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Improved protocol for the transformation of adult Citrus sinensis Osbeck ‘Tarocco’ blood orange tissues

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Abstract

The production of transgenic citrus plants from adult tissues is difficult because of low regeneration and transformation rates. To increase the transformation efficiency of adult citrus tissues, an improved protocol involving adult Citrus sinensis Osbeck ‘Tarocco’ blood orange tissues was developed. Explants were pre-incubated in a liquid medium prior to infection by Agrobacterium tumefaciens. Plant materials were also incubated on callus-induction medium supplemented with various combinations of cytokinin (Cyt) and kanamycin (Kan). An appropriate pre-incubation of the explants increased the transformation efficiency of adult tissues. During the callus-induction period, the Cyt type and Kan concentration had the largest and smallest effects on the transformation efficiency, respectively. The most effective combination of plant growth regulator and Kan for the transformation of ‘Tarocco’ blood orange tissues was 2 mg L−1 2-isopentenyl adenine and 50 mg L−1 Kan. The transformation efficiency under the optimized conditions was 11.7%. A Southern blot analysis confirmed the integration of the transgene. These results indicated that the transformation efficiency of adult citrus tissues can be enhanced by optimizing the transformation conditions.

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References

  • Ainsley PJ, Collins GG, Sedgley M (2001) Factors affecting Agrobacterium mediated gene transfer and the selection of transgenic calli in paper shell almond (Prunus dulcis Mill). J Hortic Sci Biotechnol 76:522–528

    CAS  Google Scholar 

  • Almeida WAB, Mourão Filho FAA, Pino LE, Boscariol RL, Rodriguez APM, Mendes BMJ (2003) Genetic transformation and plant recovery from mature tissues of Citrus sinensis L. Osbeck. Plant Sci 164:203–211

    Article  CAS  Google Scholar 

  • Cervera M, Juárez J, Navarro A, Pina JA, Durán-Vila N, Navarro L, Peña L (1998a) Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage. Transgenic Res 7:51–59

    Article  CAS  Google Scholar 

  • Cervera M, Navarro A, Navarro L, Peña L (2008) Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration. Tree Physiol 28:55–66

    Article  CAS  Google Scholar 

  • Cervera M, Pina JA, Juárez J, Navarro L, Peña L (1998b) Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration. Plant Cell Rep 18:271–278

    Article  CAS  Google Scholar 

  • Ding L, Li S, Gao J, Wang Y, Yang G, He G (2009) Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat. Mol Biol Rep 36:29–36

    Article  CAS  Google Scholar 

  • Domínguez A, Cervera M, Pérez RM, Romero J, Fagoaga C, Cubero J, López MM, Juárez JA, Navarro L, Peña L (2004) Characterization of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies. Mol Breeding 14:171–183

    Article  Google Scholar 

  • Dutt M, Grosser JW (2009) Evaluation of parameters affecting Agrobacterium-mediated transformation of citrus. Plant Cell Tiss Org 98:331–340

    Article  CAS  Google Scholar 

  • Fagoaga C, Rodrigo I, Conejero V, Hinarejos C, Tuset JJ, Arnau J, Pina JA, Navarro L, Peña L (2001) Increased tolerance to Phytophthora citrophthora in transgenic orange plants constitutively expressing a tomato pathogenesis related protein PR-5. Mol Breeding 7:175–185

  • Fávero P, Mourão Filho FAA, Stipp LCL, Mendes BMJ (2012) Genetic transformation of three sweet orange cultivars from explants of adult plants. Acta Physiol Plant 34:471–477

    Article  Google Scholar 

  • Ghorbel R, Dominguez A, Navarro L, Peña L (2000) High efficiency genetic transformation of sour orange (Citrus aurantium) and production of transgenic trees containing the coat protein gene of citrus tristeza virus. Tree Physiol 20:1183–1189

    Article  Google Scholar 

  • He Y, Chen S, Peng A, Zou X, Xu L, Lei T, Liu X, Yao L (2011) Production and evaluation of transgenic sweet orange (Citrus sinensis Osbeck) containing bivalent antibacterial peptide genes (Shiva A and Cecropin B) via a novel Agrobacterium-mediated transformation of mature axillary shoots. Sci Hortic 128:99–107

    Article  CAS  Google Scholar 

  • Jacq B, Lesobre O, Sangwan RS, Sangwan-Norreel BS (1993) Factors influencing T-DNA transfer in Agrobacterium-mediated transformation of sugarbeet. Plant Cell Rep 12:621–624

    Article  CAS  Google Scholar 

  • Li D, Shi W, Deng X (2002) Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene. Plant Cell Rep 21:153–156

    Article  CAS  Google Scholar 

  • Marutani-Hert M, Bowman KD, McCollum GT, Mirkov TE, Evens TJ, Niedz RP (2012) A dark incubation period is important for Agrobacterium-mediated transformation of mature internode explants of sweet orange, grapefruit, citron, and a citrange rootstock. PLoS One 7:e47426

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Peña L, Cervera M, Fagoaga C, Pérez R, Romero J, Juárez J, Pina JA, Navarro L (2004) Agrobacterium-mediated transformation of citrus. In: Curtis IS (ed) Transgenic crops of the world-essential protocols. Kluwer, Dordrecht, pp 145–157

    Chapter  Google Scholar 

  • Peña L, Cervera M, Juárez J, Navarro A, Pina JA, Navarro L (1997) Genetic transformation of lime (Citrus aurantifolia Swing.): factors affecting transformation and regeneration. Plant Cell Rep 16:731–737

    Article  Google Scholar 

  • Peng A, Chen S, Lei T, Xu L, He Y, Wu L, Yao L, Zou X (2017) Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus. Plant Biotechnol J 15:1509–1519

    Article  CAS  Google Scholar 

  • Peng A, Xu L, He Y, Lei T, Yao L, Chen S, Zou X (2015) Efficient production of marker-free transgenic ‘Tarocco’ blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker using a Cre/loxP site-recombination system. Plant Cell Tiss Org 123:1–13

    Article  CAS  Google Scholar 

  • Rodríguez A, Cervera M, Peris JE, Peña L (2008) The same treatment for transgenic shoot regeneration elicits the opposite effect in mature explants from two closely related sweet orange (Citrus sinensis (L.) Osb.) genotypes. Plant Cell Tiss Org 93:97–106

    Article  Google Scholar 

  • Villemont E, Dubois F, Sangwan RS, Vasseur G, Bourgeois Y, Sangwan-Norreel BS (1997) Role of the host cell cycle in the Agrobacterium-mediated genetic transformation of petunia: evidence of an S-phase control mechanism for T-DNA transfer. Planta 201:160–172

    Article  CAS  Google Scholar 

  • Yang L, Zhang J, Xie Y, Yang Y, Li L, Qin L, Deng Z (2009) Genetic transformation system establishment with mature internodal stem segments of Citrus sinensis L. Osbeck cv Bingtang Nonwood Forest Res 27:42–46I

  • Yasmeen A (2009) An improved protocol for the regeneration and transformation of tomato (cv Rio Grande). Acta Physiol Plant 31:1271–1277

    Article  CAS  Google Scholar 

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Acknowledgments

We thank Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac) for editing the English text of a draft of this manuscript.

Funding

This research was supported by the Science and Technology Innovation Strategy Special Fund of Guangdong Province (2018B020202009), the Earmarked Fund for China Agriculture Research System (CARS-26), the Open Project of Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education, and the National Citrus Engineering Research Center.

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Authors and Affiliations

  1. Citrus Research Institute, Southwest University, Chongqing, 400712, People’s Republic of China

    Aihong Peng, Xiuping Zou, Lanzhen Xu, Yongrui He, Tiangang Lei, Lixiao Yao, Qiang Li & Shanchun Chen

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  1. Aihong Peng
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  2. Xiuping Zou
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  3. Lanzhen Xu
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  4. Yongrui He
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  5. Tiangang Lei
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  6. Lixiao Yao
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  7. Qiang Li
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  8. Shanchun Chen
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Correspondence to Aihong Peng.

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Editor: Jessica Rupp

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Peng, A., Zou, X., Xu, L. et al. Improved protocol for the transformation of adult Citrus sinensis Osbeck ‘Tarocco’ blood orange tissues. In Vitro Cell.Dev.Biol.-Plant 55, 659–667 (2019). https://doi.org/10.1007/s11627-019-10011-9

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  • DOI: https://doi.org/10.1007/s11627-019-10011-9

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