AGRE-0017 Improvement of In Vitro Plant Cultures at Laboratory and Industrial Scales: Control of Gaseous Environment by Using Selective Membranes as Lids of Culture Containers

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ECLAIR Cluster VI - Production and Storage : Storage/Transport

Films with differant selective permeabilities to gases, M1, M2 and M3, induce either better growth, M1, or better multiplication, M2, or both better growth and multiplication, M3, than the control during the multiplication phase of Prunus root-stocks, GF 677

SUMMARY

Many types of plants, including ornamentals, horticultural crops, vegetables and fruit bushes, can be produced using techniques of cell and tissue culture, generally referred to as 'in vitro plant micropropagation'. However, the procedures used are labour intensive and costly. By examining the effects of changes in gases found in the headspace of containers sealed with membranes of varying gas permeability, a new type of container, for which a patent application has been submitted, has been designed. Results obtained at laboratory and industrial scales, with different species investigated throughout the complete propagation process (from multiplication to acclimatisation stages), show that by using containers sealed with suitable membranes it is possible to improve the multiplication, survival, growth and quality of plants and as a result decrease the contribution of labour costs per plant.

INTRODUCTION

Many types of plants, including ornamentals, horticultural crops, vegetables and fruit bushes, can be produced using techniques of cell and tissue culture, generally referred to as in vitro plant micropropagation. However, the procedures used are labour intensive, contributing to the high cost of producing plants in this way. At the same time, there is a lack of knowledge concerning the detailed physiological behaviour of plants, especially in respect of gas exchange, under the conditions used for propagation. This project, funded under the ECLAIR programme, looked at both the physiological problems and the possibility of developing automated processes in order to reduce the level of labour cost of associated with in vitro techniques.

OBJECTIVES

The first objective was to carry out a detailed analysis of components of the gas phase and to follow their evolution inside containers with different systems of closure. This information facilitated attempts to try to control the gas phase by using membranes, with a range of selective permeabilities to gases, as lids of containers and then develop disposable cheap containers. These were then assessed, using various culture conditions with a wide range of plant species (Gymnosperms, Angiosperms, Monocots, Dicots, herbaceous and woody plants), in terms of both plant behaviour and the impact of the selected procedures on the economics of the process.

WORK PROGRAMME

Analysis of the gas phase in containers (with and without permeable membranes as lids, or with other types of lids of varying tightness) implanted with in vitro plants (Prunus, Rosa and Sequoa) was carried out using gas chromatography and mass spectrometry. A range of 5 conditions was used to investigate the physical (convection, permeability, diffusion) and physiological (respiration, photosynthesis) mechanisms involved in the evolution of the headspace composition of the atmosphere in such containers as well as the influence of environmental factors (light, temperature, pressure, hygrometry, nutrient medium, plant species and culture phase). This enabled the impact on the evolution of the main gases of atmosphere of specific membranes with selective gas permeability to be determined under varying levels of air-tightness obtained by different closure systems.

RESULTS

Significant differences in the headspace composition (carbon dioxide, oxygen and nitrogen and volatiles such as ethylene) over the culture period could be associated with the use of a specific film at a specific time during either multiplication or rooting phases. An interaction was found between the culture period and the air-tightness of containers, resulting in damage to plants due to release of toxic compounds. The use of permeable membranes successfully prevented this.

From a selection of 14 microperforated and plain plastic packaging films from various companies, 8 were characterised as suitable for the purposes, where high degrees of humidity (50-100 %) and different temperatures (0-40°C) modified gas permeability properties. The chemical composition of films was also considered in terms of compatibility with other plastic materials used in the new containers, as well as light transmission properties in the 200-800 nm spectrum required for plant growth. Two types of caps were tested, as were various prototype containers, for light transmission, air-tightness of joints between cap and bottom, as well as gaseous exchanges, with different types of selective membranes. During development, handling revealed high risks of contamination at joints, as well as leaks due to incomplete sealing of membranes at the upper part of the cap. However, these problems were overcome in due course by designing various new products, including rings and hollow caps These were systematically tested in many experiments and shown to give beneficial results in many cases.

During the first two years, the range of thin membranes selected for use as lids of conventional glass containers were compared with a control system, which used the conventional thick translucent plastic screw caps. Four membranes, 2 plain and 2 microperforated, were found most suitable. However, it was shown that membrane type has to be changed between multiplication and rooting phases. This led to development of hollow caps with 3 different kinds of selective membranes sealed on plastic rings.

From results obtained at laboratory and industrial scales, with different species from multiplication to acclimatisation stages, it was confirmed that, by using suitable membranes it is possible to:

• improve multiplication rates and growth of vitroplants

• improve formation, density and quality of roots

• improve re-growth of plants ex vitro

• increase the number of quality plants during acclimatisation and reduce the acclimatisation period in the greenhouse before the transfer to outside conditions

• prolong the cold storage period of vitroplant strains without subculture over more than one year

• reduce labour costs

GENERAL CONCLUSION

Experiments performed have enabled definition of the parameters and factors which govern gas exchanges (both quantitatively and qualitatively) during in vitro propagation and thus influence the micropropagation of a number of plant species. In addition, the technical and economic benefits of using thin plastic membranes as lids of containers, to improve the quality of plants and to decrease man-power and energy needs, has been shown.

EXPLOITATION PLANS

It is expected to licence a new disposable plastic container built by THOMAS SA, for which a patent application has been made (N° 94-11402).

PARTICIPANTS

Franche Comte Foret, (France), Rijksuniversiteit Gent (RUG), Horticulture Department (Belgium), ADRIAC - UFR Sciences (France), Rose Nurseries Panos Avramis and Sons (Greece), S C Darbonne (France), COOPD'OR (France), Ets R Thomas SARL (France) and the Centre technique Interprofessionnel des Fruits et Legumes (France).