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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

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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

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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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