Selection Of Exogenous Inhibitors For Pre-harvest Sprouting And Their Inhibitorv Mechanism Research In Hybrid Rice

Due to the fact that most sterile lines of hybrid rice are less dormant,together with the application of GA in the production of rice seed,seeds sprout easily on the mother plant under rainy weather during the maturity period.This phenomenon,which is called pre-harvest sprouting(PHS),definitely leads to severe economic losses.Therefore,it's economically and empirically crucial to develop effective inhibitors for PHS and further explore the mechanism of inhibition.With Qianyou 1 and Y Liangyou 689 as experimental materials,this study firstly investigated the inhibitory effects of both eugenol and inhibitor combinations(eugenol+uniconazole+MH)on seed germination of hybrid rice;secondly explored the influences of selcected inhibitors on physiological and molecular changes during seed germination aiming to interpret their inhibition mechanism;thirdly validated the inhibitory effects of selected inhibitors on rice PHS through field trials and finally obtained three effective PHS inhibitor combinations.As an initial attempt to shed light on the inhibitory effects and mechanism of both one natural product,namely eugenol,and selected inhibitor combinations,this research provides empirical and theoretical insights for the control and inhibition of PHS during seed production of hybrid rice and other cereals.The main results are shown as follows:Physiological changes of hybrid rice Qianyou 1 seeds both during their imbibition and among different germination stages were studied.With the process of imbibition and the elongation of radical,the amount of soluble sugar and the activities of ?-amylase,POD,APX and CAT increased significantly;ABA decreased rapidly from 3 h to 18 h during seed imbibition.This study therefore showed that ?-amylase,POD,APX,CAT and ABA content were closely related to germination process of hybrid rice seeds.The effects of eugenol on germination,physiological(soluble sugar,?-amylase,POD,APX,CAT,ABA and GA3)and molecular changes(gene expression related to ABA metabolism and signaling)of Qianyou 1 seeds during the germination progress were studied.Further,the influence of GA3 on eugenol effect was also investigated.Eugenol application(?1.5 g/L)decreased the speed of seed germination.In addition,the inhibitory effect was enhanced by the increase of eugenol concentrations,while no significant inhibitory effect on seedling growth was observed.Seeds applied with eugenol showed lower activities of a-amylase,POD,APX and CAT within the first 96 h of imbibitions as compared with the control,indicating that eugenol could inhibit PHS through affecting the activities of enzymes during the process of germination.The germination rate of the seeds treated with eugenol+GA3 was ranked between those of the control and eugenol,indicating that the inhibitory effect of eugenol could be partially recovered by GA3.ABA content could be imporved by eugenol at the early stage of seed imbibitions(3 h-12 h),and GA3 could partially inhibit the increased ABA content caused by eugenol.Furthermore,eugenol and eugenol+GA3 improved GA3 content during seed imbibitions.These results suggested that the inhibitory effect caused by the increase of ABA was more than the promotive effect induced by the increase of GA3,leading to the delay of germination.According to the expression patterns of three members of OsABA80H involved in ABA catabolism and five members of OsNCED involved in ABA synthesis,OsABA80H2 and OsNCED2 were speculated to be the predominant genes affected by eugenol.Besides,eugenol might promote ABA signaling by improving the expression of OsABFl and therefore contribute to the inhibition of seed germination.Different concentrations of eugenol,uniconazole and MH were blended in different levels and finally three inhibitor combinations(A,G and I)were selected for further study.Moreover,the effects of three combinations on germination and also physiological(?-amylase,ABA and GA3)and molecular changes(gene expression related with ABA metabolism and signaling)of Qianyou 1 during seed germination were studied.Results showed that three combinations were more effective on germination inhibiting than eugenol.Inhibitory effect on seedling growth was also observed in combinations,which might be due to the inhibitory effect of uniconazole on GA biosynthesis and cell elongation.The ABA content was improved by three combinations,while GA3 was not.It suggested that the effect of the improved GA3 caused by eugenol was probably neutralized by uniconazole.At the level of gene expression,combinations A and G had inhibitory effects on gene expression of OsABA80H2,while the three combinations had no promoting effects on OsNCEDs.The ratio of 0sNCED2/OsABA80H2 from inhibitor combinations was significantly higher than that of the control,which was consistent with the result that inhibitor combinations could promote the amount of ABA.Similarly,the three inhibitor combinations might promote ABA signaling and contribute to the inhibition of seed germination through improving the expression of OsABF1.Field trials supported by manually simulated rainfall were conducted to confirm inhibition effects of eugenol and inhibitor combinations on PHS of both hybrid rice Qianyou 1 and Y liangyou 689.The ungerminated seeds harvested from control and inhibitor treatments were used for germination tests to evaluate the persistent inhibitory effects of eugenol.Eugenol and inhibitor combinations had better inhibition effects on Y liangyou 689 than Qianyou 1.The inhibitory effect of eugenol was enhanced by the increase of the concentration.As compared with the control,the PHS inhibition ratios were 43.4%(PHS rate decreased from 27.9%to 15.8%)and 80%(PHS rate decreased from 5.66%to 1.13%)respectively in Qianyou 1 and Y liangyou 689 treated by 2.0 g/L eugenol.Inhibitor combinations were more effective on hybrid rice Qianyou 1 than eugenol was,while eugenol performed better on Y liangyou 689 than inhibitor combinations did.The results of germination tests showed that,the inhibitory effect of eugenol on seed germination was totally relieved after the storage for six months.The sprouting results of field in 2015 showed that combination I(1.2 g/L eugenol+0.3 g/L uniconazole+0.4 g/L MH)had a stronger inhibitory effect(decreasing PHS rate from 13.45%to 5.31%)on Qianyou 1;combination A(0.4 g/L eugenol+0.6 g/L uniconazole+0.4 g/L MH)had a stronger inhibitory effect(decreasing PHS rate from 7.60%to 2.58%)on Y liangyou 689.The results of field trials suggested that both eugenol and three selected inhibitor combinations could strongly inhibit PHS of hybrid rice;meanwhile,they did not affect germination percentage.The selected inhibitors and their concentrations in present study could be applied to control PHS during hybrid rice seed production.