Studies On The Dynamic Changes Of Plant Hormones Content In Photoperiod-Temperature Sensitive Tassel Deficiency Line Of Maize

This study is set out to explore the effect of plant hormones on tassel morphogenesis and male fertility of photoperiod-temperature sensitive tassel deficiency line of maize and establish LC-MS analysis method of accurate and rapid determination of the content of conjugated and free abscisic acid in plant samples. Alkaline lysis method combined with LC-MS analysis were used for isolation, purification and determination of conjugated and free abscisic acid with Arabidopsis thaliana and maize as materials in this experiment. The content of indole acetic acid (IAA), abscisic acid (ABA), gibberellin (GA3) and zeatin (ZT) in leaves and tassels of maize of photoperiod-temperature sensitive tassel deficiency line 117 and its normal line N17 planted in spring and autumn were studied based on the above method. The result are showed as follows.1.Different conditions affected on determination of conjugated and free abscisic acid was studied and analyzed of leaves of Arabidopsis thaliana as materials, to which determine the optimum conditions of the hydrolysis of conjugated ABA and determine the content of conjugated ABA quickly in photoperiod-temperature sensitive tassel deficiency line of maize. Samples were extracted with the 80% precooling and acidified methanol and conjugated abscisic acid in extracts were treated by alkaline lysis method. Petroleum ether, chloroform, ethyl acetate were used for extraction. Sep-Pak C18 column chromatography was used for purification.50% methanol solution was used for elution. LC-MS was used for detection and analysis.The content of the total ABA using the 80% cold and acidified methanol solution were higher 19.42% than using the conventional 80% methanol solution when the total ABA were extracted. The pH 12.000,50℃was the best condition when alkaline lysis processing and was the most suitable extraction condition for conjugated ABA.The content of the total ABA using weak acid and weak alkali for adjusting the pH were higher 22.53% than using the conventional strong acid and strong alkali when the total ABA were extracted.The recovery rate of using petroleum ether, ethyl acetate, chloroform were quite high, in which the recovery rate of ethyl acetate was the highest, meeting the requirement of quantitative analysis. The average recovery of using LC-MS was from 80.5% to 95.2%, of which relative standard deviation (RSD) was from 1.7% to 11.8%. Sensitivity, precision and accuracy of this method are all met with the requirements of plant hormones analysis.2. The optimal extraction and determination conditions of ABA-GS and ABAMe were discussed to provide the method for further rapid determination of the content of ABA-GS and ABAMe in photoperiod-temperature sensitive tassel deficiency line of Maize. Plant tissues were firstly extracted by using 80% cold and acidified methanol solution, then the Sep-Pak Cjg solid-phase extraction (SPE) cartridges were adopted for the purification of sample extract, and finally ABA-GS and ABAMe were determined by the liquid chromatography-mass spectrometry (LC-MS) system. It was found that the accuracy was improved by using the extra standard for the estimation and correction of ABA-GS and ABAMe losses in the extraction and purification process. The detection limits for ABA-GS and ABAMe were 0.04 ng-mL-1 and 0.06 ng-mL-1, respectively, and the average recovery rate of ABA-GS and ABAMe was 91.74%,93.31%. The method was found to be reproducible and selective, yielding well isolated and easily detectable peaks for both ABA-GS and ABAMe and simplifying the time-comsuming extraction and purification. High-performance liquid chromatography (HPLC) system was employed as the control detection method for the LC-MS systems.3. The optimal extraction, purification and determination method of hormones discussed above was used to determine respectively the content of indole acetic acid (IAA), abscisic acid (ABA), gibberellin (GA3) and zeatin (ZT) in leaves and tassels of maize of photoperiod-temperature sensitive tassel deficiency line I17'and its normal line N17 planted in spring and autumn, for which exploring the effect of plant hormones on tassel morphogenesis and male fertility of photoperiod-temperature sensitive tassel deficiency line of maize. The results showed that the contents of IAA, ABA, GA3 and ZT in leaves and tassels of 117 were no significant difference between 117 and N17 of maize planted in spring. However, the contents of IAA, GA3 and ZT in the leaves of 117 were significantly lower than that of N17 in seedling stage, tasseling stage and flower seed filling stage, while ABA content was significantly higher than that of N17 planted in autumn. About the balance of plant hormones, IAA/ABA, GA3/ABA and ZT/ABA ratio of 117 in the leaves were significantly less than that of N17 planted in autumn. It is suggested that the serious loss of IAA, GA3 and ZT and excessive accumulation of ABA are the important reasons of tassel deficiency in Maize.