Abstract: Calcium (Ca 2+ ) is a universal signaling cation with a prominent role as second messenger in many different plant processes, including sexual reproduction. However, there is much less knowledge about the involvement of Ca 2+ during in vitro embryogenesis processes. In this work we performed a study of Ca 2+ levels during the different stages of microspore embryogenesis in Brassica napus , with special attention to how Ca 2+ can influence the occurrence of different embryogenic structures with different embryogenic potential. We also performed a pharmacological study to modulate Ca 2+ homeostasis during different stages of the process, using a series of Ca 2+ -altering chemicals (BAPTA-AM, bepridil, chlorpromazine, cyclopiazonic acid, EGTA, inositol 1,4,5-trisphosphate, ionophore A23187, W-7). This study shows that Ca 2+ increase can be considered as an early marker of induction of microspore embryogenesis. Besides, Ca 2+ levels are highly dynamic during microspore embryogenesis, influencing the final embryo yield. Increase of either extracellular or intracellular Ca 2+ levels improves embryo yield without altering the proportion of highly embryogenic structures formed, which suggests that elevated Ca 2+ levels increase the amount of microspores reaching the minimum Ca 2+ threshold required for embryogenesis induction. Conversely, inhibition of Ca 2+ uptake or signaling results in reduced embryogenic response. This allows to modulate embryo yield within a functional range, with lower and upper Ca 2+ thresholds beyond which embryo yield is reduced. There seems to be a relationship between Ca 2+ levels and embryo differentiation.

Abstract: Background Michelia alba DC. (Magnoliaceae), economically significant tree species in Southeast Asia, extremely low natural fruit production due to reproductive failure. This study aims to characterize the anatomical causes of this phenomenon by investigating megasporogenesis, microsporogenesis, and gametophyte development. Results Anatomical analysis revealed that microspore tetrads were arranged tetrahedrally, isobilaterally, or in T-shapes and that mature pollen was bicellular. Ovules were anatropous, forming linearly arranged megaspore tetrads; only the chalazal megaspore developed into a functional Polygonum-type embryo sac (7 cells, 8 nuclei). Critical abnormalities were identified: 1. Male gametophyte failure (>90% abortion): Premature tapetum degeneration and abnormal anther wall contraction caused microspore adhesion and pollen collapse. 2. Female gametophyte failure (>90% arrest): Most embryo sacs are arrested before maturity lacking essential cells (egg cells, synergids). Only 2 of the 200 examined sections contained mature embryo sacs. Conclusions Concurrent defects in male and female gametophyte development critically impair gametophyte functionality, explaining the low fruit set in M. alba. This study provides the first cytological elucidation of reproductive failure in this species and establishes a foundation for the conservation and breeding of endangered Magnoliaceae.

Abstract: Abstract Key message Many plant cells can be induced to regenerate in vitro. We show that successful regeneration during microspore-derived embryo culture relies in part on the ability of embryogenic cells to resolve tissue culture-induced ER stress. Abstract During Brassica napus microspore embryogenesis, the immature male gametophyte is induced by a heat stress treatment to develop into a haploid embryo. Different multicellular embryogenic structures develop in response to heat stress, each with a different potential to complete embryo development. The underlying factors that determine the ability of these initially embryogenic structures to successfully complete embryo development are not known. We show that all embryogenic structures exhibit elements of endoplasmic reticulum (ER) stress, like ER expansion and protein-filled ER cisternae, but that the ER stress response is amplified in embryogenic structures with a low potential to complete embryo development. ER stress was amplified even further by treating heat-stressed cultures with trichostatin A, a histone deacetylase inhibitor epidrug that promotes embryogenic cell formation. Pharmacological treatment of microspore-derived embryo cultures with small molecule modulators of ER stress provided further evidence for the role of ER stress in microspore embryo development. Our results suggest that (1) the inability of certain embryogenic structures to resolve their ER stress responses restricts their ability to complete embryo development, and (2) histone deacetylation enhances microspore embryogenesis in B. napus , in part through its activity as an abiotic stress inducer.

Abstract: ABSTRACT Anthers consist of various specialised cell types and play a significant role in plant reproduction. Although the molecular mechanisms underlying anther development and regulation have been extensively studied, the single‐cell transcriptional landscape and dynamic regulation during anther development in M. sativa remain largely unexplored. In the present study, we constructed the first single‐cell transcriptome atlas of M. sativa anthers to provide a comprehensive view of cell type‐specific gene expression and epigenetic modifications. The reconstruction of the developmental trajectories of tapetum and microspores led to the identification of novel genes and elucidated the regulatory networks involved in tapetum formation. Our findings revealed rapid functional transitions in tapetum during the tetrad stage, including cell formation, specialisation, and programmed cell death (PCD). Additionally, we analysed the stages at which tapetal degradation and microspore shrinkage occurred in the sterile line. Overall, this study offers valuable insights into the molecular mechanisms underlying anther development in M. sativa . Specifically, MsKIN14P was identified as a key regulator of microtubules and the cytoskeleton during mitosis, and the transcription factors ERF3 and ERF025 were shown to influence anther development through the ethylene response pathways. These findings provide an essential theoretical foundation for the development of novel male‐sterile lines and enhance the breeding capabilities of M. sativa .

Abstract: A male sterile mutant, S201, was identified in Brassica napus. Genetic analysis revealed that the male sterility trait was controlled by a recessive nuclear gene, male sterility (MS), which was stably inherited. The results of microscopy showed that the main reason for male sterility was a defect in microspore development, resulting in the absence of typical exine and mature microspores. Bulked segregant analysis (BSA) and genotyping of an F2 population showed that the MS gene was located in a 1.4 Mb region. Sequence analysis showed that the CYP704B1 gene in this region contained two non-synonymous SNPs, leading to substitutions of two amino acids. A high-throughput KASP marker was characterized to detect the presence of the ms gene in the breeding population. The data presented here indicate that the male sterile mutant S201 can be applied in rapeseed breeding by producing the male sterile line and that the KASP marker developed for male sterility will be useful in marker-assisted selection of male sterile individuals in rapeseed-breeding programs.

Abstract: Abstract Live-imaging microscopy technology has been increasingly applied for meiosis study in plants, which largely relies on the set up of a healthy ex vivo culture system for inflorescences ensuring that the captured chromosomes dynamics approaches the natural features of meiosis. Here, we report that Arabidopsis thaliana flowers cultivated in a culture medium (CCM) composed of the half-strength Murashige and Skoog basal salt, MES, Myo-inositol, sucrose and agar produce diploid microspores due to occurrence of meiotic restitution. Cytological studies revealed adjacent nuclei distribution and incomplete cytokinesis at late meiosis II in meiocytes within the CCM flowers. Immunolocalization of α-tubulin and the microtubule-associated protein MAP65-3 showed that the orientation of spindles at metaphase II and the organization of radial microtubule arrays at the tetrad stage are interfered, which explains the production of meiotically-restituted microspores. Moreover, the CCM flowers showed a gradually impaired expression of Aborted Microspores (AMS), a key transcription factor regulating tapetum development and meiotic cytokinesis. Interestingly, an increased supply of sucrose in culture medium promoted the expression of AMS and partially rescued haploid microspore formation in the CCM flowers. Taken together, this study suggests a role of sucrose in facilitating meiotic cytokinesis and gametophytic ploidy stability in plants. One-sentence summary Arabidopsis flowers cultivated in culture medium produce unreduced microspores due to interfered meiotic cytokinesis, which is partially rescued by increased sucrose supply.

Abstract: This study elucidates the key molecular features underlying the embryogenic initiation divergence between japonica rice Chongxiangjing (CXJ) and indica rice 9311 during isolated microspore culture. Comparative transcriptome analysis across critical timepoints (0, 5, and 10 days post-culture initiation) revealed that while both varieties initially exhibit comparable microspore viability, CXJ maintains transcriptional stability and activates developmental programs (e.g., hormone signaling, DNA replication, cell morphogenesis), enabling sustained callus formation. In contrast, 9311 undergoes drastic transcriptome reorganization by 5 days, characterized by maladaptive activation of stress-response pathways (glutathione metabolism, MAPK signaling, ER stress) and futile metabolic reactivation (photosynthesis, starch degradation), culminating in near-total cell death and failed callus induction. Transcription factor dynamics further explain this divergence: CXJ specifically upregulates regulators coordinating development and stress resilience (NAC, ERF, HSF, GRAS, bZIP), while 9311 exhibits detrimental upregulation of FAR1 and B3, leading to catastrophic energy misallocation. These findings identify master transcriptional networks and stress-response pathways as pivotal indicators of embryogenic initiation efficiency, providing strategic targets for enhancing indica rice microspore culture technology.

Abstract: Haploid induction (HI) through stress-treated microspore culture has gained significant attention for over half a century, yet the molecular mechanism underlying microspore fate transition for androgenesis remains poorly understood. Here, we demonstrate that microspore-specific expression of BABY BOOM (BBM) is sufficient to induce microspore cell fate transition and in vivo androgenesis in both tobacco and rice, effectively bypassing the requirement for stress treatment. We further identify BBM-activated Androgenesis Regulator 1 (BAR1) as a novel downstream effector of BBM that promotes microspore reprogramming. Remarkably, both BBM and BAR1 can replace the role of stress treatment in reprogramming microspore development and triggering androgenesis. This study reveals a conserved regulatory module governing androgenesis, providing a transformative approach to overcome long-standing limitations and enable highly efficient in vivo HI across diverse crops.

Abstract: Chinese cabbage is a cross-pollinated crop with remarkable heterosis, and male-sterile line is an important mean to produce its hybrids. In this study, a male-sterile mutant msm7 was isolated from a Chinese cabbage DH line 'FT' by using EMS-mutagenesis. Compared with the wild-type 'FT', the anthers of mutant msm7 were completely aborted, accompanied by the defects in leaf and petal development. Genetic analysis showed that a single recessive nuclear gene controlled the sterile phenotype of mutant msm7. Cytological observation indicated that the anther abortion of mutant msm7 was caused by the degenarated microspores and premature degradation of tapetum. MutMap and KASP analyses identified that BraA01g038270.3 C, encoding the large subunit of ribonucleotide reductase (RNR1) which involved in the biosynthesis of dNTPs, was the candidate gene, named BrRNR1. Compared with the wild-type 'FT', a G-A mutation occurred on the 4th exon of the BrRNR1 gene, leading to the premature termination of encoded amino acid in mutant msm7. Expression analysis indicated that the BrRNR1 gene was ubiquitously expressed in all organs and was significantly decreased in flower bud and anther of mutant msm7 compared with the wild-type 'FT'. Subcellular localization revealed that BrRNR1 was an endoplasmic reticulum localization protein. Our study is the first to characterize the function of BrRNR1 gene associated with male sterility and lays a foundation for exploring the molecular mechanism of anther abortion caused by the mutation in BrRNR1 gene of Chinese cabbage.

Abstract: Brassicas are considered the third most important source of vegetable oil globally. With the escalating production of Brassica varieties, there is growing demand for high-yielding genotypes. Doubled haploid (DH) techniques have become very popular in various Brassica breeding programs. Such DH techniques can play a significant role in plant breeding by accelerating the production of homozygous lines and increasing selection efficiency. Among these methods, isolated microspore culture stands out as the most effective, facilitating the generation of a higher number of embryos compared to conventional methods of plant breeding. Different chemical compounds such as herbicides, brassinosteroids, and polyethylene glycol have an antimitotic effect and have been found to generate DH plants and improve microspore embryogenesis in Brassica species. Colchicine and trifluralin have proven to be efficient chromosome-doubling agents as well as important supplements that can increase the rate of embryogenesis. This review serves as a comprehensive summary and effectiveness evaluation of the latest research findings in the Brassica oil crops to help increase efficiency of the future research focusing on DH methods and application of antimitotic agents in the various oilseed species of the genus Brassica.

Abstract: INDETERMINATE DOMAIN containing proteins (IDD) are plant-specific transcriptional factors with a diverse range of roles in plants. Among the 15 IDD genes in rice, a staple food crop for the world, only about half have been functionally characterized. To elucidate the function of the remaining members, we created loss-of-function mutants using the CRISPR genome editing technique. Although no mutant exhibited obvious growth phenotypes, the Osidd6 mutant was completely sterile. By genetic crossing, we showed that both the male and female gametophytes were defective in the mutant. Histochemical staining and thin sectioning revealed that microspore development was compromised, likely due to a delay in tapetum degeneration. We also showed that meiosis was impaired in the mutant, resulting in defective megaspore development. Through a series of experiments, including transcriptome analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in situ hybridization, β-glucuronidase (GUS) staining with promoter-GUS transgenic plants, yeast one-hybrid method, a dual-visible reporter assay, and transcriptional activity assay, we demonstrated that OsIDD6 is expressed in all cell types in the male and female reproductive organs and that the OsIDD6 protein directly regulates genes potentially having a role in meiosis and tapetum development. Since reproductive development is directly related to crop yield, OsIDD6 could be an important target for genetic manipulation in rice breeding.

Abstract: Reproductive, male-enriched small RNAs are present in flowering plants and animals, yet their role in plants remains underexplored. We generated dicer-like 5 ( dcl5 ) mutants in durum wheat ( Triticum turgidum ssp. durum 2n = 4× = 28; AABB), revealing temperature-sensitive genic male sterility. Loss of DCL5 depleted premeiotic and meiotic 24-nt phasiRNA production, correlating with sterility under standard growth conditions and partial fertility recovery at higher temperatures. A single functional DCL5 allele restored complete fertility, presenting a promising alternative to current methods for hybrid production. We demonstrate that premeiotic 24-nt phasiRNA biogenesis is independent of miRNA-mediated cleavage and driven by a conserved motif initiating DCL5 activity. In the dcl5 mutant developing under sterility-inducing conditions, developmental defects are observed during pollen maturation, rather than at peak 24-nt phasiRNA accumulation in premeiotic and meiotic anthers. Although no visible morphological abnormalities were apparent during early development, single-cell RNA sequencing revealed that dcl5 mutant cells exhibit transcriptional profiles distinct from those of wild-type cells, when premeiotic 24-nt phasiRNAs are accumulating at the early developmental stage. Finally, the coexpression of Argonaute ( AGO1b , AGO4a, and AGO6 ) homeologs in 24-nt phasiRNA-producing cells identifies candidate effectors and suggests a role for 24-nt phasiRNAs in transcriptional gene silencing.

Abstract: Cannabis sativa L. is a dioecious species known to produce over 1600 chemical constituents, including more than 180 cannabinoids classified into 11 structural groups. These bioactive compounds are predominantly synthesised in the glandular trichomes of female inflorescences. However, sex determination in C. sativa is influenced by both genetic and environmental factors, often leading to the development of male flowers on female plants. This unintended fertilisation reduces cannabinoid yield and increases genetic heterogeneity and challenges in medical cannabis production. Haploid and doubled haploid (DH) technologies offer a promising solution by rapidly generating homozygous lines from gametophytic (e.g., unpollinated ovaries and ovules) or sporophytic tissues (e.g., anthers and microspores) via in vitro culture or chromosome reduction during hybridisation. In land plants, the life cycle alternates between a diploid sporophyte and a haploid gametophyte generation, both capable of mitotic division to form multicellular bodies. A single genome regulates this phase transition and encodes the molecular, genetic, and epigenetic mechanisms that precisely control the developmental processes unique to each generation. While the application of haploid technology in C. sativa remains limited, through recent progress in haploid induction (HI) and CRISPR-based genome editing, the direct modification of haploid gametes or embryos enables the creation of null homozygous lines following chromosome doubling, improving genetic uniformity. Understanding the molecular mechanisms of spontaneous chromosome doubling may further facilitate the development of elite cannabis genotypes. Ultimately, enhancing the efficiency of DH production and optimising genome editing approaches could significantly increase the speed of genetic improvement and cultivar development in Cannabis sativa.

Abstract: Abiotic stress can reprogram the gametophytic pathway; the mechanisms by which floral bud pre-treatment influences microspore embryogenesis initiation remain unclear. In this study, we use bisulfite sequencing, sRNA-seq, and RNA-seq to analyze the dynamic changes in rice microspores under different cold treatment durations. Our results showed that a 10-day cold treatment is essential for CXJ microspore embryogenesis initiation. DNA methylation levels showed a slight change at CG, CHG, and CHH sites under cold treatment. The number of both hyper- and hypomethylated DMRs increased over cold treatment, with more hypermethylated DMRs at 5 and 10 dpt. Hypermethylated DMRs were more frequently in the TSS region compared to hypomethylated DMRs. The proportion of 24 nt sRNAs increased upon cold stress, with more downregulated than upregulated sRNAs at 10 dpt. The number of DMR target DEGs increased from 5 to 10 dpt. Promoter hypomethylation at the CHH site was more frequently associated with DEGs. These outcomes suggested that the RdDM pathway participates in the initiation of rice ME. GO analysis indicated that DMR target DEGs at 10 dpt were enriched in responses to chemical stimuli, biological processes, and stress responses. An auxin-related gene, OsHOX28, was further identified. Its upregulation, potentially mediated by the RdDM pathway, may play a crucial role in the initiation of rice ME. This study provides more information on epigenetic mechanisms during rice ME.

Abstract: ABSTRACT Rapeseed ( Brassica napus L.) is sensitive to high‐temperature events, particularly during the reproductive stage, which significantly affects yield. Climate change is predicted to be associated with high temperatures lasting longer than a few hours or days, and the effect of long‐term temperature stress is still poorly documented. In the current study, we investigated the impact of long‐term high‐temperature stress (HTS) on floral buds. We revealed that long‐term HTS (7–14 days) severely affects floral bud development with distinct alterations of the sporophytic anther tissue, such as tapetum, epidermis, endothecium, and stomium, with dramatic consequences on pollen viability and stigma receptivity. Comparative transcriptome and metabolome profiling upon exposure to HTS, 25°C/33°C for 11 days and control 18°C/25°C, showed 8194 and 10,786 DEGs and 636 and 696 DAMs during microsporogenesis (before late microspore, S1) and microgametogenesis (after microspore, S2), respectively. The results highlighted that prolonged HTS triggered a cascade of regulatory processes, altering the normal function and expression of genes involved in transcription regulation, signal transduction, photosynthesis and photosystem, cellular organization, and primary and secondary metabolites processing, leading to disruption of anther development and impaired pollen and stigma fertility. As a response, plants synthesize fatty acids, store them in wax, and activate phenylpropanoid and flavonoid biosynthesis pathways and amino acid metabolism as adaptive mechanisms. Thus, enhancing these pathways could help plants withstand HTS. Further investigation of genes and metabolites involved in these pathways could pave the way to develop thermotolerance B. napus toward genetic improvement.

Abstract: ABSTRACT The pollen exine serves as a protective barrier and signaling interface essential for male fertility in flowering plants. Its precise patterning depends on coordinated interactions between microspores and tapetal cells. While the CLAVATA3/EMBRYO SURROUNDING REGION‐related 19 (CLE19) peptide has been identified as a microspore‐derived “brake” that restricts tapetal activity to maintain exine developmental homeostasis, how CLE19 integrates with hormonal signaling pathways remains poorly understood. Here, we demonstrate that CLE19 attenuates brassinosteroid (BR) signaling output by engaging a defined BSL–BIN2–BES1 signaling cascade. Through quantitative phosphoproteomic analysis, we identified that CLE19 affects the phosphorylation of multiple BR signaling components, including BSL‐type phosphatases BSL1/2/3, the GSK3‐like kinase BIN2, and the transcription factor BES1. We show that CLE19 is perceived by its receptor PXL1, which directly interacts with BSL‐type phosphatases to activate the GSK3‐like kinase BIN2, leading to phosphorylation of BES1 at serine residues S219 and S223. Functional analyses using phospho‐dead and phospho‐mimic BES1 variants confirm that CLE19‐dependent phosphorylation controls BES1 nuclear export and degradation, ultimately suppressing BR‐responsive transcriptional outputs required for pollen exine patterning. Together, our findings define a peptide–hormone signaling axis that regulates transcription factor activity through post‐translational regulation, providing mechanistic insight into how developmental robustness is maintained via intercellular signal integration in plant reproduction.

Abstract: Male sterility is an important trait in heterosis utilization and marigold (Tagetes erecta L.) breeding. Currently, most male-sterile lines used in production are derived from natural mutations. ABORTED MICROSPORES (AMS) is an important gene that regulates tapetum and microspore development. Therefore, the effect of AMS on fertility was studied. TeAMS was located in the nucleus and exhibited self-activation activity. TeAMS was highly expressed in the flower buds of T. erecta. The expression of this gene in fertile plants was higher than that in sterile plants, and the expression level gradually increased with the development of flower buds. The expression level of TeAMS was highest in the flower buds with a diameter of 1.2 cm at the floret differentiation stage, while the expression level was extremely low in the flower buds with a diameter of 1.6 cm. The expression trend of TeAMS in sterile plants was opposite to that in fertile plants. At the inflorescence primordium differentiation stage, flower buds with a diameter of 0.2 cm had the highest expression level, and the stem tip had the lowest expression level. In tobacco (Nicotiana tabacum L.), overexpression of the TeAMS gene resulted in shortened floral tubes, increased thousand-seed weight, a reduced flowering period, and decreased flower numbers. The pollen viability of transgenic tobacco was significantly lower than that of the wild type, and the pollen grains were smaller and showed irregular shapes. The pollen wall was dry and shrunk. Some pollen germinal furrows were distorted, and a few were almost invisible. Silencing TeAMS resulted in a longer flowering period in tobacco, reduced thousand-seed weight, and high pollen viability. Pollen morphology in silenced lines showed no significant differences compared to the wild-type and empty vector controls. Only a few pollen grains were smaller, shriveled, and shrunken. Therefore, the TeAMS gene plays an important role in regulating the fertility of marigolds. This study provides a theoretical foundation for breeding marigold male-sterile lines.

Abstract: (Uploaded by Plazi from the Biodiversity Heritage Library) No abstract provided.