RESUMO
Introduction: MicroRNAs (miRNAs) are small endogenous non-coding RNAs that play an important role in wood formation in plants. However, the significance of the link between miRNAs and their target transcripts in wood formation remains unclear in rubber tree (Hevea brasiliensis). Methods: In this study, we induced the formation of reaction wood by artificially bending rubber trees for 300 days and performed small RNA sequencing and transcriptome deep sequencing (RNA-seq) to describe the complement of miRNAs and their targets contributing to this process. Results and discussion: We identified 5, 11, and 2 differentially abundant miRNAs in normal wood (NW) compared to tension wood (TW), in NW relative to opposite wood (OW), and between TW and OW, respectively. We also identified 12 novel miRNAs and 39 potential miRNA-mRNA pairs with different accumulation patterns in NW, TW, and OW. We noticed that many miRNAs targeted transcription factor genes, which were enriched in KEGG pathways associated with phenylpropanoid biosynthesis, phenylalanine metabolism, and pyruvate metabolism. Thus, miRNA-TF-mRNA network involved in wood formation via tension wood model were constructed. We validated the differential accumulation of miRNAs and their targets by RT-qPCR analysis and overexpressed miRNA in Nicotiana benthamiana with its potential target gene. These results will provide a reference for a deep exploration of growth and development in rubber tree.
RESUMO
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%.