Overexpression Of CdNF-YC And CdSAMDC Improves Tolerance To Multiple Stresses In Seashore Paspalum

Seashore paspalum(Paspalum vaginatum O.Swartz)is an important environmentally compatible warm-season turfgrass;which has demonstrated superior salt tolerance compared to other turfgrasses.seashore paspalum has lower drought and chilling tolerance comparable to bermudagras.Chilling and drought injury is main issue for the management of warm-season turfgrass in north china,usually causing yellowish and withering.Thus,it is pressing to improve the chilling and drought resistance of seashore paspalum by breeding technique.In this study,We developed a high-efficiency protocol for producing seashore paspalum transgenic plants mediated by Agrobacterium tumefaciens transformation,and used seed as explant for the first time.Embryogenic calli were transformed with Agrobacterium tumefaciens strain AGL1 carrying the binary vector pCAMBIA 1305.2,containing the GUS reporter and hygromycin phosphotransferase genes(Hpt).Hygromycin B(hyg B)was used as the selective agent throughout all phases of tissue culture.The minimum concentrations of hyg B were 80 and 100 mg/1 that would successfully kill 100%of non-transformed plants in regeneration and shoot stage.In the present study,we optimized several important factors that affected transformation.The most effective transformation protocol using Agrobacterium concentration of OD600=0.6,100 μM AS and 0.01%Silwet L-77 supplement in the infection medium,30 min of infection containing 5 min of sonication and 20 min of vacuum,and 2 days of co-cultivation in the dark was used for stable transformation experiments.The highest and average efficiencies of transient GUS expressing were 98.8%and 87.1%respectively after co-cultivated.In the optimised transformation system,the overall transformation efficiency reached 8%.PCR and Southern blot showed that T-DNA was successfully inserted into the seashore paspalum genome.In conclusion,we successfully established a sample and high-efficiency regeneration and Agrobacterium-mediated transformation of ’Sea Spray’.Using this system,we have further opportunities to research the functional genes or regulative gene by over expression,down-regulation or gene targeting(’knockout’)and to elucidate signal pathways which may play essential roles in the regulation of abiotic stress.In this study,nuclear transcription factor NF-YC gene from bermudagrass was integrated into the genome of the seahore paspalum using Agrobacterium-mediated transformation and the transgenic plants were generated for the first time.The transgenic plants had lower relative conductivities,higher leaf relative water content and rehydration viability comparing wild type after drought stress treatment.Expression of CdNF-YC in seahore paspalum has more tolerant to salt stress.Under high salt stress,the expression of CdNF-YC in seahore paspalum had lower leaf yellowing rate,maintained higher leaf water content,root weight,Fv/Fm,chlorophyll content and proline concentrations.Compared to the wild type,transgenic plants maintained a lower Na+concentration and a higher K+concentration after salt stress treatment.The expression of PvDREB2A,PvDREB1B,PvLEA3,PvP5CS1 and PvABI gene in transgenic plants was significantly higher than that of the control under salt and drought.NF-YC gene mediated the expression of these genes under ABA treatment expect PvDREB2A and PvDREB1B.We first obtained herbicide resistant germplasm with drought and cold resistance by overexpressing bermudagrass CdSAMDC gene and glufosinate-tolerant gene bar in seashore paspalum through Agrobacterium-mediated transformation method.SAMDC regulates responsiveness of seashore paspalum to various stresses mainly via adjusting biosynthesis and metabolism of different forms and kinds of polyamine.Under drought stress,lower relative conductivities,higher leaf relative water content and rehydration viability comparing wild type.Free Put and Spd contents,and binding and bound Put increased in seashore paspalum expressing CdSAMDC.Free Put and Spm contents significantly increased,while free Spd content decreased in transgenic plants under chilling and high salinity stresses.SAMDC improved GABA contents of transgenic lines by regulating biosynthesis and metabolism of polyamine.Moreover,the transgenic plants maintain higher photosynthesis ability and potassium content in shoot and root with higher K+/Na+ratio.The results show that expression of CdSAMDC gene from bermudagrass confers tolerance to drought and chilling and higher tolerance to salt in transgenic seashore paspalums.We obtained transgenic new germplasm expressing CdNF-YC and CdSAMDC with herbicide resistance on the basis of high-efficient Agrobacterium-mediated transformation of seashore paspalum.Drought and chilling resistance,as well as higher salinity tolerance,were notably improved in transgenic plants.