Establishment Of Efficient Determination Method For Plant Hormones And Quality And Its Application In Soybean

Plant hormones are important life regulators for crop growth and seed quality formation,and the formation of seed quality is the final feedback of different growth processes.It was found that the current plant hormones and quality detection methods were difficult to meet the demand of in-depth research.The change rules of soybean plant hormones with different time and space,different stress and chemical regulation were not clear.The chemical regulation influence of important quality compounds in soybean was still insufficient.Therefore,the establishment and application of new detection methods were great significance.In this study,based on the improvement of detection methods,it established an efficient and economic method for the detection of plant hormones in physiology,and established a fast and effective method for the determination of fatty acids and phytosterols in quality,and investigated the detection effect of methods for different test application.The method was applied to reveal the temporal and spatial changes,stress changes and chemical regulation changes of plant hormones taking main varieties in Heilongjiang Hefeng 50 and Kenfeng 16 as research objects,and to explore the effects of uniconazole regulation on the formation of soybean fatty acids and phytosterol quality.The main results and conclusions were as follows:1.The methodological ability of different detection methods for four plant hormones(ZT,IAA,GA3 and ABA)was compared.The results showed that the separation speed and sensitivity of ultra fast liquid chromatography was faster than high performance liquid chromatography,but the interference of sample matrix was remarkable due to its low sensitivity of detector.Trimethyldiazomethane derivatization combined with gas chromatography-mass spectrometry was applicable,but it was only applicable to target compounds containing carboxylic acid group.The sensitivity of LC-MS/MS was further improved,but the sample pretreatment was complicated,and the detection efficiency was greatly affected by the sample pretreatment.Online-SPE method had strong automation ability,its detection limit was 0.20 ng/ml ? 1.01ng/ml,and the relative standard deviation(RSD)of repeatability was 2.54 % ? 4.83%.However,the method was equipment dependent.2.An efficient and economic detection method based on ultra performance liquid chromatography-mass spectrometry was established.The samples were treated by vacuum freeze-drying technology,the target compounds were extracted by ultrasonic assisted solvent,the matrix was purified by improved Qu ECh ERS method,and the chromatographic separation was performed on phenomenex kinetex F5 column(50 mm 4 × 3.0 mm ID,2.6 ? m,100 ?).The target components were eluted by formic acid / water gradient system,and the mass spectrometer detector was in positive and negative simultaneous scanning mode(MRM).The four plant hormones were separated in 3 min,and the target components showed a good linear relationship in the concentration range of 0.1 ng/ml ? 100 ng/ml.the detection limit was 0.015 ng/ml ? 0.078ng/ml,and the relative standard deviation was 0.16 % ? 0.25 %.The method was simple,economical,efficient and less sample consumption.3.Based on gas chromatography combined with high-pressure transfer printing sample pretreatment,a rapid determination method of fatty acid content in soybean was established.Dielectric barrier discharge helium plasma combined with short column was used to improve the sensitivity,separation effect and analysis efficiency of the method in constant pressure separation mode.Ten fatty acids in soybean samples were detected in 30 min with high resolution,and the detection limits of target compounds were 0.105 ?g/m L ? 0.196 ?g/m L.The relative standard deviation was 1.04 % ? 1.35 %.The method required a small amount of samples,less consumption of chemical reagents,simple and rapid sample pretreatment,and acquired good reproducibility of determination results.4.A rapid method for the determination of phytosterols in soybean was established based on gas chromatography-mass spectrometry.The target compounds were extracted with isooctane,saponified with potassium hydroxide-ethanol-water system and directly analyzed without silylation derivatization after treatment.The detection sensitivity of four phytosterols in soybean samples ranged from 0.098 ?g/m L to 0.206 ?g/m L,and the relative standard deviation ranged from 1.16 % to 1.97 %.The method is simple,rapid and non-derivatization for accurately determination of phytosterols.5.The changes of plant hormones under temporal and spatial,stress and chemical regulation were investigated by using new detection method in soybean.The results showed that plant hormones changed rapidly and systematically during the daytime,and were sensitive to light and temperature.Different plant hormones showed their unique spatial and temporal characteristics in different growth stages,and their content levels varied with physiological parts and individuals.After the stress,the balance of plant hormones were rapidly broken.No matter under low temperature or drought stress,promoting plant hormones and inhibiting plant hormones showed the opposite trend,and the content of promoting plant hormones generally decreased.In the regulation of uniconazole on soybean growth,gibberellin and auxin were the key plant hormones that uniconazole playing the delaying effect.The regulation mechanism was equivalent to a kind of directional stress on plants.The effect of uniconazole could be quickly relieved by exogenous auxin and gibberellin.6.The effects of uniconazole on the quality of soybean fatty acids and phytosterols were investigated at flowering stage.The results showed that uniconazole had significant effects on fatty acids and phytosterols composition of different soybean varieties.In terms of fatty acids composition,exogenous uniconazole reduced the content of polyunsaturated fatty acids in soybean,and the regulation process may be involved in the degradation of fat.In terms of phytosterols content changes,uniconazole significantly reduced the contents of brassicasterol,stigmasterol and sitosterol in different soybean varieties,and slightly affected the phytosterol contents of different oil-bearing varieties,which showed that the effect on high oil-bearing varieties was weak.Uniconazole contributed less to quality formation than varietal genes,which was safe for soybean production.Based on the above results,this study established a high-efficiency detection method of plant hormones,fatty acids and phytosterols through technology integration innovation,and deeply revealed the change law of plant hormones and quality in soybean growth and development and chemical regulation by using the technical advantages of the new method,which provided a new method strategy and law cognition for soybean cultivation research.