International Journal of Agricultural

Science and Research (IJASR)

ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 6, Issue 5, Oct 2016, 117-122
TJPRC Pvt. Ltd

IN-VITRO MICROPROPAGATION OF A MEDICINAL

PLANT: APARAJITA (CLITORIA TERNATEA L.)
ASHISH JAISWAL1 & S. P. SINGH2
1
2

Department of Biotechnology, J.C. Bose Institute of Life Science, Bundelkhand University, Jhansi, Uttar Pradesh, India

Department of Horticulture, Institute of Agriculture Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India

ABSTRACT
Present study was undertaken to develop an in-vitro protocol for micro-propagation of Clitoria ternatea L.
The research work was carried out during the year 2016 (1st Jan to 30th June) in Tissue culture Laboratory, Department
of Horticulture, Institute of Agricultural Sciences, Banaras Hindu University Varanasi. The explants were surface
sterilized and inoculated on MS medium supplemented with different Concentrations of BAP. MS medium supplemented
win BAP (1.5 mg/L) was observed to be best for shoot formation, shoot length and regeneration frequency.
The maximum number of roots, rooting frequency and toot length was found in MS medium supplemented with IBA
(0.2 mg/L). The rooted plantlets were hardened and transferred successfully to field condition.

Received: Aug 11, 2016; Accepted: Aug 31, 2016; Published: Sep 02, 2016; Paper Id.: IJASROCT201615

INTRODUCTION
Clitoria ternatea L. which belong to the family Fabaceae is a very well known Ayurvedic medicine used

Original Article

KEYWORDS: Clitoria Ternatea L., Micro-Propagation, Regeneration Frequency, Shoot Formation

for different ailments. It is commonly called butterfly pea or conch flower or shankapushpi and in Indian
traditional medicine is known as Aparajit (Hindi), Aparajita (Bengali), and Kakkattan (Tamil). It seems to be a
native of the Caribbean, Central America and Mxico and early after the conquista was distributed to the Indian
subcontinent. In the traditional (Asian) Indian systems of medicine particularly in Ayurveda, the roots, seeds and
leaves of Clitoria ternatea L. have long been used as a brain tonic and is believed to promote memory and
intelligence.
In vitro plant regeneration has been reported in Clitoria ternatea L. through axillary shoot proliferation in
young shoot tip explants (Kalamani and Gomez, 2002), nodel segments (Rout, 2005), shoot regeneration in leaf
explants (Malabadi and Nataraja, 2001) one-step differentiations of multiple shoot buds and embryoids in
seedlings root and hypocotyl explants (Lakshmanan and Dhanalakshmi, 1990) or via somatic embroygenesis in
seedling explants (Dhanalakshmi and Lakshmanan, 1992). Not with standing, here an effort has been made to
generate information on propagation of Clitoria ternatea L. by shoot proliferation in cotyledonary node explants
derived from axenic seedings.

MATERIALS AND METHODS

Planting materials was collected from Department of Horticulture, Institute of Agriculture Science,
B.H.U., Varanasi. The fully ripe and dried pods were collected from a 2 year old climber of Clitoria ternatea L.

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Ashish Jaiswal & S. P. Singh

The seeds were removed from the pods and washed thoroughly under running tap water.
Leaves were separated from the stem and washed in 2% (v/v) Teepol (Qualigen, India) detergent solution,
Subsequently surface sterilization was conducted using 0.1% (w/v) aqueous mercuric chloride solution for 15 minutes.
After rinsing flour or five times with sterile distilled water, stems were cut transversely into 2.5 mm segments, each
containing a node.
Initiation of Culture

Sterilized explants were transferred aseptically to sterilized glass plate under the laminar flow hood.

Then a cut was given on the both basal as well as the portion of the explants to remove undesirable/dead portions
after surface sterilization.

The forceps were rinsed in 70% ethanol and were flamed and then kept for some time to get cool.
Then the lid from one test tube was removed and test tubes mouth was flamed to avoid any chance of
contamination.

Each nodal explant was then placed in an erect position in the test tube containing medium with the help of long
forceps.

The lid was finally closed carefully, flame lightly and sealed with Parafilm. The forceps were then again rinsed
with 70% alcohol to avoid any chance of contamination. The same procedure was undertaken for all the explants.

The culture tubes were then kept in the growth chamber having a temperature of 25 2C, with a photoperiod of
16 hours daylight and 8 hours night break under the cool white fluorescent light.

Establishment of Cultures

After approximately 9-10 days of inoculation, the axillary bud break was seen in some explants.
When the explants attended the stage of bud proliferation, the cultures were transferred into culture tubes
containing fresh medium.

After 21-25 days of incubation with a clean and sterilized forcep under the laminar flow hood, the initiated plants
were taken out from the test tube, medium adhered to the plants was carefully removed. The undesirable/brownish
leaves were removed from the plants and explants were taken to the culture tubes containing autoclaved
semi-solid media with the same combinations for the culture initiation.

The tubes were placed in the culture room under the standard conditions of temperature (25C) for 16/8 hours of
day/night break respectively under the cool white fluorescent light.

Rooting of the Shoots

Axillary shoots developed in cultures in the presence of cytokinin generally lack roots. To obtain full plants, the
shoots must be transferred to a rooting medium, which is different from the shoot multiplication medium, especially in its
hormonal composition. A low salt medium is satisfactory for rooting of shoots in large number of plant species.
Protocol Followed

Plantlets were taken out of the culture bottles (multiplication subculture) with the help of forceps and washed

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thoroughly with water to remove any remains of the medium.

0.1% Bavistin treatment was given to the plants in order to protect them from the fungal attack in the near future.

Plantlets were separated into single shoot by cutting their bases gently with the help of blade.

Single shoot was dipped in IBA solution (200 ppm) before planting into a hardening mixture.

After this the single shoot was carefully planted in the polybags containing sand: soil: F4M (1:1:1). After 10- 14
days of cultures on rooting media, the rooted plantets were transplanted to pots or trays for hardening prior to their
final transfer to soil.

Callus Culture
Cotyledenary node segment were used as source of explant. Explant used for callus induction was taken from
established culture of Clitoria ternatea L. The medium employed was MS basal with different concentration and
combinations of phytohormones such as NAA, Kinetic and 2, 4 - D containing media. In the media BCM (MS + 0.5 mg/L
BAP + 1mg/L 2, 4-D), rapid callus growth was observed. In case of BCM 1 (MS + 1mg/L BAP + 1mg/L IAA) initial small
globular callus was formed, from where small shoots buds had regenerated after 20-25 days. The loose jelly type callus
formation was observed in BCM2 (MS + 0.5 mg/L BAP + 2, 4-D), BCM3 (MS + 1.0 mg/L 2, 4-D) and BCM4 (MS + 2.0
mg/L 2, 4-D) which however, turned brown after a few days. After inoculation, the culture bottles were properly capped
and sealed. After labeling, these were transferred to the incubation room and incubated at 25 20C in the rack covered with
black paper.
Table 1: Response of Nodal Explants to Different Concentrations of BAP in Clitoria Ternatea L
Media Content

No. of Shoots

Shoot Length (cm)

MS + 0.0 mg/L (control)

MS + 0.5 mg/L BAP
MS + 1.0 mg/L BAP
MS + 1.5 mg/L BAP
MS + 2.0 mg/L BAP
MS + 2.5 mg/L BAP
MS + 3.0 mg/L BAP
MS + 4.0 mg/L BAP
MS + 5.0 mg/L BAP
MS + 6.0 mg/L BAP

2.40 + 0.10lm
4.82 + 0.14e
8.10 + 0.24b
10.20 + 0.33a
6.10 + 0.20c
5.19 + 0.15dc
3.70 + 0.17ghi
3.2 + 0.13f
2.40 + 0.15lm
2.25 + 0.12 lm

5.6 + 0.22c
5.50 + 0.24c
7.10 + 0.16b
8.20 + 0.28a
7.70 + 0.32b
6.90 + 0.27c
5.50 + 0.18d
5.0 + 0.22e
4.10 + 0.26e
3.20 + 0.15no

Regeneration
Frequency (%)
60
70
80
100
90
80
70
60
50
50

Each mean is based on three replicates, each of which consisted of 20 culture tubes (culture age; 6 weeks).
The alphabets indicate significant difference between means (P<0.005); comparison by DMRT.
Table 2: Response of Explants to Different Concentrations of IBA in Clitoria Ternatea L
Media Content
MS + 0.0 mg/L (control)
MS + 0.1 mg/L IBA
MS + 0.2 mg/L IBA
MS + 0.5 mg/L IBA
MS + 1.0 mg/L IBA
MS + 1.5 mg/L IBA
MS + 2.0 mg/L IBA
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Average No. of
Roots
2.5 + 0.17b
3.10 + 0.32e
6.5 + 0.39a
4.11 + 0.18b
3.50 + 0.24c
3.00 + 0.12dc
2.50 + 0.19f

Root Length
(cm)
2.9 + 0.16d
3.9 + 0.44c
6.35 + 0.42 a
4.50 + 0.15b
4.10 + 0.36bc
3.10 + 0.18e
3.00 + 0.17f

Regeneration
Frequency (%)
40
70
90
80
72
50
45
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Ashish Jaiswal & S. P. Singh

Each mean is based on three replicates, each of which consisted of 20 culture tubes (culture age; 6 weeks).
The alphabets indicate significant difference between means (P<0.005); comparison by DMRT.

RESULTS AND DISCUSSIONS

Effects of Bap on Shoot Proliferation
Shoot proliferation and establishment from cotyledonary node explants of Clitoria ternatea L. cultured on MS
basal as control and MS medium supplemented with various concentrations of BAP ranging from 0.5 mg/L to, 6.0 mg/L.
BAP was used to note its efficiency in different concentration in inducing multiple shoots. Three replicates of each
concentration were made. During initial week after inoculation, shoot initiation was very low. However, shoot initiation
was found to be started in most of the cultures initiated from 9-10 days, by showing small new shoots, which proliferate
into leaves during 25-30 days which were placed in the culture room under standard conditions of temperature (252C).
Maximum shoot regeneration frequency of 100% was observed at 1.5 mg/L BAP (Table 2). Shoot regeneration
frequency, number of shoots and shoot length were observed increasing with increase in the concentration of BAP growth
regulator up to the optimum concentration. It was noticed that when the concentration of BAP was increased from 0.5
mg/L to 1.5 mg/L, average number of shoots per culture formed, regeneration frequency and length of shoot was also
increased. By further increase in concentration of BAP average number of shoots per culture increased while regeneration
frequency and length of shoots gradually decreased.
Analysis of variance revealed that mean shoot numbers and mean shoot length were significantly affected by the
concentrations. Maximum number of shoots (10.2) and shoot length (8.2 cm) was recorded on MS medium supplemented
with 2.0 mg/L BAP.

(A)

(C)

Impact Factor (JCC): 4.8136

(B)

(D)

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In-Vitro Micropropagation of a Medicinal Plant: Aparajita (Clitoria Ternatea L)

121

(E)
Figure 1: A. Development of Axillary Shoot from Nodal Explant of Clitoria Ternatea L. B. Shoot
Multiplication and Elogation of Clitoria Ternatea L on MS Medium C. Rooting of in
Vitro-derived Shoot ofClitoria Ternatea L. D. In Vitro-raised Planlet Grown in a
Pot. E. The Callus Emerged from Nodal Explants
Effects of Auxin (IBA)
After two cycles multiplication of subculture, elongated shoots of 4-5 cm in length were excised and cultured on
MS+ Sugar 30 g/L+ Agar 8 g/L medium having different concentrations of IBA and MS + Sugar 30g/L + Agar 8 g/L
medium as control. IBA was used to note its efficiency in different concentration in inducing rooting. It was observed that
at different concentrations of IBA ranging from 0 mg/L to 2.0mg/L the rooting was induced at the basal portion of
transferred shoots.
Rooting frequency, average number of roots and root length increased with increase in the concentration of IBA
up-to the optimum concentration. At very high concentration of IBA, average number of roots per culture increased, while
regeneration frequency, length of roots gradually decreased. Shoots without treatment with IBA showed poor rooting of
micro-shoots. Initiation of rooting took place after 5-6 days of inoculation. Single and multiple roots were formed from the
base and the nodal portion and the length of the roots was 1-2 cm within 8-10 days. Analysis of variance revealed a
significant effect (P <0.05) on the frequency of cultures showing root regeneration, number of roots/shoot and mean root
length (6.0 cm) at a concentration of 0.2mg/L The higher concentrations of IBA found inhibitory for rooting of
micro-shoots of Clitoria ternatea L.
The rooted plantlets were successfully transferred from culture tubes into plastic cups containing soil and kept for
acclimatization. The acclimatized plantlets, further, transferred into pots containing mixture of garden soil and sand and
kept into greenhouse condition.

CONCLUSIONS
An efficient protocol was developed for successful micro propagation of an important medicinal plant clitoria
ternatea L.
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Kazuma, K., Kogawa, K., Noda, N., Kato, N., Suzuki, M., 2004. Identification of delphinidin 3-O-(6-O-malonyl)-glucoside3-O- glucoside, a postulated intermediate in the biosynthesis of tematin C5 in the blue petals of Clitoria ternatea (butterfly
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Kumar, K. P., Soniya, E. V., Lawrence, B., Nair, G. M., 1993. Microproragation of Clitoria ternatea L. (Papilioaceae) through
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