Background: The rice Waxy (Wx) gene plays a major role in seed amylose synthesis and consequently controls grain amylose content. Wx gene expression is highly regulated at the post-transcriptional level. In particular, the GT/TT polymorphism at the 5′splicing site of its 1st intron greatly affects this intron’s splicing efficiency and defines two predominant Wx alleles, Wx^a and Wx^b. Wx^a rice often harbours intermediate to high amylose contents, whereas Wx^b rice exhibits low to intermediate amylose contents. By deleting the Wx 1st intron using CRISPR/Cas9 technology, we generate a completely novel Wx allele and further investigate how intron removal affects Wx gene expression and rice grain amylose content. Results: CRISPR/Cas9-mediated targeted deletion of the Wx 1st intron was performed on 4 rice inbred lines: KY131 (Wx^b), X32 (Wx^b), X35 (Wx^a) and X55 (Wx^lv). Deletion of the 1st intron occurred in 8.6–11.8% of the primary transformants of these 4 inbred lines. Compared to wild-type plants, amylose content was significantly increased from 13.0% to approximately 24.0% in KY131 and X32 mutant lines, which both carried the Wx^b allele. However, no significant difference in amylose content was observed between wild-type plants and X35 and X55 mutant lines, which carried the Wx^a and Wx^lv alleles, respectively. Wx gene expression analysis of wild-type plants and mutants yielded results that were highly consistent with amylose content results. KY131 and X32 mutants accumulated increased levels of steady mRNA transcripts compared with wild-type plants, whereas steady mRNA levels were not altered in X35 and X55 mutants compared with wild-type plants. Grain quality, including appearance quality and eating and cooking quality, which are tightly associated with amylose content, was also assessed in wild-type and mutant plants, and data were presented and analysed. Conclusions: This study presents a novel and rapid strategy to increase amylose content in inbred rice carrying a Wx^b allele. Our data strongly suggest that the 1st intron of the Wx gene regulates Wx gene expression mainly at the post-transcriptional level in rice. This finding is in contrast to a previous hypothesis suggesting that it influences Wx gene transcription. In addition, removal of the first intron generates a completely novel Wx allele. Further studies on this new Wx allele will provide invaluable insights into the regulation of Wx gene expression, which will help researchers engineer new Wx alleles to facilitate the breeding of rice cultivars with better eating and cooking quality.

Rice (Oryza sativa) is one of the world’s major staple foods. However, stable rice production is constrained by various biotic and abiotic and stresses. Breeding and cultivation of rice varieties with resistance to multiple pathogens and environmental stresses is the most effective strategy to mitigate the adverse effect of pathogen attacks and abiotic stresses. Recently, researchers have focused on the exploitation of CRISPR/Cas9 technology to manipulate some negative defense-regulator genes to generate rice varieties with broad-spectrum resistance against rice pathogens. In this study, four negative regulator genes of rice blast, OsMads26, OsBsr-1, OsELF3-2 and OsERF922, were selected as CRISPR/Cas9 targets. By simultaneously knocking out all four genes via CRISPR/Cas9 technology, we created three mads26/bsr-1/elf3-2/erf922 quadruple knockout mutants. Our results demonstrated that all quadruple mutants exhibited much higher resistance not only to rice blast and bacterial blight but also to drought and salt stresses than the wildtype. Interestingly, grain yield of all three quadruple mutants was also drastically increased by 17.35% to 21.95%. Therefore, this study provides a novel strategy to rapidly improve rice varieties with broad-spectrum resistance to pathogens, elevated tolerance to abiotic stresses and enhanced yield potential.

Rice blast and bacterial blight cause severe harm to rice production, and the breeding of resistant varieties guarantees the safety of rice production. Meanwhile, multigene pyramiding breeding based on molecular marker-assisted selection is a crucial approach for rice breeding to combat multiple diseases. This study aimed to develop accurate and efficient PARMS markers for rice blast resistance genes Pita, Pigm, and Pi2, and bacterial blight resistance gene Xa23. A systematic genotyping analysis of the resistant alleles of these 4 genes was performed on 384 major cultivated varieties in production. The results showed that only 5.21% of the varieties harbored more than two resistant alleles simultaneously. Using traditional breeding strategies in combination with the developed PARMS markers, the high-quality three-line male sterile line Ruanfeng A (pyramiding Pita and Pigm) and the strong restorer line Gui 610 (pyramiding Pi2 and Xa23) were bred. Crossing these lines produced a new hybrid rice variety, Ruanfengyou 610. Ruanfengyou 610 pyramids 4 resistance genes (Pita/Pigm/Pi2/Xa23), exhibits resistance to both rice blast and bacterial blight, has prominent heterosis and excellent grain quality, and has strong application potential, which is of great significance for ensuring the safety of rice production.

Black rice contains substantial levels of anthocyanins, a class of bioactive compounds recognized for their health-promoting properties that constitute an important determinant of nutritional and functional quality. In this study, we cloned rice TRANSPARENT TESTA 2 (OsTT2), a regulatory gene for anthocyanin biosynthesis that is located on chromosome 3 with a 331 bp segmental duplication in its promoter region. OsTT2 encodes an R2R3-MYB transcription factor that localizes to the nucleus. Interestingly, OsTT2 significantly influences plant height, panicle length, total grain number per plant, grain length, and grain width. Although no significant differences were detected in the number of panicles per plant or 1000-grain weight, the presence of a functional OsTT2 gene substantially increases overall rice yield. We found that OsTT2 binds to the promoter region of OsMED15a, which activates the OsMED15a–OsNAC024 pathway, thereby modulating grain length and width. Our findings provide new resources and novel approaches for the genetic improvement of rice anthocyanin biosynthesis, quality, and yield.

Shoot regeneration is an important prerequisite for many plant transformation and genome-editing protocols, but responses can vary among commercial cultivars. We screened 43 Luffa cultivars, recorded as 21 ridged and 22 smooth types, on four MS-based media using seedling-derived cotyledon and cotyledonary-axis explants. At a common six-week endpoint, 10,433 of 10,517 explants formed callus (99.20%), 2074 showed browning (19.72%), and 353 produced a recognizable small shoot bud and/or young leaves (3.36%; descriptive 95% Wilson CI: 3.03–3.72%). Twenty-five cultivars were shoot-positive on at least one medium, whereas 18 were negative on all four. Cuihua2212 had the highest pooled response (60/250 (24.0%)). A cultivar-clustered, denominator-weighted binomial-family generalized estimating equation gave a global medium Wald χ²(3) of 7.48 (p = 0.058). In six exploratory pairwise comparisons, C exceeded D (OR: 2.51, 95% CI: 1.30–4.88; BH-adjusted q = 0.039). The medium-adjusted ridged/smooth OR was 2.54 (95% CI: 1.04–6.17), with substantial uncertainty and sensitivity to cultivar composition. Frequent callus formation, therefore, coincided with infrequent visible shoot recovery under the conditions tested. The screen identifies candidates for independent regeneration optimization; it does not establish a callus-derived shoot pathway, species-wide differences or transformation competence.