In hybrid plants, heterosis often produces large, vigorous plants with high yields; however, hybrid seeds are generated by costly and laborious crosses of inbred parents. Apomixis, in which a plant produces a clone of itself via asexual reproduction through seeds, may produce another revolution in plant biology. Recently, synthetic apomixis enabled clonal reproduction of F₁ hybrids through seeds in rice (Oryza sativa), but the inheritance of the synthetic apomixis trait and superior heterotic phenotypes across generations remained unclear. Here, we propagated clonal plants to the T₄ generation and investigated their genetic and molecular stability at each generation. By analyzing agronomic traits, as well as the genome, methylome, transcriptome, and allele-specific transcriptome, we showed that the descendant clonal plants remained stable. Unexpectedly, in addition to normal clonal seeds, the plants also produced a few aneuploids that had eliminated large genomic segments in each generation. Despite the identification of rare aneuploids, the observation that the synthetic apomixis trait is stably transmitted through multiple generations helps confirm the feasibility of using apomixis in the future.

Cadmium (Cd) is a heavy metal that poses harm to both plants and humans. OsHMA3, a member of the heavy metal ATPase (HMA) family, plays a crucial role in sequestering Cd into the vacuoles of roots, thereby limiting its accumulation in rice grains. However, the response of rice plants to Cd under complete loss-of-function of OsHMA3 remains unclear. In this study, we successfully generated OsHMA3 null mutants in an indica variety 93 − 11 using CRISPR/Cas9 technology. A Cd resistance experiment revealed that the Oshma3 mutants exhibited increased sensitivity to Cd compared to the wild-type at a tested concentration of 10 µM CdCl₂. Furthermore, the seedlings of Oshma3 mutant lines displayed inhibited plant growth in the presence of 1 µM Cd, specifically suppressing aboveground growth. As expected, knockout lines of OsHMA3 showed lower Cd accumulation in roots but higher concentrations in shoots compared to wild-type plants, highlighting the role of OsHMA3 in root-to-shoot Cd translocation. We further performed RNA sequencing analysis on wild-type and Oshma3 plants under control and Cd treatment conditions and found that differentially expressed genes were mainly enriched in metal ion binding, integral component of the membranes, and biosynthesis pathways for secondary metabolites triggered by exposure to Cd. When grown in a paddy field, the Oshma3 mutants exhibited shorter plant height, lower seed setting rate, and higher Cd accumulation in grains compared to wild-type plants. Our results indicate that knockout of OsHMA3 in the 93 − 11 variety increases sensitivity to Cd and inhibits plant growth.

In vivo doubled haploid (DH) production based on crossing heterozygous germplasm with mtl haploid inducer lines promises to transform modern rice (Oryza sativa) breeding. However, this technology is limited, as haploid inducers and pollen acceptors have asynchronous heading dates. To address this obstacle, we developed a panel of multiple-heading-date mtl haploid inducer lines that produce pollen for more than 35 days. We edited the MTL gene in a hybrid rice with the CRISPR-Cas9 system. We then selected transgene-free homozygous mutants in the T₁ generation and reproduced to T₄ generation by single-seed descent method. We obtained 547 mtl haploid inducers with diverse heading dates (from 73 to 110 days) and selected 16 lines comprising a core population with continuous flowering. The seed-setting rate and haploid induction rate (HIR) of the core panel were 4.0–12.7% and 2.8–12.0%, respectively. Thus, our strategy of using multiple-heading-date mtl haploid inducers could accelerate the use of in vivo DH technology in rice breeding.

Background/Objectives: BABY BOOM (BBM), a transcription factor from the APETALA2 (AP2) protein family, plays a critical role in somatic embryo induction and apomixis. BBM has now been widely applied to induce apomixis or enhance plant transformation and regeneration efficiency through overexpression or ectopic expression. However, the structural and functional evolutionary history of BBM genes in plants is still not well understood. Methods: The protein sequences of 10 selected plant species were used to locate the branch of BBM-Like by key domain identification and phylogenetic tree construction. The identified BBML genes were used for further conserved motif identification, gene structural analysis, miRNA binding site prediction, cis-acting element prediction, collinear analysis, protein–protein interaction network construction, three-dimensional structure modeling, molecular docking, and expression pattern analysis. Results: A total of 24 BBML proteins were identified from 10 representative plant species. Phylogenetic relationship analysis displayed that BBML proteins from eudicots and monocots were divided into two clusters, with monocots exhibiting a higher number of BBMLs. Gene duplication events indicated that whole genome/segmental duplication were the primary drivers of BBML genes’ evolution in the tested species, with purifying selection playing a key role during evolution processes. Comparative analysis of motif, domains, and gene structures revealed that most BBMLs were highly evolutionarily conserved. The expression patterns of BBML genes revealed significant tissue specificity, particularly in the root and embryo. We also constructed protein–protein interaction networks and molecular docking models to identify functional pathways and key amino acid residues of BBML proteins. The functions of BBMLs may differ between monocots and eudicots, as suggested by the functional enrichment of interacting proteins. Conclusions: Our research delved into the molecular mechanism, evolutionary relationships, functional differentiation, and expression patterns of BBML genes across plants, laying the groundwork for further investigations into the molecular properties and biological roles of BBMLs.

Production of homozygous inbred lines is crucial in rice breeding programs. Doubled haploid (DH) technology enables the efficient development of pure lines and significantly shortens the breeding cycle compared to conventional methods. This review summarizes recent advances in the four key steps of DH technology mediated by haploid inducers in rice: (1) exploration of haploid induction genes and development of high-efficiency haploid inducers, (2) rapid identification of haploids using molecular and morphological markers, (3) chromosome doubling of haploid plants through colchicine treatment, and (4) application of DH technology in rice breeding. Furthermore, current challenges and issues associated with each step are discussed. With ongoing advancements and the resolution of existing limitations, DH technology based on haploid inducers is anticipated to become a fundamental and widely adopted tool in rice breeding.