Investigation into the Problems of Initiation and Maintenance of Cinchona ledgeriana Suspension Cultures

J.PlantPhysiol. Vol. 132.pp. 184-189{1988}

Investigation into the Problems of Initiation and Maintenance of Cinchona ledgeriana Suspension Cultures ALAN

H. SCRAGd), EUNICE J. ALLAN2) and PHILLIP MORRIS 3)

1) Wolfson Institute of Biotechnology, University of Sheffield, Sheffield S10 2TN 2) Division of Bacteriology, School of Agriculture, University of Aberdeen, 531 King Street, Aberdeen AB91 UD 3) University College of Wales, Welsh Plant Breeding Station, Plas Gogerddan, Aberystwyth, Dyfed SY23 3EB Received June 12, 1987 . Accepted August 31, 1987

Summary The establishment and maintenance of suspension cultures Cinchona ledgeriana Moens have proved difficult but cultures have been established in Gamborg's 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.

Key words: Cinchona ledgeriana Moens, suspension culture, pH Introduction Plant cell culture has been regarded as an alternative supply of fine chemicals to those extracted from whole plants. Both the antimalarial and bittering agent quinine and the antiarrhythmic quinidine, derived from the bark of Cin· chona spp. have been suggested as possible plant cell culture products (Curtin, 1983; Fowler, 1984). In our experience cell cultures of Cinchona ledgeriana Moens have proved difficult to maintain when compared with other plant cell lines. Sustained cultures of Cinchona spp. have been reported but culture protocol and conditions have apparently been modified from those typically used with plant cell cultures. Thus, media replacement at seven day intervals (Staba and Chung, 1981), frequent subculture at high inoculation densities (Koblitz et aI., 1983; Wijnsma et aI., 1985), supplementation with 10% (v/v) coconut milk (Morris and Fowler, 1982) and 1 % (w/v) polyvinylpyrrolidone (Anderson et aI., 1982) have been used. Both Hunter et a1. (1982) and Parr et a1. (1984) have reported the establishment of suspension cultures of C. ledgeriana in a defined medium but no information concerning subculture procedures was given. The subculture protocols described above prove inconvenient for experimental investigations and are presumably re© 1988 by Gustav Fischer Verlag, Stuttgart

sponsible for the lack of information on the growth kinetics of Cinchona spp. cell suspensions. The use of undefined medium components such as coconut milk is restrictive, e.g. when studying the effects of plant growth regulators or is unsuitable for mass cultivation in terms of cost and availability. Polyvinylpyrrolidone has, however, been used as an antioxidant to prevent necrosis by polyphenol oxidation in both callus (Hu and Wang, 1984), and suspension cultures of Cinchona spp. (Anderson et aI., 1982). This paper describes the possible reasons for the loss of viability of Cinchona suspension cultures and the advantages and disadvantages of polyvinylpyrrolidone in the initiation and maintenance of suspension cultures of C. ledgeriana. Material and Methods Callus cultures were established from Cinchona ledgeriana Moens seeds and maintained as described previously (Scragg et al., 1986). Suspension cultures were initiated by adding 1 g wet weight of friable callus to 50 ml Gamborg's B5 medium (Gamborg et aI., 1968) supplemented with 2 % glucose, 1 mg .1- 1 2,4-D, 0.1 mg .1- 1 kinetin, and other additions as described in the text. The volume was gradually increased over the initial subcultures until a culture volume of 120 ml was obtained. Incubation was at 25°C in 250 ml Erlenmeyer flasks containing 100 ml medium shaken at 150 rpm on

Cmchona ledgenana suspension cultures a New Brunswick G-10 shaker in subdued light. The suspension cultures were subcultured on a two week cycle using an inoculum of 20 ml of culture to 100 ml fresh medium. Cultural parameters, i.e. wet weight, dry weight, viability, and medium composition were determined as described by Stafford et al. (1984). Growth indices were calculated from the following formula: · d (GI) G rowt h III ex

=

Final dry wt. - Initial dry wt. . . Initial dry wt.

Alkaloids were extracted from freeze-dried cells, by 2 h soxhlet extraction with 50 ml methanol. The methanol extract was dried, resuspended in 2 ml methanol and analysed using a Waters Associates high performance liquid chromatograph (HPLC) with a Model 440 dual channel detector. A radial cartridge (8 mm x 10 em) packed with /LBondapak C18 was used at ambient temperature, the absorbance being monitored at 254 nm and 280 nm. The solvent system used was methanol and water containing 5 % acetic acid with a non-linear gradient of 20 to 45 % methanol III 20 min being suitable for separating quinine and quinidine. QuantificatIOn of quinine and quinidine was achieved by HPLC with their identity confirmed by thin layer chromatography in a solvent system of chloroform: methanol: 6N ammonia (85: 14 : 1) on silica gel plates against authentic standards. Chemicals used were generally from Fisous, BDH or Sigma and where possible were «AR» grade. Polyvinylpyrrolidone of 40,000 MW was used. Coconut milk was prepared using the method of Street (1977).

Results Suspension cultures of C. ledgeriana used in this study have been successfully initiated and maintained in BS medium containing 10% coconut milk or 1 % PVP. Attempts at initiating suspension cultures from callus or maintaining growth from already established suspension cultures in medium lacking either coconut milk or PVP all failed. This failure appeared to be associated with a drop in pH. Table 1 shows the pH and cell viability estimate during initiation of suspension cultures in Gamborg's BS medium containing a number of zwitterionic biological buffers. Friable callus (200 mg) was added to the medium and the pH

Table 1: Initiation of C. ledgerzana suspension cultures in buffered media. Media ComEosltion BS BS B5 B5 BS B5 B5 B5 B5

+ + + + + + + +

CM MES PVP HEPES EPPS TES PIPES pho>phate buffer

Time (day) Day 21 Growth Day 7 Day 14 EH viabilit:/: EH viabiltt:/: EH viabilit:/: 0 0 4.S8 + S.2S + 4.60 ++ 5.15 ++ 5.2 +++ + 5.82 ++ 4.90 +++ 5.70 ++ + 5.60 ++ 5.20 ++ 5.40 +++ ++ 4.60 ++ 4.75 + 4.80 + + 5.20 +++ 5.05 ++ 4.95 ++ + 4.07 + 4.05 ++ 4.90 ++ + 5.70 ++ 5.60 + 5.50 + + 4.50

+

4.95

+

0

0

Fnable callus (A8-S2) 200mg wet weight was added to 100ml Gamborg's B5 medIum supplemented WIth varIOus buffers (100 mM). The initial pH was adjusted to 5.8 with NaOH prior to sterilization. Samples were removed at weekly intervals for pH and viablltty determinations. Viability estimates: + 50%; + + 50 - 80%; + + + 80 - 100%. At day zero VIability was + + + .

185

and viability estimated at weekly intervals. It can be seen that a low pH (i.e. approx. 4.5) was found in the control, coconut milk supplemented, and HEPES, TES, and phosphate buffered media after an incubation period of 7 days after which time the medium pH remained relatively constant. The viability of the control and phosphate buffered cultures was low within 7 days of initiation and declined to zero within 21 days. The viability remained high, and indeed gradually increased, only in the cultures maintained in medium containing either coconut milk, PVP, or MES buffer. Table 2 shows the effects on viability and pH of the reduction or omission of certain ions from the BS medium during initiation suspension. The omission of phosphate or calcium from the medium resulted in a rise in pH above the initial pH of 5.8, but the viability was low and in the case of calcium, viability was zero by 21 days. The omission or reduction in magnesium concentration to 0.1 mM in the medium resulted in an initial drop in pH and loss of viability after 4 weeks. Only in the medium where the sodium phosphate concentration was reduced to 0.11 mM from 1.1 mM was both pH and viability maintained, but viability was lost after two subcultures. It was clear from Tables 1 and 2 that suspension culture initiation was associated with a rapid drop in pH, and that viability was only sustained in the presence of coconut milk, MES, or PVP. The effect of addition of PVP or MES on the growth and pH of established cultures is shown in Fig. 1. A 14d old suspension culture grown in B5 medium containing 1 % (w/v) PVP was filtered and three inoculation densities of 1, 2, and 3 g wet weight used to inoculate 100 ml BS medium containing 100mM MES or 1 % (w/v) PVP. The medium containing MES was adjusted to pH7.0 prior to inoculation. At all three inoculation densities the MES containing medium maintained the pH in the region of 7.0 for 21 days, but although viability was kept high, cell growth was poor in all cases as can be seen from the growth indices. In contrast in the medium containing 1 % (w/v) PVP the pH decreased to 4.0 in the first two days, subsequently increased slowly to 5.5. Despite this drop in pH, considerably better cell growth was obtained at all three inoculation densities. Titration again 0.1 M hydrochloric acid shows that supplementation of 100ml BS medium with coconut milk or poly-

Table 2: The effect of media composition on the initiation of suspension cultures of C. ledgeriana. Media GB5 control - Mg + 1/10Mg - P0 4 + 1/ 10 P0 4 - Ca - ammonium sulphate

Day 7 Day 14 Day 21 Day 28 pH viability pH viability pH viablltt:/: pH viabilit:/: 4.8 4.6 4.8 8.0 5.4 7.1

+ ++ + + + +

4.6 5.4 5.8 5.6 5.7 5.5

+ ++ + ++ +

4.7 5.3 5.9 5.6 6.0 5.5

4.8

+

5.9

+

6.5 0

+

+ ++ + + ++

5.1 0 4.6 0 5.6 0 6.2 + 6.1 ++

0

Friable callus (A8-S2) 200mg wet weIght was added to 100ml Gamborg's B5 medium where salts had been reduced or omitted. The initial pH was adJusted to 5.8 WIth NaOH pnor to sterilization. Samples were removed at weekly intervals for pH and viability determinations. V lability estimates: + 50%; + + 50 - 80%; + + + 80 - 100%. At day zero vlablltty was + + + .

ALAN H. SCRAGG, EUNICE J. ALLAN and PHILLIP MORRIS

186

A.

80 G.I. 0

<:> .... o OJ

E

:::>

'0

> :I: Co

<:

60

B

ttl OJ

G.I. 4'61

l:

65-60 60-55

5·5-50 50-45 45-40 40-35 35-3-0 pH Increments

Fig. 2: The relative buffering capacites of BS medium when supplemented with either PVP or coconut milk. 100 ml of medium were filtrated against a 0.1 M Hel solution and the volume of acid required to change the pH by an increment of 0.5 pH unit noted. The filtration was over the range of pH6.5-3.0 (0) BS medium; (0 ) BS medium containing 1 % (w/v) PVP; (_) BS medium containing 5% (w/v) PVP; (0 ) BS medium containing 10% (v/v) coconut milk.

10

20 Time (days)

Fig. 1: The changes in pH during the growth of C. ledgeriana suspension cultures in the presence of 100 mM MES (A) and 1 % (w/v) PVP (B). An established suspension culture growing on medium supplemented with 1 % (w/v) PVP was harvested by filtration after 14 days growth and various wet weights of cells used to inoculate flasks containing 100 ml BS medium containing either MES or PVP. The pH of the medium containing MES was adjusted to 7.0, and the medium containing PVP was adjusted to 5.8 prior to autoclaving. The experiment was run in triplicate, sampled at intervals, and the mean values are shown. (0 - -0 ) 1 g wet weight inoculum; (.--.) 2 g wet weight inoculum; (0--0) 3 g wet weight inoculum. Growth indices (G.!.) were calculated as described in Material and Methods.

vinylpyrrolidone increased its ability to resist changes in pH (Fig. 2). Although the supplements do not act as true buffers their ability to resist pH change was marked, with coconut milk being the most resistant followed by 5 % (w/v) PVP and 1 % PVP respectively. The B5 medium itself had little buffering capacity. Interestingly, much greater volumes of acid were required to cause a 0.5 unit decrease in pH at pH 5.0 and below for 10 % (v/v) coconut and 5 % (w/v)

PVP. The limited buffering capacity of PVP is again shown in Fig. 3 where the drop in pH was followed during the subculture of an established culture growing in medium containing either 1 % or 5 % PVP. The culture pH dropped slowly in the medium containing 5 % PVP and only reached a minimum pH of 4.2, whereas the medium containing 1 % PVP dropped rapidly to a pH value of 3.6. Unfortunately the presence of 5 % PVP produces a very viscous medium which in our experience caused the production of long, «strangled»

60

10

20

Time (days)

Fig. 3: The pH changes during the growth of C. ledgeriana suspensions in BS medium containing 1 % and 5% (w/v) PVP. The pH of the medium was adjusted to 5.8 prior to autoclaving. Duplicate flasks were inoculated with 3 g wet weight of filter harvested cells and samples removed at intervals for pH estimation. (0 - -0 ) medium containing 1 % (w/v) PVP; ( . - - . ) medium containing 5 % (w/v) PVP.

cells which grew poorly leading to eventual cell death. Hunter (personal communication) has however successfully used 5 % PVP for sustained suspension cultures.

187

Cinchona ledgeriana suspension cultures

~

L.

15

150

10

100

5

50

~

'-

»

o

30

20

10 Time days

15

Fig. 4: The growth of C. ledgeriana suspension cultures in medium supplemented with 1 % PVP or 10% coconut milk. A: 100 ml of B5 medium containing 1 % PVP and 2 % glucose was inoculated with 3 gwet weight C. ledgeriana cells (A8-S2 lined) and shaken at 150 rpm in a 250 ml flask at 25°C. Samples were removed at intervals and wet weight and dry weight estimated. Bars represent standard errors exceeding 5% of the mean (n = 3). (0 - -0 ) wet weight g'l-\ (e--e) dry weight g·l-I. B: 100ml of B5 medium containing 10 % coconut milk and 2 % sucrose was inoculated with 20 ml of a C. ledgenana suspension culture (PM line). Samples were removed and wet and dry weights estimated (0 - -0 ) wet weightg .1- 1; (e--e) dry weightg .1- 1•

1---~\

1---+ I I "t-..___•

150

100

~~

01

..... .J:::

01

OJ

~

.....QJ

5

50

3

V~

A growth curve of C. ledgeriana suspension culture in medium containing 1 % PVP is shown in Fig. 4 a. Growth was balanced in terms of dry and wet weights with a growth rate (p. medium) of 0.11 d -I, a doubling time of 6.3d, with a maximum biomass yield of 8.95 g ·1 - I. This represents a yield of 47 % of the glucose added. Similar kinetics were found in medium supplemented with 10 % (v/v) coconut milk (Fig. 4 b) although the cell line was different and sucrose was used as the carbon source. Growth was again balanced with a growth rate (p.) of 0.10d - l , a doubling time of 6.9d, and a final biomass yield of 10 g .1- 1 , representing a yield of 50%. The use of PVP in the culture medium of suspension cultures of C. ledgeriana is however not without its problems. Having established a suspension culture of C. ledgeriana giving good growth in the presence of 1 % (w/v) PVP the cells were analysed for the presence of quinine and quinidine.

10

20

30

40

Time days

These were detected at low levels, 0.005 - 0.03 % in flask grown suspension cultures and 0.0009 % in bioreactor cultured cells (Allan and Scragg, 1986). The initial extraction method used for quinine and quinidine was soxhlet extraction with methanol followed by chloroform extraction under acid and alkaline conditions in order to remove interference from contaminating anthraquinones and pigments. At a later stage this was replaced by the use of Waters SepPak columns to remove the pigments and anthraquinones, a procedure that was considerably quicker. However, with the second method the yields of quinine and quinidine were drastically reduced. It was observed that PVP could not be completely removed from harvested cells, and was extracted into the solvent during soxhlet treatment. It appeared that PVP bound quinine and quinidine and was retained by the Sep-Pak columns. This can be seen in Fig. 5 where quinine and quinidine have been mixed with a 1 % PVP solution and

188

ALAN H. SCRAGG, EUNICE]' ALLAN and PHILLIP MORRIS

A

E

c o

LJl N

B

QJ

u

C III

.c 'o Vl

~

c

Time(min) Fig. 5: HPLC analysis of quinine and quinidine extracted by various methods in the presence and absence of PVP. Quinine and quinidine at a concentration of 2 mg . ml- 1 were added to a solution of 1 % PVP which was either passed through a Sep-Pak column or extracted by the acid/alkali chlorofrom method as described under Methods a, control; b, Sep-Pak treated; c, acid/alkali treated.

passed through a Sep-Pak column. The same experiment has been repeated using the normal acid alkali extraction (Fig. 5). The Sep-Pak treatment in the presence of PVP removed all the added quinine and quinidine whereas the acid alkali treatment gave 94 % and 83 % yields of quinine and quinidine respectively. There was also a reduction in PVP containing samples applied directly to the HPLC columns indicating a similar type of binding to that found for the Sep-Pak system. These observations were also confirmed by thin layer chromatography. Samples of quinine and quinidine mixed with a 1 % PVP solution streaked and failed to give any spots. In contrast the acid alkali extraction in the presence of 1 % PVP gave clear spots corresponding to quinine and quinidine. Discussion

In general the initiation and maintenance of suspension cultures of Cinchona has proved difficult. The loss of viabil-

ity on initiation of suspension cultures, appears to be associated with a drop in pH. This drop in pH can be reduced by supplementing the medium with buffers, but of the buffers tried only MES maintained viability. Other additions that have proved successful in maintaining viabilities have been coconut milk or PVP. The rapid drop in pH upon addition of Cinchona callus to liquid medium may be a result of a hydrogen ion efflux. The addition of Cucumis callus to medium of various pH values caused the pH to be changed within 24 hours to 4.5-4.8 (Skirvin et al., 1986). This type of efflux has been observed for Acer pseudoplatanus and Phaseolus vulgaris cultures (Fisher and Albersheim, 1974) and has been related to the uptake of certain ions, probably ammonium or nitrate (Martin and Rose, 1976). Table2 indicates that the uptake of phosphate and calcium may also be the ions involved. The ability of both PVP and coconut milk to maintain viability when added to culture medium is presumably associated with their buffering ability. Fig. 2 shows that although neither coconut milk or PVP are true buffers they have the ability to resist changes in pH. Even in the presence of PVP the pH of the medium drops rapidly after the subculturing of a suspension culture before rising as a result of cell growth. Both coconut milk and PVP function by stopping the pH dropping too low. The other buffer MES was able to maintain a stable pH but was unable to support growth in suspension cultures. This may be due to MES directly inhibiting growth or that coconut milk and PVP have another feature such as the ability to inhibit polyphenol oxidase (Hu and Wang, 1984). This is unknown at present. Whatever the full mechanism(s), addition of PVP or coconut milk to the culture medium allowed good growth of Cinchona suspensions with doubling time of 6.3 and 6.9 days and yields of 8.95 and 10.6 g .1- 1 respectively. The addition of PVP to the culture medium does however cause problems when the suspension cultures were analysed for the presence of quinine and quinidine. PVP binds quinine and quinidine which interferes with the small SepPak columns, stops their running on TLC plates and probably reduces the efficiency obtained in HPLC analysis. The method of acid/alkali extraction with chloroform was found to separate PVP from the alkaloids, PVP being found at the aqueous chloroform interface, giving yields of between 94- 83 % of the added quinine and quinidine respectively. References ALLAN, E. ]. and A. H. SCRAGG: Comparison of the growth of Cinochona ledgeriana Moens suspension cultures in shake flasks and 7 litre air-lift bioreactors. Biotec. Letters 8,635-638 (1986). ANDERSON, L. A., A. T. KEENE, and]. D. PHILLIPSON: Alkaloid production by leaf organ, root organ, and cell suspension cultures of Cinchona ledgeriana. Planta Medica 46,25-27 (1982). CURTIN, M. E.: Harvesting profitable products from plant tissue culture. Biotechnology 1, 649-657 (1983). FISHER, M. L. and P. ALBERSHEIM: Characterization of a H+ efflux from suspension-cultured plant cells. Plant Physiol. 53, 464-468 (1974). FOWLER, M. W.: Plant cell culture: Natural products and industrial application. Biotechnology and Genetic Engineering Reviews 2, 41-67 (1984).

Cinchona ledgeriana suspension cultures GAMBORG, O. L., R. A. MILLER, and K. OJIMA: Nutrient requirements of suspension cultures of soybean root cells. Expl. Cell Res. 50, 151-158 (1968). Hu, C. Y. and P. J. WANG: Meristem, shoot tip and bud cultures. In: EVANS, D. A., W. R. SHARP, P. V. AMMIRATO, and Y. YAMADA (eds.). Handbook of plant cell culture, pp. 177 - 226, Macmillan Inc., New York, London (1984). HUNTER, C. S., D. V. MCCALLEY, and A. J. BARRACLOUGH: Alkaloids produced by cultures Cinchona ledgerzana L. In: FUJIWARA, A. (ed.) Proc. 5th IntI. Congo Plant Tissue and Cell Culture, pp. 317-318, Maruzen Co. Ltd., Tokyo (1982). KOBLITZ, H., D. KOBLITZ, H.-P. SCHMAUDER, and D. GROGER: Studies on tissue cultures of the genus Cinchona L. alkaloid production in cell suspension cultures. Plant Cell Rep. 2, 122-125 (1983). MARTIN, S. M. and D. ROSE: Growth of plant cell (Ipomoea) suspension cultures at controlled pH levels. Can. J. Bot. 54, 1264-1270 (1975). MORRIS, P. and M. W. FOWLER: Growth characteristics and pigment production by cell suspension cultures of Cinchona ledgeriana. Poster. Soc. Exptl. Biology (1982). PARR, A. J., J. 1. SMITH, R. J. ROBINS, and M. J. C. RHODES: Apparent free space and cell volume estimation: A non-destructive method

189

for assessing the growth and membrane integrity/viability of immobilised plant cells. Plant Cell Rep. 3, 161-164 (1984). SCRAGG, A. H., P. MORRIS, and E. J. ALLAN: The effects of plant growth regulators on growth and alkaloid formation in Cinchona ledge· riana callus culture.J. Plant Physio!. 124, 371- 377 (1986). SKIRVIN, R. M., M. C. CHU, M. L. MANN, H. YOUNG, J. SULLIVAN, and T. FERMANIAN: Stability of tissue culture medium pH as a function of autoclaving time, and cultured plant material. Plant Cell Rep. 5, 292-294 (1986). STABA, E. J. and A. C. CHUNG: Quinine and quinidine production by Cinchona leaf, root, and unorganised cultures. Phytochemistry 20,2495-2498 (1981). STAFFORD, A., L. SMITH, and M. W. FOWLER: Regulation of product synthesis in cell cultures of Catharanthus roseus (L.) G. Don. Plant Cell Tissue Organ Culture 4, 83 - 94 (1984). STREET, H. E.: In: H. E. STREET (ed.), Plant Tissue and Cell Culture pp. 11- 30. 2nd Edition, Blackwell Scientific Publications, Oxford, London, Edinburgh, Melbourne (1977). WI]NSMA, R., J. T. K. A. Go, 1. N. VAN WEERDEN, P. A. A. HARKES, R. VERPOORTE, and A. BAERHEIM-SVENDSEN: Anthraquinones as phytoalexins in cell and tissue cultures of Cinchona spp. Plant Cell Rep. 4, 241- 244 (1985).