Inducement and cultivation of novel red Cyclocarya paliurus callus and its unique morphological and metabolic characteristics

Cyclocarya paliurus (C. paliurus) is a unique plant growing in China with many bioactive components, but is endangered due to the great difficulties in seed germination and asexual propagation. Plant tissue culture can be used to overcome the resource shortage which severely limits the development and utilization of this plant. In the present paper, five typical types of C. paliurus callus lines with different morphologies, including a novel red callus line (CR) and a common light yellow-green callus line (CYG), were obtained after a long time induction and cultivation. Three triterpenoids (betulinic acid, oleanolic acid and ursolic acid) and two polyphenols ((+)-catechin and procyanidin B1) were all detected in both CR and CYG, but differed greatly in content. The contents of total triterpenoids and total polyphenols in CR were all significantly higher than that in CYG. However, anthocyanins (cyanidin-3-O-galactoside and cyanidin-3-O-glucoside) were only found in CR. Both the growth rate and biomass increment of CYG were slightly higher than that of CR. The results of high throughput RNA sequencing indicated that the expression of the triterpenoid biosynthesis pathway genes, such as HMGS (hydroxymethylglutaryl-CoA synthase), HMGR (hydroxymethylglutaryl-CoA reductase), FPS (farnesyl diphosphate synthase), SS (squalene synthase), SE (squalene epoxidase) and β-AS (beta-incense resin synthase), significantly increased in CR compared with CYG, which led to a marked rising of triterpenoid contents. Similarly, the up-regulated expression of polyphenol biosynthesis pathway genes, including CHI (chalcone isomerase), N3D (naringenin 3-dioxygenase), F3′H (flavonoid 3′-monooxygenase), DFR (dihydroflavonol 4-reductase), and 3GalT (galactosyltransferase), improved the polyphenol contents in CR, especially anthocyanin content. The decreased transcription factor expression of MYC2 and ERF003 resulted in a high triterpenoid content by increasing the expression of pathway genes in the CR. Meanwhile, the up-regulated MYB7, MYB1, bHLH122, WD40, ERF105 and down-regulated MYB44, MYC2, ERF003 enhanced the polyphenol content of the CR by stimulating the expression of the related pathway genes. RT-qPCR (quantitative real-time PCR) determination further validated the above-mentioned differentially expressed genes analysis of RNA-sequencing.