Unveiling the Molecular Mechanisms of Browning in Camellia hainanica Callus through Transcriptomic and Metabolomic Analysis

<i>Camellia hainanica</i> is one of the camellia plants distributed in tropical regions, and its regeneration system and genetic transformation are affected by callus browning. However, the underlying mechanism of <i>Camellia hainanica</i> callus browning formation remains largely unknown. To investigate the metabolic basis and molecular mechanism of the callus browning of <i>Camellia hainanica</i>, histological staining, high-throughput metabolomics, and transcriptomic assays were performed on calli with different browning degrees (T1, T2, and T3). The results of histological staining revealed that the brown callus cells had obvious lignification and accumulation of polyphenols. Widely targeted metabolomics revealed 1190 differentially accumulated metabolites (DAMs), with 53 DAMs annotated as phenylpropanoids and flavonoids. Comparative transcriptomics revealed differentially expressed genes (DEGs) of the T2 vs. T1 associated with the biosynthesis and regulation of flavonoids and transcription factors in <i>Camellia hainanica</i>. Among them, forty-four enzyme genes associated with flavonoid biosynthesis were identified, including <i>phenylalaninase</i> (<i>PAL</i>), <i>4-coumaroyl CoA ligase</i> (<i>4CL</i>), <i>naringenin</i> via <i>flavanone 3-hydroxylase</i> (<i>F3H</i>), <i>flavonol synthase</i> (<i>FLS</i>), <i>Chalcone synthase</i> (<i>CHS</i>), <i>Chalcone isomerase</i> (<i>CHI</i>), <i>hydroxycinnamoyl-CoA shikimate transferase</i> (<i>HCT</i>), <i>Dihydroflavonol reductase</i> (<i>DFR</i>), <i>anthocyanin reductase</i> (<i>LAR</i>), <i>anthocyanin synthetase</i> (<i>ANS</i>), and <i>anthocyanin reductase</i> (<i>ANR</i>). Related transcription factors <i>R2R3-MYB</i>, <i>basic helix-loop-helix</i> (<i>bHLH</i>), and <i>WRKY</i> genes also presented different expression patterns in T2 vs. T1. These results indicate that the browning of calli in <i>Camellia hainanica</i> is regulated at both the transcriptional and metabolic levels. The oxidation of flavonoids and the regulation of related structural genes and transcription factors are crucial decisive factors. This study preliminarily revealed the molecular mechanism of the browning of the callus of Camellia hainanensis, and the results can provide a reference for the anti-browning culture of <i>Camellia hainanica</i> callus.