In vitro organogenesis protocol for Rauvolfia serpentina - an endangered

medicinal plant
Keywords:
Medicinal plants, in vitro, Rauvolfia serpentine, Benzyl amino purine, Kinetin.
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution, and
reproduction in all medium, provided the original work is properly cited.
Authors:
Singh K
1
and Dash M
2
.


Institution:
1. Department of Botany,
Subas Science College,
Patia, Bhubaneswar.

2. School of Biotech
Sciences, Trident Academy
of Creative Technology,
Infocity, Patia,
Bhubaneswar.




Corresponding author:
Dash M.












Email:
manasi_dash @yahoo.com.





Web Address:
http://plantsciences.info/
documents/PS0028.pdf.
Dates:
Received: 27 Mar 2012 Accepted: 01 May 2012 Published: 21 May 2012
Article Citation:
Singh K

and Dash M.
In vitro organogenesis protocol for Rauvolfia serpentina - an endangered medicinal
plant.
Journal of Research in Plant Sciences (2012) 1: 083-088
Original Research
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Journal of Research in Plant Sciences
ABSTRACT:

Rauvolfia serpentina commonly known as sarpagandha is a
pharmacologically important medicinal plant containing numerous alkaloids with
antibacterial, antidysentric and antidotal properties. The present study reports an
efficient in vitro regeneration protocol by using nodal explants for this species. The
sterilization technique was first standardized using ethyl alcohol, mercuric chloride

and sodium hypochlorite with hot water and without hot water treatment. 100%
aseptic culture was obtained when the explants were treated with hot water (at 50
0
C
for 10 minutes) and 0.1% mercuric chloride. The aseptic cultures were inoculated in to
culture medium with different concentrations of growth regulators. Higher explants
response (78.33%) and higher multiple shoot formation from Rauvolfia serpentina
nodal explants was observed in the medium supplemented with BAP (1mg/l) + KIN
(1mg/l) + GA
3
(0.5mg/l).

083-088 | JRPS | 2012 | Vol 1 | No 1
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INTRODUCTION
The availability and relatively cheaper cost of
medicinal plants makes them more attractive as
therapeutic agents when compared to modern medicines
(Agbor and Ngogang, 2005; Agbor et al., 2005a). Hence
nowadays medicinal plants have become important for
the treatment of different disease conditions, such as
diabetes, malaria, anaemia (Fola, 1993). Genetic
biodiversity of traditional medicinal herbs and plants is
continuously under the threat of extinction as a result of
growth-exploitation, environment-unfriendly harvesting
techniques loss of growth habitats and unmonitored trade
of medicinal plants. There is thus an urgent need to
develop and implement regeneration/ conservation
strategies for over exploited medicinal plant species.
Rauvolfia serpentina (apocyanaceae) is also
known as serpentwood. It comprises over 170 species
distributed in the tropical and subtropical parts of the
world including 5 species native to India. In India, it is
cultivated in the states of Uttar Pradesh, Bihar, Tamil
Nadu, Orissa, Kerala, Assam, West Bengal and Madhya
Pradesh. It is an erect perennial shrub generally 15-45
cm high, but growing upto 90cm under cultivation
(Figure. 1). Among the different species of Rauvolfia, R.
serpentina is preferred for cultivation because of higher
reserpine content in the root. The root is bitter, acrid,
laxative, anthelmintic, diuretic and sedative. Over 200
alkaloids have been isolated from the plant of which 1.4-
3% alkaloids are present in the root possessing
pharmacological importance. The alkaloids are
classsified into 3 groups, viz, reserpine, ajmaline and
serpentine groups. Reserpine group (comprising
reserpine, rescinnamine, deserpine etc) act as
hypotensive, sedative and tranquillising agent. Ajmaline,
ajmalicine, ajmalinine, iso-ajmaline etc of the ajmaline
group stimulate central nervous system, respiration and
intestinal movement with slight hypotensive activity.
Serpentine group (comprising serpentine, sepentinine,
alstonine etc) is mostly antihypertensive (Husain, 1993
and Iyengar, 1985). Extracts of the roots possess anti
bacterial properties and are valued for the treatment of
intestinal disorders, particularly diarrhea and dysentery.
Mixed with other plant extracts, they have been used in
the treatment of cholera, colic and fever. Root extracts of
this species is used as an antidote to the bites of
poisonous reptile like snakes. Genetic resources are
renewable, provided they are well managed (Das, 2008).
Rauvolfia is highly valuable but endangered plant (Sudha
and Seeni, 2006; Sharma and Chandel, 1992). Due to its
irrational and uncontrolled use it has become an
endangered species in India. Seed propagation is the best
method for raising commercial plantation but seed
germination is very poor and variable from 10-74%. For
fast multiplication of this plant, development of suitable
techniques is required not only to save it from becoming
extinct but also to enable large-scale cultivation.
Micropropagation using in vitro techniques will address
to the problem of mass scale production and cultivation
to meet the market demands. It will also help in
production of disease and virus free planting material for
the interested farmers. Protocols has been standardized
for the in vitro culture of many medicinal plants like
Artemisia annua and Passiflora foetida (Ganesan and
Singh and Dash, 2012
084 Journal of Research in Plant Sciences (2012) 1: 083-088
Figure 1. A fully grown plant of
R.serpentina bearing fruits
Paulsamy, 2011 and Komathi et al., 2011). It is therefore
imperative that for mass multiplication of this plant the
in vitro culture procedure has to be developed so as to
obtain the phytochemicals in a large scale without
damaging the natural growing population. Hence the
present research included standardization of in vitro
culture technique of Rauvolfia serpentina.

MATERIALS AND METHODS
The application of tissue culture technology for
the establishment of in vitro regeneration of the study
species has been done by following the methods as
described below.
Source of plant material
Healthy plant material of Rauvolfia serpentina
(Figure 1) were obtained from medicinal plant nursery,
Bhubaneswar.
Explant preparation and sterilization
The plant materials were thoroughly washed
under running tap water for 10-20 minutes for removing
dust and microorganisms present on the surface of the
explants. Internodes, nodes and leaves were used as
source of explants. These were initially treated with
0.2% (w/v) bavistin for 10 minutes and washed with
sterile double distilled water for 3-4 times. Then the
explants were surface sterilized with different sterilants
like ethyl alcohol (EtOH), mercuric chloride (HgCl2)
and sodium hypochlorite (NaOCl) at various
concentration and durations under the laminar flow
cabinet. The effect of hot water treatment (500C for 10
minutes) was also studied along with the sterilants. The
concentration of the sterilants varied from 0.1% to 1.0%
(V/V) depending on the toxicity of the chemical. The
explants were immersed in a beaker containing the
particular disinfectant at a specific concentration as
indicated in Fig 2a and 2b and for a specific time period.
After the treatment the explants were washed thrice with
sterile double distilled water to remove all the traces of
the chemicals from the explants. The duration of the
treatment varied from 1-10 minutes as mentioned in
Fig 2.
Culture media preparation and culture conditions
Double distilled water was used for preparing the
culture medium. The nutrient medium basically consists
of inorganic nutrients, carbon source, vitamins, irons and
amino acids. The chemicals were weighed accurately in
electronic weighing balance. All the stock solutions were
prepared in and stored in well-stoppered sterilized bottles
and preserved in refrigerator at 4C. Specific quantity of
the stock solutions of the chemicals (Murashige and
Skoog, 1962) and growth regulators were pipetted out
onto a one liter beaker and required sucrose was added.
The final volume was made up with distilled water and
the pH was adjusted to 5.8-5.9 with 0.1N NaOH or 0.1N
HCL using a pH meter. The stock solution of cytokinin
(BAP, KIN) was prepared by dissolving 10mg of 6-
benzyl amino purine (BAP), kinetin (6-
furfurylaminopurine) in 1ml of 0.1N Hydrochloric acid
(HCL) and the volume was made up to 10ml by adding
sterile distilled water. The different concentrations were
used before autoclaving.
The explants were inoculated into Murashige and
Skoog (MS) culture medium (1962) supplemented with
7gm/l agar with pH 5.8. MS culture medium was
supplemented with different combination of growth
regulators for shoot induction and proliferation. The
media were autoclaved at 15 lb/inches2 pressure at
1210C for 20 minutes. The autoclaved medium in the
culture tubes were cooled allowed to solidify and stored
in dark for further use. The inoculations were done after
three days to ensure that the bottles were free from
contamination.
All the cultures were maintained in the culture
room at 250C20C temperature in 16 hour photoperiod.
The effects of shoot formation in Rauvolfia serpentina
was studied with different concentration of BAP, KIN
and GA3.

Singh and Dash, 2012
Journal of Research in Plant Sciences (2012) 1: 083-088 085


The elongated shoots were transferred to MS
medium supplemented with different auxin like IAA,
IBA and NAA at different concentration for root
induction. The rooted plantlets were removed from
culture tubes and transferred for hardening. The well
developed healthy explants were removed from the
culture flask and were thoroughly washed in running tap
water to remove the adhering nutrient medium
completely without causing damage to roots for which
the protocol of Bhojwani and Razdan (1983) was
followed. During hardening process, initially the
plantlets were kept inside the green house for
acclimatization. Then the plantlets were exposed to the
natural environmental conditions.
Data analysis
The present investigation on in vitro propagation
of the study species consisted of three replication for
each treatment and each replication consisted of mean
data of 10 test-tube cultures. Analysis of variance
(ANOVA) was carried out and tested against students t-
test at 5% level.

RESULTS AND DISCUSSIONS
Standardization of in vitro culture technique for
Rauvolfia was carried out for mass multiplication of the
plant. The salient features of findings are presented
below.
Standardization of sterilization technique to obtain
aseptic culture
It was observed that the nodal explants when
treated with 0.1% mercuric chloride (HgCl
2
) for 5
minutes gave 90% aseptic cultures in absence of warm
water treatment (Figure 2a). The sterilization efficiency
of 100% was achieved when the explants were treated
with 0.1% HgCl
2
for 5 minutes preceded by warm water
treatment for 10 minutes (Figure 2b).
Effects of hormones on organogenesis in Rauvolfia
The MS media fortified with 2 mg/l BAP and 1
mg/l KIN induced shoots within 3 days from nodal
Singh and Dash, 2012
086 Journal of Research in Plant Sciences (2012) 1: 083-088
Growth regulators
(mg/l)
Days to shoot
Initiation
% Response
BAP KIN GA
3

1 0 0 12.66 63.33
2 0 0 14.66 60.00
1 1 0 15.00 60.00
1 1 0.5 3.66 78.33
2 1 0 3.00 63.33
2 2 0 6.33 60.00
2 2 0.5 11.66 6.33
0 1 0 9.33 73.33
MEAN 9.54 65.20
C.V[E] 0.02 0.03
C.D[P=0.05] 0.78 87.14
Table 1. Effect of different concentrations of
growth hormones on organogenesis of the species;
Rauvolfia nodal explants
Figure 2a. Effect of different sterilizing agents
without hot water treatment on explants response of
Rauvolfia species
Figure 2b. Effect of different sterilizing agents
(with hot water treatment ) on explants respone of
Rauvolfia species
explants of Rauvolfia serpentina (Table 1). The mean
explants response varied significantly among the
treatments. Highest explant response (78.33) to shoot
induction was observed when the MS media was
fortified with BAP (1mg/l) + KIN (1mg/l) and GA
3
(0.5mg/l).The same media also resulted in high number
of multiple shoots (4 shoots per explant) (Plate 3).
Similar kind of findings has been reported by Sudha and
Seni (2006), Faisal et al., (2005), Mathur et al., (1987)
and Sharma and Chandel (1992). Plant regeneration by
direct somatic embryogenesis has also been achieved by
various researchers. Sudha and Seeni (2006) used MS
media containing NAA (0.5mg/l) to obtain somatic
embryogenesis in Rauvolfia micrantha Hook. F.
Similarly Faisal et al., (2005) were successful in
inducing shoot buds in Rauvolfia tetraphylla using MS
media containing TDZ (0.5-10
m
M). They obtained 18
shoots per explant. Mathur et al., (1987) also had
developed a tissue culture protocol for Rauvolfia.
The tissue culture of medicinal plants has a wide
range of industrial applications (Ghosh, 2005). Explant
sterilization is a major step in culture establishments.
Proper concentration of sterilizing agent is a key factor
(Roy and Saha, 1997). This was determined by applying
different concentration of HgCl
2
in the present
investigation. The present paper describes a prime and
easy-to-use protocol for large scale production of
plantlets of R. serpentina through node culture and the
method is useful for the ex situ conservation of this
species as well. In addition, the findings of the present
investigation provide a baseline data for further research
in this species.

CONCLUSION
The tissue culture of medicinal plants has a wide
range of industrial applications (Ghosh, 2005). Explant
sterilization is a major step in culture establishments.
Proper concentration of sterilizing agent is a key factor
(Roy and Saha, 1997). This was determined by applying
different concentration of HgCl2 in the present
investigation. The sterilization efficiency of 100% was
achieved when the explants were treated with 0.1%
HgCl2 for 8 minutes preceded by warm water treatment
for 10 minutes. The MS media fortified with 2 mg/l BAP
and 1 mg/l KIN induced shoots within 3 days from nodal
explants of Rauvolfia. Highest explant response (78.33)
to shoot induction was observed when the MS media was
fortified with BAP (1mg/l) + KIN (1mg/l) and GA3
(0.5mg/l).The MS media fortified with BAP (2mg/l) and
KIN (1mg/l) result in high number of multiple shoots.
The present paper describes a prime and easy-to-use
protocol for large scale production of plantlets of R.
serpentina through node culture and the method is useful
for the ex situ conservation of this species as well. In
addition, the findings of the present investigation provide
a baseline data for further research in this species.
Singh and Dash, 2012
Journal of Research in Plant Sciences (2012) 1: 083-088 087
Figure 3: Effect of growth hormones on multiple shooting in Rauvolfia serpentina nodal explants
A. Shooting in MS medium containing BAP (1mg/l)+ KIN(1mg/l)
B. Shooting in MS medium containing BAP (2mg/l)+KIN(1mg/l)
C. Higher degree of multiple shoots in MS medium containing BAP (2mg/l)+KIN (2mg/l)+GA
3
(0.5mg/l)
D. Shooting in MS medium containing BAP (2mg/l)+KIN (2mg/l)
Singh and Dash, 2012
088 Journal of Research in Plant Sciences (2012) 1: 083-088
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