Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (9): 1723-1734.doi: 10.3864/j.issn.0578-1752.2022.09.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Optimization of Callus Genetic Transformation System and Its Application in FtCHS1 Overexpression in Tartary Buckwheat

ZHAO HaiXia(),XIAO Xin,DONG QiXin,WU HuaLa,LI ChengLei,WU Qi*()   

  1. College of Life Science, Sichuan Agricultural University, Ya’an 625014, Sichuan
  • Received:2021-11-01 Revised:2021-12-30 Online:2022-05-01 Published:2022-05-19
  • Contact: Qi WU E-mail:zhaohaixia@sicau.edu.cn;wuqi@sicau.edu.cn

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Abstract:

【Objective】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. 【Method】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. 【Result】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). 【Conclusion】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.

Key words: tartary buckwheat, callus, genetic transformation, chalcone synthase gene

Supplementary Table 1

Callus induction and subculture media"

Supplementary Table 2

Primer sequences"

Fig. 1

Callus induction of cotyledons and hypocotyls upon different treatments C1-C25: Twenty-five different combinations of callus induction hormones (attached table 1); Distinct alphabets represent significant differences (P<0.05). Error bars represent ±SDs. The same as below"

Fig. 2

Tartary buckwheat callus subculture A: The 5 day and 25 day callus under the subculture treatments-S4; B: The callus proliferation rate under different subculture treatments; C: The callus proliferation coefficient upon different treatments. S1-S9: Nine different subculture hormone combinations (attached table 1)"

Fig. 3

Induction of callus infected by different Agrobacterium tumefaciens A: Hypocotyl phenotypes after 7-day infection of Agrobacterium tumefaciens GV3101/C58C1; B: The transformation efficiency of three treatments numbered C19/C20/C22 infected by different Agrobacterium tumefaciens (GV3101/C58C1)"

Fig. 4

Induction, subculture and identification of FtCHS1-overexpressing tartary buckwheat calli A: Schematic structure of pCHF3-FtCHS1-YFP; B: The phenotype of hypocotyl for 5-day infection by Agrobacterium GV3101. #1, #2 and #3 represent different strains; C: PCR determination of transgenic callus; D: Fluorescence detection in callus; E: Proliferation (subculture) rate of different tartary buckwheat calli; F: The expression of FtCHS1 in transgenic calli and control group calli; G: Callus proliferation coefficient after subculturing for 0-5 d, 10-15 d, 15-20 d and 20-25 d. ** P<0.01; *P<0.05. The same as below"

Fig. 5

Effect of FtCHS1 overexpression on flavonol/anthocyanin accumulation in calli A: The flavonol (rutin, kaempferol, quercetin and myricetin) contents in transgenic calli; B: Total anthocyanin content in transgenic calli"

Fig. 6

Expression analysis of flavonoid synthesis related genes in overexpressed FtCHS1 callus A: FtCHSs gene; B: Flavonoid metabolic branch enzyme genes; C: SG7 subfamily transcription factors genes and SG4-like gene subfamily transcription factor"

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