Conservation of Piper mullesua Buch-Ham.: An important … · 2013. 7. 22. · TECHNOFAME- A Journal of Multidisciplinary Advance Research 61 Vol.2 No. 1, 61-66 (2013) Received: June

TECHNOFAME- A Journal of Multidisciplinary Advance Research

61

Vol.2 No. 1, 61-66 (2013)

Received: June 2012; Accepted: April.2013

Conservation of Piper mullesua Buch-Ham.: An important medicinal plant

from Arunachal Pradesh through in vitro culture strategies

Purnima Dubey and Padmanabh Dwivedi1

Laboratory of Plant Tissue Culture, Department of Botany, Rajiv Gandhi University, Itanagar-

791112, Arunachal Pradesh 1 Reader, Department of Crop Physiology, BHU, Varanasi, Uttar Pradesh

Abstract

Arunachal Pradesh, one of the hot spots of biodiversity harbours a rich diversity of medicinal

plant species. Over the centuries, the collection of many important medicinal plant species for

commercial purpose and traditional practice has exerted tremendous pressure on their existing

population in wild. Many species of medicinal plants growing in natural habitats are becoming scarce

followed by poor regeneration. Consequently, their unlimited collection as raw materials may lead to

the complete disappearance and extinction of certain species and varieties. Such deterioration is

further augmented due to unplanned and ruthless exploitation by pharmacological industries,

biopiracy and deforestation. The endangered medicinal plants which are also slow propagating and

thus having low regenerative ability require rapid clonal multiplication through tissue culture

strategies. Piper mullesua Buch-Ham., an important medicinal plant from Arunachal Pradesh is facing

threat owing to over-exploitation by the commercial establishments and local people for their

medicinal utility, is taken up for in vitro clonal propagation and multiplication. Micropropagation

protocol was developed for this plant and the in vitro regenerated plantlets were successfully

transferred to the botanic garden of the University.

Key words: Conservation, micropropagation, Piper mullesua.

Introduction

Arunachal Pradesh is a global

biodiversity hotspot harbouring a rich

diversity of medicinal plants. There are

120 ethnic communities in this state. These

communities still almost fully depend on

nature for their livelihood. The

unsustainable utilization of natural

resources through the practice of Jhuming

(Shifting cultivation), hunting, fishing,

trapping and also gathering plants and

animals for medicines, ornaments,

decoration and supplementing food by the

aborigines has led to the depletion of the

natural resources. Harbouring a rich

diversity of medicinal plants, Arunachal

Pradesh is facing tremendous pressure on

their existing population, which are being

collected for commercial purpose and

traditional practice. Many species of

medicinal plants are becoming scarce due

to seed dormancy coupled with poor

regeneration. Consequently, their

unlimited collection as raw materials may

lead to the complete disappearance and

extinction of certain species and varieties.

Such deterioration is further augmented

due to unplanned and ruthless exploitation

by pharmacological industries, biopiracy

and deforestation.


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The endangered medicinal plants which

are also slow propagating and thus having

low regenerative ability require rapid

clonal multiplication through tissue culture

strategies. Piper mullesua Buch-Ham., an

important medicinal plant from Arunachal

Pradesh facing threat owing to over-

exploitation by the commercial

establishments and local people for its

medicinal utility, was taken up for in vitro

clonal propagation and multiplication.

Micropropagation protocol was developed

for this plant and the in vitro regenerated

plantlets were successfully transferred to

the Botanic Garden of Rajiv Gandhi

University, Itanagar.

Piper mullesua Buch-Ham.,

commonly known as Pahari peepal,

belongs to the family Piperaceae. It is

highly distributed in subtropical

Himalayas from Shimla to Bhutan upto the

height of 1500 m in Khasi hills and in

Nilgiris. In Arunachal Pradesh, it is found

in Dibang Valley (Roing), Lohit

(Hayuliang, Wakro), West Siang (Along)

and East Siang (Pasighat). The plant is

economically important because almost

each and every part of the plant is

medicinally useful. The stem portion is

crushed and applied for toothaches, the

fruits are used for curing headaches and

stomach aches, it is also used as spice. The

roots also possess medicinal value and are

used in treatment of asthma, bronchitis,

dyspepsia and anorexia. Chemical

isolation from the hexane fraction of

alcoholic extracts of Piper mullesua shows

the presence of Myristin (4-Methoxy-6{2-

propynyl} 1, 3-bengodioxole) [5]

.Due to

the excessive utilization of plant by local

people of the area for medicinal purpose,

especially the bark part, the population of

Piper mullesua is declining. The plant is

also used by commercial establishment

companies of India like Dabur for

preparation of certain digestive tablets e.g.

Hajmola. In addition, owing to some

religious purpose, the seed is consumed by

the aborigines which has led to the less

availability of seed and hence, decline in

the plant population.

In spite of the immense utility and

limited work on germplasm conservation

of this species; the in vitro culture of Piper

mullesua Buch-Ham., has highly been

called for, thus necessitating the need of

micropropagational study.

Material and methods

Plant material and Culture media:

The explants used for culture

initiation were collected from the

seedlings, obtained from State Forest

Research Institue, Itanagar, Arunachal

Pradesh and established in the garden of

the University. The nodal segment was

used as culture explant; small stem twigs

were collected and the leaves were

removed, the nodal explants were washed

in agitated solution of liquid detergent for

15 min and later on washed in running tap

water for 1 hour. Surface sterilization in

case of Piper mullesua was done with 0.1

% mercuric chloride for 2 min. Explants

were thoroughly washed with double

distilled water followed by explants

trimming into 1.0-1.5 cm long size. This

was followed by further treatment with

0.1% ascorbic acid for 8 min, 0.2%

ampicillin and 0.5% bavistin together for 5

min. Explants were thoroughly washed

with sterile double distilled water after


63

every treatment and finally, were

inoculated onto the nutrient medium. The

basal medium consisted of the mineral

salts and organic nutrients of MS

medium [3], 3% sucrose and 0.8% agar.

Depending upon the experiment, the

basal medium was variously

supplemented with combinations of

different growth regulators such as Kn,

BAP, IBA, IAA and NAA at different

concentrations (0.5-2.0 mg/l). All of the

supplements were added to the molten

agar, and the pH of the medium was

adjusted to 5.8, before autoclaving it at

121°C at a pressure of 15 psi for 15 min

in culture tubes (150 x 25mm) or 100 ml

conical flasks. The cultures were

maintained at 25± 2⁰ C under 16-h

photoperiod with a light intensity of

3000 lux provided through Philips cool-

white fluorescent tubes and with 60-70%

relative humidity. Shoot growth was

periodically observed. There were 10

explants per treatment and the

experiments were repeated thrice.

Acclimatization and establishment of

plants in soil

Four-to-six week-old regenerants

with well-developed roots were removed

from the culture tubes and washed free of

agar. They were then dipped in 0.2%

bavistin for 2 min and subsequently

transplanted into plastic trays containing

sterilized soil and river sand (1:1). The

tray containing plantlets were covered

with a transparent polythene lid to

maintain high humidity, and the

microcuttings were moisted twice every

day. Following three 3 weeks at 24± 2⁰C

under a 16 hr photoperiod, the lid was

removed and the plantlet were

transferred singly to earthen pots

containing sterile sand, soil and humus

(1:2:1) at ambient room temperature

(28±2⁰C) with indirect sunlight . After 2

months, the well acclimatized plantlets

were planted outside in the nature and

finally transferred to the Botanic Garden

of the University.

Results and discussion

In Piper mullesua, shoot

induction was not found in MS

(Murashige and Skoog) basal medium

even after four weeks of culture similar

to the findings in Gloriosa superba L. [2]

.After four weeks of culture, of the

various plant growth regulators tried,

best response in terms of % of shoots

induced (60.7± 2.5) was observed in MS

medium supplemented with 1.5 mg/l

BAP (Fig.1 A). At this concentration an

average of 2.4 ± 0.3 shoots/explants was

produced. However, in terms of both the

parameters studied viz. % of shoot

induced/ culture (56.3± 1.5) and no. of

shoots produced/explant (4.2± 0.2), the

combination of 0.75 mg/l each of Kn +

BAP in MS medium was found best

(Table 1) (Fig.1 B) [4]. Rooting in Piper

mullesua was very difficult owing to the

semi woody nature of the plant. After

several trials, and combination of auxins,

supplementationof ½ MS with 1.0 mg/l

IAA showed good number of root

formation (2 roots/ shoot) 30 days after

culture (data not shown). IAA also

reported effective for root induction in


64

Table 1: Effect of growth regulators in MS basal medium on shoot induction and number of

shoots per culture established from nodal explant of Piper mullesua after 4 weeks of culture.

Growth regulators (mg/l) Percentage of cultures with

induced shoots (%)

Number of Shoots per explants

Control Kn - -

0.5 10.7 ± 1.3 1.8 ± 0.2

1.0 20.9 ± 2.0 1.7± 0.4

1.5 30.1 ± 2.3 2.1 ± 0.2

2.0 25.2 ± 1.7 2.0± 0.4

BAP

0.5 45.3 ± 1.6 1.7± 0.2

1.0 50.6 ± 2.2 1.9± 0.3

1.5 60.7± 2.5 2.4± 0.3

2.0 55.8 ± 1.8 2.1± 0.2

Kn + BAP

0.25+ 0.25 48.6 ± 1.5 2.5± 0.4

0.50 + 0.50 55.3 ± 1.7 3.8±0.3

0.75 + 0.75 56.3 ± 1.5 4.2±0.2

1.00 + 1.00 50.1 ± 1.6 3.4 ± 0.3


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Fig.1 A, B, C: In vitro shoot induction and multiplication in Piper mullesua. D: The mother

plant in nature

Tectona grandis [1]

.Rooted

microshoots were acclimatized and

transferred to the garden behind the

laboratory. 50% survivality observed after

30 days of field transfer.The result of the

present study thus provides a promising

protocol for the propagation of Piper

mullesua through in vitro culture strategies

on commercial scale as well for the

conservation of their superior genetic

strains.

References

1. Aini, A.S.N., Goh,B.L. and

Ridzuan,R. (2009). The effects of

different indole-3-butyric acid (IBA)


66

concentrations, two light regimes of

in vitro rooting and acclimatization

of in vitro teak (Tectona grandis L.f)

plantlets. African Journal of

Biotechnology. 8 (22): 6158-6161.

2. Hassan, A.K.M.S. and Roy,S.K.

(2005). Micropropagation of

Gloriosa superba L. through high

frequency shoot proliferation. Plant

Tissue Culture 15 (1): 67-74.

3. Murashige, T. and Skoog, F. (1962).

A revised medium for rapid growth

and bioassays with tobacco tissue

cultures. Physiolgia Plantarum, 15:

473-497.

4. Roy, S. K., Islam, M. S. and

Hadiuzzaman, S. (1998).

Micropropagation of Elaeocarpus

robustus Roxb. Plant Cell Rep.17:

810–813.

5. Srivastava, S., Gupta, M.M.,

Prajapati, V., Tripathi, A.K., and

Sushil Kumar (2002). Insecticidal

activity of myristicin from P.

mullesua. Pharmaceutical Biol. 39,

226-229

Documents

Conservation of Piper mullesua Buch-Ham.: An important … · 2013. 7. 22. · TECHNOFAME- A Journal of Multidisciplinary Advance Research 61 Vol.2 No. 1, 61-66 (2013) Received: June