2. Ceropegia attenuata

Full text

1 23
Journal of Plant Biochemistry
and Biotechnology
ISSN 0971-7811
Volume 20
Number 2
J. Plant Biochem. Biotechnol.
(2011) 20:276-282
DOI 10.1007/
s13562-011-0059-0
Micropropagation and in vitro flowering
of endemic and endangered plant
Ceropegia attenuata Hook
Jaykumar J. Chavan, Mansingraj
S. Nimbalkar, Avinash A. Adsul, Sharad
S. Kamble, Nikhil B. Gaikwad, Ghansham
B. Dixit, Rajaram V. Gurav, et al.

1 23
Your article is protected by copyright and
all rights are held exclusively by Society for
Plant Biochemistry and Biotechnology. This
e-offprint is for personal use only and shall
not be self-archived in electronic repositories.
If you wish to self-archive your work, please
use the accepted author’s version for posting
to your own website or your institution’s
repository. You may further deposit the
accepted author’s version on a funder’s
repository at a funder’s request, provided it is
not made publicly available until 12 months
after publication.

ORIGINAL ARTICLE
Micropropagation and in vitro flowering of endemic
and endangered plant Ceropegia attenuata Hook
Jaykumar J. Chavan &Mansingraj S. Nimbalkar &Avinash A. Adsul &
Sharad S. Kamble &Nikhil B. Gaikwad &Ghansham B. Dixit &Rajaram V. Gurav &
Vishwas A. Bapat &Shrirang R. Yadav
Received: 8 January 2011 / Accepted: 10 May 2011 / Published online: 21 May 2011
#Society for Plant Biochemistry and Biotechnology 2011
Abstract Factors affecting in vitro propagation were
evaluated for Ceropegia attenuata Hook., an endemic and
endangered plant having ornamental potential but a limited
reproductive capacity. Rapid shoot multiplication from
nodal explants was established using varying concentrations
of cytokinins and auxins either alone or in combinations. The
highest frequency of shoot induction was achieved when
nodal explants were inoculated on Murashige and Skoog (MS)
medium supplemented with 13.31 μM 6-benzylaminopurine
with a mean of 12.9±0.5 shoots per explant. High concen-
trations of TDZ (6.81–11.35 μM) and KN (6.78–11.61 μM)
resulted in stunted and vitrified shoots. Factors implicated in
the promotion of floral transition of the C. attenuata have
been identified which are 4-amino-3, 5, 6-trichloropicolinic
acid (picloram), 6-benzylaminopurine, sucrose and photope-
riod. The highest frequency of flowering (100%) was
obtained when axillary shoot explants were transferred to
MS medium supplemented with picloram (4.14 μM)
within 4 weeks of culture. Transfer of in vitro regen-
erated shoots to half strength MS medium with 2.46 μM
indole-3-butyric acid (IBA) showed maximum root
induction. The in vitro grown plantlets were successfully
acclimatized in the glasshouse with 85% of survival and
showed normal development. The developed protocol
provided a simple, cost-effective approach for the conserva-
tion of endangered plant C. attenuata for replenishing its
declining populations.
Keywords Ceropegia attenuata .Conservation .
Endangered .Fruiting .In vitro flowering .
Micropropagation .Picloram
Abbreviations
BAP 6-benzylaminopurine
IAA indole-3-acetic acid
IBA indole-3-butyric acid
Kinetin 6-furfurylaminopurine
TDZ thidiazuron
Picloram 4-amino- 3, 5, 6-trichloropicolinic acid
½ MS half-strength MS medium
Introduction
The genus Ceropegia L. (Asclepiadaceae) represented by
approximately 200 species which are usually tuberiferous
erect herbs and climbers distributed in tropical and
subtropical Asia, Africa, Australia and in Canary and
Pacific Islands (Anonymous 1992; Bruyns 2003). Most of
the Ceropegia species have morphologically unique flowers
with beautiful architecture and a few of the species are
ornamentals and are cultivated in Europe and United States
(Hodgkiss 2004; Reynolds 2006). In Indian Subcontinent,
majority of the Ceropegia species are endemic to Western
Ghats (Nayar and Sastry 1987). The root tubers of many
J. J. Chavan :M. S. Nimbalkar :A. A. Adsul :S. S. Kamble :
N. B. Gaikwad :G. B. Dixit :R. V. Gurav :V. A. Bapat :
S. R. Yadav
Department of Botany, Shivaji University,
Kolhapur, MS, India 416 004
V. A. Bapat
Department of Biotechnology, Shivaji University,
Kolhapur, MS, India 416 004
J. J. Chavan (*)
Laboratory of Cytogenetics and Plant Breeding,
Department of Botany, Shivaji University,
Kolhapur, MS, India 416 004
e-mail: jaychavansu@gmail.com
J. Plant Biochem. Biotechnol. (July–Dec 2011) 20(2):276–282
DOI 10.1007/s13562-011-0059-0
Author's personal copy

Ceropegia species are storehouse of starch, sugars, gum,
albuminoids, fats, crude fiber and other valuable phytocon-
stituents which are routinely used in traditional Indian
Ayurvedic drug preparations for treating diarrhea and
dysentery. The starchy tubers are a good source of nutritive
tonic (Kirtikar and Basu 1935). This pharmacological
importance of the genus Ceropegia is mainly due to the
presence of a pyridine alkaloid called ‘cerpegin’(Sukumar
et al. 1995).
The habitat degradation, overexploitation of root tubers
and anthropogenic activities are major threats to the
survival of this plant. Besides, the scarcity of pollinators
and poor seed setting are other major constraints in the
natural propagation, leading to a continuous depletion of its
natural population. Establishment of an efficient protocol of
micropropagation is necessary to ensure large-scale multi-
plication and preservation of this plant. Since it is a
seasonal plant, generally vegetative growth is seen only
after first rain by sprouting of dormant underground tubers.
However, the species is difficult to propagate by conven-
tional methods of cuttings and seedlings because of poor
seed setting and limited seed viability. These difficulties
could be overcome using alternative effective plant tissue
culture techniques. The induction of multiple shoots
through axillary bud proliferation is now recognized as a
useful technique for propagation, in vitro conservation and
reestablishment of threatened plant species back into the
wild (Patil 1998; Desai et al. 2003; Bapat et al. 2008;
Karuppusamy et al. 2009). Only a few reports are available
on the micropropagation of Ceropegia species (Patil 1998;
Beena et al. 2003; Karuppusamy et al. 2009; Chandore et
al. 2010; Chavan et al. 2011).
Considering the IUCN status of the Ceropegia species,
in the present investigation an attempt has been made to
establish an efficient protocol of micropropagation and in
vitro flowering of C. attenuata which would be beneficial
in conservation of such plant species.
Materials and methods
Plant material and surface sterilization
Plants were collected from Sindhudurg District, Maharashtra,
India and maintained in the greenhouse of Botanical
Garden of Department of Botany, Shivaji University,
Kolhapur (India). Actively growing shoots of mature
plant of Ceropegia attenuata Hook were used to initiate
the cultures. The shoot segments were defoliated and
thoroughly washed under running tap water for 20 min to
remove soil and other superficial contamination. Standard
decontamination procedure was adopted using detergent,
Tween 20 (5% v/v) for 10 min. The explants were treated
with Bavistin (0.5% w/v) for 3 min and washed thoroughly
with sterile distilled water. Thereafter, under aseptic con-
ditions, explants were disinfected in 70% (v/v) alcohol for
30 s, and 0.1% (w/v) mercuric chloride for 5 min. Each
treatment was followed by 3–4 rinses with sterile distilled
water. The surface sterilized shoots were separated into single
node pieces and were aseptically cultured on the shoot
induction medium.
Media and culture conditions
MS basal salts (Murashige and Skoog 1962) and vitamins
supplemented with 3% (w/v) sucrose and solidified with
0.2% gelrite was used in all the experiments. The pH of the
medium was adjusted to 5.8±0.1 using 0.1 N NaOH or
0.1 N HCl, prior to the autoclaving at 121°C for 15 min.
All chemicals used for the experiments were of analytical
grade (Sigma, Hi-media, Qualigenes or SD Fine Chemicals,
India). The cultures were maintained in a growth room at
25±2°C with a 16-h photoperiod with 33 μmol m
−2
s
−1
light intensity from cool white fluorescent tubes (Philips
India Ltd., Mumbai). All the cultures were subcultured on
the fresh medium after every 4 weeks. For in vitro flower
bud induction, different photoperiod cycles were examined.
Shoot induction and multiplication
Nodal segments were placed on MS medium with 6-
benzylaminopurine (BAP, 4.44-22.19 μM), thidiazuron
(TDZ, 2.27–11.35 μM) and kinetin (KN, 2.32–9.29 μM)
at different concentrations either singly or in combination
with indole-3-acetic acid (IAA, 0.57–11.42 μM) for
multiple shoot induction and proliferation. MS medium
without any growth hormone was served as a control. The
percent regeneration, number of shoots per explant and
mean shoot length were recorded after 4 weeks of culture.
Effect of picloram, BAP, sucrose and photoperiod
on in vitro flowering
Depending on the objective of experiment performed, MS
medium was supplemented with different concentrations
and combinations of picloram, BAP and sucrose to study their
effects on in vitro flowering. The apical and axillary buds
from in vitro regenerated microshoots were used as explants.
Picloram (0.41–10.35 μM), BAP (4.44–22.19 μM) and
sucrose (87–233 mM) were tested for the flower bud
induction. Picloram was added alone whereas; BAP and
sucrose were used at different concentrations and combina-
tions. In the first step, single concentration of BAP was kept
constant and all sucrose concentrations (87–233 mM) were
varied. Similarly, all the BAP concentrations (4.44–
22.19 μM) were added keeping a constant concentration of
J. Plant Biochem. Biotechnol. (July–Dec 2011) 20(2):276–282 277
Author's personal copy

sucrose. Elongated shoots were subcultured on the fresh
medium of same composition after every 4 weeks for further
propagation and flowering. Different photoperiod cycles were
also tested for the in vitro flower bud induction and
development. Regenerated apical shoots of C. attenuata were
cultured on MS salts supplemented with picloram (4.14 μM)
and were placed under each of six photoperiods: dark, 4, 8,
12, 16 and 20-h light for flower bud induction. All the
cultures were placed in the culture room and maintained at
25± 2°C. Measurement of in vitro flowering was recorded by
observing the number of flower buds per culture and percent
response after 4 weeks of culture.
Rooting of micropropagules
Microshoots from shoot clusters were carefully separated
and axillary nodes (3–4 cm long with two pairs of fully
expanded leaves) were transferred to half-strength MS
medium supplemented with various root inducing growth
hormones. Efficacy of IBA (0.49–12.26 μM), IAA (0.57–
14.27 μM) and BAP (8.87 μM) were checked for root
induction. Data on percentage of root induction, average
number of roots and average root length were recorded after
4 weeks of culture.
Plant acclimatization
Plantlets with well developed shoots and roots were
removed from the culture medium carefully, washed gently
under running tap water and transferred to small plastic pots
containing a potting mixture. Special efforts were taken
during hardening process of C. attenuata by using different
substrates such as, sterile garden soil, river sand and coco
peat either individually or in combinations. Potted plantlets
were covered with transparent polythene bags to retain
humidity and were watered every 3 days with ½ MS
medium without sucrose for 2 weeks. These plastic pots
were kept under diffuse light (16-h photoperiod) conditions.
Polythene bags were opened after 2 weeks in order to
acclimatize plants to field conditions. After 4 weeks,
acclimatized plants were transferred to pots containing
normal garden soil and maintained in a natural condition.
Experimental design and data analysis
The cultures were observed periodically and morphological
changes were recorded at regular intervals. All experiments
were repeated twice with 20 replicates per treatment. The
experiments were conducted in a completely randomized
design and the means and the standard error (SE) were
compared. Data were subjected to analysis of variance
(ANOVA) and comparisons of means were made with the
Dunnett multiple comparison test.
Results and discussion
Role of exogenous growth hormones on shoot
multiplication
Nodal explants cultured on MS basal media supplemented
with growth hormone combinations at different concen-
trations showed shoot induction and proliferation according
to the concentration of cytokinins and auxins (Table 1). MS
medium without growth hormone served as a control,
which did not induce shoots from nodal explant. Medium
was supplemented with BAP alone at different concen-
trations produced healthy axillary shoots and has been
reported as most effective hormone during shoot induction
and multiplication. Maximum shoots were obtained on MS
medium fortified with BAP (13.31 μM), branched repeatedly
giving an average number of 12.9 shoots per node (Fig. 1a).
The BAP was more effective than other cytokinins and
auxins for shoot induction and proliferation in other
threatened and medicinal plants (Patil 1998; Beena et al.
2003; Karuppusamy et al. 2009;Chavanetal.2011). MS
medium supplemented with KN (4.46 μM) showed good
response (95%) with an average of 3.5 shoots per explant
and also gave shoots with longer internodes. However, TDZ
did not enhance shoot induction significantly and
produced maximum of 2.4 shoots per explant at
4.54 μM concentration. High concentrations of TDZ
resulted in formation of stunted and vitrified shoots.
Similar response was also observed in the shoot multiplication
of a few other Ceropegia species (Beena et al. 2003;
Karuppusamy et al. 2009).
A combination of BAP (13.31 μM) and IAA (2.85 μM)
was found effective for axillary bud proliferation, which
developed average of 7.3 shoots per node explant and the
developed shoots were healthy, strong and elongated
(Fig. 1b). A synergistic effect of BAP in combination
with an auxin has been demonstrated in Ceropegia species
(Beena et al. 2003; Chavan et al. 2011). The shoot
multiplication rate was highest on MS medium with BAP
(13.31 μM) when compared to all other tested growth
hormones as evident from our work. Therefore, BAP
(13.31 μM) was used for subsequent subcultures and
multiplication experiments. Repeated subcultures enhanced
shoot multiplication rate (18 shoots per explant) up to 4
th
subculture, then declined afterwards. Shoot number,
shoot height and leaf number increased with sub
culturing up to the fourth subculture and then remained
constant or decreased. The micropropagated shoots did
not exhibit leaf and shoot tip abscission in contrast to
other reports (Patnaik and Debata 1996;Beenaetal.
2003). Enhanced shoot multiplication in subsequent
subculture was reported in this plant which is in
accordance with earlier reports in Asclepiadaceae mem-
278 J. Plant Biochem. Biotechnol. (July–Dec 2011) 20(2):276–282
Author's personal copy

Growth regulators (μM) % Response Mean shoots /
explant ± S.E
Mean shoot length
(cm) ± S.E
BAP TDZ KN IAA
Growth hormone free –– –
4.44 90 2.5± 0.3** 5.0 ±0.5**
8.87 95 2.4± 0.2** 7.4 ±0.5**
13.31 95 12.9± 0.5** 7.0± 0.5**
17.74 100 3.4± 0.2** 8.2 ± 0.3**
22.19 70 2.1± 0.4** 5.4 ±0.4**
2.27 85 1.4± 0.2** 5.2 ±0.6**
4.54 90 2.4± 0.2** 6.1 ±0.6**
6.81 85 1.8± 0.2** 5.2 ±0.7**
9.08 85 1.4± 0.2** 5.1 ±0.6**
11.35 70 0.9± 0.2** 4.5±0.7**
2.32 90 2.3± 0.2** 6.5 ±0.6**
4.46 95 3.5± 0.3** 6.5 ±0.6**
6.78 85 2.2± 0.3** 5.6 ±0.7**
9.29 65 1.9± 0.4** 4.4 ±0.8**
11.61 75 1.5± 0.3 ** 7.2± 0.7**
13.31 0.57 80 2.7± 0.4** 5.9± 0.7**
13.31 2.85 100 7.3± 0.4** 7.7± 0.3**
13.31 5.71 75 2.1± 0.3** 5.8± 0.8**
13.31 8.56 80 1.6± 0.2** 5.6± 0.7**
13.31 11.42 70 1.1± 0.2** 5.1± 0.8**
Table 1 Effect of different plant
growth hormones on shoot
multiplication of Ceropegia
attenuata Hook on
MS medium
Values represents mean±SE
from 20 replicates per treatment
and all the experiments were
repeated twice. The values are
significantly different **P≤0.01
when compared with Dunnett
multiple comparison test.
Fig. 1 Ceropegia attenuata
Hook: aShoot multiplication
(MS+BAP 13.31 μM),
bMultiple shoots (MS+BAP
13.31 μM+IAA 2.85 μM),
cFlowering (Picloram
4.14 μM), dRooting
(½MS+IBA 2.46 μM), eFollicle
formation (Picloram 4.14 μM),
fHardened plants, gTwo month
old plant under green house
conditions. Scale bar= 1cm
J. Plant Biochem. Biotechnol. (July–Dec 2011) 20(2):276–282 279
Author's personal copy

bers (Patnaik and Debata 1996; Karuppusamy et al. 2009;
Chavan et al. 2011).
In vitro flowering
Regulative role of picloram during in vitro flowering
Picloram is one of the potent synthetic auxin useful for the
initiation and maintenance of callus, suspension cultures
and induction of somatic embryos from various plant
tissues (Valverde et al. 1987; Hagen et al. 1990). The first
flower appeared after 2 weeks of transfer of plants on the
new medium and ten to fifteen flowers per explant were
developed within the next 2 weeks. Among the different
picloram concentrations tested, MS medium supplemented
with picloram (4.14 μM) was found more effective for in
vitro flower bud induction and maturation of in vitro
flowers (Fig. 2). Induction of flowers was observed on MS
medium containing different picloram concentrations, but
the picloram requirements for in vitro flowering was
variable. In vitro flowering has been reported for Ceropegia
species (Patil 1998; Britto et al. 2003; Nair et al. 2007)
where effect of spermine, GA
3
, 6-benzylaminopurine and
sucrose were tested on in vitro flowering. However, none of
these reports have used 4-amino- 3, 5, 6-trichloropicolinic
acid (picloram) for in vitro flower bud induction and
development. Maximum flower bud induction (100%)
was observed on the same media composition with an
average of 11.4 flower buds per explant (Fig. 1c). Within
10–15 days of flower formation, most of the floral buds
either turned brown or dropped during their development.
After the flowering, follicle formation (Fig. 1d) occurred
within 15 days on the same media however, further
increase in concentration of picloram resulted decreased
flower bud induction (Fig. 2), but was characterized by
induction of roots on the basal cut ends of shoot. When the
picloram was withdrawn from the medium, no flowering
was observed, indicating an in vitro flower bud induction
effect of picloram in C. attenuata.
Effect of BAP and sucrose concentrations on in vitro
flowering
MS medium without growth hormones failed to induce the
flower buds and the plants on this medium remains stunted.
Media supplemented with BAP alone usually resulted in the
production of multiple shoots. In a similar way, various
sucrose concentrations individually above 87 mM resulted
in decrease in the regularity of leaf initiation. Zhang (2007)
reported in the regenerated shoots of Perilla frutescens,
BAP with ammonium nitrate promoted such flowering.
Likewise, a combination of BAP and GA
3
promoted in
vitro flowering of Ceropegia bulbosa var bulbosa (Britto et
al. 2003), while in Ceropegia jainii, addition of BAP with
spermine induced floral buds (Patil 1998). In our work,
BAP stimulated flowering in combination with sucrose,
thus proving that BAP is a key component for the in vitro
flowering in accordance with other report on Ceropegia
species (Nair et al. 2007). The most significant results of
flower bud induction are highlighted in the Fig. 2.MS
medium with BAP (4.44 μM) in combination with 87 mM
sucrose started the flower bud initiation. Maximum response
of cultures (100%) producing flower buds and number of
flowers (6.3) per microshoot were observed in MS
medium supplemented with 17.72 μM BAP in combina-
tion with 175 mM sucrose at 16-h photoperiod (Fig. 2).
Flowering ability decreased with further increase or
decrease in BAP as well as sucrose concentration.
Subsequently, MS medium supplemented with BAP
(22.19 μM) treatment in combination with 175 mM
sucrose, induced 4.8 floral buds per shoot. At lower
Fig. 2 Effect of different
concentrations of picloram
(in μM) with 87 mM sucrose
and BAP (in μM) with 175 mM
sucrose on in vitro flowering of
Ceropegia attenuata Hook. GH
free: Growth hormone free;
P: Picloram (4-amino- 3, 5,
6-trichloropicolinic acid);
BAP: 6-benzylaminopurine
280 J. Plant Biochem. Biotechnol. (July–Dec 2011) 20(2):276–282
Author's personal copy

concentration of BAP (4.44 μM), the rate of flower bud
induction was very poor. The production of flowering
shoots continued for more than 35–40 days (Fig. 3).
Effect of photoperiod on in vitro flowering
Light period or day length is the most predictable factor in
plants to time their reproduction (Bernier and Perilleux
2005). The present work also suggested that specific
photoperiod was essential for in vitro flowering in C.
attenuata cultures. Different photoperiod cycles influ-
enced the in vitro flowering of C. attenuata (Table 2).
The 16-h photoperiod was found to be most effective for
flowering which showed highest percentage (100%) of
cultures producing about 10–12 flower buds per explant
on MS medium supplemented with picloram (4.14 μM).
Flower bud induction was not observed when cultures were
kept in the dark. Importance of photoperiod for in vitro
flowering has been reported for Lycopercon esculentum,
exposed to 16-h photoperiod (Sheeja and Mandal 2003).
Root induction and recovery of complete plants
Elongated shoots (3–4 cm) were excised from regenerating
cultures and were transferred to half strength MS medium
with varied concentrations of growth hormones. In all the
treatments, except control, roots were developed from the
basal cut end of the shoot within 15 days. Rooting
percentage, number of roots per shoot and length of roots
were affected significantly among the treatments (Table 3).
Among the three growth hormones tested, IBA was found
to be most effective for root induction. After being cultured
for 4 weeks, microshoots produced roots of various sizes
and lengths. The highest number of roots per shoot (8–10)
and the greatest root length (2.2 cm) were obtained when
the half-strength MS medium was incorporated with
2.46 μM IBA (Fig. 1e). Effectiveness of IBA in root
induction has been reported in other rare plant species (Patil
1998; Faisal et al. 2005). Significant root induction was
also observed when half-strength MS medium enriched
with IAA (2.85 μM), produced 4–5 roots per shoot. Thin
and delicate roots were noticed when the medium was
supplemented with higher concentrations of IAA. When the
microshoots were cultured on MS medium supplemented
Fig. 3 Effect of different planting substrates during acclimatization of
Ceropegia attenuata Hook
Table 2 Effect of different photoperiods on in vitro flowering of
Ceropegia attenuata Hook recorded after 4 weeks of culture on MS
medium containing picloram (4.14 μM)
Photoperiod (h) % of cultures producing
flower buds
Number of flowers
per culture±S.E
Dark ––
4––
8 25 0.7± 0.3*
12 45 2.4± 0.7*
16 100 11.3± 0.6**
20 55 3.5± 0.9**
Values represents mean±SE from 20 replicates per treatment and all
the experiments were repeated twice. The values are significantly
different **P≤0.01 when compared with Dunnett multiple comparison
test.
Table 3 Effect of different plant growth hormones on root induction
of Ceropegia attenuata Hook on half strength MS medium
Growth regulators (μM) % Response Average roots/
shoot±S.E
Average root
Length (cm)±S.E
IBA IAA BAP
Growth hormone free –– –
0.49 80 3.1± 0.4** 1.3 ±0.2**
2.46 100 9.8± 0.5** 2.2 ±0.2**
4.90 85 4.2± 0.4** 1.3 ±0.3**
7.36 80 2.8± 0.3** 1.7 ±0.2**
9.80 75 1.9± 0.3** 1.4 ±0.2**
12.26 60 1.7± 0.4** 1.2 ±0.3**
0.57 85 2.3± 0.2** 1.9 ±0.3**
2.85 90 4.7± 0.4** 1.8 ±0.2**
5.71 80 3.8± 0.4** 1.3 ±0.2**
8.56 80 2.6± 0.3** 1.4 ±0.2**
11.42 65 1.7± 0.3** 1.0± 0.2**
14.27 65 1.7± 0.3** 1.2 ±0.2**
0.49 8.87 85 2.5 ± 0.3** 1.4± 0.2**
2.46 8.87 95 5.7 ± 0.4** 1.9± 0.2**
4.90 8.87 80 3.7 ± 0.4** 1.6± 0.2**
7.36 8.87 70 2.2 ± 0.4** 1.5± 0.2**
9.80 8.87 75 2.8 ± 0.4** 1.5± 0.2**
Values represents mean±SE from 20 replicates per treatment and all
the experiments were repeated thrice. The values are significantly
different **P≤0.01 when compared with Dunnett multiple comparison
test.
J. Plant Biochem. Biotechnol. (July–Dec 2011) 20(2):276–282 281
Author's personal copy

with IBA (2.46 μM) in combination with BAP (8.87 μM)
showed a synergistic effect on in vitro rooting and produced
an average of 6 roots per shoot. It has been shown that
combination of auxins and cytokinins are more effective in
the induction of roots in Huernia hystrix (Amoo et al. 2009)
and the present results showed much similarity. Application
of IBA along with BAP during the root induction phase had
a clear beneficial effect on root production compared to
IAA treatments. Callus formation was occurred when the MS
medium was supplemented with increasing concentrations of
IAA.
Plantlets with well developed shoots and roots were
transferred to small plastic pots containing different types
of substrates. A combination of autoclaved river sand and
coco peat was the most effective substrate for the
acclimatization of in vitro regenerated plantlets (Fig. 2),
were revived within 20 days (Fig. 1f). One month after
transferring, the survival rate of the plantlets was highest
(85%) and the plants grew vigorously and exhibited true
characters similar to the field grown ones (Fig. 1g) and
could be transplanted at their natural surroundings.
The successful micropropagation procedure described
here is a simple and cost-effective protocol for the rapid
multiplication and conservation of endangered medicinal
plant C. attenuata. The Indian species of this genus possess
ornamental features which could be preserved and com-
mercialized through tissue culture techniques. In vitro
studies especially flower bud induction provided a model
system to study the role of hormones and photoperiod at
molecular and cellular level and developmental studies for
conducting microbreeding.
Acknowledgments Authors are thankful to Department of Biotech-
nology (DBT), Government of India for providing financial assis-
tance. VAB thankful to Council of Scientific and Industrial Research
(CSIR), New Delhi for Emeritus Scientist Fellowship. Authors are
also thankful to Head, Department of Botany, Shivaji University,
Kolhapur for providing necessary laboratory facilities.
References
Amoo SO, Finnie JF, Staden JV (2009) In vitro propagation of
Huernia hystrix: an endangered medicinal and ornamental
succulent. Plant Cell Tiss Org Cult 96:273–278
Anonymous (1992) Ceropegia Linn. (Asclepiadaceae), The wealth of
India. Vol. 3. New Delhi, (CSIR), 448
Bapat VA, Yadav SR, Dixit GB (2008) Rescue of endangered plants
through biotechnological applications. Nat Acad Sci Lett
31:201–210
Beena MR, Martin KP, Kirti PB, Hariharan M (2003) Rapid in vitro
propagation of medicinally important Ceropegia candelabrum.
Plant Cell Tiss Org Cult 72:285–289
Bernier G, Perilleux C (2005) A physiological overview of the
genetics of flowering time control. Plant Biotech 3:3–16
Britto SJ, Natarajan E, Arockiasamy DI (2003) In vitro flowering and
shoot multiplication from nodal explants of Ceropegia bulbosa
Roxb var bulbosa. Taiwania 48(2):106–111
Bruyns PV (2003) Three new succulent species of Apocynaceae
(Asclepiadoideae) from Southern Africa. Kew Bull 58:427–
435
Chandore AN, Nimbalkar MS, Gurav RV, Bapat VA, Yadav SR
(2010) An efficient micropropagation protocol for multiplication
and restoration of Ceropegia fantastica Sedgw: a critically
endangered plant species. Curr Sci 99:1593–1596
Chavan JJ, Nimbalkar MS, Gaikwad NB, Dixit GB, Yadav SR (2011)
In vitro propagation of Ceropegia spiralis Wight—an endemic
and rare potential ornamental plant of peninsular India. Proc Nat
Acad Sci India Sect B 81:120–126
Desai NS, Suprasanna P, Bapat VA (2003) Conservation status and in
vitro multiplication of Frerea indica Dalz. an endemic and
endangered plant from Western Ghats of Maharashtra, India.
Physiol Mol Biol Plants 9(2):265–268
Faisal M, Ahmad N, Anis M (2005) Shoot multiplication in Rauvolfia
tetraphylla L. using thidiazuron. Plant Cell Tiss Org Cult
80:187–190
Hagen SR, Letourneau D, Muneta P, Brown J (1990) Initiation and
culture of potato tuber callus tissue with picloram. Plant Growth
Regul 9:341–345
Hodgkiss RJ (2004) http://www.succulent-plant.com/ceropg.html
Karuppusamy S, Kiranmani C, Aruna V, Pullaiah T (2009) In vitro
conservation of Ceropegia intermedia—an endemic plant of
south India. Afr J Biotechnol 8:236–239
Kirtikar KR, Basu BD (1935) Indian medicinal plants 3, (M/ s Bishen
Singh Mahendrapal Singh, New Delhi), India, 1638
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue culture. Physiol Plant 15:473–497
Nair AK, Naik DD, Pandit SS (2007) High frequency in vitro flowering
in six species of Ceropegia. J Plant Biol 50(3):374–377
Nayar MP, Sastry ARK (1987) Red data book of Indian plants, vol 1
& 2. Botanical Survey of India, Kolkata
Patil VM (1998) Micropropagation studies in Ceropegia spp. In Vitro
Cell Dev Biol Plant 34:240–243
Patnaik J, Debata BK (1996) Micropropagation of Hemidesmus
indicus (L.) R. Br. through axillary bud culture. Plant Cell Rep
15:427–430
Reynolds S (2006) http://www.sagereynolds.com/cero/clist.com
Sheeja TE, Mandal AB (2003) In vitro flowering and fruiting in
tomato (Lycopersicon esculentum Mill.). Asia Pac J Mol Biol
Biotechnol 11(1):37–42
Sukumar E, Gopal RH, Rao RB, Viswanathan S, Thirugnanasambantham
P, Vijayasekaran V (1995) Pharmacological actions of cerpegin, a
novel pyridine alkaloid from Ceropegia juncea. Fitoterapia 66
(5):403–406
Valverde R, Arias O, Thorpe TA (1987) Picloram-induced somatic
embryogenesis in pejibaye palm (Bactris gasipaes H.B.K.). Plant
Cell Tiss Org Cult 10:149–156
Zhang T (2007) In vitro flowering of Perilla frutescens. In Vitro Cell
Dev Biol Plant 43:91v94
282 J. Plant Biochem. Biotechnol. (July–Dec 2011) 20(2):276–282
Author's personal copy