Cell Suspension Culture of Picrasma quassioides: the Development of a rapidly growing, Shear Resistant Cell Line capable of Quassin Formation

JPlantPhystol. Vol. 132.pp.176-183(1988}

Cell Suspension Culture of Picrasma quassioides: the Development of a rapidly growing, Shear Resistant Cell Line capable of Quassin Formation E. J.

ALLAN

l

),

A.

H. SCRAGG, and K. PUGH

Wolfson Institute of Biotechnology, University of Sheffield, Sheffield S10 2TN

I) Division of Bacteriology, School of Agriculture, University of Aberdeen, 531 King Street, Aberdeen AB9 1UD. Received June 12, 1987· Accepted September 3,1987

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 p.g' g dry weight -1). The growth of P. quassioides 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.

Key words: Picrasma quassioides Bennett, bioreactors, quassin, suspension culture. Abbreviations: FDA = fluorescein diacetate, TLC = thin layer chromatography, UV = ultraviolet, HPLC = high pressure liquid chromatography.

Introduction An increasing interest in plant derived drugs, used particularly as traditional medicinals, has developed over the last twenty years (Phillipson, 1985). The bitter principles (quassinoids) produced by the Simaroubaceae lie within this category. Products from approximately 15 simaroubaceous species have been used extensively in folk medicine while their active principle and other novel quassinoids have been identified only recently, as a result of developments in physical chemistry (Polonsky, 1973 a). At least 50 quassinoids have been identified (Polonsky, 1973 b) with some being shown to be antiamoebic (Chan et al., 1986), antileukaemic (Kupchan and Streelman, 1976; Cordell, 1978), and insecticidal (Odjo et al., 1981). This chemical diversity and medicinal potential makes these compounds attractive targets for plant cell biotechnology. © 1988 by Gustav Fischer Verlag, Stuttgart

Little information is, however, available concerning in vitro studies of the Simaroubaceae. Successful cell and tissue culture of Ailanthus altissima has been undertaken, but no quassinoids were isolated (Anderson et al., 1983). An enzyme linked immunosorbent assay for the bitter principle quassin and closely related metabolites was developed by Robins et al. (1984) so as to simplify routine analysis of tissue cultures, but no data on tissue culture has been reported. The bitter principle quassin is a degraded triterpenoid and is produced by several simaroubaceous species. Commercial quassin is a mixture of the bitter principles derived from quassia, the heartwood of Picrasma excelsa (Picraena excelsa) or of Quassia amara (Surinam quassia) and consists of various mixtures of quassin, its hemiketal neoquassin and 18-hydroxyquassm. This investigation has studied the development of cell culture of the simaroubaceous tree, Picrasma quassioides Bennett

Cell Culture of Plcrasma

over a two year period. Details of growth kinetics i? 0.1 litre, 1.0 litre shake flasks, a 7 litre air-lift and 3 litre stIrre?tank bioreactor are described along with reports of quassm production.

Material and Methods Imtiatton and growth of suspension cultures

Suspension cultures of Pzcrasma quasstoldes Bennett were initiated from stock callus material growing on semi-solid Gamborg's B5 medium (Gamborg et a!., 1968) supplemented with 10% (v/,:,) co.conut milk (Scragg and Allan, 1986). The parental call~s mate:lal dId not produce quassin, neoquassin or 18-hydroxyquassm. MedIUm D consisting of Gamborg's B5 (Gamborg et a!., 1968), wit~ 2% gluco~e and supplemented with the plant growth regulators: mdolebutync acid (1.0 mg .1- 1) and N 6 (.,:l2-isopentenyl)adenine riboside (0.5 ~g: 1- I) was used throughout this survey. Suspension cultures were mltiated by transferring callus (1-2g wet weight) into 50ml of medium D and adding a further 50 ml media after 14 days of growth. All suspension cultures were incubated at 25°C in continuous subdued light on an orbital shaker (New Bruns:-,ick, G-I0) at .150 rpm and were subcultured, unless stated otherwIse, at 14 day mtervals using an inoculum ratio of 3: 10 (v/v), representing approximately 1.5 g wet weight cells. Newly initiated cultures were transferred, after 7 subcultures (i.e. 14 weeks), into fluted 250ml flasks (4, 1.5cm indentations) which encouraged aggregate breakdown. The cellli.ne was maintained in these flasks for 34 weeks (17 subcultures) wIth transfer into normal Erlenmeyer flasks for experimental work, and subsequently maintained in normal Erlenmeyer flasks. Th~ majority of experimental work described was undertaken on thIS latter, less aggregated cell suspension. Wet weight subculture was accomplished by filtering the cultures through 105 ftm nylon mesh (Simon Ltd, Stockport, England) using gentle suction. Growth was determined by taking wet and dry weight measurements using the method of Stafford and Fowler (1983). Viability was assessed by staining cells with fluorescein diacetate accordi~g ~o the method of Widholm (1972). Cultures were grown m the alr-hft fermenter (71 working volume) as described by Morns et a!. (1985). Growth temperature was 25°C and aeration at 11 min - I. The stirred-tank bioreactor (31 working volume) was of the standard design fitted with a six bladed impeller of 8.3 cm diameter. The bioreactor diameter was 15 cm. The bioreactor was sterilised empty, and sterile medium and cells added later. The impeller speed was 150 rpm, temperature 25°C, and aeration rate was 100 mI· min - I. Growth rate (ft) was calculated over the period of increasing dry weight using linear regression analysis. Doubling tIme (td) was calculated using the equation, td = 0.693/ ft. Results are shown as mean ± standard error. Standard errors less than 5 % of the mean are not shown in the figures. Investigation znto aggregatIOn and shear stress sensitivity

Size dlstnbutions of cell suspensions were determined by gently washing 100 ml volumes through a range of nylon meshes of known pore size (Simon Ltd, Stockport, England). These were then drie.d to a constant dry weight. Results are presented as the weIght retamed on each filter mesh as a percentage of the total dry weight. The cells were exposed to shear stress in the same 31 stirred-tank bioreactor as the cells were grown. The cell suspension was exposed to fixed impeller speeds at a temperature of 25°C and aeration rate of 100 mI· min -I. At intervals sterile samples were removed, wet and dry weights estimated and 3 x 2 g wet weight aliquots used to inoculate 100 ml of medium in 20 ml flasks. After 14 days incubation

177

growth was estimated by wet and dry measurements. The average and maximum shear rates were estimated using the formula of Metzner et a!. (1957). In addition to the analytical results presented, stock cell suspension cultures were routinely analysed on every passage for quassin.

Extraction, detectIOn and quantificatIOn of quassin

Quassin was analysed by soxhlet extraction (3 h) of 100 - 200 mg lyophilised cells with dichloromethane containing .10 % Jv/v) methanol. Quassin in the medium was extracted by shakmg with an equal volume of chloroform, removing the chlorofo~m phase and reducing to dryness. The residue was resuspended m methanol. Quantitative analysis was achieved by reverse and/or normal phase HPLC (Water Associates and Shimadzu LC-4A). Reverse phase HPLC was achieved using a radial cartridge (8 mm x 10 em) packed with ftBondapak C18 in a solvent system of methanol, water and nheptane sulphonic acid (45: 55 : 5) with a gradient of 45 to 65 % methanol in 20 min. A radial cartridge (8mmx 10cm) packed with 5 ftm silica was used for normal phase HPLC with isocratic elution (2 mI· min - I) in a solvent system of 2 % methanol in dichloromethane. Detection was by UV absorbance at 254 nm and 280 nm for reverse phase HPLC and at 254 nm for normal phase HPLC. Authentic standards of commercial quassin (Bush Boake Allen, London, England, and Koch Light Laboratories, Colnbrook, ~ng­ land) and purified quassin (gift of Cadbury-Schweppes, Readmg, England) were used. Thin layer chromatography (TLC) was on Kieselgel60F254 plates (Merck, West Germany) in chloroform containing 3 % (v/v) methanol. Quassin quenches at 254 nm but does not fluoresce at 360 nm. Quassin spots turn brown/black in colour after heating (approx. 10 min at 60°C) when sprayed with 0.5 % ceric sulphate in 2M sulphuric acid. UV spectral data (Pye-Unicam SP8-500) and mass spectroscopy (V.G. Micromass 70-70F) confirmed :he authenticity of quassin in a pooled samples collected by preparative HPLC. The identity of quassin was subsequently accepted on the basis of HPLC retentIon times (approx. 8 and 3.7 min on reverse/ normal phase HPLC respectively; a 254/280 nm absorbance ratio of approx. 3.10) and characteristics on thin layer chromatograms as described above.

Results

Growth of P. quassioides suspension cultures was initially very poor. Growth rate after six subcultures was 0..058 days-I (td = 12.0 days) with only 7.25±3.37g dry welght cells being obtained after 24 days culture (Fig. 1 a). The high standard error resulted from the culture being highly aggregated. Subsequent transfer into fluted flasks for 17 subcultures produced faster growth rates and an apparent reduction in the degree of aggregation. Thus, 21 subcultures (42 weeks) after initiation a growth rate of 0.102 days-I (td = 6.8 days) was obtained (Fig. 1 b). Both these cultures were assayed at intervals throughout their growth cycle for the presence of quassin but none was detected in these or routine analysis. The stock lines cultural history and the changes in growth rate, doubling time and biomass levels are shown in Fig. 2 and Table 1 respectively. The effect of varying the concentration of glucose on the growth of the suspension culture after 27 subcultures is shown in Fig. 3. With 2 % (w/v) glucose a growth rate of 0.142 days-I (td = 4.89 days) was obtained with a biomass

178

E.}.

ALLAN,

A. H. SCRAGG, and K. PUGH

Table 1: Growth rate, doubling time and quassin formation by Pi· crasma quassioides suspension cultures.

A.

Inoculum age

Growth rate days-I

100 ml cultures L6 0.058 L21 0.102 L27 0.142 L35 0.138 L43 0.143 L48 0.239 1,000 ml cultures L4 (L42) 0.152 L9 (L47) 0.123

Doublmg time Maximum blOmass days g .1- 1 time (days)

I'g' g -

Quassm 1 time (days)

12.0 6.8 4.89 5.03 4.85 2.9

7.4 6.8 9.1 8.1 10.0 11.1

24 12 21 21 18 14

0 0 165 30

0 0 0 0 10 31

4.56 5.63

13.62 13.57

15 19

145

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10 Time days

Fig. 1: The growth of P. quassioides suspension cultures in 250 ml flasks (100 ml medium) after six (A) and twenty one (B) subcultures. The latter culture had been maintained in fluted flasks for 15 subcultures.

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cultures l.nl.tl,ated (2Id)

1

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SO growth in stl.rred tank bioreactor

L52 (present)

Fig. 2: The cultural history of the P. quassioides suspension culture.

yield of 9.1±0.29g dry weight I-I. The biomass yields were much greater when the cells were grown on 5, 10 and 15 % glucose. Thus the biomass increased to 25 g .1- 1 at 28 days with 10% glucose, at 15% (w/v) the maximum biomass was reduced, 23.0 g .1- 1, and was only reached after a much longer growth period of 50 days (results not shown). The maximum growth rate obtained was with 5 % glucose (0.173 days -I, td = 4 days), with the rates being reduced above this concentration. No quassin was detectable in cultures analysed at 0, 7, 14, 21 and 28 days after inoculation for all glucose concentrations.

Cell Culture of Picrasma Table 2: The effect of inoculum potential on the growth characteristics of P. quassioides suspension cultures (culture volume = 0.11). Inoculum Potential (g'wet weight)

Duration of lag Phase (d)

1.0ge 1.5 2.0 3.0 4.0

'" (h -11

(x10- ) 3.05 6.6444.4 5.74 5.74 5.14

12 6 4 4

td (h- 1)

227.3 120.72 120.72 134.83

179

200

Max biomass obtained g .1- 1) 8.6 8.3 8.1 10.5 9.8

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Investigations into the egfect of inoculataon densities on growth were undertaken on the 35th subculture after initiation. Compared to the experiment investigating the effect of glucose on growth (i.e. subculture 27) there was very little change in growth rates. However, it is evident (Table 2) that at lower inoculation densities a long lag phase occurred, although once growth commences the growth rate was similar irrespective of inoculation density. It is evident from Table 2 and Fig. 3 that routine subculture, being carried out at fortnightly intervals, was in fact subculturing cells that were in the early exponential growth phase. In consideration of this, the subculture regime was changed to a period of 18 d with a fresh weight inoculum (rather than volume) of 2.0 g wet weight per 100 ml culture medium. Since this time regime was inconvenient for a regular subculture it was, after six subcultures, increased to a 21 d period. Growth curves undertaken at this stage showed that this represented the period at the end of the growth phase (in terms of dry weight). Sometime after changing subculture regimes, low levels of quassin (approx. 0.01 %) were detected (Fig. 4). In addition, by subculture 48 growth rates had also changed (Fig. 5) with p., = 0.239 d -1 (td = 2.9 d) with low levels of quassin still being produced.

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Fig. 5: The growth and quassin formation by a suspension culture of P. quassioides at subculture 48. 0, dry weight g .1- 1; . , wet weight g .1- 1; 6, quassin J-tg' g dry weight -1.

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It was observed that on initiation the suspension culture was highly aggregated and hense initially fluted flasks were used. Consequently and even after the return of the culture to normal Erlenmeyer flasks the degree of aggregation continued to reduce. The change in aggregation as the suspension culture developed can be seen in Fig. 6 where suspensions at 29 and 45 subcultures have been analysed. Thus over a period of 32 weeks there was over a 15 % reduction in aggregates of greater than 600 /Lm in diameter. The growth of P. quassioides in volumes greater than 100 ml proved difficult initially with many cells losing viability shortly after subculture. However by using high inoculum densities (50 g wet cells into 1,000 ml) growth was achieved. In consideration of the results from 0.11 cultures, a cell line, on medium D, was initiated from stock lines maintained in 100 ml culture volumes with a 14 day subculture regime, but for these larger culture volumes a 21 day subculture period was initiated. The ease of initiation of this 1.01 culture compared to similar earlier trials suggested that the cells were becoming tolerant to shear. Fig. 7 a shows the growth and quassin formation of P. quas· sioides suspension cultures grown in 1 litre of medium, after 4 subcultures in 21 shake flasks. The growth rate was 0.152 days-I (td = 4.56 days) and the maximum biomass of 13.62 g .1- 1 was obtained after 15 days. These values are comparable to the growth and biomass levels found in the smaller flasks (Table 1) at equivalent subculture number. No quassin was detected throughout the growth cycle. How-

ever,S subcultures later (15 weeks), the growth experiment was repeated and this time quassin was detected at low levels (145/Lg·g dry weight-I) in a number of samples (Fig. 7b). This subculture period parallels the subculture period at which quassin had begun to be detected in the smaller (100 ml) cultures (Fig. 1). The growth rate 0.123 days-I (td = 5.63 days) and maximum biomass of 13.57g·1- 1 at day 19 were similar to those of the previous culture. Similar difficulties of obtaining growth were encountered when cells were cultivated in the 71 air-lift bioreactor. Cells failed to grow (in terms of increase in wet and dry weights) in some 90 % of the investigations (total 20) although viability in terms of FDA staining of the inoculated cells was maintained. However, by maintaining inocula in 1 litre culture volumes rather than scaling up from 0.11 cultures on each run, growth was obtained in a 7 litre air-lift bioreactor (Fig. 8). Long lag phases of up to 14 days were found and the growth rate on medium D with 2 % (w/v) glucose was 0.118 days-I (td = 5.85 days) which were lower than those of shake flasks cultures. The size distribution of P. quassioides suspensions grown in the air-lift bioreactor has been compared with those found in 250ml (100ml culture), and 2 litre (1 litre culture) shake flasks. A wider range of filter meshes were used in these determinations. After 14 days cultivation in the 7 litre air-lift bioreactor (at this stage the culture was still in the lag growth phase) the suspension was less aggregated with approximately 40 % of the biomass consisting of aggregates of a diameter of between 105 to 200/Lm (Fig. 9 a) than the suspension grown in a 2 litre flask (Fig. 9 b). The 2 litre flask had an aggregate distribution where over a third of the suspension consisted of aggregates greater than or equal to 1.00 mm in diameter. Plant cells in suspensions have been regarded as sensitive to shear stress, a feature that has been used to explain the dif-

15

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Fig. 8: The growth of P. quassioides suspension cultures in a 71 airlift bioreactor using either 2 % glucose (.) and 5 % glucose (0).

Cell Culture of Picrasma

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80 105 140 ZOO 250 355 530 600 710 1000

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Fig. 10: The loss of viability of P. quassioides suspension cultures as measured by dry weight achieved after 14 days growth in shake flasks. 0, culture treated at 440 rpm, average shear rate 73 sec - 1 at subculture 27; 0, culture treated at 1,000 rpm, average shear rate 167 sec- I at subculture 48.

150

15

ficulties in cultivating plant cells in bioreactors. The effect of shear stress on the ability of P. quassioides cells to grow and divide, has been determined using a 3 litre stirred-tank bioreactor. A suspension culture of P. quassioides at subculture 27 (54 weeks) was exposed to 440 rpm (an average shear rate of 73 sec -I) and failed to grow after 2.5 h of exposure (Fig. 10). Approximately a year later at subculture 48, the suspension was again tested as it had an improved growth rate and was less aggregated. The suspension was exposed to 1,000 rpm (average shear rate 167 seC I). Although the culture lost some 20 % of its dry weight no loss in viability was observed (Fig. 10). This apparent enhanced resistance to shear stress suggested that the P. quassioides suspension had changed such that it could be grown successfully in stirred-tank bioreactors and may also help to explain the easier initiation of 1 litre cultures. Half a litre of 21-day-old cells were inoculated into 2.51 of medium in a 31 stirred-tank bioreactor. The results are shown in Fig. 11. The culture grew successfully with a growth rate of 0.2 days-I (td = 3.5 days) and a final biomass yield of 8.81 g .1- 1 after 6 days. Thus, growth in the stirred tank bioreactor was much faster than in the air-lift bio-

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182

E. J. ALLAN, A. H . SCRAGG, and K. PUGH

reactor, and unlike growth in shake flasks has no lag phase. Indeed the growth rate was much higher than that generally found in shake flask cultures while the maximum biomass obtained was lower. Discussion The P. quassioides cell suspension used in this investigation has been maintained in the same growth media for 2 years. However, its growth characteristics and the ability to accumulate quassin have changed in this time. The transfer of cells into fluted flasks caused the suspension to be visibly less aggregated and subsequently the growth rates improved (Fig. 2). The alteration of the inoculation density to 2 g wet weight cells to 100 ml medium and a 21 day subculture cycle continued to improve growth. The maximum growth rate obtained was 0.239 days-l (td = 2.9 days) with a biomass yield of 11 g . 1- I, values comparable to those found for well established cultures such as Catharanthus roseus. No quassin was detected in the suspension culture grown in 250 ml or 21 shake flasks until subculture 43 and 36 respectively. It appears unlikely that quassin had been present in the early cultures below the detection limits since it was not detected in extracts of large amounts of cells. Therefore, the appearance of quassin, although in low yields of approximately 0.01 % dry weight may be due to the induction of quassin biosynthesis. Initial experiments where quassin was fed to cells (results not shown) indicate that there is a rapid turnover of quassin which may be responsible for its absence in early culture and low fluctuating levels in those producing quassin. Induction of quassin synthesis may be associated with the selection of the more rapidly growing cells which had occurred over repeated subcultures. Stafford et a1. (1985) showed that the stage in the growth cycle at which subculture occurs has a marked effect on serpentine yields in C. roseus, and this may also be the case for triterpenoid synthesis in P. quassioides. However, this does not explain why such a delay occurred prior to the onset of quassin synthesis (27 weeks in the 1.01 flasks), although the aggregated nature of the culture may have some influence. The increases in growth rates have been accompanied by a reduction in the size of aggregates in the suspension (Fig. 5). Aggregates have been proposed as reason for the synthesis of certain products in suspension cultures as large aggregates produce their own microenvironments in their interiors (Schuler, 1981). Perhaps the reduction in culture aggregates has triggered quassin accumulation. The P. quassioides suspension cultures show typical behaviour in response to various glucose concentrations and inoculation densities. The best growth rate was found with 5% (wlv) glucose and the maximum biomass with 10% glucose. Increasing the inoculum size decreased the lag phase but had little effect on the growth rates. Initial difficulty was encountered cultivating P. quassioides in volumes greater than 100 m!. It is thought that the attainment of good growth at larger culture volumes is associated with the stock cell line becoming more resistant to shear stress (Fig. 10). The growth rates and biomass yields in 100 ml and 11 cultures were comparable when grown on 2 %

glucose. Similar difficulties have been found with the cultivation of P. quassioides in a 7 litre air-lift bioreactor. Eventually a successful run was obtained but there was no obvious reason for this since subsequent runs were unsuccessful. No growth experiments were undertaken in air-lift bioreactors after subculture 36. The cultures in the 7 litre air-lift bioreactor were considerably less aggregated than the equivalent shake flask culture. The reduction of aggregation may be due to a higher shear in the bioreactor than in the shake flask, which may also be responsible for the long lag phase. The P. quassioides culture proved to be sensitive to shear stress at subculture 27 (Fig. 10 b). When tested approximately a year later at subculture 48 the P. quassioides cells were resistant to a higher shear stress over five hours. In addition the growth rate had improved considerably and the cells proved capable of growth in a stirred-tank. Growth of these more resistant cells in an airlift bioreactor has not been undertaken. The growth rates and biomass yields in the stirred-tank were greater than with those obtained in shake flasks and did not have any lag phase. The suspension culture has developed shear resistance over a period of a year in which the growth rate has also improved. Whether these two parameters are linked is unclear, but it may explain the initial difficulties of cultivation in 2 litre flasks and 7 litre air-lift bioreactor. Over the period of two years representing some 50 subcultures a highly aggregated, shear sensitive, non-quassin producing suspension has developed into a rapidly growing, less aggregated resistant culture capable of producing low levels of quassin. Acknowledgements The authors would like to thank Cadbury-Schweppes and Plant Science Ltd. for the support of this research. The authors are also most grateful to Mrs. Helen Woodhead and Dr. J. B. Sheridan (Cadbury-Schweppes) for many helpful discussions, assistance and hard work concerning HPLC analysis. Cadbury-Schweppes also provided purified samples of quassin and we are endebted to Dr. W. A. Laurie for mass spectra determination.

References ANDERSON, L. A., A. HARRIS, and J. D. PHILLIPSON: Journal of Natural Products 46,374-378 (1983). CORDELL, G. A.:In: REINHOLD, L., L. REINHOLD, J. B. HARBORNE, and T. SWAIN (eds.). Progress Biochemistry Vol. 5, 273-316. Pergamon Press, Oxford, New York (1978). CHAN, K. L., M. J. O'NEILL, J. D. PHILLIPSON, and D. WARHURST: Planta Medica 2, 105-107 (1986). GAMBORG, O. L., R. A. MILLER, and K. OJIMA: Experimental Cell Research 50, 151-158 (1968). KUPCHAN, S. M. and D. R. STREELMAN: Journal of Organic Chemistry, 41, No. 21, 3481-3482 (1976). METZNER, A. B. and R. E. OTTO: A. I. Ch. E.Journal 3, 3 -11 (1957). MORRIS, P., A. H . SCRAGG, N . J. SMART, and A. STAFFORD: In: DIXON, R. A. (ed.). Plant Cell Culture: A Practical Approach. 127 -167. IRL Press, Oxford (1985). ODJO, A., J. PlART, J. POLONSKY, and M. ROTH: Academie des Sciences Comptes Rendus, Paris. 293, 241-244 (1981). PHILLIPSON,]. D.: The Pharmaceutical Journal, 334-340 (1985). POLONSKY, J.: Recent Advances in Phytochemistry 6, 31-64 (1973 a).

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