Plant Science 131 (1998) 77 – 83

Agrobacterium-mediated genetic transformation in hot chilli

(Capsicum annuum L. var. Pusa jwala)

M. Manoharan, C.S. Sree Vidya, G. Lakshmi Sita *

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India

Received 1 April 1997; received in revised form 14 October 1997; accepted 14 October 1997

Abstract

A protocol for regeneration and genetic transformation has been established for chilli (Capsicum annuum L. var.
Pusa jwala). High frequency regeneration of shoot buds from cotyledonary leaves was achieved with Murashige and
Skoog’s (MS) medium supplemented with 0.5 mg/l thidiazuron. Elongation of shoot buds and subsequent rooting was
obtained on half-strength MS medium with 0.5 mg/l IAA. The Agrobacterium tumefaciens strain EHA 105 carrying
a binary vector plasmid pBI 121 has been used for transformation. The cotyledonary explants from in vitro grown
shoots were cocultivated for 2 days. Shoot buds were produced on the regeneration medium containing kanamycin
(50 mg/l) and cefotaxime (400 mg/l). The shoot buds were elongated and rooted in the presence of kanamycin (25
mg/l). The transgenic nature of the regenerated plants were confirmed by the histochemical staining of GUS,
polymerase chain reaction (PCR) and Southern hybridization analyses of nptII gene. © 1998 Elsevier Science Ireland
Ltd.

Keywords: Regeneration; Thidiazuron (TDZ); Genetic transformation; b-Glucuronidase (GUS); Neomycin phospho-
transferase (NPT II); Chilli (Capsicum annuum L).

1. Introduction ducing them gradually into plants of important

Recent advances in genetic engineering of
plants have evoked great interest in developing
modern technology for crop improvement. In
many countries efforts have been made at cloning
genes for use potential in agriculture and intro-
* Corresponding author. E-mail: sitagl@mcbl.iisc.ernet.in
crop species. Consequently, many transgenic
plants have been successfully produced which
showed remarkable results such as resistance to
chemicals, pests and disease [1].
Chilli is a spice cum vegetable of commercial
importance. In India, the production of chilli has
declined from 779 thousand tonnes during 1992–
93 to 730 thousand tonnes during 1993–94, the
overall percentage in decrease is − 6.25. Conse-

0168-9452/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved.

PII S 0 1 6 8 - 9 4 5 2 ( 9 7 ) 0 0 2 3 1 - 8
78 M. Manoharan et al. / Plant Science 131 (1998) 77–83
quently, the export of chilli fell during the 1994 –
95 season [2]. There are many factors which con-
tributed to this decline. The most important
among them are the diseases caused by viruses,
bacteria, fungi and insects. Spraying of fungicides
and pesticides can control the diseases to some
extent, however, effective resistance against sev-
eral destructive pathogens is still not possible.
Efforts are being made to produce disease resis-
tant plants through genetic engineering.
Genetic transformation through Agrobacterium
tumefaciens is now a routine procedure for intro-
ducing foreign genes into many plant species in-
cluding several vegetable crop plants such as
tomato, brinjal, Brassica etc. [3 – 5]. The two most
important pre-requisites for the success of the
method are the availability of a plant regeneration
system from the explants and suitable method for
transformation. Although chilli belongs to the
Solanaceae family, whose members are easily
amenable to tissue culture and transformation
practices, it is highly recalcitrant. Several reports
of plant regeneration either through organogene-
sis [6–16] or embryogenesis [17,18] are evident.
However, these reports are genotype-specific and
consequently, the regeneration protocol as well as
viable transformation has to be established for
each commercial cultivar for exploiting the poten-
tial of genetic engineering. Recently transforma-
tion has been reported [19,20] in sweet pepper
with viral coat protein gene and herbicide resis-
tant gene.
In this study, we show the successful establish-
ment of plant regeneration and genetic transfor-
mation with marker genes (GUS and NPT II)
from the cotyledonary leaves of chilli var. Pusa
jwala.
2. Materials and methods
2.1. Establishment of aseptic plants
Seeds of Capsicum annuum L. cv. Pusa jwala
were surface disinfected with the fungicide,
Bavistin, for 1 h and rinsed repeatedly with run-
ning tap water. The seeds were sterilized for 2 min
in 0.1% HgCl2 and then rinsed in several changes
of sterile distilled water. The seeds were germi-
nated in a magenta box containing solidified MS
basal medium [21].
2.2. Culture condition and media
The cotyledonary leaves from 3 week old
seedlings were used as explants and inoculated on
the MS medium with 2% sucrose and supple-
mented with cytokinins (BAP, TDZ) and auxin
(IAA) either singly or in combination. The
medium was adjusted to pH 5.7 with 1 N HCl or
1 N NaOH phyta gel (0.2%, Sigma) or agar agar
(0.8%, BDH) was used to solidify the medium.
The shoot buds were transferred to shoot elonga-
tion and rooting (SER) medium containing half-
strength MS medium with 0.5 mg/l IAA for
further elongation and rooting.
2.3. Establishment of plants into soil
The plantlets were transferred to liquid medium
consisting of half strength MS salts for hardening
of roots for a week. Rooted plants measuring
about 7–10 cm were transferred to paper cups
filled with autoclaved soilrite and covered with
plastic bags. Pots were kept in the culture room
for 1 week before being transferred to soil.
2.4. Bacterial strain and 6ector
The disarmed hypervirulent A. tumefaciens
strain EHA 105 carrying the binary plasmid pBI
121 (Fig. 1) was used as a vector system. The
T-DNA contains the neomycin phosphotrans-
ferase II (NPT II) gene, driven by the nopaline
synthase (NOS) promoter and terminator se-
quences, which provides resistance to kanamycin
and was used as selectable marker, and the b-glu-
curonidase (GUS) gene, driven by the cauliflower
mosaic virus (CaMV) 35S promoter, was used as
reporter gene. Bacterial strain was grown
overnight in a Luria broth (LB) medium with
appropriate antibiotics and collected in the log
phase, when the absorbance at 550 nm was be-
tween 0.4 and 0.8.
 M. Manoharan et al. / Plant Science 131 (1998) 77–83 79

Fig. 1. Plasmid pBI 121 binary vector map 0.7 kb PCR fragment of nptII gene (approximately indicated as —) was used as probe
for Southern hybridization.
2.5. Transformation and regeneration
The cotyledonary leaves were precultured in the
regeneration medium (MS+ 0.5 mg/l TDZ) for 2
days. Then, the explants were infected for 2 – 5
min with Agrobacterium suspension, blotted dry
with Whatman No. 1 filter paper and returned to
the regeneration medium for co-cultivation. Fol-
lowing the 48 h co-cultivation, the explants were
washed with MS liquid medium. Subsequently,
the explants were placed on selection medium,
which is the regeneration medium, as described
above, complemented with 50 mg/l kanamycin
and 400 mg/l cefotaxime. The explants were trans-
ferred to fresh selection medium after 15 days of
culture. The explants with putative transgenic
shoot buds were transferred to SER medium with
25 mg/l kanamycin and 200 mg/l cefotaxime. Af-
ter hardening The transgenic plants were trans-
ferred to soil.
2.6. Histochemical GUS assay
The histochemical GUS assay was conducted
essentially as described by Jefferson et al. [22].
Leaf segments were immersed in an X-Gluc solu-
tion consisting of 2 mM X-Gluc, 100 mM Tris –
HCl, pH 7.0, 50 mM NaCl, 2 mM potassium
ferricyanide and 0.1% (v/v) Triton X-100. Tissues
were stained overnight in dark at 37°C. The tis-
sues were cleared through ethanol series to re-
move chlorophyll. Assayed tissues were observed
under a microscope and photographed.
2.7. Polymerase chain reaction (PCR)
For PCR analysis, DNA was isolated from
leaves according to Edwards et al. [23]. Two
primers of the NPT II gene were used: 5%-GAG
GCT ATT CGG CTA TGA CTG-3% and 5%-
ATC GGG AGG GGC GAT ACC GTA-3%.
Expected size of the fragment was 700 bp. PCR
amplification was carried out in 50 ml contain-
ing 1 ml of the DNA solution, 200 mM dNTPs,
2.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl
pH 9.0, 0.1% (v/v) Triton X-100, 0.25 mM of
each primer and two units of Taq DNA poly-
merase. DNA was subjected to 35 cycles of 1
min at 92, 55 and 72°C. Amplified DNA frag-
ments were electrophoresed on 0.8% agarose
ethidium bromide gel and observed under ultra-
violet.
2.8. Southern hybridization analysis
Genomic DNA was extracted by the method
of Dellaporta et al. [24]. About 10 mg of DNA
from transformed and non transformed plants
and 1 mg DNA of plasmid pBI 121 were di-
gested with HindIII separated by electrophoresis
through a 0.8% agarose gel and transferred onto
Hybond–Nylon membrane. Hybridizations were
performed according to Sambrook et al. [25]. A
0.7 kb PCR fragment of the nptII gene was
used as a probe. Probe was labelled with 32P
dCTP (Amersham) using a random-primed la-
belling kit (Amersham).
80 M. Manoharan et al. / Plant Science 131 (1998) 77–83

Table 1
Effect of growth regulators on shoot bud formation from the cotyledonary leaves of Capsicum annuum L. var. Pusa jwala

Hormone concentration No. of explants cul- No. of explants responded Regeneration frequency (%) (mean 9S.D.)
(mg/l) tured
BAP TDZ IAA

2.0 1.0 33 15.0 45.4 90.8

5.0 1.0 32 21.0 65.6 91.3
10.0 1.0 37 27.0 78.9 91.2
20.0 1.0 35 24.0 68.5 91.6
0.1 33 7.0 21.2 92.1
0.2 33 13.0 39.3 92.1
0.5 33 30.0 90.9 92.4
1.0 32 27.0 84.3 92.6
2.0 0.5 1.0 32 23.0 71.8 92.1
5.0 0.5 1.0 33 21.0 63.6 91.2
10.0 0.5 1.0 33 19.0 57.5 91.6
20.0 0.5 1.0 30 17.0 56.5 9 1.8

Observations were made after 1 month of culture.

All experiments were repeated thrice

3. Results and discussion
3.1. Regeneration
The composition of the media used in the re-
generation of shoot buds and their frequency of
induction from the cotyledonary explants are
shown in Table 1. On the media containing BAP
and IAA, the maximum regeneration frequency of
72.9% was obtained with 10 mg/l BAP and 1 mg/l
IAA. Our results are consistent with those in
previous reports in which BAP and IAA were
used successfully to regenerate shoot buds [6 –
14,16]. However, the highest frequency of 90.9%
of shoot buds regeneration was obtained in TDZ
medium (0.5 mg/l). On the media containing vari-
ous concentrations of TDZ (0.1 – 1.0 mg/l), callus
formation was found to be less frequent and the
buds induced on the cotyledons were green and
vigorous. Our observations show that on media
with BAP and IAA, callus formation interferes
with shoot buds formation and that could be the
reason for less frequency induction of shoot buds.
Similar regeneration was reported in pepper
plants by organogenesis from explants treated
with TDZ [15]. TDZ has a high efficiency in
stimulating cytokinin-dependent shoot regenera-
tion from a wide variety of plants [26,27]. On the
medium containing TDZ, BAP and IAA, the
frequency of shoot buds formation decreased with
the increase in BAP concentration due to callus
growth.
Although efficient shoot buds induction was
observed in the present study and those of others
[6–16], elongation of shoot buds into long shoots
has been a consistent problem. The shoot buds
which formed at the cut end of the distal part of
the cotyledonary leaves after 30–40 days of cul-
ture (Fig. 2A), were transferred to shoot elonga-
tion and rooting medium (SER). The SER
contain half-strength MS supplemented with 0.5
mg/l IAA. After 90 days of culture, both shoot
elongation and rooting were observed simulta-
neously (Fig. 2B,C). The transfer of shoot buds to
the medium containing GA (1 mg/l) and Kinetin
(1 mg/l) leads to excessive callus growth and did
not support shoot bud elongation and rooting
(data not shown). By transferring the shoot buds
to SER, a total of 30 normal plants were ob-
tained. Normally one, and occasionally two, elon-
gated shoots were produced from the total of ten
to 60 shoot buds per explant (observed under
stereo microscope). Only vigorous grown shoot
buds after transfer to SER produced elongated
 M. Manoharan et al. / Plant Science 131 (1998) 77–83
Fig. 2. Regeneration of plants from the cotyledonary leaves of
chilli (Capsicum annuum L. var. Pusa jwala). A: Initiation of
shoot buds; B: Elongation of shoos; C: Rooted plants; D: In
vitro flowering; E: Hardening of the in vitro grown plants; and
F: Plants in the soil.
shoots. More number of vigorous grown shoot
buds were obtained in TDZ medium than the
medium containing BAP and IAA or combination
Fig. 3. Genetically transformed chilli (Capsicum annuum L. var. Pusa jwala) plants. A: Kanamycin resistant transgenic plants; B:
Transgenic plants in the soil; C: Untransformed leaf tissue assayed for GUS activity (control); and D: Transgenic leaf tissue showing
characteristic blue colour of the GUS gene.
81
of TDZ, BAP and IAA. Liu et al. [28] failed to
induce rooting whereas Ebida et al. [13] rooted
the rossette buds and transferred to soil where the
shoots are elongated. In our study, both elonga-
tion and rooting were obtained before transferring
to soil. More than 95% of the elongated shoots
were rooted. The plants were transferred to soil
after hardening (Fig. 2E) and started producing
flowers and normal sized and shaped fruits (Fig.
2F). Interestingly, in vitro flowering was also ob-
served (Fig. 2D). However, there was no fruit set.
3.2. Transformation
For transformation, around 200 cotyledonary
leaves were co-cultivated with Agrobacterium for
48 h. The shoot bud induction was observed after
15 days of inoculation on the selection medium.
The explants with shoot buds (about 5%) were
subcultured in the fresh selection medium. After
40–45 days culture, the putatively transformed
shoot buds were transferred to the SER medium
containing 25 mg/l kanamycin and 200 mg/l cefo-
taxime. Of the ten explants with shoot buds, only
four elongated shoots with roots were obtained
(Fig. 3A). These putative transgenics were subse-
quently transferred to soil (Fig. 3B).The trans-
82 M. Manoharan et al. / Plant Science 131 (1998) 77–83

23 kb size indicating the integration of nptII gene

Fig. 4. PCR analysis of kanamycin resistant transgenic plants
showing the presence of an expected 0.7 kb DNA fragment of
nptII gene. Lane 1: l HindIII marker; Lane 2: Positive control
(pBI 121); Lane 3: negative control (untransformed); Lanes
4–7: Transgenic plants; and Lane 8: fX 174 molecular weight
marker digested with HaeIII.
in to the genome of transgenic plants. To confirm
further, DNA from transgenic plants and plasmid
DNA was digested with HindIII, which cuts once
in the plasmid. Hybridization results clearly show
variation in pattern among transgenic plants, indi-
cating independent transformation events, and
differed from the 13 kb band corresponding to the
plasmid DNA disgested with HindIII. No hy-
bridization signals were observed in genomic
DNA isolated from non-transformed plants (Fig.
5.) Thus, genomic DNA blot hybridization data
confirmed the introduction of nptII gene in the
genome of transgenic chilli plants. Although it is
desirable to have segregation data of the trans-
genic plants it has not been done since only
marker genes were used.
The results presented above demonstrate the
potential of genetic transformation for the intro-
duction of useful traits such as disease resistance
in chilli.

genic nature of these plants were confirmed by

histochemical localization of GUS gene and PCR
and Southern analyses of nptII gene. Histochemi-
cal assay of the leaves for GUS activity, after
staining with X-Gluc demonstrated the presence
of blue coloured cells typical of the gusA gene
(Fig. 3D). Leaf tissues from control plants did not
show GUS activity (Fig. 3C). PCR was used to
demonstrate the presence of T-DNA in the trans-
genic plants. Two specific primers derived from
nptII gene sequences were used to detect a 0.7 kb
fragment. The amplified DNA samples were elec-
trophoresed on 0.8% agarose gel. As shown in
Fig. 4, the 0.7 kb fragment co-running with the
amplified product from pBI 121 could be detected
from the transgenic plants but not from non-
transformed plant. Southern hybridization was
also carried out in order to further confirm the Fig. 5. Southern blot analysis of total DNA isolated from
T-DNA integration. A 0.7 kb PCR fragment of transgenic plants. DNAs were digested with HindIII probed
the nptII gene was used as a probe. We obtained with 0.7 kb PCR fragment of the nptII gene. Lane 1: Undi-
hybridization signal in the undigested positive gested pBI 121 binary vector; Lane 2: pBI 121 digested with
HindIII; Lanes 3 – 6: Undigested transgenic DNA; Lane 7:
control at 13 kb.(Two bands corresponding to Undigested untransformed DNA; Lane 8: Untransformed
two forms of plasmid). However, undigested DNA digested with HindIII; Lanes 9 – 12: Transgenic DNA
DNA from transgenic plants gave signal at above digested with HindIII
 M. Manoharan et al. / Plant Science 131 (1998) 77–83
Acknowledgements
The corresponding author is grateful to Council
of Scientific and Industrial Research (CSIR) for
sanctioning the project entitled ‘In vitro develop-
ment of transgenic plants in tomato and Cap-
sicum’.
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