IN VITRO PROPAGATION OF CLITORIA TERNATEA L.
21
BIOL. LETT. 2004, 41(1): 21–26
Available online at http://www.biollett.amu.edu.pl
Effect of cytokinins and auxins on micropropagation
of Clitoria ternatea L.
GYANA RANJAN ROUT
Plant Biotechnology Division, Plant Tissue Culture Laboratory, Regional Plant Resource Centre,
Bhubaneswar – 751 015, Orissa, India; e-mail: [email protected] / [email protected]
(Received on 16th February 2004; Accepted on 2nd June 2004)
Abstract: In vitro shoot multiplication from nodal explants of Clitoria ternatea was standardized by
using MS (Murashige & Skoog) medium supplemented with a cytokinin (BA or Kin). Inclusion of NAA
in the culture medium along with BA promoted a higher rate of shoot multiplication. The mean number
of shoots per explant after 4 weeks of culture was maximum (5.21) on the MS medium supplemented
with 2.0 mg l-1 BA and 0.25 mg l-1 NAA. The elongated shoots rooted within 7-8 days in ½ strength MS
medium supplemented with 0.25 mg l-1 of NAA and 2% (w/v) sucrose. About 85% of the rooted plantlets
survived acclimatization and transfer to the greenhouse. The results will facilitate the conservation and
propagation of this important medicinal plant.
Key words: growth regulators, in vitro, medicinal plant, shoot multiplication
Abbreviations: BA – 6-benzylaminopurine; Kin – kinetin; IAA – indole-3-acetic acid; NAA –
1-naphthaleneacetic acid; IBA – indole-3-butyric acid; MS medium – MURASHIGE & SKOOG (1962)
medium
INTRODUCTION
Clitoria ternatea L. belongs to the family Fabaceae and is distributed in tropical Asia, Philippine Islands and Madagascar. It is an ornamental perennial climber
with conspicuous blue or white flowers, and in India it is commonly called butterfly
pea (ANONYMOUS 1988). It is a highly palatable forage legume, generally preferred
by livestock over other legumes. It is also used as a cover crop or green manure. The
root is used in the treatment of various diseases, like indigestion, constipation, arthritis and eye ailments. It is also employed in cases of ascetics, enlargement of the
abdominal viscera, sore throat, skin diseases, etc. (ANONYMOUS 1988, MORRIS 1999).
The root, stem and flower are recommended for the treatment of snake-bite and scorpion-sting. The extract of C. ternatea was found to have anxiolytic, antidepressant,
anticonvulsant and antistress properties (JAIN et al. 2003). The United State Devel-
22
Gyana Ranjan Rout
opment Agency (USDA) intends to conserve C. ternatea along with other 16 leguminous species with potentially useful phytochemicals (MORRIS 1999). However,
pharmaceutical companies largely depend upon material collected from natural stands.
Due to unrestricted large-scale exploitation of this natural resource, coupled with
limited cultivation and insufficient attempts for its replenishment, the wild stock of
this species has been markedly depleted, so now it is listed as a rare species by the
International Union for Conservation of Nature and Natural Resources (IUCNNR)
(http://foia.state.gov, PANDAY et al. 1993). Propagation from seed is unreliable due
to poor germination and death of many young seedlings under natural conditions
(ANONYMOUS 1988). In vitro culture is an alternative method for conservation and
propagation of this species, but no report on this subject has been published so far.
Hence this study aimed at development of an efficient protocol for micropropagation of C. ternatea.
MATERIALS AND METHODS
Plant material
Elongated shoots (5–6 cm) were collected from greenhouse-grown plants of
Clitoria ternatea L. and brought to the laboratory with cut ends dipped in distilled
water. Stems without leaves were washed in a 2% (v/v) (Teepol; Qualigen, India)
detergent solution. Subsequently, surface disinfection was performed in a 0.1%
(w/v) aqueous mercuric chloride solution for 15 min. After rinsing 4–5 times with
sterile distilled water, stems were cut into smaller segments (ca. 0.25 cm long), each
with one node, which were used as explants.
Culture medium and culture conditions
Nodal explants were placed on semisolid MS medium (MURASHIGE & SKOOG
1962) supplemented with different concentrations of BA (0.0, 0.5, 1.0, 1.5, 2.0, 2.5,
3.0, 3.5 and 4.0 mg l-1) or Kin (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 mg l-1) for shoot
proliferation. Next, NAA or IAA (0.0, 0.25, 0.5, 1.0 mg l-1) along with different
concentrations of BA (0, 1.5, 2.0, 2.5, 3.0 mg l-1) were added to MS medium with
3% (w/v) sucrose for shoot multiplication. The pH of media was adjusted to 5.8 with
0.1 N NaOH or 0.1 N HCl prior to adding agar 0.8% (w/v) (Qualigen, India). Routinely, 20 ml of molten medium were dispensed into culture tubes (25 × 150 mm),
which were then plugged with non-absorbent cotton wrapped in one layer of cheesecloth. Media were steam-sterilized at 121ºC and 1.05 Kg cm-2 for 15 min. The cultures were incubated under 16 h photoperiod in cool white fluorescent light
(55 µmol m-2s-1) (Phillips, India) and maintained at a constant temperature of 25 ± 2ºC.
The culture was maintained by regular subculturing at 4-week intervals on fresh media
of the same composition.
Induction of rooting and acclimatization
Elongated shoots (1–2 cm long) were excised from the culture and transferred
to ½ strength semisolid MS medium supplemented with different concentrations of
IBA and/or NAA (0.1, 0.25 and 0.5 mg l-1) and 2% (w/v) sucrose for root initiation.
One excised shoot was cultured in each tube (25 × 150 mm) with 15 ml of the medium. All the cultures were incubated at 25 ± 2ºC under 16 h photoperiod with cool
IN VITRO PROPAGATION OF CLITORIA TERNATEA L.
23
white fluorescent lamps, as above. After 4 weeks, the percentage of shoots forming
roots and the number of roots per shoot were assessed. Rooted micropropagules were
thoroughly washed to remove the adhering gel and planted in 2.5-cm earthen pots
containing a mixture of soil, sand and dry cow-dung manure (1 : 1 : 1, w/v) and kept
in the greenhouse for acclimatization for 4 weeks. The plants were watered at 2-day
intervals and were supplied with ¼ strength MS inorganic solution two times, twice
a week before transfer to the open field. The survival rate was recorded 1 month
after transfer to pots.
Statistical analysis
Each experiment was repeated 3 times, with 20 cultures per treatment. The mean
percentage of cultures producing multiple shoots and mean number of shoots per
culture were recorded after 4 weeks. The percentage of rooting and average number
of roots per shoot were recorded after 2-weeks. The data were analysed statistically
by the Duncan’s multiple range test (HARTER 1960). Means followed by the same
letter within columns were not significantly different at P < 0.05.
RESULTS AND DISCUSSION
There was no sign of growth when nodal explants were cultured on media
without cytokinin or auxin. By contrast, shoot proliferation and elongation were
observed within 1 week of culture on MS medium supplemented with varying concentrations of BA or Kin (Table 1). At higher concentrations of BA or Kin, the growth
Table 1. Effects of different concentrations of cytokinins (BA, Kin) added to the MS medium on shoot
proliferation from nodal explants of Clitoria ternatea after 4 weeks of culture
BA (mg l-1)
Kin (mg l-1)
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.5
1.0
2.0
2.5
3.0
3.5
4.0
% of growing explants (mean ± SE)*
0
0
52.8 ± 1.2 d
70.2 ± 1.4 i
76.6 ± 2.0 j
75.8 ± 1.0 j
68.6 ± 0.8 h +
62.4 ± 1.2 g +
60.6 ± 1.4 f +
0
26.2 ± 0.9 a
38.8 ± 1.0 b
46.2 ± 1.2 c
54.4 ± 1.4 e +
54.2 ± 0.8 e +
52.6 ± 1.0 d +
* mean of 3 replications of 20 cultures each; + stunted growth
Means followed by the same letter were not significantly different at P < 0.05 (Duncan’s multiple comparison test)
24
Gyana Ranjan Rout
of the shoots was stunted. Shoot proliferation was poorer on media enriched with
Kin than on those enriched with BA. On media with BA alone (1.5–2.5 mg l-1), shoot
elongation was faster (Fig. 1A). At higher concentrations of either BA or Kin in the
culture medium, basal callusing of the growing shoots was observed. Cytokinin formulations were earlier shown to be critical for shoot elongation of many other plant
species, including medicinal plants (JHA & JHA 1989, SHARMA et al. 1993, CHEN et
al. 1995, SAXENA et al. 1998, ROUT et al. 2000). Inclusion of either NAA or IAA in
the culture medium along with BA favoured shoot multiplication. In general, shoot
multiplication was more rapid on media with BA + NAA than with BA + IAA. There
were differences between treatments in both the percentage of cultures with multiple
shoots and the mean number of shoots per culture. Many authors report that cytokinin is required in optimal quantity for shoot proliferation in many genotypes but
inclusion of a low concentration of auxin along with cytokinin increases the rate of
shoot multiplication (TSAY et al. 1989, SHARMA et al. 1993, SHARMA & SINGH 1997,
SHASANY et al.1998, ROUT et al. 2000). The maximum percentage of multiple shoots
(85.6%) after 4 weeks of culture was observed on media containing 2.0 mg l-1 BA,
0.25 mg l-1 NAA and 3% sucrose (Fig. 1B, Table 2). A higher concentration of
either NAA (³ 0.5 mg l-1) or BA (³ 2.5 mg l-1) in the culture medium inhibited the
growth of the shoots and stimulated small callusing at the basal end. The number of
Table 2. Effects of different concentrations of cytokinin (BA) and auxins (NAA, IAA) added to the medium
(MS + 3% sucrose) on shoot multiplication from nodal explants of Clitoria ternatea after 4 weeks of
culture
BA
(mg l-1)
NAA
(mg l-1)
IAA
(mg l-1)
% of explants with multiple
shoots (mean ± SE)*
Mean number of shoots/explant
(mean ± SE)*
0
1.5
2.0
2.5
2.0
2.5
2.0
2.5
3.0
2.0
2.5
3.0
2.0
2.5
2.0
2.5
0
0.25
0.25
0.25
0
0
0.5
0.5
0.5
0
0
0
1.0
1.0
0
0
0
0
0
0
0.25
0.25
0
0
0
0.5
0.5
0.5
0
0
1.0
1.0
0
74.2 ± 1.2 j
85.6 ± 1.0 l
80.2 ± 1.6 k
64.8 ± 1.1 g
68.2 ± 1.2 h
72.8 ± 1.4 i +
74.4 ± 1.0 j +
68.0 ± 0.8 h +
60.6 ± 1.2 f +
64.2 ± 1.3 g +
56.6 ± 1.0 e +
50.6 ± 1.2 c +
52.4 ± 1.3 d +
46.2 ± 0.9 a +
48.0 ± 1.0 b +
0
3.78 ± 0.8 h
5.21 ± 0.6 j
4.61 ± 0.7 i
3.56 ± 0.6 f
3.82 ± 0.8 h
3.11 ± 0.4 d
3.52 ± 0.8 f
3.66 ± 1.0 g
3.10 ± 0.6 d
3.41 ± 0.5 e
2.86 ± 0.7 c
2.82 ± 0.5c
2.76 ± 0.6 b
2.73 ± 0.8 b
2.62 ± 0.6 a
* mean of 3 replications of 20 cultures each; + basal callusing
Means followed by the same letter within columns were not significantly different at P < 0.05 (Duncan’s
multiple comparison test)
25
IN VITRO PROPAGATION OF CLITORIA TERNATEA L.
multiple shoots per explant varied from 2.62 to 5.21 between treatments. The rate of
multiplication increased as the number of subcultures increased (every subculture was
made at 4-week intervals). This was probably due to adaptation of the explants to in
vitro conditions. Similar observations have been reported for Picrorhiza kurroa (UPADHYAY et al. 1989) and Gentiana kurroo (SHARMA et al. 1993). ROUT et al. (1999)
demonstrated a significant improvement in shoot multiplication rate by subculturing
Plumbago zeylanica at 4-week intervals.
Elongated shoots were excised and placed in ½ strength MS medium supplemented with various concentrations of NAA and/or IBA for induction of rooting. MS
medium without growth regulators did not promote root induction; roots were observed in media containing NAA and/or IBA with 2% sucrose. However, optimal
rooting and growth of microshoots were observed in medium containing 0.25 mg l1
NAA with 2% sucrose 7–8 days after transfer, without intervening callus (Fig. 1C,
Table 3). The percentage of shoots forming roots and the number of roots per shoot
significantly varied depending on concentrations of IBA and NAA. The maximum
Table 3. Effect of different concentrations of auxins (NAA, IBA) added to the medium (½ strength MS
+ 2% sucrose) on rooting response of Clitoria ternatea after 2 weeks
NAA
(mg l-1)
IBA
(mg l-1)
0
0.10
0.25
0.50
0
0
0
0.10
0.25
0.1
0
0
0
0
0.10
0.25
0.50
0.10
0.10
0.25
% of shoots rooted
(mean ± S.E)*
0
66.8 ± 1.2 c
90.2 ± 1.6 g
78.4 ± 1.6 + f
58.4 ± 1.0 a
64.8 ± 1.2 b
68.4 ± 1.6 + d
70.6 ± 1.0 e
72.4 ± 1.2 + e
68.6 ± 1.3 + d
No. of roots/shoot
(mean ± S.E)*
Days to rooting
0
1.4 ± 0.6 a
3.6 ± 0.8 c
3.2 ± 0.5 c
1.6 ± 0.8 a
2.6 ± 0.6 b
2.2 ± 0.8 b
2.3 ± 0.6 b
2.2 ± 0.4 b
1.6 ± 0.6 a
0
10
7–8
9 − 10
10 – 11
8–9
9 – 10
8−9
9 – 10
10 – 11
* mean of 3 replications of 20 cultures each; + basal callusing at the cut end
Means followed by the same letter within columns were not sigificantly different at P < 0.005 (Duncan’s
multiple comparison test)
percentage of rooting (90.2%) was obtained in medium containing 0.25 mg l-1 NAA.
Root development was, however, slow at higher concentrations of NAA or IBA.
Rooted plantlets were transferred into pots (see: Methods). The survival rate
was 85% after 1 month. The acclimatized plants showed normal growth (Fig. 1D).
In conclusion, an efficient protocol for micropropagation of an important
medicinal plant, C. ternatea, was developed in this study by testing various concentrations of growth regulators and culture conditions. The results will make the conservation and propagation of the species much easier. This can lead to the synthesis
and extraction of active compounds from its root.
26
Gyana Ranjan Rout
Acknowledgement: I wish to thank the Department of Forest and Environment, Government of Orissa,
for providing the necessary facilities.
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POPULATION DYNAMICS OF ERIOPHYOIDS
101
Fig. 1: Micropropagation of Clitoria ternatea: (A) development of axillary shoots from a nodal explant
on MS medium supplemented with 2.0 mg l-1 BA after 1 week of culture; (B) shoot multiplication and
elongation on MS medium supplemented with 2.0 mg l-1 BA, 0.25 mg l-1 NAA and 3% sucrose, after 4
weeks of culture; (C) rooting of in vitro derived shoot on ½ MS + 0.25 mg l -1 NAA + 2% sucrose, after
8 days of culture; (D) in vitro derived plantlet grown in a pot