Liquid Culture Systems for in vitro Plant Propagation

pp 165-195

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

  • M. BerthoulyAffiliated withCentre de Coopération Internationale en Recherche Agronomique pour le Développement — Amis (CIRAD-AMIS) CIRAD
  • , H. EtienneAffiliated withCentre de Coopération Internationale en Recherche Agronomique pour le Développement-Cultures Pérennes (CIRAD-CP) CIRAD

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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