Temporary immersion system: a new concept for use liquid medium in mass propagation

  • M. Berthouly
  • H. Etienne


Mass propagation of plants by tissue culture is labour intensive and costly. Gelling agents have many drawbacks: they are not inert medium components and do not enable easy automation for commercial mass propagation. So liquid culture systems are considered to have advantages, e.g. culture conditions are much more uniform, media can be changed easily. The use of liquid medium for in vitro culture has many advantages and has been the subject of many studies over many years. It has also frequently been considered an ideal technique for mass production as it reduces manual labor and facilitates changing the medium composition. Techniques and culture vessels of varying complexity have been developed as a result of studies.

The major disadvantage of a liquid medium is hyperhydricity, which is a severe physiological disorder. So we considered that to compensate for this problem it would be necessary to expose the plant to the liquid medium intermittently rather than continuously. For this the bioreactors previously developed are not suitable as they are mainly adapted to bacterial culture and do not take into account the specific requirements of plant cells and tissues, such as sensitivity to shear forces, mechanical damages or foam formation in bubble aerated bioreactors.

So temporary immersion systems for plant micropropagation have been described and grouped into 4 categories according to operation: i) tilting and rocker machines, ii) complete immersion of plant material and renewal of nutrient medium, iii) partial immersion and a liquid nutrient renewal mechanism, iiii) complete immersion by pneumatic driven transfer of liquid medium and without nutrient medium renewal. The positive effects of temporary immersion on micropropagation are indicated for shoot proliferation and microcuttings, microtuberization and somatic embryogenesis. Immersion time, i.e. duration or frequency, is the most critical parameter for system efficiency. Optimizing the volume of nutrient medium and the volume of container also substancially improves efficiency, especially for shoot proliferation. Temporary immersion also generally improves plant tissue quality. It results in increased shoot vigour and quantity of morphologically normal somatic embryos. Hyperhydricity, which seriously affects cultures in liquid medium, is eliminated with these culture systems or controlled by adjusting the immersion times.

Plant material propagated by temporary immersion performs better during the acclimatization phase than material obtained on semi-solid or liquid media. Successful regeneration of Solanum tuberosum microtubers and Coffea arabica somatic embryos produced in temporary immersion bioreactors after direct sowing on soil has been demonstrated. As was predicted, when using liquid medium for micropropagation, several investigations have confirmed large gains in efficiency from temporary immersion. The parameters most involved in reducing production costs are, firstly a large reduction in labour, followed by a reduction in shelving area requirement and the number of containers used, along with better biological yields. Scaling up embryogenesis and shoot proliferation procedures involving temporary immersion systems are now taking place, in order to commercialize this process. To improve this system as well in research as in commercial production, CIRAD has developed a new simple and specific apparatus for plant tissue culture using temporary immersion in liquid medium.

Key words

acclimatization bioreactor hyperhydricity organogenesis shoot proliferation somatic embryogenesis temporary immersion 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Aitken-Christe J & Davies HE (1988) Development of a semi-automated micropropagation system. Acta Hort. 230: 81–87Google Scholar
  2. Aitken-Christie J, Jones C & Bond S (1985) Wet and waxy shoots in radiata pine micropropagation. Acta Hortic. 166: 93–100Google Scholar
  3. Aitken-Christie J & Jones C (1987) Towards automation: radiata pine shoot hedges in vitro. Plant Cell, Tiss. Org. Cult. 8: 185–196Google Scholar
  4. Akita M & Takayama S (1994) Stimulation of potato (Solanum tuberosum L.) tuberization by semicontinuous liquid medium surface level control. Plant Cell Rep. 13: 184–187Google Scholar
  5. Alvard D, Côte F & Teisson C (1993) Comparison of methods of liquid medium culture for banana micropropagation. Effects of temporary immersion of explants. Plant Cell, Tiss. Org. Cult. 32: 55–60Google Scholar
  6. Berthouly M, Dufour M, Alvard D, Carasco C, Alemano L & Teisson C (1995) Coffee micropropagation in a liquid medium using the temporary immersion technique. In: ASIC Publishers (eds) 16th International Scientific Colloquium on Coffee, Kyoto, Japon (pp. 514–519). Vevey, SwitzerlandGoogle Scholar
  7. Berthouly M & Etienne H (1999) Somatic embryogenesis of coffee. In: Jain SM, Gupta PK & Newton RJ (eds) Somatic Embryogenesis in Woody Plants, Vol. 5 (pp. 259–288). Kluwer Academic Publishers, LondonGoogle Scholar
  8. Cabasson C, Alvard D, Dambier D, Ollitrault P & Teisson C (1997) Improvement of Citrus somatic embryo development by temporary immersion. Plant Cell, Tiss. Org. Cult. 50: 33–37Google Scholar
  9. Chu I (1995) Economic analysis of automated micropropagation. In: Aitken-Christie J, Kozai T, Smith MAL (eds) Automation and Environmental Control in Plant Tissue Culture (pp. 19–27). Kluwer Academic Publischers, DordrechtGoogle Scholar
  10. Connor AJ & Meredith CP (1984) An improved polyurethane support system for monitoring growth in plant cell cultures. Plant Cell, Tiss. Org. Cult. 3: 59–68Google Scholar
  11. Debergh P (1988) Improving mass propagation of in vitro plantlets. In: Kozai T (ed) Horticulture in High Technology Era (pp. 45–57). International Symposium on High Technology in Protected Cultivation, TokyoGoogle Scholar
  12. Debergh P, Harbaooui Y & Lemeur R (1981) Mass propagation of globe artichoke (Cynara scolymus) Evaluation of different hypotheses to overcome vitrification with special reference to water potential. Physiol. Plant. 53: 181–187Google Scholar
  13. Escalant J-V, Teisson C & Côte F (1994) Amplified somatic embryogenesis from male flowers of triploid banana and plantain cultivars (Musa spp.). In Vitro Cell. Dev. Biol. 30: 181–186Google Scholar
  14. Escalona M, Lorenzo JC, González B, Daquinta M, Fundora Z, Borroto CG, Espinosa D, Arias E & Aspiolea ME (1998) New system for in vitro propagation of pineapple (Ananas comosus (L.) Merr). Pineapple News 5: 5–7Google Scholar
  15. Escalona M, Lorenzo JC, González B, Daquinta M, González JL, Desjardins Y & Borroto CG (1999) Pineapple (Ananas comosus L. Merr) micropropagation in temporary immersion systems. Plant Cell Rep. 18: 743–748Google Scholar
  16. Etienne H, Berger A & Carron MP (1991) Water status of callus from Hevea brasiliensis during induction of somatic embryogenesis. Physiol. Plant. 82: 213–218Google Scholar
  17. Etienne H, Bertrand B, Anthony F, Côte F & Berthouly M (1997a) L’embryogenèse somatique: un outil pour l’amélioration génétique du caféier. In: ASIC Publishers (eds) 17th International Scientific Colloquiun on Coffee, Nairobi, Kenya (pp. 457–465). Vevey, SwitzerlandGoogle Scholar
  18. Etienne H, Lartaud M, Michaux-Ferrière N, Carron MP, Berthouly M & Teisson C (1997b) Improvement of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.) using the temporary immersion technique. In Vitro Cell. Dev. Biol.-Plant. 33: 81–87Google Scholar
  19. Etienne-Barry D, Bertrand B, Vásquez N & Etienne H (1999) Direct sowing of Coffea arabica somatic embryos mass-produced in a bioreactor and regeneration of plants. Plant Cell Rep. 19: 111–117Google Scholar
  20. Gawel NJ & Robacker CD (1990) Somatic embryogenesis in two Gossypium hirsutum genotypes on semi-solid versus liquid proliferation media. Plant Cell, Tiss. Org. Cult. 23: 201–204Google Scholar
  21. Hamilton R, Pederson H & Chin CK (1985) Plant tissue culture on membrane rafts. Bio Techniques. March/April, p. 96Google Scholar
  22. Hammerschlag F (1982) Factors affecting establishment and growth of peach shoots in vitro. HortScience 17: 85–86Google Scholar
  23. Harris RE & Mason EB (1983) Two machines for in vitro propagation of plants in liquid media. Can. J. Plant Sci. 63: 311–316Google Scholar
  24. Harris RE & Stevenson JH (1982) In vitro propagation of Vitis. Vitis 21: 22–32Google Scholar
  25. Hussey G (1986) Problems and prospects in the in vitro propagation of herbaceous plants. In: Withers LA & Alderson PG (eds) Plant Tissue Culture and its Agricultural Applications (pp. 69–84). Butterworths, BostonGoogle Scholar
  26. Jiménez E, Pérez J, Gil V, Herrera J, García Y & Alonso E (1995) Sistema para la propagación de la caña de azucar. In: Estrade M, Riego E, Limonta E, Tellez P & Fuente J (eds) Avances en Biotecnología Moderna, Vol 3 (pp. 11–20). Elfos Scientiae, CubaGoogle Scholar
  27. Jones AM & Petolino JF (1988) Effects of support medium on embryo and plant production from cultured anthers of soft-red winter wheat. Plant Cell, Tiss. Org. Cult. 12: 243–261Google Scholar
  28. Kitto SL (1997) Commercial micropropagation. Hort. Sci. 32: 1012–1014Google Scholar
  29. Krueger S, Robacker C & Simonton W (1991) Culture of Amelanchier x grandiflora in a programmable micropropagation apparatus. Plant Cell, Tiss. Org. Cult. 27: 219–226Google Scholar
  30. Liu CM, Xu Z-H & Chua N-H (1993) Auxin polar transport is essential for the establishment of bilateral symmetry during early plant embryogenesis. The Plant Cell 5: 621–630PubMedGoogle Scholar
  31. Lorenzo JC, Gonzalez BL, Escalona M, Teisson C, Espinosa P & Borroto C (1998) Sugarcane shoot formation in an improved temporary immersion system. Plant Cell, Tiss. Org. Cult. 54: 197–200Google Scholar
  32. Maene L & Debergh P (1985) Liquid medium additions to established tissue cultures to improve elongation and rooting in vivo. Plant Cell, Tiss. Org. Cult. 5: 23–33Google Scholar
  33. Monette PL (1983) Influence of size of culture vessel on in vitro proliferation of grape in a liquid medium. Plant Cell Tiss. Org. Cult. 2: 327–332Google Scholar
  34. Noriega C & Söndahl MR (1993) Arabica coffee micropropagation through somatic embryogenesis via bioreactors. In: ASIC publishers (eds) 15th International Scientific Colloquium on Coffee, Montpellier, France (pp. 73–81). Vevey, SwitzerlandGoogle Scholar
  35. Reuther G (1985) Principles and application of the micropropagation of ornemental plants. In: Schäfer-Menuhr A (ed) In Vitro Techniques: Propagation and Long-Term Storage (pp. 1–14). Martinus Nijhoff, DordrechtGoogle Scholar
  36. Simonton W & Robacker C (1988) Alternative system for micropropagation. Amer. Soc. Agr. Engineers, Technical Paper No 88-1028Google Scholar
  37. Simonton W, Robacker C & Krueger S (1991) A programmable micropropagation apparatus using cycled medium. Plant Cell, Tiss. Org. Cult. 27: 211–218Google Scholar
  38. Sluis CJ & Walker KA (1985) Commercialization of plant tissue culture propagation. Intl. Assoc. Plant Tiss. Cult. Newsl. 47: 2–12Google Scholar
  39. Smith DR (1985) Pinus radiata. In: Bajaj YPS (ed) Trees. Biotechnology in Agriculture and Forestry, Vol 1 (pp. 274–291). Springer Verlag, BerlinGoogle Scholar
  40. Smith MAL & Spomer LA (1995) Vessels, gels, liquid media and support systems. In: Aitken-Christie J, Kozai T & Smith MAL(eds) Automation and Environmental Control in plant tissue culture (pp. 371–405). Kluwer Academic Publishers, DordrechtGoogle Scholar
  41. Söndhal SP, Nakamicra F, Medince Filho MP, Carvalho A, Fazuoli LC & Caslo WM (1989) Coffee Handbook of Plant Cell Culture, Vol III, Chap. 21Google Scholar
  42. Stevenson JH & Harris RE (1980) In vitro plantlet formation from shoot tip explants of Fuchsia hybrida cv. Swingtime. Can. J. Bot. 58: 2190–2192Google Scholar
  43. Teisson C & Alvard D (1995) A new concept of plant in vitro cultivation liquid medium: temporary immersion. In: Terzi M et al. (eds) Current Issues in Plant Molecular and Cellular Biology (pp. 105–110). Kluwer Academic Publishers, DordrechtGoogle Scholar
  44. Teisson C & Alvard D (1999) In vitro production of potato microtubers in liquid medium using temporay immersion. Potato research 42: 499–504Google Scholar
  45. Teisson C, Alvard D, Lartaud M, Etienne H, Berthouly M, Escalona M & Lorenzo JC (1999) Temporary immersion for plant tissue culture. In: Plant Biotechnology and In vitro Biology in the 21st Century, Proceedings of the IXth International Congress of Plant Tissue and Cell Culture, Section H: Novel micropropagation methods (pp. 629–632). JerusalemGoogle Scholar
  46. Tisserat B, Jones D & Galletta PD (1993) Construction and use of an inexpensive in vitro ultrasonic misting system. Hort. Technology 3: 75–79Google Scholar
  47. Tisserat B & Vandercook CE (1985) Development of an automated plant culture system. Plant Cell, Tiss. Org. Cult. 5: 107–117Google Scholar
  48. Wardle K, Dobbs EB & Short KC (1983) In vitro acclimatization of aseptically cultured plantlets to humidity. J. Amer. Soc. Hort. Sci. 108: 386–389Google Scholar
  49. Wataad A, Raghothana KG, Kochba M, Nissim A & Caba V (1997) Micropropagation of Spathiphyllum and Syngonium is facilited by use of interfacial membrane rafts. HortScience 32: 307–308Google Scholar
  50. Weathers PJ, Cheetham RD & Giles KL (1988) Dramatic increases in shoot number and lengths for Musa, Cordyline and Nephrolepsis using nutrient mists. Acta Hort. 230: 39–44Google Scholar
  51. Weathers PJ & Giles KL (1988) Regeneration of plants using nutrient mist culture. In vitro Cellular and Development Biology 24: 727–732Google Scholar
  52. Zamarripa A, Ducos JP, Tessereau H, Bollon H, Eskes AB & Petiard V (1991) Developpement d’un procédé de multiplication de masse en masse du caféier par embryogenèse somatique en milieu liquide. In: ASIC Publishers (eds) 14th International Scientific Colloquium on Coffee, San Francisco, US (pp. 392–402). Vevey, SwitzerlandGoogle Scholar
  53. Ziv M, Meir G & Halevy AH (1983) Factors influencing the production of hardened glaucous carnation plantlets in vitro. Plant Cell, Tiss. Org. Cult. 2: 55–65Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • M. Berthouly
    • 1
  • H. Etienne
    • 2
  1. 1.Centre de Coopération Internationale en Recherche Agronomique pour le Développement — Amis (CIRAD-AMIS) CIRADMontpellier Cedex 5France
  2. 2.Centre de Coopération Internationale en Recherche Agronomique pour le Développement-Cultures Pérennes (CIRAD-CP) CIRADMontpellier Cedex 5France

Personalised recommendations