In order to induce unreduced pollens, microsporogenesis and male flower bud (MFB) development were compared in rubber tree clone RRIM 600. We observed strong asynchronism in different MFBs in an inflorescence. Asynchronism of microsporogenesis in different microsporangiums from a MFB was also observed. The relationship between microsporogenesis and external morphology was examined, which was used to estimate microsporogenesis stages of MFBs. Unreduced pollen was successfully induced by high temperature exposure in this study, with the highest production ratio of about 20.17% at 44 °C. Our findings showed that diplotene to metaphase I may be the most effective stage for unreduced pollen induction, and 42–44 °C may be the suitable treatment temperature in rubber trees. Thus, microsporogenesis of MFBs has been elucidated in detail in the rubber tree clone RRIM 600 and will provide a reference for future breeding studies of rubber trees.

Catalase (CAT) is one of the key enzymes involved in antioxidant defense systems and mainly scavenges H₂O₂ and plays a vital role in plant growth, development, and various adverse stresses. To date, a systematic study of the CAT gene family in rubber tree has not been reported. In this study, five HbCAT gene family members were identified from the rubber tree genome, and these were mainly clustered into two subfamilies. Gene structure and motif analysis showed that exon-intron and motif patterns were conserved across different plant species. Sequence analysis revealed that HbCAT proteins contain one active catalytic site, one heme-ligand signature sequence, three conserved amino acid residues (His, Tyr, and Asn), and one peroxisome-targeting signal 1 (PTS1) sequence. Fragment duplication is a selection pressure for the evolution of the HbCAT family based on Ka/Ks values. Analysis of cis-acting elements in the promoters indicated that HbCAT gene expression might be regulated by abscisic acid (ABA), salicylic acid (SA), and MYB transcription factors; furthermore, these genes might be involved in plant growth, development, and abiotic stress responses. A tissue-specific expression analysis showed that HbCATs gradually increased with leaf development and were highly expressed in mature leaves. Gene expression profiling exhibited the differential expression of the HbCATs under cold, heat, drought, and NaCl stresses. Our results provide comprehensive information about the HbCAT gene family, laying the foundation for further research on its function in rubber tree.

Eight triploids were screened among offspring of the rubber tree clone GT1 × different clones by flow cytometry and chromosome counting. Twenty-five simple sequence repeat (SSR) markers were screened to identify the origin of 2n gametes, to determine the male parents of these triploids, and to evaluate the mechanism of 2n gamete formation using band configurations and microsatellite DNA allele counting peak ratios (MAC-PR). The results showed that 2n gametes originated from the maternal rubber tree clone GT1, contributing the extra genome copy present in the triploids. It was confirmed that GT1 is able to produce a 2n megagametophyte spontaneously. Many male parents were shown to provide pollen for formation of triploid rubber trees, including clones RRIC 103, Yunyan 277-5, and three other clones. The second division restitution (SDR) was likely the main mechanism involved in formation of megagametophytes in GT1, as the rate of maternal heterozygosity restitution (HR) of all eight triploids varied from 27.78% to 75.00%, with a mean of 51.46%, and all 25 markers varied from 0% to 100%, with a mean of 51.69%. Elucidation of the origin and formation of 2n gametes will help optimize further sexual hybridization of polyploid rubber trees.