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.

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.

The pericarp of Macadamia integrifolia represents a promising but underexplored source of functional flavonoids. To systematically elucidate their biosynthesis and enhance the industrial potential of this by-product, we conducted integrated transcriptomic and metabolomic profiling of pericarps across five developmental stages (50, 80, 110, 140, and 170 days after flowering). Our analysis reveals, for the first time, a distinct temporal shift in both gene expression and metabolite accumulation. Early stages were characterized by high expression of PAL, 4CL, CHS, and FLS, coupled with abundant flavonols and anthocyanins. In contrast, late stages exhibited upregulation of CHI and F3’5’H, redirecting the metabolic flux toward flavanones and isoflavones. This dynamic profile was closely associated with jasmonate and gibberellin signaling pathways and was likely regulated by key transcription factors (MYB, NAC, bHLH). These findings provide a multi-omics framework that elucidates the temporal flavonoid biosynthesis in macadamia pericarp, thereby laying the groundwork for its future industrial valorization.

Color serves as a crucial visual signal for attracting pollinating insects and directly affects the fruit set rate in woody crops. This study investigated the molecular mechanisms underlying flower color formation in macadamia. The results demonstrated that darker flower colors were associated with higher fruit set rates: the rates for purple, pink, pinkish-white, and white flowers were 2.78, 1.99, 1.35, and 1.31, respectively. High-throughput sequencing identified 1359 differentially accumulated metabolites, including benzoic acid, 4-hydroxybenzaldehyde, and isorhamnetin. Transcriptional regulators such as ERF, MYB, and WRKY were significantly up-regulated in darker flowers. KEGG analysis revealed two key metabolic pathways, in which genes including HCT (shikimate hydroxycinnamoyl transferase) and F3GalTase (flavonol 3-O-galactosyltransferase), as well as related metabolites such as p-coumaric acid, chlorogenic acid, and myricetin, showed higher expression levels in darker flowers. Anthocyanin content was highest in pink and pinkish-purple varieties (462.79 and 446.35 μg/g, respectively), and lower in white and light pink varieties (140.52 and 167.97 μg/g). In conclusion, flower color intensity is positively correlated with both fruit set rate and anthocyanin content. Genes involved in the flavonoid and phenylpropanoid pathways, along with transcription factors such as WRKY and MYB, collectively regulate flower color formation. This study provides a theoretical basis for macadamia flower color breeding.

Palmitoleic acid (C16:1) is rare among woody oil crops yet accumulates to high levels in macadamia kernels, where its inter-accession variation and relationship with fruit traits remain poorly characterized. We profiled 22 fatty acids by GC-MS, quantified crude fat, crude protein and total soluble sugar by NIRS, and measured nine fruit traits in 139 accessions. The major fatty acids ranked as C18:1 > C16:1 > C16:0. C16:1 varied widely among accessions (mean 10.66%, CV = 27.7%, range 2.78–19.05%), about 12-fold the analytical RSD, whereas C18:1 was stable (CV = 5.3%). C16:1 was strongly negatively correlated with C18:1 (r = −0.921, p < 0.001) and positively correlated with C16:0 (r = 0.534). Correlation, partial correlation and PCA consistently indicated that fatty acid composition was independent of fruit traits; after controlling for C18:1, C16:1 retained only a weak positive association with single dry kernel weight (r = 0.170, p < 0.05). Entropy-weighted TOPSIS with five indicators selected five accessions combining high C16:1 with desirable fruit traits. These accessions provide breeding material for C16:1-enriched oil, and the independence of C16:1 from fruit traits means that both can be improved simultaneously.

To characterize the transcriptomic dynamics underlying shell hardening in Macadamia integrifolia, RNA sequencing was performed on pericarp and shell tissues collected at three key developmental stages. A total of 18 libraries were generated, yielding 125.19 Gb of clean data. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that husk-associated DEGs were mainly enriched in photosynthesis-related functions, whereas shell-associated DEGs were enriched in phenylpropanoid biosynthesis, secondary metabolism, and cell wall organization. Weighted gene co-expression network analysis (WGCNA) identified module M8 as being strongly associated with the middle and late stages of shell development. Genes in this module were mainly enriched in phenylpropanoid metabolism, redox-related processes, and extracellular region functions. The hub genes within M8 included those encoding a glutaredoxin family protein, a WNK-type protein kinase, a tRNA methyltransferase-related protein, an EFR3 membrane-associated protein, a pectin modification-related protein, and a WAT1-related protein. Collectively, these results reveal clear tissue-specific transcriptomic patterns during pericarp and shell development and identify candidate co-expression networks that may contribute to shell hardening.