Romanian Biotechnological Letters Vol. 15, No.

2, 2010
Copyright © 2010 University of Bucharest Printed in Romania. All rights reserved
ORIGINAL PAPER
.

Callogenesis Capability and Calli Somaclonal Variation of Costmary

(Tanacetum balsamita L.)
Received for publication, November 13, 2009
Accepted, March 25, 2010

ATEFEH MOHAJJEL SHOJA1, MOHAMMAD BAGHER HASSANPOURAGHDAM2*,

MAHMOUD KHOSROWSHAHLI3, ALI MOVAFEGHI4
1
Department of Biology, Rabe-Rashidi Institute, Tabriz, Iran.
2
Department of Horticultural Sciences, Faculty of Agrictlture, University of Maragheh,
Maragheh 55181-83111, Iran.
3
Department of Animal Biology, Faculty of Natural Sciences, Tabriz University,Tabriz, Iran.
4
Department of Plant Biology, Faculty of Natural Sciences, Tabriz University,Tabriz, Iran.
Corresponding author's E-mail: hassanpouraghdam@gmail.com, Phone: +989144038472

Abstract
Tanacetum balsamita L. (Costmary) has long history of use as an important medicinal herb for
treatment of many diseases. The objective of the present study was to establish an in-vitro tissue
culture protocol for calli production and somaclonal variation study of calli derived from different
plant parts of costmary. Nodal segments, petiole and leaf disc explants were employed for calli
production on 6 different MS media supplemented with different combinations of 2,4-D (1 and 2
mg/L) and BAP (0.2, 0.5 and 1 mg/L) and two magnesium ion concentrations (185 and 370 mg/L).
Callus production was significantly higher on MgMS basal medium containing 1 mg/L 2,4-D + 1
mg/L BAP. To assess somaclonal variation, genomic DNA was extracted from calli of fourth
subculture. Ten RAPD primers were used to analyze genetic variation between callus samples. The
results showed different amplification patterns between callus samples from different subcultures.

Keywords: Tanacetum balsamita L., tissue culture, callus, somaclonal variation, RAPD

Introduction
The genus Tanacetum is one of about 100 genera in the tribe Anthemideae and family
Asteraceae. The basic chromosome number of the species in the tribe Anthemideae is 2n=18
[1]. Tanacetum balsamita L. is a perennial herbal plant that can grow up to 80 cm. Its
flowering tops are widely used as diuretic, flatulent, appetizer, aphrodisiac, vermifugal,
emmenagogue and for migraine [2]. There are limited studies on tissue culture and
micropropagation of Tanacetum species except for one or two cosmopolitan species
producing compounds of economic value [3-5]. Higher plants, as source of medicinal
compounds, continue to play a dominant role in human health [6]. Members of the
Anthemideae have been occupied an important place in the traditional system of medicine all
over the world [3]. A number of studies have been conducted with the aim of inducing calli
production from a number of explant sources. This trend has been experienced in many
species of the Anthemideae [3]. The techniques of plant tissue culture in medicinal plants
include the following sequential stages or developments: selection of desired plant among
wild counterparts with high-metabolite producing capability, in-vitro culture for callogenesis,
which involves the selection and stabilization of explants with high calli production potential;
industrial scaling-up of calli with probably high expected medicinal material, mass calli
cultivation in bioreactors, and extraction and purification of the sought compounds [7].
Previously, the effect of Mg ion with different concentrations has been investigated on
organized chamomile cultures and it was found that Mg enhanced both the biomass formation
and essential oil production of organized cultures [8]. Furthermore, magnesium ion is
indispensable for plant growth and development [8,9]. Somaclonal variation, a common
phenomenon in plant cell and tissue cultures, includes all types of variations that can be
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 ATEFEH MOHAJJEL SHOJA, MOHAMMAD BAGHER HASSANPOURAGHDAM,
MAHMOUD KHOSROWSHAHLI, ALI MOVAFEGHI

present among plants or cells derived from “in vitro” cultures [10].
RAPD (Randomly Amplified Polymorphic DNA) analysis using PCR in association with
short primers of arbitrary sequence has been demonstrated to be sensitive and useful method
in detecting variation among individuals [10].
The present experiment was carried out to establish a protocol for calli production in
T. balsamita L. different explants based on MS medium as well as to assay somaclonal
variation between subcultures of calli derived from petiole explants.

Material and Methods

Plant material: Leaf, petiole and nodal cuttings were afforded from mother
Tanacetum balsamita L. plants at the active growing period. Donor plants were under grown
in the ambient greenhouse conditions (16 h photoperiod, 500 μmol m-2s-1), mean 250C
temperature and 45-50% relative humidity. Plant materials were surface-sterilized by 2.5%
sodium hypochlorite solution followed by three times rinsing with sterilized distilled water.
The leaf, petiole and nodal cuttings were placed on MS basal medium containing 2% sucrose
supplemented with different combinations of BAP; 0.2, 0.5 and 1 mg/L and 2,4-D - 1 and 2
mg/L. Six variants of MS basal medium as MS+ Mg (MS+370 mg/L MgSO4), MS+Mg/2
(MS + 185mg/L MgSO4), MS/2 (1/2 Macro elements), Mg+MS/2 (MS/2+370 mg/L MgSO4),
Mg/2+MS/2 (MS/2+185 mg/L MgSO4) were used for calli induction from the explants. All
media were solidified with 0.8% agar. The pH was adjusted to 5.8 prior to autoclaving at
1210C for 20 min. Explants were incubated in growth chamber under 24±20C and 16/8
photoperiod. After callus induction and its initial growth, calli were repeatedly subcultured at
4-6 week intervals.
Analysis of tissue culture data: For determining the growth parameters after 60
days, fresh and dry weight of calli and calli growth index were calculated. To do so, we
defined 4 levels of callogenesis for calli produced on surface and margins of explants as 4:
more than 75%, 3: between 50-75%, 2: between 25-50%, 1: between 0-25% and 0: lack of
callogenesis. Dry weight of calli was evaluated by drying fresh calli in an air forced oven at
600C for 36 hrs. Calli growth index was determined according to the below equation:
Calli growth index = (final dry weight- initial dry weight)/ initial dry weight: where
initial dry weight is the mean dry weight of calli just before first subculture and final dry
weight means dry weight of calli after final subculture.
Somaclonal variation analysis by RAPD: DNA was isolated from costmary petioles
calli after first, second, third and fourth months of culture, following CTAB
(Hexadecyltrimethyl ammonium bromide) extraction protocol [11].
Genomic DNA of mother plant was extracted according to previous method as well. DNA
was quantified using a biophotometer [Eppendorf spectrophotometer] and purity established
from 260:280 nm ratios. Reactions for RAPD analysis were set up using the following ten
10-mer primers: 580:(5'-GCGATAGTCC-3'), 590: (5'-CCGGCATGTT-3'), 558: (5'-
CGATATCCG-3'), 586: (5'-CCGGTTCCAG-3'), 599: (5'-CAAGAACCGC-3'), 498: (5'-
GACAGTCCTG-3'), 493: (5'-CCGAATCACT-3'), 485: (5'-AGAA TAGGGC-3'), 559: (5'-
GAGAATCACG-3'), and 688: (5'-GCAGGAGCGT-3'). Polymerase chain reactions were
performed in a Biometra PCR system thermocycler. Cycling reactions were as follows: 940C
5 min, 40 cycle of (940C 1 min, 370C 1 min, 720C 2 min) followed by 720C for 5 min. All
cycling conditions included a hot start. A totall of 10 μL of PCR products were analyzed by
agarose gel electrophoresis. 2% agarose gel was run with 1 μL ethidium bromide. DNA was
visualized using a 320 nm UV transilluminator.

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 Callogenesis Capability and Calli Somaclonal Variation of Costmary (Tanacetum balsamita L.)

Statistical Analysis: Experiments were organized according to a factorial experiment

based on CRD with 3 replications. Data were subjected to variance analysis by SPPS 11.5
statistical software. Mean comparisons were carried out by Duncan's multiple rang test at
probability level of 0.01. Data of RAPD were analyzed by GenAlex 6 and STATISTICA
softwares.

Results and Discussion

Interactive effects of culture medium and explant type were statistically significant
(p< 0.01) for calli fresh weight, callogenesis and calli growth index (Table 1).
The highest mean for calli growth index, calli fresh weight and callogenesis of both leaf and
petiol explants belonged to the Mg+MS culture medium, supplemented with 1 mg/L 2,4-D
and 1 mg/L BAP. Leaf and petiole explants were superior to the nodal explants regarding the
callogenesis, growth index, and calli fresh weight.
Table 1. Mean comparison for calli traits of costmary explants in different culture media containing 1mg/L 2,4-
D + 1mg/L BAP .

Culture Calli Calli

Explant Callogenesis
media fresh weight (g) growth index
A 393b 1.3b 1.5a
‘Leaf’ B 400a 1.7a 1.5a
C 333b 1.4b 1.5a
A 350b 1.3b 1.4b
‘Petiole’ B 400a 1.8a 1.5a
C 353b 1.2b 1.5a
Different letters in columns show significant difference based on Duncan's multiple range test at P≤ 0.01
A (MS medium ), B (Mg + MS medium) and C (Mg/2 + MS medium).

This is the first report on the in-vitro culture of costmary. Callus induction was
established successfully on the MS, Mg+MS and Mg/2+MS culture media (Table 1). The
callus induction in Mg+MS medium showed a higher biomass, expressed in growth index and
callogenesis compared with those grown on MS and Mg/2+MS media. These factors were not
shown for the MS/2, Mg+MS/2 and Mg/2+MS/2 media because of their non-significant
effects. Previous studies on chamomile tissue cultures have shown the positive effect of
magnesium ion on the biomass and calli growth [8]. Addition of MgSO4 to the solid and
liquid MS media favorably affected the biomass and essential oil production of chamomile
hairy root cultures [8,9]. In the present research this effect has been proven clearly. Tissue
culture technology enabling the rapid production of a large quantity of uniform plants from a
single plant with good genetic potential is gaining importance in recent times [12]. Synergetic
effects of plant growth regulators play an important role in callus induction and cell
differentiation [13]. The difference in the callogenesis of different explants may be a result of
differences in their intrinsic potential. In general, an increase of auxin levels such as 2,4-D in
the medium stimulates dedifferentiation of the cells [13]. Costmary showed the best callus
induction in the MgMS basal medium with the combination of 1 mg/L of both BAP and 2,4-D.
In the present study, a total of 10 arbitrary 10-mer primers were screened to detect
somaclonal variation using DNA samples of donor mother plant and calli of 4 different
petiole subcultures. A total of 66 bands were generated with these primers. The profiles of
amplified RAPD markers (Figure 2) showed that the donor plant and calli of petiole’s
different subcultures had different banding patterns that classified them in 3 groups (Figure
1).
5122 Romanian Biotechnological Letters, Vol. 15, No. 2, 2010
 ATEFEH MOHAJJEL SHOJA, MOHAMMAD BAGHER HASSANPOURAGHDAM,
MAHMOUD KHOSROWSHAHLI, ALI MOVAFEGHI
Tree Diagram for 5 Variables
Unweighted pair-group average
Dissimilarities from matrix

M1

M2

M3

M4

M5S

6 8 10 12 14 16 18 20 22
Linkage Distance

Figure 1. Phylogenetic tree of RAPD-PCR for in vitro culture derived calli of costmary.
M1, M2, M3, M4 and M5 refer to first, second, third and forth subcultures of petiole and mother plant respectively.

Figure 2. DNA pattern of Tanacetum balsamita L. calli based on PCR-RAPD analysis using 10 different ten-mer primers.
Lanes 1-4: first, second, third and forth subcultures of petiole and Lane 5: mother plant respectively.
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 Callogenesis Capability and Calli Somaclonal Variation of Costmary (Tanacetum balsamita L.)

These results indicate that there was a variance among the mother plant and calli of
first, second, third and fourth subcultures. It was concluded that calli of the different
subcultures were not reliable in producing genetically similar pattern to the mother plant, but
Rout (2006) reported that somaclonal variation in the callus cultures can be a promising
source for creating new cultivars in plants and for isolating cell lines with high capacity of
producing desired secondary compounds [14].
In conclusion, the current experiment was an initial step towards the mass production
of calli from costmary different explants. Leaf and petiol were the preferential explants with
acceptable results. However, selection of desired somaclones from different calli and
monitoring of high valued compounds need further and detailed investigation.

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