Skip to main content
SpringerLink
Log in

High efficient transgenic plant regeneration from embryogenic calluses of Citrus sinensis

  • Original Papers
  • Published:
Biologia Plantarum

Abstract

Transformation and high efficient regeneration of transgenic plants from embryogenic calluses of Bingtang sweet orange [Citrus sinensis (L.) Osbeck] was reported. Embryogenic calluses were inoculated with Agrobacterium tumefaciens strain EHA105, harboring the binary Ti plasmid pROK II and carrying a neomycin phosphotransferase II (NPTII) gene, an intron β-glucuronidase (GUS) gene and the Arabidopsis APETALA1 (AP1) gene. Transformation treatment was with inoculation time of 30 min, co-culture of 3 d at 23 °C and supplementation of the co-culture medium with 2 mg dm−3 acetosyringone (AS). Kanamycin (50 mg dm−3) was effective to inhibit the growth of non-transformed calluses while it did not affect the transformed ones. The total number of transformed callus lines was 7 with 100 % embryo induction. High efficient regeneration of the transgenic embryos (88 % with 4–5 shoots per embryoid) was realized within 3 months. Integration of the transgene into the citrus genome was confirmed by histochemical GUS staining, polymerase chain reaction (PCR) analysis with AP1-specific primer and Southern blot hybridization with a 712 bp PCR fragment of AP1 as the probe.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

AS:

acetosyringone

BA:

6-benzylaminopurine

Cef:

cefotaxime

Cm:

chloromycetin

GUS:

β-glucuronidase

Kin:

kinetin

Km:

kanamycin

LB medium:

Luria-Bertani medium

MT:

Murashige and Tucker

NAA:

α-naphthaleneacetic acid

NPTII:

neomycin phosphotransferase II

PCR:

polymerase chain reaction

References

  • Aráujo, S.S., Duque, A.S.R., Santos, D.M.M.F., Fevereiro, M.P.S.: An efficient transformation method to regenerate a high number of transgenic plants using a new embryogenic line of Medicago truncatula cv. Jemalong.-Plant Cell Tissue Organ Cult. 78: 123–131, 2004.

    Article  Google Scholar 

  • Bespalhok Filho, J.C., Kobayashi, A.K., Pereira, L.F.P., Galvao, R.M., Vieira, L.G.E. Transient gene expression of β-glucuronidase in citrus thin epicotyl transversal sections using particle bombardment.-Braz. Arch. Biol. Technol. 46: 1–6, 2003.

    Article  CAS  Google Scholar 

  • Cervera, M., Orgeta, C., Navarro, L., Pena, L.: Generation of citrus plants with the tolerance-to-salinity gene HAL2 from yeast.-J. Hort. Sci. Biotechnol. 75: 26–30, 2000.

    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: 177, 2003.

    Google Scholar 

  • Domínguez, A., Cervera, M., Pérez, R.M., Romero, J., Fagoaga, C., Cubero, J., López, M.M., Juárez, J.A., Navarro, L., Peña, L.: Characterisation 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. Breed. 14: 171–183, 2004.

    Article  Google Scholar 

  • Fleming, G.H., Olivares-Fuster, O., Fatta Del-Bosco, S., Grosser, J.W.: An alternative method for the genetic transformation of sweet orange.-In Vitro cell. dev. Biol. Plant 36: 450–456, 2000.

    Article  CAS  Google Scholar 

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

    PubMed  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 a juice quality related pectin methylesterase gene.-Plant Cell Rep. 24: 482–486, 2005.

    Article  PubMed  CAS  Google Scholar 

  • Gutiérrez, E.M.A., Luth, D.E., Moore, G.A.: Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of Citrus Tristeza Virus.-Plant Cell Rep. 16: 745–753, 1997.

    Article  Google Scholar 

  • Jefferson, R.A.: Assaying chimeric genes in plants: the Gus gene fusion system.-Plant mol. Biol. 5: 387–405, 1987.

    CAS  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.

    PubMed  CAS  Google Scholar 

  • Mitić, N., Nikolić, R., Ninković, S., Miljuš-Djukić, J., Nešković, M.: Agrobacterium-mediated transformation and plant regeneration of Triticum aestivum L.-Biol. Plant. 48: 179–184, 2004.

    Article  Google Scholar 

  • Molinari, H.B.C., Bespalhok, J.C., Kobayashi, A.K., Pereira, L.F.P., Vieira, L.G.E.: Agrobacterium tumefaciens-mediated transformation of Swingle citrumelo (Citrus paradisi Macf. × Poncirus trifoliata L. Raf.) using thin epicotyl sections.-Sci. Hort. 99: 379–385, 2004.

    Article  CAS  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.-Proc. 1st Citrus Symp. 1155–1161, 1969.

  • Niedz, R.P., McKendree, W.L., Shatters. R.J.: Electroporation of embryogenic protoplasts of sweet orange (Citrus sinensis (L.) Osbeck) and regeneration of transgenic plants.-In Vitro cell. dev. Biol. Plant 39: 586–594, 2003.

    Article  CAS  Google Scholar 

  • Pena, L., Cervera, M., Juarez, J., Navarro, A., Pina, J.A., Navarro, L.: Genetic transformation of lime: factor affecting transformation and regeneration.-Plant Cell Rep. 16: 731–737, 1997.

    Article  CAS  Google Scholar 

  • Pena, L., Martin-Trillo, M., Juarez, J., Pina, J.A., Navarro, L., Martinez-Zapater, J.M.: Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time.-Nature Biotechnol. 19: 263–267, 2001.

    Article  CAS  Google Scholar 

  • Pena, L., Perez, R.M., Cervera, M., Juarez, J.A., Navarro, L.: Early events in Agrobacterium-mediated genetic transformation of citrus explants.-Ann. Bot. 94: 67–74, 2004.

    Article  PubMed  CAS  Google Scholar 

  • Perez-Molphe-Balch, E., Ochoa-Alejo, N.: Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes-transformed tissues.-Plant Cell Rep. 17: 591–596, 1998.

    Article  CAS  Google Scholar 

  • Pillitter, L.J., Walling, L.L., Lovatt, C.A.: Regulation of flowering in the Washington navel orange: floral genes.-Proc. int. Soc. Citricult. 1: 201–204, 2003.

    Google Scholar 

  • Yu, C.H., Huang, S., Chen, C., Deng, Z.A., Ling, P., Gmitter, F.G., Jr.: Factors affecting Agrobacterium-mediated transformation of sweet orange and citrange.-Plant Cell Tissue Organ Cult. 71: 147–155, 2002.

    Article  CAS  Google Scholar 

Download references

Author information

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

    Y. X. Duan, W. W. Guo, H. J. Meng, N. G. Tao, D. D. Li & X. X. Deng

Authors
  1. Y. X. Duan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. W. W. Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H. J. Meng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. N. G. Tao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D. D. Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. X. X. Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to W. W. Guo.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Duan, Y.X., Guo, W.W., Meng, H.J. et al. High efficient transgenic plant regeneration from embryogenic calluses of Citrus sinensis . Biol Plant 51, 212–216 (2007). https://doi.org/10.1007/s10535-007-0043-7

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10535-007-0043-7

Additional key words

Search

Navigation