Optimized Callus Subculture and Multi-Omics Analysis Reveal Somatic Embryogenesis Bottlenecks in the Cold-Resistant Rubber Tree Clone GT1

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.