To develop a novel tool for functional verification and molecular breeding in tartary buckwheat, this study focused on establishing and optimizing an efficient callus genetic transformation system.

Callus induction factors including different explants, ratios of diverse growth regulators, and Agrobacterium tumefaciens types were systematically evaluated using “Xiqiao No. 2” as the derived plant. We further overexpressed FtCHS1, a key enzyme gene involved in the biosynthesis of tartary buckwheat flavonoids in obtained calli to validate the optimized genetic callus transformation system. The positive transgenic lines were confirmed by PCR and fluorescent observation. Subsequently, the content of anthocyanins and metabolites in flavonol branch pathway were determined by UV spectrophotometry and High Performance Liquid Chromatography (HPLC), respectively. Furthermore, quantitative real-time PCR was performed to analyze expression levels of genes involved in flavonoid synthesis, in order to compare the differences between the FtCHS1-overexpressed calli and the control.

The optimal explant was hypocotyls and the optimal induction medium was the Murashige and Skoog (MS) medium supplemented with the addition of 0.8 mg·L^-1 6-BA (6-Benzylaminopurine) and 3.5 mg·L^-1 2,4-D (2,4-Dichlorophenoxyacetic acid). The induction rate of calli grown on the above medium reached up to 72%. Moreover, the optimized subculture medium containing MS with the additives of 3 mg·L^-1 6-BA and 1 mg·L^-1 KT (Kinetin) increased the percentage and coefficient of callus proliferation to 98% and 1.09, respectively. Additionally, the best Agrobacterium tumefaciens in the transformation process was GV3101, and the transformation efficiency was up to 31.3%. The functional analysis of FtCHS1 overexpressing in transgenetic calli demonstrated that: (1) The accumulations of kaempferol and quercetin in transgenic calli overexpressing FtCHS1 were dramatically higher than those in control groups (P<0.01), and anthocyanin, rutin and myricetin contents were also remarkably higher (P<0.05); (2) Overexpression of the exogenous FtCHS1 did not affect the expression levels of 5 endogenous orthologous genes FtCHSs in the transgenic calli (P>0.05), whereas genes encoding key enzymes of the flavonoid synthesis pathway, such as FtCHI, FtF3H, FtFLS1, FtFLS2, FtFLS3, and FtDFR1, were up-regulated (P<0.05); (3) FtMYB5 and FtMYB6, the transcription factor genes that specifically positively regulated the flavonol synthesis, were up-regulated, while FtMYB8, a suppressor gene of anthocyanin synthesis, was down-regulated (P<0.05).

In this study, the callus genetic transformation system of tartary buckwheat was successfully established from “Xiqiao No. 2”. FtCHS1 overexpression in the transgenic calli up-regulated genes related to flavonoid synthesis, resulting in flavonoids accumulation.