Improved The Stress Tolerance In Alfalfa Through Introducing An Tonoplast H~+-pyrophosphatase Gene AVP1 From Arabidopsis Thaliana

Salinity and drought are two major abiotic factors limiting crop production and forageyield.In particular,with ingravescent fresh water scarcity,drought and soil salinity havebeen threatening the development of agriculture and animal husbandry,and even thesecurity of whole ecosystem in the arid and semi-arid areas of northwest China.Alfalfa(Medicago sativa L.) is one of the most important legume forages and acts a veryimportant role in the agriculture and animal husbandry,and the ecological construction inthe arid and semi-arid areas of northwest China.However,most alfalfa cultivars aredifficult to maintain high forage yield in saline and arid soils since they are sensitive to saltand drought.Breeding enhanced salt and drought resistant alfalfa cultivars is one ofnecessary ways for increasing forage yield of alfalfa pastures,decreasing irrigation,andreusing salinity land,in the arid and semi-arid areas of northwest China.Salt and droughttolerance of alfalfa can be improved by conventional breeding method,but it often takes along time and is readily limited by narrow genes resources within species.Recentdevelopments in transgenetic technology have provided an efficient tool for improvingalfalfa,and will trigger an important direction of alfalfa stress tolerance breeding.It hasbeen confirmed that vacuolar H⁺-pyrophosphatase (H⁺-PPase) plays important roles invacuolar Na⁺ sequestration and auxin fluxes.The overexpressions of H⁺-PPase confersome model plants and crops enhanced growth and tolerance to salinity,water-deficit andlow-Pi stress conditions,suggesting that the H⁺-PPase genes have potential benefits inimproving stress tolerance of alfalfa.We have previously established an Agrobacterium-mediated transformation systemfor alfalfa cultivars.In this study,to generate transgenic alfalfa lines with enhanced stresstolerance,we comprehensively assessed the tolerance of five cultivars which are widelysowed in northwest China through subordinate function values analysis using relevantagricultural and physiological indicators together,and selected the alfalfa cultivar withwell salt and drought tolerance.Then an H⁺-PPase gene AVP1 from Arabidopsis wasintroduced into this cultivar based upon the transformation system,which was furthermodified and evaluated.Finally,the stress tolerance of transgenic alfalfa was assayed.Themain results of this study are as following:1.Compared with 0 mM NaCl,200 mM NaCl reduced significantly the shoot dryweight and shoot K⁺/Na⁺ ratio,and enhanced the leaf MDA content and relative membranepermeability in all five alfalfa cultivars,but the degree of either reduction or enhancementdiffered between these cultivars.Through comprehensively combining above fourindicators,the salt tolerance of the five cultivars decreased in the order:Rambler,XinjiangDaye,Sandili,Gannong No.3,and Longdong.Similarly,compared with well watered condition,after drought stress treatment,the shoot dry weight,leaf osmotic potential andrelative water content were significantly dropped,and the leaf relative membranepermeability was significantly increased in all five alfalfa cultivars,however the changerange of these indicators are different between five cultivars.Through comprehensiveassessment combining above four indicators,the drought tolerance of the five cultivarsdecreased in the order:Longdong,Xinjiang Daye,Gannong No.3,Sandili,and Rambler.According to analysis of above two comprehensive assessment results,we found that thecultivar Xinjiang Daye possessed well both salt and drought tolerance,and it thus wasselected as a potential germplasm material to further improve its stress tolerance in thisstudy.2.Based upon our previous transformation system,we modified the Agrobacteriuminfection and kanamycin (Kan) selection procedures:the Agrobacterium infection wasperformed under normal pressure for 12 min;kanamycin resistant somatic embryos andplants were selected by 75 and 40 mg·L^-1 Kan after 2 weeks when somatic embryos androots were induced,respectively.This adjusted transformation system took about 19 weeksto get a transformation efficiency (number of PCR-positive plants produced per onehundred infected hypocotyls.) of about 2.1%.3.This study firstly transformed an H⁺-PPase gene A VPI from Arabidopsis intoalfalfa cultivar Xinjiang Daye and obtained transgenic plants.PCR amplification indicatedthat A VPI was integrated into the chromosomal DNA of alfalfa.RT-PCR analysis inrandom eight transgenic lines showed that A VPI expressed in all tested lines,but itstranscript levels are different between eight lines.Compared with other lines,line 1 andline 8 showed the highest and lowest expression level ofA VPI,respectively.4.The overexpression of AVPI enhanced salt and drought tolerance in transgenicalfalfa.After 10 days in the presence of 200 mM NaCl,wild type plants exhibited growthreduction,chlorosis,even die,whereas transgenic plants continue to grow well.Moreover,After 6 days of drought stress,wild-type plants showed chlorosis,whereas transgenicalfalfa still maintained normal growth until day 8 after subjected to water-deficit stress.After rewatered,wild-type plants showed irreversible chlorosis and die,but plants fromtransgenic lines survived and resumed normal growth.The examinations of physiologyshowed that transgenic alfalfa accumulate more Na⁺,K⁺ and Ca²⁺ in leaves and roots thanwild type,which resulted in lower leaf osmotic potential in transgenic plants;thus theirleaves retained more water during drought stress than wild-type plants.Furthermore,undersalt or water-deficit stress,transgenic alfalfa displayed significantly lower leaf MDAcontent and relative membrane permeability,significantly higher net photosynthetic rateand root activity compared with wild-type plants.These results suggested that theoverexpression ofA VPI increased sequestration of Na^-1 into vacuole and uptake capacity of other cations in the transgenic alfalfa.These mechanisms might on the one hand reduce thedeleterious effects of excess Na⁺ in the cytosol under salt stress and steady intracellular ionhomeostasis,on the other hand,enhance the osmoregulation capacity of cells,thus increasethe water retention and maintain the turgor of transgenic alfalfa under salt or droughtstress.5.Pot experiments showed that the overexpression of AVPI resulted in increased rootproliferation and shoot development in transgenic alfalfa.Under normal condition,orlong-term salt/drought treatments,transgenic alfalfa always displayed enhanced growththan wild-type.The plants of two transgenic lines grown in well water soil for 60 days(cutting at 20th day) exhibited 15.5%-21.6% higher shoot height and 27.8%-35.2% highershoot dry weight than wild-type plants.Similar results were observed in long-term salt ordrought treatments.These phenotypes should be ascribed to that transgenic alfalfadeveloped more robust root systems,and thus got higher root biomasses and root/shootratios compared with wild-type under either normal condition or salt/drought stress.Therobust root systems allowed transgenic alfalfa to take up greater amounts of water andnutrient during the imposed salt or water deficit stress,thus also contributed to enhance saltand drought tolerance of transgenic plants.6.The overexpression of AVPI increased the barren tolerance in transgenic alfalfa.Transgenic plants outperformed wild-type plants when challenged with inphosphorus-deficient condition (10μM Pi) for 21 days:the root biomass,shoot biomassand root/shoot ratio of transgenic plants from two transgenic lines were 54.6%-96.5%,20.7%-39.7%,and 23.5%-41.2% higher,respectively,than that of wild-type plants.Undereither normal or restrictive Pi condition,the toltal Pi content per plant (μmol·plant^-1) in twotransgenic lines were 1.3-1.4 fold (under 1 mM Pi) and 1.3-1.8 fold (under 10μM Pi) incomparison that of wild-type;the normalized Pi content (μmol·g^-1 DW) was also higher intransgenic alfalfa than in wild-type plants,suggesting that transgenic plants absorbed morePi and thus grown accordingly.