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%.

Previously, we have realized the CRISPR/Cas9-RNP and plasmid mediated protoplast transient transformation genome editing in the rubber tree ( Hevea brasiliensis ), but no gene editing plants were acquired due to the bottleneck of genetic transformation. In present study, antibiotic sensitivity tests against kanamycin, hygromycin and basta were analyzed for embryo screening, the results demonstrated that 10 mg/L hygromycin is the best for transformation. Then Agrobacterium mediated transformation of H. brasiliensis embryos was carried out using a pCAMBIA1300-based CRISPR/Cas9 vector targeting Phytoene desaturase gene ( HbPDS ). High-throughput sequencing of T0 generation positive embryos which were used as regeneration materials in typical transformation procedure showed that more than 90% T0 edited embryos are chimeric with a 3.2% editing efficiency. A T0 embryo with 9.8% edited cells was sliced into small pieces for one more cycle embryogenesis to produce T1 generation embryos in order to improve the ratio of homozygous embryos. Subsequently, next-generation sequencing (NGS) demonstrated that 29 out of 33 T1 embryos were edited, nearly 50% of which were found homozygous. At last, besides four chimeric plantlets with partial albino leaves, four plantlets with complete albino phenotype were regenerated from the 29 T1 generation edited embryos, among which one is a homozygous mono-allelic mutant and the other three are homozygous bi-allelic mutants. NGS demonstrated that the threshold for the proportion of edited cells with expected albino phenotype is between 70-85%. Additionally, Tail-PCR indicate that the T-DNA was inserted into different genome positions in the four homozygous edited plantlets, combined with the different genotypes are considered, the four homozygous plantlets can be confirmed as independently derived from single transformed cells. Overall, this is the first edited rubber trees with expected phenotype reported publicly, which shows the potential in genetic improvement of H. brasiliensis by CRISPR/Cas9 gene editing, and subculture of T0 positive transformed somatic embryos into T1 generation is proved to be an effective and necessary procedure to produce homozygous transgenic plantlets. This study presents a significant advancement in transgenic and gene editing for rubber tree.

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.