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Indian Journal of Plant PhysiologyAn International Journal of PlantPhysiology ISSN 0019-5502Volume 18Number 4 Ind J Plant Physiol. (2014) 18:388-391DOI 10.1007/s40502-014-0062-2
Glass beads in liquid media: an alternativematrix for in vitro root induction ofCephaelis ipecacuanha A. Richard
Ved Prakash Pandey, Shanoli Ghosh,Elizabeth Cherian & Abraham Patani
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SHORT COMMUNICATION
Glass beads in liquid media: an alternative matrix for in vitro rootinduction of Cephaelis ipecacuanha A. Richard
Ved Prakash Pandey • Shanoli Ghosh •
Elizabeth Cherian • Abraham Patani
Received: 10 April 2013 / Accepted: 26 December 2013 / Published online: 6 February 2014
� Indian Society for Plant Physiology 2014
Abstract Cephaelis ipecacuanha shoots were rooted in
different concentrations of auxins on solid Murashige and
Skoog (Physiol Plant 15:473–497, 1962) media and liquid
MS media with glass beads. Glass beads were used as an
alternative source of support matrix in liquid culture media
to replace agar. The highest rooting response was observed
on liquid MS medium with glass beads containing
2 mg l-1 a-naphthalene acetic acid within 15–20 days.
Shoot elongation was also found to be better compared to
solid MS medium. Rooted shoots were successfully hard-
ened and established in the soil. Hence, an easy, reliable
and reproducible protocol was developed for in vitro
rooting of Ipecac on liquid MS medium with glass beads,
which is biologically inert and reusable.
Keywords Cephaelis ipecacuanha � Micropropagation �Auxins � Root induction � Support matrix
Introduction
Cephaelis ipecacuanha commonly known as Ipecac, a
member of the family rubiaceae, which is rich in alkaloids,
mainly emetine and cephaeline, is usually propagated by
stem or root cutting as growth is slow from the seeds
(Yonzone and Chatterjee 1986). An efficient rooting
treatment yields a high percentage of rooted shoots. In
tissue culture-raised plants, a high quality root system is
necessary for acclimatization (Welander 1985; Zimmer-
man 1981).
Agar which is frequently used as a gelling agent is the
most expensive constituent of plant tissue culture media
(Bhattacharya et al. 1994). The use of liquid media in tissue
culture is often described as a means of reducing the cost of
micropropagation (Alvard et al. 1993). In our earlier
studies, the in vitro growth of Rauwolfia serpentina has
been compared in solid and liquid media (Pandey et al.
2007, 2010). Most of in vitro studies on Ipecac have used
solid media for rooting of shoots (Ideda et al. 1988; Jha and
Jha 1989; Yoshimatsu and Shimomura 1991; Yamuna et al.
1993; Chaudhuri and Jha 2008). Rooting has also been
achieved from leaf segment of Ipecac on both solid and
liquid MS media and studied for its alkaloid content (Te-
shima et al. 1988). To our knowledge, there has been no
comparative study on rooting of C. ipecacuanha from
shoots in both liquid and solid media. In our present
experiment, we have replaced agar with glass beads as an
alternative support matrix in liquid medium, and compared
the in vitro rooting response of C. ipecacuanha.
Nodal explants of Ipecac (1–1.5 cm in length) were col-
lected from the plantation of Dr. Patani Scientific and
Industrial Research, Andheri (E), Mumbai, India. The
explants were washed in running tap water for 10 min and
then washed with one drop of Tween 80 and two drops of
Dettol in 150 ml distilled water for 20 min, followed by
surface sterilization with 0.1 % (w/v) mercuric chloride
(HgCl2) for 10 min and by washing four times with sterile
distilled water. The sterile explants were aseptically trans-
ferred to Murashige and Skoog (1962) medium at pH 5.6
adjusted prior to autoclaving at 121 �C and 1.06 kg cm-2 for
20 min, supplemented with growth regulators, 3 % sucrose
and 0.8 % agar. The cultures were incubated at 25 ± 2 �C
under 16 h light/8 h dark photoperiod (40.5 lmol m-2 s-1).
V. P. Pandey (&) � S. Ghosh � E. Cherian � A. Patani
Dr. Patani Scientific and Industrial Research, PSIR Building,
Inga Complex, Mahakali Road, Andheri (E), Mumbai 400093,
India
e-mail: psir79@gmail.com
123
Ind J Plant Physiol. (October–December 2013) 18(4):388–391
DOI 10.1007/s40502-014-0062-2
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The explants were inoculated for shoot multiplication on
media as described by Chaudhuri and Jha (2008). After
four subcultures, the shoots were inoculated on both solid
and liquid media for comparing the rooting response. In
liquid media, glass beads (2 mm diameter) were used as
support matrix by replacing agar. The glass beads were
soaked overnight in 1 % (v/v) Teepol and washed with
distilled water and dried in hot air oven prior to use. In each
test tube (25 9 150 mm) about 20 g glass beads were
added with 10 ml of liquid media. The shoots were inoc-
ulated on MS media containing different auxins, viz.,
indole-3-acetic acid (IAA), 3-indole butyric acid (IBA),
para-amino benzoic acid (PABA) and a-naphthalene acetic
acid (NAA) at concentrations of 1, 2, 3, 5, 7 and
10 mg l-1. The auxin concentrations were kept same for
both solid and liquid media. Effects of supporting materials
and auxins on percentage of root formation, number of
roots per shoot and days required for root induction were
recorded for 80–85 days from the date of inoculation.
The rooted shoots were transplanted into plastic cups
containing a mixture of steam sterilized garden soil, ver-
miculite and sand (1:1:1), and grown under laboratory
conditions (25 ± 2 �C) of regulated humidity and tem-
perature for 2 weeks. The plants were kept under shade for
4 weeks and then placed under full sunlight. All the
experiments were repeated thrice and each treatment con-
sisted of 10 explants. Mean values were assessed by using
t test and statistical significance of differences are shown
by Duncan’s multiple range test with a probability of
P B 0.05.
In the preliminary experiment, nodal explants cultured
on solid MS medium containing 8 mg l-1 kinetin ?
0.5 mg l-1 NAA ? 200 mg l-1 adenine showed 25–30
shoots per explant (Fig. 1a), whereas, Chaudhuri and Jha
(2008) recorded maximum of 12.5 shoots in the same
combination of media. We observed that with the pro-
gression of the number of subcultures, the number of shoot
proliferation increased. Similar response was also observed
in banana by Akbar and Roy (2006). The media devoid of
growth regulators failed to produce response for rooting in
both type of cultures. However, as shown in Table 1, root
induction occurred on solid MS media containing 1 mg l-1
IAA, after 45–50 days and in liquid media, after 30–35 days
of inoculation. Among all the concentration of auxins,
NAA gave optimum results with highest rooting response,
number of roots and shoot length. The number of days
required for root induction in NAA containing media was
also very less in both types of cultures. NAA (2 mg l-1) in
liquid media with glass beads was found to give the best
rooting response (96 %) with highest number of roots (71)
and shoot length (9.5 cm) in 15–20 days as compared to
solid media (Table 1; Fig. 1b, d). Similarly, the other auxin
concentrations of liquid media also resulted in fast rooting
response than the agar gel media. It was noticed that the
penetration ease of roots in liquid medium as compared to
the solid medium leads to faster root induction. Higher
concentrations of NAA in solid medium resulted in callus
growth along with roots (Fig. 1c), but in liquid medium the
same concentrations failed to produce callus (Table 1). The
presence of glass beads in liquid media could be a reason
for the inhibition of callus formation. Agar has been
reported to have a number of drawbacks that negatively
affect culture growth and differentiation. This is because
the low uptake of nutrients in the solid medium may lead to
lower nutrient availability to the plants and hence a
reduction in growth rate (Debergh 1983; George 1993;
Scholten and Pierik 1998). In our experiment, statistical
analysis of rooting also showed that liquid media was more
efficient than solid media with statistical significance. The
plantlets that were sufficiently healthy with new growth
(Fig. 1e) were subsequently transferred to larger pots and
gradually acclimatized to outdoor conditions. Flowering of
the plant was observed within 1 year of hardening to field
condition (Fig. 1f).
Fig. 1 In vitro propagation of C. ipecacuanha. a Multiple shoots on
MS medium containing 8 mg l-1 kinetin, 0.5 mg l-1 NAA and
200 mg l-1 adenine, b rooting response on solid MS medium
containing 2 mg l-1 NAA, c callus and root induction on MS solid
medium containing 10 mg l-1 NAA, d rooting response on liquid MS
medium with glass beads containing 2 mg l-1 NAA, e hardening of
rooted plant, f flowering of 1 year old plant
Ind J Plant Physiol. (October–December 2013) 18(4):388–391 389
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Maintenance of cultures in liquid media is a common
practice for many plant systems and has been found to be
more convenient than agar gel media. The rate of con-
tamination is also reduced as subculturing takes place only
in the form of addition of sterile liquid media. The use of
glass beads as a support matrix to the explants affords
better aeration. The glass beads can be easily removed and
re-used after sterilization. The chances of root damage or
the presence of agar that remains on the roots, leads to
unwanted bacterial and fungal contamination. MacLeod
and Nowak (1990) reported no differences in regeneration
capability and observed a 60 % saving on media compo-
nents by replacing agar with glass beads. Though, it may be
debated, whether media cost really contributes significantly
to the total cost (George 1996), however it may be noted
that the agar powder ideally used in plant tissue culture
media, is an expensive commodity.
Conclusion
A protocol has been developed to replace the agar with
glass beads for in vitro rooting of C. ipecacuanha plantlets.
Glass beads as a support matrix of liquid medium was
found to produce better results in rooting with minimal
time period as compared to solid medium. It is also useful
in producing Ipecac plantlets in lesser time. This method of
replacing agar with glass beads could also be used to
evaluate the effect on shoot multiplication for cost-effec-
tive and large scale production of disease free Ipecacuanha
plants for commercial cultivation.
Acknowledgments We wish to express our thanks to Dr. George
Patani of PSIR, Mumbai, for his guidance and encouragement during
this research and Dr. Pramod Kumar Pandey of CIFE, Mumbai, for
his help in the statistical analysis.
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Table 1 Effect of various
concentrations of different
auxins on MS solid and liquid
medium for adventitious root
formation of in vitro C.
ipecacuanha shoots
Values followed by the same
letter in each column were not
significantly different at
P B 0.05� Callus growth
* Each value represents the
mean ± SE
Growth regulators
(mg l-1)
Media Rooting
response (%)*
No. of roots
per shoot*
Shoot length
(cm)*
Days required
for rooting
IAA (1) Solid 36.67 ± 2.35a 2.50 ± 0.76a 2.52 ± 0.36a 45–50
Liquid 26.67 ± 1.75b 4.27 ± 1.38b 4.58 ± 0.22b 30–35
IAA (5) Solid 90.00 ± 0.00a 11.27 ± 1.18a 4.43 ± 0.24a 60–65
Liquid 93.33 ± 1.75b 21.03 ± 1.96b 7.62 ± 0.19b 35–40
NAA (2) Solid 93.33 ± 0.58a 62.03 ± 4.02a 8.38 ± 0.48a 30–35
Liquid 96.67 ± 0.84b 71.27 ± 4.42b 9.50 ± 0.34b 15–20
NAA (5) Solid 90.00 ± 1.52a �11.13 ± 1.35a 4.71 ± 0.31a 50–55
Liquid 80.00 ± 1.52b 20.17 ± 2.17b 6.10 ± 0.19b 25–30
NAA (10) Solid 80.00 ± 3.03a �13.37 ± 2.34a 3.68 ± 0.36a 65–70
Liquid 90.00 ± 1.52b 14.27 ± 1.20b 5.04 ± 0.16b 30–35
PABA (5) Solid 53.33 ± 1.75a 6.03 ± 1.65a 4.08 ± 0.18a 50–55
Liquid 60.00 ± 1.52b 15.07 ± 2.48b 6.20 ± 0.14b 35–40
PABA (10) Solid 33.00 ± 0.85a 6.20 ± 1.87a 2.98 ± 0.32a 55–60
Liquid 86.67 ± 0.86b 18.10 ± 2.04b 6.45 ± 0.13b 45–50
IBA (5) Solid 56.67 ± 1.28a 4.33 ± 0.98a 3.59 ± 0.22a 70–75
Liquid 83.33 ± 0.88b 21.27 ± 2.22b 5.82 ± 0.13b 25–30
IBA (10) Solid 36.67 ± 1.75a 3.43 ± 1.08a 4.93 ± 0.44a 80–85
Liquid 50.00 ± 2.63b 11.30 ± 2.14b 6.52 ± 0.12b 40–45
390 Ind J Plant Physiol. (October–December 2013) 18(4):388–391
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