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Research Article
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Induction of Morphogenetic Callus and Multiple Shoot Regeneration in Ceropegia pusilla Wight and Arn. |
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R. Kondamudi,
V. Vijayalakshmi
and
K. Sri Rama Murthy
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ABSTRACT
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This study was undertaken to evaluate the most suitable concentration of plant growth regulators and perfect explant (node, internode and thin cell layer explants-TCLs) for callus induction and subsequent organogenesis in an endangered medicinal Ceropegia pusilla. The best callus induction was found on the MS medium supplemented with 6-benzylaminopurine (BAP) 13.32 μM L-1 + 2, 4-dichlorophenoxy acetic acid (2,4-D) 0.45 μM L-1 from TCLs. After the initiation of the callus, it was immediately transferred to MS medium supplemented with BAP along with other auxins like 2, 4-D, Indol-3-Acetic Acid (IAA), Indole-3-Butyric Acid (IBA), Naphthalene Acetic Acid (NAA). The regenerative calli were raised on the MS medium supplemented with 1.13 μM L-1 of 2,4-D. Whereas, the organogenic calli was raised on the medium containing 22.7, 40.86, 45.4 μM L-1 of Thidiazuron (TDZ) induced 37.54±0.29, 37.12±0.18 and 34.32±0.17 shoots, respectively. On the media containing BAP 13.32 + IBA 0.49 to 1.23 μM L-1 micro shoots rooted best and 75% of the shoots were survived. The plantlets were established, acclimatized and thrived in green house conditions with 80%. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of bio active constituents of this medicinal plant.
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INTRODUCTION
Ceropegia L. is an old world tropical genus containing about 200 species
of which 48 Ceropegia species found in India (Bruyns,
2003). Twenty eight species of Ceropegia are endemic to the Peninsular
India (Ansari, 1984; Ahmedulla and
Nayar, 1986). The existence of the Ceropegia species has become restricted
to remote pockets in the Himalayas and the Western Ghats, two biodiversity hot
spots. Regrettably, the Ceropegia genus has now been added to the list
of Indian endangered plants (Botanical Survey of India, 2002).
Ceropegia pusilla is an annual herb grown wildly in South India and is
in endangered category (Nayar and Sastry, 1987; Madhav,
2004; Walter and Gillett, 1998). The root tubers contain
an alkaloid called Ceropegin (Nadkarni, 1976), consumed
after cooking (Mabberley, 1997). The root tubers also
contain starch, sugars, gum, albuminoids, fats, crude fiber and valuable constituents
in many traditional Indian Ayurvedic drug preparations that are active against
many diseases especially diarrhea and dysentery. The Ceropegin was an analgesic
drug, tranquilizer and known to use against ulcers, inflammation etc., (Adibatti
et al., 1991). Optimization for this plants flowering (in
vivo/in vitro) is an important issue because of the commercial value
of this beautiful flower in the market. It is important to prevent the extinction
of C. pusilla for its taxonomic and ethanobotanical importance as well
as for the fact that it can be used as root stock for propagation of C. pusilla,
however the in situ conservation effort has had the limited impact on
halting the decline in the population. It is therefore, necessary to establish
ex-situ conservational methods like micropropagation as supplementary
measures. The present in vitro propagation study was taken to develop
a method for multiplication of this endangered progenitor species.
MATERIALS AND METHODS
Ceropegia pusilla Wight and Arn. (Asclepiadaceae) was collected from
the Shevaroy hill ranges, Tamil nadu and the voucher specimen was deposited
in the herbarium of Department of Biotechnology, Montessori Mahila Kalasala,
Vijayawada Andhra Pradesh, India. Plant material was collected during November
and December 2007, due to scarcity of the plant material, the work was started
only with single plant. The nodes were excised from the one month-old plant
to initiate in vitro cultures. The nodes containing axillary buds were
washed in the running tap water, followed by a fungicide and bactericide each
0.3% for 10 min and with 5% tween 20 (v/v) for 4 min. Then, with surface disinfectant
0.1% HgCl2 (w/v) for 2 min, after repeated washes in double distilled
water, the sterilized segments were then washed thoroughly with sterilized distilled
water, cut into appropriate sizes and cultured on nutrient medium.
Before placing onto MS medium (Murashige and Skoog, 1962)
solidified with agar 0.9% (w/v) HiMedia Laboratories Pvt. Ltd. Mumbai and different
growth regulators (BAP, Kn, TDZ, 2,4-D, IAA, IBA and NAA) at different concentrations
either alone or in combinations were added to the medium. In the present study,
all the media were autoclaved at 121°C and 15 lbs pressure for 20 min after
adjustment of the pH to 5.7±2 with 1 N NaOH and 1 N HCl. To study the
callogenesis, about 6-7 TCLs (3 from each side) were taken from either side
of the in vitro grown plant nodes. All the in vitro cultures were
maintained at 24±2°C and illuminated for 16 h with fluorescent light
(18-24 μmol/m/sec) followed by 8 h dark period and the relative humidity
was about 60-80% within the 250 mL bottles and 25x150 mm culture tubes covered
with the aluminum foil. When the hormones failed to induce a specific response
(callus, somatic embryos and direct organogenesis) at the end of the first cycle,
it was marked as inappropriate combination. Twenty cultures were raised for
each treatment and all experiments were repeated thrice. Cytokinins were tested
individually to estimate the callogeny, caullogeny and organogenesis of the
nodal/TCL explants.
In vitro regenerated shoots were inoculated for rooting on full strength medium supplemented with IAA, IBA and NAA in combination with BAP and sucrose. The rooted shoots were washed with distilled water to remove the traces of the medium. The in vitro rooted plantlets were transferred to vessels containing autoclaved vermiculite and sand in 1:1 ratio. Those vessels were covered initially with polythene bags to maintain humidity and placed in a mist chamber. After every alternative day, quarter strength MS medium salt solution was supplied to the plantlets. After two weeks of growth, the complete plants were established, acclimatized and thrived in green house conditions. Statistical analysis Experiments were set in the Completely Randomized Block Design (CRD). The 25x1.5 cm test tubes formed a replicon, 20 such replicants were provided for every culture. Similarly 20 replicates were provided during the trials on shoot organogenesis from callus derived axillary shoot and root induction was from microshoots. However, media in these cases were dispensed in Erlenmayors flasks of 250 mL capacity, 30 mL of media was poured in each flask. The parameters studied were percentage of explant that under went callusing average number of shoots regenerated from each callus, average number of shoots developed for axillary bud, percentage of shoots from which roots developed, average number of shoots that developed per shoot and mean root length, data were subjected to Analysis of Variance (ANOVA) and comparison among mean of treatments were made by Tukeys HSD test with p≤0.05 was considered to be statistical significant, using statistical software Graphpad instat. RESULTS AND DISCUSSION
Effect of growth regulators on the callus formation: The combination
of BAP and NAA induced an excellent amount of callus from the nodes of Ceropegia
pusilla and the morphology of the callus was friable, cream to yellow colored
and nodular in its nature (Table 1). It was observed that
the 2, 4-D at any concentration will stimulate the TCLs callus proliferation
together with BAP. The medium supplemented with 2, 4-D in the range of 0.45
to 4.52 μM L-1 together with BAP 13.32 μM L-1
had the ability to produce the excellent embryogenic callus (Fig.
1A). While the explants cultured on the medium supplemented with 2, 4-D
1.13 μM L-1 + BAP 13.32 μM L-1 had optimum effect
on organogenesis. Since, 2, 4-D is said to show unorganized growth regulation
and is supportive for the organogenesis together with the cytokinins as in the
findings of Jager and van Staden (1996). In this study,
all the media were supplemented with cytokinin (constant), the necessity of
the cytokinin for shoot initiation was well established as in the findings of
Beck and Componetti (1983).
Effect of auxins and BAP on efficiency of callus formation: Induction
of callus from the Thin Cell Layer (TCL) explants of Ceropegia pusilla
were initiated on the medium supplemented with various concentrations of auxins
and cytokinins.
Table 1: | Callus
induction from stem explants of Ceropegia pusilla cultured on MS
medium supplemented with various hormonal concentrations |
 |
YC:
Yellowish cream; N: Nodular; F: Friable |
Table 2: | Induction
of callus from the TCLs on the medium containing different concentrations
of auxins in combination with BAP |
 |
Values
with same letters was not significantly different. E: Embryogenic; NE:
Non embryogenic; YC: Yellowish cream; N: Nodular; C: Compact; F: Friable;
G: Greenish; W: Whitish; NP: Non proliferating; SC: Slight callus; SRC:
Slight rhizoidal callus; MRC: Moderate rhizoidal callus |
Callus was observed from TCL explants on MS medium containing combinations
of BAP, 2, 4-D, IAA, IBA and NAA. The callusing response of most of these PGRs
has been studied while, multiplication of Gymnema sylvestre (Reddy
et al., 1998); Hemidesmus indicus (Siddique
et al., 2003). The callus was induced at the periphery of the explants
by culturing the TCLs for 2-3 weeks on almost all the media tested (Fig.
1A). It was observed that the tTCLs are immediately ready to produce an
extensive callus, the variation in the callusing of different tissue layers
was observed clearly. However, the degree of the callus formation varied with
the treatments.
The young stem derived callus is highly viable, whereas the callus derived
from the leaf bits was soft and could not be maintained beyond a second or third
sub cultures similar observations also found in Tylophora indica (Rao
and Narayanaswamy, 1972); Ceropegia jainii, C. bulbosa var.
bulbosa and C. bulbosa var. lushii (Patil,
1998). On the contrary, in Ceropegia candelabrum (Beena
and Martin, 2003); Decalepis hamiltonii (Giridhar
et al., 2004) produced callus from the leaf and internodal explants.
Later, same callus has produced somatic embryos too. Whereas, Neetha
et al. (2005) obtained his embryogenic callus from roots and leaves
of Hemidesmus indicus. The optimum callusing was observed on the MS
medium fortified with BAP 13.32 μM L-1 + 2,4-D 0.45 μM
L-1, as in case of Ceropegia sahyadrica (Nikam
and Savant, 2007). The 2, 4-D is the principle auxin to induce the callus,
it was best observed in case of other taxa too, Ceropegia species viz.,
C. jainii, C. bulbosa var bulbosa and C. bulbosa
var lushii (Patil, 1998); Gymnema sylvestris
(Gopi and Vatsala, 2006; Roy et
al., 2008). Whereas, the other auxin also used to induce the callus
in Tylophora indicia (Faisal, 2003); 2, 4-D +
2iP were used to induce the callus in Pergularia daemia (Kiranmai
et al., 2008).
The callus produced on the medium containing BAP 4.44 μM L-1
+ 2, 4-D 0.45 μM L-1 + NAA 2.26 μM L-1. The
2, 4-D 0.45 μM L-1 had the ability to produce hairy roots (99%).
The texture of the callus depends on the concentration and type of growth regulators.
The callus formed on the media augmented with IAA and NAA quite often showed
fine hairy mass on the surface of calli while the calli induced on the 2,4-D
were soft, pale yellow and slight morphogenetic, similar findings were noticed
in Ceropegia sahyadrica (Nikam and Savant, 2007).
The combination of BAP with NAA and 2, 4-D had the organogenic ability for
certain extent, The TCLs are cultured on the medium supplemented with BAP and
NAA and produced an excellent callus, the callus is very competent and friable
in its nature. The regeneration of shoot primordia on the callus were observed
clearly (Table 3) on the medium containing BAP13.32 μM
L-1 along with 2,4-D 1.13 μM L-1, IBA 0.49 μM
L-1, NAA 5.37 μM L-1. Dissimilar observations were
noticed in case of Gymnema sylvestris internodes while inducing the callus
(Roy et al., 2008). It was observed that the
media supplemented with IBA and IAA had less callusing ability when compared
to 2, 4-D and is non embryogenic too. But, the ability of these media to produce
the considerable number of shoots (4) was recorded (Table 2).
Whereas, on the medium containing NAA along with BAP was found to produce good amount of callus with rhizoids like structures. The concentration of NAA and the rooting ability are directly proportional to one another up to 0.53 to 5.37 μM L-1 along with BAP 13.32 μM L-1 (constant). The embryogenic nature of the callus clumps was quite well, considerable organogenesis was observed on the media supplemented with 2, 4-D 1.13 μM L-1 along with BAP13.32 μM L-1 (Fig. 1B). Table 3: | Effect
of cytokinins on the regeneration of Ceropegia pusilla |
 |
Data
indicate Mean±SD of the mean following by the same letter was not
significantly different by the Tukey-Kramer multiple comparisons test
at 0.05% probability. Twenty replicates were used per treatment experiments
were repeated thrice |
Effect of TDZ on induction of organogenesis from the nodes: The aim
of the present study was to study the activity of TDZ in in vitro regeneration
of Ceropegia pusilla. Even, TDZ is very useful to induce the callus.
TDZ at 22.7, 40.86 and 45.4 μM L-1 had the maximum ability to
induce organogenesis in the nodes, almost all the concentrations of TDZ resulted
in the organogenesis, some of the concentration had the tendency to support
the growth of the tubers, the protocol we generated is a reproducible protocol
for the commercial companies.
All the media initially have a propensity to produce callus, later on it was noticed that only the 2, 4-D had ability to produce friable and light green colored callus. TCLs are quite active in callogenesis on the MS medium supplemented with BAP in combination with different auxins to induce callus, organogenesis and somatic embryos.
Effect of BAP, KN and TDZ on shoot regeneration: Among these three hormones,
studied, first two are not much effective in induction of the shoots or for
the organogenesis. The tested BAP concentrations 2.22, 2.66 and 3.10 μM
L-1 supports to induce multiple shoots, i.e., 5.40±0.19, 5.24±0.12
and 4.60±0.05 shoots, respectively, more or less similar number of shoots
were reported in Ceropegia hirsute (Nikam et al.,
2008).
KIN had very low percentage of response, even high levels also not able to
induce multiple shoots 3.19, 2.19 μM L-1 KIN had some response
to induce 2.28±0.15, 2.19±0.11 shoots, respectively. Among the
cytokinins tested, KIN was less effective than BAP or TDZ for multiple shoot
induction. The present results are in agreement with previous reports on
C. bulbosa and C. jainii revealed that the BAP alone can induce
axillary shoot multiplication from nodal segments (Patil,
1998). On the other hand, a synergistic effect of a range of growth regulators
in combination with BAP for shoot regeneration was well documented for members
of Asclepiadaceae viz., C. candelabrum (Beena et
al., 2003), Holostemma ada kodien (Martin,
2002) and Hemidesmus indica (Sreekumar et al.,
2000). Holostemma annulare (Sudha et al.,
1998; Martin, 2002) and Leptadenia reticulate
(Arya et al., 2003). In other cases, the superior
activity of BAP compared to other cytokinins was reported in many members of
this family, i.e., Gymnema sylvestre (Komalavalli
and Rao, 2000) and Anisomelus indica (John et
al., 2001). The response on the media containing BAP/KIN was considered
as very low response when compared to the TDZ concentrations. The stimulatory
effect of TDZ on bud breaks and shoot regeneration has been reported earlier
by Singha and Bhatia (1988). In this investigation,
all the shoots regenerated on TDZ supplemented media are very small and can
not be counted as it is because of their compact origin format. Although, high
concentrations of TDZ induced more number of shoot buds, but failed to elongate.
The formation of stunted shoots on TDZ supplemented medium has been reported
earlier by Preece and Imel (1991), which could be a
result of the phenyl group in TDZ.
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Fig. 1: |
Morphogenesis of Ceropegia pusilla. (A) Induction of callus from the nodes
of Ceropegia pusilla on MS media
containing 2, 4-D 0.45 µM LG1 along with BAP 13.32 µM LG1. (B)
Organogenesis from the callus of C. pusilla
on the media containing 2, 4-D 1.13 µM LG1 along with BAP13.32 µM
LG1. (C) Morphogenesis of the callus to
shoots on the media supplemented with TDZ 22.7 µM LG1. (D) Induction
of multiple shoots from the nodes of
C. pusilla on the media supplemented with TDZ 22.7 µM L 1. (E) Induction
of roots from the shoots on MS
medium containing BAP 13.32 + IBA 1.23 µM LG1. (F) Healthy transplanted
plantlet in soil after 15 days |
Among the three cytokinins tested, it was TDZ which shown an excellent outstanding
response showing its superiority over the other two cytokinins i.e., BAP and
KIN. The TDZ combination is well suitable for the shoot regeneration and organogenesis.
The optimum number 37.54±0.29 shoots were induced on the medium containing
22.7 μM L-1 of TDZ (Fig. 1C, D).
Where as the medium containing 40.86 μM L-1 TDZ had the second
highest number of shoots 37.12±0.18 and at 45.4 μM L-1
TDZ induced considerable number (34.32±0.17) of shoots. Superior activity
of TDZ on the BAP was observed in the findings of Hussain
et al. (2008). The explants cultured on either TDZ or BA differentiated
multiple shoots, even though the highest number of shoots per explant was recorded
on TDZ (Hussain et al., 2007, 2008;
Huetteeman and Preece, 1993; Murthy
et al., 1998; Thomas, 2007; Preece
and Imel, 1991; Pradhan et al., 1998). Fiola
et al. (1990) and Malik and Saxena (1992)
had an opinion that the TDZ has been shown to promote shoot regeneration with
efficiency comparable to or greater than that of cytokinins. On the contrary,
among the cytokinins tested, BAP was found more effective than others in including
shoot development and multiple shoot induction in Andrographis paniculata
(Purkayastha et al., 2008). The ability of TDZ
to induce high shoot regeneration efficiency in plant tissue has been reported
for a number of species (Thomas and Philip, 2005; Landi
and Mezzetti, 2006).
The combinations of media which was responsible for the organogenesis possessed
stunted shoots were transferred to the medium containing 9.12 μM L-1
KIN and 8.88 μM L-1 BAP. All the plants showed considerable
variation in the length in accordance with Samuel et
al. (2009). Many combinations of auxins along with BAP 13.32 μM
L-1 and will not induce the rooting. But on the medium containing
BAP 13.32 μM L-1 + IBA 0.49 μM L-1 to 1.23 μM
L-1 micro shoots rooted best (Fig. 1E).
Micro shoots with well-developed root system were directly transferred to small
pots containing sterile vermiculite and coco peat in (1:1) ratio rejuvenated
growth within 20 days. Survival rate of the plantlets is 80% and plantlets successfully
established in the field exhibited morphology similar to that of mother plants
(Fig. 1F). About two weeks, the pots were placed in a mist
chamber, where gradual decrease in the humidity was taken place.
CONCLUSION
In conclusion, the present study reported successful micropropagation protocol
that can be employed in the propagation of endemic taxa Ceropegia pusilla
and helps in conservation and domestication. There by minimizing the pressure
on wild populations of the valuable flora of the forest. The TCLs here employed
to reduce the negative effect of the latex on explant - medium contact. Creating
genetically modified variety will also be possible from this callus (Gamborg
and Phillips, 1995). This in vitro study will help future workers
on developing related manipulations
ACKNOWLEDGMENT The authors extend their gratitude to the Council of Scientific and Industrial Research, New Delhi of their financial assistance.
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