Effect And Mechanism Of Fertility Alteration Of Thermo-sensitive Genic Male Sterile Lines Induced By Low Temperature In Rice (Oryza Sativa Indica) Inhibited By Chemical Substances

Rice(Oryza sativa L.)is one of the most important grain crops in the world and plays an important role in global food security.Utilizing hybrid rice seeds in agricultural production has achieved a historic leap in Chinese agriculture.Grain production has achieved a double leap in quantity and quality.During two-line hybrid rice seed production,thermo-sensitive genic male sterile(TGMS)rice line is vulnerable to unpredictable climate change to alter fertility to produce false hybrids(self-pollination).That is the bottleneck in the development of two-line hybrid rice seed production in China.How to effectively prevent the fertility alteration induced by low temperature during the sensitive period of TGMS has become an urgent problem in the production of two-line hybrid rice.Currently,given the limitation of using male sterile lines,it is highly desirable to explore appropriate solutions to solve the issue of fertility alteration of TGMS rice lines under low temperature.Exploring the mechanism of how chemical substances inhibiting fertility alteration induced by low temperatre,which provides practical experience and theoretical basis for two-line hybrid rice seed production.In the preliminary research,proteomics,physiological and biochemical analysis were used to reveal the mechanism of fertility alteration of TGMS under low temperature inducement.Enhanced antioxidant pathway,increased protein synthesis and degradation,more sufficient carbohydrate and energy supply,disintegrated anther wall,increased putrescine and spermine content,weakened ethylene synthesis were contributed to fertility alteration.Moreover,exogenous ethephon treatment could inhibit the fertility alteration of TGMS during low temperture inducement.In this study,from both production application and theoretical research,screening chemical substances on the optimization of spraying period,using concentration,so as to find effectively chemical substances that inhibiting fertility alteration,easy to operation and moderate price.Transcriptomics and physiological approach were applied to their young panicles to preliminary study on the molecular mechanism of chemical substances inhibiting the fertility alteration of TGMS rice under low temperature(22.0℃)inducement.The main results are as follows:1.Fertility alteration of different sterile starting temperature TGMS rice Jing 4155S,Shen 08S and 1892S were induced by natural low temperature in winter,Hainan.In the sensitive period of TGMS,Ⅳ-Ⅵ phase of young panicle differentiation,chemical substances MGBG,D-Arg,polyamines,GABA and ethephon with different concentrations were sprayed on the leaves of TGMS rice,with a total of 3 times.The pollen staining rate and the bagged seed setting rate were used to evaluate using efficiency of chemical substances.There is no significant difference in spraying MGBG and polyamines.While 5 mmol/L D-Arg,5 mmol/L and 10 mmol/L GABA,1200 mg/L and 2400 mg/L ethephon treatment significantly decreasd the pollen staining rate and the bagged seed setting rate of TGMS rice Jing 4155S and Shen 08S.That is to say,chemical substances D-Arg,GABA and ethephon treatment could significantly inhibit the fertility alteration of TGMS rice induced by low temperature.2.Fertility alteration of different sterile starting temperature TGMS rice Guangzhan 2S,1892S,Jing 4155S and Y58S were induced by natural low temperature in winter,Hainan.In the sensitive period of TGMS,Ⅳ-Ⅵ phase of young panicle differentiation,chemical substances,(10 mmol/L GABA+1200 mg/L ethephon+NF100),were sprayed in different ways.The pollen staining rate and the bagged seed setting rate were used to evaluate using efficiency of chemical substances.It found that different combinations of chemical substances could significantly inhibit fertility alteration of TGMS induced by low temperature,in which treatment group 4(pre-stage multi-spray ethephon,late-stage multi-spray GABA)has the best effect on inhibiting fertility alteration.Spraying "920",semifinished product of Gibberellin,at an appropriate time,the sensitivity of gibberellin of TGMS was investigated after xogenous substance treatment.It was found that the treatment of different combinations of chemical substances significantly decreased the height of the plants,but there is non-necked phenomenon.That is,the total exposure of the kernels did not affect.Sowing date,artificial supplementary pollination during flowering,full outcrossing,statistics on seed setting rate found that treatment group 3(spraying the same amount at every time)and treatment group 4(pre-stage multi-spray ethephon,late-stage multi-spray GABA)had no significant influence on outcrossing rate.While treatment group 2(pre-stage multi-spray GABA,late-stage multi-spray ethephon)significnatly decreased the outcrossing rate.3.After the chemical substances MGBG,D-Arg,polyamine,GABA and ethephon treatment,the chlorophyll content of the leaves of the first and second leaves was determined.It found that chemical substances MGBG,D-Arg,polyamines and GABA treatment have no significant influence on the chlorophyll content of the second leaf.After the second and third spraying treatment,the high concentration of ethephon(2400 mg/L)significantly reduced the chlorophyll content of the second leaf.chemical substances MGBG,D-Arg,polyamine and GABA had no significant effect on plant morphology.While high concentration of ethephon(2400 mg/L)significantly reduced plant height and showed leaf stiffness.After the seeds matured,the seeds in the bags was harvested and found that chemical substances treatment had no significant effect on the 1000-grain weight of the seeds.4.The cold-irrigation tank with low temperature(22.0℃)induced the fertility alteration of TGMS rice Jing 4155S.Simultaneously,2400 mg/L ethephon,10 mmol/L D-Arg and water(control)were sprayed respectively.Then,transcriptomic analysis was performed with the treated young panicles.The fertile pollen grains of Jing 4155S were detected in the water treated group but not in the two groups treated with chemical substances,respectively.These results indicated that fertility alteration occurred only in water treated group but not in chemical substance treated group during the low temperature inducement.Compared to the water-treated group,there were 1026 in ST-inducement and 977 DEGs in LT-inducement identified in D-Arg-treated group,whereas 636 DEGs in ST-inducement and 229 DEGs in LT-inducement identified in the ethephon-treated group,respectively.compared between the D-Arg-treated group and ethephon-treated group,there were 524 and 569 DEGs identified in ST-inducement and LT-inducement,respectively.The differentially expressed genes were mainly enriched in RNA transport,mRNA surveillance pathways and biosynthesis of secondary metabolites,etc.Genes with differentially expressed transcription factors,genes involved in ethylene synthesis,carbohydrates,cell synthesis and lipid metabolism may be involved in the process of inhibiting fertility alteration of TGMS rice induced by low temperature.