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

Montoro Pascal, Rattana Wiparat, Pujade-Renaud Valérie, Michaux-Ferrière Nicole, Monkolsook Yupa, Kanthapura Reena, Adunsadthapong Saisunee. 2003. Production of Hevea brasiliensis transgenic embryogenic callus lines by Agrobacterium tumefaciens: roles of calcium. Plant Cell Reports, 21 (11) : 1095-1102.

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

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Résumé : 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 bêta-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.

Mots-clés Agrovoc : Hevea brasiliensis, Agrobacterium tumefaciens, transformation génétique, calcium, milieu de culture, cal, transfert de gène, antibiotique, néomycine

Mots-clés complémentaires : GUS

Classification Agris : F30 - Génétique et amélioration des plantes

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