Glass beads in liquid media: an alternative

matrix for in vitro root induction of

Cephaelis ipecacuanha A. Richard
Ved Prakash Pandey, Shanoli Ghosh,
Elizabeth Cherian & Abraham Patani

Indian Journal of Plant Physiology

An International Journal of Plant
Physiology
ISSN 0019-5502
Volume 18
Number 4
Ind J Plant Physiol. (2014) 18:388-391
DOI 10.1007/s40502-014-0062-2

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Author's personal copy

Ind J Plant Physiol. (OctoberDecember 2013) 18(4):388391
DOI 10.1007/s40502-014-0062-2

SHORT COMMUNICATION

Glass beads in liquid media: an alternative matrix for in vitro root

induction 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 15:473497, 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 1520 days.
Shoot elongation was also found to be better compared to
solid MS medium. Rooted shoots were successfully hardened 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

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

necessary for acclimatization (Welander 1985; Zimmerman 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 (Teshima 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 (11.5 cm in length) were collected 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 transferred 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).

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Ind J Plant Physiol. (OctoberDecember 2013) 18(4):388391

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 inoculated 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 8085 days from the date of inoculation.
The rooted shoots were transplanted into plastic cups
containing a mixture of steam sterilized garden soil, vermiculite and sand (1:1:1), and grown under laboratory
conditions (25 2 C) of regulated humidity and temperature 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 consisted of 10 explants. Mean values were assessed by using
t test and statistical significance of differences are shown
by Duncans 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 2530
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 progression 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 4550 days and in liquid media, after 3035 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 1520 days as compared to
solid media (Table 1; Fig. 1b, d). Similarly, the other auxin
concentrations of liquid media also resulted in fast rooting

389

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

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).

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390
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

Ind J Plant Physiol. (OctoberDecember 2013) 18(4):388391

Growth regulators
(mg l-1)

Media

Rooting
response (%)*

IAA (1)

Solid

36.67 2.35a
b

IAA (5)
NAA (2)
NAA (5)
NAA (10)

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

Shoot length
(cm)*

Days required
for rooting

2.50 0.76a

2.52 0.36a

4550

4.58 0.22b

3035

Liquid

26.67 1.75

4.27 1.38

Solid

90.00 0.00a

11.27 1.18a

4.43 0.24a

6065

Liquid

93.33 1.75

21.03 1.96

7.62 0.19

3540

Solid

93.33 0.58a

62.03 4.02a

8.38 0.48a

3035

Liquid

96.67 0.84b

71.27 4.42b

9.50 0.34b

1520

Solid

90.00 1.52a

11.13 1.35a

4.71 0.31a

5055

Liquid

80.00 1.52b

20.17 2.17b

6.10 0.19b

2530

Solid

80.00 3.03

3.68 0.36

6570

Liquid

90.00 1.52b

14.27 1.20b

5.04 0.16b

3035

PABA (5)

Solid
Liquid

53.33 1.75a
60.00 1.52b

6.03 1.65a
15.07 2.48b

4.08 0.18a
6.20 0.14b

5055
3540

PABA (10)

Solid

33.00 0.85a

6.20 1.87a

2.98 0.32a

5560

IBA (5)
IBA (10)

13.37 2.34

Liquid

86.67 0.86

6.45 0.13

4550

Solid

56.67 1.28a

4.33 0.98a

3.59 0.22a

7075

Liquid

83.33 0.88b

21.27 2.22b

5.82 0.13b

2530

Solid

36.67 1.75a

3.43 1.08a

4.93 0.44a

8085

Liquid

50.00 2.63b

11.30 2.14b

6.52 0.12b

4045

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 contamination 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 components 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-effective and large scale production of disease free Ipecacuanha
plants for commercial cultivation.

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No. of roots
per shoot*

18.10 2.04

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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