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Regeneration of transgenic citrus plants from the trimmed shoot/root region of etiolated seedlings

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Biologia Plantarum

Abstract

Transformation and high efficient regeneration of transgenic plants from the trimmed etiolated shoot/root region (TESRR) of Anliucheng sweet orange [Citrus sinensis (L.) Osb.] seedling was reported. A visual green fluorescent protein (GFP) marker gene was introduced to evaluate transformation efficiency by using the explants from TESRR and epicotyls. The transformation protocol was: infection 20 min, co-culture 3 d, selection culture 30 d, and rooting 15 d. Out of a total of 288 sprouted shoots obtained from TESRR, 34 shoots (11.8 %) yielded GFP expression. In contrast, only 2 (3.0 %) of the 67 sprouted shoots from epicotyl transformation yielded GFP expression. In all plants showing the green fluorescence an expected 500 bp GFP fragment was proved by PCR analysis. Southern blot analysis further confirmed the integration of GFP gene into citrus genome. Transgenic plantlets were obtained within 80 d using the TESRR, compared within 150 d by using epicotyls.

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Abbreviations

TESRR:

trimmed etiolated shoot/root region

AS:

acetosyringone

BA:

6-benzyladenine

GFP:

green fluorescent protein

GUS:

β-glucuronidase

IBA:

indole butyric acid

KIN:

kinetin

MT medium:

Murashige and Tucker (1969) medium

NAA:

α-napthaleneacetic acid

NPT II:

neomycin phosphotransferase II

References

  • Ballester, A., Cervera, M., Peña, L.: Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination. — Plant Cell Rep. 26: 39–45, 2007.

    Article  CAS  PubMed  Google Scholar 

  • Cai, X.D., Liu, X., Guo W.W.: GFP expression as an indicator of somatic hybrids between transgenic Satsuma mandarin and calamondin at embryoid stage. — Plant Cell Tissue Organ Cult. 87: 245–253, 2006.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Cheng, Y.J., Guo, W.W., Yi, H.L., Pang, X.M., Deng, X.X.: An efficient protocol for genomic DNA extraction from citrus species. — Plant mol. Biol. Rep. 21: 117a–117g, 2003.

    Article  Google Scholar 

  • Chiu, W.L., Niwa, Y., Zeng, W.K., Hirano, T., Kobayashi, H., Sheen, J.: Engineered GFP as a vital reporter in plants. — Curr. Biol. 6: 325–330, 1996.

    Article  CAS  PubMed  Google Scholar 

  • Dominguez, A., Fagoaga, C., Navarro, L., Moreno, P., Peña, L.: Regeneration of transgenic citrus plants under non selective conditions results in high-frequency recovery of plants with silenced transgenes. — Mol. Genet. Genomics 267: 544–556, 2002.

    Article  CAS  PubMed  Google Scholar 

  • Duan, Y.X., Guo, W.W., Meng, H.J., Tao, N.G., Li, D.D., Deng, X.X.: High efficient transgenic plant regeneration from embryogenic calluses of Citrus sinensis. — Biol. Plant. 51: 212–216, 2007a.

    Article  CAS  Google Scholar 

  • Duan, Y.X., Liu, X., Fan, J., Li, D.L., Wu, R.C., Guo, W.W.: Multiple shoot induction from seedling epicotyls and transgenic citrus plant regeneration containing the green fluorescent protein gene. — Bot. Stud. 48: 165–171, 2007b.

    Google Scholar 

  • EI-Shemy, H., Khalafalla, M., Fujita, K., Ishimoto, M.: Improvement of protein quality in transgenic soybean plants. — Biol. Plant. 51: 277–284, 2007.

    Article  Google Scholar 

  • Ghorbel, R., Juarez, J., Navarro, L., Peña, L.: Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants. — Theor. appl. Genet. 99: 350–358, 1999.

    Article  Google Scholar 

  • Guo, W.W., Cheng, Y.J., Deng, X.X.: Regeneration and molecular characterization of intergeneric somatic hybrids between Citrus reticulata and Poncirus trifoliata. — Plant Cell Rep. 20: 829–834, 2002.

    Article  CAS  Google Scholar 

  • Guo, W.W., Duan, Y.X., Olivares-Fuster, O., Wu, Z.C., Arias, C.R., Burns, J.K., Grosser, J.W.: Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing the juice quality related pectin methylesterase gene. — Plant Cell Rep. 24: 482–486, 2005.

    Article  CAS  PubMed  Google Scholar 

  • Guo, W.W., Grosser, J.W.: Somatic hybrid vigor in Citrus: direct evidence from protoplast fusion of an embryogenic callus line with a transgenic mesophyll parent expression the GFP gene. — Plant Sci. 168: 1541–1545, 2005.

    Article  CAS  Google Scholar 

  • Haseloff, J., Siemering, K.R., Prasher, D.C., Sarah, H.: Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. — Proc. nat. Acad. Sci. USA 94: 2122–2127, 1997.

    Article  CAS  PubMed  Google Scholar 

  • Khawale, R.N., Singh, S.K., Garg, G., Baranwal, V.K., Ajirlo, S.A.: Agrobacterium-mediated genetic transformation of Nagpur mandarin (Citrus reticulata Blanco). — Curr. Sci. 91: 1700–1705, 2006.

    CAS  Google Scholar 

  • Kobayashi, S., Uchimiya, H.: Expression and integration of a foreign gene in orange (Citrus sinensis Osb.) protoplasts by direct DNA transfer. — Jap. J. Genet. 64, 91–97: 1989.

    Article  Google Scholar 

  • Li, D.D., Shi, W., Deng, X.X.: 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, 2002.

    Article  CAS  Google Scholar 

  • Li, D.D., Shi, W., Deng, X.X.: Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene. — Tree Physiol. 23: 1209–1215, 2003.

    CAS  PubMed  Google Scholar 

  • Moore, G.A., Jacono, C.C., Neidigh, J., Lawrence, S.D., Cline, K.: Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants. — Plant Cell Rep. 11: 238–242, 1992.

    Article  CAS  Google Scholar 

  • Murashige, T., Tucker, D.P.H.: Growth factor requirements of citrus tissue culture. — In: Chapman, H.D. (ed.): Proceedings of the 1st Citrus Symposium. Pp. 1155–1161. Riverside 1969.

  • Olivares-Fuster, O., Duran-Vila, N., Navarro, L.: Green fluorescent protein as a visual marker in somatic hybridization. — Ann. Bot. 89: 491–497, 2002.

    Article  CAS  PubMed  Google Scholar 

  • Orbovic, V., Grosser, J.W.: Citrus. — Methods mol. Biol. 344: 177–189, 2006.

    CAS  PubMed  Google Scholar 

  • Peña, L., Cervera, M., Juarez, J., Navarro, A., Pina, J.A., Duran-Vila, N.: Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants. — Plant Cell Rep. 14: 616–619, 1995.

    Article  Google Scholar 

  • Suwanaketchanatit, C., Piluek, J., Peyachoknagul, S., Huehne, P.S.: High efficiency of stable genetic transformation in Dendrobium via microprojectile bombardment. — Biol. Plant. 51: 720–727, 2007.

    Article  CAS  Google Scholar 

  • Uzelac, B., Ninkovic, S., Smigocki, A., Budimir. S.: Origin and development of secondary somatic embryos in transformed embryogenic cultures of Medicago sativa. — Biol. Plant. 51: 1–6, 2007.

    Article  Google Scholar 

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan, 430070, P.R. China

    D. L. Li, B. Tan, Y. X. Duan & W. W. Guo

Authors
  1. D. L. Li
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  2. B. Tan
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  3. Y. X. Duan
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  4. W. W. Guo
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Corresponding author

Correspondence to W. W. Guo.

Additional information

Acknowledgements: This research was financially supported by the national NSF of China, the Ministry of Education (20060504014), the National 863 project of China (2007AA10Z182), and the NSF of Hubei province of China (2008CDA069).

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Li, D.L., Tan, B., Duan, Y.X. et al. Regeneration of transgenic citrus plants from the trimmed shoot/root region of etiolated seedlings. Biol Plant 53, 578–582 (2009). https://doi.org/10.1007/s10535-009-0105-0

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  • DOI: https://doi.org/10.1007/s10535-009-0105-0

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