Scientia Horticulturae 119 (2009) 325329

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Screening of vincristine yield in ex vitro and in vitro somatic embryos derived

plantlets of Catharanthus roseus L. (G) Don.
Junaid Aslam a,b, Abdul Mujib a,*, Seikh A. Nasim a, Maheshwar Prasad Sharma a
a
b

Cellular Differentiation and Molecular Genetics Section, Department of Botany, Hamdard University, New Delhi 110062, India
Plant Tissue Culture Laboratory, Dubai Pharmacy College, Al-Muhaisanah 1, Al-Mizhar, P.O. Box 19964, Dubai, United Arab Emirates (U.A.E.)

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 13 April 2008
Received in revised form 11 August 2008
Accepted 15 August 2008

Catharanthus roseus L. (G.) Don. (Apocynaceae) is an important dicotyledonous medicinal plant as it is the
sole source of vincristine and vinblastine that are used against a variety of cancers. Quantitative
estimation of vincristine was carried out using high performance liquid chromatography (HPLC) in
various in vitro grown tissues; calluses (embryogenic and non-embryogenic callus), embryogenic stages
(proliferated, matured and germinated embryos), somatic embryo derived plantlets (leaf, root and whole
plant) and leaves of ex vitro developed plantlets. The yield in those in vitro and ex vitro-developed tissues
was monitored for 30 weeks. Except at an early lag period, vincristine production was detected up to 20
25 weeks old plant samples. Vincristine content was very high in leaf callus and germinated embryos.
Leaves of in vitro raised plants showed higher amount of vincristine when compared to ex vitro-developed
leaves of similar age. Vincristine production was tissue specic and age dependent that was discussed in
detail in this present communication.
2008 Elsevier B.V. All rights reserved.

Keywords:
Apocynaceae
Catharanthus roseus
Embryogenic callus
High performance liquid chromatography
Somatic embryogenesis
Vincristine

1. Introduction
The genus Catharanthus comprises of eight species, which are
subshrubs (3090 cm long), generally upright or decumbent,
produce white latex and a strong pungent smell when damaged
(Anonymous, 1992). The C. roseus has a pantropic distribution,
naturalized in continental Africa, America, Asia, Australia, South
Europe and on some islands in the Pacic Ocean. It is a rich source
of alkaloids; more than 130 different compounds have so far been
reported (Verpoorte et al., 1993; Van der Heijden et al., 2004), in
which vincristine (Fig. 1) is highly important. Vincristine is mainly
used to treat against acute leukemia, Hodgkins disease, rhabdomyosarcomas, neuroblastoma and other lymphomas (Mukherjee
et al., 2001; Van der Heijden et al., 2004). Traditionally, C. roseus is
propagated through seed, which leads to genetic segregation and
decline in uniform yield of dimeric alkaloid (Venkataramaiah et al.,
1980; Chand and Singh, 1999, 2004). Structurally, vincristine and
vinblastine (another anticancerous compound, present in the same
plant) are bisindole alkaloids with vindoline attached to tetracyclic
indole, carbomethoxyvelnamine. These two compounds are very
similar in structure and display nearly the same kind of action by
inhibiting cell division at metaphase. Isolation of this substance

* Corresponding author. Tel.: +91 11 26059683x5542; fax: +91 11 26059663.

E-mail address: amujib3@yahoo.co.in (A. Mujib).
0304-4238/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.scienta.2008.08.018

from wild plant is impeded by low productivity, is therefore very

expensive. Thus, synthetic preparations have been attempted to
produce these alkaloids in several laboratories at an affordable
rate. Although total synthesis of vinblastine has been achieved in a
number of cases synthesis of vincristine has not been accomplished except a recent report of stereo-selecting coupling of
demethyl vindoline with an eleven membered carbomethoxyverbanamine precursor (Kuboyama et al., 2004). This de novo
synthesis of vincristine was described to be the rst synthesis of
its kind, which was not dependent on derivatization of vinblastine
or on semi-synthesis with vindolinethe other two ways of
syntheses, previously reported (Kuboyama et al., 2004). As the
yield is not always good, a comprehensive multidisciplinary
approach has long been attempted in order to improve the content
(Verpoorte et al., 1993; Moreno et al., 1995; Van der Heijden et al.,
1989, 2004). In plant tissue culture, different tissues of this plant
have been used to establish cultures and the content of dimeric
alkaloid was analyzed (Hirata et al., 1990; Batra et al., 2005), but
the yield of this compound is still not very high. The technique of
somatic embryogenesis has been used and reported in a wide
range of plant species (Thorpe, 1995; Mujib and Samaj, 2006) but
the same embryonic tissue has never been utilized in alkaloid
enrichment programme as somatic embryogenesis report in C.
roseus is relatively new (Junaid et al., 2006, 2007a,b). In the present
study we quantied the level of vincristine from various in vitro
raised tissues and a comparison was made with the plants grown

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326

2.60 mM GA3. Part of the matured embryos was oven dried for
extraction and the other half was cultured for germination/plantlet
conversion.
Matured SEs were cultured on MS that was added with
optimized level of BAP (2.24 mM) for ensuing quick germination
(Junaid et al., 2006). Plantlets, roots, etc. developed from in vitro
germinated embryos were separately dried for alkaloid extraction
purpose.
2.5. Quantication of vincristine by HPLC and sample preparation

Fig. 1. Chemical structure of vincristine.

ex vitro. This protocol may be used in pharmaceutical industries to

synthesize enhanced level of vincristine.
2. Materials and methods
2.1. Plant material, sterilization, in vitro seed germination and culture
condition
Catharanthus roseus L. (G). Don. was collected from the Jamia
Hamdard (Hamdard University, New Delhi) herbal garden, was
identied by one of our experts (Dr. M.P. Sharma, Plant taxonomist,
Department of Botany) and used as experimental material. The
voucher specimen has been deposited in the Herbarium of the
same Department, Faculty of Science, Jamia Hamdard (Hamdard
University, New Delhi, India).
Various explants (leaf, nodal segments and root) and unripe
fruits were used as explants. The surface sterilization of explants
and the method of in vitro seed germination were discussed earlier
(Junaid et al., 2006). Germination medium (Murashige and Skoog,
1962) contained all the components of MS and 3% sucrose but
without organic compounds and plant growth regulators. Germinated seedlings were grown until they reached 2 cm in length; the
hypocotyls were later excised for culture.
The medium pH was adjusted to 5.8. The medium was sterilized
in an autoclave for 15 min at 121 8C. Cultures were incubated at
25  2 8C under 16h photoperiod with cool white uorescent
illumination (100 mmol m 2 s 1 PFD).
2.2. Induction of callus
The initial procedure of establishment of callus was carried out
as described earlier (Junaid et al., 2006). The explants were
cultivated on MS supplemented with optimized concentration of
2,4-D (6.96 mM).
2.3. Somatic embryo (SE) initiation and proliferation
The embryogenic callus was cultured on medium added
with 3% sucrose and optimized concentrations of 5.37 mM
NAA + 6.72 mM BAP for embryo initiation and further proliferation
(Junaid et al., 2006). Half of the proliferated culture was processed
for alkaloid extraction and the rest of the embryos were
maintained in medium for maturation.

The content of vincristine in eld-grown plants and in vitro

cultures were estimated using vincristine (SigmaAldrich) as
standard.
One gram each of various harvested in vitro cultural sample and
eld grown Catharanthus plant were dried separately at 45 8C for a
week and pulverized in a mortar and pestle. Dried material was
shaken in 20 ml methanol for 24 h, evaporated up to 2 ml and then
20 ml of (0.5)N H2SO4 (Hi-media Lab., India) was added. Solution
was made alkaline by adding 25% ammonium hydroxide (Hi-media
Lab., India) solution (pH 912) and nally extracted three times
with chloroform (Hi-media Lab., India) and evaporated up to
dryness. The left residue was reconstituted in 4 ml methanol (Himedia Lab., India). The methanol soluble fraction of each sample
was further diluted to 1:10 and processed for HPLC.
2.6. Standard vincristine and standard curve
Two milligram of vincristine sulphate with over 97% purity
available from SigmaAldrich (St. Louis, U.S.A.) was processed in the
same way similar to sample preparation. It was prepared in
methanol and 1 ml of this solution was diluted to 10 ml with
methanol to get 100 mg ml solutions, the solution was used for
application on TLC plate for preparation of standard plot. The UV
spectrum of standard vincristine solution in methanol (50 mg ml)
was recorded using a UV spectrophotometer (UV-1601, Shimadzu,
Japan) for authentication. The lmax (220 nm) obtained was matched
with that as reported for standard vincristine (Chu et al., 1996).
Stock solution 1 mg ml of vincristine was prepared in methanol.
From the stock solution, dilution of 220 ml was taken in triplicate
and analyzed independently by HPLC and a standard curve was
plotted between concentration and peak area. The injected
quantities showed good linearity. The data of peak area vs.
vincristine concentration were treated by linear least-square
regression and the regression equation thus obtained from standard
curve was used to estimate vincristine in different samples.
2.7. High performance liquid chromatography (HPLC) and
quantication of vincristine in different samples
For HPLC analysis, A Merck LiChro CART C18 column
(125 mm  4 mm 5 mm) and the solvent systems (acetonitrile
and methanol; 3:1) were used at a constant ow rate of 1 ml min
with 2300 pressure. A PDA detector was employed for detection of
peaks, set at wavelength of 220 nm and bandwidth of 5. All the
analyses were performed with three replicates.
Samples of in vitro culture and eld grown plant, 20 ml each,
were applied in triplicate for quantication of vincristine. The
alkaloid was quantied by using regression equation of calibration
curve.

2.4. SE maturation and germination

2.8. Statistical analysis
SE maturation was made in optimized medium that was
discussed at length in earlier report (Junaid et al., 2006). The
medium basically amended with 3% maltose and optimized

The inuence of various days intervals at vincristine yield was

analyzed by one-way analysis of variance (ANOVAs). Values are

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327

Fig. 2. Vincristine yield in callus induced from different explants. Data were scored up to 30 weeks.

means of ve replicates from two experiments, and the presented

mean values were separated using Duncans Multiple Range Test
(DMRT) at p  0.05.
3. Result and discussion
Various ex vitro and in vitro developed plantlets and plant parts
obtained from normal and somatic embryogenic pathways were
used for the quantication of vincristine. Of the different explants
(leaf, nodal segment, root and hypocotyls of in vitro germinated
seeds), only hyocotyl of in vitro germinated seed produced friable
embryogenic callus; calluses from other sources were nonembryogenic which were compact in nature. Quantitative
estimation of vincristine showed a difference in yield in
embryogenic and non-embryogenic callus; but in both cases,
vincristine started to appear after 10 weeks onwards with almost
linear increase up to 25 weeks. Among non-embryogenic calluses,
maximum vincristine was observed in leaf callus followed by nodal
callus; while in root callus vincristine was altogether absent, even
in tissue that was 30 weeks old. In leaf callus, maximum vincristine
was reported in 20 weeks old culture; thereafter the yield became
stable (Fig. 2). Similarly, SEs at various developmental stages
(proliferation, maturation and germination) were isolated, dried
and processed for vincristine estimation. In all the stages
vincristine detection was observed 10 weeks onwards. Maximum
vincristine was quantied from germinated embryos followed by
matured and proliferated embryos (Fig. 3).
Quantitative estimation of ex vitro and in vitro raised plantlets
also showed a difference in vincristine yield. In eld grown
plants, a linear increase in vincristine yield was noted for about
20 weeks, later it started to decline, but in SE derived plants, the

synthesis of alkaloid continued to increase for another few

weeks, 25 weeks to be precise (Fig. 4). Leaves of in vitro raised
plant showed maximum vincristine when compared to in vivo
developed leaves (Fig. 5). It is rather difcult to point out a single
mechanism by which in vitro raised tissues or plantlets
overproduce alkaloids, the synthesis is reported to be under
control by several factors at different level instead (Moreno et al.,
1995; Zhao et al., 2001). The balanced medium components,
carbon source, photoperiod, the cellular stresses (biotic and
abiotic) created during culture and other factors regulate the
synthesis of alkaloids in culture (Rocha et al., 2005; Bhat et al.,
2008). The PGRs (especially the 2,4-D and other members of
auxin) represent another class of compounds, supplemented to
medium for inducing specic morphogenetic response, also
cause and sometimes add stress to the cultivated cells. At the
same time, the same groups of compounds act as signaling
molecule in inuencing biochemical pathways underlying
enhanced level of alkaloid synthesis.
We screened vincristine yield in various in vitro and ex vitro
developed plantlets or tissues raised through conventional and
somatic embryogenesis pathways. In present investigation, we
found that vincristine production was tissue specic and age
dependent. It was earlier conrmed that the synthesis of
secondary metabolites being associated with the degree of
differentiation of individual cell or tissues (Pande et al., 2002;
Ma et al., 2006). The results observed in the present study is very
similar to the study that dealt with other diametric alkaloid
vinblastine (Datta and Srivastava, 1997), where increased
accumulation of vinblastine was noted as the seedlings grew,
reached a steady state when the plants became older (three
months old or more). Reports on dimeric alkaloids are very limited

Fig. 3. Vincristine yield in different developing stages of somatic embryogenesis. Data were scored up to 30 weeks.

J. Aslam et al. / Scientia Horticulturae 119 (2009) 325329

328

vincristine that can be enriched to a level of acceptability, even

industrially, if in vitro mutagenesis, other biotechnological
techniques and semi-synthetic method be integrated.
Acknowledgement
Author (Dr. Junaid Aslam) gratefully acknowledges Dr. Javed
Ahmads contribution, to provide AFMI fellowship during the
course of present investigation.
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g

Fig. 4. Variation in vincristine yield (mg dry wt.) in eld grown and somatic
embryo derived plantlets. Means with common letters are not signicantly
different at p  0.05, according to DMRT.

Fig. 5. Variation in vincristine yield (mg g dry wt.) in various ex vitro and in vitro
developed plant parts. Means with common letters are not signicantly different at
p  0.05, according to DMRT.

and analysis was mostly conned to in vitro raised aerial plant

parts derived through organogenesis (Miura et al., 1988), the
present work is therefore a new of its kind. We also noted that the
dimeric alkaloid was always found more in leaf callus compared to
intact plant and the level decreased, as the cultures were old.
Similarly, in other medicinal plants a certain degree of differentiation was considered essential for the secondary metabolites
biosynthesis (Ma et al., 2006; Bhat et al., 2008). In other
observations, dimeric alkaloid was isolated from in vitro raised
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the preconditions for the biosynthesis of more complex dimeric
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vincristine was extracted from leaf callus and germinating
embryos; that too in older tissues. We also feel that this
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