Anther-derived somatic embryogenesis is a valuable approach in rubber tree (Hevea brasiliensis) breeding; however, its effectiveness is highly influenced by the developmental stage of the microspores. The present investigation focused on male flower buds of the cultivar Reyan 73397 at successive developmental stages to examine the relationship between visible bud characteristics and internal microspore development, assess how microspore developmental stage affects callus induction and somatic embryo formation, and identify the stage with the greatest embryogenic potential. Cytological observations distinguished six well-defined phases of microspore development, spanning from microspore mother cells to fully mature pollen grains, each reliably linked to particular bud diameters, coloration, and anther morphology. Anthers corresponding to each developmental phase were cultured in vitro, and their ability to initiate callus and produce somatic embryos was systematically evaluated. Anthers containing uninucleate microspores exhibited the highest rates of both callus formation and somatic embryogenesis, with the early-uninucleate stage showing the strongest response. This stage consistently matched flower buds measuring 1.42–1.57 mm in transverse diameter and displaying a green to yellowish-green appearance. In contrast, anthers collected at the microspore mother cell and tetrad stages did not produce embryogenic responses. Histological evidence has indicated that both callus and somatic embryos originate from diploid somatic tissues of the anther wall, particularly connective parenchyma cells, rather than from microspores themselves. Based on these findings, a rapid, non-destructive selection method integrating bud diameter, bud color, and sieve-based size separation was developed to identify responsive explants efficiently. Overall, this study defines the optimal developmental window for anther culture in rubber trees, verifies the somatic origin of embryogenic tissues, and provides a practical morphological and cytological basis for improving anther culture efficiency in rubber tree breeding programs.

The rubber tree (Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg.) is the predominant global source of natural rubber; however, its cultivation is largely confined to tropical regions due to chilling sensitivity. Although the chilling-tolerant clone GT1 holds considerable promise for expanding cultivation latitudes and serving as a genetic transformation recipient, its utility is severely compromised by a recalcitrant somatic embryogenesis (SE) capacity relative to the elite clone Reyan 7-33-97 (R73397). Despite prior media optimization efforts, an efficient callus subculture system for GT1 remains elusive, and the molecular basis of its SE recalcitrance is poorly characterized. Here, we establish a repeated GT1 callus subculture system using a pulsed plant growth regulators regimen and integrated comparative transcriptomics, whole-genome bisulfite sequencing (WGBS), and Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) with Tn5 footprinting to elucidate the regulatory bottlenecks distinguishing GT1 from R73397. Our results suggest that, although the GT1 callus has not completely lost the expression of genes that characterize the tendency to develop into embryos, it does not complete the morphogenetic transition into globular embryos, possibly due to the reduced levels of transcription factors critical for embryo patterning. The epigenetic analyses indicate that the differential expression of these regulators is not tightly coupled with DNA methylation or chromatin accessibility, but reveal distinct transposable element silencing mechanisms between the two genotypes. Furthermore, construction of a hierarchical TF-DNA binding network predicts the positions of several transcription factors, which have previously been reported to positively regulate somatic embryogenesis within the gene regulatory network. Collectively, this work overcomes the technical bottleneck associated with multiple subcultures of GT1 callus tissue and provides a multi-omics framework for identifying the precise genetic targets to facilitate SE in chilling-tolerant rubber tree breeding.