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Production of Hevea brasiliensis transgenic embryogenic callus lines by Agrobacterium tumefaciens: roles of calcium

  • Genetic Transformation and Hybridization
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Abstract

A procedure has been established for Agrobacterium tumefaciens-mediated genetic transformation of Hevea brasiliensis embryogenic friable calli. Precultivation of tissues on a CaCl2-free maintenance medium dramatically enhanced the transient activity of the reporter gene, gusA encoding β-glucuronidase (GUS). The increase was first noticed in highly active cells (undifferentiated or/and embryogenic), in tissues precultured for 2–8 weeks. Beyond 8 weeks of preculture, GUS activity increased again, but this time in tissues consisting of differentiated cells accumulating polyphenols. Out of five Agrobacterium strains cocultivated with CaCl2-free precultured tissues, only inoculation with EHA105pC2301 led to high transient GUS activity. Paromomycin proved more effective than kanamycin for the selection of transformed cells, as it inhibits the growth of non-transformed cells more radically. Five paromomycin-resistant callus lines were established. The presence of gusA and neomycin phosphotransferase (nptII) genes in the plant genome was confirmed by DNA amplification, and by Southern hybridization. These results confirmed that A. tumefaciens is an effective system for mediating stable transformation of rubber tree calli with a low copy number of transgenes. Transgenic callus lines constitute a useful tool for studying genes of interest on a cellular level and for regenerating transgenic rubber trees.

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Abbreviations

DM:

Decontamination medium

FM :

Fresh matter

GUS :

β-Glucuronidase

LSD:

Least significant difference

MM :

Maintenance medium

NBB :

Naphthol blue-black stain

PAS :

Periodic acid-Schiff stain

PCR:

Polymerase chain reaction

RGR :

Relative growth rate

SDS :

Sodium dodecyl sulphate

TCL :

Transgenic callus line

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Acknowledgements

This work was supported by Franco-Thai co-operation between CIRAD, DTEC, Department of Agriculture/Rubber Research Institute of Thailand, Kasetsart University and the French Embassy in Bangkok. We would like to thank Associate Professor Vichai Haruthaithanasan, Director of KAPI-KU, and Associate Professor Klanarong Sriroth, Head of CSTR unit, for hosting the Rubber Tree Genetic Engineering Research Programme at KAPI. We also thank Dr. Marc-Philippe Carron and Dr. Ludovic Lardet (CIRAD, France) for providing friable callus lines, Ms. Kanikar Therawattanasuk (RRIT, Thailand) for providing rubber tree fruits, Dr. Richard Jefferson (CAMBIA, Australia) for providing the pCAMBIA2301 vector and the EHA105 Agrobacterium tumefaciens strain. Finally, the authors are grateful to Mrs. Dolores Triaire for her technical assistance.

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Authors and Affiliations

  1. CIRAD-CP, Rubber Programme, BP 5035, 34032, Montpellier, France

    P. Montoro & V. Pujade-Renaud

  2. KAPI, Kasetsart University, 10900, Bangkok , Thailand

    W. Rattana & Y. Monkolsook

  3. BIOTROP, CIRAD, BP 5035, 34032, Montpellier, France

    N. Michaux-Ferrière

  4. DORAS Centre, KAPI, Kasetsart University, 10900, Bangkok , Thailand

    R. Kanthapura & S. Adunsadthapong

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  1. P. Montoro
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  2. W. Rattana
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  3. V. Pujade-Renaud
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  4. N. Michaux-Ferrière
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  5. Y. Monkolsook
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  6. R. Kanthapura
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  7. S. Adunsadthapong
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Correspondence to P. Montoro.

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Communicated by P. Debergh

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Montoro, P., Rattana, W., Pujade-Renaud, V. et al. Production of Hevea brasiliensis transgenic embryogenic callus lines by Agrobacterium tumefaciens: roles of calcium. Plant Cell Rep 21, 1095–1102 (2003). https://doi.org/10.1007/s00299-003-0632-7

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  • DOI: https://doi.org/10.1007/s00299-003-0632-7

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