Alan H. Scragg

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.

Growth of Catharanthus roseus suspensions for maximum biomass and alkaloid accumulation

Three methods of cultivating Catharanthus roseus for biomass and secondary product accumulation have been examined. Batch, fed-batch, and draw-fill methods have been used in both shake flasks and bioreactors. In addition, cultures have been grown to high biomass levels (30 g l−1) using 6% (w/v) sucrose. At present the most effective culture method for biomass and serpentine accumulation is batch culture using 6% (w/v) sucrose.

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.

Natural Products from Higher Plants and Plant Cell Culture

The economic importance of plants is immense with plants being used as a source of food, construction materials, fabrics and paper and fuel. In addition, plants contribute to a wide array of chemicals used in industry including pharmaceuticals, foods, flavours, dyes, pigments and agrochemicals. The potential for chemical synthesis within the plant kingdom is vast, something of the order of 2 × 104 structures are known from plants. New chemical structures reported from plants each year are currently running in excess of 1500.

Large-scale cultivation of Helianthus annuus cell suspensions

Abstract Suspension cultures of Helianthus annuus have been cultivated in volumes of 100 ml to 80 l in a variety of vessels, including shake flasks, air-lift, and stirred-tank bioreactors. In general, the growth rates and biomass yields were similar, with no bioreactor showing a distinct advantage. Scale-up from 100 ml to 80 l could be achieved within 40 days using three 10-day subcultures. The determination of oxygen uptake rates indicated that any reduction in growth rate did not appear to be due to oxygen supply .

Growth and alkaloid production in bioreactors by a selected Catharanthus roseus cell line

Abstract A cell line of Catharanthus roseus has been selected from a culture derived from a long-established cell line C87. The original cell line C87 was capable of linked growth and serpentine accumulation using one medium. The selected line C. roseus ID1 retained many of the culture characteristics of the parent line, such as rapid growth and increased alkaloid formation at high sucrose concentration, but serpentine accumulation was not linked to growth. A more significant difference was shown in that the selected line retained the ability to accumulate serpentine when grown in bioreactors, whereas the original cell line lost this ability upon scale-up. However, the accumulation of serpentine by the C. roseus ID1 cell line in bioreactors was reduced with increasing subculture number. This trend was reversed by maintaining the stock lines in the dark and only switching to light conditions during scale-up.

Cell suspension culture of Picrasma quassioides: the development of a rapidly growing, shear resistant cell line capable of quassin formation

Summary Picrasma quassioides Bennett ( Simaroubaceae ) produces the tetracyclic triterpenoid quassin. The development of a suspension culture of P. quassioides capable of growing in bioreactors and which produces low levels of quassin is described. The suspension culture was initially highly aggregated and slow growing (doubling time 12 days) but after 2 years cultivation and a number of changes in subculture methods a less aggregated and faster growing (doubling time, 2.9 days) suspension has been achieved. In addition to an increased growth rate the cultures have begun to produce low levels of quassin (30 – 150 μg . g dry weight -1 ). The growth of P. quassioide s suspension cultures in volumes above 100 ml was initially difficult to establish, but after a year growth in 1 litre volumes was achieved followed a year later by growth in a stirred-tank bioreactor. This cell line has been shown to have developed shear resistance in this time.

Production of the triterpenoid quassin in callus and cell suspension cultures of Picrasma quassioides Bennett

Plant cell and suspension cultures have been established from stem cuttings of Picrasma quassioides Bennett. The effect of 244 different types/concentrations of plant growth regulators on growth and quassin accumulation in callus tissue was investigated. Best growth, in terms of wet/dry weight after four weeks growth, was obtained on B5 media supplemented with 2% glucose, 10% coconut milk, 0.5 mg.l−1 zeatin riboside and 1.5 mg.l−1 IBA. The highest yields of quassin (0.014–0.018%) were detected on this same media supplemented with 1.0 mg.l−1 IBA and varying concentrations of zeatin riboside. Suspension cultures were easily established on B5 media supplemented with 2% glucose, 1.0 mg.l−1 2,4-D and 0.5 mg.l−1 kinetin. The carbon source had a marked effect on quassin accumulation with 0.32% quassin being detected when cells were grown in 2% galactose. This is comparable to the highest reported quassin yield for the whole plant.

The Effects of Plant Growth Regulators on Growth and Alkaloid Formation in Cinchona ledgeriana Callus Culture

Summary Callus cultures of Cinchona ledgeriana were established from seeds obtained from Kenya on Gamborgs B5 medium containing 2% glucose, 1 mg l −1 2,4-D and 0.25 mg l -1 kinetin. The effect of 240 combinations of auxins and cytokinins on growth and alkaloid production was determined. Best callus growth was obtained in the presence of NAA and IpAR, 2,4-D and kinetin, NAA and zeatin riboside, and IBA and zeatin. Quinidine was detected from several of these calli with a maximum yield of 0.053 %. Quinine was seldom detected and then only in non-quantifiable amounts.

Investigation into the problems of initiation and maintenance of Cinchona ledgeriana suspension cultures

Summary The establishment and maintenance of suspension cultures Cinchona ledgeriana Moens have proved difficult but cultures have been established in Gamborgs B5 medium supplemented with 10 % coconut milk or 1 % polyvinylpyrrolidone (PVP). The loss of viability in cultures without these supplements is associated with a sustained drop in pH. Both coconut milk and PVP increase the resistance of B5 medium to changes in pH which may explain their success in maintaining viability in cultures of Cinchona . PVP does, however, have its limitations as it appears to bind both quinine and quinidine which prevents the detection of products in Cinchona cultures.