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Plant cell culture is viewed as a potential means of producing useful plant products such that conventional agriculture, with all its attendant problems and variables, can be circumvented. These problems include: environmental factors (drought, floods, etc.), disease, political and labour instabilities in the producing countries (often Third World countries), uncontrollable variations in the crop quality, inability of authorities to prevent crop adulteration, losses in storage and handling. Thus, the production of useful and valuable secondary metabolites in large bioreactors located in the consuming country is an attractive proposal. Additional advantages of such processes include: controlled production according to demand and a reduced and requirement.
However, this technology is still being developed and despite the advantages outlined above, there are a variety of problems to be overcome before it can be adopted on a wide scale for the production of useful plant secondary metabolites. The success of Mitsui Petrochemical Industry Co. Ltd. in Japan in producing shikonin on a commercial scale from Lithospermum erythrorhizon cultivations and that of Nitto Denko Co. Ltd. also in Japan in mass production of Panax ginseng or ginseng cells using 20 kL tanks have demonstrated that many of the problems can be overcome with perseverance. The economic feasibility of these processes is another question and this will be dealt with in a separate section.
In theory, it is anticipated that such large scale suspension cultures will be suitable for industrial production of useful plant chemicals such as pharmaceuticals and food additives, in a manner similar to that of microbial fermentation.
Nevertheless, there are some significant differences between microbial and plant cell cultures that must be considered when attempting to apply plant cell cultures to the available technology. Table 1 shows a comparison of some of the characteristics of plant and microbial cultures of relevance to fermentation.
This table serves to demonstrate some of the problems that can be encountered with plant cell cultures. The sensitivity to shear is due both to the large size of the cells and to the relatively inflexible cellulose cell wall. Thus, with normal blade impellers the cells may twist which will inhibit mitoses and, for this reason, air-lift fermentors are recommended by some researchers. The large size of the plant cell contributes to its comparatively high doubling time (12 h - several days), which thus prolongs the time required for a successful fermentation run.
The vacuole is the major site of product accumulation, and since product secretion is uncommon, the high metabolite yields seen in microorganisms that secrete product (thereby removing product inhibition of biosynthesis) cannot be expected. There is some ongoing research on membrane permeabilization of plant cells which may serve to relieve the constraints of product inhibition by facilitation of leakage into the extracellular medium. If this would also permit recycling of the biomass (e.g. via immobilization) it would help reduce production costs (1). The low aeration requirement for plant cells is an advantage over microbial cultures in general. In addition, the high cost of running a fermentation vessel over several weeks should be considered, although media costs are much less than those of animal cell cultures.
Characteristic of Microbial and Plant Cell Relevant to Fermentation
|Size||2 u||>10 u|
|Duplication time||<1 hour||days|
|Aeration||1-2 vvm||0.3 vvm|
|Production phase||Uncoupled||Often growth-linked|
|Medium cost ($)
Source: Zenk, M.H., Plant Cell Culture Conference, Oyez Sci, Tech. Serv. (1982)
In addition to the problems outlined above, concerned with fermentation technology, there are also considerable hurdles to be overcome at the biochemical level. The two major problems concern poor expression of products and instability of cell lines.
Cultured plant cells often produce reduced quantities and different profiles of secondary metabolites when compared with the intact plant and these quantitative and qualitative features may change with time. The poor product expression is often attributed to a lack of differentiation in cultures (2). On the other hand, there are cases of cultures that over-produce metabolites compared with the whole plant (Table 2).
There are a number of examples of cultured cells producing metabolites not observed in the plant, eg. Lithospermum erythrorhizon cultures have been observed to synthesize rosmarinic acid (3). It has become apparent that the choice of original plant material having high yields of the desired phytochemical may be important in establishing high-yielding cultures (4). Furthermore, the need to repeatedly screen for high-producing lines (due to inherent instability of cell lines) has been emphasized, although the nutritional composition of the medium is also important (2). Thus, a variety of approaches are being investigated by many researchers to increase productivity of useful plant metabolites in plant cell cultures as seen in another section in this review.
Secondary Metabolites Produced in High Levels
by Plant Cell Cultures
|YIELDS (% DRY WT)||
|Rosmarinic acid||Coleus blumeii||15||3||s|
|Benzylisoquinoline Alkaloids||Coptis japonica||11||5 - 10||s|
|Berberine||Coptis japonica||10||2 - 4||s|
|* s = suspension; c = callus|