The Journal of Horticultural Science and Biotechnology

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In vitro organogenesis of Abutilon indicum (L.)

Sweet from leaf derived callus and assessment of
genetic fidelity using ISSR markers

Sen Seth & Jogeswar Panigrahi

To cite this article: Sen Seth & Jogeswar Panigrahi (2018): In�vitro organogenesis of Abutilon
indicum (L.) Sweet from leaf derived callus and assessment of genetic fidelity using ISSR markers,
The Journal of Horticultural Science and Biotechnology, DOI: 10.1080/14620316.2018.1447314

To link to this article: https://doi.org/10.1080/14620316.2018.1447314

Published online: 14 Mar 2018.

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THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY, 2018
https://doi.org/10.1080/14620316.2018.1447314

In vitro organogenesis of Abutilon indicum (L.) Sweet from leaf derived callus
and assessment of genetic fidelity using ISSR markers
Sen Setha and Jogeswar Panigrahib
a
Plant Biotechnology Laboratory, School of Life Sciences, Sambalpur University, Sambalpur, Odisha, India; bDepartment of
Biotechnology, Central University of Rajasthan, Kishangarh, Rajasthan, India

ABSTRACT ARTICLE HISTORY

This study reports on in vitro regeneration of Abutilon indicum plantlets through callus Accepted 27 February 2018
mediated organogenesis. The leaf explants implanted on Murashige and Skoogs (MS) med-
KEYWORDS
ium supplemented with 4.52 µM 2, 4-Dicholorophenoxy acetic acid (2,4-D) and 8.88 µM 6 Malvaceae; Murashige and
Benzyladenine (BA) showed highest response (70.3%) for callus proliferation, but these callus Skoogs (MS) medium;
did not showed any morphogenetic differentiation on the same medium even after 12 weeks. 6-Benzyladenine (BA);
Whereas, subsequent sub-culture of this green proliferated callus on MS medium added with inter simple sequence
2.68µM α-Napthalene acetic acid (NAA), 8.88µM BA and 543 µM Adenine sulphate showed repeat (ISSR); shoot bud;
the highest frequency (62.2%) of multiple shoot-buds production and also elongation of leaf explants
shoots. Well developed shoots were efficiently rooted in vitro on half strength MS medium
supplemented with 7.38 µM Indole-3-butyric acid (IBA). Seventy per cent of in vitro regener-
ated plantlets were successfully established in garden and were morphologically alike to the
donor plants. The genetic homogeneity of these in vitro regenerated plantlets was also
affirmed by inter simple sequence repeat (ISSR) analysis using eight ISSR primers. This
standardised in vitro organogenesis protocol supplements a good platform for the conserva-
tion of A. indicum germplasms and also caters for the needs of the herbal industry.

Introduction phenols have also been isolated and characterised

from this plant (Abdul rahuman, Gopalkrishnan,
Abutilon indicum L. Sweet belongs to the family
Venkatesan, & Geetha, 2008; Kashmir, Yasmin,
Malvaceae is a sub-shrub, predominantly found in
Ahmad, & Mohy-ud-Di, 2009; Sharma & Ahmad,
tropical and subtropical regions (Chopra, Nair, &
1989).
Chopra, 1956). The plant parts are used in conven-
Mostly this plant is propagated through seed, but low
tional system of medicine (Prajapati, Purohit,
percentage of seed germination and poor viability of
Sharma, & Kumar, 2003), such as roots are used to
seedlings restrict its propagation on a large scale. The
cure uterine haemorrhagic discharges and barks are
natural population of this potent medicinal plant has
used as antihelmintic, febrifuge and astringent
been subjected to genetic erosion because of the over-
(Mohite, Shelar, Raje, Babar, & Sapkal, 2012). In the
utilisation by herbal industries in many countries.
Siddha system of medicine, these plant-based extracts
Hence, there is absolute need to develop methods for
have been used as a remedy for jaundice, ulcer,
its propagation in large-scale either in vivo or in vitro as
leprosy and piles (Yoganarasimhan, 2000). A. indi-
reported for several medicinal plants (Rout,
cum is used in many Asian countries as anti-inflam-
Samantaray, & Das, 2000; Briskin, 2000). In vitro orga-
matory and antiemetic (Chopra et al., 1956; Kirtikar
nogenesis is quite useful for rapid mass multiplication
& Basu, 1975). The plant has been reported for anti-
and production of superior genotypes. This technique
oxidant, antibacterial, antifungal, hypoglycaemic,
has many advantages over other conventional propaga-
antimalarial and hepatoprotective activities (Abdul
tion methods, and is cost effective and favourable for
Rahuman, Gopalakrishnan, Venkatesan, & Geetha,
large scale production of desirable medicinal plants.
2008; Kashmiri, Yasmin, Ahmad, & Mohy-ud-Din,
There are few reports on in vitro callusing, organogen-
2009; Mata, Nakkala, & Sadras, 2015; Mehta, Neogi,
esis and somatic embryogenesis were already made in
Kotra, & Mall, 1997; Seetharama, Chalageri, Setty, &
this species (Nataraja & Patil, 1984; Rout, Mishra, Das,
Bheemachar, 2002). The herbal extract of this plant is
& Sahoo, 2009; Seth, Rath, Rout, & Panigrahi, 2017).
a major constituent of Diabecon tablet used for the
The in vitro organogenesis was reported only from
diabetes (Kundu & Chatterjee, 2010). Alkaloids, fla-
nodal explants, and there is no report on the use of
vonoids, steroids, terpenoids, linoleic acid and

CONTACT Jogeswar Panigrahi drjpanigrahi@gmail.com Department of Biotechnology, Central University of Rajasthan, NH-8, Bandarsindri-
305817, Kishangarh, Rajasthan, India
© 2018 The Journal of Horticultural Science & Biotechnology Trust
2 S. SETH AND J. PANIGRAHI
leaf explants on in vitro organogenic differentiation till
date. Even, clonal fidelity of these plantlets generated
from nodal explants via in vitro organogenesis has not
been tested either at molecular level or at chromosome
level. Genetic variability often arise during in vitro
callus mediated organogenesis as a manifestation of
epigenetic influences or the changes in the genome
induced by in vitro culture conditions (Larkin &
Scowcroft, 1981). Hence, the genetic fidelity of in vitro
regenerated plantlets needs to be assessed. Various
DNA markers have been used for the clonal fidelity
assessment of the true to type plantlets regenerated in
vitro (Shasany, Shukla, & Khanuja, 2007) either indivi-
dually or in conjugation with cyto-morphology. Among
various DNA markers, Random amplified polymorphic
DNA (RAPD; Williams, Kubelik, Livak, Rafalski, &
Tingey, 1990), Inter simple sequence repeat (ISSR)
(Zietkiewicz, Rafalski, & Labuda, 1994), Simple
sequence repeat (SSR) (Morgante & Olivieri, 1993)
and Start codon targeted polymorphism (SCoT)
(Collard & Mackill, 2009) have been used predomi-
nantly for the assessment of clonal fidelity of in vitro
raised plantlets. ISSR involves amplification of DNA
segments between 2 identical SSR regions using di- or
tri- or tetra-nucleotide SSR motifs with or without 1 to 3
nucleotide anchors as primers. ISSR markers are super-
ior over other random markers, because it overcomes
many of the technical limitations of random markers
due to its high reproducibility and simplicity (Goulão &
Oliveira, 2001). Further, it reveals the specificity of SSR
markers without any sequence information in priori.
In the present study, a protocol for mass multi-
plication of A. indicum via callus mediated organo-
genesis using leaf as explants was established, and the
genetic homogeneity of in vitro raised plantlets using
ISSR markers were reported.
Materials and methods
Source of explants, media composition and culture
conditions
About 20 saplings of 20-weeks-old were collected
from adjoining villages of Sambalpur University and
were self-pollinated, and successfully grown in the
experimental garden. The homogeneity of these
plants was assessed using morphogenetic parameters
and DNA profiling studies (data not shown). Young
apical leaves were obtained from 6 plants of 30-days-
old and were used as explants. Leaves were washed
thoroughly in running tap water, and then surface
sterilised with 50% (v/v) ethanol for 30 seconds.
Afterwards, these leaves were treated with 0.05% (w/
v) HgCl2 solution (Himedia, India) for 2 min. and
followed by thorough washing in sterile double dis-
tilled water. The leaves were sliced into 10 mm2 and
were aseptically cultured on MS basal medium
(Murashige & Skoog, 1962) with 3% sucrose and
0.8% agar. All media were supplemented with acti-
vated charcoal (200 mgl−1), ascorbic acid (11.54µM)
throughout the study. pH of the medium was
adjusted to 5.8 prior to autoclaving at 1.06 kg cm−2
at 121°C for 15 min. The cultures were incubated at
25 ± 2°C under 16 hour photoperiod with a light
intensity of 48 µmol m−2 s−1 provided by cool,
white fluorescent lamps (Surya Roshini Pvt. Ltd.,
India).
Callus induction
The sterilised leaf explants (~ 50mm2) from a 30-
days-old plant were implanted on MS medium for-
tified with 2, 4-Dichlorophenoxyacetic acid (2, 4-D;
2.26–4.52 µM), α-Naphthalene acetic acid (NAA;
2.68–5.37 µM), 6-Benzyladenine (BA; 4.44–
17.76 µM) and Kinetin (KIN; 4.64–18.58 µM) either
individually or in combinations. Cultures were main-
tained in culture tube (150 X 25 mm) under the
above- mentioned conditions. The cultures were
incubated in darkness for a few days until profuse
callus induction. The induced calluses were gradually
sub cultured onto the same medium at regular inter-
val of 2 weeks and the subculture passage was main-
tained up to fifth generation. The morphological
observations were taken after 4 weeks in terms of
the percentage of explants responded for callus
induction and fresh weight of callus in each passage
of subculture. The effects of subculture on fresh
weight of callus and its morphology was also
observed among the PGR combinations after
4 weeks of each sub culture (Table 3).
Multiple shoot induction from callus
The green compact callus clumps (̴ 200 mg) obtained
from callusing medium of each subculture and further
cultured onto MS medium supplemented with 543 µM
adenine sulphate (Ads) and different concentrations of
BA (4.44–13.32 µM) and/or KIN (4.64–13.94 µM) in
combination with NAA (1.07–5.37 µM) and incubated
up to 10 weeks. Each individual adventitious shoots
were further excised and subcultured in culture tubes
(150x25 mm2) containing same medium for growth
and development. Cultures were incubated under
same conditions as mentioned earlier. The data on
regeneration frequency, average number of shoot
buds produced per callus clump and length of shoots
at the end of fouth week were recorded for each
subculture.
In vitro rooting
The in vitro derived well-developed shoots were
further cultured on half strength MS medium with
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY
different concentration of Indole-3-butyric acid (IBA;
2.46–14.76 µM), NAA (2.68–16.11 µM) and Indole-3-
acetic acid (IAA; 2.85–17.12). All cultures were main-
tained under the same conditions as mentioned ear-
lier. The data on rooting response, the number of
roots per shoot and length of root were recorded at
the end of 2 weeks of culture.
Acclimatisation of plantlets
The in vitro regenerated plantlets with well developed
shoot and roots were washed gently with sterile dou-
ble-distilled water under controlled environment to
remove excess semi-solid media. Subsequently the
plantlets were transferred to plastic pots containing
varying combinations of autoclaved garden soil, sand
and vermi-compost. The pots were irrigated with ½
MS liquid medium for 1 week followed by sterile
distilled water. The potted plantlets were maintained
in culture room for initial period of 2 weeks for
gradual acclimatisation, and subsequently the well
developed plantlets were shifted to experimental gar-
den. The percentage of plantlets successfully estab-
lished in soil was also recorded after 4 weeks of
transplantation in the experimental garden.
Genetic fidelity assessment using ISSR markers
Genomic DNA was isolated from young leaves
(~1.2 g) of 6 donor plants and 30 in vitro raised
plantlets using Cetyl-trimethyl ammonium bromide
(CTAB) method with few modifications (Seth, Rath,
Rout, & Panigrahi, 2017). The purification of crude
DNA was done using RNAse (10mg/ml) and protei-
nase K (20 mg/ml) treatment followed by single wash
of phenol: chloroform: isoamyl alcohol (25:24:1 v/v/
v) and 3 washes of chloroform: isoamyl alcohol
(24:1 v/v). The upper aqueous phase was removed
after centrifugation and mixed with 1/10th volumes of
3M sodium acetate. DNA was precipitated by adding
2.5 volume of chilled ethanol then pelleted, dried in
vacuum and dissolved in 10 mM Tris and 1mM
EDTA (T10E1) buffer. The genomic DNA concentra-
tion was determined by using UV-Vis spectrophot-
ometer (UV 1, Thermo Electron Corporation,
England) with T10E1 buffer as blank. The quantifica-
tion of purified DNA was visually confirmed by using
0.8% agarose gel along with diluted uncut lambda
DNA as the standard. The DNA from 6 donor plants
and 30 in vitro regenerated plantlets were equilibrated
to a concentration of 20 ng/µl using T10E1 buffer.
Ten synthesised ISSR primers from the set 100/9
(University of British Columbia, Vancouver, Canada)
were tested for ISSR marker analysis, each reaction
carried out in a mixture of 25 µl containing 20 ng of
genomic DNA as template, 50 picomole primer, 2.5µl
of 10X assay buffer [100mM Tris-Cl, pH 8.3; 0.5M
3
KCl; 0.1% (w/v) gelatin], 1.5mM MgCl2, 200µM of
each dNTPs, 1.0 units Taq DNA polymerase
(Bangalore Genei Pvt. Ltd., Bangalore, India). The
amplification was performed in thermal cycler
(Veriti 96, Applied Biosystem, USA) programmed
with initial denaturation at 94°C for 5 min; 40 cycle
of denaturation at 94°C for 30 sec, primer annealing
at 43–52°C for 45 sec and elongation at 72°C for
2 min; and final elongation at 72°C for 5 min. The
amplified products were electrophoresed using 1.4%
agarose gel containing ethidium bromide solution
(0.5µgml−1) using TAE (40 mM Tris acetate; 2 mM
EDTA) buffer at constant 60 V for about 3 hour. A
gel-loading buffer (20% sucrose; 0.1 M EDTA, 1%
SDS; 0.25% bromophenol blue; 0.25% xylene cyanol)
was used as tracking dye. The size of the amplified
fragments were estimated using 250 bp Step-up DNA
ladder (Bangalore Genei Pvt. Ltd., Bangalore, India)
as molecular weight marker and TL120 software
(Version 1.2). The PCR reactions and electrophoresis
of amplified products were repeated twice for their
reproducibility and stability.
Statistical analysis
The data on callus inducing response, fresh weight of
callus, mean number of shoots per callus and the shoot
length, rooting response, the number of roots per
shoot, length of root and percentage of plantlet estab-
lished in soil were recorded. Each treatment consists of
5 replicated culture vessels and was repeated thrice.
The experiment was arranged in a completely rando-
mised block design and the standard error was calcu-
lated. The mean value (of all percentage data) of each
treatment were normalised by converting to arcsin
angular transformed values (Y) in degrees by using
the formulae “Y = sin−1√P/100. Subsequently, the
mean values were statistically compared using
Duncan’s multiple range test (DMRT) (Harter, 1960)
at p = 0.05 level of significance. For this, SPSS V 16.0.1
software was used with parameters – one way ANOVA
and homogeneity of variance.
Results and discussion
Effects of PGRs on callus induction
In vitro organogenic differentiation depends upon
the concentration and combinations of exogeneous
plant growth regulators (PGRs) added on to the
nutrient medium with respect to endogeneous
PGRs, in particular auxin and cytokinin, present
in the explant, and also on the acquisition of the
explant to organogenic competence. In this study,
leaf explants from 30-days-old A. indicum plant
were implanted on callus induction medium sup-
plemented with various concentration of NAA
4 S. SETH AND J. PANIGRAHI

Table 1. Effect of plant growth regulators (NAA, 2, 4-D, BA cytokinins (Sugiyama, 1999; Phillips, 2004).
and KIN) on callus induction from leaf explants of A. indicum Analysis of variance also revealed that the callusing
cultured on MS medium at the end of fourth week#. response of the explant and the growth of callus
PGRs concentration (µM) *Leaf Explants Fresh weight of
Response (%) callus (mg/tube) were significantly affected by the combination and
NAA 2,4-D BA KIN (Mean ± SE) (Mean ± SE) concentration of exogeneous PGRs (Table 1). The
Control 0.0 0.0 proliferated callus masses were gradually sub-cul-
- 2.26 4.44 - 34.9 ± 0.53h 345.0 ± 0.54g
- 2.26 8.88 - 41.9 ± 0.29ij 651.6 ± 0.68i tured up to fifth passage on the same medium at
- 2.26 13.32 - 35.3 ± 0.31h 274.8 ± 0.58ef regular interval of 2 weeks, but it did not show any
- 2.26 17.76 - 23.9 ± 0.52cde 214.2 ± 0.58a
- 4.52 4.44 - 41.4 ± 0.5ij 651.8 ± 0.58i
organogenic or embryogenic differentiation even
- 4.52 8.88 - 70.3 ± 0.46l 886.8 ± 0.73k after 10 weeks of culture and sub culture. This
- 4.52 13.32 - 42.9 ± 0.39ij 652.0 ± 0.54i might be due to the cell quiescence of the explants
- 4.52 17.76 - 22.9 ± 0.63bc 273.0 ± 1.00def
- 2.26 - 4.64 23.9 ± 0.39cde 272.8 ± 1.15de as well as callus mass with respect to perception of
- 2.26 - 9.29 43.0 ± 0.43j 775.2 ± 0.58j PGRs (Phillips, 2004). The proliferation of callus,
- 2.26 - 13.94 34.2 ± 0.64fgh 273.6 ± 0.68def
- 2.26 - 18.58 23.9 ± 0.52cde 265.4 ± 0.92b on the MS medium added with 4.52 µM 2, 4-D and
- 4.52 - 4.64 33.2 ± 0.44fg 354.2 ± 1.20h 8.88 µM BA, in term of fresh weight was also
- 4.52 - 9.29 45.2 ± 0.39k 774.8 ± 0.37j
- 4.52 13.94 34.7 ± 0.57gh 354.8 ± 0.49h evaluated for each passage of subculture and the
- 4.52 - 18.58 23.2 ± 0.69cd 265.2 ± 0.86b fresh weight of callus during the passage of 5 sub-
2.68 - 4.44 - 23.9 ± 0.53cde 266.2 ± 0.80bc
2.68 - 8.88 - 34.7 ± 0.57gh 345.8 ± 0.73g
cultures was at par (Table 3).
2.68 - 13.32 - 25.4 ± 0.38cde 266.6 ± 1.66bc
2.68 - 17.76 - 23.9 ± 0.63cde 268.4 ± 1.12c
5.37 - 4.44 - 24.6 ± 0.38de 275.8 ± 0.97f Proliferation of shoot buds from callus
5.37 - 8.88 - 32.8 ± 0.43f 355.4 ± 0.60h
5.37 - 13.32 - 32.7 ± 0.46f 355.6 ± 0.51h As a consequence of non-responsiveness, the green and
5.37 - 17.76 - 23.7 ± 0.52cde 265.2 ± 0.86b
2.68 - - 4.64 21.5 ± 0.43b 266.2 ± 0.97bc proliferated callus masses were subsequently transferred
2.68 - - 9.29 24.2 ± 0.15cde 272.6 ± 1.32de to MS medium fortified with 543 µM Adenine sulphate
2.68 - - 13.94 22.9 ± 0.71bc 271.2 ± 0.91d
2.68 - - 18.58 23.1 ± 0.76cd 266.4 ± 1.20bc (Ads), NAA (1.07–5.37 µM) with BA (4.44–13.32 µM) or
5.37 - - 4.64 9.5 ± 0.65a 214.0 ± 0.63a KIN (4.64–13.94 µM) for organogenic differentiation.
5.37 - - 9.29 32.7 ± 0.46f 356.0 ± 0.95h
5.37 - - 13.94 23.7 ± 0.38cde 265.6 ± 1.02b
The initiation of shoot meristemoids observed on the
5.37 - - 18.58 9.5 ± 0.65a 213.8 ± 0.73a callus surface after 1 week of sub culture in these medium
*The percentage values are converted into arcsin angular transformed (Figure 1b). This might be due to PGRS triggered entry of
value prior to DMRT analysis using SPSS (version 16.0.1).#The means the quiescent cells of the callus into cell cycle and cana-
with a column having the same letter are not statistically significant
(p = 0.05) lised cell division to determine the differentiation of
specific organ primordia and shoot buds, which will be
later on differentiated into multiple shoots (Sugiyama,
(2.68–5.37 µM), 2, 4-D (2.26–4.52 µM), BA (4.44–
1999; Phillips, 2004). The highest frequency of shoot
17.76 µM) and KIN (4.64–18.58 µM) either alone
multiplication (62.2 ± 0.81) observed on MS medium
or in combination, and the explants underwent
supplemented with 8.88 µM BA and 2.68 µM NAA
callogenesis within 1 week of implantation into
with maximum 17.8 ± 0.66 number of shoot buds per
the medium (Table 1). The calli were mostly
callus with average shoot length of 12.4 ± 0.35 cm
green and compact, and best callusing response
(Figure 1c; Table 2). On comparison, 16.6 ± 0.39 number
(70.3 ± 0.46%) was obtained on MS medium for-
of shoot buds per callus was noticed on MS medium
tified with 4.52 µM 2, 4-D and 8.88 µM BA
supplemented 9.29 µM KIN and 2.68 µM NAA with
(Figure 1a) with a maximum 886.8 ± 0.73 mg of
average shoot length of 11.6 ± 0.12cm. It is to be noted
fresh callus weight per explant. However, fresh
here that, without addition of 543 µM adenine sulphate to
callus weight ranges from 213.8 to 886.8 mg per
either of the multiplication medium, these growth regu-
explant and their morphology also differs from
lator combinations were also ineffective for shoot bud
combination to combination of PGRs. The
induction (data not shown). The additive effects of ade-
response of leaf explants for callusing was superior
nine sulphate on in vitro shoot multiplication have
on MS medium supplemented with 2,4-D and BA
reported in different medicinal plant species
as compared to the combination of 2,4-D and KIN
(Nandagopal & Ranjitha Kumara, 2006; Bantawa, Roy,
(Table 1). Similar to our findings, 2, 4-D along
Ghosh, & Mondal, 2009). This might be probably due to
with BA or KIN play significant role on callus
the impediment of exogenous adenine sulphate, added to
induction in different plant species of family
the medium, on the degradation of cytokinins by feed-
Malvaceae (McLean, Lawrence, & Reichert, 1992;
back inhibition or by competing with the metabolites
Rout, Mishra, Das, & Sahoo, 2009; Yang, Hidaka,
involved in cytokinin anabolism (VanStaden,
Masaki, & Uozumi, 1995), and this might be attrib-
Zazimalova, & George, 2008). Among 2 cytokinins
uted to acquisition organogenic competence by the
employed in presence of adenine sulphate for the shoot
explants on exposure to these auxins and
multiplication, BA was comparatively better than KIN
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 5

Figure 1. In vitro organogenesis in Abutilon indicum. (a) Development of callus from the leaf explant on MS medium fortified
with activated charcoal (200 mgl−1), ascorbic acid (11.54µM), 2,4-D (4.52µM) and BA (8.88 µM) at the end of 4th week; (b)
Subculture of callus showing emergence of shoot buds on MS medium with activated charcoal (200 mgl−1), ascorbic acid
(11.54µM), NAA (2.68µM) and BA (8.88 µM); (c & d) Proliferation of shoot buds showing development of apical shoot and leaves
during first (c) and fifth (d) subculture; (e) Individual adventitious shoots cultured on MS medium with PGRs for proliferation and
development; (f) Rooting of elongated shoots on half strength MS medium with IBA (7.38 µM) and (g) Acclimatisation of shoots
on plastic pots containing soil: sand: vermi-compost (1:1:1; v/v). Scale bar in ‘a’, ‘b’, ‘c’ and ‘d’ is 5 mm, in ‘e’ is 10 mm, in ‘f’5 mm
and in ‘g’ is 4 cm, respectively.

and this superiority of BA over KIN has been reported in
various plant species of the family Malvaceae (Agrawal
et al., 1997; Herath, Suzuki, & Hattori, 2004; Sivanesan &
Jeong, 2007; & Christensen, Sriskandarajah, Serek, &
Müller, 2008). Shoot regeneration frequency was also
varied from 17 to 62% with respect to different concen-
trations and combinations of BA, KIN and NAA. These
finding also demonstrated that 2.68 µM NAA is quite
optimum along with different concentrations of BA or
KIN for shoot multiplication (Table 2). The low concen-
tration of auxin requirement was also well documented in
several plant species, including Rhinacanthus nasutus
(Cheruvathur, Sivu, Pradeep, & Thomas, 2012), Sida
cordifolia (Sivanesan & Jeong, 2007) and Trichodesma
indicum (Mahesh & Jeyachandran, 2013). This exogen-
ous application of auxin causes asymmetric distribution
of auxin which is necessary for de novo initiation of organ
primordia under culture conditions (Zhao, Su, Cheng, &
Zhang, 2008). Although there was no effect of sub-culture
upto fifth passage on callus growth in term of fresh weight
of callus, the passage of subculture have varied effect
on shoot regeneration frequency and average number
shoots per callus clump of A. indicum implanted on
MS media added with 8.88 µM BA, 2.68 µM NAA and
543 µM adenine sulphate (Table 3). The regeneration
frequency and average number of shoots were almost
at par up to third passage of subculture whereas the
subculture did not influence the length plantlets
regenerated in vitro. During fourth and fifth passage
of subculture the regeneration frequency and average
number of shoots were significantly reduced (Figures
1d and 2b). Previous reports in different medicinal
species also substantiated our present findings on the
effects of subculture on in vitro regeneration of A.
indicum (Ahmed & Anis, 2014; Ramadevi, Ugraiah,
& Pullaiah, 2012; Tiwari, Tiwari, & Singh, 2001). Each
individual adventitious shoots were further separated
and subcultured in the same medium (Figure 1e) was
successful grown into well developed shoots
(Figure 1f).
6 S. SETH AND J. PANIGRAHI

Table 2. Effects of different concentrations and combinations ium, which composed of half strength MS salts sup-
of BA, KIN and NAA on the shoot multiplication from leaf plemented with different concentrations of IBA,
derived callus of A. indicum cultured on MS medium at the NAA and IAA alone for root induction (Table 4).
end of 4th week#.
Root induction was observed from the well devel-
Plant growth Average
regulators (μM) *Regeneration number of Shoot oped shoots, after 3 to 5 days of sub-culture.
frequency (Mean shoots length (cm) However, complete root development has taken
BA NAA KIN ± SE) (Mean ± SE) (Mean ± SE)
almost 2 weeks. A maximum of 17.6 roots per
0.0 0.0 0.0 0.0 0.0 0.0
4.44 1.07 - 19.7 ± 0.46bc 8.39 ± 0.78abc 5.06 ± 0.12bc shoot was observed on half strength MS medium
4.44 2.68 - 36.6 ± 0.70g 13.6 ± 0.75f 7.87 ± 0.21d supplemented with 7.38 µM IBA (Figure 1(f)).
4.44 5.37 - 18.2 ± 0.57ab 9.55 ± 0.65c 5.67 ± 0.19c
8.88 1.07 - 36.9 ± 0.33g 13.9 ± 0.46fg 7.99 ± 0.24d Among the 3 auxins, IBA showed better response
8.88 2.68 - 62.2 ± 0.81j 17.8 ± 0.66i 12.4 ± 0.35g for rhizogenesis as compared to that of NAA and
8.88 5.37 - 39.7 ± 0.50h 15.5 ± 0.53gh 9.83 ± 0.23e
13.32 1.07 - 28.5 ± 0.50e 9.24 ± 0.45bc 5.55 ± 0.18c IAA. IBA showed its superiority over other auxins
13.32 2.68 - 37.2 ± 0.41g 12.6 ± 0.50ef 8.03 ± 0.27d for adventitious rooting in many species, and is
13.32 5.37 - 17.4 ± 0.54a 7.54 ± 0.81ab 5.73 ± 0.15c
- 1.07 4.64 17.8 ± 0.66a 8.02 ± 0.67abc 5.09 ± 0.32bc
probably due to its existence in conjugated form,
- 2.68 4.64 26.0 ± 0.53d 9.92 ± 0.54cd 8.20 ± 0.21d stability and consistently weak auxin activity, and
- 5.37 4.64 20.8 ± 0.44c 9.24 ± 0.45bc 5.37 ± 0.18bc insensitivity to auxin degrading enzymes (Stefancic,
- 1.07 9.29 28.8 ± 0.25e 11.5 ± 0.47de 10.1 ± 0.19e
- 2.68 9.29 52.2 ± 0.67i 16.6 ± 0.39hi 11.6 ± 0.12f Stampar, & Osterc, 2005; Ludwig-Müller, Vertocnik,
- 5.37 9.29 34.3 ± 0.60f 11.8 ± 0.55e 8.04 ± 0.17d & Town, 2005). Many previous reports also sub-
- 1.07 13.94 17.2 ± 0.67a 8.02 ± 0.67abc 4.42 ± 0.21a
- 2.68 13.94 28.4 ± 0.81e 11.5 ± 0.47de 5.37 ± 0.18bc stantiate the potency of IBA for root induction in
- 5.37 13.94 18.0 ± 0.58ab 7.17 ± 0.59a 4.86 ± 0.17ab different Malvaceous plant species (Sivanesan &
*The percentage values are converted into arcsin angular transformed value Jeong, 2007; Ganesh & Jayabalan, 2006). Root length
prior to DMRT analysis using SPSS (version 16.0.1).#The means with a
column having the same letter are not statistically significant (p = 0.05) varied significantly from 6.4 to 13.7 cm with respect
to type and concentration of auxins tested here. The
survival rate of in vitro regenerated plants was
Rooting and acclimatisation almost 70% (Figure 1(g)), and was obtained by
transferring in vitro rooted shoots to plastic pots
Regenerated shoots were subsequently transferred
containing garden soil, sand and vermi-compost
from shoot multiplication medium to rooting med-

Table 3. Effect of the passage of subcultures on callus growth and shoot multiplication in A. indicum*.
Shoot multiplication of green compact callus
clump of A. indicum cultured on MS+ 543 µM
Ads +8.88 µM BA and 2.68 µM NAA
Average
Fresh weight of callus of A. indicum subcultured on Regeneration number of Shoot
Passage of Sub culture of callus derived in MS+ 4.52 µM 2, 4-D and 8.88 µM BA frequency# shoots# length (cm) #
vitro growing leaf explants (Mean ± SE) (Mean ± SE) (Mean ± SE) (Mean ± SE)
1st 886.8 ± 0.73a 62.2 ± 0.81a 17.81 ± 0.66ab 12.36 ± 0.35a
2nd 878.6 ± 18.24a 64.04 ± 0.74a 18.58 ± 0.71a 12.94 ± 0.23ab
3rd 881.4 ± 10.33a 62.92 ± 1.04a 18.39 ± 0.68a 13.31 ± 0.36ab
4th 878.6 ± 13.55a 58.74 ± 1.12b 15.96 ± 0.56b 13.43 ± 0.26b
5th 885.2 ± 8.28a 54.71 ± 1.64c 15.96 ± 0.56b 13.37 ± 0.34ab
#
The values are converted into arcsin angular transformed value prior to DMRT analysis using SPSS (version 16.0.1). *The means with a column having
the same letter are not statistically significant (p = 0.05)

Figure 2. Graphical representation showing the influence of subculture on callus growth (a), regeneration frequency, average
number of shoots per callus clump and shoot length (b) in A. indicum.
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 7

Table 4. Effect of auxins on rooting response of excised are quite efficient and reliable for genetic fidelity
shoots, root number and root length in A. indicum cultured
assessment. Thus, different types of DNA markers
on half strength MS medium at the end of 2nd week#.
including ISSR were employed to assess the genetic
Growth regulators * Rooting Number of Root length
concentration (µM) Response (%) roots per shoot (cm) (Mean ±
stability of in vitro organogenesis derived plantlets in
IBA NAA IAA (Mean ± SE) (Mean ± SE) SE) several medicinal species, such as, Rhinacanthus nasu-
2.46 - - 28.9 ± 0.34 12.3 ± 0.72 gh 10.1 ± 0.57 de tus (Cheruvathur et al., 2012); Ceropegia evansii
cd

4.92 - - 42.5 ± 0.59 h 13.4 ± 0.31 hi

9.3 ± 0.48 d
(Cheruvathur, Sivu, Pradeep, & Thomas, 2015) and
7.38 - - 57.5 ± 1.13 j 17.6 ± 0.80 j
13.7 ± 0.31 h Hylocereus undatus (Fan et al., 2013). During the pre-
9.84 - - 39.0 ± 0.86 g 13.4 ± 0.31 hi
11.2 ± 0.39 ef sent study, 10 ISSR 8 primers were screened for genetic
12.30 - - 30.2 ± 0.77 d 11.8 ± 0.55 fgh
10.7 ± 0.36 de
14.76 - - 23.2 ± 0.83 b 10.6 ± 0.38 defg
7.8 ± 0.56 bc fidelity assessment among the 30 in vitro regenerated
- 2.68 - 27.8 ± 0.56 c 8.4 ± 0.78 abc
8.7 ± 0.38 cd plantlets and 6 donor plants. A total of 61 numbers of
- 5.37 - 36.9 ± 0.68 f 8.5 ± 0.37 abc
8.7 ± 0.38 cd
- 8.05 - 48.9 ± 0.56 i 13.9 ± 0.46 i
12.6 ± 0.5 gh scorable amplified fragments were produced by
- 10.74 - 36.4 ± 0.61 f 11.5 ± 0.47 efg
10.1 ± 0.39 de responsive ISSR primers tested here (Table 6). The
- 13.42 - 27.1 ± 0.72 c 8.8 ± 0.45 abcd
7.1 ± 0.59 ab
- 16.11 - 18.4 ± 0.86 a 8.0 ± 0.67 a
6.4 ± 0.30 a banding pattern was quite homogeneous among the
- - 2.85 27.1 ± 0.72 c 10.2 ± 0.63 cdef
8.8 ± 0.34 cd donor plant(s) and the in vitro regenerated plantlets.
- - 5.70 34.9 ± 0.98 ef 12.3 ± 0.90 ghi
10.9 ± 0.36 de
- - 8.56 42.9 ± 0.43 h 13.9 ± 0.46 i
12.2 ± 0.43 fg
But, the number fragments amplified by these primers
- - 11.41 33.7 ± 0.63 e 9.9 ± 0.54 bcde
10.7 ± 0.35 de and the size of amplified fragments were varied among
abcd
- - 14.27 28.6 ± 0.61 8.8 ± 0.45 7.0 ± 0.55 ab themselves. The size of amplified fragments ranged
cd

- - 17.12 18.7 ± 0.94 a

8.0 ± 0.67 a
7.2 ± 0.44
*The percentage values are converted into arcsin angular transformed value
prior to DMRT analysis using SPSS (version 16.0.1).# The means with a
column having the same letter are not statistically significant (p = 0.05)
(1:1:1, v/v/v) mix (Table 5). The plant to soil estab-
lishment took almost 4 weeks and the acclimatised
plantlets were then successfully grown in experi-
mental garden, which are morphologically similar
to their donor plants
ISSR analysis of in vitro regenerants
In spite of morphological similarity, genetic variation
among the in vitro regenerated plantlets was also
detected in plant species, which could possibly
detected by using cyto-morphological and molecular
marker analysis. Among these, DNA based markers
ab from 155 to 2585 bp. Primer UBC 868 amplified a
maximum of 11 monomorphic fragments, whereas
primer UBC 840 exhibited the least (3) numbers of
monomorphic bands (Figure 3). The homogeneous
ISSR marker profiling substantiated the reliability of
the protocol established in vitro organogenic differen-
tiation of leaf explants in A. indicum.
Conclusion
An in vitro propagation protocol was established for A.
indicum via callus mediated organogenesis of leaf
explants, and data on effects of PGRs (auxins, cytoki-
nins and growth adjuvants) on callus induction, regen-
eration of shoots and rooting of A. indicum plantlets
was also obtained. The genetic stability of regenerated
plantlets also confirmed using ISSR marker analysis.
This established protocol would not only be applicable

Table 5. Effect of different composition of soil, sand and vermicompost on acclimatisation and survivability of in vitro
regenerated plantlets in A. indicum at the end of 4th week*.
* Survivability (%)
Composition (V/V) No. of plantlets tested for acclimatisation (Mean ± SE)
Autoclaved soil: sand (1:1) 50 47.80 ± 2.45 a
Garden soil: sand:vermicompost (1:1:1) 50 70.00 ± 7.14 b
Vermicompost:soil (1:1) 50 51.60 ± 5.88 ab
Autoclaved soil:sand:vermicompost (1:1:1) 50 57.20 ± 6.53 ab
*Means with a column having the same letter are not statistically significant (p = 0.05) according to Duncan’s multiple range test (SPSS V 16.0)

Table 6. Response of ISSR primers* used for the assessment of genetic fidelity in vitro regenerated plantlets of A. indicum.
Primer Sequence (5ʹ-3ʹ) Annealing Temperature (°C) Number of amplified fragments Ranges (bp)
UBC 835 [(AG)8]YC AGAGAGAGAGAGAGAGYC 54.0 9 450–2410
UBC 840 [(GA)8]YT GAGAGAGAGAGAGAGAYT 48.0 3 690–1470
UBC 861 [(ACC)6] ACCACCACCACCACCACC 55.0 8 610–1890
UBC 865 [(CCG)6] CCGCCGCCGCCGCCGCCG 72.0 9 570–2500
UBC 868 [(GAA)7] GAAGAAGAAGAAGAAGAA 52.0 11 360–2385
UBC 873 [(GACA)4] GACAGACAGACAGACA 52.0 7 355–2585
UBC 808 [(AG)8C] AGAGAGAGAGAGAGAGC 52.0 7 590–2030
UBC 807 [(AG)8T] AGAGAGAGAGAGAGAGT 52.0 7 155–2355
Total 61 155–2585
*No polymorphism revealed by all the eight responded primers
8 S. SETH AND J. PANIGRAHI
Figure 3. Genetic fidelity assessment showing the ISSR profile of Donor plant (D) and 30 in vitro regenerated plantlets
generated by primers UBC-865(a), UBC-868(b) and UBC-873(c).
as platform for rapid multiplication and conservation
of elite clones, secondary metabolite production and
genetic improvement, but also provides a useful experi-
mental system for studying regulatory mechanisms of
growth and development in A. indicum.
Acknowledgments
The author (SS) acknowledges gratitude to University
Grants Commission (UGC), India [Grant number F-14-2
(SC)/2010(SA-III)] for Rajiv Gandhi National Fellowship.
Authors also grateful to the Vice Chancellor, Sambalpur
University, Odisha, India for providing necessary facilities
for this work.
Disclosure statement
No potential conflict of interest was reported by the authors.
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