Plant Cell Tiss Organ Cult (2010) 102:129134

DOI 10.1007/s11240-010-9712-x

RESEARCH NOTE

Efficient regeneration and antioxidant potential in regenerated

tissues of Piper nigrum L.
Nisar Ahmad Hina Fazal Bilal Haider Abbasi
Muhammad Rashid Tariq Mahmood
Nighat Fatima

Received: 12 November 2009 / Accepted: 11 February 2010 / Published online: 6 March 2010
Springer Science+Business Media B.V. 2010

Abstract The organogenic potential and antioxidant

potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper)
were investigated. Callus induction and shoot regeneration
were induced from leaf explants of potted plants cultured
on MS medium supplemented with different plant growth
regulators. The best callogenic response was observed on
explants cultured for 30 days on MS medium supplemented
with either 0.5 or 1.5 mg l-1 6-benzyladenine (BA) ?
1.0 mg l-1 a-naphthaleneacetic acid. Subsequent transfer
of the callogenic explants onto MS medium supplemented
with 1.5 mg l-1 BA ? 1.0 mg l-1 gibberellic acid (GA3)
achieved 85% shoot organogenesis after 30 days of culture.
The maximum number (7.2) of shoots/explant was recorded
for explants cultured in MS medium supplemented with
1.0 mg l-1 BA. Following the transfer of shoots to an
elongation medium, the longest shoots (5.4 cm) were
observed on MS medium supplemented with 1.0 mg l-1 BA
? 1.0 mg l-1 GA3. The elongated shoots were rooted on MS
medium supplemented with different concentrations of

N. Ahmad  B. H. Abbasi (&)  N. Fatima

Department of Biotechnology, Faculty of Biological Sciences,
Quaid-i-Azam University, Islamabad 45320, Pakistan
e-mail: bhabbasi@qau.edu.pk
H. Fazal  T. Mahmood
Department of Plant Sciences, Quaid-i-Azam University,
Islamabad 45320, Pakistan
M. Rashid
Department of Botany, Gomal University, Dera Ismail Khan,
Pakistan
H. Fazal
Pakistan Council of Scientific and Industrial Research (PCSIR)
Laboratories Complex, Peshawar 25100, Pakistan

indole butyric acid. An assay of the antioxidant potential of

the in vitro-grown tissues revealed that the antioxidant
activity of the regenerated shoots was significantly higher
than that of callus and the regenerated plantlets.
Keywords Antioxidant  6-Benzyladenine 
Gibberellic acid  Organogenesis  Piper nigrum
Abbreviations
BA
6-Benzyladenine
2,4 D 2,4 Dichlorophenoxyacetic acid
DPPH 1, 1-Diphenyl-2-picrylhydrazyl
GA3
Gibberellic acid
IBA
Indole butyric acid
MS0
MS medium without plant growth regulators
NAA
a-Naphthaleneacetic acid
PGRs Plant growth regulators

Piper nigrum L. (black pepper) is a flowering vine in the

family Piperaceae that is primarily cultivated for its fruit,
which is usually dried and used as a spice and seasoning
(Joseph et al. 1996). Black pepper is native to South India and
is extensively cultivated there and elsewhere throughout the
tropical regions. Dried ground pepper is one of the most
common spices in European cuisine and its descendants,
having been known and prized since antiquity for both its
flavour and its use as a medicine. The spiciness of black
pepper is due the presence of the chemical, piperine (1-piperoylpiperidine), which is a nitrogenous pungent substance
and a major alkaloid in black pepper. Piperine has been
shown to have anti-inflammatory, analgesic, anticonvulsant,
anti-ulcer and antioxidant activities and cytoprotective and

123

130

anti-depressant effects (DHooge et al. 1996; Bai and Xu

2000; Gupta et al. 2000; Selven-diran et al. 2003; Lee et al.
2005). In a recent study, Wattanathorn et al. (2008) demonstrated that piperine also affects mood and cognitive disorder. Piperine can dramatically increase the absorption of
selenium, vitamin B and b-carotene as well as other nutrients
(Bhardwaj et al. 2007).
The germplasm of P. nigrum conserved in natural
repositories is under serious threat from various environmental stresses. In addition, germplasm conservation in a
seed bank is not pragmatic due to heterozygous nature
induced through cuttings (Nair and Gupta 2006). In vitro
propagation methods provide powerful tools for the mass
multiplication and germplasm conservation of this economically important species. To date, there have been no
reports on the successful in vitro regeneration of P. nigrum
from leaf explants of potted plants. However, several
protocols for regeneration and somatic embryogenesis in
P. nigrum from shoot tips and nodal explants have been
published (Philip et al. 1992; Bhat et al. 1995; Joseph et al.
1996; Nair and Gupta 2006).
Kapoor et al. (2009) recently demonstrated that the
protective effect of piperine is most likely due to its antioxidant activity. Although a few reports on the antioxidant
activity of cultivated P. nigrum are available in the literature (Singh et al. 2008; Topal et al. 2008; Aziz et al. 2009;
Misharina et al. 2009 ), there seems to be no mention of the
influence of organogenesis on antioxidant activity in
P. nigrum. In the study reported here, we established an in
vitro system for the production of P. nigrum from leaf
explants of potted plants and conducted 1, 1-diphenyl-2picrylhydrazyl (DPPH8)-based antioxidant assays to evaluate the antioxidant activity of the main secondary
metabolites in different in vitro-derived tissues.
Leaves were collected from potted plants of P. nigrum
maintained in the greenhouse. Leaves were sterilized
according to the method of Abbasi et al. (2010a). Briefly,
the leaves were immersed first in 70% (v/v) ethanol for 60 s
and then in a 0.2% (w/v) mercuric chloride (HgCl2) solution
for approximately 2 min, followed by three rinses with
sterile distilled water. These surface-sterilized explants
were placed on MS (Murashige and Skoog 1962) medium
containing 30 g l-1 sucrose, and solidified with 8 g l-1
agar (Agar Technical LP0013; Oxoid, Hampshire, England). Different plant growth regulators (PGRs) were added
to the medium, and the pH was adjusted to 5.8. All media
were autoclaved at 121C for 20 min. All cultures were
maintained in a growth room at 25 1C under a 16/8-h
light/dark photoperiod with light provided by cool-white
fluorescent tubes at an intensity ranging from approximately 40 to 50 lmol m-2 s-1 (Abbasi et al. 2010a).
For regeneration, leaf explants were cut into reasonably
sized pieces, and placed onto MS medium supplemented

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Plant Cell Tiss Organ Cult (2010) 102:129134

with either 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (BA) or gibberellic acid (GA3) or with BA in
combination with 1 mg l-1a-naphthaleneacetic acid (NAA)
or 1 mg l-1 GA3. MS basal medium (MS0) without any PGR
was used as a control. About five to six explants were incubated in a 100-ml Erlenmeyer flask containing approximately 25 ml of medium. After about 4 weeks of culture, the
percentage callus induction was recorded. Yellowish-green
callus was excised and transferred to fresh MS medium
containing a similar composition of PGRs for organogenesis.
Data on percentage shooting, number of shoots per explant
and mean shoot length were recorded after 5 weeks following subculture of the callus cultures. Elongated shoots
were transferred to MS0 or MS containing indole butyric
acid (IBA) for rooting. After 5 weeks, the rooted plantlets
were removed, washed with water to remove any remaining
medium and transferred to a potting soil mixture where they
acclimatized and were grown under controlled conditions.
The antioxidant activity of the regenerated tissues, such
as callus, regenerated shoots and regenerated plantlets, was
determined using the method of Amarowicz et al. (2004)
with some modifications. Briefly, 10.0 mg of dried plant
tissue was dissolved in 4 ml of methanol and then added to
a methanol solution of DPPH (1 mM, 0.5 ml). The mixture was vortexed for 15 s and then left to stand at room
temperature for 30 min. The absorbance of the resulting
solution was read on a spectrophotometer at 517 nm.
A methanol solution of DPPH8 that had decayed and hence
no longer exhibited a purple colour [i.e. 2 mg of butylated
hydroxyanisole (BHA) dissolved in 4 ml of methanol with
0.5 ml of DPPH8 solution added] was chosen for background correction instead of pure methanol. The radical
scavenging activity was calculated as the percentage of
DPPH8 discolouration using the equation
% scavenging DPPH free radical 100  1  AE =AD
where AE is the absorbance of the solution when the extract
has been added at a particular level and AD is the absorbance of the DPPH8 solution with nothing added.
For the statistical analysis, each treatment consisted of
five culture flasks and data were collected from treatments
carried out in triplicate. Analysis of variance and Duncans
multiple range test were used to compare the means of the
different treatments.
The overall objective of current research was to develop
an efficient regeneration protocol for P. nigrum from leaf
explants (Fig. 1) and to evaluate the antioxidant activity of
the regenerated plant tissues. Several protocols for plant
regeneration have been reported for different species of
Piper, including P. nigrum (Philip et al. 1992; Bhat et al.
1995; Joseph et al. 1996; Kelkar et al. 1996; Kelkar and
Krishnamurthy 1998; Madhusudhanan and Rahiman

Plant Cell Tiss Organ Cult (2010) 102:129134

2000Nair and Gupta 2006). However, to the best of our

knowledge, there has been no published report on successful regeneration from leaf explants of P. nigrum due to
the recalcitrance of leaf tissue to in vitro conditions.
There has been mention of endogenous microbial contamination causing severe setbacks to the in vitro establishment of aseptic cultures of P. nigrum (Philip et al.
1992; Bhat et al. 1995). To avoid such issues, we used the
valuable protocol of Abbasi et al. (2010a) to decontaminate
leaf explants and observed no contamination in subsequent
experiments. The application of this decontamination protocol significantly decreased the levels of contamination by
approximately 75% (B5%; data not shown). In many of the
protocols used for decontaminating leaf explants, the
explants have shown a sensitivity to the decontaminating
agents (Eeswara et al. 1999; Debnath 2009; Makunga et al.
2003; Atak and Celik 2009). However, we did not observe
any inhibitory effect of the decontamination protocol on
regeneration (data not shown).
The effects of various PGRs, such as BA, GA3, 2,4-D
alone or BA in combination with 1 mg l-1 GA3 or 1 mg l-1
NAA on indirect organogenesis were evaluated (Figs. 2, 3, 4,
5). The leaf explants of P. nigrum used in our study
responded to all of the PGRs used (Fig. 1). The best callus
induction was recorded on MS medium supplemented with
0.5 mg l-1 BA alone (93%) and with 1.5 mg l-1 BA ?
1.0 mg l-1 NAA (90%; Fig. 1). Callus induction on medium
containing only 2, 4-D (42%) or GA3 (32%) was significantly
lower than that in medium containing the other PGRs, and no
callus induction was observed on MS0 medium. However,
the addition BA to GA3-containing medium enhanced callus
induction (70%) to levels comparable to those obtained with
1.0 mg l-1 BA alone and 2.0 mg l-1 BA ? 1 mg l-1 NAA.
In a recent study on Silybum, we reported that the addition of
NAA to medium containing BA/GA3 enhanced callus
induction (Abbasi et al. 2010a). Bhat et al. (1995) did not
observe any response of leaf explant of P. nigrum to PGRs;
however, other species of Piper have been successfully
regenerated from leaf explants on a similar medium. The
findings of Philip et al. (1992) on shoot-tip explants of
P. nigrum are in agreement with our data. Madhusudhanan

131

and Rahiman (2000) reported that the addition of activated

charcoal to the culture medium induced callus formation
but failed to produce regenerants from leaf explants of
P. nigrum. Differences in the data among these studies are
likely due to (pre-)existing genetic diversity between
explants (Abbasi et al. 2010b).
Callogenesis is considered to be a significant feature of
indirect organogenesis and essential for research on biologically active molecules in medicinal plant species
(Abbasi et al. 2007, 2010a). Data on organogenesis was
determined after 5 weeks of subculture. The best percentage shooting was recorded for explants cultured on medium containing the combination of 1.5 mg l-1 BA ?
1 mg l-1 GA3 (85%). However, the combination
2.0 mg l-1 BA ? 1 mg l-1 GA3 produced a shooting
percentage of 78%, which was similar that obtained on
medium containing 0.5 mg l-1 BA (75%). In contrast, the
addition of NAA to medium already containing BA significantly inhibited shooting. Similar values have been
reported for Capsicum species (Rubluo and Barroso 1992).
However, in another study on Silybum, Abbasi et al.
(2010a) made different observations regarding the incorporation of auxin in cytokinin-containing medium. Bhat
et al. (1995) obtained a maximum number of differentiated
shoot buds for P. longum at low concentrations of BA;
nonetheless, BA was more effective than kinetin. Philip
et al. (1992) found that BA at concentrations [5 mg l-1
suppressed the proliferation and growth of shoots in shoottip cultures of P. nigrum. We recorded 7.2 shoots/explant
on medium containing 1.0 mg l-1 BA. Based on our
results and those reported earlier, the overall response of
medicinal plant species to BA is quite positive (Kelkar and
Krishnamurthy 1998; Liu et al. 2003). Kelkar and Krishnamurthy (1998) found that moderate concentrations of BA
induced the optimum number of shoots per explant (6.1) in
P. colubrinum. In our study, the incorporation of BA in
medium already containing GA3 produced a reasonable
number of shoots/explant (Fig. 3), while the presence of 2,
4-D was not beneficial for either callogenesis or shoot
organogenesis (Figs. 2, 3, 4, 5). The addition of NAA to
the medium significantly inhibited shoot organogenesis.

Fig. 1 Plant regeneration in Piper nigrum L. a Callus b regenerated shoots c shoot multiplication d shoot elongation e rooting f regenerated
plantlets

123

Plant Cell Tiss Organ Cult (2010) 102:129134

c
cd
e

-1

2.0BA+GA3
1.5 BA+GA3
1.0 BA+GA3
0.5 BA+GA3
2.0 GA3
1.5 GA3
1.0 GA3
0.5 GA3
2.0BA+NAA
1.5 BA+NAA
1.0 BA+NAA
0.5 BA+NAA
2.0 BA
1.5 BA
1.0 BA
0.5 BA
2.0 2,4-D
1.5 2,4-D
1.0 2,4-D
0.5 2,4-D
MS0
0

Plant Growth Regulators (mg l )

-1

Plant Growth Regulators (mg l )

132

ef

h
g
bc
a
b
de

d
d

bc
a

h
gh

g
f

10

20

30

40

50

60

70

80

90

100

2.0BA+GA3
1.5 BA+GA3
1.0 BA+GA3
0.5 BA+GA3
2.0 GA3
1.5 GA3
1.0 GA3
0.5 GA3
2.0BA+NAA
1.5 BA+NAA
1.0 BA+NAA
0.5 BA+NAA
2.0 BA
1.5 BA
1.0 BA
0.5 BA
2.0 2,4-D
1.5 2,4-D
1.0 2,4-D
0.5 2,4-D
MS0

bc
bc
cd
gh
h
g

cd

a
c
ij
i

h
gh

-1

a
bc

d
h

i
h
h

gh

e
d
c
b

i
i
10

h
h

20

30

40

50

60

2.0BA+GA3
1.5 BA+GA3
1.0 BA+GA3
0.5 BA+GA3
2.0 GA3
1.5 GA3
1.0 GA3
0.5 GA3
2.0BA+NAA
1.5 BA+NAA
1.0 BA+NAA
0.5 BA+NAA
2.0 BA
1.5 BA
1.0 BA
0.5 BA
2.0 2,4-D
1.5 2,4-D
1.0 2,4-D
0.5 2,4-D
MS0

ab

70

80

90

100

d
g

de

hi

c
c
d
f

h
f

e
d

g
h
hi
i
0

Fig. 4 Effects of various concentrations of 2,4-D, BA, GA3, BA ?

1 mg l-1 NAA and BA ? 1 mg l-1 GA3 on the number of shoots per
explant in P. nigrum. Data were collected after 5 weeks of subculture
to MS media with a similar composition of plant growth regulators.
Values are the means of five replicates. Columns with the same lowercase letters are not significantly different at P \ 0.05

Plant Growth Regulators (mg l )

-1

Plant Growth Regulators (mg l )

ij

ab

Number of shoots/explant

Fig. 2 Effects of various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (BA) or gibberellic acid (GA3)
alone, BA ? 1 mg l-1a-naphthaleneacetic acid (NAA) and BA ? 1
mg l-1 GA3 on percentage callus induction of P. nigrum. Data were
collected after 4 weeks of culture. Values are the means of five
replicates. Columns with the same lower-case letters are not
significantly different at P \ 0.05

fg
f

% Callus Induction

2.0BA+GA3
1.5 BA+GA3
1.0 BA+GA3
0.5 BA+GA3
2.0 GA3
1.5 GA3
1.0 GA3
0.5 GA3
2.0BA+NAA
1.5 BA+NAA
1.0 BA+NAA
0.5 BA+NAA
2.0 BA
1.5 BA
1.0 BA
0.5 BA
2.0 2,4-D
1.5 2,4-D
1.0 2,4-D
0.5 2,4-D
MS0

Mean Shoot Length (cm)

% Shooting

Fig. 3 Effects of various concentrations of 2,4-D, BA or GA3 alone,

BA ? 1 mg l-1NAA and BA ? 1 mg l-1 GA3 on percentage shooting
in P. nigrum. Data were collected after 5 weeks of subculture to MS
media with a similar composition of plant growth regulators. Values
are means of five replicates. Columns with the same lower-case letters
are not significantly different at P \ 0.05

Kelkar et al. (1996) found that the combination of BA ?

2,4-D induced an optimum shooting response in other
species of Piper. Shooting was recorded for all of the PGRs
tested in our experiments (Figs. 3, 4, 5). Data on mean
shoot length (Fig. 5), however, showed that the incorporation of BA into medium containing GA3 produced the
longest shoots (5.4 cm), and in contrast to the data on
number of shoots/explant, the addition of NAA to medium
containing BA increased mean shoot length. Similar
observations were reported by Makunga et al. (2003) for
Thapsia spp. However, Lucchesini et al. (2009) found
optimum shoot length for Echinacea angustifolia on

123

Fig. 5 Effects of various concentrations of 2,4-D, BA, GA3 alone,

BA ? 1 mg l-1 NAA and BA ? 1 mg l-1 GA3 on mean shoot length
in P. nigrum. Data were collected after 5 weeks of subculture to MS
media with a similar composition of plant growth regulators. Values
are the means of five replicates. Columns with the same lower-case
letters are not significantly different at P \ 0.05

medium containing only BA. Magyar-Tabori et al. (2010)

concluded from their work that the type and concentration
of cytokinins that result in optimum shoot production is
largely dependent on genotype, and interactions have
proven to be significant.
Shoots grown on shoot organogenesis medium were
transferred to MS0 and MS medium supplemented with
different concentrations of IBA for rooting (Table 1).
Rooting frequency was found to be directly positively
correlated with increasing concentrations of IBA in the
medium. The optimum percentage rooting (89%), number
of roots/shoot (7.8) and root length (11.5 mm) were
obtained shoots cultured on medium containing 2 mg l-1

Plant Cell Tiss Organ Cult (2010) 102:129134

133

Table 1 Effects of different concentrations of indole butyric acid on

percentage rooting, number of roots per shoot and approximate root
length after 20 days of culture on rooting media
Indole butyric
acid (mg l-1)

Rooting
(%)

Number of
roots/shoot

Root length
(mm)

0.5

40 d

2.1 d

5.1 d

1.0

62 c

4.6 c

6.9 b,c

1.5

78 b

6.2 b

8.3 b

2.0

89 a

7.8 a

11.5 a

Values followed by different letters are significantly different at

P \ 0.05

IBA. We obtained healthy roots (Fig. 1e). White roots

appeared after 15 days of culture, turning brown thereafter.
In contrast, Philip et al. (1992) obtained rooted plantlets
through shoot-tip cultures of P. nigrum on medium containing 1 mg l-1NAA. In a study on another Piper species,
rooting was achieved on medium containing indole acetic
acid (IAA) (Kelkar et al. 1996). It would therefore appear
that Piper species are not recalcitrant to commonly used
auxins for rooting. However, some medicinal species have
shown rooting in MS0 (Abbasi et al. 2010a). Rooted
plantlets were successfully transferred to pots for further
growth under controlled conditions (Fig. 1f).
Regenerated plantlets are able to accumulate secondary
metabolites similar to those found in the mother plant
(Shilpa et al. 2010). The antioxidant potential of the
regenerated tissues of P. nigrum was determined using the
DPPH8 free radical (Fig. 6). The shoot culture was found to
have a significantly higher antioxidant potential than the
other regenerated tissues. Singh et al. (2004) reported that

Antioxidant activity (%)

100

80

60

b
c

40

20

R
eg
an ene
tle ra
ts ted
Pl

eg

al

lu

en
e
sh rat
oo ed
ts

Fig. 6 Antioxidant activity in regenerated tissues of P. nigrum. The

activity was determined using 1, 1-diphenyl-2-picrylhydrazyl (DPPH)
as free radical. Values are the means standard deviation of triplicate
experiments. Means with the same lower-case letters are not significantly different at P \ 0.05

the antioxidant activity of Piper extracts is comparable to

that of butylated hydroxyanisole and butylated hydroxytoluene. Irradiation and steam have been reported to be
damaging to the antioxidant activity of piperine (Waje
et al. 2008). Andarwulan and Shetty (1999) found that
differentiated tissues of Pimpinella anisum had a higher
antioxidant activity and higher total phenolic contents. We
also found a lower antioxidant activity in callus cultures.
However, Abbasi et al. (2010a) recently reported that in
their system, the callus culture of Silybum had a higher
antioxidant activity. This variation in data demonstrates
that different components are accumulated during different
growth phases. Hence, the regeneration protocol reported
here obtained a higher content of physiologically active
antioxidants. This protocol can be scaled up to bioreactor
level to produce chemically consistent P. nigrum plantlets.
Acknowledgements The authors acknowledge the support of
Higher Education Commission (HEC) of Pakistan and University
Research Fund (URF) of Quaid-i-Azam University (QAU), Islamabad, Pakistan.

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