Short Communication

The Effects of Plant Growth Regulators on Growth and

Alkaloid Formation in Cinchona ledgeriana Callus Culture
ALAN H. SCRAGG, PHILUP MORRIS and EUNICE J. ALLAN
Wolfson Institute of Biotechnology, University of Sheffield, Sheffield, S10 2TN, United
Kingdom
Received October 7,1985 Accepted January 2,1986

Summary
Callus cultures of Cinchona ledgeriana were established from seeds obtained from Kenya on
Gamborg's B5 medium containing 2 % glucose, 1 mg 1-1, 2,4-D and 0.25 mg I-I 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 rnA 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.

Key words: Cinchona ledgeriana Moms, callus growth, auxin and cytokinin levels, cultural
conditions, alkaloid production, plant growth regulators.

Introduction
The two Cinchona species C. ledgeriana Moens and C. succirubra Pavon ex
Klotzsch (c. pubescens Vahl) are the source of pharmaceutically important compounds; the anti-malarial quinine and the anti-arrhythmic quinidine. In addition,
quinine is used as a bittering agent in soft drinks. Therefore, the use of plant cell cultures of Cinchona spp. as an alternative supply of these compounds is of commercial
interest.
Several laboratories have succeeded in establishing callus cultures of Cinchona spp.
e.g. Wiryowidagdo et al. (1980); Staba and Chung (1981). Mulder-Krieger et al.
(1982a, b) and Verpoorte et ai. (1984) have reported, along with Morris and Fowler
(1982), details of growth and optimisation of media constituents. In addition, Verpoorte et aI., (1984) have compared alkaloid in calli obtained from different explant
tissues and further studied the effect of plant growth regulators on stem callus of C.
pubescens (Mulder-Krieger et aI., 1982 b, 1984). Quinine yields of 0.0112 %, reported
Abbreviations: 2,4-D = 2,4-dichlorophenoxyacetic acid, NAA = naphthalene acetic acid,
IAA = indole-3-acetic acid, IBA = indolebutyric acid, IpA = N6(~2-isopentenyl) adenine,
IpAR = N6(~2-isopentenyl) adenosine, 6BA = 6-benzyladenine, Z = zeatin, ZR = zeatin riboside, K = kinetin, KR = Kinetin riboside.

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

by the latter authors for callus cultures of Cinchona spp., are lower than the highest yield
of 0.04% found in suspendion cultures (Hunter et aI., 1982) which, in turn, are both
much lower than that found in the bark of mature Cinchona trees (Chatterjee, 1974).
Cultural conditions, especially the quality and quantity of phytohormones can
have a profound effect on both growth and secondary product formation (Zenk et
al., 1977). Here, we report an extensive survey of the effect of plant growth regulators
on both growth and production of quinine and quinidine by callus cultures of C. led-

genana_

Materials and Methods

Callus cultures were established from Cinchona ledgeriana Moens seeds obtained from
Kenya. The rough husk was removed and the seed washed in ethanol before being surface sterilized in 15 % Domestos (Lever Limited, U.K.) for 30 mins. After several washings in sterile
distilled water the seeds were transferred to solid B5 medium (Gamborg et aI., 1968) supplemented with coconut milk (10%), 2% sucrose, 0.8% agar, Imgl- 1 2,4-D and 0.lmgl- 1 kinetin. Once callus was initiated it was subcultured every 4 - 6 weeks on B5 media with 2 %
glucose rather than sucrose as carbon source and without supplementation with coconut milk.
Calli used in the experiments were subjected to a single passage on each experimental medium.
All cultures were incubated at 25C in subdued light.
The effect of cytokinins on callus gowth was tested by using one of seven cytokinins at concentrations ranging from 0 to 1.0 mg 1- I in the presence of one of four auxins at a fixed concentration of 1 mgl- 1 Thus 125 different combinations of phytohormone type and concentration
were investigated while the effect of auxins on growth and production was tested by maintaining the cytokinin concentration at 0.5 mg 1- I and varying the type and concentration of
auxin (0; 0.1; 0.25; 0.5 and 1.0 mgl- 1). A total of 240 different types of plant growth regulators
and concentrations were studied.
Sterile plastic boxes (Sterilin) containing 25 wells (5 x 5) were used and media (2 ml) was added
to each well with the required concentration of filter-sterilized auxin and cytokinin. Five wells
were used for each cultural variation with each well being inoculated with approximately 50 mg
wet weight of callus. As far as possible callus of a similar type in terms of age, colour, and
friability was used for the inocula. The boxes were covered with polythene to retain moisture
and incubated for 4 weeks by which time the calli were in exponential growth phase. The calli
were weighed separately for wet weight and combined, dried for 48 hours at 60C for dry
weights.
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 chromatography system (HPLC) with a Model 440
dual channel detector. A radial cartridge (8 mm x 10 cm) packed with p.Bondapak 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 in 20 mins being suitable for separating quinine and quinidine. Quantification
of quinine and quinidine was thus achieved by HPLC with their identity confirmed by thin
layer chromatography in a solvent system of chloroform: methanol: ammonia, 6N (85: 14: 1)
on silica gel plates against authentic standards.

Results
Effect ofphytohormones on growth
Fig. 1 shows the growth of C. ledgeriana callus at varying cytokinin concentrations
and fixed auxin concentrations. All seven cytokinins supported growth in the pres-

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Vol. 124. pp. 371-377 (1986)

Callus culture of Cinchona ledgeriana

373

IpA

6BA

10

o.

6BA

10

Fig. 1: The effect of various cytokinin concentrations on dry weight-yields of C. ledgeriana callus at fixed auxin concentrations of (A) 1.0mgl- 1 2,4-D; (D) 1.0mgl- 1 IAA; (C) 1.0mgl- 1
NAA; (D) 1.0mgl- 1 IBA. The calli were grown for 4 weeks as described in Materials and
Methods. The cytokinin concentrations used were 0, 0.1, 0.25, 0.5 and 1.0mgl- l .
Dotted line represents IpAR.

ence of 2,4-D/ffiA/NAA although extensive aerial root formation occurred with the
latter. Growth was relatively poor in the presence of IAA. In the presence of 1 mg I-I
2,4-D all seven cytokinins supported growth but none showed a dose response to increasing concentration of cytokinin. The growth of callus at zero cytokinin concentration may be due to carry over of cytokinin. In the presence of 2,4-D best growth

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Vol. 124. pp. 371-377 (1986)

374

ALAN H.
11
2
10

SCRAGG, PHILUP

-A.

6BA

~~ ~ ~
I<R

I~ ~

IpA'lpAR

2
0

~~

MORRIS and EUNICE J. AlLAN

ZR

2
0

8
6
4
2

IpA

~
"

Ii

1
12
10
8
6
4
2

14
12
10

KR

6BA

..h

IpA,lpAR

~~]~

14
12

Ie

8
6

~~

14
12
10
8
4
2

ZR

6BA

KR

IpA: lpAR

..D;

14
1
10
8
6
4
2

14
12
10
8
6
4
2

.~

~~
ZR

~ ~

Fig. 2: The effect of various auxin concentrations on dry weight yields of C. ledgeriana callus at
fixed cytokinin concentrations of 0.5 mg I-I. The calli were grown for 4 weeks as described in
Materials and Methods. The auxin concentrations used were 0, 0.1, 0.25, 0.5 and 1.0 mg 1-1, (A)
varying the 2,4--D concentration; (B) varying the IAA concentration; (C) varying the NAA concentration; (D) varying the rnA concentration.
Dotted line represents IpAR.
was obtained with kinetin, with IAA and IDA it was zeatin, and with NAA it was
IpAR. The best overall growth was with combinations of 2,4-D and kinetin or NAA
and IpAR. The callus colour and structure varied from pale yellow and friable in the
presence of 2,4--D to hard and brown under adverse conditions such as IAA with a red
colouration being occasionally found.

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Callus culture of Cinchona ledgeriana

375

In Fig. 2 conditions were reversed with 115 hormonal combinations being investigated. All seven cytokinins gave good growth, in terms of dry weight, in the presence
of 2,4-D with all but zeatin riboside and IpA showing no growth at zero values of
2,4-D. However, no auxin exhibited a dose response so that the higher concentrations
of 2,4-D only stimulated growth in the presence of zeatin. As observed previously
IAA only supported limited growth with zeatin and IpA. NAA concentrations
yielded a higher dry weight particularly with zeatin, zeatin riboside and IpAR than
that obtained with 2,4-D. Here again no dose response was observed with the auxin
but no growth was observed at zero auxin concentration. A similar result was found
with varying concentrations of IBA except that growth was observed at zero concentrations with 6BA, IpAR and zeatin riboside.
In the presence of 2,4-D best growth was obtained with kinetin and IpA, with
NAA it was with zeatin and zeatin riboside, and with IBA it was zeatin. The best
overall growth was obtained with IBA and zeatin or NAA and zeatin riboside.
Table 1: Quinine (JLg/g) (a) and quinidine (JLg/g) (b) present in Cinchona ledgeriana callus grown
in the presence of fixed auxin (1 mg I-I) and various concentrations of cytokinins.
Auxin
(mg I-I)
1.0

Cytokinins

Cytokinin concentration (mg I-I)

0

0.25

0.50

kinetin
IpA

+
0

+
0

0
0

kinetin
zeatin

5.6
0

136.6
0

0
+

0
+

IAA

zeatin
zeatin riboside
IpA
IpAR

0
0
0
0

0
0
42
0

531
34
42
+

453
67
111
+

0
84
53
+

NAA

kinetin
kinetin riboside
6BA

0
0
0

0
0
0

25
0
35

12
26
24

8
35
18

(a)
2,4-D
IAA
(b)
2,4-D

0.75
0
+

1.00
0
0
5.5
+

+ alkaloid present in trace amounts.

Formation ofquinine and quinidine

Calli grown on 240 phytohormonal variations were analysed for the presence of
quinine and quinidine. Trace amounts of quinine were only detected when calli were
grown in the presence of 2,4-D and kinetin or in lAA plus IpA (Table 1 a). Analysis
of cell extracts from calli grown in the presence of a fixed cytokinin concentration
and various auxin concentrations showed that low levels of quinidine (1-100 Itg/ g
freeze dried cells) were only detected in calli grown on 0.5mgl- 1 IBA and 0.251.0 mg I-I NAA. Quinidine production was more evident when calli were grown at a
fixed auxin concentration of 1 mg 1- I and varying cytokinin concentrations (Table

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ALAN H. SCRAGG, PHILUP MORRIS and EUNICE]. ALuN

1 b), with a maximum. quinidine concentration of 531 p.g/ g being found when calli
were grown on Gamborg's BS supplemented with 1 mg 1-1 IAA and 0.5 mg 1-1
zeatin.

Discussion
The initiation of C. ledgeriana callus on Gamborg's BS medium containing 2 %
sucrose, 1 mgl- I 2,4-D and 0.1 mgl- I kinetin by germinating sterile seeds presented
little problem in comparison with previous attempts where browning and subsequent death occurred (Hunter, 1979; Morris and Fowler, 1982). The addition of
coconut milk used in previous work (Morris and Fowler, 1982) was also found not to
be essential.
It can be concluded from the survey of the effect on growth of various concentrations of auxins and cytokinins that C. ledgeriana callus can grow under a wide range
of conditions. This is perhaps not unexpected as other cultures of C. ledgeriana have
been grown on Murashige and Skoog's (M & S) medium containing IBA and 6BA or
2,4-D and 6BA (Staba and Chung, 1981), M & S medium containing 2,4-D and kinetin (Anderson et al., 1982) and C. pubescens on M & S medium containing 2,4-D
and 6BA, or IBA and 6BA (Mulder-Krieger et aI., 1982 a). In contrast, Hunter et al.,
(1982) have used BS medium containing 2,4-D and 6BA or kinetin.
Only trace amounts of quinine were detected in calli grown on Gamborg's BS
media supplemented with a wide range of auxins and cytokinins. Similar levels of
quinine (0.0008 % on a fresh weight basis) have been detected in stem material of
shoot cultures of C. ledgeriana (Robins et al., 1984) which contrasts with levels of
0.4% quinine detected in the bark of l-year-old C. ledgeriana plants (Scragg, unpublished results). Many calli did however produce quinidine particularly when grown
on NAA and higher concentrations of 6BA. The maximum quinidine yield of
0.053 % was found for calli grown on BS media with 0.5 mg 1-1 zeatin and 1 mg 1-1
IBA. This yield of quinidine is much higher than that reported by Mulder-Krieger et
al. (1982 b) and Verpoorte et al. (1984) for callus cultures and is also higher than that
found in suspension cultures (Hunter et al., 1982; Anderson et al., 1982).
In these experiments alkaloids were extracted from calli harvested in the exponential phase growth which may not represent the optimum time for alkaloid accumulation. However, the low yields of quinoline alkaloids detected indicate that even
a broad range of phytohormones may not be effective in either triggering the production or allowing the accumulation of the alkaloids. In view of these results future
research may therefore be more justified in investigating the regulatory mechanisms
of the quinoline alkaloid pathway and the accumulation of alkaloids in the cell.
Acknowledgements
The authors wish to express their thanks to Cadbury-Schweppes for support of this work
and to Miss Karen Pugh and Mrs. H. Woodhead for technical assistance.

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