| The seed gradually loses its dormancy in the first stage after harvest,and the germination ability of the seed gradually increases until it reaches a peak.Thereafter,the dried seeds gradually deteriorate during storage,and the germination ability of the seeds gradually decreases until the life capacity is completely lost,eventually led to germination failure.According to the incomplete statistics of the Ministry of Agriculture,the aging and mildew of rice seeds caused by improper storage account for 8%of the total storage every year,causing serious losses.People usually prolong the storage time of seeds by changing the external storage conditions in production practice,but it also consumes a lot of manpower and financial resources.However,it is a practical technical to improve the seed storage resistance by improving the genetic characteristics of the seeds.Previous studies have shown that the massive degradation of lipids is the main cause of rice aging,and lipoxygenase is a key enzyme that caused lipid degradation.Therefore,it can significantly slow down the rate of rice aging by inhibiting the content of lipoxygenase in rice seeds and activity.For this purpose,the Ningjing 1 was used as a recurrent parent to create a batch of transgenic lines that interfered with lipoxygenase.This study focused on the further screening and identification of two families with XO3 and XO6 which have obvious interference effects,and obtained families with stable inheritance.qRT-PCR analysis showed that the expression level of OsLOX2、OsLOX3 in XO3 and XO6 was significantly lower than that in the Ningjingl;the phenotypic identification showed that the seed vigor of XO3 and XO6 families was significantly higher than that of wild type after stored in natural conditions for 10 months and artificial aging treatment for 8 days;TTC staining showed that the content of TTCH(toluene)in XO3 and XO6 was significantly higher than that of Ningjing 1;the determination of lipoxygenase in the seeds revealed that the disturbed families were significantly lower than the recipients;the analysis of quality indicators indicated that the changes of amylose,total starch and gel consistency of seeds in disturbed families before and after aging treatment were smaller than those of control Ningjing 1;the field plot comparison experiment demonstrated that most of the main agronomic traits in the disturbed families were no significant differences compared with recurrent parent;The gene editing of OsLOX1,OsLOX2 and OsLOX3 was carried out by using CRISPR/Cas9 technology,and transgenic homozygous plants with various editing sites were obtained.Among them,the resistance of the CRISPR/Cas9-LOX1 T2 seed was significantly enhanced compared with the recurrent parent Ningjing 4.Seed storage tolerance is a quantitative trait controlled by multiple genes.Many QTLs related to seed storage tolerance have been reported and some stable QTLs have been detected.The indica variety Kasalath showed a extreme resistant phenotype,the qSS-9Kas has been verified in our laboratory is derived from the main QTL that controls seed storage stability in Kasalath.In this study,the storage-resistant SL36(chromosome segment substitution lines)that carrys qSS-9Kas was used as donor parent and "Ningjing 4" was used as recurrent parent by MAS(Molecular Marker-Assisted Breeding).through many years of continuous hybridization,backcrossing and self-crossing,stable improved families were obtained.Genotype analysis showed that qSS-9Kas was successfully transferred into the improved line and could be inherited stably.Malondialdehyde(MAD)content detection showed that the MAD content in the improved seed after aging was significantly lower than that in the wild type;phenotypic identification found that after many years of storage and accelerated aging,the seed vigor of the improved lines was higher than that of the wild type;in the TTC staining,more seeds were stained in the improved lines and the TTCH content was significantly higher than that of the Ningjing 4;the determination of agronomic traits showed that most of the main agronomic traits and apparent amylose content were not significantly different from Ningjing 4;phenotypic identification found that after many years of storage and accelerated aging,the seed vigor of the improved lines was higher than that of the Ningjing 4;in the TTC staining,more seeds were stained in the improved lines and the TTCH content was significantly higher than that of the Ningjing 4;the determination of agronomic traits showed that most of the main agronomic traits and apparent amylose content were not significantly different from Ningjing 4. |
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