The Journal of Horticultural Science and Biotechnology

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An improved micropropagation protocol for

the recalcitrant plant Capsicum – a study with
ten cultivars of Capsicum spp. (C. annuum, C.
chinense, and C. frutescens) collected from diverse
geographical regions of India and Mexico

Sk Moquammel Haque & Biswajit Ghosh

To cite this article: Sk Moquammel Haque & Biswajit Ghosh (2018) An improved micropropagation
protocol for the recalcitrant plant Capsicum – a study with ten cultivars of Capsicum spp.
(C. annuum, C. chinense, and C. frutescens) collected from diverse geographical regions of
India and Mexico, The Journal of Horticultural Science and Biotechnology, 93:1, 91-99, DOI:
10.1080/14620316.2017.1345331

To link to this article: https://doi.org/10.1080/14620316.2017.1345331

Published online: 12 Jul 2017.

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THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY, 2018
VOL. 93, NO. 1, 91–99
https://doi.org/10.1080/14620316.2017.1345331

An improved micropropagation protocol for the recalcitrant plant Capsicum – a

study with ten cultivars of Capsicum spp. (C. annuum, C. chinense, and C. frutescens)
collected from diverse geographical regions of India and Mexico
Sk Moquammel Haque and Biswajit Ghosh
Plant Biotechnology Laboratory, Department of Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata, India

ABSTRACT ARTICLE HISTORY

Capsicum spp. is a commercially important crop of the Solanaceae family, well-known for its Accepted 12 June 2017
multipurpose use as a vegetable, spice, medicinal and ornamental plants. The genus
KEYWORDS
Capsicum is a recalcitrant species in terms of in vitro morphogenesis and plant regeneration. Bhut Jolokia; Capsicum; Chili
An efficient method was developed for multiple shoot regeneration in 10 cultivars of pepper; cytokinin;
Capsicum collected from diverse geographical regions of India and Mexico. Seeds germinated micropropagation;
in vitro on a half-strength Murashige and Skoog (MS) medium supplemented with 3.0 % spermidine
sucrose. Nodes of the in vitro germinated seedlings were used as explant for micropropaga-
tion. The combination of the 6-benzylaminopurine, indole-3-acetic acid, and spermidine was
found to be the best for multiple shoot induction. However, the optimum responcse varied
accompanied by different cultivers with maximum 8.9 ± 0.52 (Capsi-10) to 15.3 ± 0.69 (Capsi-
5) multiple shoot per explant. Depending on the cultivar, multiplied shoots were successfully
rooted with maximum 18.4 ± 0.20 (highest for Capsi-9) to 36.8 ± 0.29 (highest for Capsi-5)
roots per shoot on half-strength MS medium supplemented with 2.0 mg l−1 indole-3-butyric
acid, 1.0 mg l−1 α-naphthalene acetic acid, and 1.5 mM spermidine. Finally, the micropropa-
gated plantlets were acclimatized with 40.0–86.7 % survival rate, depending on different
cultivars.

Introduction ‘world’s hottest Chili pepper’ (Bosland & Baral,

2007). Capsaicin (8-methyl-N-vanillyl-6-nonena-
The genus Capsicum (common name: Chili peppers)
mide) is an alkaloid substance present in the fruits
is a commercially important crop of the Solanaceae
of Capsicum, which makes Chili hot to taste (Das,
family cultivated worldwide for spices, vegetables, as a
Kundu, & Ghosh, 2015; Kundu, Das, & Ghosh,
colouring agent, and for medicinal intention (Ochoa-
2015a; Ferri, Gruarin, Barbieri, & Tassoni, 2017).
Alejo & Ramirez-Malagon, 2001). A few varieties of
Immeasurable genetically divers Capsicum cultivars
Capsicum are also cultivated for horticultural pur-
are grown in various agro-climatic zones of India; in
poses (Kehie, Kumaria, & Tandon, 2012, 2013;
particularly, the state of West Bengal has the richest
Kumar et al., 2007). This genus includes 38 species,
diversity in a Chili ecosystem (Paul, Das, Sarkar, &
of which only six are cultivated; among them, the
Ghosh, 2013). For the last few years our laboratory
major ones are C. annuum, C. chinense, and C. fru-
has been involved in the collection of Capsicum spp.
tescens (Ramchiary, Kehie, Brahma, Kumaria, &
from different parts of India and abroad, their ex situ
Tandon, 2014). The Indian germplasm is mainly
conservation and various other aspects like extraction
represented by two species, C. annuum and C. frutes-
and quantification of capsaicin, modulation of pun-
cens with a number of cultivars (Thul et al., 2009);
gency, examining antioxidant property and major
moreover a few cultivars of C. chinense are also culti-
bioactive compounds, in vitro flowering, fruiting and
vated widely in the North-East regions of India
karyological studies (Paul et al., 2013; Das et al., 2015;
(Bosland & Baral, 2007; Kehie et al., 2012, 2013).
Kundu et al., 2015a, Kundu, Das, Haque, & Ghosh,
The fruits of C. chinense and C. frutescens are extre-
2015b; Haque, Paul, & Ghosh, 2016a, 2016b).
mely pungent and generally used as a source of pun-
In vitro clonal propagation of high-yielding elite
gency in food, while those of C. annuum are usually
genotypes to produce good quality planting materials
known as sweet peppers and used in curried dishes or
with higher rates of multiplications is an advantage of
preparation of pickle. The ‘ghost pepper’ or ‘Bhut
modern plant biotechnology (Haque & Ghosh, 2014).
Jolokia’ (Capsicum chinense Jacq. cultivar) is one of
Since Capsicum plant reproduces via sexual reproduc-
the hottest Chili pepper, and it won the title of
tion only, the natural vegetative propagation is totally

CONTACT Biswajit Ghosh ghosh_b2000@yahoo.co.in

© 2017 The Journal of Horticultural Science & Biotechnology Trust
92 S. M. HAQUE AND B. GHOSH

absent; in vitro propagation is the only alternative way 1.04 kg cm–2 pressure and 121°C for 18 min. All cultures
to produce large quantities of true-to-type plants of the were incubated at 22 ± 2°C under the 16-h light and 8-h
elite genotype that do not depend on seasons. Gunay dark cycle with the light intensity of 50 μmol m–2 s–1
and Rao (1978) first reported the in vitro regeneration provided by Philips cool-white fluorescent lamps in an
of Capsicum over three decades ago; since then, several environment-controlled culture room.
researchers have attempted to develop and improve the
in vitro methods for propagating Capsicum spp. Plant materials
(Bairwa, Kachhwaha, & Kothari, 2012; Barrales-López, All the 10 cultivars of Capsicum spp. (Capsi-1 to 10)
Robledo-Paz, Trejo, Espitia-Rangel, & Rodríguez-De La used in the present experiment along with their scien-
O, 2015; Christopher & Rajam, 1994; Dabauza & Peña, tific name, local name/cultivar, area of collection, geo-
2001; Kehie et al., 2012, 2013; Kundu et al., 2015b; graphic locations are summarized in Table 1. Fresh
Mohamed & Alsadon, 2011; Orlińska & Nowaczyk, and ripened red chili was collected from fields or local
2015; Sanatombi & Sharma, 2008a, 2008b). But unlike markets of seven different regions in India (Figure 1).
other Solanaceous species, Capsicum has been a recal- The seeds of these ripened fruits were used for in vitro
citrant species in terms of in vitro organogenesis and culture establishment. Seeds of the Mexican cultivars
plant regeneration (Kothari, Joshi, Kachhwaha, & were collected from the Chile Pepper Institute, New
Ochoa-Alejo, 2010; Máthé, Hassan, & Kader, 2015). It Mexico State University, Mexico (Figure 1).
has been challenging to establish efficient, reliable, and
broad-spectrum protocols for members of this recalci- Surface sterilization and in vitro seed germination
trant genus Capsicum (Kehie et al., 2013). However, all The seeds of all 10 cultivars were surface-sterilized in
of the previous reports deal with any one or very few 2.5 % (w/v) systematic fungicide (Bavistin®) for
cultivars of Capsicum. Considering this, the present 12 min, then in a 5.0 % (v/v) liquid detergent
study was designed with 10 cultivars belonging to (tween-20) for 5 min, then in 0.1 % (w/v) mercuric
three different species of Capsicum (C. annuum, C. chloride (HgCl2) for 10 min, and rinsed three times
chinense, and C. frutescens), and the addition of spermi- (5 min each) in sterile distilled water to remove traces
dine (SDN) with the aim of improving the micropro- of HgCl2. Then, surface-sterilized seeds were inocu-
pagation methods which would possibly help in lated aseptically on half-strength of the MS basal
understanding the in vitro regeneration of this recalci- medium and kept in a dark chamber for germination.
trant genus.

Multiple shoot regeneration

Materials and methods Nodes of about 0.5–0.7 cm were excised from in vitro
grown seedlings (4.5–5.0 cm height with 3–4 nodes) and
Culture media, culture conditions, and plant
used as explants for the induction of multiple shoots. The
materials
explants were implanted in the MS basal medium sup-
Media preparation and culture conditions plemented with different concentrations of 6-benzylami-
A half-strength Murashige and Skoog (1962) (MS) med- nopurine (BAP) or Kinetin (KIN) alone. The effect of
ium supplemented with 0.75 % (w/v) agar-agar and 1.5 SDN was evaluated in another set of experiments. Five
% (w/v) sucrose was used for seed germination; and a different concentrations (1.0, 1.5, 2.0, 2.5, and 3.0 mM) of
full-strength MS medium supplemented with 3.0 % (w/ SDN were assessed in combination with the optimum
v) sucrose, 0.75 % (w/v) agar-agar and different concen- concentration (6.0 mg l–1) of BAP or KIN (Table 2). After
trations and combinations of plant growth regulators detecting the optimistic role of SDN, SDN was supple-
(PGRs) and SDN were used for micropropagation mented for all further experiments in combination with
experiment. The media were adjusted to pH 5.6 before different concentrations and combinations of BAP, KIN,
the addition of agar-agar. The media were autoclaved at and indole-3-acetic acid (IAA) (Table 3). At every 4-week

Table 1. Detail of the ten cultivars of Capsicum spp. used in this experiment.
Collection area /Source
Code name Local name (cultivar) Species District and State Country Geographic location (latitude & longitude)
Capsi-1 Bhut Jolokia C. chinense Silchar, Assam India 24°49′N 92°48′E
Capsi-2 Dhani Lanka C. frutescens Nadia, West Bengal India 23°24′N 88°30′E
Capsi-3 Kull Lanka C. frutescens Cooch Behar, West Bengal India 26°22′N 89°29′E
Capsi-4 Beldanga Lanka C. annuum Murshidabad, West Bengal India 24°08′N 88°16′E
Capsi-5 Jhanti Lanka C. annuum Howrah, West Bengal India 22°36′N 87°92′E
Capsi-6 Bullet Lanka C. annuum Malda, West Bengal India 25°00′N 88°09′E
Capsi-7 Acchar Lanka C. annuum Shamshabad, Hyderabad India 17°15′N 78°23′E
Capsi-8 Serrano C. annuum The Chile Paper Institute, Las Cruces Mexico 32°16′N 106°44′W
Capsi-9 deArbol C. annuum The Chile Paper Institute, Las Cruces Mexico 32°16′N 106°44′W
Capsi-10 NuMex Mirasol C. annuum The Chile Paper Institute, Las Cruces Mexico 32°16′N 106°44′W
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 93

Figure 1. Fruits of seven Indian cultivars of Capsicum and three packets of seeds of the Mexican cultivars of Capsicum.

interval, the cultures were sub-cultured in their respective Statistical analysis

fresh media. The data were collected after 60 days of
In the present experimental design, each treatment of
implantation.
micropropagation and rooting was repeated three
times with 10 explants per treatment. All data were
In vitro root inductions subjected to one-way analysis of variance (ANOVA)
using SPSS software for Windows (IBM® SPSS, ver-
For root induction, multiplied shoots were separated into
sion 21.0, Chicago, IL). After conducting an ANOVA,
single ones (about 2.5–3.0 cm) and cultured on the half-
the means were further separated using Tukey’s test
strength MS media supplemented with different concen-
(Haynes, 2013) at P ≤ 0.05.
trations and combinations of indole-3-butaric acid
(IBA), α-naphthalene acetic acid (NAA), and SDN. The
data were collected after 30 days of implantation. Results
The seeds of all 10 cultivars were germinated aseptically
Acclimatization of regenerated plants (Figure 2(a)). However, the days required for the germi-
Rooted plantlets (about 5–6 cm) were transferred to nation (5–14 days) as well as the percentage of germina-
small earthen pots containing ‘Soilrite-mix™’ (sterile, tion (42.2–93.3 %) varied with different cultivars of
chemically inert horticultural graded perlite marketed Capsicum. After germination, the seedlings were trans-
by Keltech Energies Ltd., Bangalore, India) and cov- ferred to the MS basal medium without any PGR and
ered with transparent polythene bags to maintain grown up to 4.5–5.0 cm with 3–4 nodes (Figure 2(b)).
90–99 % relative humidity, and they were kept in
25 ± 2°C temperature and 16-h photoperiod for
Effect of different PGRs and spermidine on
30 days. A total 150 plants, i.e. 15 from each of the
multiple shoot induction
10 cultivars (Capsi-1 to 10) were planted for acclima-
tization. The data were collected after 30 days of Out of two cytokinins, BAP proved to be more effective
plantation. than KIN for multiple shoot induction. Depending on
 94

Table 2. Effect of Spermidine (SDN) on in vitro shoot multiplication of ten different cultivars of Capsicum spp. cultured on MS medium suplimented with 6 mg l−l BAP or KIN. (Data collected after 60
days of implantation).
Total number of shoots induced per nodal explant after 60 days of implantation
BAP mg l–l KIN mg l–l SDN mM Capsi-1 Capsi-2 Capsi-3 Capsi-4 Capsi- 5 Capsi-6 Capsi-7 Capsi-8 Capsi-9 Capsi-10
6.0 - - 8.6 ± 0.56d 8.2 ± 0.38de 7.7 ± 0.42de 5.3 ± 0.36c 9.1 ± 0.48de 4.5 ± 0.52bcd 5.6 ± 0.42a 4.9 ± 0.26bc 4.0 ± 0.37b 5.5 ± 0.40bc
6.0 - 1.0 10.2 ± 0.45e 9.3 ± 0.55def 8.6 ± 0.51ef 5.9 ± 0.51d 10.4 ± 0.51ef 5.0 ± 0.62cd 6.3 ± 0.58b 6.2 ± 0.38cde 5.1 ± 0.48cd 6.6 ± 0.47d
6.0 - 1.5 10.9 ± 0.62ef 9.6 ± 0.51ef 9.0 ± 0.63f 6.1 ± 0.42d 10.7 ± 0.37ef 5.4 ± 0.54d 6.8 ± 0.36c 7.2 ± 0.44de 5.3 ± 0.54d 7.0 ± 0.36de
6.0 - 2.0 11.8 ± 0.73f 10.4 ± 0.42f 9.2 ± 0.65f 6.5 ± 0.52de 11.2 ± 0.68f 5.7 ± 0.51de 7.4 ± 0.25d 7.4 ± 0.54e 5.4 ± 0.62d 7.3 ± 0.52de
6.0 - 2.5 11.4 ± 0.71ef 10.1 ± 0.41ef 8.9 ± 0.58ef 6.9 ± 0.44e 10.5 ± 0.49ef 6.1 ± 0.48e 6.4 ± 0.38abc 6.8 ± 0.43de 5.0 ± 0.51cd 7.6 ± 0.38e
S. M. HAQUE AND B. GHOSH

6.0 - 3.0 9.6 ± 0.59de 8.7 ± 0.47de 8.2 ± 0.55e 5.8 ± 0.41cde 9.7 ± 0.36e 5.5 ± 0.42d 5.9 ± 0.45ab 5.4 ± 0.51c 4.3 ± 0.35bc 6.3 ± 0.55cd
- 6.0 - 4.3 ± 0.52a 3.8 ± 0.56a 4.1 ± 0.46a 3.6 ± 0.35a 5.1 ± 0.42a 3.3 ± 0.36a 5.7 ± 0.41a 3.7 ± 0.35a 3.2 ± 0.40a 4.2 ± 0.62a
6.0 1.0 6.6 ± 0.62bc 4.9 ± 0.34b 5.0 ± 0.44b 4.5 ± 0.62b 5.8 ± 0.28ab 3.8 ± 0.52abc 6.3 ± 0.36b 4.6 ± 0.28b 3.8 ± 0.43b 5.0 ± 0.56b
- 6.0 1.5 7.1 ± 0.43c 5.5 ± 0.51bc 5.4 ± 0.50bc 4.7 ± 0.51bc 5.9 ± 0.51ab 4.0 ± 0.29b 6.7 ± 0.33c 4.9 ± 0.34bc 4.2 ± 0.24bc 5.5 ± 0.50bc
- 6.0 2.0 7.7 ± 0.24cd 6.1 ± 0.38bc 6.9 ± 0.33d 5.1 ± .28c 6.2 ± 0.61b 4.3 ± 0.36bc 7.3 ± 0.56d 5.5 ± 0.26c 4.4 ± 0.15bcd 5.8 ± 0.57c
- 6.0 2.5 6.8 ± 0.31bcd 5.1 ± 0.25b 5.8 ± 0.45bc 5.0 ± 0.53bcd 5.8 ± 0.57ab 4.5 ± 0.42bc 6.4 ± 0.43abc 5.1 ± 0.29bc 4.0 ± 0.26b 6.0 ± 0.51c
- 6.0 3.0 5.4 ± 0.45b 4.0 ± 0.42a 4.3 ± 0.39a 4.2 ± 0.46b 5.3 ± 0.54a 4.8 ± 0.37b 6.0 ± 0.38ab 4.2 ± 0.32ab 3.3 ± 0.39a 5.2 ± 0.46abc
Each value represents the mean ± standard error, n = 30 (3 sets, 10 samples in each set). Mean followed by the same letters in each column are not significantly different at P ≤ 0.05 according to Tukey’s multiple range tests.

Table 3. Effect of cytokinin and auxin on in vitro shoots multiplication of ten different cultivars of Capsicum spp. cultured on MS medium suplimented with 2 mM spermidine. (Data collected after
60 days of implantation).
Total number of shoots induced per nodal explant after 60 days of implantation
BAP mg l–l KIN mg l–l IAA mg l–l Capsi-1 Capsi-2 Capsi-3 Capsi-4 Capsi- 5 Capsi-6 Capsi-7 Capsi-8 Capsi-9 Capsi-10
- - - 1.7 ± 0.21a 1.6 ± 0.34a 1.9 ± 0.27a 1.5 ± 0.08a 1.8 ± 0.17a 1.6 ± 0.08a 1.2 ± 0.06a 1.1 ± 0.04a 1.3 ± 0.03a 0a
2.0 - - 6.2 ± 0.57c 5.4 ± 0.33c 4.5 ± 0.34b 3.3 ± 0.25ab 4.3 ± 0.32bc 2.9 ± 0.06b 3.5 ± 0.15bc 3.6 ± 0.23b 2.6 ± 0.12ab 2.2 ± 0.33b
4.0 - - 9.4 ± 0.48de 7.3 ± 0.25d 6.8 ± 0.54bc 4.2 ± 0.34b 8.8 ± 0.41ef 4.2 ± 0.15bc 5.7 ± 0.35cd 5.8 ± 0.27cd 4.2 ± 0.35bc 4.9 ± 0.32cd
6.0 - - 11.8 ± 0.73ef 10.4 ± 0.42f 9.2 ± 0.65cd 6.5 ± 0.52cd 11.2 ± 0.68g 5.7 ± 0.51cd 7.4 ± 0.25e 7.4 ± 0.54de 5.4 ± 0.62cd 7.3 ± 0.52e
8.0 - - 10.7 ± 0.65e 9.7 ± 0.41ef 8.3 ± 0.53c 7.3 ± 0.36de 9.4 ± 0.82f 7.1 ± 0.55de 9.2 ± 0.26fg 7.1 ± 0.62de 6.3 ± 0.33de 8.8 ± 0.15f
10.0 - - 8.9 ± 0.45d 6.3 ± 0.36cd 6.1 ± 0.51bc 6.4 ± 0.25cd 8.1 ± 0.67e 7.8 ± 0.38e 8.6 ± 0.24f 4.8 ± 0.54bcd 4.8 ± 0.54bcd 6.4 ± 0.24de
- 2.0 - 4.1 ± 0.32bc 3.3 ± 0.17b 2.6 ± 0.22a 2.7 ± 0.45ab 3.4 ± 0.56b 2.5 ± 0.56ab 2.9 ± .52b 2.2 ± 0.05ab 2.2 ± 0.35ab 2.7 ± 0.26b
- 4.0 - 6.3 ± 0.36c 4.8 ± 0.25bc 5.1 ± 0.28b 3.9 ± 0.26b 4.3 ± 0.25bc 3.1 ± 0.45b 5.2 ± 0.25cd 4.1 ± 0.28bc 3.5 ± 0.12b 4.2 ± 0.55c
- 6.0 - 7.7 ± 0.24cd 6.1 ± 0.38cd 6.9 ± 0.33bc 5.1 ± .28bc 6.2 ± 0.61cd 4.3 ± 0.36bc 7.3 ± 0.56e 5.5 ± 0.26cd 4.4 ± 0.15bc 5.8 ± 0.57d
- 8.0 - 8.6 ± 0.41d 7.5 ± 0.32de 8.4 ± 0.42c 6.6 ± 0.62cd 8.1 ± 0.28e 4.9 ± 0.25c 8.5 ± 0.27f 6.4 ± 0.56d 5.8 ± 0.27cde 7.1 ± 0.68e
- 10.0 - 9.2 ± 0.52de 6.8 ± 0.29cde 8.8 ± 0.47cd 7.1 ± 0.56de 8.9 ± 0.60ef 5.6 ± 0.58cd 7.8 ± 0.85ef 7.0 ± 0.57de 5.5 ± 0.36cd 6.5 ± 0.15de
6.0 - 0.5 13.3 ± 0.56fg 11.9 ± 0.62g 11.9 ± 0.84e 6.8 ± 0.45cd 12.7 ± 0.84gh 6.7 ± 0.48d 9.9 ± 0.56g 9.7 ± 0.48fg 8.1 ± 0.25f 11.3 ± 0.81gh
8.0 - 0.5 12.5 ± 0.74f 11.2 ± 0.74fg 11.3 ± 0.78de 7.6 ± 0.26de 10.8 ± 0.87fg 7.4 ± 0.69de 11.2 ± 0.25gh 9.2 ± 0.75f 8.4 ± 0.28fg 11.9 ± 0.48h
6.0 - 1.0 14.2 ± 0.68g 13.5 ± 0.95h 12.5 ± 0.93ef 7.7 ± 0.61de 15.3 ± 0.69i 7.2 ± 0.80de 10.9 ± 0.98gh 10.1 ± 0.59g 8.2 ± 0.42f 10.0 ± 0.85g
8.0 - 1.0 13.0 ± 0.72fg 12.1 ± 0.75gh 11.4 ± 0.68de 8.0 ± 0.58e 13.1 ± 0.61h 9.4 ± 0.73f 12.7 ± 0.46i 9.5 ± 0.82fg 8.9 ± 0.52g 10.4 ± 0.74gh
6.0 - 1.5 13.4 ± 0.46fg 10.3 ± 0.68f 12.0 ± 0.77e 8.9 ± 0.45ef 11.6 ± 0.40g 9.1 ± 1.03ef 11.1 ± 0.78gh 9.3 ± 0.58f 7.8 ± 0.68ef 9.6 ± 0.56fg
8.0 - 1.5 11.7 ± 0.54ef 9.4 ± 0.71ef 11.6 ± 0.71de 9.8 ± 0.76f 10.2 ± 0.36fg 10.9 ± 0.83g 12.1 ± 1.20hi 8.6 ± 0.94ef 8.3 ± 0.56g 10.2 ± 0.93g
6.0 - 2.0 11.6 ± 0.83ef 6.6 ± 0.52cd 10.7 ± 0.63d 8.2 ± 0.59e 8.3 ± 0.70e 8.0 ± 0.56e 9.4 ± 0.56 8.1 ± 0.77e 6.1 ± 0.51de 8.3 ± 0.58ef
8.0 - 2.0 10.3 ± 0.66e 6.2 ± 0.64cd 9.4 ± 0.72 8.5 ± 0.67ef 8.0 ± 0.35e 9.2 ± 0.60ef 9.7 ± 0.84 7.5 ± 0.56de 6.7 ± 0.48e 9.1 ± 0.47f

Each value represents the mean ± standard error, n = 30 (3 sets, 10 samples in each set). Mean followed by the same letters in each column are not significantly different at P ≤ 0.05 according to Tukey’s multiple range tests.
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 95

Figure 2. In vitro micropropagation of different Capsicum cultivars. In vitro seed germination in Capsi-4 (Panel a). In vitro grown
seedling of Capsi-2 (Panel b). In vitro shoot multiplication in Capsi-8 (Panel c), Capsi-6 (Panel d), Capsi-7 (Panel e), Capsi-9 (Panel f),
Capsi-3 (Panel g), Capsi-10 (Panel h), Capsi-1 (Panel i), Capsi-5 (Panel j).

different cultivars, the best results were noted between 6 optimum concentration of auxins had enhanced the
and 8 mg l−l BAP. The SDN in combination with the rate of in vitro root induction in all 10 cultivars
optimum concentration of cytokinin had significantly (Table 4 and Figure 3(a–h)); for example, in Capsi-5
enhanced the growth and multiplication rate of all 10 just 30.2 ± 0.33 roots were induced in the presence of
cultivars (Table 2). Above than the optimum concen- IBA (2.0 mg l−1) plus NAA (1.0 mg l−1), but these
tration of PGRs, a negative effect was observed and the numbers were improved to 36.8 ± 0.29 when 1.5 μM
number of multiplied shoots reduced in all 10 cutivars SND was added along with optimum PGRs.
(Table 3). Overall trends indicate that IAA at low con- Depending upon the cultivar, highest 18.4 ± 0.20
centration in combination with optimal BAP and SDN (maximum for Capsi-9) to 36.8 ± 0.29 (maximum
had enhanced the multiplication rate significantly for Capsi-5) number of roots were induced within
(Table 3). However, the maximum number of multi- 30 days of culture in a half-strength MS medium
plied shoots differed along with different cultivars from containing 2.0 mg l−1 IBA, 1.0 mg l−1 NAA, and
8.9 ± 0.52 (maximum for Capsi-9) to 15.3 ± 0.69 (max- 1.5 mM SDN.
imum for Capsi-5) shoots per explant. This noticeable
difference in the optimum response of different culti-
vars could be because of the recalcitrant nature of the Acclimatization of regenerated plants
genus Capsicum (Figure 2(c–j)). The micropropagated plants of all 10 cultivars were
acclimatized successfully (Figure 3(i–l)), but the sur-
vival rates varied from 40.0 to 86.7 %, depending on
Effect of different auxins and spermidine on in
different cultivars (Figure 4). A maximum 86.7 % (13
vitro root induction
out of 15 plants survived) plants survived in Capsi-5
Multiplied shoots had successfully rooted in the half- followed by 80.0 % (12 out of 15 plants survived) in
strength MS media supplemented with IBA alone or Capsi-2. The lowest survival rate 40.0 % (6 out of 15
in combination with NAA. The best results were plantlets survived) was noted in Capsi-9 and Capsi-
obtained in the presence of 2.0 mg l−1 IBA plus 10. During these hardening periods plantlets grew
1.0 mg l−1 NAA. The SDN in combination with the about 4.0–5.5 cm with two to four new leaf
96 S. M. HAQUE AND B. GHOSH

formation. Overall trends indicated that the survival

Table 4. Effect of IBA, NAA and Spermidine (SDN) on in vitro root induction of ten different cultivars of Capsicum spp. cultured on half strength MS medium. (Data collected after 30 days of

17.7 ± 0.25def
13.6 ± 0.26de

15.0 ± 0.54de
5.8 ± 0.42bc

10.4 ± 0.29cd
18.3 ± 0.38ef
19.4 ± 0.24ef
12.5 ± 0.35d

13.1 ± 0.35d
16.8 ± 0.26e
0.4 ± 0.14a

9.2 ± 0.36c

8.3 ± 0.30c

20.9 ± 0.18f
Capsi-10
rates of the local cultivars are better than the Mexican

Each value represents the mean ± standard error, n = 30 (3 sets, 10 samples in each set). Mean followed by the same letters in each column are not significantly different at P < 0.05 according to Tukey’s multiple range tests.
cultivars (Figure 4). Thereafter, the acclimatized
plants were ready for transfer to field condition.

8.3 ± 0.61bcd

12.5 ± 0.48de

12.8 ± 0.35de
5.1 ± 0.36bc

9.8 ± 0.62cd

10.1 ± 0.51cd
17.3 ± 0.57ef

16.5 ± 0.54ef
3.7 ± 0.71b

11.4 ± 0.58d
15.2 ± 0.62e
6.9 ± 0.65c

18.4 ± 0.20f
Discussion
Capsi-9

The genus Capsicum is a recalcitrant plant in terms of in

0a

vitro cell, tissue, and organ culture (Kothari et al., 2010;

Máthé et al., 2015). Because of its high genotypic depen-
14.8 ± 0.29cde

19.3 ± 0.51def
16.1 ± 0.28de
6.5 ± 0.25bc

7.4 ± 0.26bc
10.2 ± 0.36cd
12.8 ± 0.34cd
10.7 ± 0.15cd

20.1 ± 0.64ef
13.6 ± 0.24d

14.6 ± 0.28d
dence and recalcitrant nature, the in vitro growth of
0.7 ± 0.18a

9.7 ± 0.57c

22.6 ± 0.53f
Capsi-8

Capsicum is comparatively slower than other members

of the Solanaceae (Kehie et al., 2012; Ochoa-Alejo &
Ramirez-Malagon, 2001). The aim of the present study
was to obtain a general method for the multiplication of
Total number of roots induced per shoot after 60 days of implantation

17.5 ± 0.52cde
18.2 ± 0.36de
9.6 ± 0.25bc
14.1 ± 0.51cd

30.3 ± 0.21fg

29.4 ± 0.28fg
25.3 ± 0.24ef

24.4 ± 0.26ef

32.2 ± 0.59g
31.7 ± 0.64g
21.6 ± 0.35e
1.4 ± 0.33a

11.2 ± 0.56c

28.0 ± 0.15f

10 different cultivars of Capsicum. Our results showed

Capsi-7

that BAP play a crucial role in multiple shoot induction in

Capsicum and it proved to be better than KIN for that
purpose. BAP has been reported to be more efficient than
15.9 ± 0.51cde
19.3 ± 0.33def

21.2 ± 0.36def

other cytokinins for inducing multiple shoots in many

16.8 ± 0.41de
7.6 ± 0.42bc
9.2 ± 0.35bc
12.5 ± 0.26cd

13.5 ± 0.25cd

23.8 ± 0.34ef

21.5 ± 0.21ef
5.5 ± 0.30b
0.5 ± 0.23a

10.3 ± 0.35c

24.4 ± 0.26f
Capsi-6

other plants like Tylophora, Aloe, Ipomoea, Withania

(Haque & Ghosh, 2013a, 2013b; Cheruvathur,
Abraham, & Thomas, 2015; Sivanandhan et al., 2015),
including Capsicum (Sanatombi & Sharma, 2008a). The
33.7 ± 0.14gh

34.3 ± 0.25gh
20.9 ± 0.25de

20.5 ± 0.37de
13.8 ± 0.62cd

30.2 ± 0.33fg
17.6 ± 0.24d

36.8 ± 0.29h

31.4 ± 0.18g
22.3 ± 0.12e
2.6 ± 0.12a
12.2 ± 0.26c

27.1 ± 0.21f

26.4 ± 0.28f

present findings showed that the propagation rate of

Capsi-5

Capsicum was significantly increased when IAA was

added in a low concentration along with the optimum
concentration of BAP. These findings totally corroborate
with some of the previous reports on Bacopa, Tylophora,
15.4 ± 0.21def
6.2 ± 0.32bc

10.3 ± 0.54cd

9.7 ± 0.33cd
18.1 ± 0.21ef

17.7 ± 0.31ef
11.7 ± 0.42d

16.3 ± 0.16e
7.2 ± 0.38c
13.0 ± 0.5de
8.9 ± 0.5cd

19.2 ± 0.25f
19.6 ± 0.29f
Capsi-4

Aloe (Haque & Ghosh, 2013a, 2013c; Baskaran, Kumari,

Naidoo, & Van Staden, 2015) including Capsicum spp.
0a

(Mohamed & Alsadon, 2011; Orlińska & Nowaczyk,

2015) where BAP along with IAA proved to be the best
18.1 ± 0.23cde

19.7 ± 0.52de
7.5 ± 0.24bc

8.8 ± 0.14bc
13.5 ± 0.25cd

24.4 ± 0.46ef

23.9 ± 0.36ef

PGR combination for micropropagation. Our findings

16.2 ± 0.41d
22.6 ± 0.50e

22.3 ± 0.25e
0.6 ± 0.15a

10.3 ± 0.32c

11.2 ± 0.12c

26.3 ± 0.34f
Capsi-3

revealed that the different cultivars of Capsicum gave

optimum results in different concentrations of PGR, i.e.
the response is genotype dependent. Similar findings
were reported in previous studies on Capsicum spp.
24.1 ± 0.46def
22.6 ± 0.34de

21.9 ± 0.25de
10.4 ± 0.42bc

10.7 ± 0.25bc
16.8 ± 0.35cd
15.0 ± 0.31cd

27.3 ± 0.48ef

28.8 ± 0.26ef

29.2 ± 0.42ef
25.5 ± 0.41e
1.8 ± 0.24a

13.5 ± 0.51c

31.7 ± 0.62f

(Orlińska & Nowaczyk, 2015; Sanatombi & Sharma,

Capsi-2

2008a).
Polyamines occur universally in plants, especially in
actively growing tissues, and are considered plant growth
31.6 ± 0.38efg

19.5 ± 0.54de

regulators influencing several physiological and develop-

9.5 ± 0.34bc

17.3 ± 0.12cd

33.3 ± 0.52fg

32.4 ± 0.65fg
26.7 ± 0.14ef
18.4 ± 0.51d

35.2 ± 0.35g
2.1 ± 0.23a

13.2 ± 0.52c

20.1 ± 0.2de

29.2 ± 0.56f

35.5 ± 0.1g
Capsi-1

mental processes (Anwar, Mattoo, & Handa, 2015).

Polyamines play a crucial role in the growth and devel-
opment of higher plants by influencing cell division, stem
elongation, root growth, and many other functions
SDN mM

(Podwyszyn’ska, Kosson, & Treder, 2015). Ethylene is

1.0
1.5
2.0
2.5
-
-
-
-
-
-
-
-
-
-

synthesized by plants during certain stages of develop-

ment and its accumulation during in vitro culture acts as
NAA mg l–l

an inhibitor of many plant regeneration systems, includ-

0.5
1.0
1.5
2.0
1.0
1.0
1.0
1.0

ing Capsicum (Orlińska & Nowaczyk, 2015). The exo-

-
-
-
-
-
-

genous application of polyamines will possibly decrease

implantation).

the assembly of unwanted ethylene and increase the

IBA mg l–l

morphogenesis in plant tissues. Chi, Lin, Lee, and Pua

(1994) had also described the enhancing role of polya-
1.0
1.5
2.0
2.5
3.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0

mines in the shoot regeneration of Brassica campestris.

-
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 97

Figure 3. In vitro rooting and acclimatization of different Capsicum cultivars. In vitro rooting in Capsi-1 (Panel a), Capsi-7 (Panel
b), Capsi-10 (Panel c), Capsi-4 (Panel d). In vitro regenerated plants with well developed root system ready for acclimatization in
Capsi-9 (Panel e), Capsi-3 (Panel f), Capsi-2 (Panel g), Capsi-5 (Panel h). Acclimatization of regenerated plants of Capsi-8 (Panel i),
Capsi-3 (Panel j), Capsi-4 (Panel k), Capsi-6 (Panel l).

Capsicum, which clearly have a certain impact particu-

larly when Capsicum is discredited for its recalcitrant
behaviour.
The aim of the present study was to establish a
common protocol for the high frequency of in vitro
rooting in 10 different cultivars of Capsicum. In gen-
eral, the in vitro rooting of regenerated shoots or
shoot buds is difficult in Capsicum species
(Christopher & Rajam, 1994). The promotion of in
vitro rooting by exogenous auxin, mainly IBA and
NAA is well documented in several plant species
(Saraswathi et al., 2016; Zribi, Bayoudh, & Haouala,
2015). In Capsicum spp. in vitro rooting was induced
in the presence of IBA or IAA or NAA individually,
but their combined effect was not studied (Sanatombi
Figure 4. Survival percentage of the regenerated plants of
ten different cultivars of Capsicum after 30 days of & Sharma, 2008a). Our result suggested that a com-
acclimatization. bination of IBA and NAA gave optimum results.
These findings corroborate with the findings of
Saraswathi et al. (2016) during establishment of a
Our results showed the exogenous application of SDN cost-effective micropropagation method of banana,
had improved the multiple shoot induction rate in where maximum rooting was achieved in the pre-
Capsicum along with the growth and elongation of the sence of the combination of IBA and NAA. The
multiplied shoots. The similar optimistic role of polya- supportive role of SDN on root induction in
mines, especially SDN, on the growth and development Capsicum was not checked by any authors of the
of plants was previously reported by several authors previous reports, but SDN was examined in combi-
Kumar et al., 2007; Haque & Ghosh, 2016. However, nation with auxins which proved to be much bene-
the present protocol had standardized on 10 different ficial. Our findings revealed that the stimulating effect
cultivars belonging to three different species of of IBA and NAA was further improved by
98 S. M. HAQUE AND B. GHOSH

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