Int. J. Agric. Environ & Biotech. Vol-2 (No.

1) 83 to 86 : (March 2009) BIOTECHNOLOGY

Factors influencing callus induction in the

medicinal plant, Nothapodytes
nimmoniana (Grah.) Mabb.
Indramohan Singh, N. Kumaravadivel, R. Gnanam, L. Arul and K. Rajamani

Department of Plant Molecular Biology & Biotechnology, Centre for Plant Molecular Biology, TNAU, Coimbatore, India
Department of Plant Breeding & Genetics, College of Agriculture & Research Institute, TNAU, Madurai, India
Department of Spices & Plantation Crops, Faculty of Horticulture, TNAU, Coimbatore, India.

E-mail : inder191@gmail.com

Received : 1st January 2009 in final form : 21st February 2009

Published : March, 2009.

Abstract
Studies were carried out on callus induction in Nothapodytes nimmoniana. The callus induction protocol was standardized using leaves,
cotyledons and whole seeds as explants. The maximum callus induction of up to 90% was noticed when tender leaf explants were cultured in MS
medium supplemented with Picloram (2.0 mg L-1), BAP (0.5 mg L-1) and GA3 (0.5 mg L-1). Callus fresh weight was maximum, when leaf explants
were cultured in MS medium supplemented with Picloram at 2.0 mg L-1. The cotyledon explants recorded a maximum of 81% callus induction in
MS medium supplemented with picloram at a concentration of 2.0 mg L-1 in combination with BAP at 0.2 mg L-1.

Keywords : As the demand for the plant derived pharmaceutical compounds is

Callus, Nothapodytes, Picloram, BAP
Introduction
Nothapodytes nimmoniana (Grah.) Mabb. (Synonyms,
Nothapodytes foetida and Mappia foetida) belonging to family
Icacinaceae, is an important anti-cancer medicinal plant. It is an
endangered medicinal tree from Western Ghats of India. Because of
destructive harvesting and habitat loss, the population of this species
has declined by 50-80%. Recently the tree has been assigned a
vulnerable status. (Padmanabha, 2006). It is an excellent source of
quinoline alkaloids, Camptothecin (CPT) and 9-
methoxycamptothecin (9-OMeCPT) which are used clinically as
such or after derivation as anticancer agents for the treatment
of solid tumors (Govindachari and Viswanathan, 1972).
Camptothecin was first reported in the Chinese tree Camptotheca
acuminata (Wall and Wani, 1996) and later discovered in
Nothapodytes nimmoniana. The stem, wood, bark, seeds, leaves and
various organs of the tree are rich sources of alkaloids. In C.
acuminata, the camptothecin content was 0.005% of dried plant as
against 0.140.24% in N. nimmoniana (Puri, 1999). ). There have been
very few reports of in vitro propagation from Nothapodytes
nimmoniana which also showed anti bacterial and anti viral
properties besides more pronounced anti cancer activities.
increasing, possibilities for mass production need to be explored. In
vitro grown plant cells and tissues, in addition to the in vivo parts of
the plant have been used extensively for the production of secondary
metabolites. In vitro propagation of plants holds tremendous
potential for the production of high-quality plant-based medicines. In
vitro culture techniques such as micro propagation and cell culture
are important to select and multiply and conserve the endangered
medicinal plants like Nothapodytes nimmoniana. The cell culture
techniques enable the production of secondary metabolites from
callus. Callus/ cell cultures in suspensions produced through
bioreactors hold a great promise for large scale production of
secondary metabolites. Hence, the present study was carried out with
the aim of developing a reliable and repeatable in vitro callus/cell
culture system so that the extraction of secondary metabolites from
the callous/cells and the economic feasibility can be assessed
subsequently.
Materials and Methods
Collection of Plant materials
The plant materials were collected from Ooty and Yercaud of Tamil
Nadu during the months of October to March as seedlings, leaves
(both mature and immature) and seeds. The seedlings were allowed
to grow in the green house. The fresh leaves from the seedlings were

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Int. J. Agric. Environ & Biotech.
also used for in vitro culture studies.
Culture media preparation
MS basal nutrient medium (Murashige and Skoog, 1962) was used
for callus induction studies. The macronutrients, minor and micro
nutrients as well as vitamins were taken from stock solutions
according to the concentration of the basal medium and mixed to
known quantity with distilled water. Myo-inositol (100 mg L-1) and
sucrose (30 g L-1) were separately added in the solution and mixed
well. The growth regulators at required concentrations were added
and homogenized. The volume was made up to one litre after the pH
of the medium was adjusted to 5.6-5.8 using 0.1 N HCl or 0.1 N
NaOH. The solidifying agent Agar (8 g L-1) was dissolved at the time
of boiling with constant stirring. The medium was then dispensed to
sterilized culture tubes (20 ml per tube) and bottles (50 ml per bottle)
of suitable size and capacity. The culture tubes and the bottles were
plugged with non-absorbent cotton and plastic autoclaveable caps
respectively. The media in tubes and bottles was sterilized in an
autoclave at a temperature of 1210C and at a pressure of 15 pounds per
square inch for 20 minutes (Dodds and Roberts, 1982). The media
was also dispensed in sterilized petri-dishes in a laminar flow
cabinet, as per the requirement.
Explants sterilization
The leaf explants were first washed in running tap water followed by
treatment with Twenty 20 emulsifier solution (2-3 drops in 100 ml
distilled water) for five minutes. After the distilled water wash for 2-3
times, the explants were taken to the laminar air flow chamber for
further sterilization. Then, the leaf explants were surface sterilized
with 2.5 per cent NaOCl (NaClO4) for three to five minutes followed
by washing three to four times with sterile distilled water. The seeds
were first washed in running tap water after removing the hard seed
coat followed by pre-soaking in distilled water for 24 hours. Then the
seeds were treated with 1 ppm bavistin solution for 15-20 minutes.
Then the seeds were treated with 70 per cent ethanol for 45-60
seconds followed by treatment with 0.1 per cent HgCl2 solution for 3-
5 minutes and washed four to five times with sterile distilled water in
laminar air flow.
Inoculation of explants
The leaves were divided into smaller bits (approx. 1-2 cm2 in size)
and inoculated in petri dishes containing the media for callus
induction. Seeds were dissected into two halves and the embryos
were removed. The dissected cotyledons were inoculated onto the
media. The leaf cultures and cotyledon cultures were incubated in
dark at a temperature of 25+2º C at a relative humidity of 60-70%.
The best treatment was decided based on the callus induction
(expressed in percent after 21 days) and callus fresh weight
(expressed in 'g' per tube after 6-7 weeks). Callus induction was
calculated using the following formula:
Number of explants showing callus induction
Callus induction (%) = x 100
Total number of explants inoculated
After 6-7 weeks of inoculation, all the test tubes and petri dishes from
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each treatment, containing calli and the medium were weighed. After
removal of the callus for sub-culturing, the weight of the test tubes
and petri dishes with media alone was again taken. The mean of the
difference in weights gave the calli fresh weight for each treatment.
The best treatment was assessed based on the fresh weight of callus.
Results and Discussion
In callus induction experiments, surface sterilization of leaves with
2.5% NaOCl for 6 min recorded minimum contamination with
maximum survival of explants (Table. 1). Exposure to 0.1 per cent
HgCl2 resulted in maximum survival and minimum contamination
for seed and cotyledon explants. Tender leaves showed
comparatively higher percentage of callus induction (26.79%) than
mature leaves (23.96%) (Fig. 1). Callus induction was the best (90%)
with tender leaves as explants when cultured in MS medium
supplemented with Picloram (2.5 mg L-1) and BAP (0.5 mg L-1) and
GA3 (0.5 mg L-1) (Table2). The highest amount of callus (81 %) was
obtained with Picloram (2.0 mg L-1) + BAP (0.2 mg L-1) combination
with dissected cotyledons as explants (Table 3). Callus growth and
fresh weight of callus were at their best (>150 mg) in MS medium
supplemented with picloram at 2.0 mg L-1. When compared to intact
seeds, the dissected cotyledons responded well to different media
combinations for callus induction.
In the present study, callus induction was the best when tender leaves
were cultured in medium supplemented with Picloram, BAP and
GA3. There are not many reports of callus induction from leaves of N.
nimmoniana, but in the present study successful use of N.
nimmoniana leaves for callus induction was achieved. For callus
induction, successful use of picloram alone and with cytokinin and
GA3 combinations was also reported earlier (Ciddi and Shuler, 2000;
Sundravelan, 2004). Sundravelan , (2004) used comparatively high
concentrations of BAP and GA3 in N. foetida, but in the present study
BAP and GA3 at 0.5 mg L-1 each were found to be optimum to obtain
90% callus induction from tender leaves. Similarly the concentration
of Picloram at 2.5 mg L-1 in the above formulation was found to be
optimum for callus induction. Picloram alone at a level of 2.0 mgL-1
recorded 87.5% callus induction from tender leaves ( Fig. 2). Tender
leaves recorded better callus induction than mature leaves. This may
be due to the developmental stage of explant. It was reported that
explants taken from newly originated organs were more capable of
growing in vitro and young meristematic tissues were most suitable
for establishing callus cultures (George, 1993). In the present study,
highest amount of callus (81%) was obtained from dissected
cotyledons with picloram (2.0 mg L-1) and BAP (0.2 mg L-1)
combination. Thengane, (2003) reported highest callus induction
(72.97%) from the same explants with 2, 4-D (0.5 mg L-1) and BAP
(0.2 mg L-1) combination. Except the present study, perhaps no earlier
report of using picloram for callus induction from cotyledon and seed
explants exists. The calli generated in the present study was shiny
white and transparent . Further studies are required to refine the
protocol and to develop creamy white and friable calli and
regenerate plants through organogenesis.
Hence, the present study shows that developmental stage of leaf
explants as well as type of explants (cotyledon and intact seeds) affect
the extent of callus induction in Nothapodytes nimmoniana. Further
studies are needed to optimize the in vitro culture as well as correct
stage for extracting the medicinal compounds in order to get the
maximum quantity without disrupting the natural occurrence of the
plant.
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Factors influencing nimmoniana (Grah.) Mabb.

Table 1: Effect of different sterilants in reducing Table.2: Effect of growth regulators on callus
contamination and maximizing survival induction from leaf explants

S. Explants Name of the Concentration Mean Mean S. Plant growth Concentration Amount
contamination survival No. regulator (mg L–1) of callus
No. sterilants percentage percentage
1. Tender 1. MS basal
Ethanol 70 25 80
leaves 2. 1.0 + 0.2 -
2, 4-d + BAP
Ethanol + HgCl2 70 + 0.1 10 60
1.5 + 0.2 -
Ethanol + NaOCl 70 + 2.5 10 90
Mature 2.0 + 0.2 +
Ethanol 70 20 80
2.
leaves
Ethanol + HgCl2 70 + 0.1 10 60 2.5 + 0.2 +

Ethanol + NaOCl 70 + 2.5 10 80 3.0 + 0.2 -

Seeds and Ethanol 70 35 95 3. Picloram + BAP 1.0 + 0.2 -
cotyledons +
3. Ethanol + HgCl2 70 + 0.1 5 80 1.5 + 0.2
Ethanol + NaOCl 70 + 2.5 15 80 2.0 + 0.2 ++

2.5 + 0.2 ++
Fig.1: Effect of developmental stages of leaves
on callus induction 3.0 + 0.2 +

4. Picloram + 2.0 + 0.5 + 0.5 ++

BAP + GA3
2.5 + 0.5 + 0.5 +++

3.0 + 0.5 + 0.5 ++

*60 days after inoculation
Amount of Callus = Callus mean fresh weight
(FW) is symbolized as follows:
+: Callus FW ~ 50-100 mg.
++: Callus FW ~ 100-150 mg.
+++: Callus FW > 150 mg.
: Absence or negligible amount of callus formation.

Table.3: Effect of Growth regulators on callus induction

from seed and cotyledon explants.
Plant Concentration Amount of callus
S. growth
No. regulators (mg L )
-1
Cotyled on Seed
Fig.2: Effect of different auxins on callus explants explants
induction from leaf explants T0 MS Basal - - -
T1 2, 4-D 1.5 - -
T2 2.0 + -
T3 2.5 + +
T4 3.0 + -
T5 Plcloram 1.5 - -
T6 2.0 ++ -
T7 2.5 ++ +
T8 3.0 + -
T9 1.0 + 0.2 - -
T10 2.0 + 0.2 + +
T 2, 4-D + BAP
11 3.0 + 0.2 + +
T12 1.0 + 0.2 - -
T13 2.0 + 0.2 +++ +
T14 Plcloram + BAP 3.0 + 0.2 ++ +
Amount of Callus = Callus mean fresh weight
(FW) is symbolized as follows:
+: Callus FW ~ 50-100 mg.
++: Callus FW ~ 100-150 mg.
+++: Callus FW > 150 mg
- : Absence or negligible amount of callus formation.

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Factors influencing nimmoniana (Grah.) Mabb.
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