance of turfgrasses.The lowest and highest LT50 was observed in manilagrasss,and seashore paspalum,respectively,suggestingthat manilagrass has thestrongest cold tolerance,and seashore paspalum has the weakest cold tolerance among the tested turfgrass.We also oberved the period that resulted resulted in plant death under freezing(-7?).Plants were acclimated for one week at 5?,follwed by placing in a growth chamber with-7?.Seashore paspalum showed death after 20 hours of freezing treatment,while centipedegrass,hybrid bermudagrass and manilagrass showed death at 78,120,and 168 hours of freezing treatment,respectively.Proline and malondialdehyde(MDA)contents and antioxidant enzyme activities were also determined.Proline and MDA contents were significantly increased in all turfgrasses after cold acclimation.Superoxide dismutase(SOD)and peroxidase(POD)activities of were also significantly increased after cold acclimation.The results indicated that antioxidant defense system is induced to remove reactive oxygen species during cold acclimation.There was no significant difference in MDA content among four turfgrasses species before cold acclimation,while higher level of MDA in seashore paspalum as compared with the other three grasses after cold acclimation.There was no significant difference in proline content among four turfgrasses before cold acclimation.The highest proline content was oberved in manilagrass,while the lowest proline content was observed in seashore paspalum.Lower activities of SOD and POD was observed in manilagrass than the other three turfgrass.The results showed that a series of physiological changes occurred in turfgrass cells to resist the damage of low temperature under cold stress.2.Screening cold tolerant mutants of seashore paspalum induced by EMSFor screening an effective concentration and time of EMS treatment,seashore paspalum calli were treated with 0.1%,0.4%,0.8%and 1.2%of EMS for two or five hours,the survival rate of calli was statistically collected after two-month subculture.The results showed that the survival rate was close to 50%when calli were treated with 0.8%of EMS for 5 hours.Thus this treatment condition was generally used in the following study.A large number of seashore paspalum calli were treated using the above condition,and the survival calli were further regenerated to plants.The regenerated plants were transplanted to plug trays with soil(perlite:vermiculite:soil nutrition= 1:2:4)in the greenhouse.A total of 1000 regenerated plants were produced,among them 15 lines showed dwarf,stem stout or branching irregular phenotypes.Cold tolerant plant lines were screened by selecting survival plants after freezing treatment.A total of 240 survival plants were harvested after freezing treatment for 24 hours at-7?.The survival plants were recovered in greenhouse for 2 weeks and exposed to a second round of screening.Thirty-six survival plants were harvested plants were treated at-8? for 24 hours.In order to verify cold tolerance of mutants,we measured the LT50 of 2 well-grown mutant lines and its wild type plants.The results showed that the LT50 of wild type is-2.16?,while the LT50 of A1 and A2 were-5.26? and-6.18?,respectively,which were significantly lower than that of the wild type and proved that the cold tolerance of these lines were improved obviously.