The basic helix–loop–helix (bHLH) transcription factor family is the second-largest transcription factor family in plants. Members of this family are involved in the processes of growth and development, secondary metabolic biosynthesis, signal transduction, and plant resistance. Loropetalum chinense var. rubrum is a critical woody plant with higher ornamental and economic values, which has been used as ornamental architecture and traditional Chinese herbal medicine plants. However, the bHLH transcription factors in Loropetalum chinense var. rubrum (L. chinense var. rubrum) have not yet been systematically demonstrated, and their role in the biosynthesis of anthocyanin is still unclear. Here, we identified 165 potential LcbHLHs genes by using two methods, and they were unequally distributed on chromosomes 1 to 12 of the genome of L. chinense var. rubrum. Based on an evolutionary comparison with proteins from Arabidopsis and Oryza sativa, these bHLH proteins were categorized into 21 subfamilies. Most LcbHLHs in a particular subfamily had similar gene structures and conserved motifs. The Gene Ontology annotation and Cis-elements predicted that LcbHLHs had many molecular functions and were involved in processes of plant growth, including the biosynthesis of flavonoids and anthocyanins. Transcriptomic analysis revealed different expression patterns among different tissues and cultivars of L. chinense var. rubrum. Many LcbHLHs were expressed in the leaves, and only a few genes were highly expressed in the flowers. Six LcbHLHs candidate genes were identified by bioinformatics analysis and expression analysis. Further Real-time quantitative PCR analysis and protein interaction network analysis showed that LcbHLH156, which is one of the candidate proteins belonging to the IIIf subfamily, could interact with proteins related to anthocyanin synthesis. Therefore, LcbHLH156 was transiently expressed in L. chinense var. rubrum to verify its function in regulating anthocyanin synthesis. Compared with the control group, red pigment accumulation appeared at the wound after injection, and the total anthocyanin content increased at the wound of leaves. These results lay a foundation for the research of the regulation mechanism of leaf colors in L. chinense var. rubrum and also provide a basis for the function of the LcbHLH family.

Loropetalum chinense var. rubrum is a common landscape plant. Because of the preference for the ‘red leaf’ trait, the ‘flower color’ trait was ignored in breeding, and the genetic diversity of L. chinense var. rubrum was replaced by a few genotypes. Therefore, the potential value of flower color traits was affected, and the genetic diversity was not fully exploited. In this study, we utilized a cultivar with mosaic-colored flowers and leaves and observed 15 phenotypic traits in its flower organs and its progeny, as well as a total of 60 different flower color materials. We analyzed the variation characteristics and diversity of flower colors, and qualitatively described and preliminarily classified flower colors using the ISCC-NBS and CIELab systems to explore the correlation with the flower color phenotype from the distribution and content of anthocyanins. The phenotypic diversity of the flower organs was enriched, and genetic differences in flower color were significant for the L.chinense var. rubrum, with most of the 15 phenotypic traits showing significant positive correlations, and the most critical phenotypes are sepal, petal and nectary color, as well as petal length. According to the CIELab color system and anthocyanin content and distribution characteristics, it is considered more accurate and convenient to classify the color phenotypes of L. chinense var. rubrum flowers into five categories. In this study, for the first time, the flower organ phenotype of L. chinense var. rubrum has been systematically analyzed, and it is explored as a reference method for breeding new cultivars of a diverse range of colors in L. chinense var. rubrum.

The friability of callus is closely associated with its genetic transformation efficiency, and optimizing induction and transformation conditions is essential for establishing an efficient transformation system. In this study, we developed a high-efficiency friable callus induction and Agrobacterium-mediated transformation system for Chrysanthemum ‘Jimba’. Three plant growth regulator (PGR) combinations—6-Benzylaminopurine (6-BA) + Naphthaleneacetic Acid (NAA), 6-BA + 2,4-Dichlorophenoxyacetic Acid (2,4-D), and Thidiazuron (TDZ) + 2,4-D—were evaluated for their effects on callus morphology, proliferation, and transformation efficiency. The optimal PGR combination was identified as 1.0 mg/L 6-BA + 0.4 mg/L NAA, which produced highly friable calli with a loose structure, rapid proliferation, and the highest nuclear-to-cytoplasmic ratio. The optimal subculture time for maintaining friability and high proliferation was the 7th week, while the best Agrobacterium infection conditions were OD600 = 0.5 with a 10 min infection period, which achieved a transformation efficiency of 91%. This optimized protocol provides an efficient and rapid transformation method for future gene function studies using callus transformation.

Challenges significantly hinder the sustainable cultivation of tea chrysanthemum, leading to imbalances in soil nutrients, the accumulation of allelopathic phenolic acids, reduced enzymatic activity, and disruptions in rhizosphere microbial communities. To explore potential mitigation strategies, this study systematically evaluated the integrative effects of exogenous methyl jasmonate (MeJA, 0–400 (μmol L⁻¹)) on both soil environmental parameters and plant growth performance under continuous cropping conditions. The results revealed that treatment with 100 (μmol L⁻¹) MeJA significantly enhanced plant height, canopy width, flower number, and fresh flower weight. Concurrently, it improved soil organic matter content, the available nitrogen levels, and redox stability while increasing the activity of key enzymes, including polyphenol oxidase, urease, and catalase. Notably, this treatment markedly reduced the accumulation of allelopathic phenolic acids, such as p-hydroxybenzoic acid and vanillic acid. High-throughput sequencing further demonstrated that 100 (μmol L⁻¹) MeJA optimized the composition of soil microbial communities, increasing the abundance of beneficial taxa, such as nitrogen-fixing and phosphate-solubilizing bacteria, while suppressing pathogenic fungi. Metabolomic analysis showed that this concentration of MeJA activated stress-resistance metabolic pathways involving flavonoids and terpenoids while downregulating degradation-related processes, thereby supporting enhanced plant resilience at the metabolic level. Collectively, these findings demonstrate that an appropriate concentration of exogenous MeJA can effectively alleviate continuous cropping obstacles in Chrysanthemum morifolium, providing both theoretical insights and practical guidance for its eco-friendly and efficient cultivation.

The basic helix-loop-helix (bHLH) transcription factor family is the second-largest transcription factor in plants. Members of this family are involved in the process of growth and development, secondary metabolic biosynthesis, signal transduction, and plant resistance. Loropetalum chinense var. rubrum is a critical woody plant with higher ornamental and economic values, which used as ornamental architecture and traditional Chinese herbal medicine plant. However, the bHLH transcription factor in the L. chinense var. rubrum (Loropetalum chinense var. Rubrum) have yet to be systematically demonstrated, and their role in anthocyanin biosynthesis remains secret. Here, we identified 165 potential LcbHLHs genes by two methods, and they were unequally distributed on chromosomes 1 to 12 of the L. chinense genome. Based on a phylogenetic comparison with proteins from Arabidopsis, these bHLH proteins were categorized into 21 subfamilies. Most LcbHLHs in a given subfamily had similar gene structures and conserved motifs. The gene ontology annotation and cis-elements predicted that the LcbHLHs had many molecular functions and were involved in plant growth processes, including flavonoid and anthocyanin biosynthetic processes, etc. Transcriptomic analysis revealed different expression patterns among different tissues and cultivars in L. chinense. Many LcbHLHs were expressed in leaves, and only a few genes were highly expressed in flowers. Six LcbHLH genes differentially expressed between species and periods in color variation, which may play a key role in anthocyanin synthesis. These further functional predictions of LcbHLHs were established by quantitative real-time PCR analysis and protein-protein interaction network. This study identified the six potential bHLH genes related to anthocyanin in L. chinense and created a solid framework for future research on the function and evolution of bHLHs.

Wild orchid populations are declining with intensified habitat fragmentation posing severe challenges to germplasm conservation. As an important ornamental Orchidaceae species, Cymbidium ensifolium has abundant germplasm resources and frequent natural and artificial hybridization. Long-term natural evolution and anthropogenic disturbance have led to complex genetic backgrounds and ambiguous phylogenetic relationships hindering accurate germplasm identification, elite resource excavation, and selective breeding. As a distinctive variety, Cymbidium ensifolium var. susin has great breeding potential. Clarifying its phenotypic and genetic characteristics is crucial for accelerating breeding progress. In this study, phenotypic determination, Hyper-seq reduced-representation genome sequencing, SNP/InDel genotyping, genetic diversity analysis, and core collection construction were used to evaluate the genetic diversity, population differentiation, and core germplasm screening of 13 Cymbidium ensifolium var. susin accessions. The results showed significant phenotypic differences and rich genetic variation among tested materials. Based on highly weighted floral traits, accessions were divided into three major phenotypic groups. At the molecular level, 963,239 SNP and 182,399 InDel loci were identified and mainly distributed in intergenic regions, followed by introns and exons. A phylogenetic tree was constructed from SNP loci combined with principal component and phenotypic clustering analyses. This study preliminarily clarified the genetic structure of pure-heart Cymbidium ensifolium var. susin, showing a distinct geographical pattern: “high consistency in Fujian and Guangdong; strong differentiation in Southwest China; and a transitional gradient in Central China”. Meanwhile, six core germplasm accessions were screened in this study, which provides a solid theoretical basis and material support for the conservation of pure-heart Cymbidium ensifolium var. susin accessions, variety improvement, hybrid parent selection, and molecular marker-assisted breeding. This is of great significance for promoting the innovation of Chinese orchid germplasm resources and the high-quality development of the industry.