Skip to main content
SpringerLink
Log in

Organogenesis and Agrobacterium tumefaciens-mediated transformation of Eucalyptus saligna with P5CS gene

  • Original Papers
  • Published:
Biologia Plantarum

Abstract

The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes Δ1-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots were subsequently cultured on an elongation medium (BE), then transferred to a rooting medium and finally transplanted to pots and acclimatized in a greenhouse. For genetic transformation, a binary vector carrying P5CSF129A and uidA genes, both under control of the 35SCaMV promoter, was used. Leaves were co-cultured with Agrobacterium tumefaciens in the dark on CI medium for 5 d. The explants were transferred to the selective callogenesis inducing medium (SCI) containing kanamycin and cefotaxime. Calli developed shoots that were cultured on an elongation medium for 14 d and finally multiplied. The presence of the transgene in the plant genome was demonstrated by PCR and confirmed by Southern blot analysis. Proline content in the leaves was four times higher in transformed than in untransformed plants while the proline content in the roots was similar in both types of plants.

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

BAP:

benzylaminopurine

BE:

bud elongation medium

BM:

bud multiplication medium

CI:

medium for callogenesis induction from leaf explants

G/MR:

germination and microcutting rooting medium

GA3 :

gibberellic acid

IAA:

indole-3-acetic acid

NAA:

naphthaleneacetic acid

OI:

medium for organogenesis induction from cotyledonary explants

PFD:

photon flux density

SCI:

selective callogenesis induction medium

SE:

shoot elongation medium

SI:

medium for shoot induction from leaf explants

TDZ:

thidiazuron

References

  • Alves, E.C.S.C., Xavier, A., Otoni, W.C.: Organogênese de explante foliar de clones de Eucalyptus grandis × Eucalyptus urophylla. — Pesq. agropec. Bras. 39: 421–430, 2004.

    Google Scholar 

  • Barrueto-Cid, L.P., Machado, A.C.M., Carvalheira, S.R.C., Brasileiro, A.C.M.: Plant regeneration from seedling explants of Eucalyptus grandis × E. urophylla. — Plant Cell Tissue Organ Cult. 56: 17–23, 1999.

    Article  Google Scholar 

  • Bates, L.S., Waldren, R.P., Teare, I.D.: Rapid determination of free proline for water stress studies. — Plant Soil 39: 205–207, 1973.

    Article  CAS  Google Scholar 

  • Brody, J.R., Kern, S.E.: Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. — BioTechnology 36: 214–216, 2004.

    CAS  Google Scholar 

  • Cho, M.A., Moon C.Y., Liu J.R., Choi P.S.: Agrobacteriummediated transformation in Citrullus lanatus. — Biol. Plant. 52: 365–369, 2008.

    Article  CAS  Google Scholar 

  • Dibax, R., Eisfeld, C.deL., Cuquel, F., Koehler, H., Quoirin, M.: Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis. — Scientia Agr. 62: 406–412, 2005.

    Google Scholar 

  • Doyle, J.J., Doyle, J.L.: Isolation of plant DNA from fresh tissue. — Focus 12: 13–15, 1990.

    Google Scholar 

  • Fantini, M., Jr., Graça, M.E.C.: Propagação in vitro de Eucalyptus saligna. — In: 6 Congresso Florestal Brasileiro. Pp. 373–378. Sociedade Brasileira de Silvicultura, São Paulo 1990.

    Google Scholar 

  • Gleeson, D., Walter, M.A.L., Parkinson, M.: Overproduction of proline in transgenic hybrid larch (Larix leptoeuropaea (Dengler) cultures renders them tolerant to cold, salt and frost. — Mol. Breed. 15: 21–29, 2005.

    Article  CAS  Google Scholar 

  • Hervé, P., Jauneau, A., Pâques, M., Marien, J.N., Boudet, A.M., Teulières, C.A.: Procedure for shoot organogenesis in vitro from leaves and nodes of an elite Eucalyptus gunnii clone: comparative histology. — Plant Sci. 161: 645–653, 2001.

    Article  Google Scholar 

  • Ho, C.K., Chang, S.H., Tsay, J.Y., Tsai, C.J., Chiang, V.L., Chen, Z.Z.: Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants. — Plant Cell Rep. 17: 675–680, 1998.

    Article  CAS  Google Scholar 

  • Hong, Z., Lakkineni, K., Zhang, Z., Verma, D.P.S.: Removal of feedback inhibition of Δ1-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. — Plant Physiol. 122: 1129–1136, 2000.

    Article  CAS  PubMed  Google Scholar 

  • Hood, E.E., Gelvin, S.B., Melchers, L.S., Hoekema, A.: New Agrobacterium helper plasmids for gene transfer to plants. — Transgen. Res. 2: 208–218, 1993.

    Article  CAS  Google Scholar 

  • Hur, J., Jung, K.H., Lee, C.H.: Stress-inducible OSP5CS2 gene is essential for salt and cold tolerance in rice. — Plant Sci. 167: 417–426, 2004.

    Article  CAS  Google Scholar 

  • Kavi-Kishor, P.B.K., Hong, Z., Miao, G.-H., Hu, C.A.A., Verma, D.P.S.: Overexpression of Δ1-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. — Plant Physiol. 108: 1387–1394, 1995.

    Google Scholar 

  • Konstantinova, T., Parvanova, D., Atanassov, A., Djilianoiv, D.: Freezing tolerant tobacco, transformed to accumulate osmoprotectants. — Plant Sci. 163: 157–164, 2002.

    Article  CAS  Google Scholar 

  • Lainé, E., David, A.: Regeneration of plants from leaf explants of micropropagated clonal Eucalyptus grandis. — Plant Cell Rep. 13: 473–476, 1994.

    Article  Google Scholar 

  • Le Roux, J.J., Van Staden, J.: Micropropagation of Eucalyptus species. — J. hort. Sci. 26: 199–200, 1991.

    Google Scholar 

  • Maggio, A., Miyazaki, S., Veronese, P., Fujita, T., Ibeas, J.I., Dasm, B., Narasimhan, M.L., Hasegawa, P.M., Joly, R.J., Bressan, R.A.: Does proline accumulation play an active role in stress-induced growth reduction? — Plant J. 31: 699–712, 2002.

    Article  CAS  PubMed  Google Scholar 

  • Mullins, K.V., Llewellyn, D.J., Hartney, V.J., Strauss, S., Dennis, E.S.: Regeneration and transformation of Eucalyptus camaldulensis. — Plant Cell Rep. 16: 787–791, 1997.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Nanjo, T., Fujita, M., Seki, M., Kato, T., Tabata, S., Shinozaki, K.: Toxicity of free proline revealed in an Arabidopsis T-DNA-tagged mutant deficient in proline dehydrogenase. — Plant Cell Physiol. 44: 541–548, 2003.

    Article  CAS  PubMed  Google Scholar 

  • Quoirin, M., Quisen, R.: Advances in genetic transformation of Eucalyptus species. — In: Franche, C. (ed.): Molecular Biology of Tropical Plants. Pp. 41–56. Research Signpost, Kerala 2006.

    Google Scholar 

  • Sambrook, J., Maniatis, T., Fritsch, E.F.: Molecular Cloning: a Laboratory Manual. 2nd Ed. — Cold Spring Harbor Press, New York 1989.

    Google Scholar 

  • Sanatombi, K., Sharma, G.J.: In vitro plant regeneration in six cultivars of Capsicum spp. using different explants. — Biol. Plant. 52: 141–145, 2008.

    Article  CAS  Google Scholar 

  • Saritha, K.V., Naidu, C.V.: Direct shoot regeneration from leaf explants of Spilanthes acmella. — Biol. Plant. 52: 334–338, 2008.

    Article  CAS  Google Scholar 

  • Savouré, A., Hua, X.J., Bertauche, N., Van Montagu, M., Verbruggen, N.: Abscisic acid-independent and abscisic acid-dependent regulation of proline biosynthesis following cold and osmotic stresses in Arabidopsis thaliana. — Mol. Genet. Genom. 254: 104–109, 1997.

    Article  Google Scholar 

  • Sawahel, W.A., Hassan, A.H.: Generation of transgenic wheat plants producing high levels of the osmoprotectant proline. — Biotech. Lett. 4: 721–725, 2002.

    Article  Google Scholar 

  • Sreedhar, R.V., Venkatachalam, L., Thimmaraju, R., Bhagyalakshmi, N., Narayan, M.S., Ravishankar G.A.: Direct organogenesis from leaf explants of Stevia rebaudiana and cultivation in bioreactor. — Biol. Plant. 52: 355–360, 2008.

    Article  CAS  Google Scholar 

  • Zhang, C.S., Lu, Q., Verma, D.S.P.: Removal of feedback inhibition of Δ1-pyrroline-5-carboxylate synthetase, a bifunctional enzyme catalyzing the first 2 steps of praline biosynthesis in plants. — J. biol.Chem. 270: 20491–20496, 1995.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank EMBRAPA Florestas, Colombo, PR, Brazil, for providing seeds, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação Araucária, Paraná, Brazil for grants to R.D.

Author information

Authors and Affiliations

  1. Department of Plant Science, Federal University of Parana, Curitiba, 80035-050, PR, Brazil

    R. Dibax, C. Deschamps & J. C. Bespalhok Filho

  2. Plant Biotechnology Laboratory, IAPAR, Londrina, 86001-970, PR, Brazil

    L. G. E. Vieira, H. B. C. Molinari & M. K. F. De Campos

  3. Department of Botany, Federal University of Parana, P.O. Box 19031, 81531-990, Curitiba, PR, Brazil

    M. Quoirin

Authors
  1. R. Dibax
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C. Deschamps
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. C. Bespalhok Filho
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L. G. E. Vieira
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. H. B. C. Molinari
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M. K. F. De Campos
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M. Quoirin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. Quoirin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dibax, R., Deschamps, C., Bespalhok Filho, J.C. et al. Organogenesis and Agrobacterium tumefaciens-mediated transformation of Eucalyptus saligna with P5CS gene. Biol Plant 54, 6–12 (2010). https://doi.org/10.1007/s10535-010-0002-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10535-010-0002-6

Additional key words

Search

Navigation