Comparative histology, transcriptome, and metabolite profiling unravel the browning mechanisms of calli derived from ginkgo (Ginkgo biloba L.)

Gingko biloba accumulates high levels of secondary metabolites of pharmaceutical value. Ginkgo calli develop a typical browning that reduces its regenerative capacity and thus its usefulness. To elucidate the browning mechanism, histological, transcriptomic, and metabolic alterations were compared between green and browning calli derived from immature ginkgo embryos. Histological observations revealed that browning calli had a more loosely arranged cell structure and accumulated more tannins than in green calli. Integrated metabolic and transcriptomic analyses showed that phenylpropanoid metabolism was specifically activated in the browning calli, and 428 differentially expressed genes and 63 differentially abundant metabolites, including 12 flavonoid compounds, were identified in the browning calli compared to the green calli. Moreover, the expression of flavonol synthase ( FLS ) and UDP-glucuronosyl-transferase ( UGT ) genes involved in the flavonoid pathway was more than tenfold higher in browning calli than in green calli, thus promoting biosynthesis of flavonol, which serves as a substrate to form glycosylated flavonoids. Flavonoid glycosides constituted the major coloring component of the browning calli and may act in response to multiple stress conditions to delay cell death caused by browning. Our results revealed the cellular and biochemical changes in browning callus cells that accompanied changes in expression of browning-related genes, providing a scientific basis for improving ginkgo tissue culturability.