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"

[1] ZHANG L J, LI X X, MA B, GAO Q, DU H, HAN Y H, LI Y, CAO Y H, QI M, ZHU Y X, LU H W, MA M C, LIU L L, ZHOU J P, NAN C H, QIN Y J, WANG J, CUI L, LIU H M, LIANG C Z, QIAO Z J. The tartary buckwheat genome provides insights into rutin biosynthesis and abiotic stress tolerance. Molecular Plant, 2017, 10(9): 1224-1237.
doi: 10.1016/j.molp.2017.08.013
[2] ZHANG X M, ZHAO L, LARSON-RABIN Z, LI D Z, GUO Z H, NITABACH M N. De Novo sequencing and characterization of the floral transcriptome of Dendrocalamus latiflorus (Poaceae: Bambusoideae). PLoS ONE, 2012, 7(8): e42082.
doi: 10.1371/journal.pone.0042082
[3] GAO F, YAO H P, ZHAO H X, ZHOU J, LUO X P, HUANG Y J, LI C L, CHEN H, WU Q. Tartary buckwheat FtMYB10 encodes an R2R3-MYB transcription factor that acts as a novel negative regulator of salt and drought response in transgenic Arabidopsis. Plant Physiology and Biochemistry, 2016, 109: 387-396.
doi: 10.1016/j.plaphy.2016.10.022
[4] ZHOU H, WANG K L, WANG F R, ESPLEY R V, REN F, ZHAO J N, OGUTU C, HE H P, JIANG Q, ALLAN A C, HAN Y P. Activator- type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation. New Phytologist, 2018, 221(4): 1919-1934.
doi: 10.1111/nph.15486
[5] YAO P F, ZHAO H X, LUO X P, GAO F, LI C L, YAO H P, CHEN H, PARK S U, WU Q. Fagopyrum tataricum FtWD40 functions as a positive regulator of anthocyanin biosynthesis in transgenic tobacco. Journal of Plant Growth Regulation, 2017, 36(3): 755-765.
doi: 10.1007/s00344-017-9678-6
[6] LÜ B B, WU Q, WANG A H, LI Q, DONG Q X, YANG J J, ZHAO H X, WANG X L, CHEN H, LI C L. A WRKY transcription factor, FtWRKY46, from tartary buckwheat improves salt tolerance in transgenic Arabidopsis thaliana. Plant Physiology and Biochemistry, 2020, 147: 43-53.
doi: 10.1016/j.plaphy.2019.12.004
[7] YAMANE Y. Induced differentiation of buckwheat plants from subcultured calluses in vitro. Japanese Journal of Genetics, 1974, 49(3): 139-146.
[8] LEE S Y, KIM Y KY, UDDIN M R, PARK N I, PARK S U. An efficient protocol for shoot organogenesis and plant regeneration of buckwheat (Fagopyrum esculentum Moench.). Romanian Biotechnological Letters, 2009, 14(4): 4524-4529.
[9] KWON S J, HAN M H, HUH Y S, ROY S K, LEE C W, WOO S H. Plantlet regeneration via somatic embryogenesis from hypocotyls of common buckwheat (Fagopyrum esculentum Moench.). Korean Journal of Crop Science, 2013, 58(4): 331-335.
doi: 10.7740/kjcs.2013.58.4.331
[10] HAN M H, KAMAL A H, HUH Y S, JEON A Y, BAE J S, CHUNG K Y, LEE M S, PARK S U, JEONG H S, WOO S H. Regeneration of plantlet via somatic embryogenesis from hypocotyls of tartary buckwheat (Fagopyrum tataricum). Australian Journal of Crop Science, 2011, 5(7): 865-869
[11] HOU S Y, SUN Z X, HU B L, WANG Y G, HUANG K S, XU D M, HAN Y H. Regeneration of buckwheat plantlets from hypocotyl and the influence of exogenous hormones on rutin content and rutin biosynthetic gene expression in vitro. Plant Cell Tissue and Organ Culture, 2015, 120(3): 1159-1167.
doi: 10.1007/s11240-014-0671-5
[12] JOVANKA M D, MIRJANA N, SLAVICA N, RADOMIR C. Agrobacterium-mediated transformation and plant regeneration of buckwheat (Fagopyrum esculentum Moench.). Plant Cell Tissue & Organ Culture, 1992, 29(2): 101-108.
[13] KOJIMA M, ARAI Y, IWASE N, SHIROTORI K, SHIOIRI H, NOZUE M. Development of a simple and efficient method for transformation of buckwheat plants (Fagopyrum esculentum) using Agrobacterium tumefaciens. Bioscience Biotechnology & Biochemistry, 2000, 64(4): 845-847.
[14] KIM H S, KANG H J, LEE Y T, LEE S Y, KO J A, RHA E S. Direct regeneration of transgenic buckwheat from hypocotyl segment by agrobacterium-mediated transformation. Korean Journal of Crop Science, 2001, 46(5): 375-379.
[15] CHEN L H, ZHANG B, XU Z Q. Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na(+)/H(+) antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum). Transgenic Research, 2008, 17(1): 121-132.
doi: 10.1007/s11248-007-9085-z
[16] 丰明, 陈庆富, 葛维德, 薛仁风. 抗旱调控基因DREB2A转化辽荞5号的研究. 东北农业科学, 2019, 44(4): 29-36.
FENG M, CHEN Q F, GE W D, XUE R F. Research on the drought-resistant regulatory genes DREB2A transforming the ‘Liao 5 Buckwheat’. Journal of Northeast Agricultural Sciences, 2019, 44(4): 29-36. (in Chinese)
[17] 李占旗. 苦荞离体再生体系的建立和遗传转化研究[D]. 西安: 西北大学, 2007.
LI Z Q. Establishment of plant regeneration system and genetic transformation of tartary buckwheat[D]. Xian: Northwest University, 2007. (in Chinese)
[18] SUN X M, CHEN X, DENG Z X, LI Y D. A CTAB-assisted hydrothermal orientation growth of ZnO nanorods. Materials Chemistry & Physics, 2003, 78(1): 99-104.
[19] HUANG Y J, WU Q, WANG S, SHI J Q, DONG Q X, YAO P F, SHI G N, XU S X, DENG R Y, LI C L, CHEN H, ZHAO H X. FtMYB 8 from tartary buckwheat inhibits both anthocyanin/proanthocyanidin accumulation and marginal trichome initiation. BMC Plant Biology, 2019, 19(1): 263.
doi: 10.1186/s12870-019-1876-x
[20] 高飞. 苦荞光应答转录因子FtMYB5对黄酮合成的调控[D]. 成都: 四川农业大学, 2017.
GAO F.Tartary buckwheat light-induced transcription factor FtMYB 5 involved in flavonoid biosynthesis regualtion[D]. Chengdu: Sichuan Agricultural University, 2017. (in Chinese)
[21] YAO P E, HUANG Y J, DONG Q X, WAN M, WANG A H, CHEN Y W, LI C L, WU Q, CHEN H, ZHAO H X. FtMYB6, a Light-Induced SG7 R2R3-MYB transcription factor, promotes flavonol biosynthesis in tartary buckwheat (Fagopyrum tataricum). Journal of Agricultural and Food Chemistry, 2020, 68(47): 13685-13696.
doi: 10.1021/acs.jafc.0c03037
[22] 王成龙. 苦荞毛状根的诱导及高频再生体系的建立[D]. 成都: 四川农业大学, 2015.
WANG C L. Induction hairy roots and established high-frequency plant regeneration system tartary buckwheat (Fagopyrum tataricum Gaertn.)[D]. Chengdu: Sichuan Agricultural University, 2015. (in Chinese)
[23] 王官凤, 吕兵兵, 王安虎, 赵海霞, 王晓丽, 吴琦, 陈惠, 李成磊. 苦荞抗旱相关转录因子基因FtWRKY10的克隆及功能鉴定. 农业生物技术学报, 2020, 28(4): 629-644.
WANG G F, LÜ B B, WANG A H, ZHAO H X, WANG X L, WU Q, CHEN H, LI C L. Cloning and functional identification of drought resistance related transcription factor gene FtWRKY10 from tartary buckwheat (Fagopyrum tataricum). Journal of Agricultural Biotechnology, 2020, 28(4): 629-644. (in Chinese)
[24] LI Q, WU Q, WANG A H, LV B B, DONG Q X, YAO Y J, WU Q, ZHAO H X, LI C L, CHEN H, WANG X L. Tartary buckwheat transcription factor FtbZIP83 improves the drought/salt tolerance of Arabidopsis via an ABA-mediated pathway. Plant Physiology and Biochemistry, 2019, 144: 312-323.
doi: 10.1016/j.plaphy.2019.10.003
[25] 伍小方, 高国应, 左倩, 赵辉, 张凯旋, 严明理, 周美亮. FtMYB1转录因子调控苦荞毛状根黄酮醇合成的机理研究. 植物遗传资源学报, 2020, 21(5): 1270-1278.
WU X F, GAO G Y, ZUO Q, ZHAO H, ZHANG K X, YAN M L, ZHOU M L. Deciphering the functional basis of FtMYB1 transcription factor in flavonol biosynthesis of tartary buckwheat hairy root. Journal of Plant Genetic Resources, 2020, 21(5): 1270-1278. (in Chinese)
[26] 卢晓玲, 何铭, 张凯旋, 廖志勇, 周美亮. 苦荞鼠李糖基转移酶FtF3GT1基因的克隆与转化毛状根研究. 作物杂志, 2020(5): 33-40.
LU X L, HE M, ZHANG K X, LIAO Z Y, ZHOU M L. Study on the cloning and transformation of rhamnose transferase FtF3GT1 gene in tartary buckwheat. Crops, 2020(5): 33-40. (in Chinese)
[27] 翁文凤, 伍小方, 张凯旋, 唐宇, 江燕, 阮景军, 周美亮. 过表达FtbZIP5提高苦荞毛状根黄酮积累及其耐盐性. 作物杂志, 2021(4): 1-9.
WENG W F, WU X F, ZHANG K X, TANG Y, JIANG Y, RUAN J J, ZHOU M L. The overexpression of FtbZIP5improves accumulation of flavonoid in the hairy roots of tartary buckwheat and its salt tolerance. Crops, 2021(4): 1-9. (in Chinese)
[28] WANG C L, DONG X N, DING M Q, TANG Y X, ZHU X M, WU Y M, ZHOU M L, SHAO J R. Plantlet regeneration of tartary buckwheat (Fagopyrum tataricum Gaertn.) in vitro tissue cultures. Protein and Peptide Letters, 2016, 23(5): 468-477.
doi: 10.2174/0929866523666160314152317
[29] 侯思宇, 王欣芳, 杜伟, 冯晋华, 韩渊怀, 李红英, 刘龙龙, 孙朝霞. 苦荞WOX家族全基因组鉴定及响应愈伤诱导率表达分析. 中国农业科学, 2021, 54(17): 3573-3586.
HOU S Y, WANG X F, DU W, FENG J H, HAN Y H, LI H Y, LIU L L, SUN Z X. Genome-wide identification of WOX family and expression analysis of callus induction rate in tartary buckwheat. Scientia Agricultura Sinica, 2021, 54(17): 3573-3586. (in Chinese)
[30] AHMAD F, DANNY G. Agroinfiltration of intact leaves as a method for the transient and stable transformation of saponin producing Maesa lanceolate. Plant Cell Reports, 2012, 31(8): 1517-1526.
doi: 10.1007/s00299-012-1266-4
[31] VARGAS-GUEPWARAL C, VARGAS-SESURA C, VILALTAVILALOBOS J, FERERALF P, GATIOAARIAS A. A simple and efficient agroinfiltration method in coffee leaves (Coffea arabica L.): Assessment of factors affecting transgene expression. 3 Biotech, 2018, 8(11): 471.
doi: 10.1007/s13205-018-1495-5
[32] BONDT A D, EGGERMONT K, PENWNCKX I, GODERIS I, BROEKAERT W F. Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): An assessment of factors affecting regeneration of transgenic plants. Plant Cell Reports, 1996, 15(7): 549-554.
doi: 10.1007/BF00232992
[33] FEINBAUM R L, AUSUBEL F M. Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene. Molecular and Cellular Biology, 1988, 8(5): 1985-1992.
[34] KROL A R, LENTING P E, VEENSTRA J, MEER I M, KORS R E, GERATS A G, MOL J N M, STUITJE A R. An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature, 1988, 333(6176): 866-869.
doi: 10.1038/333866a0
[35] LOTKOWSKA M E, TOHGE T, FERNIE A R, XUE G P, BALAZADEH S, BERND M R. The Arabidopsis transcription factor MYB112 promotes anthocyanin formation during salinity and under high light stress. Plant Physiology, 2015, 169(3): 1862-1880.
[36] SUN Z X, HU B L, HOU S Y, LIU R H, WANG L, HAO Y R, HAN Y H, ZHOU M L, LIU L L, LI H Y. Tartary buckwheat FtMYB31 gene encoding an R2R3-MYB transcription factor enhances flavonoid accumulation in tobacco. Journal of Plant Growth Regulation, 2020, 39(2): 564-574.
doi: 10.1007/s00344-019-10000-7
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