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IntroductionTwo-line hybrid rice based on Photoperiod/thermo-sensitive genic male sterile (P/TGMS) lines has been developed and applied widely in agriculture due to the freedom in making hybrid combinations, less difficulty in breeding sterile lines, and simpler procedures for breeding and producing hybrid seed. However, there are certain risks associated with hybrid seed production; if the temperature during the P/TGMS fertility-sensitive period is lower than the critical temperature, seed production will fail due to self-pollination. In a previous study, we found that the issue of insufficient purity of two-line hybrid rice seed could be initially addressed by using the difference in tolerance to β-triketone herbicides (bTHs) between the female parent and the hybrid seeds.MethodsIn this study, we further investigated the types of applicable herbicides, application methods, application time, and the effects on physiological and biochemical indexes and yield in rice.ResultsThe results showed that this method could be used for hybrid purification by soaking seeds and spraying plants with the bTH benzobicylon (BBC) at safe concentrations in the range of 37.5-112.5 mg/L, and the seeds could be soaked in BBC at a treatment rate of 75.0 mg/L for 36-55 h without significant negative effects. The safe concentration for spraying in the field is 50.0-400.0 mg/L BBC at the three-leaf stage. Unlike BBC, Mesotrione (MST) can only be sprayed to achieve hybrid purification at concentrations between 10.0 and 70.0 mg/L without affecting yield. The three methods of hybrid seed purification can reach 100% efficiency without compromising the nutritional growth and yield of hybrid rice. Moreover, transcriptome sequencing revealed that 299 up-regulated significant differentially expressed genes (DEGs) in the resistant material (Huazhan) poisoned by BBC, were mainly enriched in phenylalanine metabolism and phenylpropanoid biosynthesis pathway, it may eliminate the toxic effects of herbicides through this way.DiscussionOur study establishes a foundation for the application of the bTH seed purification strategy and the three methods provide an effective mechanism for improving the purity of two-line hybrid rice seeds.

Remobilization of pre-anthesis NSCs (non-structural carbohydrates) is significant for effective grain filling in rice (Oryza sativa L.). However, abundant starch particles as an important component of NSCs are still present in the leaf sheath and stem at the late stage of grain filling. There are no studies on how bioengineering techniques can be used to improve the efficiency of NSC remobilization. In this study, RAmy1A was expressed under the senescence-specific promoter of SAG12, which was designed to degrade starch in the leaf sheath and stem during grain filling. RAmy1A mRNA successfully accumulated in the leaf, stem, and sheath of transgenic plants after anthesis. At the same time, the starch and total soluble sugar content in the leaf, stem, and leaf sheath were obviously decreased during the grain-filling period. The photosynthetic rate of transgenic lines was higher than that of the wild types by an average of 4.0 and 9.9%, at 5 and 10 days after flowering, respectively. In addition, the grain-filling rate of transgenic lines was faster than that of the wild types by an average of 26.09%. These results indicate an enhanced transport efficiency of NSCs from source tissues in transgenic rice. Transgenic rice also displayed accelerated leaf senescence, which was hypothesized to contribute to decreased grain weight.

Genetic diversities or favorable genes within distantly related species are the important resources for crop genetic improvement and germplasm innovation. Spike-Stalk injection method (SSI) has long been applied in rice genetic improvement by directly introducing genetic materials from non-mating donor species, while its inheritance patterns and the underlying mechanisms are poorly elucidated. In this study, a rice variant ERV1 with improved yield-related traits was screened out in the way of introducing genomic DNA of Oryza eichingeri (2n=24, CC genome) into RH78 (Oryza sativa L. 2n=24, AA genome) using SSI method. Genome-wide comparison revealed that the genomic heterozygosity of ERV1 was approximately 8-fold higher than RH78. Restriction-site associated DNA sequencing technology (RAD-seq) and association analysis of the ERV1 inbred F2 population identified 5 quantitative trait loci (QTLs) regions responsible for these yield-related traits, and found that genomic heterozygosity of ERV1 inbred lines was significantly lower than ERV1, while spontaneous mutation rate of the ERV1 inbred lines was significantly higher than ERV1. Our results preliminarily uncovered the inheritance patterns of SSI variant rice, and the potential genomic regions for traits changes, which yielded novel insights into the mechanisms of SSI method, and may accelerate our understanding of plant genome evolution, domestication, and speciation in nature.