Flower development is a vital developmental process in the life cycle of woody perennials, especially fruit trees. Herein, we used transcriptomic, proteomic, and hormone analyses to investigate the key candidate genes/proteins in loquat (Eriobotrya japonica) at the stages of flower bud differentiation (FBD), floral bud elongation (FBE), and floral anthesis (FA). Comparative transcriptome analysis showed that differentially expressed genes (DEGs) were mainly enriched in metabolic pathways of hormone signal transduction and starch and sucrose metabolism. Importantly, the DEGs of hormone signal transduction were significantly involved in the signaling pathways of auxin, gibberellins (GAs), cytokinin, ethylene, abscisic acid (ABA), jasmonic acid, and salicylic acid. Meanwhile, key floral integrator genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and floral meristem identity genes SQUAMOSA PROMOTER BINDING LIKE (SPL), LEAFY (LFY), APETALA1 (AP1), and AP2 were significantly upregulated at the FBD stage. However, key floral organ identity genes AGAMOUS (AG), AP3, and PISTILLATA (PI) were significantly upregulated at the stages of FBE and FA. Furthermore, transcription factors (TFs) such as bHLH (basic helix-loop-helix), NAC (no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF1/2) and cup-shaped cotyledon (CUC2)), MYB_related (myeloblastosis_related), ERF (ethylene response factor), and C2H2 (cysteine-2/histidine-2) were also significantly differentially expressed. Accordingly, comparative proteomic analysis of differentially accumulated proteins (DAPs) and combined enrichment of DEGs and DAPs showed that starch and sucrose metabolism was also significantly enriched. Concentrations of GA₃ and zeatin were high before the FA stage, but ABA concentration remained high at the FA stage. Our results provide abundant sequence resources for clarifying the underlying mechanisms of the flower development in loquat.

Loquat (Eriobotrya japonica Lindl.) is one of the most important subtropical evergreen fruit trees. However, due to the lack of widely applicable genetic transformation platforms, the research about gene functional characterization and molecular mechanisms is largely confined. In this study, the efficient protocol of protoplast isolation (the enzyme solution composed of 2.4% macerozyme R-10, 4.8% cellulase RS, dissolved in a 0.6 M mannitol solution) and the method of protoplast purification (CPW solution containing 5% sucrose and 11% mannitol) have been achieved with protoplast yields of 12.6 × 10⁶/g·FW, reaching a viability rate of up to 91%. A protoplast transient gene expression system has been established with an efficiency of approximately 40% using GFP reporter gene. Using this reliable and efficient system, the protein localization characteristics of transcription factor EjDELLA, EjbHLH79, and marker gene OsPHT4 were also utilized for further analysis. To our knowledge, this is the first report on establishing an efficient system for protoplast isolation, purification, and transformation of loquat mesophyll. The system reported here will definitely promote rapid progress in breeding, genetic transformation, and molecular research.

Polyploidy plays a significant role in loquat breeding, particularly in triploid breeding for seedless fruit production. Currently, loquat polyploid breeding primarily relies on natural seedling selection and sexual hybridization approaches. In this study, unfertilized ovules from four loquat varieties were in vitro cultured. Gynogenesis and embryoid regeneration were achieved in ‘Xingning 1’ and ‘Huabai 1’, with ‘Xingning 1’ demonstrating the highest gynogenesis efficiency (21.63%). Flow cytometry and chromosome counting revealed that the obtained embryoid lines included haploid, diploid, tetraploid, hexaploid, and chimeric ploidy types. Further characterization of ‘Xingning 1’-derived embryoid lines through SSR markers and whole-genome resequencing confirmed that the haploid, diploid, tetraploid, and hexaploidy embryoid originated from haploid–somatic chimeras, diploid, doubled diploid and tripled diploid, respectively. Metabolic analysis showed a positive correlation between ploidy level and the content of both soluble sugars and organic acids. This study explored a novel platform for polyploid induction in loquat and may provide methodological insights for improvement of other perennial fruit trees.