Callus browning is a major drawback to lotus callus proliferation and regeneration. However, the underlying mechanism of its formation remains largely unknown. Herein, we aimed to explore the metabolic and molecular basis of lotus callus browning by combining histological staining, high-throughput metabolomics, and transcriptomic assays for lotus callus at three browning stages. Histological stained brown callus cross sections displayed severe cell death symptoms, accompanied by an obvious accumulation of polyphenols and lignified materials. Widely targeted metabolomics revealed extensively decreased accumulation of most detected flavonoids and benzylisoquinoline alkaloids (BIAs), as well as a few phenolic acids, amino acids and their derivatives in callus with browning symptoms. Conversely, the contents of most detected tannins were significantly increased. Subsequent comparative transcriptomics identified a set of differentially expressed genes (DEGs) associated with the biosynthesis and regulation of flavonoids and BIAs in lotus. Notably, callus browning was coupled with significantly up-regulated expression of two polyphenol oxidase (PPO) and 17 peroxidase (POD) encoding genes, while the expression of ethylene associated genes remained at marginal levels. These results suggest that lotus callus browning is primarily controlled at the level of metabolism, wherein the oxidation of flavonoids and BIAs is crucially decisive.
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