WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Geetha et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 3, Issue12, 1266-1274. Research Article ISSN 2278 – 4357

ESTABLISHMENT OF AN EFFICIENT EXPLANT SURFACE

STERILIZATION PROTOCOL FOR IN VITRO
MICROPROPAGATION OF SALACIA CHINENSIS L., AN
ENDANGERED ANTI-DIABETIC MEDICINAL PLANT

Majid BN1, Roopa G1, Sampath KKK2, Kini RK2, Prakash HS2, S Abbagani3, Kiani
Mehdi3, Geetha N1*

1
Echo-Biotech Lab, DOS in Biotechnology, Manasagangotri, University of Mysore, Mysore-
570006. Karnataka, India.
2
DOS in Biotechnology, Manasagangotri, University of Mysore, Mysore-570006. Karnataka,
India.
3
Department of Biotechnology, Kakatiya University, Warangal -506002, Telangana, India.

Article Received on
08 Oct 2014,
Revised on 21 Nov 2014,
Accepted on 25 Nov 2014
*Correspondence for Author
Dr. Geetha N.
Echo-Biotech Lab, DOS in
Biotechnology,
Manasagangotri, University of
Mysore, Mysore-570006.
Karnataka, India.
effective for maximum survival percentage (96%) in nodal explants. The described method
has this potential to be successfully employed for micropropagation and in vitro conservation
of S. chinensis.
ABSTRACT
This paper describes the most efficient explant surface sterilization
protocol for in vitro micropropagation of an endangered medicinal
plant, Salacia chinensis. Healthy, undamaged collected explant sources
were washed twice with running tap water. The explants then were pre-
washed by concentrated dishwasher gel (3-4 drops in 100 ml sterile,
distilled water) for 5 min followed by rinsing five times in sterile,
distilled water. Surface sterilization with 70% ethanol for 1 min
followed by 1% sodium hypochlorite (NaOCI) (+ 2-3 drops of Tween
20) for 15 min proved most effective for maximum survival percentage
(99%) in leaf explants whilst 70% ethanol washing for 2 min, followed
by 0.1% mercuric chloride (HgCl2) for 5 min proved to be more

KEYWORDS: Celastraceae, Salacia chinensis, anti-diabetic medicinal plant,

micropropagation, surface sterilization.

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INTRODUCTION
Salacia chinensis L. (Syn. S. prinoides DC.), commonly known as Saptrangi, family
of Hippocrateaceae, which has since been incorporated into the Celastraceae family, is a
woody climbing shrub, an endangered medicinal plant naturally found in tropical Africa, Sri
Lanka and southern regions of India. The roots, rootbarks, stems, dried parts and water
extraction of the whole plant have been extensively used in the ayurvedic system of Indian
[1, 2]
traditional medicine and in countries in Southwest Asia to treat a variety of ailments. Its
roots have biologically active compounds, such as triterpenes, phenolic compounds,
[3, 4]
glycosides and coloring agents, which show various medicinal properties. The plant and
[5-10]
its extracts have been evaluated for number of activities like anti-diabetic , anti-
hyperlipidemic [2], anti-inflammatory [11], tonic [12] and blood purifier. [13]

Due to the lack of proper cultivation practices, destruction of plant habitats, excessive and
indiscriminate collection of medicinal plants for supplement of global demands on herbal
medicine, many medicinal plants like S. chinensis are severely threatened. Therefore, in order
to conserve and rapidly propagate the rare and endangered medicinal plants, advanced
biotechnological methods of culturing plant cells and tissues like micropropagation methods
are employed. [14]

The in vitro conservation of medicinal plants comprises of selection of explants, aseptic

culture establishment, multiplication of microshoots; rooting followed by acclimatization of
the plantlets. Amongst these stages, the challenging step is standardization of sterilization of
explants for aseptic culture establishment. On an average, losses due to contamination under
in vitro conditions average between 3-15% in majority of commercial and scientific plant
[15]
tissue culture laboratories , the majority of which is caused by bacterial, fungal, and yeast
contaminants [16]. Various environmental factors such as source of explants, plant species, age
and other climatic changes are the reasons for explant contaminations. [17]

There are no reports on surface sterilization of S. chinensis, which is the prerequisite for
further tissue culture techniques like organogenesis for any genetic manipulation and for
conservation of this endangered medicinal plant. The most decisive step in explant
preparation for further processes is keeping the explant alive overcoming the problem of
contamination. Therefore, in this investigation, an attempt has been made to establish an
efficient surface sterilization protocol for the in vitro multiplication of S. chinensis, using

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different types of sterilizing agents and varying their concentrations and duration of exposure
as a best alternative for conservation of this endangered, medicinally important plant.

MATERIALS AND METHODS

Explant source
The experiments were carried out at the Plant Cell and Tissue Culture Lab, Echo-Biotech
group, DOS in Biotechnology, University of Mysore, Mysore. As tissue culture success
always depends on the types and age of explant, young and healthy leaves and nodal
segments (2.5 cm) containing axillary buds as our explants sources were collected from three-
year-old S. chinensis plants that are maintained in the Botanical Garden of DOS in
Biotechnology, University of Mysore, Mysore.

Surface sterilization
Initially the healthy, undamaged collected explant sources were washed twice in running tap
water. The explants were then pre-washed with concentrated dishwasher gel (3-4 drops in
100 ml sterile, distilled water) (Vim, Double power concentrated gel, India) for 5 min
followed by rinsing five times in sterile, distilled water. Thereafter, the explants were
submerged in 70% (v/v) ethanol for 1-2 minutes; alcohol was decanted by washing the
explants with sterile, distilled water and surface sterilized with different concentrations of
mercuric chloride (HgCl2) (SD Fine-Chem. Limited, India) (0.01, 0.1, and 0.5%) (w/v) for 5
min and with different concentrations of sodium hypochlorite (NaOCI) (HiMedia, India) (0.5,
1.0 and 2.0 %) (w/v) plus Tween 20 (2-3 drops in 100 ml sterile, distilled water) for 15 min
separately. For better contact of the sterilizing agents and explants, they were placed on the
incubator orbital shaker (GeNei, India). The solutions were removed and surface sterilized
explants were washed thoroughly with sterile, distilled water for four times under aseptic
conditions in a laminar airflow hood and placed on a sterilized petridish covered with
autoclaved filter paper to remove excess moisture present on the surface of explants.
Procedure of surface sterilization of different explants has been shown through a flow chart
(Fig. 1).

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Fig. 1: Procedure of explant surface sterilization of Salacia chinensis.

In the present experiment, explants after sterilization were cut into suitably-sized pieces (1.5
and 2 cm for leaf and nodal segments respectively) and were inoculated onto MS [18] medium
with 3% sucrose and 0.8% agar (HiMedia, India). The pH of the medium was adjusted to 5.7
before autoclaving at 121oC at 1.06 kg cm-2 for 20 minutes. Cultures were then kept at
temperature of 25 ± 2oC, air humidity of 55% and a 16-h photoperiod with irradiance of 125
µmol m-2 s-1 supplied by cool-white florescent tubes (Philips, India). Data on contamination
and survival percentage were recorded. The following formula has been applied to
calculation of contamination percentage.

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RESULTS AND DISCUSSION

[19-21]
Plant tissue culture always has been linked with microbial contamination.
Multiplication of microbes, their competition with explant growth for nutrients and altering
the culture environment e.g. pH by releasing chemicals inhibit explants growth or cause
death. [19, 22] To avoid this contamination, the explants must be gently surface sterilized before
inoculation on to tissue culture medium. In order to avoid phenols oozing from cut tissues
and browning of explants, antibrowning treatment was carried out with 0.3% activated
charcoal. Washing with 70% ethanol and then decantation of alcohol, enhances the contact of
sterilizing agents (HgCl2 and NaOCI). To decontaminate the explants we have used different
types of sterilizing agents in various ranges of concentrations. The surface sterilized explants
used in this experiment are illustrated in Fig. 2 (b-c).

Fig. 2 (a-d): (a) Salacia chinensis plant in Botanical Garden of DOS in Biotechnology;
(b) A plate of one week old leaf explants on basal medium; (c) Leaf explants from Fig
2(b) 26 days after inoculation on MS medium; & (d) A batch of surface sterilized
explants plated on MS medium.

There were differences between the explant responses to various types and concentrations of
sterilization agents. 1% NaOCI was found to be proper and effective sterilizing agents for
leaf explants in S. chinensis. Over sterilization increased the tissue mortality of explants, and

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hence to overcome to this problem we have optimized the concentration of sterilizing agents
and duration of the sterilization. In case of S. chinensis, surface sterilization with 70% ethanol
for 1 min followed by NaOCI (1% + 2-3 drops of Tween 20) for 15 min proved most
effective for maximum survival percentage in leaf explants whilst 70% ethanol washing for 2
min, followed by 0.1% HgCl2 sterilization for 5 min proved to be more effective for
maximum survival percentage in axillary bud explants (Table 1, Fig.3 and Fig.4). In
conclusion, this is a simple, reproducible and affordable surface sterilization protocol for in
vitro propagation of S. chinensis by explant sources (especially leaf and nodal buds) for
further goals and mainly saving this medicinally important species from extinction.

Table. 1. Standardization of surface sterilization for various explants in S. chinensis by

HgCl2 and NaOCI.
70% alcohol Nodal explants Leaf explants
Sterilizing Concentrations
exposure % % % % % %
agent (%)
duration (min) CON SUR MOR CON SUR MOR
Control 0.0 0.0 71.0 20.0 9.0 78.0 7.0 15.0
1.0 52.0 36.0 12.0 55.0 33.0 12.0
HgCl2 0.01
2.0 43.0 51.0 6.0 47.0 32.0 21.0
1.0 9.0 89.0 2.0 0.0 93.0 7.0
HgCl2 0.1
2.0 3.0 96.0 1.0 0.0 86.0 13.0
1.0 3.0 70.0 27.0 7.0 71.0 22.0
HgCl2 0.5
2.0 1.0 60.0 39.0 5.0 52.0 43.0
1.0 23.0 74.0 3.0 13.0 86.0 11.0
NaOCI 0.5
2.0 24.0 69 7.0 11.0 82.0 7.0
1.0 7.0 81.0 12.0 1.0 99.0 0.0
NaOCI 1.0
2.0 3.0 87.0 10.0 1.0 85.0 14.0
1.0 13.0 85.0 2.0 3.0 79.0 18.0
NaOCI 2.0
2.0 6.0 77.0 17.0 2.0 82.0 16.0
% CON- % Contamination, % SUR- % Survival, % MOR- % Mortality (Browning or blacking of explants).

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Fig. 3: Standardization of surface sterilization for various explants in Salacia chinensis

by HgCl2.

Fig. 4: Standardization of surface sterilization for various explants in S. chinensis by

NaOCI.

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ACKNOWLEDGMENT
The authors acknowledge the support from Ministry of Human Resource Development and
University Grant Commission under Institution of Excellence (IOE) scheme awarded to the
University of Mysore.

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