The recurring growth of bacterium in newly developed resistant cells and a minimal level of bacterial infection rate are the main limiting factors of Agrobacterium-mediated transformation experiments in Hevea brasiliensis. The current study aimed to optimize crucial factors of the transformation protocol in order to obtain an efficient transformation experimental model for Hevea using cotyledonary somatic embryos as explants. Transformation conditions such as antibiotic concentration, preculture duration, Agrobacterium concentration, sonication and cocultivation conditions were analyzed using the binary vector pCAMBIA2301. Transient transformation was confirmed by GUS histochemical staining. The best transformation efficiency was observed when the explants were not cultured on a preculture medium that contained acetosyringone at a level of 100 μM. The best results were obtained using a bacterial density of 0.45 at OD 600 nm, 50 s of sonication of explants in a bacterial liquid culture and a total incubation time of 18 min in the same bacterial suspension. Transmission electron microscopical analysis confirmed the impacts of sonication on bacterial infection efficiency. Cocultivation conditions of 22 °C and 84 h of darkness were optimal for the transfer of T-DNA. Agrobacterium was eliminated with 500 mg/L of timentin, and the selection of transformants was performed using 100 mg/L of kanamycin in the selection medium. The presence of transgene was confirmed in the resistant embryos by polymerase chain reaction (PCR). The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest into the Hevea genome for the breeding of this economically important plant species in the future.

The rubber tree (Hevea brasiliensis) is the most important commercial plant for producing natural rubber. Immature seed inner integument and anther-derived somatic embryogenesis techniques play a crucial role in the in vitro large-scale propagation and genetic transformation of the rubber tree. However, somatic embryogenesis is highly genotype-dependent, that is, only a limited number of H. brasiliensis genotypes, such as CATAS73397, CATAS917, and PB260, can be efficiently induced by somatic embryogenesis and used for large-scale propagation or transformation. The genotype dependence of the somatic embryogenesis technique is a conundrum for the application of Hevea biotechnology in most commercially important cultivars, such as Reken628 and CATAS879. Previous studies have shown that several somatic embryogenesis regulators can overcome genotype dependence and enhance the transformation and regeneration efficiency of recalcitrant plants and cultivars. In this review, we first describe the relevant successful applications of somatic embryogenesis technology in seedling production and genetic modification of H. brasiliensis. Second, we discuss the genotype dependence of somatic embryogenesis as the major challenge currently. Third, we summarize the recent significant advances in the understanding of the molecular mechanisms underlying somatic embryogenesis in other plants. Finally, we suggest a roadmap for using somatic embryogenesis regulatory genes to facilitate genotype-dependent somatic embryogenesis technology in H. brasiliensis.

To overcome rubber tree (RT) tissue culture explant source limitations, the current study aimed to establish a new Hevea brasiliensis somatic embryogenesis (SE) system, laying the technical foundation for the establishment of an axillary-bud-based seedling regeneration system. In this study, in vitro plantlets of Hevea brasiliensis Chinese Academy of Tropical Agricultural Sciences 917 (CATAS 917) were used as the experimental materials. Firstly, the optimum conditions for axillary bud swelling were studied; then, the effects of phenology, the swelling time of axillary buds (ABs), and medium of embryogenic callus induction were studied. Plantlets were obtained through somatic embryogenesis. Flow cytometry, inter-simple sequence repeat (ISSR molecular marker) and chromosome karyotype analysis were used to study the genetic stability of regenerated plants along with budding seedlings (BSs) and secondary somatic embryo seedlings (SSESs) as the control. The results show that the rubber tree’s phenology period was mature, and the axillary bud induction rate was the highest in the 2 mg/L 6-benzyladenine (6-BA) medium (up to 85.83%). Later, 3-day-old swelling axillary buds were used as explants for callogenesis and somatic embryogenesis. The callus induction rate was optimum in MH (Medium in Hevea) + 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) + 1.5 mg/L 1-naphthalene acetic acid (NAA) + 1.5 mg/L Kinetin (KT) + 70 g/L sucrose (56.55%). The regenerated plants were obtained after the 175-day culture of explants through callus induction, embryogenic callus induction, somatic embryo development, and plant regeneration. Compared with the secondary somatic embryo seedling control, axillary bud regeneration plants (ABRPs) were normal diploid plants at the cellular and molecular level, with a variation rate of 7.74%.

Cold stress limits the growth and productivity of Hevea brasiliensis, the primary source of natural rubber. This study investigated early transcriptomic responses in Reyan ‘7-33-97’ seedlings exposed to 4 °C, 10 °C, and 15 °C for 1, 2, and 4 h with room temperature (25 °C) as the control. RNA sequencing identified 9894 differentially expressed genes (DEGs), with the most significant transcriptional changes observed at 10 °C, indicating that genes to resist cold stress could not be mobilized at 4 °C, resulting in poor cold resistance of the rubber tree. KEGG enrichment analysis of DEGs between 10 °C (2 h) and 4 °C (2 h) revealed that genes involved in tryptophan metabolism (HbKynL.x1, HbKynL, HbCLP1, HbCLP2) and carbon metabolism (TCH4, XTH23), which contribute to cell wall modification, exhibited higher expression at 10 °C. Gene Ontology enrichment analysis highlighted significant involvement of “thylakoid,” “photosystems,” and “photosynthetic membrane,” alongside molecular functions such as “xyloglucan transferase activity” and “transcriptional regulator activity.” The interacting network of key pathways, including carbon metabolism (ko01200) and carbon fixation (ko00710) pathways, was sorted out, highlighting their integration with plant hormone signal transduction. Complex signaling networks, including MAPK, and kynurenine pathways coordinate the expression of cold-responsive genes and protective proteins, and it was confirmed and speculated that there is crosstalk response in cold defense mechanisms. Furthermore, 61 DEGs were associated with antioxidant processes, including major catalase and peroxidase enzymes. Our study shows that rubber trees physiological activities that respond to low-temperature signals cannot be carried out normally at 4 °C. The newly discovered metabolic pathway and the reason for abnormal cold signal transduction at low temperatures are the focus of future research on cold resistance.

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.