The Physiological, Gene Expression And Ultrastructure Responses Of Populus Euphratica Explants To Salt Stress In Vitro Culture

Populus euphratica Oliv. is a unique tree species used for afforestation on salinity and desert in the north-west China,and play important role to maintain ecological stability. It has strong salt resistance because of physiological characteristics. In this research, Populus euphratica Oliv. buds in vitro culture as experiment material, cell membrane permeability, osmotic adjustment substance content determination and oxidation resistance system were determined to salt stress. In addition, CuZn-SOD and PtAPX2 gene expression and ultrastructure were studied. Therefore, salt-tolerance mechanism was explored from physiology and biochemistry, gene expression and ultrastructure. The results do not only breed a salt resistance species early and to furtherly provide theory basis for exploring salt-tolerace ability in Populus euphratica. The main results were as follows:1.Response of physiologyThe growth of Populus euphratica was inhibited by salt stress, decreased relative water content?RWC?,chlorophyll content?Chl?,Chl/Cxc ratio with the increasing of salt concentration.The relative electrical conductivity?REC? and MDA content were obviously increased with moderate and high dose of salinity?150²00mmol/L?,REC content increased by 46.8%, 55.2% than the control?10d?,MDA content increased by 42.7%, 63.2% than the control?10d?. Ascensional range was rised 10 d more than 5d under salt stress.Soluble protein content was reached a maxium with moderate dose of salinity?150mmol/L?, increased by 72.8%?5d?and 47.6%?10d? than the control. soluble sugar content was significantly increased, was similar to that of control under other NaCl treatment. The high level of proline was found under 50mmol/L NaCl treatment, the cells could enchance the osmotic adjustment capacity.O₂^·- and H₂O₂ content were incrased under NaCl treatment,especially were obviously increased with moderate and high dose of salinity?150²00mmol/L? in Populus euphratica. The SOD activity were incresaed first and then decreased,and was higher than the control. The POD activity was reached a maxium under 150mmol/L NaCl treatment. The CAT activity was significantly increased than the control with low and high dose of salinity?50¹50mmol/L?. The APX activity was reached a maxium under 100mmol/L NaCl treatment, was similar to that of control with mo- derate and high dose of salinity?150²00mmol/L?. The GR activity was significantly increased with low dose of salinity?50¹00mmol/L?, were 1.8 times?5d? and 1.48 times?10d?of the control.ASA content was decreased first and then increased,increased by 26.9%?5d? and 47.3%?10d? than the control. The GSH content was decreased under 5 days salt stress, and was increased first and then decreased under 10 days salt stress.The above analysis shows that the Populus euphratica were accumulation of osmotic agents, such as soluble protein, soluble sugar, which may help to increase the concentration of cytoplasm and decrease the osmotic potential. A series of protective enzyme activities and antioxidants were increased, which will alleviate the O2-and H2O2 damage to their own.2.Gene expressionSemi-quantitative PCR and real-time fluorescent quantitative PCR have the same results. CuZn-SOD gene expression was significantly increased under 24⁴8h salt treatment. PtAPX2 gene expression was significantly incresaed under 36⁷2h salt treatment.The SOD activity were increased first and then decreased, reached a maxium under 48 h. CuZn-SOD, PtAPX2 relative expression quantity were correlated with SOD and APX activity?>48h?. The above analysis shows that CuZn-SOD gene was expressed before PtAPX2. Enzyme activity had significantly positive correlations with antioxidase gene expression?>48h?. Antioxidase gene expression?CuZn-SOD, PtAPX2? resulted in the salt resisitance in Populus euphratica.3.UltrastructureThe structure did not change significantly under 100mmol/L NaCl treatment. The structure was obviously damaged under 200mmol/L NaCl treatment. The density of leaf stomata, open stomata proportion and stomatal apertura were decreased, wax layer was increased of epidermal cells. Vascular tissue area was decreased, and cell cavity of leaf was increased. The cell membrane of mesophyll cell was invaginated. Moreover, chloroplast, starch grains content were decreased. Thylakoid lamella was loosely arranged, expansed, twisted. Osmiophilic granule were increased. The above analysis shows that the structure was obviously damaged under 200mmol/L NaCl treatment, which exceed their salt resistance.In conclusion, Populus euphratica was responsed to salt stress, such as physiology, antioxidase gene expression, ultrastructure. Suitable dose of salinity?< 150mmol/L?had a certain tolerance and adaptive capacity to salt stimulation. Salt-tolerance ability had significantly positive correlations with antioxidase gene expression?>48h?. The structure was obviously damaged NaCl treatment?> 200mmol/L?.