From Micropropagation to Microponics (part II)

by Maciej Hempel

Originally published in: Practical Hydroponics & Greenhouses 1994, May/June: 17-20

Recent experiments on photosynthesis in vitro have changed our approach to micropropagation (see From Micropropagation to "Microponics"). Novel in vitro propagation systems, where plants are capable of photosynthesis, have been proposed and implemented in research laboratories. This new approach to micropropagation will revolutionise the plant propagation industry in the near future. To illustrate the importance of incoming changes, I will briefly review some experimental work related to the subject.

Micropropagation matured as an industry in the late 1970s and the early 1980s. It was giving astonishing economic results in propagation of ornamental plants and other crops where prices for planting material are high. However, it was very labour consuming and, in many situations, caused physiological changes in propagated plants (see Reasons for High Production Costs of Traditional Micropropagation). During the eighties, research workers started to look for reasons of anomalies in the anatomy and physiology of in vitro grown plants. The most important from today's perspective were investigations on photosynthesis.

Japanese workers led by Dr. Kozai have established a leading role in this domain and built up foundations of autotrophic tissue culture systems. His team confirmed a theory that chlorophyll containing plantlets can grow in vitro on media without sugar if the standard micropropagation environment is modified to enable photosynthesis. They grew carnation plantlets in tubes covered with translucent caps enabling gas exchange (Kozai and Iwanami, 1988). Plantlets were placed on the standard micropropagation medium, containing different amounts of sucrose in carbon dioxide enriched and non-enriched environment. Growth of plantlets was similar on sugar-free and sugar-containing media in the carbon dioxide enriched environment (Fig. 1).

Much subsequent research followed, confirming the above findings for many different plants (carnation, chrysanthemum, cymbidium, primula, potato, strawberry, etc.). It has been established that plants are not only able to grow in vitro on sugar-free media at high light intensity and close to normal carbon dioxide concentration, but their quality and acclimatisation capabilities increase.

Figure 1

The Japanese scientists proposed the photoautotrophic tissue culture system (PTCS) as a novel approach to micropropagation (Fujiwara et al., 1988). The basic idea behind it is to grow plants in large containers where air content (carbon dioxide, oxygen, relative humidity) and composition of culture solution could be controlled. Strawberry plantlets grew better in PTCS than in standard tissue culture conditions, i.e. tubes covered with caps enabling gas exchange (Fig. 2). Mineral salt mixture at half-strength according to Murashige and Skoog (1/2 MS) was used for both of the systems but with 2% of sucrose and 0.8% of agar in the case of traditional tissue culture.

The photoautotrophic tissue culture system has unquestionable advantages over traditional tissue culture methods of propagation (see Advantages of Autotrophic Micropropagation). The most promising is expected reduction in production cost due to larger culture vessels, simpler culture solutions, elimination of losses caused by culture contaminations and mutations, etc. However, propagation is still conducted in containers which will be difficult to access by robots if automation of cutting is to be introduced. Also container and media preparation and maintenance would be costly if sterile culture conditions are to be maintained. It is obvious that containers can be abandoned

 Figure 2
and plantlets can be grown in larger structures where control of air composition and light would cost less per plantlet produced. Nutrient supply can be provided by hydroponic technologies such as nutrient film technique or ebb and flow systems. It has been confirmed ( Kozai et al., 1988) that plantlets cultured in tubes covered with translucent film enabling carbon dioxide exchange, can grow better on hydroponic nutrient solution than on a traditional tissue culture medium (1/2 MS) (Fig. 3).

Going one step further in the simplification of mass-propagation, we can abandon the sterility of the production environment. The last thirty or so years of practical micropropagation taught us how difficult is to free plants from "latent" bacteria in culture. Almost all micropropagation laboratories around the world propagate plants colonised by such bacteria. They are harmless to plants in normal culture conditions in vitro and, also, after transfer to the greenhouse. Micropropagation managers learned that it is not economically viable to eliminate all microorganisms during micropropagation. The important ones are those which are pathogenic during normal cultivation or limit propagation efficiency in vitro by colonising culture media. Therefore, propagating plants in an autotrophic culture system, we should worry only about pests and diseases.

Figure 3

Carnation or chrysanthemum mother stock is a good example of a similar propagation system. However, its culture environment is controlled to a much lesser extent. Such mother stock used to be established from plants derived from tissue culture and maintained in highly protected greenhouses which are constantly monitored for the presence of pests and diseases. This culture system has been used with good results in the production of cuttings for the last half century.

It is evident that traditional tissue culture is necessary only for the elimination of pathogens from stock plants and, also very importantly, induction of juvenility in propagated plants. It is because of juvenility that we can achieve high efficiency of the propagation process in vitro, through enhanced branching and rooting. Traditional plant propagators have been taking advantage of this phenomenon during propagation by stooling or softwood cuttings. Enhanced branching of carnation mother plants obtained by the tissue culture method has been known about for years. The productivity of stock plants decreases by 10 cuttings per year per plant, for each subsequent generation after tissue culture.

Recently, horticulturists have been moving away from chemicals such as pesticides and growth regulators towards environmental factors, i.e. predatory organisms, day and night temperature differential, carbon dioxide enrichment, etc. in controlling plant production. The photoautotrophic tissue culture is the latest step in this direction in the plant propagation domain. Nevertheless, we are presently able to go one step further and transfer mass-propagation to non-sterile, though highly controlled, environment. Propagation can be conducted in specialised greenhouses or growth chambers (plant factories) with restricted access and regular screening for pathogens. A hydroponic culture method is ideal for this type of propagation because nutrient solutions can be adjusted to specific stages of production, light and carbon dioxide levels and even time of day. We can call this type of cultivation "microponics" because of the small dimensions of the plants.

Microponics would permit the elimination of some obstacles of PTCS such as the need for specialised sterile culture containers and the high costs of cooling if artificial light of high intensity is to be applied in a closed environment. The introduction of robots in the microponic environment will be also more feasible due to open culture space and, therefore, easier access to handled plants (see Benefits of Microponics).

The advent of microponics will not reduce the importance of traditional tissue culture and PTCS. The former is indispensable at the beginning of the propagation process for the elimination of plant pathogens (bacteria, mycoplasmas, viruses) when shoot meristems are placed in culture. It is a routine treatment in freeing plants from viruses and mycoplasmas. After screening for pathogens, individual pathogen-free propagules can be transferred to more open propagation systems, such as PTCS to ensure the build up of diseases-free stock in vitro. Plantlets produced with application of PTCS are much more vigorous and easier to acclimatise than traditional tissue culture plants. They would be an ideal source of high quality plantlets for the establishment of a microponic production system where, in a "plant factory" environment, millions of plants can be produced at much lower cost than in a micropropagation laboratory.

However, many questions must be answered before microponics can become a reality. We know that the idea is economically and technologically viable but we must learn how to control the development of specific species and cultivars by the use of environmental factors. We must also maintain stock in a juvenile stage for as long as possible in order to take full advantage of small plant dimensions (high productivity from an expensive production area) and enhanced branching and rooting.

The author would appreciate any comments on the above subject and would be happy to participate in any enterprise leading to the practical development of microponics.

References

Fujiwara, K., Kozai, T., Watanabe, I. 1988. Development of photoautotrophic tissue culture system for shoot and/or plantlets at rooting and acclimatisation stages. Acta Horticulture 230: 153-158

Kozai, T., Iwanami, Y. 1988. Effects of carbon dioxide enrichment and sucrose concentration under high photon fluxes on plantlet growth of carnation (Dianthus caryophyllus L.) in tissue culture during the preparation stage. J. Japan. Soc. Hort. Sci. 57(2): 279-288

Kozai, T., Kubota, C., Watanabe, T. 1988. Effects of basal medium composition on the growth of carnation plantlets in auto- and mixotrophic tissue culture. Acta Horticulture 230: 159-166



Reasons for High Production Costs of Traditional Micropropagation





Advantages of Autotrophic Micropropagation


but it has disadvantages such as:




Benefits of Microponics