See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/271833353

Micropropagation of Rauvolfia serpentina - a potent endangered medicinal

plant

Article · July 2010

CITATIONS READS

0 2,015

1 author:

PE Rajasekharan
ICAR-Indian Institute of Horticultural Research
237 PUBLICATIONS   653 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Adaptation and Development of Medicinal Plant Technologies for Income Generation, Agricultural Diversification and Export Promotion View project

Development of complementary conservation strategies for horticulture PGRs (recalcitrant seed, pollen and in vitro material) View project

All content following this page was uploaded by PE Rajasekharan on 16 June 2015.

The user has requested enhancement of the downloaded file.

IN VITRO REGENERATION AND CONSERVATION OF RAUVOLFIA
SERPENTINA(L.) BENTH. EX KURZ. A CRITICALLY
ENDANGERED MEDICINAL PLANT SPECIES

P. E. Rajasekharan 2*, S. R. Ambika1 and S. Ganeshan 2

1
Physiology Laboratory, Department of Botany, Bangalore University,
Jnanabharathi, Bangalore 560 056 India. 2In Vitro and Cryopreservation
Laboratory, Division of Plant Genetic Resources, Indian Institute of
Horticultural Research, Hessaraghatta Lake, Bangalore 560 089, India.
(*Request for off prints; E-mail: pers@iihr.kar.nic.in)

Abstract
R. Serpentina plants were collected from Western Ghats and relocated to ex situ conditions
in Bangalore and were maintained in glass house and field. Plantlets were successfully
regenerated from shoot cultures of Rauvolfia serpentina initiated from auxiliary meristems.
MS medium containing 4.44 µM BA and NAA 0.54µM NAA resulted in best shoot
proliferation. Low temperature storage of vitroplants appeared highly promising because
vitroplants exhibited normal health even after one year. Shoot tips and single nodal cuttings
were used for in vitro multiplication. Excised shoots were rooted on half-strength MS
supplemented with 1.0 mg/litre each of IBA and IAA. Within 3 weeks rooting was 100%.
After acclimatization 95% of the plantlets survived. The in vitro generated shoots were
rooted and later transferred to the soil. Experiments carried out under reduced culture
conditions; vitroplants could be conserved for durations ranging from 6 to 12 months with
one intervening subculture.

Key Words: Ex situ conservation, endangered species, reduced culture condition

Introduction

Rauvolfia serpentina is an important medicinal plant to treat insomnia, hypochondriasis,

ailments of the central nervous system and high blood pressure. R. serpentina is
economically important because of the presence of reserpine and rescinnamine group of
alkaloids used in the allopathic systems for the treatment of hypertension, cardiovascular
diseases and as a sedative tranquilizing agent. Interestingly, the powdered root of Rauvolfia
serpentina has been in use in India for at least 2,000 years to treat mental illness
(Srivastava, 1995). Reserpine is considered a sympathomimetic agent, one that targets the
sympathetic nervous system. Reserpine has been found to lower blood pressure in
remarkably low oral doses. In Ayurvedic system of medicine, Rauvolfia serpentina roots is
part of complex formulation for treatment of insomnia, epilepsy, asthma, acute stomach
ache and painful delivery of child besides controlling high blood pressure and insanity
(Trivedi, 1995). The plant is widely distributed in India but nowhere gregarious. Natural
populations are encountered in widely separated localities having different habitat
conditions. It grows as an under shrub in the sub-Himalayan tract, Karnataka and Goa and
eastern and western Ghats up to an attitude of 1200 m.

Over 95 per cent of the medicinal plants used by the Indian pharmaceutical industry are
today collected from the wild (FRLHT, 1997). Over 70 per cent of the plant collections
involve the use of roots, bark, wood, stem and in some areas the whole plant, leading to
destructive harvesting. Rauvolfia serpentina is also no exception. If not carefully
monitored, this practice could lead to the depletion of genetic stocks and ultimately to the
diversity of medicinal plants. Simultaneously it is also important to look at the ex-situ
conservation of medicinal plants through In vitro studies are required to be taken up in this
species due to the following reasons.
Plants from the tropics are worth between $ billion to $ 47 billion, annually, to the global
pharmaceutical industry sarpagandha alone, (Rauvolfia serpentina), is the base for $ 260
million worth trade in hypertension and schizophrenia drugs. Large quantities of roots of
this plant were collected from forest areas during sixties, leading to a great depletion of
natural stock in the country (Sarin,1986). At present Rauvolfia serpentina reserves are
becoming increasingly exhausted while the requirements of pharmaceutical industry of
importing countries in rauanatine, reserpine, ajmalicine etc cannot be met (Kunakh and
Akhimova, 1989)
Due to the nonavailability of R.serpentina commercial material is often adulterated with the
stems of the drug, roots of other Rauvolfia species mainly R.canescens Linn. and
Clerodendrum species. However the roots of R.serpentina can be easily distinguished from
the others based on the surface character of the root. Despite their wide geographical
distribution and edaphic tolerance, Rauvolfia serpentina species have not lent themselves to
easy cultivation due to various factors, which influence their propagation, growth and
development and also their alkaloid content (Anonymous, 1969). Cloning of elite plants
would be advantageous for increased alkaloid production (Raja and Heble, 1996)
Poor seed viability and very low germination percentage that may be ascribed largely to the
presence of cinnamic acid derivatives in the seeds (Mitra, 1976) which could be an
inhibiting factor.

For rare species that are in decline, in situ conservation may not be adequate protection
(Rajora and Mossier, 2000). Tissue and cell culture and cryopreservation provide excellent
avenues and opportunities for ex situ conservation of these genetic resources. Low
temperature storage of in vitro stock material commonly used for conservation of plant
germplasm (Pruski et al, 2000). This method, if properly adjusted to specific genotypes,
can substantially reduce labour and media costs The present paper deals with in vitro
regeneration and conservation of R.serpentina , as a possible approach to conserve genetic
diversity of this species.

Materials and methods

1.Collection
Wild plants were collected from the Western Ghat region of Kerala and brought to
Bangalore and established ex situ and used for studies.
2.Culture initiation: Shoot tips and single node explants were used for optimizing in vitro
regeneration protocols. Shoot tips and nodes were washed thoroughly in running water to
remove soil and other dust particles. Followed by washing with dilute detergent, ‘Teepol’,
for 5 minutes. Explants were then sterilized inside the laminar flow by using 0.01 %
mercuric chloride for 5 minutes. Following this, the explants were washed with sterile
double distilled six times to get rid of mercuric chloride traces retained by the explant. The
explants were rinsed with sterile distilled water 5-6 times. The surface sterilization
procedure of explants described yielded 90% aseptic cultures.
3.Culture medium and conditions: The medium was supplemented with 3% w/v sucrose
and gelled with 6.5w/v bacteriological grade agar, of Himedia, India. The pH of the
medium was adjusted to 5.8 prior to autoclaving. Routinely,15 ml of molten medium was
dispensed into sterile 25X150 cm tubes. For each treatment, 10 replicates were used and
the all the experiments were repeated thrice. The cultures were incubated at 25±20C with
50 to 55% RH and a 16 h photoperiod, provided by cool ‘Philips’ white fluorescent light.
4.Regeneration and multiplication: Nodes and shoot tips of wild plants cultured in MS
medium (1962) supplemented with 4.44 µm BA and 0.54 µm NAA produced 5-6 shoots
within sixty days. The explants exhibited moderate callusing at the cut end after a culture
period of one month. At the time of sub-culturing newly formed shoots were separated and
transferred to fresh medium, shoots continued to proliferate through 5 subcultures with an
average of 20 shoots per transfer at higher and lower concentrations of the cytokinins. Even
though the latter induced slight callusing, the hormonal regime did not adversely affect
shoot multiplication and growth.
5.Rooting: well developed shoots were excised from tube and implanted individually on
root induction medium containing ½ MS medium in combination with IAA (5.71µM
+IBA4.9 µM). Rooting was observed in all explants within one month.
6.Hardening
Rooted vitropnts removed from tube and washed, transferred to polybag with soilrite and
clipped and kept in a chamber with light for one month after one month removed from
chamber and kept in glass house for one month and followed by this transferred to earthen
pot.

The following treatments were imposed for optimizing in vitro storage conditions

MEDIUM CULTURE OBSERVATIO

CONDITIONS NS ECORDED
FMS SCC (L+25 0C) PH, NOS, ,NO
HMS SCC (L+250 C) OF NODES,
QMS SCC (L+250 C) INTL
FMS+M-1G SCC (L+250 C)
FMS RCC (LL+100 C)
FMS RCC (LL+150C)
SCC- STANDARD CULTURE CONDITIONS PH- PLANT HEIGHT; NOS- NUMBER OF SHOOTS;
NOR- NUMBER OF ROOTS,RCC: REDUCED CULTURE CONDITIONS;M- 1 G:MANNITOL
ONE GRAM/L MEDIUM
INTL-INTERNODAL LENGTH IN CM; NN- NUMBER OF NODES
FMS-FULL STRENGTH MS; HMS-HALF STRENGTH MS;QMS: QUARTER STRENGTH MS;
LL-LOW LIGHT INTENSITY (2.97 MICROEINSTEIN/M2/SEC).

Parameters considered determining conservation gain:

1. Longer duration of storage
2. Sustainable post storage recovery in terms of nodes and shoots
3. Root shoot ratio
4. Reducing subculture frequency
7.Statistical analysis
The data was analysed using factorial CRD. Analysis of variance was performed on the
mean values.

RESULTS
A method to regenerate callus free shoots from nodal and shoot tip explants of R.serpentina
was successfully developed. Shoot tips were found better for multiplication of vitroplants.
Production of large number of plants was accomplished in glass bottles. Increasing
concentrations of auxins and cytokinins induced callusing at the root-shoot interface at the
expense shoot formation.
Response of vitroplants to storage conditions: the results were presented in figures one to
four.

1.FMS SCC (L+25 0C):

After six months, a maximum vitroplant height of 6.24 was recorded. The mean number
of shoots per explant averaged to 5.6. The internodal length was increased from 0.38 to
0.64 cm and mean number of nodes recorded was 3.6.
2.HMS SCC (L+250 C):
After three months, vitroplant height reached full tube length of 11 cm and hence, the
experiment could not be extended till the end of six months. The number of shoots
averaged to 1.8. Internodal length increased from 0.93 to 1.3.
3.QMS SCC (L+250 C):
After three months, vitroplant height reached 11.58 cm the number of shoots per explant
averaged 1.6. Internodal length averaged to 1.74 and the mean number of nodes recorded
was 3.
4.FMS+M-1G SCC (L+250 C):
Vitroplants conserved averaged to a height of 11.78 after six months. While number of
shoots averaged to 1. Internodal length and number of nodes averaged to 1.12 and 2
respectively.
5.FMSRCC (LL+100 C):
When cultures in FMS were relocated to 100C after 2 to 3 weeks of establishment under
SCC an average maximum vitroplant height of 4.64 was recorded. The average number of
shoots formed was 1.8. Internodal length and number of nodes averaged too 0.64 and 2
respectively.
6.FMSRCC (LL+150C)
The average maximum vitroplant height of 6.05 was reached at 15 0 C the average
number of shoots formed was 0.8 at the end of six months. Internodal length and number
of nodes averaged to 1.12 and 2 respectively.
All treatments were highly significant (p=0.01)

DISCUSSION

In numerous in vitro multiplication systems, it has been observed that variant rates increase
with the number of multiplication cycles or with the length in culture (Cote et al, 2001). So
it is advisable to limit the multiplication rate to minimal whenever in vitro conservation
method is employed for a given species. in vitro conservation strategies aim a reducing the
growth rate of cultures and avoiding frequent subculture. In the results pertaining to
conservation in vitro, it was possible to maintain vitroplants healthy under normal growth
conditions, provided with optimal media and elimination of accidents. In tropical species
this could be useful and minimize the need for low temperature requirement, help in being
cultured for instant multiplication and exchange.
In the results pertaining to conservation in vitro, it was possible to maintain vitroplants
healthy under normal growth conditions. In the present study R.serpentina could be kept at
SCC for 6 months to one year, prior to first sub-culture or transfer.
From the results, it is evident that there is conservation gain in terms of plant height among
the low temperature treatments (10 and 150C). Addition of osmoregulator (mannitol) had
very little effect on vitroplant height; in fact ½ and ¼, MS induce rapid growth, within 3
months. Height of vitroplant reached 11.78 cm after six months.
There is conservation gain in terms of plant height among the low temperature treatments
(10 and 150C).

The response of shoot number per explant showed a reducing trend in all treatments, the
multiple shoot formation is restricted only under low temperature and mannitol treatments.
Shoot and nodal cultures of Rauvolfia serpentina have been successfully conserved for
over 15 months at 15 0C, while 100 C and 50 C were found deleterious to growth of cultures
(Sharma and chancel, 1992). These in vitro conserved cultures showed shoot multiplication
comparable to the cultures maintained at 250 C and the plantlets obtained from these
conserved cultures could be established in soil and no morphological differences were
observed in these plants.

In slow growth studies, the aim is to reduce the requirement of sub culture without causing
any damage to the tissue. It can be achieved by one method or by a combination of
methods involving manipulations of factors such as temperature, light and addition of
osmoticum. The technique has the benefits of being inexpensive and requires least of
specialized equipment or expense (Grout, 1995).
Maximum conservation gain was obtained for Rauvolfia serpentina at 100C +low light in
FMS, where the vitroplant height recorded was least. Increasing storage temperature to
150C supports less shoot formation, whereas at 100C, conservation gain in terms of increase
in number of shoots enhanced post storage recovery was observed, besides having an
optimal differentiation and axillary bud formation. There was no effect of low temperature
on vitroplant height in this species, instead there was eight fold increase in vitroplant
height at low temperature. The increase in number of shoot was only two fold. Number of
nodes was increased to the extent of ca. 30 percent over the same period, and a two-fold
increase in Internodal length was also recorded.

Tropical species are stored in the range of 10 to 22 0C (Chandel et al., 1996). The method is
very simple, easy to use and may be applied to wide range of genotypes. Low temperature
induces slow growth by lowering the overall cell metabolic activity, which remains more or
less uniform throughout the culture period (Bhat, 1993). During storage of plantlets,
photosynthetic and re-growth abilities would have to be maintained, but growth suppressed
(Kubota and Kozai, 1995). It was reported that broccoli plantlets were successfully stored
for 6 weeks at 50c under 2-µmol m-2 S-1 PPFD (Photosynthetic Photon Flux Density).
Plantlets stored in darkness-expressed loss of dry weight and lost the ability to regrow.
They also found that plantlets kept at the PPFD level of the light compensation points
maintained their initial dry weight and retained their photosynthetic and re-growth abilities.
 LITERATURE CITED

Cote

Foundation for the Revitalisation of Local Health Traditions. 1997. Conserving a National
Resource. Need for a National Policy and National Programme on Medicinal Plants
Conservation Draft of Madras Consultation (unpublished).

Ihllahi,I. Akram, M. and Krans, L.1988. In vitro studies on Rauvolfia for mass propagation
and alkaloid synthesis. Pakistan J. Sci. Ind. Res.31: 114-117.

Jha, AK. 1996Medicinal Plants : Poor Regulation Blocks Conservation.. Economic and
Political Weekly. December 23, 1996. pp 3,270.

Kunakh, V. A and Alkhimova, A. G. 1989. Rauvolfia serpentina in vitro cultures and

production of Ajmalicine in:Biotechnology in Agriculture and Forestry.Vol.7
Medicinal and Aromatic Plants II Y.P.S.Bajaj (Edt.) Sringer Verlag, Berlin.pp.398-
416
Mathur, A., Mathur, A. K., Kukreja, A. K., Ahuja, P. S. and Tyagi, B. R. 1987.
Establisment and multiplication of colchi-atotetraploids of Rauvolfia serpentina L.
Benth ex Kurz. through tissue culture Plant Cell Tissue Org. Cult.129-134.
Murashige and Skoog.1962.

Patil, V. M and M. Jayanthi. 1997 .Micropropagation of two species of Rauvolfia

(Apocynaceae) Curr.Sci.,72:961-965.
Roja, G. and M. R. Heble.1996. Indole alkaloids in clonal propagules of Rauvolfia
serpentina benthe ex kurz Plant Cell Tissue and Organ Cult. 44: 111-115.
Sarkar, K. P., Islam, A ., Islam, R., Hoque A. and O. I. Joarder.1996. In vitro propagation
of Rauvolfia serpentina through tissue culture. Planta Medica 62:358-59.
Sarin, Y. K. 1986. Rauvolfia serpentina Benth: A re- assessment of resources in
India.pp.385- In: Plantation crops: Opportunities and constraints Vol. H. L.
Srivastava, B. Vartya and K .K. G..Menon. ( eds. ) Hindustan Lever Research
Foundation. Oxford and IBH, New Delhi.Sharma, N .and Chandel, K. P. S.(1992a).
Low temperature storage of Rauvolfia serpentina Benth ex Kurz.-An endangered ,
endemic medicinal plant. Pl.Cell Rep11:200-203.
Sarin, Y. K. 1977.Cultivation and utilization of Rauvolfia serpentina in India. In cultivation
and utilization of medicinal and aromatic plants (ed. C.K.Atal an B.M.Kapur)RRL
Jammu
Srivastava, P. S. and D. Pande .1998. In Vitro propagation and conservation of medicinal
plants.In Plant tissue culture and molecular biology: application and prospects.
Narosa Publishing House, New Delhi.pp:254-281
Sudha ,C.G. and S. Seeni. 1996. In vitro propagation of Rauvolfia micrantha, a rare
medicinal plant Plant Cell Tiss.Org.Cult. 44: 243-248.
 Srivastava, J, J Lambert, N Vietmeyer. 1995. Medicinal Plants. A Growing Role in
Development. The World Bank, Washington, DC

Trivedi, K.C.1995. Sarpagandha. In Advances in Horticulture vol.11-Medicinal and

aromatic plants (Eds.K.L.Chadha and R.Gupta)Malhotra Publishing House New
Delhi 110 064.454-466.

View publication stats