Micropropagation of Costus speciosus (Koen.) Sm. Using Nodal Segment Culture

Abstract

Nodal segments of Costus speciosus (Koen.) Sm. containing single axillary buds were cultured on Murashige and Skoog medium (MS medium) supplemented with plant growth regulators for inducing plantlets. For breaking of axillary bud dormancy, nodal segments were cultured on 40-70gl-1 sucrose or 1-13 µM adenine sulphate (AdS) supplemented MS basal medium containing 5 µM 6-benzylaminopurine (BAP) and 1µM α-naphthalene acetic acid (NAA). The nodal segments cultured on 1-13 µM AdS, 5 µM BAP, 1 µM NAA and 50gl-1 sucrose showed simultaneous production of shoots and roots while those cultured on 5 µM BAP, 1 µM NAA and 40-70gl-1 sucrose produced shoots only. The most effective media for breaking axillary bud dormancy was 5 µM BAP, 1 µM NAA, 50 gl-1 sucrose and 10 µM AdS supplemented medium. The propagules from 40-70gl-1 sucrose produced roots in shoot multiplication medium, i.e.,10 µM AdS, 1 µM NAA, 50gl-1 sucrose and 3-11 µM BAP supplemented medium. The best response for shoot multiplication was on 10 µM AdS, 1 µM NAA, 50gl-1 sucrose and 7 µM BAP. The well-rooted shoots were hardened and transferred to the soil where they showed 95% survival rate. Results show that axillary bud can be used for micropropagation of Costus speciosus.

Full text

Page 1

Available online at www.notulaebiologicae.ro
Not Sci Biol 2 (1) 2010, 58-62
Print ISSN 2067-3205; Electronic 2067-3264
Notulae Scientia Biologicae
Micropropagation of Costus speciosus (Koen.) Sm. Using Nodal
Segment Culture
Kshetrimayum PUNYARANI, G. Jitendra SHARMA
Manipur University, Department of Life Sciences, Imphal-795003, India; punyaraniksh@gmail.com; gjs1951@yahoo.com
Abstract
Nodal segments of Costus speciosus (Koen.) Sm. containing single axillary buds were cultured on Murashige and Skoog medium (MS
medium) supplemented with plant growth regulators for inducing plantlets. For breaking of axillary bud dormancy, nodal segments were
cultured on 40-70gl-1 sucrose or 1-13 µM adenine sulphate (AdS) supplemented MS basal medium containing 5 µM 6-benzylaminopurine
(BAP) and 1µM α-naphthalene acetic acid (NAA). The nodal segments cultured on 1-13 µM AdS, 5 µM BAP, 1 µM NAA and 50gl-1
sucrose showed simultaneous production of shoots and roots while those cultured on 5 µM BAP, 1 µM NAA and 40-70gl-1 sucrose
produced shoots only. The most effective media for breaking axillary bud dormancy was 5 µM BAP, 1 µM NAA, 50 gl-1 sucrose and 10
µM AdS supplemented medium. The propagules from 40-70gl-1 sucrose produced roots in shoot multiplication medium, i.e.,10 µM AdS,
1 µM NAA, 50gl-1 sucrose and 3-11 µM BAP supplemented medium. The best response for shoot multiplication was on 10 µM AdS,
1 µM NAA, 50gl-1 sucrose and 7 µM BAP. The well-rooted shoots were hardened and transferred to the soil where they showed 95%
survival rate. Results show that axillary bud can be used for micropropagation of Costus speciosus.
Keywords: costus speciosus, micropropagation, adenine sulphate, axillary bud
Introduction
About 150 species of Costus speciosus (Koen.) Sm.
(family: Costaceae) have been reported from the tropical
regions of the world (Deb, 1983). The decoction of roots
of Costus speciosus are prescribed in urinary complaints and
are stimulant, tonic, and antihelmintic. Rhizome extract,
paste and juice are applied to cure white leprosy, juice
poured in earache, eaten with sugar to expel intestinal
worms, cooked and eaten as laxative (Prakash and Meh-
rotra, 1996). The root extract acts as an astringent, aph-
rodisiac, depurative, purgative and is useful in catarrhal
fever, cough, skin diseases and snake bites (Khanna et al.,
1977; Rathore and Khanna, 1978; Rastogi and Mehrotra,
1991). The rhizomes can be used as an alternative source
of diosgenin (Chopra et al., 1956; Dasgupta and Pandey,
1970). The main advantage of Costus speciosus over the two
species of Dioscorea is that C. speciosus grows abundantly
in the plains, whereas both D. prazeri and D. deltoidea
grow only at high altitudes of the Himalayas (Dasgupta
and Pandey, 1970). Eremanthin and costunolide isolated
from Costus speciosus possessed hypoglycemic and hypo-
lipidemic activities (Eliza et al., 2009 a and b). However,
the existence of this species has been threatened due to de-
forestation, jhum cultivation, habitat disturbance for con-
version of wetland ecosystem into agricultural land and
uncontrolled plucking/uprooting of these plants. Shoot
multiplication in Costus speciosus have been achieved using
shoot tips (Chaturvedi et al., 1984) and rhizome thin sec-
tions (Malabadi et al., 2005). Micropropagation through
rhizomatous eyes/ buds/ shoot tips or rhizome thin sec-
tions has disadvantages: the uprooted rhizomes usually fail
to survive after rhizomatous eyes/ shoot tips are decapi-
tated, and establishment of in vitro culture is usually dif-
ficult due to higher contamination. The present investiga-
tion, therefore, is an attempt to develop mass propagation
protocol using nodal segments of stem. Micropropagation
using nodal segments overcomes many disadvantages of
rhizomatous eye/bud/shoots tip culture. In nodal seg-
ment culture, contamination is less, mature stem which
have produced seeds are used and one mature stem usually
contain 15-20 axillary buds in comparison to 4-5 rhizoma-
tous eyes in one rhizome.
Materials and methods
Stems of C. speciosus (Koen.) Sm. were collected from
the Experimental Field of Manipur University. The leaf
sheaths covering the stem were removed and then the
stems were washed under running tap water for 15 mins.
The stems were then cut into segments, each segment con-
taining one axillary bud. The nodal segments were surface
decontaminated in 0.1% HgCl2 (10 mins.) before rinsing
with sterilized distilled water. Nodal segments were cul-
tured in 250 ml conical flasks (Borosil), each containing
15 ml of MS medium supplemented with various plant
growth regulators (PGRs) and sucrose concentration. The
pH of the media was adjusted to 5.8 before being auto-
Received 21 November 2009; accepted 10 January 2010

Page 2

Punyarani K. and G. J. Sharma / Not Sci Biol 2 (1) 2010, 58-62
59
bud (Pilate et al., 1989). Langridge et al. (1989) demon-
strated with transgenic plant which contained the genes
for bacterial luciferage (LUX A and LUX B) under the
control of an auxin responsive promoter that auxin con-
tent of the axillary bud increases after shoot apex were de-
capitated. Hence, it was possible to break bud dormancy
in cultured nodal segments due to separation from shoot
apex. The position of the nodal buds on the stem did affect
bud-break (Fig. 1). Nodal segments taken from the upper
and middle portion of the stem showed higher bud-break
compared to the nodal segments taken from lower por-
tion of the stem. In Flytrigia repens (quackgrass), the ab-
scissic acid (ABA) level which is usually high in dormant
lateral buds declined to 20% of control level within 24 hrs
after the rhizomes were decapitated (Pearce et al., 1995).
Hence, the higher bud-break frequency in nodal segments
from middle portion of stem may be due to lower level of
ABA, in comparison to lower portion of stem; also they
are more mature than the upper portion of the stem.
The explants cultured on MS basal medium did not
show initiation of bud-break. Success was achieved when
the nodal segments were cultured on MS medium supple-
mented with 1 µM NAA, 5 µM BAP and 30-70 gl-1 su-
crose. After 3 weeks, the highest percentage of shoot in-
duction was observed in the presence of 50 gl-1 sucrose
(Tab. 1, Fig. 2 a). However, increased percentage of dif-
ferentiated buds along with roots was observed in MS
medium supplemented with 1 µM NAA, 5 µM BAP, 50
gl-1 sucrose and 1-13 µM AdS (Tab. 2, Fig. 2 b). The high-
est percentage was found in 1 µM NAA, 5 µM BAP, 50
gl-1 sucrose and 10 µM AdS supplemented medium (Tab.
2, Fig. 2 b). The length of the plantlets increased as the
concentration of AdS increased. However, 13 µM AdS was
inhibitory, as decreased in shoot length was observed after
achieving maximum length in 1 µM NAA, 5 µM BAP, 50
gl-1 sucrose and 10 µM AdS (Tab. 2, Fig. 2b). The average
number of roots decreased as the concentration of AdS in-
creased (Tab. 2). The maximum number of roots was on 1
µM NAA, 5 µM BAP, 50 gl-1 sucrose and 1µM AdS supple-
mented media, but the axillary bud failed to differentiate
to shoots. Therefore, 50g l-1 sucrose and 10 µM AdS were
considered as optimal for bud-break in C. speciosus.
claved at 121°C for 20 min and 15 lbs sq inch-1 pressure.
All cultures were incubated at 25 ± 2°C under 16 h (day/
night) photoperiod with light supplied by white fluores-
cent tubes (3000 lux). Subculturing was carried out after
every 4 weeks by trimming-off leaves and roots. For break-
ing the dormancy of the bud, the nodal segments were
cultured on MS medium supplemented with 5 µM BAP,
1 µM NAA, 50 gl-1 sucrose and 1-13 µM AdS or 5 µM
BAP, 1 µM NAA and 30-70 gl-1 sucrose. The cultures were
maintained for induction of bud-break. The freshly initi-
ated plantlets/ shoots were subcultured on MS medium
(Murashige and Skoog, 1962) supplemented with 10 µM
AdS, 1 µM NAA, 50 gl-1 sucrose and 3-11 µM BAP for
shoot multiplication.
Each treatment had 10 replicates and data were record-
ed after 4 weeks. For bud break induction, it was repeated
three times while for shoot multiplication experiment, it
was repeated two times. All the data were scored after four
weeks of culture. Data were analyzed for significance us-
ing ANOVA and the differences contrasted using Tukey’s
comparison tests at 5% probability test. All statistical
analysis was performed using the SPSS statistical software
package.
Results and discussion
In natural habitats, the axillary buds of Costus specio-
sus are dormant. The present investigation has shown that
cutting the stem into segments and culturing them on
suitable medium supplemented with suitable PGRs can
break the dormancy of the bud. Literature surveys have
revealed many possible reasons for this. Auxin makes the
shoot apex a sink for cytokinin from the roots and decapi-
tation increases the accumulation of cytokinin in axillary
Tab. 1. Effect of MS medium supplemented with different
concentration of sucrose on bud-break in the single node
segment culture of Costus speciosus
Sucrose(gl-1)
30
40
50
60
Response
bud enlarged
Bud-break (%)
51.12
57.36
69.05
61.48
bud differentiated to shoots
bud differentiated to shoots
bud differentiated to shoots
bud differentiated to shoots
but the leaves turned colorless
7053.31
Fig. 1. Effect of teh position of the node in the stem on bud-
break

Page 3

Punyarani K. and G. J. Sharma / Not Sci Biol 2 (1) 2010, 58-62
60
The possible growth regulatory effect caused by ade-
nine was first noted by Bonner and Huagen-Smith (1939).
It could induce bud formation in both tobacco stem seg-
ments and elm and tobacco callus in vitro (Bonner et al.,
1939; Skoog and Tsui, 1948; Jacquiot, 1951; Miller and
Skoog, 1953). In Nicotina tabacum, medium containing
Kn and AdS showed marked increase in the activities of
two enzymes of the oxidative pentose phosphate pathway,
compared to their activities in non-shoot forming medi-
um (Scott et al., 1964). Hence, it may be inferred that pres-
ence of BAP, NAA and AdS in the medium increases en-
zymes of the oxidative pentose phosphate pathway which
provide activation energy for breaking bud dormancy
through sucrose metabolism. After 4 weeks, propagules
from 5 µM BAP, 1 µM NAA and 30-70 gl-1 sucrose and
from 1µM NAA, 5 µM BAP, 1-13 µM AdS and 50 gl-1
sucrose supplemented MS medium were cultured on MS
medium supplemented with 10 µM AdS, 1 µM NAA, 50
gl-1 sucrose and 3-11 µM BAP for shoots multiplication.
The average number of multiple shoot increased as the
concentration of BAP increased from 3-7 µM. However,
further increase was inhibitory (Tab. 3 and 4, Fig. 2 c and
d). Maximum average number of shoots, in both cases, was
achieved in 7 µM BAP (Tab. 3 and 4). BAP has almost
the same effect on shoot multiplication of the propagules
Tab. 2. Effect of MS medium supplemented with different
concentration of adenine sulphate (AdS) and 50 gl-1 sucrose on
bud–break frequency of single node segment of Costus speciosus
Treatment
with
AdS (µM)
Bud–
break
(%)
Response
Length of the
plantlets (cm)
(mean ± SE)
No.of roots
(mean ± SE)
171.50
bud failed to
differentiate
but rooting
was observed
bud
differentiated
to plantlets
bud
differentiated
to plantlets
bud
differentiated
to plantlets
bud
differentiated
to plantlets
-8.70ab± 0.26
4 78.532.12a ± 0.167.70b ± 0.34
783.13 2.62ab ± 0.14 6.70b ± 0.37
10 85.733.94c ± 0.11 4.40a ± 0.37
13 82.533.24b ± 0.063.40a ± 0.48
Means followed by same letters are not significantly different
at p < 0.05, according to Tukey’s comparison test
Fig. 2. Micropropagation of Costus speciosus; (a) Axillary bud-break in MS + 50gl¯1 sucrose; (b) Axillary bud-break with roots in
MS + 10µM Ads; (c) Proliferation of propagules from 1-13 µM Ads in MS + 3 µM BAP; (d) Proliferation of propagules from 1-13
µM Ads in MS + 7 µM BAP; (e) Transplanted plantlets established on soil

Page 4

Punyarani K. and G. J. Sharma / Not Sci Biol 2 (1) 2010, 58-62
61
derived from 40-60 gl-1 sucrose and 1-13 µM AdS supple-
mented medium. Although, there was no significant dif-
ference between the average shoot numbers of two types
of propagules, the plantlets produced from propagules of
AdS supplemented medium were healthier and average
number of leaves was also higher (Tab. 3 and 4). This is
in agreement with the findings on Brassica campestris in
which adding adenine sulphate to medium containing Kn
and IBA did not increase shoot multiplication, but shoot
weight increased, leaves were dark green and the cultures
were healthy (Paek et al., 1987).
During bud-break and shoot multiplication experi-
ment, rooting was observed only on medium supplement-
ed with AdS in conjunction with BAP and NAA. The
propagules from 40-60gl-1 sucrose supplemented medium
were successfully rooted when transferred on to medium
supplemented with 10 µM AdS, 1 µM NAA and 3-11 µM
BAP. The maximum average number of roots was found
in 3 µM BAP treatment (Tab. 3). In the case of propagules
from 1-13 µM AdS supplemented medium, further in-
crease in average number of roots was observed, when
transferred on to a medium supplemented with 10 µM
AdS, 1 µM NAA, 3-11 µM BAP (Tab. 4). Start and Cum-
ming (1976) have reported positive effect of AdS on root-
ing in Saintpaulia ionantha in the same trend as shown in
the current results. The plant survival touches about 95%
when acclimatization is carried out by transferring the
plantlets to potting mixture (Fig. 2 e).
Conclusions
Nodal segment culture provides new technique for
micropropagation of Costus speciosus. It also overcomes
many disadvantages of rhizomatous eye/bud or shoots tip
culture, while one mature stem usually contain 15-20 ax-
illary buds in comparison to 4-5 rhizomatous eye in one
rhizome. Further induction of multiple shoot from plant-
lets resulted in regeneration of a large number of plantlets
from a single stem. The plant survival touches about 95%
when acclimatization is carried out by transferring the
plantlets to potting mixture.
References
Bonner, D. M. and A. J. Haagen-Smith (1939). Leaf growth
factors II. The activity of pure substances in leaf growth.
Proc. Nat. Acad. Sci. U.S.A. 25:184-188.
Bonner, D. M., A. J. Haagen-Smith and F. W. Went (1939). Leaf
growth hormones. 1. A bio-assay and source for leaf growth
factors. Bot. Gaz. 101:128-144.
Chaturvedi, H. C., P. Misra and M. Jain (1984). Proliferation
of shoot tips and clonal multiplication of Costus speciosus in
long term culture. Plant Sci. Letts. 35:67-71.
Chopra, R. N., S. L. Nayer and I. C. Chopra (1956). Glossary of
Indian medicinal plants. New Delhi: CSIR Publication and
Information Directorate. 78-79.
Dasgupta, B. and V. B. Pandey (1970). A new Indian source of
diosgenin (Costus speciosus). Cell. Molec. Life Sci. 26:475-
476.
Deb, D. B. (1983). The Flora of Tripura State. Buddlejaceae-
Gramineae (Poaceae). Today and Tomorrow’s Printers and
Publishers. Vol. II.
Eliza, J., P. Daisy, S. Ignacimuthu and V. Duraipandiyan (2009a).
Normo-glycemic and hypolipidemic effect of costunolide
isolated from Costus speciosus (Koen.) Sm. in streptozotocin
induced diabetic rats. Chem. Biol. Interact.179:329-334.
Eliza, J., P. Daisy, S. Ignacimuthu and V. Duraipandiyan (2009b).
Antidiabetic and antilipidemic effect of eremanthin from
Costus speciosus (Koen.) Sm. in STZ induced diabetic rats.
Chem. Biol. Interact. 182:62-72.
Jacquiot, C. (1951). Action of meso-inositol and of adenine on
bud formation in the cambium tissue of Ulmus campestris
cultivated. In vitro Compt. Rend. Acad. Sci. Paris. 233:815-
817.
Khanna, P., G. L. Sharma, A. K. Rathore and S. K. Manot (1977).
Effect of cholesterol on in vitro suspension tissue cultures of
Costus speciosus (Keon.) Sm. Dioscorea floribunda, Solanum
aviculare and Solanum xanthocarpum. Ind. J. Exptl. Biol.
15:1025-1027.
Langridge, W. H. R., K. J. Fitzgerald, C. Koncz, J. Schell and
Tab. 4. Effect of different concentration of BAP on shoot
multiplication and rooting of propagules of Costus speciosus
from 1-13 µM AdS enriched medium in MS medium
supplemented with 50gl-1 sucrose
Treatment
with
BAP
(µM)
3
5
7
9
11
Mean followed by the same letters are not significantly different
at p < 0.05, according to Tukey’s comparison test
No. of
shoots
(Mean± SE)
No. of roots
(Mean ± SE)
Rooting rate
(Mean ±SE)
No. of leaves
(Mean ± SE)
0.90a±0.28
2.00b±0.21
3.60ab ± 0.27
2.70b ± 0.34
1.40a ± 0.31
12.80ab ± 0.33
11.50ab ± 0.50
9.82b ± 0.48
8.88b ± 0.54
7.30a ± 0.37
3.20ab ± 0.09
2.87ab ± 0.13
2.45b ± 0.12
2.22b ± 0.14
1.82a ± 0.10
9.80b ± 0.33
8.76a ± 0.38
8.58a ± 0.34
8.06a ± 0.35
7.39a ± 0.36
Tab. 3. Effect of different concentration of BAP on shoot
multiplication and rooting of propagules of Costus speciosus
from 40-60 gl-1 sucrose enriched medium in MS medium
Treatment
with BAP
(µM)
3
5
7
9
11
Mean followed by the same letters are not significantly different
at p < 0.05 ,according to Tukey’s comparison test
No. of shoots
(mean ± SE)
No. of roots
(mean ± SE)
No. of leaves
(mean ± SE)
1.20a ± 0.25
1.80a ± 0.26
3.30b ± 0.25
2.20b ± 0.30
1.60a ± 0.34
3.60b ± 0.27
3.10b ± 0.23
2.20a ± 0.25
1.90a ± 0.23
1.50a ± 0.34
2.17a ± 0.23
2.96a± 0.20
3.23a ± 0.24
3.25a ± 0.22
3.16a ± 0.24

Page 5

Punyarani K. and G. J. Sharma / Not Sci Biol 2 (1) 2010, 58-62
62
A. A. Szalay (1989). Dual promoter of Agrobacterium
tumefaciens mannopine synthase genes is regulated by plant
growth hormones. Proc. Natl. Acad. Sci. USA 86:3219-
3223.
Malabadi, R. B., G. S. Mulgand and K. Nataraja (2005). Effect
of Triacotanol on the micropropagation of Costus speciosus
(Koen.) Sm. using rhizome thin sections. In vitro Cell Dev.
Biol.-Plant. 41:129-132.
Miller, C. and F. Skoog (1953). Chemical control of bud
formation in tobacco stem segments. Am. J. Bot. 40:768-
773.
Murashige, T., F. Skoog (1962). A revised medium for rapid
growth and bioassays with tobacco tissue cultures. Physiol.
Planta. 15:473-497.
Paek, K. Y., S. F. Chandler and T. A. Thorpe (1987). In vitro
propagation of Chinese cabbage from seedling shoot tips. J.
Am. Soc. Hort. Sci. 112:841-845.
Pearce, D. W., J. S. Taylor, J. M. Robertson, K. N. Harker and E.
J. Daly (1995). Changes in abscisic acid and indole-3-acetic
acid in axillary buds of Elytrigia repens released from apical
dominance. Physiol. Planta. 94:110-116.
Pilate, G., B. Sotta, R. Maldiney, L. M. Jacques, Sossountzov
and E. Miginiac (1989). Absccisic acid, IAA and cytokinin
changes in buds of Pseudotsuga menziesii during bud
quiescence release. Physiol. Planta. 76:100-106.
Prakash, V. and B. N Mehrotra (1996). Zingiberaceae of
India: Biological screening and ethnobotanical diversity:
In Proceedings of the second Symposium on the family
Zingiberaceae, South China Institute of Botany. 229-237.
Rastogi, R. P. and B. N. Mehrotra (1991). Compendium
of Indian medicinal plants. p.81-84, vol.2 (1970-1979).
Lucknow, India: Central Drug Research Institute (CDRI).
Rathore, A. K. and P. Khanna (1978). Production of diosgenin
from Costus speciosus (Koen.) Sm. and Solanum nigram L.
suspension cultures. Curr. Sci. 47:870-871.
Scott, K. J., J. Daly and H. H. Smith (1964). Effects of
indoleacetic acid and kinetin on activities of enzymes of the
hexose monophosphate shunt in tissue cultures of Nicotiana.
Plant Physiol. 39:709-712.
Skoog, F. and C. Tsui (1948). Chemical control of growth and
bud formation in tobacco and callus cultured. In vitro. Am.
J. Bot. 35:782-787.
Start, N. G. and B. G. Cumming (1976). In vitro propagation of
Saintpaulia ionantha Wendl. HortScience 11:204-206.