Study For Resistance Mechanism Of Watermelon Under Different Temperature Stress At Cell Level

Using autopolyploid watermelon as materials, the following experiments have been carried on. First, protoplasts from leaves were generated by enzyme hydrolysis and preparation conditions were optimized; Then protoplasts were treated under different temperatures and cell membrane hydraulic conductivity was determined to evaluation the cell membrane stability; Secondly, Ca2+concentration changes in cells were observed by using fluorescent probe combined with microscopy. Thirdly, with direct and indirect immunofluorescence microscopy, the structural changes of microfilament and microtubules were observed. Last, the resistance formation in autopolyploid watermelon on cell level was proposed.The main results are as follows:(1) The optimum preparation conditions for watermelon leaf protoplasts were:2.7%cellulase+0.78%macerozyme dissolved in0.4mol/L mannitol,80rpm,120min;(2) Cell membrane permeability of different ploidy watermelon were determined under different temperatures. At15℃and20℃, cell membrane permeability of diploid increase was significantly lower than tetraploid, but at high temperature of30℃to40℃, result was opposited. This indicates that diploid with better tolerance of low temperature, whereas the tetraploid is with better tolerance on high temperature;(3) The optimized Ca2+fluorescent probe loading method for watermelon leaf protoplasts is:10mol/L fluorescent dye,25℃, in the dark for2h;(4) Ca2+concentration in cells were measured under different buffer.5℃treatment can increase intracellular Ca2+concentration. And most Ca2+are from extracellular;(5) By comparing the change of different ploidy watermelon protoplast at different temperatures, the following results were found. Ca2+concentration in cells were more stable in diploid than tetraploid at15℃-20℃. And opposite result was obtained when treated by30℃-40℃.This indicate that tolerant one can maintain more stable intracellular Ca2+concentration, and vise verse;(6) With indirect immunofluorescence method, microtubule of watermelon Leaves were observed. And the optimized protocol is:3.7%paraformaldehyde+1%glycerol+2%tritonX-100, fixl.5h, hydrolize15min. Microtubule of diploid depolymerized at5℃,30min which is more profound than tetraploid. high temperature challenge produce opposite results. Microtubule depolymerize severely over treatment in both diploid and tetraploid. This shows that the prompt response of microtubule is related with tolerance;(7) Optimized the microfilaments observation protocol of suspension cells of watermenlon:cell fixed in1.85%paraformaldehyde+1%glycerol+2%tritonX-100for1.5h, then hydrolize5min. Similar phenomena were obtain compared with microtubular change after30min treatment. However, microfilaments depolymerized more severe than microtubular.In conclusion, plasma membrane, cytoskeleton and Ca2+are with close relationship.the one which is more tolerance always show the characters of stable plasma membrane, prompt cytoskeleton response and less intracellular Ca2+fluctuation. and vise verse.