Optimization of in vitro adventitious shoot regeneration and genetic transformation systems in blueberry (Vaccinium corymbosum) using a heterogeneous Ruby1 gene

Abstract

Blueberry is an economically important fruit crop, and demand for it is rising. Despite being essential for advancing precision breeding of blueberries, genetic transformation platforms remain limited for transgenic applications. In this study, we used the widely cultivated blueberry cultivar ‘Legacy’ (Vaccinium corymbosum) as the experimental material to establish an efficient adventitious shoot-regeneration system and an Agrobacterium-mediated transformation protocol. Adventitious bud development was promoted via a two-step protocol: induction on WPM medium with 2 mg/L TDZ and 0.5 mg/L NAA, followed by elongation on WPM medium with 3 mg/L ZT. Younger leaves from the shoot tips were selected as explants for the regeneration system. Wounding treatment, placing the abaxial (back) side in contact with the medium, and a 12-d dark treatment at the initial regeneration stage significantly improved the adventitious shoot regeneration rate. Building upon this regeneration system, an Agrobacterium-mediated genetic transformation protocol was developed using a vector carrying the reporter genes enhanced green fluorescent protein (Egfp) and CgRuby1, an anthocyanin-regulating transcription factor from purple pummelo (Citrus grandis). The optimal parameters included culturing Agrobacterium-mediated in liquid medium, preparing the Agrobacterium suspension with an OD₆₀₀ of 0.8, a 1-h vacuum infiltration, 6 d of co-cultivation, and selection on adventitious shoot regeneration medium containing 10 mg/L kanamycin, which enhanced the regeneration rates of resistant shoots. Polymerase chain reaction (PCR) analysis confirmed that transgenic plants carrying CgRuby1 were achieved with a final transformation efficiency of 6.5%. Phenotypic analysis revealed that the transgenic plants accumulated significantly higher anthocyanin levels in their leaves than the wild-type plants. In addition, key blueberry anthocyanin biosynthetic genes, including VcCHS, VcFHT, VcDFR, VcANS, and VcUFGT, were markedly upregulated in the transgenic lines. Taken together, we have successfully established an efficient transformation system that may hold great potential for functional characterization of genes involved in various processes.

Keywords

Blueberry / Adventitious shoot regeneration / Agrobacterium-mediated transformation / Anthocyanin synthesis / Ruby1

References

[1] Bechtold N, Bouchez D. In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. In: Potrykus I, Spangenberg G, editors. Gene Transfer to Plants. Berlin, Heidelberg: Springer; 1995.p.19–23.

[2] Bierhuizen MFA, Westerman Y, Visser TP, Dimjat W, Wognum AW, Wagemaker G. Enhanced green fluorescent protein as selectable marker of retroviral-mediated gene transfer in immature hematopoietic bone marrow cells. Blood, 1997, 90: 3304-3315

[3] Brevis PA, Bassil NV, Ballington JR, Hancock JF. Impact of wide hybridization on highbush blueberry breeding. J Am Soc Hortic Sci, 2008, 133: 427-437

[4] Butelli E, Licciardello C, Zhang Y, Liu JJ, Mackay S, Bailey Pet al.. Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges. Plant Cell, 2012, 24: 1242-1255

[5] Butelli E, Garcia-Lor A, Licciardello C, Las Casas G, Hill L, Recupero GRet al.. Changes in anthocyanin production during domestication of Citrus. Plant Physiol, 2017, 173: 2225-2242

[6] Butelli E, Licciardello C, Ramadugu C, Durand-Hulak M, Celant A, Reforgiato Recupero Get al.. Noemi controls production of flavonoid pigments and fruit acidity and illustrates the domestication routes of modern citrus varieties. Curr Biol, 2019, 29: 158-164

[7] Cao X, Liu Q, Rowland LJ, Hammerschlag FA. GUS expression in blueberry (Vaccinium spp.): factors influencing Agrobacterium-mediated gene transfer efficiency. Plant Cell Rep, 1998, 18: 266-270

[8] Chhabra G, Sharma M, Kalia A, Kaur A, Sandhu JS. Monitoring dissociation of chimerism through real-time PCR and scanning electron microscopy following in planta transformation of rough lemon (Citrus jambhiri Lush.). Plant Biotechnol Rep, 2024, 18: 497-506

[9] Debnath SC. A two-step procedure for adventitious shoot regeneration on excised leaves of lowbush blueberry. In Vitro Cell Dev Biol Plant, 2009, 45: 122-128

[10] Desfeux C, Clough SJ, Bent AF. Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol, 2000, 123: 895-904

[11] Dutt M, Mahmoud LM, Weber KC, Satpute A, Stanton D, Qiu WMet al.. Anthocitrus: evaluation of anthocyanin accumulating “Mexican” lime fruits produced by overexpressing the Ruby transcription factor gene from Citrus sinensis “Moro”. Plant Cell, Tissue and Organ Culture (PCTOC), 2023, 155: 283-296

[12] Geier T. Chimeras: properties and dissociation in vegetatively propagated plants. in: Shu QY, Forster BP, Nakagawa H, editors. Plant mutation breeding and biotechnology. Wallingford and Rome : CAB International and Food and Agriculture Organization of the United Nations (FAO); 2012. p. 191–201.

[13] Gupta V, Estrada AD, Blakley I, Reid R, Patel K, Meyer MDet al.. RNA-Seq analysis and annotation of a draft blueberry genome assembly identifies candidate genes involved in fruit ripening, biosynthesis of bioactive compounds, and stage-specific alternative splicing. GigaScience, 2015, 4 s13742

[14] Hada A, Krishnan V, Mohamed Jaabir MS, Kumari A, Jolly M, Praveen Set al.. Improved Agrobacterium tumefaciens-mediated transformation of soybean [Glycine max (L.) Merr.] following optimization of culture conditions and mechanical techniques. In Vitro Cell Dev Biol, 2018, 54: 672-688

[15] Hao SY, Zhang YY, Li RD, Qu PY, Cheng CZ. Agrobacterium-mediated in planta transformation of horticultural plants: current status and future prospects. Sci Hortic, 2024, 325 112693

[16] He G, Zhang R, Jiang SH, Wang HH, Ming F. The MYB transcription factor RcMYB1 plays a central role in rose anthocyanin biosynthesis. Hortic Res, 2023, 10 uhad080

[17] Huang D, Wang X, Tang ZZ, Yuan Y, Xu YT, He JJet al.. Subfunctionalization of the Ruby2-Ruby1 gene cluster during the domestication of citrus. Nat Plants, 2018, 4(11): 930-941

[18] Hwang HH, Yu MD, Lai EM. Agrobacterium-mediated plant transformation: biology and applications. Arabidopsis Book, 2017, 15 e0186

[19] Koes R, Verweij W, Quattrocchio F. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci, 2005, 10: 236-242

[20] Li HX, Wang SW, Zhai LL, Cui YH, Tang GL, Huo JWet al.. The miR156/SPL12 module orchestrates fruit colour change through directly regulating ethylene production pathway in blueberry. Plant Biotechnol J, 2024, 22: 386-400

[21] Lin TY, Walworth A, Zong XJ, Danial GH, Tomaszewski EM, Callow Pet al.. VcRR2 regulates chilling-mediated flowering through expression of hormone genes in a transgenic blueberry mutant. Hortic Res, 2019, 6 96

[22] Liu JX. Agrobacterium-mediated genetic transformation of different blueberry varieties. [Master’s thesis]. Beijing: Beijing Forestry University; 2018. https://doi.org/10.26949/d.cnki.gblyu.2018.000207.

[23] Marcotrigiano M. Origin of adventitious shoots regenerated from cultured tobacco leaf tissue. Am J Bot, 1986, 73: 1541-1547

[24] Oh CJ, Woo JK, Yi KU, Park YC, Lee HY, Kim MJet al.. Development of molecular markers for genotyping of Ruby, a locus controlling anthocyanin pigment production in Citrus with its functional analysis. Sci Hortic, 2021, 289 110457

[25] Padavannil A, Jobichen C, Yang QH, Seetharaman J, Velazquez-Campoy A, Yang Let al.. Dimerization of VirD2 binding protein is essential for Agrobacterium induced tumor formation in plants. PLoS Pathog, 2014, 10 e1003948

[26] Qin XJ, Hu J, Xu GH, Song HF, Zhang LY, Cao YB. An efficient transformation system for fast production of VcCHS transgenic blueberry callus and its expressional analysis. Plants, 2023, 12 2905

[27] Qin XJ. Optimization of leaves regeneration and genetic transformation system of several blueberry varieties. [Master’s thesis]. Beijing: Beijing Forestry University; 2020. https://doi.org/10.26949/d.cnki.gblyu.2020.000241.

[28] Qiu DL, Wei XY, Fan SF, Jian DW, Chen JJ. Regeneration of blueberry cultivars through indirect shoot organogenesis. J Am Soc Hortic Sci. 2018;53:1045–9. https://doi.org/10.21273/HORTSCI13059-18.

[29] Ramsay NA, Glover BJ. MYB–bHLH–WD40 protein complex and the evolution of cellular diversity. Trends Plant Sci, 2005, 10: 63-70

[30] Shah T, Andleeb T, Lateef S, Noor MA. Genome editing in plants: advancing crop transformation and overview of tools. Plant Physiol Biochem, 2018, 131: 12-21

[31] Sinelli N, Spinardi A, Egidio DV, Mignani I, Casiraghi E. Evaluation of quality and nutraceutical content of blueberries (Vaccinium corymbosum L.) by near and mid-infrared spectroscopy. Postharvest Biol Technol. 2008;50:31–6. https://doi.org/10.1016/j.postharvbio.2008.03.013.

[32] Song GQ, Chen QX. Overexpression of the MADS-box gene K-domain increases the yield potential of blueberry. Plant Sci, 2018, 276: 22-31

[33] Song GQ, Hancock JF. Recent advances in blueberry transformation. Int J Fruit Sci, 2012, 12: 316-332

[34] Song GQ, Sink KC. Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.). Plant Cell Rep, 2004, 23: 475-484

[35] Song GQ, Walworth AE, Hancock JF. Stability of transgenes in blueberry. Int J Fruit Sci, 2012, 12: 333-341

[36] Song GQ, Walworth A, Lin TY, Chen QX, Han XM, Zaharia ILet al.. VcFT-induced mobile florigenic signals in transgenic and transgrafted blueberries. Hortic Res, 2019, 6: 105

[37] Song YP, Luo WB, Wu Y, Li XJ, Albert NW, Zhang YHet al.. A callus-derived regeneration and Agrobacterium-mediated gene transformation developed for bilberry, Vaccinium myrtillus. Plant Cell Tissue Organ Cult, 2023, 154: 177-187

[38] Song GQ, Sink KC. Agrobacterium-mediated transformation of highbush blueberry (Vaccinium corymbosum L.) cultivars. In: Wang K, editor. Agrobacterium protocols. New York: Springer; 2015. p. 37–44.

[39] Song GQ. Blueberry (Vaccinium corymbosum L.). In: Wang K, editor. Agrobacterium protocols. New York: Springer; 2015. p. 121–31.

[40] Tague BW, Mantis J. In planta Agrobacterium-mediated transformation by vacuum infiltration. In: Salinas J, Sanchez-Serrano JJ, editors. Arabidopsis Protocols. Totowa: Humana Press; 2006. p. 215–23.

[41] Thilmony R, Dasgupta K, Shao M, Harris D, Hartman J, Harden LAet al.. Tissue-specific expression of Ruby in Mexican lime (C. aurantifolia) confers anthocyanin accumulation in fruit. Front Plant Sci, 2022, 13 945738

[42] Walworth A, Song GQ. The cold-regulated genes of blueberry and their response to overexpression of VcDDF1 in several tissues. Int J Mol Sci, 2018, 19: 1553

[43] Wang GD, Liu MM, Liu GZ, Bao ZL, Ma FF. Establishment and optimization of agrobacterium-mediated transformation in blueberry (Vaccinium species). Sci Hortic, 2022, 304 111258

[44] Ward ER, Barnes WM. VirD2 protein of Agrobacterium tumefaciens very tightly linked to the 5' end of T-strand DNA. Science, 1988, 242: 927-930

[45] Xu WJ, Dubos C, Lepiniec L. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant Sci, 2015, 20: 176-185

[46] Yang WT, Zhai HH, Wu FM, Deng L, Chao Y, Meng XWet al.. Peptide REF1 is a local wound signal promoting plant regeneration. Cell, 2024, 187: 3024-3038

Funding

Sichuan Provincial Science and Technology Plan Project, China(2021YFYZ0023)

Program on Industrial Technology System of National Soybean(CARS-29)

RIGHTS & PERMISSIONS

The Author(s)