Michael W. Fowler

Plant cell biotechnology: A perspective

Abstract The use of plant cell cultures as a commercial source of secondary products such as medicinals, flavours and pigments is discussed. Environmental factors affecting yield, including medium components and culture conditions, are briefly summarized, followed by a consideration of the kinetics of growth and productivity in plant cell suspensions. Intrinsic factors affecting the capacity for secondary product accumulation are dealt with in some depth, including the physiological, biochemical and genetic limitations. The final part of the review deals with the inherently variable nature of plant cell cultures, and the use of this property in establishing high-yielding cell lines. The mechanisms underlying this variability are discussed, distinguishing genetic and ‘epigenetic’ components of variation. Finally, a brief reference is made to the problems associated with culture instability from the commercial point of view.

Regulation of product synthesis in cell cultures of Catharanthus roseus (L) G. Don

Cell suspension cultures of the Madagascan Periwinkle Catharanthus roseus (L) G. Don were maintained on Gamborgs B5 medium and their growth monitored by measuring cellular fresh and dry weight, cell number and mitotic activity. Samples of cells of different ages and physiological states were subcultured onto an alkaloid production medium and their rates of growth and alkaloid accumulation measured over a period of 30–45 days. In two experiments the rate of biomass accumulation was directly related to the rate of cellular serpentine accumulation. Possible mechanisms underlying this phenomenon are discussed in relation to the properties of cells comprising the inocula.

Effect of scale-up on serpentine formation by Catharanthus roseus suspension cultures

Abstract A culture of Catharanthus roseus has been developed that is capable of growth-linked serpentine formation. Two separate cell lines of this culture, C87 and C87N, were grown in air-lift bioreactors of 7, 30, and 80 liter working volume. Good growth was obtained with both cell lines in all vessels, with better growth rates at the higher volumes. In contrast, serpentine formation was very low when either cell line was grown in any of the vessels when compared with shake flasks. The reason for this loss of alkaloid formation does not appear to be associated with either bioreactor type or cell line.

The supply of reducing power for nitrite reduction in plastids of seedling pea roots (Pisum sativum L.)

Plastids were separated from extracts of pea (Pisum sativum L.) roots by sucrose-density-gradient centrifugation. The incubation of roots of intact pea seedlings in solutions containing 10 mM KNO3 resulted in increased plastid activity of nitrite reductase and to a lesser extent glutamine synthetase. There were also substantial increases in the activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenases. No other plastid-located enzymes of nitrate assimilation or carbohydrate oxidation showed evidence of increased activity in response to the induction of nitrate assimilation. Studies with [1-14C]-and [6-14C]glucose indicated that there was an increased flow of carbon through the plastid-located pentose-phosphate pathway concurrent with the induction of nitrate assimilation. It is suggested that there is a close interaction through the supply and demand for reductant between the pathway of nitrite assimilation and the pentose-phosphate pathway located in the plastid.

A physical method for the separation of various stages in the embryogenesis of carrot cell cultures

Abstract A technique is described for the separation of near homogenous samples of globular, heart and torpedo embryoids from carrot cell cultures. The level of contamination by other cell types is low, of the order of 5–10%. The different embryoid types may be separated in bulk, and retain the full viability for further growth and development. The technique also removes senescent embryoids.

Effect of carbon and nitrogen growth limitation upon nutrient uptake and metabolism in batch cultures of Catharanthus roseus (L) G. Don

Cell suspension cultures of the Madagascan Periwinkle, Catharanthus roseus (L). G. Don were grown as batch cultures in two different types of media; in one medium the limiting nutrient was inorganic nitrogen, and in the other it was carbon. The response of the cells to these growth-limiting conditions was monitored by measuring cellular fresh weight, dry weight and protein accumulation, cell viability, medium sugar and nitrate levels, and the activities of certain intracellular enzymes throughout growth in batch culture. The enzymes investigated were glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), hexokinase (EC 2.7.1.40), phosphofructokinase (EC 2.7.1.11), nitrate reductase (EC 1.6.6.1), glutamate dehydrogenase (EC 1.4.1.2) and glutamine synthetase (EC 6.3.1.2). The effect of culturing the cells under different nutritional regimes was apparent in all aspects of growth; only some enzyme activities were unaffected. Cell viability remained at a high level for several days after growth limitation in both types of culture. The possibility that protein degradation in nitrogen-limited batch cultures is under very stringent control is discussed.

A flat-sided photobioreactor for culturing microalgae

A V-shaped flat-sided photobioreactor (working volume: 2 litre) was designed and used to grow the microalga Porphyridium cruentum for biopolymer production. Mixing of culture in the bioreactor was provided by a mixture of air/CO2 supplied through sintered glass in the form of fine bubbles from the base of the bioreactor. The advantages of this flat-sided photobioreactor are the high surface-to-volume ratio, efficient mixing, low shear force, low cost and absence of wall growth.

Growth and secondary product formation of a selected Catharanthus roseus cell line

A cell line Catharanthus roseus ID1 has been selected from the original C87 cell line, which lost its ability to synthesize serpentine and ajmalicine, by picking out autofluorescent cell aggregates. The selected cell line responded to alterations in cultural conditions, such as decrease in temperature or increase in sucrose concentration, in a similar manner to the C. roseus line C87, by increasing the formation of serpentine. Serpentine production was not linked to growth, in contrast to the original cell line. The long-term stability of serpentine formation was followed with the selected cell line, as the original line C87 lost this ability over a 6-year period of culture. The newly selected cell lines ability to produce serpentine declined during initial subculture and fluctuated thereafter. Growth of cultures in the light restored the production of serpentine to its original level; however, continued culture in the light resulted in a decline in production and finally death of the cell line. Maintaining the cell line in the dark and switching to growth in the light for serpentine production appeared to retain the high-yield characteristics.

Process strategies for plant cell cultures

Plants synthesize a tremendously wide range of chemical structures, many of which have been used over the centuries to the benefit of mankind. Today, with attention again turning to the plant kingdom as a source of drugs, food additives, perfumes and so on, a further dimension has been added to traditional methods of synthesis of these products: the use of cultured plant cells. Although plant cell culture technology is perhaps still in its infancy compared with other aspects of biotechnology, an extensive information and experience base is being established for the development of plant cell processes. The establishment of such a foundation is important for the application of plant cell technology in the years ahead.

Regulation of product synthesis in cell cultures of Catharanthus roseus. V. Long-term maintenance of cells on a production medium

Three unselected cell lines of C. roseus maintained on a growth-associated alkaloid production medium were studied over a period of 2 to 5.5 years for the stability of alkaloid production (serpentine and ajmalicine). Large fluctuations in the total alkaloid content of 20-day-old cells were found for all three cell lines at each subculture over a two-year period. Growth rates increased during prolonged subculture and one cell line became unproductive after five years culture. By selection of small autofluorescent aggregates, high alkaloid production was restored in this cell line, while the parent line was found to be unresponsive to alkaloid induction treatments. The instability in both alkaloid production and spectrum and the loss of alkaloid productivity are discussed in relation to the selection pressures present during long-term maintenance of cell suspension cultures.