| Nitraria sibirica Pall(N.sibirica)is a deciduous small shrub and belongs to Nitraria L.genus of Zygophyllceae family,its abilities of salt tolerance and alkali tolerance are very strong,and its ecological value,medicinal value and additional economic value are very high,so its exploitation and application are promising.In this dissertation,with the desert halophytes N.sibirica from the Baicheng desert areas of Jilin province as the study material,the study aims to analysis the germination characteristics of N.sibirica seeds,built the suspension cell culture system and the protoplast preparation technique,and then provide plant and cellular materials for further research and application of N.sibirica;the primary responses of membrane ionized flow and membrane potential in N.sibirica seedling roots under salt,alkali and osmosis stress as well as its relevance to resistance mechanism were investigated with Microelectrode ion flux estimation(MIFE)and Patch-Clamp technique.This study has important theoretical significance to clarify the mechanism of plant resistance to salt and alkali as well as guide plant resistance breeding,it also promotes the exploitation and application of N.sibirica.The research works of this dissertation includes the following four parts.1.Dormancy and germination characteristics of N.sibirica seedsThis study aims to break the seed dormancy and improve the germination rate of the N.sibirica seeds.N.sibirica seeds were treated with soaking in warm water,storing with moisture sand,and immersing seed in H2SO4(98%)and gibberellic acid(GA),then the germination rate,germination vigor,and germination peak period of the seeds were determined.The results are as follows:The germination rate and germination vigor were 30%and 10%under soaking in warm water.The seeds began to germinate on the day 7 and reached the germination peak on the day 10.The germination rate and germination vigor were 55.5%and 26.5%under storing with moisture sand for 37 d.The seeds began to germinate on the day 2 and reached the germination peak on the day 10.The germination rate and germination vigor were 86.8%and 60.3%under storing with moisture sand for 95 d.The seeds began to germinate on the day 2 and reached the germination peak on the day 7.The germination rate and germination vigor were both 90.0%under being treated with 98%H2SO4 for 2 h.The seeds began to germinate on the day 1 and reached the germination peak on the day 4.Seeds after being treated with 98%H2SO4 for 2 h were sowed on the dark medium of MS+0.5g/mL BA+0.5g/mL GA,and the germination rate and germination vigor both reached to 98.1%.The seeds began to germinate on the day 1 and reached the germination peak on the day 4.The dormancy of N.sibirica seeds is caused by hard seed vessels.These results suggest that the best way of breaking the seed dormancy is first treated with 98%H2SO4 for 2 h and then cultured in the dark medium of MS+0.5g/mL BA+0.5g/mL GA,which could effectively break the dormancy of hard seeds and reach a high germination rate.2.N.sibirica cell suspension culture:establishment,characterization and applicationThe objectives of this study include induction and multiplication of callus,establishment of a suspension cell line,and isolation of protoplasts from cell suspensions.Murashige and Skoog(MS)medium was used for callus induction from mature seeds of N.sibirica.Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mg L-1 6-benzylaminopurine(6-BA)and 1.0 mg L-1 2,4-dichlorophenoxy(2,4-D)acetic acid.Suspension cultures of N.sibirica were initiated by transferring friable calli to the same liquid multiplication medium.Characterization of the suspension culture was assessed based on fresh mass,dry mass,cell viability and pH value of the culture.A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium,including a lag phase,an exponential growth phase,a stationary phase,and a negative acceleration phase.The effect of factors such as pre-plasmolysis,enzyme combination,enzymolysis time and mannitol concentration,on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures.The maximum yield(9.799×106 cells/g)and highest viability(79.97%)of protoplast were reached when approximately 1 g of cell suspension(cultured for 6 days)was inoculated for 12 h in cell and protoplast washing solution made of 0.8 mol L-1 mannitol mixture solution,cellulose onozuka R-10 2%(w/v),hemicellulose 0.2%,macerozyme R-10 1%,and pectolyase Y-23 0.5%.Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10(P<0.05).3.Study on response characteristics of ion flow in N.sibirica under salt,alkali and osmosis stressTo investigate the primary responses of ion flow in N.sibirica under salt,alkali and osmosis stress as well as its relevance to resistance mechanism,the characteristics of ion flow at the 12 micro area of N.sibirica seedling roots stressed by 100 mM NaCl,50 mM Na2CO3 and 175 mM mannitol for 1h were detected and analysed with MIFE respectively.The results are as follows:In the early stages,N.sibirica response to salt and alkali stress included limiting Na influx,reducing K+efflux,increasing H+ efflux and reducing the Na+/K+ value,which enhance its ability to resist salt and alkali;Na+influx mainly located in 200?800 ?m root cap region,K+ efflux mainly located in 1400 ?m root extension region,H+ efflux mainly located in 1400?2000 ?m root extension region.The main different of response to salt stress and alkali stress in ion flow speeds was shown,the speed of Na+ influx under alkali stress(-375.159 pmol cm-2 s-1)was higher than that under salt stress(-214.104 pmol cm-2 s-1),the speed of K+efflux under alkali stress(143.142 pmol cm-2 s-1)was higher than that under salt stress(48.575 pmol cm-2 s-1),the speed of H+efflux under alkali stress(87.683 pmol cm-2 s-1)was lower than that under salt stress(2686.485 pmol cm-2 s-1),and the value of Na+/K+under alkali stress(1.411)was larger than that under salt stress(1.226).As a result of alkali stress,the Increase of Na+influx,the Increase of K+efflux and the decrease of H+efflux was revealed by contrast with salt stress,so the damage to N.sibirica of alkali stress was greater than that of salt stress.These results suggested that the plasma H+-ATPase activities of N.sibirica under salt stress and alkali stress could have significant differences,which lead to H+ efflux and the reconstruction of plasma proton gradient as well as restoring the membrane potential depolarization caused by the salt stress;at the same time,Na+ efflux could be increased through H+-ATPase activating plasma membrane Na+/H+antiporter(SOS1),so the damage caused by salt stress would be relieved.In the early stages,the responses to salt and alkali stress of N.sibirica and Phaseolus radiates L.had both significant differences,the values of Na+/K+ of N.sibirica under salt and alkali stress were both less than those of Phaseolus radiates L.under salt and alkali stress,these results suggested that the lower Na+/K+values and the ionic balance in the cell could be maintained by response of ion flow in N.sibirica under salt and alkali stress,so N.sibirica have stronger resistance to salt and alkali.The response characteristics of ion flow in N.sibirica under osmosis stress were different from those of salt and alkali stress,Na+ efflux,K+influx and H+efflux were revealed,Na+ efflux mainly located in 1700 ?m root extension region,K+ influx mainly located in 800?m root cap region,H+ efflux mainly located in 1700?m root extension region,and the location of H+ efflux in N.sibirica under osmosis stress was same with those under salt and alkali stress.In the early stages,the responses to osmosis stress of N.sibirica and Phaseolus radiates L.had significant differences,Na+efflux,K+efflux and H+efflux were revealed,and the speed of K+influx in N.sibirica under osmosis stress was significantly higher than that in Phaseolus radiates L.under osmosis stress.these results suggested that the lower Na+/K+ values and the ionic balance in the cell could be maintained by response of ion flow in N.sibirica under osmosis stress,which avoid the damage to the plant roots by the high osmotic stress,so N.sibirica have stronger resistance to osmosis.4.Study on response characteristics of membrane potential in N.sibirica under salt and alkali stress as well as its relevance to plasma membrane H+-ATPaseTo investigate the primary responses of membrane potential(Em)in N.sibirica under salt and alkali stress as well as its relevance to plasma membrane(PM)H+-ATPase and H+ ion flow,the characteristics of Em and H ion flow of N.sibirica seedling roots stressed by 100 mM NaCl and 50 mM Na2CO3 were detected and analysed with Patch-Clamp technique and MIFE respectively.The results are as follows:Em depolarization of N.sibirica seedlings roots stressed instantaneously by different concentration of KCl,NaCl and Na2CO3 were all detected,and Em depolarization included fast response stage and slow response stage,the fast reaction stage was the key period for Em response.The stimulus thresholds were determined according to the depolarization amplitudes and times of fast response stage,then N.sibirica seedling roots were stressed instantaneously by the stimulus thresholds(160 mM KCl,100 mM NaCl and 50 mM Na2CO3)respectively,the depolarization amplitudes were 92.0 mV,91.9 mV and 53.0 mV respectively,and the depolarization times were 6.085s,29.571s and 5.987s.The resting potential(Er)of N.sibirica seedlings roots in the test liquid(control group)was 156.252 mV,the depolarization was detected with VV preprocessing,and Er was-136.399 mV;hyperpolarization was detected with FF preprocessing,and Er was-228.311 mV.Thoese results suggested that VV and FF could cause the change of Em by influencing the PM H+-ATPase activity,and PM H+-ATPase could involve in the formation of Er.After pretreating with VV(NSVVC1)and FF(NSFFC1),the Em changes of N.sibirica seedlings roots stressed instantaneously by 100 mM NaCl were analyzed for 30s respectively,the contribution rates of PM H+-ATPase in the process of Em depolarization were calculated by comparing with the control group(NSC1),the results showed the contribution peak values of NSVVC1 and NSFFC1 were reached 20.326 mV and 13.011 mV in 5 s and 6 s respectively,and the contribution rates were 14.173%and 9.073%respectively;after 30 s,PM H+-ATPase was no longer play a role in Em depolarization.These results suggested that the primary responses of Em in N.sibirica under salt stress could be associated with PM H+-ATPase activity,and H+-ATPase could participate in the response process of Em.After pretreating with VV(NSVVC2)and FF(NSFFC2),the Em changes of N.sibirica seedlings roots stressed instantaneously by 50 mM Na2C03 were analyzed for 30s respectively,the contribution rates of PM H+-ATPase in the process of Em depolarization were calculated by comparing with the control group(NSC2),the results showed the contribution peak values of NSVVC2 and NSFFC2 were reached 13.337 mV and 7.238 mV in 5 s and 1 s respectively,and the contribution rates were 26.039%and 14.131%respectively;after 30 s,PM H+-ATPase was no longer play a role in Em depolarization.These results suggested that the primary responses of Em in N.sibirica under alkali stress could be associated with PM H+-ATPase activity,and H+-ATPase could participate in the response process of Em.Then the H+ion flows of N.sibirica seedling roots stressed by 100 mM NaCl were detected with MIFE,the H+efflux rate of NSCl was 4.558pmol cm-2 s-1;comparing with NSCl,the H+efflux rate of NSFFCl increased before salt stress,and significantly increased after salt stress,the rate were reached 24.685pmol cm-2 s-1;the H+efflux rate of NSVVC1 reduced before salt stress,while the H+ influx was detected after salt stress,the rate were reached-3.392pmol cm-2 s-1.These results suggested the primary responses of Em in N.sibirica under salt stress could be associated with PM H+-ATPase and H+ ion flow. |
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