Improvement of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.) using the temporary immersion technique

  • H. Etienne
  • M. Lartaud
  • N. Michaux-Ferriére
  • M. P. Carron
  • M. Berthouly
  • C. Teisson
Developmental Biology/Morphogenesis


A culture procedure using temporary immersion in a liquid medium was tested for somatic embryogenesis of Hevea brasiliensis (Müll. Arg.). Embryogenic callus was placed under regeneration conditions, either on a gelled medium (Phytagel, Sigma, St. Louis, MO) or in a container designed for temporary immersion. The latter technique has some advantages over the use of a gelled medium during both the early steps of somatic embryogenesis, i.e., embryo development, and later on, i.e., during maturation, desiccation and germination. Somatic embryo production in a liquid medium was three to four times greater than on a semi-solid medium: 400 embryos/g fresh weight under the best embryogenesis induction conditions. Somatic embryogenesis had to be initiated on a gelled medium before the embryogenic callus was transferred to temporary immersion, and the amounts of 3,4- dichlorophenoxyacetic acid and N6-benzyladenine had to be reduced. Temporary immersion resulted in substantially more consistent, synchronized somatic embryo development, reducing the number of abnormal embryos by half and stimulating germination. All of the late events could be carried out in the temporary immersion container. Effective drying conditions were achieved after 12 wk without immersion and without selection of the embryos. Temporary immersion during germination greatly stimulated root development (+60%) and epicotyl emergency (+35%), combined with increased synchronization and a substantially reduced workload.

Key words

rubber tree liquid culture embryo development plant regeneration 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Akita, M.; Takayama, S. Stimulation of potato (Solanum tuberosum L.) tuberization by semi continuous liquid medium surface level control. Plant Cell Rep. 13:184–187; 1994.Google Scholar
  2. Aitken-Christie, J.; Jones, C. Towards automation: radiata pine shoot hedges in vitro. Plant Cell Tissue Organ Cult. 8:185–196; 1987.CrossRefGoogle Scholar
  3. Alvard, D.; Cote, F.; Teisson, C. Comparison of methods of liquid medium culture for banana micropropagation. Effects of temporary immersion of explants. Plant Cell Tissue Organ Cult. 32:55–60; 1993.CrossRefGoogle Scholar
  4. Ammirato, P. V.; Styer, D. J. Strategies for large scale production of somatic embryos in suspension culture. In: Zaitlin, M.; Day, P.; Hollaender, A., ed. Biotechnology in plant science: relevance to agriculture in the 1980’s. New York: Academic Press; 1985:161–178.Google Scholar
  5. Cabasson, C. Régénération de Citrus deliciosa Ten. par embryogenèse somatique en milieu liquide. Fusions somatiques et essais de transformation génétique. Thèse Université Montpellier II; 1993.Google Scholar
  6. Carron, M. P.; Etienne, H.; Lardet, L., et al. Somatic embryogenesis in rubber (Hevea brasiliensis Müll. Arg.). In: Jain, S.; Gupta, P.; Newton, R., ed. Somatic embryogenesis in woody plants, Vol. 2. The Netherlands: Kluwer Academic Publishers; 1995a:117–136.Google Scholar
  7. Carron, M. P.; Etienne, H.; Michaux-Ferrière, N., et al. Somatic embryogenesis in rubber tree (Hevea brasiliensis Müll. Arg.). In: Bajaj, Y. P. S., ed. Biotechnology in agriculture and forestry, Vol. 30. Somatic embryogenesis and synthetic seed I. Berlin: Springer-Verlag Heidelberg; 1995b:353–369.Google Scholar
  8. Connor, A.; Meredith, C. P. An improved polymethane support system for monitoring growth in plant cell cultures. Plant Cell Tissue Organ Cult. 3:59–68; 1984.CrossRefGoogle Scholar
  9. Debergh, P. Effects of agar brand and concentration on the tissue culture medium. Physiol. Plant. 59:270–276; 1983.CrossRefGoogle Scholar
  10. Debergh, P.; Harbaooui, Y.; Lemeur, R. Mass propagation of globe artichoke (Cynara scolymus)/evaluation of different hypotheses to overcome vitrification with special reference to water potential. Physiol. Plant. 53:181–187; 1981.CrossRefGoogle Scholar
  11. Debergh, P. C.; Maene, L. J. A scheme for commercial propagation of ornamental plants by tissue culture. Scientia Hort. 14:335–345; 1981.CrossRefGoogle Scholar
  12. El Hadrami, I.; Carron, M. P.; d’Auzac, J. Influence of exogenous hormones on somatic embryogenesis in Hevea brasiliensis. Ann. Bot. 67:511–515; 1991.Google Scholar
  13. Escalant, J. V.; Teisson, C.; Côte, F. Amplified somatic embryogenesis from male flowers of triploid banana and plantain cultivars (Musa sp.). In Vitro Cell. Dev. Biol. 30P:181–186; 1994.Google Scholar
  14. Etienne, H.; Berger, A.; Carron, M. P. Water status of callus from Hevea brasiliensis during induction of somatic embryogenesis. Physiol. Plant. 82:213–218; 1991.CrossRefGoogle Scholar
  15. Etienne, H.; Montoro, P.; Michaux-Ferrière, N., et al. Effects of desiccation, medium osmolarity, and abscisic acid on the maturation of Hevea brasiliensis somatic embryos. J. Exp. Bot. 44:1613–1619; 1993.CrossRefGoogle Scholar
  16. Faure, A. Embryons somatiques de Vitis rupestris et embryons zygotiques de Vitis sp.: morphologie, histochimie et développement. Can. J. Bot. 68:2305–2315; 1990.Google Scholar
  17. Fisher, D. B. Protein staining of ribboned epon sections for light microscopy. Histochemie 16:92–96; 1968.PubMedCrossRefGoogle Scholar
  18. Goebel-Tourand, I.; Mauro, M.-C.; Sossountzov, L., et al. Arrest of somatic embryo development in grapevine: histological characterization and the effect of ABA, BAP and Zeatin in stimulating plantlet development. Plant Cell Tissue Organ Cult. 33:91–103; 1993.CrossRefGoogle Scholar
  19. Homès, J. La préparation des tissus végétaux pour l’observation au microscope électronique à balayage. Bull. Soc. Roy. Bot. Belg. 108:219–231; 1975.Google Scholar
  20. Hussey, G. Problems and prospects in the in vitro propagation of herbaceous plants. In: Withers, L. A.; Alderson, P. G., ed. Plant tissue culture and its agricultural applications. Boston: Butterworths; 1986:69–84.Google Scholar
  21. Lelu, M. A.; Bastien, C.; Klimaszewska, K., et al. An improved method for somatic plantlet production in hybrid larch (Larix × leptoeuropaea): Part 1. Somatic embryo maturation. Plant Cell Tissue Organ Cult. 36:107–115; 1994.CrossRefGoogle Scholar
  22. Maene, L.; Debergh, P. Liquid medium additions to established tissue cultures to improve elongation and rooting in vivo. Plant Cell Tissue Organ Cult. 5:23–33; 1985.CrossRefGoogle Scholar
  23. Michaux-Ferrière, N.; Grout, H.; Carron, M. P. Origin and ontogenesis of somatic embryos in Hevea brasiliensis. Am. J. Bot. 79:174–180; 1992.CrossRefGoogle Scholar
  24. Montoro, P.; Etienne, H.; Michaux-Ferrière, N., et al. Callus friability and somatic embryogenesis in Hevea brasiliensis. Plant Cell Tissue Organ Cult. 33:331–338; 1993.CrossRefGoogle Scholar
  25. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–479; 1962.CrossRefGoogle Scholar
  26. Pétiard, V.; Ducos, J. P.; Florin, B., et al. Production en masse et conservation des embryons somatiques. Le sélectionneur français 43:65–75; 1993.Google Scholar
  27. Preil, W.; Beck, A. Somatic embryogenesis in bioreactor culture. Acta Hort. 289:179–192; 1991.Google Scholar
  28. Roberts, A. V.; Smith, E. F. The preparation in vitro of chrysanthemum for transplantation to soil. I. Protection of roots by cellulose plugs. Plant Cell Tissue Organ Cult. 21:129–132; 1990.CrossRefGoogle Scholar
  29. Simonton, W.; Robacker, C.; Krueger, S. A programmable micropropagation apparatus using cycled liquid medium. Plant Cell Tissue Organ Cult. 27:211–218; 1991.CrossRefGoogle Scholar
  30. Stuart, D. A.; Strickland, S. G.; Walker, K. A. Bioreactor production of alfalfa somatic embryos. HortScience 22:800–803; 1987.Google Scholar
  31. Tautorus, T. E.; Fowke, L. C.; Dunstan, D. I. Somatic embryogenesis in conifers. Can. J. Bot. 69:1873–1899; 1991.Google Scholar
  32. Tautorus, T. E.; Lulsdorf, M. M.; Kikcio, S. I., et al. Bioreactor culture of Picea mariana Mill. (black spruce) and the species complex Picea glauca-engelmannii (interior spruce) somatic embryos. Growth parameters. Appl. Microbiol. Biotechnol. 38:46–51; 1992.Google Scholar
  33. Teisson, C.; Alvard, D. A new concept of plant in vitro cultivation liquid medium: temporary immersion. In: Terri, M.; Cella, R.; Falavigna, A., ed. Current issues in plant molecular and cellular biology. Dordrecht/Boston/London: Kluwer Academic Publishers; 1995:105–110.Google Scholar
  34. Wheathers, P. J.; Giles, K. L. Regeneration of plants using nutrient mist culture. In Vitro Cell. Dev. Biol. 24:727–732; 1988.CrossRefGoogle Scholar
  35. Ziv, M.; Meir, G.; Halevy, A. H. Factors influencing the production of hardened glaucous carnation plantlets in vitro. Plant Cell Tissue Organ Cult. 2:55–65; 1983.CrossRefGoogle Scholar

Copyright information

© Society for In Vitro Biology 1997

Authors and Affiliations

  • H. Etienne
    • 1
  • M. Lartaud
    • 1
  • N. Michaux-Ferriére
    • 1
  • M. P. Carron
    • 1
  • M. Berthouly
    • 1
  • C. Teisson
    • 1
  1. 1.Laboratoire BIOTROPCIRAD (Centre de Coopération Internationale en Recherche Agronomique pour le Développement)Montpellier CedexFrance

Personalised recommendations