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 K1 and Dash M2.

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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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 500 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) + GA3 (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 4°C. 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 250C±20C 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 student’s 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 (HgCl2) 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% HgCl2 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 GA3

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 GA3

(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 (0.5-10mM). 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 HgCl2 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)+GA3(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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