The Analysis Of Comprehensive Traits And Quality In T₁ Generation Alfalfa Co-overexpressing ZxNHX And ZxVP1-1 From Zygophyllum Xanthoxylum

Abiotic stresses,such as soil salinity,drought and nutrient deficiency,are major limiting factors for plant growth,develpment and distribution.Abiotic stresses cause around 50%of major crop yield worldwide annually.Alfalfa?Medicago sativa L.?is a perennial legume forage throughout the world and plays important roles in the production of animal husbandry.Over last ten years,the demand of alfalfa as high quality forage has increased dramatically in China,resulting in a rapid expansion of its planting area.However,alfalfa cultivation areas are mainly in the arid and semi-arid regions in northern China,where the shortage of water resources,soil salinization and infertility are very serious.The cultivation of alfalfa on a large scale will inevitably cause the contradiction between forage crops and food crops because of limited arable land and water resources,which is not beneficial to the healthy development of alfalfa industry and brings tremendous pressure on the fragile ecological environments.Therefore,developing alfalfa cultivars suitable to grow on saline,arid and nutrient-deficient marginal lands is necessary to promote alfalfa production and environmental improment in China.However,genetic potential for stress resistance in alfalfa became very limited because of long-term cultivation,which has increased the difficulty of cultivating more resistant varieties through traditional breeding methods.Using excellent gene resources from stress tolerant xerophytes to improvement alfalfa will break the boundary among various species in traditional breeding.In the previous study,we have already identified the tonoplast Na⁺/H⁺antiporter gene,ZxHHX,and the H⁺-pyrophosphatase gene,ZxVP1-1,from the xerophyte Zygophyllum xanthoxylum,and co-transformed them into alfalfa.The T₀ generation alfalfa showed stronger salt and drought tolerance and significantly improved growth rate.It is not known whether the excellent stress-resistance or forage quality is stably inherited in transgenic offspring lines.Therefore,based on previous work,the stress tolerance,growth performance and forage quality of T1 generation alfalfa co-overexpressing Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 were investigated systematically in this work and main results are obtained as follows:1.T₁ generation alfalfa lines were successfully obtained that stably inherited Zygophyllum xanthoxylum ZxNHX and ZxVP1-1.2.T₁ generation alfalfa lines inherited salt and drought tolerance from T₀ generation lines.Under salt stress?200 mmol/L NaCl?and drought stress?30%FWC?,T₁ generation alfalfa showed better growth and accumulated more biomass and more Na⁺and K⁺than the wild type.3.Co-overexpression of ZxNHX and ZxVP1-1 enhanced T₁ generation alfalfa resistance to low-phosphorus.Under low-phosphorus treatment?5?mol/L Pi?,although the growth of both T₁generation alfalfa and wild type were significantly inhibited,but T₁ generation alfalfa exhibited a higher height and biomass and stronger root systems than wild type.The total phosphorus content of T₁ generation alfalfa plants was significantly higher that wide type under control?1 mmol/L Pi?,low-Pi or field conditions.4.Co-overexpression of ZxNHX and ZxVP1-1 enhanced the growth of the T₁ generation alfalfa.Under greenhouse conditions?no treatment?,T₁ generation alfalfa exhibited a faster growth than the wild type in shoot height,root length and dry weight.Under field condition,T₁generation alfalfa has higher net photosynthetic rate and shoot height,and more branches and biomass than the wild type.5.T₁ generation alfalfa co-overexpressing ZxNHX and ZxVP1-1 exhibited improved forage quality.Under greenhouse conditions?no treatment?,T₁ generation alfalfa accumulated more crude protein and crude fat than wild type;under salinity,drought and low-Pi,the quality indicators of T₁ generation alfalfa were better than wild type.Similarly,T₁ generation alfalfa exhibited a better forage quality than the wild type under field condition.Above results laid a solid foundation for developing new alfalfa cultivars with improved yield,stress resistance,forage quality in northern China.