AsPac J. Mol.
2014
Mol. Biol.
Biol.Biotechnol.
Biotechnol.
Vol. 22 (1), 2014
Vol. 22(1) : 137-144
AgNO3 and GA3 in in vitro Naga chilli propagation
137
Effect of silver nitrate and gibberellic acid on in vitro regeneration,
flower induction and fruit development in Naga Chilli
G. Bora1,2*, H. K. Gogoi1, P. J. Handique2
Division of Biotechnology, Defence Research Laboratory, Tezpur-784001, Assam, India
2
Department of Biotechnology, Gauhati University, Guwahati-781014, India
1
Received 24th September 2013 / Accepted 18th February 2014
Abstract. In vitro flower induction and fruit development are rarely achieved in the genus Capsicum. This communication
reports for the first time the induction of in vitro flower induction and fruit development in Bhot jolokia or Naga Chilli under
the influence of silver nitrate (AgNO3) and gibberellic acid (GA3). The greatest number of multiple shoots from a single shoot
apical meristem was induced in MS medium fortified with BAP (45µML-1), NAA (5.5µML-1) and AgNO3 (35µML-1). Half
strength MS medium supplemented with AgNO3 at a concentration of 34µML-1 and GA3 at 28µML-1 was found to be the
optimum concentration for in vitro flower induction, and AgNO3 at 32µML-1 added to half strength MS was found best for
in vitro fruit development. In vitro regenerated shoots were provided with rooting medium containing MS medium, IBA and BAP at
3.5μML-1 and 2.5μML-1, respectively. Healthy rooted plantlets were transferred to potting substrate where half strength MS
medium was enhanced with soil and vermicompost in equal volumes (1:1) for hardening in the poly house. 72.6% of all
acclimatised plants were successfully transferred to the open field.
Keywords: In vitro flower induction, In vitro fruit development, In vitro shoot induction, Naga Chilli.
INTRODUCTION
Pungency of flavour is the characteristic for which all
Capsicum plants are commercially valued. This pungent trait
lies in the seed of the fruit and the adjacent placenta. Naga
Chilli is one of the world’s hottest chillis, belonging to the
genus Capsicum and the Nightshade family, Solanaceae. This
rare type of chilli is endemic to Assam and the nearby North
Eastern region of India (Bhagowati and Changkija, 2009).
This chilli is best known for its peculiar aroma and unique
heat. In the year 2007 it gained a name and widespread fame
for being the world’s hottest chilli with an exceptionally high
Schoville Heat Unit (SHU) value of about 10,41,427. Since
then this chilli is discussed frequently in the world media.
Capsicum species are well-known for possessing
many medicinal properties. Chillies contain numerous
chemicals including steam-volatile oil, fatty oils,
capsaicinoids, carotenoids, vitamins, protein, fibre and
mineral elements (Bosland and Votava, 2000; Sanatombi
and Sharma, 2007). Chilli peppers are found to be most
effective in curing gastric problems and stomach ulcers.
Capsicum oleoresin contained in pepper sprays behaves as
a non-lethal repellent to both animal and human targets
(De, 2003) and serves as a valuable riot control agent and
self defence tool (Sanatombi and Sharma, 2007). Chilli
extract has an analgesic effect against many painful
conditions such as post-herpetic neuralgia, diabetic
neuropathy, osteoarthritis and mouth sores (Nelson,
1994; Rains and Bryson, 1995). Apart from this, chillies
also possess in vitro anti-tumour-promoting activities and
synergistic anti-mutagenic properties (de Meija et. al.
1998; Maoka et.al., 2001). Despite having such valuable
medicinal properties, very little scientific work has been done on
Capsicum and particularly on Naga Chilli.
Tissue culture is an excellent way to maintain the desired
pungency and yield true-to-the-type fruit. But Capsicum
species are highly recalcitrant to micropropagation
techniques (Sharma et.al., 2008). Even though there
are several reports of in vitro regeneration of different
domesticated species of Capsicum (Christopher and Rajam,
1994; Szasz et al., 1995; Christopher and Rajam, 1996;
Ramirez -Malagon and Ochoa-Alejo, 1996; Steinitz et.al.,
1999; Bodhipadma and Leung, 2003; Venkataiah et.al.,
2003, Vinod et.al., 2005; Kumar Anil and Nair, 2004;
Khan et al., 2006; Peddaboina et.al., 2006; Sanatombi and
Sharma, 2006; Sanatombi and Sharma, 2007; Sanatombi
and Sharma, 2008; Sharma et. al. 2008; Ashrafuzzaman et al.,
* Author for correspondence: Ms. Geetashree Bora, Christian Colony, Jail
Road, Borbheta, Jorhat-785004, Assam, India. Tel.: - 91-7896851882,
Email - [email protected].
138
AsPac J. Mol. Biol. Biotechnol. Vol. 22 (1), 2014
AgNO3 and GA3 in in vitro Naga chilli propagation
MATERIALS AND METHODS
2009; Kothari et al., 2010; Song et al., 2010; Otroshy et.al.,
2011; Kumari Maya et al., 2012, Gogoi et al., 2014), there
are no such reports of tissue culture of Naga Chilli to date.
In vitro flower induction and fruit development are
rarely observed in chilli plants (Tisserat and Galleta, 1995;
Sharma et. al. 2008). Gibberellic Acid (GA3) has been
reported by several authors to facilitate in vitro flower
development in different crops (Rkhis et.al., 2006;
Ziv and Naor, 2006). GA3 is known to regulate many
physiological processes in plants including seed
germination, shoot development and elongation, and flower
development. However for flowers to be induced in vitro
there are other requirements including genetic, hormonal
and trophic factors (Rkhis et.al., 2006). The response of plant
species towards GA3 is also dependent on genotype, type of
explants used, media composition, and culture conditions
including temperature and photoperiod (Rkhis et.al., 2006).
Another important compound that has records of
being a good inducer of flowering and fruiting is
silver nitrate (AgNO3). Silver nitrate enhances flower
induction by suppressing the action of ethylene
(Beyer, 1976; Sharma et.al, 2008). It also has a profound
effect on multiple shoot induction. The regeneration
potential of Naga Chilli using GA3 and AgNO3 is
investigated in this study. This communication reports
for the first time the occurrence of in vitro flower induction
and fruit development in Naga Chilli using shoot tips as
explant material.
Plant material. Seeds of Naga Chilli “Bhot Jolokia” grown
in the greenhouse of the Defence Research Laboratory,
Tezpur, were selected for the present investigation. Healthy
seeds were placed under running tap water for 15-20
minutes and treated with 0.1% Bavistin solution (w/v)
for another 15-20 minutes. Seeds were thoroughly
washed with distilled water 3-4 times and subjected to
decontamination by rinsing with 70% alcohol for 5-10
seconds, followed by surface sterilisation with an aqueous
solution of 0.1 (w/v) HgCl2 for 3 minutes, followed by
thorough rinsing with sterile distilled water 4-6 times to
remove any traces of HgCl2. Seeds were aseptically
inoculated onto basal MS medium (Murashige and Skoog,
1962), without supplementing any plant growth regulators
(PGRs), which are not required for germination of seeds.
The basal medium was supplemented with 3% (m/v) sucrose
(Himedia, Mumbai, India) and 0.8% (m/v) agar was used as
a gelling agent. The pH of the medium was adjusted to 5.8
and autoclaved at 1.06 kg cm-2 and 121°C for 15 minutes
prior to inoculation. The cultures were incubated at 25±2°C
and 16 hour photoperiod using cool white fluorescent tubes.
In vitro shoot induction.
Healthy apical meristems
(1.5-2cm in length) were aseptically trimmed from a
15 day old mother culture and inoculated onto
proliferation medium consisting of MS basal medium
supplemented with BAP (45µML-1), NAA (5.5µML-1) and
AgNO (35µML-1), respectively (Figure 1(a)). Data for
number of multiple shoot buds induced was collected from
four-week-old cultures and the increases in shoot length are
represented in Table 1 and Figure 1(b).
Table 1. Effect of various media components on in vitro regeneration, multiple shoot induction and
shoot length in Naga Chilli.
Sl.No.
Treatments
MS+ BAP, NAA
and AgNO3 (µM/L)
1
2
3
4
5
6
7
8
9
10
20.0+3.0+10.0
25.0+3.5+15.0
30.0+4.0+20.0
35.0+4.5+25.0
40.0+5.0+30.0
45.0+5.5+35.0
50.0+6.0+40.0
55.0+6.5+45.0
60.0+7.0+50.0
65.0+7.5+55.0
No. of shoot
forming explant
Mean±SE
11.0±2.62a
20.4±2.67b
28.3±2.9c
35.7±4.13d
43.4 ± 1.89e
49.1 ± 1.28f
40.9 ± 2.76e
31.6 ± 3.23g
20.8± 4.02b
14.5 ± 4.37h
No. of multiple
shoots/explant
Mean±SE
1.6 ± 0.69a
2.6 ± 0.51b
3.2 ± 0.63c
3.7 ± 0.67c
4.7 ± 0.67d
6.3 ± 0.94e
4.9 ± 1.10d
4.1 ± 1.10d
3.2 ± 1.03c
2.1 ± 0.56b
Shoot length
(cm)
Mean±SE
2.6 ± 0.33a
3.7 ± 0.21b
4.4 ± 0.24c
5.2 ± 0.39d
5.9 ± 0.37e
7.0 ± 0.46f
5.9 ± 0.37e
5.1 ± 0.63d
4.3 ± 0.49c
3.2 ± 0.60g
SE(Means) = 1.40 SE(Means) = 0.36 SE(Means) = 0.19
NB. Means followed by the same letters are not significantly different at p=0.05.
AsPac J. Mol. Biol. Biotechnol. Vol. 22 (1), 2014
AgNO3 and GA3 in in vitro Naga chilli propagation
In vitro flower induction and fruit development. For
in vitro flower induction, healthy shoot buds were selected
from six-week-old cultures and transferred into half strength
MS medium supplemented with AgNO3 (34 µML-1) and
GA3 (28µML-1). In vitro flowering was recorded after 35 days
of inoculation and data presented in Table 2 and Figure 1 (c)
and (d). Terminal shoots with in vitro flowers were carefully
transferred to half strength MS medium supplemented with
AgNO3 (32µML-1) and cultures were subjected to 16/8 h
light/dark cycle for fruit development (Figure 1 (e)).
In vitro root induction and hardening.
Shoots
successfully raised in vitro were provided with rooting
medium in which auxin was combined with cytokinin.
For in vitro root induction, MS medium was modified by
adding IBA (3.5µML-1) and BAP (2.5µML-1) and data
were collected from four-week-old cultures to see the
number of roots generated in vitro (Table 3., Figure 1 (f )).
Rooted plantlets were carefully taken out from the rooting
medium and washed gently with tap water to remove
any traces of medium. These plantlets were transferred to
perforated plastic cups containing half strength MS mixed
with soil and vermicompost (1:1) and kept in the poly
house for hardening (Figure 1(g)). The tops of the cups were
covered with polythene bags perforated with a few holes
for an initial 10-15 days. For this period, to maintain highly
humid conditions, plantlets were watered regularly, after
which humidity was reduced by removing the polythene
bags. Six week old hardened plants were finally transferred to
the open field. The percentage plant survival was recorded.
RESULTS
Multiple shoot induction in vitro. Naga Chilli seeds used
for the present experiment were found to exhibit very high
germination frequency (95%) when inoculated onto MS
basal medium. From germination until the appearance of
the first single node took between 18 and 23 days. Healthy
shoot apices cultured onto proliferation medium gave
the best results when BAP (45µML-1), NAA (5.5µML-1)
and AgNO3 (35µML-1) were added to MS basal medium
(Table 1 and Figure 1 (a) and (b)). Concentration
of AgNO3 was very crucial through the entire experiment
in determining the number of multiple shoots induced.
A slight increase or decrease in its concentration greatly
affected multiple shooting. A mean of 6.3 shoots per
explant was obtained when AgNO3 (35 µML-1) was added to
shoot induction medium. This was taken to be the optimum
concentration and used for the rest of the experiment.
The same hormonal combination was also found to be
effective in increasing the shoot length wherein a mean
of 7 cm increase in shoot length was recorded (Table 1).
Any alteration in this combination negatively
affected both multiple shoot induction and shoot length.
In vitro flower and fruit induction. The greatest number
of flowers was obtained when the two key components were
combined together: a mean of 7.2 flowers per explant was
recorded when AgNO3 (34µML-1) and GA3 (28µML-1) were
added to MS medium (Table 2 and Figure 1 (c) and (d)).
Table 2. Effect of different concentrations of GA3 and AgNO3 on flower and fruit induction in vitro.
Sl. No.
1
2
3
4
5
Treatments
No. of flower buds/
MS+ GA3 and
explants
AgNO3 (µM/L)
Mean±SE
26 + 0
28 + 34
30 + 0
0 + 32
0 + 30
10.4±1.26a
13.3±1.15b
10.5±1.65a
12.5±1.77b
8.8 ± 1.03c
139
No. of flowers/
explant
Mean±SE
3.6 ± 0.96a
7.2 ± 0.91b
4.6 ± 1.07c
6.7 ± 1.25b
3.6 ± 1.07a
SE(Means) = 0.62
SE(Means) = 0.46
Means followed by the same letters are not significantly different at p=0.05.
No. of fruits/
explants
Mean±SE
2.9 ± 0.99a
5.2 ± 0.63b
3.2 ± 1.54a
5.3 ± 1.33b
2.1 ± 0.99a
SE(Means) = 0.51
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AsPac J. Mol. Biol. Biotechnol. Vol. 22 (1), 2014
AgNO3 and GA3 in in vitro Naga chilli propagation
Figure 1. In vitro flowering and fruiting of Naga Chilli under the influence of AgNO3 and GA3. a) In vitro regeneration of
shoots on MS medium supplemented with BAP (45µM/L), NAA (5.5µM/L) and AgNO3 (35 µM/L), b) Induction of multiple
shoots, c) In vitro flower induction on MS with AgNO3 (34 µM/L) and GA3 (28 µM/L), d) Fully bloomed flower under in vitro
condition, e) In vitro fruit induction, f ) Initiation of roots on MS supplemented with IBA (3.5 µM/L) and BAP (2.5µM/L),
g) In vitro regenerated plantlets transferred to potting material containing half strength MS medium, and soil and
vermicompost (1:1:1).
Flower induction was significantly reduced when one of
these components was omitted from the medium. It was
observed that AgNO3 (32µML-1) alone was also effective in
producing a sufficient number of flowers per explant (6.7 ±
1.25b) but its effect increased in the presence of GA3 (Table
2).
Application of GA3 in the absence of AgNO3 was not
seen to be very effective in inducing a greater number of
flowers in this experiment. A mean of 4.6 flowers per explant
was recorded on addition of GA3 (30µML-1) to the flower
induction medium
In vitro induction of fruit development has been a rare
phenomenon in Naga Chilli. A mean of 5.3 fruits per
explant was obtained when AgNO3 (32µML-1) alone was
added to growth medium. In vitro development of fruit was
also recorded when AgNO3 (34µML-1) and GA3 (28µML-1)
AsPac J. Mol. Biol. Biotechnol. Vol. 22 (1), 2014
AgNO3 and GA3 in in vitro Naga chilli propagation
Table 3. Effect of different concentrations of IBA and BAP
on root induction and root length in vitro
Sl. Treatments
No MS+ IBA
and BAP
(µM/L)
1
2
3
4
5
6
2.0+2.5
2.5+2.5
3.0+2.5
3.5+2.5
4.5+2.5
5.0+2.5
No. of roots/
regenerated
shoot
Mean±SE
Root length
(cm)
29.3±4.98a
40.1±6.60b
50.3±7.18c
62 .0±7.13d
50.3 ± 7.95c
37.3 ±8.32b
3.8 ± 1.27a
5.5 ± 0.46a
7.5 ± 1.96b
11.6 ± 3.13c
16.03 ± 2.49d
12.54± 2.70c
SE(Means) = 3.18
SE(Means) = 0.98
Means followed by the same letters are not significantly
different at p=0.05
were combined together (Table 2 and Figure 1 (e)). When
GA3 (30µML-1) alone was added to the medium, a mean of
3.2 fruits per explant was recorded. Fruits developing in vitro
were all healthy and remained in that condition for 6-7 days.
In vitro rooting and hardening of plantlets in the poly
house. For effective in vitro root induction in Naga Chilli,
various combination of IBA (ranging from 2 to 5µML-1)
were tested, keeping the concentration of BAP constant at
2.5 µML-1. The greatest number of roots (62.0±7.13) per
shoot was generated at concentrations of IBA of and BAP of
3.5µML-1 and 2.5µML-1, respectively (Table 3 and Figure 1
(f )). It was observed that as a root inducing hormone, IBA
was not able to induce roots profusely when it was applied
to the medium alone. The greatest increase in root length
(to a length of 16.03 ± 2.49 cm) was seen at treatment
combinations of 4.5µML-1 IBA and 2.5µML-1 BAP
(Table 3).
In vitro rooted plants were transferred to the poly
house for hardening after 25-30 days of initial rooting.
Hardening was carried out on substrate comprising half
strength MS, soil and vermicompost in a 1:1:1 ratio. A
maximum of 450 in vitro-raised plantlets were transferred to
the poly house and maintained there for 30-40 days under
appropriately humid conditions. Finally, 327 healthy plants
were transferred to the open field and yielded a survival rate
of 72.6%.
Experimental design and statistical analysis.
All
experiments were conducted with a Completely
Randomised Design (CRD) and repeated thrice, with each
treatment having 10 replicates. Significant differences between
treatments was calculated through One-Way ANOVA and
differences among treatment means were based on Least
Square Difference (LSD) and considered significant when
P<0.05
141
DISCUSSION
Seeds of the genus Capsicum are fairly recalcitrant to
in vitro germination. Unless provided with a rich and
appropriate source of nutrients, they fail to
germinate in vitro. Seeds of Naga Chilli obtained from the
Defence Research Laboratory (DRL) in Tezpur showed
a good germination potential when cultured onto
full strength MS basal medium without any growth
hormones: 95% germination was recorded. Even dried seeds
were found to be equally effective when soaked overnight
in luke warm water prior to inoculation the following day.
The medium used for in vitro regeneration of
Naga Chilli was supplemented with different
concentrations of AgNO3 in combination with BAP and
NAA. In the said investigation, BAP along with NAA
could also initiate multiple shoots but the number of
shoots per explant increased significantly in the presence
of AgNO3. A mean of 6.3 healthy multiple shoot buds
were regenerated from each explant when AgNO3
(35µML-1) was added to shoot induction medium along
with BAP (45µML-1) and NAA (5.5µML-1) (Table 1).
Growth of the shoot also increased significantly to a
maximum of 7cm in length at this particular hormonal
combination. Upon altering the concentrations of BAP and
AgNO3 a drastic negative change in the growth pattern was
observed both in terms of shoot bud formation and shoot
length. To date several regeneration protocols have been
standardised for different species of Capsicum. Although the
morphogenetic potential of AgNO3 was well documented
in different plant systems such as Coffea (Giridhar et al.
2003; Giridhar et al. 2004), Decalepis (Obul Reddy et al.
2001), and Vanilla (Giridhar et al. 2001), the effect of the
component was for the first time investigated here in the
in vitro regeneration of Naga Chilli.
In vitro flowering and fruiting are rarely observed
in the genus Capsicum. Tisserat and Galletta (1995)
reported in their investigation in vitro flowering and
fruiting in C. frutescens upon culturing shoot tips in a
polycarbonate container coupled to an automated plant
tissue culture system, and Sheeja and Mandal (2003)
have investigated the effect of BAP on flower bud
initiation in tomato. Sharma et al. (2008) reported the
effect of AgNO3 and CoCl2 on in vitro flowering in
C. frutescens. In the present investigation it was observed
that in vitro induction of flower and fruit development
are primarily controlled by AgNO3 and GA3, respectively.
Cytokinins contained in the medium stimulate
flowering in vitro on shoots of C. annuum
(Tomaszewska–Sowa et al., 2002). The present results
confirm the activity of these growth regulators in control
of plant flowering. As components of the signalling system,
they transfer information to shoot meristems about changes
in the morphogenetic programme (Galoch et al., 1996).
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AsPac J. Mol. Biol. Biotechnol. Vol. 22 (1), 2014
Addition of AgNO3 along with GA3 to flower and fruit
induction media significantly increased the number of
flower buds forming as well as fruit development. The
maximum number of flower buds produced per shoot
(7.2 ± 0.91) was produced when 34µML-1 AgNO3 was
provided in combination with 28µML-1 GA3 (Table 2).
AgNO3 is a potent flower inducer, as evident from the
work of Giridhar et al. in the case of coffee (2003) and
Sharma et al. (2008) in C. frutescens. It was able to produce
a mean of 6.2 flowers per explant i the current study when
applied alone at a concentration of 32µML-1. Off white
flowers (with 5 sepals and 5 petals) remained healthy and
in an open condition for almost 15 days. A very high dose
(above 34µM) not only reduced the number of flowers
developing but also the leaf size. A concentration lower
than 30µML-1 did not produce any flower at all. AgNO3
is considered to be a powerful ethylene inhibitor
(Beyer, 1976), and ethylene is known to inhibit the action
of an enzyme called S-adenosyl methionine decarboxylase
which is responsible for production of polyamines that
promote flower induction. Thus the application of silver
nitrate promotes in vitro flowering by inhibiting
ethylene production. The effect of AgNO3 was also elucidated
during the process of in vitro fruit induction. A mean of 5.3
fruits per explant was recorded when AgNO3 (32µML-1) was
added to the medium. The fully matured fruit attained a
diameter of approximately 4 cm and girth of about 5.8 cm
and remained healthy for almost 1 week before starting to
deteriorate.
Another potent regulator of flower and fruit induction
both in vitro and in vivo is GA3. Induction of floral bud
development in response to the application of GA3
has been recently reported in C. annuum by Kumari
Mira et al. (2012). The effect of GA3 was here investigated
in Naga Chilli. It was able to induce a mean of 4.6 flowers
per explant when applied alone at a concentration of 30
µML-1 whereas when coupled with AgNO3 (28µML-1),
profuse flowering was recorded (7.2 ± 0.91).
The healthy shoots regenerated in vitro were transferred to
root induction medium containing full strength MS fortified
with BAP and IBA in different combinations, as represented
in Table 3. The combination of IBA (3.5µML-1) and BAP
(2.5µML-1) yielded the highest number of roots (62.0±7.13)
from a single regenerated shoot. The roots regenerated
in vitro were healthy, elongated and branched. However
roots attained a maximum length of 16.03 ± 2.49 cm when
4.5µML-1 of IBA was added along with 2.5µML-1 of BAP
to MS medium. Higher doses of IBA generated shorter
and thinner roots whereas doses lower than 3µM produced
very few roots. IBA was found to be an efficient auxin in
promoting rooting in Chilli, as has been reported previously
(Peddaboina et al., 2006; Sanatombi et al., 2007; Kumari
Maya et al., 2012). However, other reports have suggested
that IAA has a more profound effect on in vitro rooting in
Capsicum (Bodhipadma and Leung, 2003; Siddique and
Anis, 2006).
AgNO3 and GA3 in in vitro Naga chilli propagation
The in vitro rooted Naga Chilli plantlets were
transferred to poly house for hardening and
acclimatization. For that purpose half strength MS
medium with several potting substrates were tested, of
which soil and vermicompost in a 1:1 ratio gave the best
results. Inclusion of biofertilizer in the soil not only
improved the soil status but also the health of the plants and
increased survival (72.6%). This observation complies with
the earlier report submitted by Verma et al. (2013) where
the authors acclimatised in vitro regenerated plantlets of
C. annuum cv. California Wonder in potting mixture
containing soil, sand and biofertilizer in the form of
farmyard manure.
CONCLUSION
The present investigation brought into focus for the first
time an efficient in vitro regeneration protocol using shoot
apical meristems. The importance of AgNO3 for various
processes including in vitro shoot regeneration and flower
and fruit induction has been elucidated. Naga Chilli, being
one of the hottest chillies, has tremendous economic and
research potential as food components, medicines and
pharmaceuticals. Tissue culture of Naga Chilli is still in its
infancy. The present investigation opens a way to further
improve results through micropropagation techniques.
ACKNOWLEDGEMENTS
We thank the Director, Defence Research Laboratory
(DRL), Tezpur, Assam for providing showing keen interest
in the work and providing a DRDO fellowship. The DRL
and Department of Biotechnology, Gauhati University are
duly acknowledged for providing the necessary facilities and
permission for the work.
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