A simple and efficient Agrobacterium-mediated procedure for transformation of tomato 423

A simple and efficient Agrobacterium-mediated procedure for

transformation of tomato

MANOJ K SHARMA1, AMOLKUMAR U SOLANKE1, DEWAL JANI1, YOGENDRA SINGH2 and ARUN K SHARMA1,*
1
Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India
2
Institute of Genomics and Integrative Biology, Delhi 110 007, India
*Corresponding author (Fax, +91-11-24119430; Email, arun@genomeindia.org)

We describe a highly efficient and reproducible Agrobacterium-mediated transformation protocol applicable to

several varieties of tomato (Solanum lycopersicum, earlier known as Lycopersicum esculentum). Conditions such as
co-cultivation period, bacterial concentration, concentration of benzyl amino purine (BAP), zeatin and indole acetic
acid (IAA) were optimized. Co-cultivation of explants with a bacterial concentration of 108 cells/ml for three days on
2 mg/l BAP, followed by regeneration on a medium containing 1 mg/ml zeatin resulted in a transformation frequency
of 41.4%. Transformation of tomato plants was confirmed by Southern blot analysis and β-glucuronidase (GUS) assay.
The protocol developed showed very high efficiency of transformation for tomato varieties Pusa Ruby, Arka Vikas
and Sioux. The optimized transformation procedure is simple, efficient and does not require tobacco, Petunia, tomato
suspension feeder layer or acetosyringone.

[Sharma M K, Solanke A U, Jani D, Singh Y and Sharma A K 2009 A simple and efficient Agrobacterium-mediated procedure for transformation
of tomato; J. Biosci. 34 423–433]

1. Introduction 1991). Since the first report of Agrobacterium-mediated

Manipulation of the plant genome by introducing foreign
genes has become a core tool in plant biology. Targets
include enhancement in productivity by increasing resistance
to abiotic and biotic stresses as well as fundamental
studies such as identification and characterization of key
regulatory genes. Plant transformation methods in use
employ Agrobacterium, microprojectile bombardment,
microinjection and electroporation of protoplasts (Otoni
et al. 2003). Among these, Agrobacterium-mediated plant
transformation is the most extensively used method. It
exploits the natural ability of Agrobacterium to transform
plants to complete its own life cycle (Otoni et al. 2003).
Tomato, a member of the Solanaceae family, is a major
vegetable crop and consumed all over the world (Mueller et
al. 2005). It has relatively small DNA content and is a genetic
model for crop improvement (Arumuganathan and Earle
tomato transformation (McCormick et al. 1986), there have
been many reports of tomato being engineered for a variety
of purposes, including characterization of gene function,
production of insect- and disease-resistant plants, herbicide
tolerance, improved fruit quality, delay in fruit ripening,
production of foreign proteins and improved transformation
protocol (Davuluri et al. 2005; Fillatti et al. 1987; Janssen
et al. 1998; Lin et al. 2004; Park et al. 2003; Youm et al.
2008). In these studies, cotyledons or leaves have been
used for Agrobacterium-mediated tomato transformation.
Transformation efficiency has been calculated in various
ways in the literature. This has been expressed as per cent
explants regenerating on selection medium (Madhulatha
et al. 2007), per cent co-cultivated explants showing
shoot regeneration on selection medium (Patil et al. 2002)
or per cent co-cultivated explants producing transgenic
plants representing independent transformation events

Keywords. Agrobacterium; regeneration; tomato; transformation

Abbreviations used: AMV, alfalfa mosaic virus; BAP, 6-benzylaminopurine; DMSO, dimethyl sulphoxide; GUS, β-glucuronidase; IAA,
indole-3-acetic acid; MS, Murashige and Skoog; OD, optical density; PCR, polymerase chain reaction; SDS, sodium dodecyl sulphate

http://www.ias.ac.in/jbiosci J. Biosci. 34(3), September 2009, 423–433, © Indian

J. Biosci. 34(3),ofSeptember
Academy 423
Sciences 2009
424 Manoj K Sharma et al.
(Frary and Earle 1996; Park et al. 2003; Sun et al. 2006).
Transformation efficiency values reported in the literature
are 8% (Vidya et al. 2000), 7–37% (Ling et al. 1998), 9%
(Roekel et al. 1993), 11% (Frary and Earle 1996), 14%
(Hamza and Chupeau 1993), 20% (Park et al. 2003), 20%
(Qiu et al. 2007), 25% (Hu and Phillips 2001) and 28–48%
(Sun et al. 2006). In this article, only those reports have
been included where transformation efficiency has been
calculated as per cent co-cultivated explants producing
independent transformation events and where the presence
of a transgene has been validated by polymerase chain
reaction (PCR) or Southern analysis.
In our study, three tomato cultivars largely cultivated
in India were studied. Sioux is an American variety which
gives reliably high yields in hot humid weather. Arka Vikas
performs very well in the Indian subcontinent and has been
developed from an American variety Tip Top by pure line
selection (Lopez et al. 1996). The Indian cultivar Pusa
Ruby is the most widely grown in India and Nepal (Lohar
and Peat 1998) and is ideal for processing (Adhiguru et al.
2004). Using cotyledon explants and Agrobacterium strain
LBA4404, the Indian cultivar Pusa Ruby has previously
been reported to have a transformation frequency of 8%
(Vidya et al. 2000) and 14.2% (Raj et al. 2005). Although
generation of transgenic tomato plants has been reported
for cv. Arka Vikas (Mythili et al. 2005), transformation
efficiency has not been discussed, and there is no available
report for transformation of cv. Sioux. In this study, we
describe a protocol for improved transformation efficiency
by Agrobacterium-mediated transformation of tomato
varieties Pusa Ruby, Arka Vikas and Sioux without the use
of a feeder layer or acetosyringone.
2. Materials and methods
2.1 Growth conditions
A growth room maintained at 28 ± 1°C was used for seed
germination and maintenance of pre-culture, co-cultivation
and plant regeneration. Agrobacterium culture was carried
out in a rotary shaker maintained at 28°C. Autoclavable
flasks, culture tubes, scalpel blades, forceps, MQ water,
Whatman paper and other equipment required for tissue
culture were used after autoclaving for 20 min at 121°C and
15 lb/inch2 pressure. Cultures were grown under fluorescent
lights of 40 W (Philips India Ltd), providing a fluence rate
of 100 μmol m-2 s-1.
Cotyledons from 10-day-old plants were used as
explants. To generate explant material, seeds of Solanum
lycopersicum var. Pusa Ruby, Arka Vikas and Sioux were
treated with 5% teepol detergent for 15 min with vigorous
shaking, followed by washing under running tap water for
3–4 h. Seeds were then surface-sterilized with 4% sodium
J. Biosci. 34(3), September 2009
hypochlorite solution for 12 min with intermittent shaking,
followed by 4–6 washes with sterile water. Finally, seeds
were germinated on seed germination medium in glass
culture bottles (12.5 cm height, 6.5 cm diameter). Ten days
after seed inoculation, seedlings were used for preparation
of explants. After 10 days of growth, cotyledons were cut
at the tip as well as at the base and middle segments of
approximately 0.7 cm × 1.0 cm in size were placed on pre-
culture medium with their adaxial surface in contact with the
medium. Explants were handled gently with flat forceps to
avoid any injury to them.
The effects of five bacterial cell densities, two different
co-cultivation time periods and five different plant growth
regulator combinations on transformation efficiency were
studied. To study the influence of these treatments on plant
regeneration, three independent experiments were performed
for each factor with a minimum of 50 explants in each
experiment. All plantlets regenerated from a single callus
were labelled as clones to indicate that they represented a
single transformation event. To estimate the variation in
transformation efficiency among independent experiments for
each treatment, the standard error was calculated. Data were
examined for statistical significance using t-distribution. The
optimized protocol was performed with a minimum of 150
explants from each variety for transformation with a foreign
gene, in at least two independent experiments.
2.2 Culture media
The composition of various media is described in table 1.
Components of Murashige and Skoog (MS) medium
(Murashige and Skoog 1962), B5 medium (Gamborg et al.
1968), antibiotics and growth regulators were purchased
from Sigma-Aldrich (St Louis, MO, USA) and stored at the
prescribed temperature. Media components were mixed and
the pH adjusted to 5.8 using 1 M KOH prior to addition of
0.8% w/v plant tissue-culture grade agar powder (HiMedia
Laboratories Pvt. Ltd. Mumbai, India) and autoclaved.
The growth regulators BAP and zeatin were dissolved in
dimethyl sulphoxide (DMSO) and IAA was dissolved in
absolute ethanol. The antibiotics kanamycin and cefotaxime
were prepared in MQ (Millipore Corporation, USA) water
and filter sterilized, whereas rifampicin was dissolved
in methanol. Antibiotics and growth regulators were
added to the autoclaved medium after it cooled to ~50°C.
Agrobacterium strain AGL1 was grown in YEM medium
containing 100 mg/l rifampicin and 50 mg/l kanamycin.
2.3 Bacterial strain and plasmids
Agrobacterium tumefaciens strain AGL1 was used for plant
transformations. Agrobacterium used for co-cultivation
 A simple and efficient Agrobacterium-mediated procedure for transformation of tomato 425

carried either the pCTBE2L (figure 1a) or pRINASE2L replaced with a BamHI–HindIII fragment consisting of the
(figure 1b) construct. The binary vector pCTBE2L 35S promoter with the double enhancer and AMV leader
was derived from the modified plant expression vector sequence, generating pCTBE2L. To design pRINASE2L,
pCAMBIA2301 (http://www.cambia.org/daisy/bioforge_ the LeMADS-RIN gene was amplified from a cDNA pool
legacy/3724.html), expressing the ctxB gene encoding of tomato fruit prepared from total RNA and cloned in
cholera toxin B subunit along with the uidA reporter gene, pBluescriptSK(+) to generate the vector pBSKRIN. Total
both driven by the 35S promoter (Jani et al. 2002). To design RNA was isolated using TRI reagent (Sigma-Aldrich)
the pCTBE2L vector, first the DNA fragment consisting of from tomato fruits. Random hexamer as well as an oligo
the 35S promoter with double enhancer and alfalfa mosaic dT primer and M-MuLV reverse transcriptase (Fermentas)
virus (AMV) leader sequence was excised from plasmid were used for generating a cDNA pool. This cDNA pool
pFF53-6 as a NcoI-HindIII fragment. The NcoI site was was directly used for amplification of the LeMADS-RIN
then destroyed to create a blunt end and the fragment cloned cDNA. The sequence of the forward primer (RINF) used
into pBluescriptSK(+) at SmaI-HindIII sites to generate the for amplification of the LeMADS-RIN gene was 5´-ATA
vector pBSKE2L. The 35S promoter controlling expression GAGCTCATGGGTAGAGGGAAAGTAGAA-3´ and that
of the ctxB gene in the modified pCAMBIA vector was of the reverse primer (RINR) was 5´-ATAGGATCCTCA
Table 1. Components of media used in various transformation and regeneration experiments
Seed germination Pre-culture and co-cultivation Washing Selection * Rooting
MS salts** 0.5x 1x 1x 1x 1x
***
B5 vitamins 0.5x 1x 1x 1x 1x
Sucrose (g/l) 15.0 30.0 30.0 30.0 30.0
Agar (% w/v) 0.8 0.8 0 0.8 0.8
BAP (mg/l) 0 2 0 0* 0
Kanamycin (mg/l) 0 0 0 100 100
Cefotaxime (mg/l) 0 0 0 500 500
*In the medium, various combinations of hormones were used. For details see table 3.
**Murashige and Skoog 1962
***Gamborg et al. 1968
BAP, 6-benzylaminopurine

35S ter nos ter

BamH I Catalase intron R Border
a L Border Eco RI Sac I
Hind III nos ter

npt II 35S ER ctxB KZ 35SE2L 35S uidA

pCTBE2L

35S ter nos ter

BamH I Catalase intron R Border
b L Border Eco RI Sac I
Hind III nos ter

npt II 35S LeMADSRIN-AS 35SE2L 35S uidA

pRINASE2L

Figure 1. Diagrammatic representation of T-DNA regions of plant expression vectors. (a) pCTBE2L, (b) pRINASE2L. nptII, neomycin
phosphotransferase gene; ctxB, cholera toxin B subunit gene from Vibrio cholerae; RINAS, Solanum lycopersium MADS box ripening
inhibitor gene in antisense orientation; uidA, β-glucuronidase gene; 35S, CaMV 35S promoter; 35SE2L, CaMV 35S promoter with double
enhancer and leader sequence from alfalfa mosaic virus; KZ, Kozak sequence; ER, endoplasmic reticulum retention signal; nos ter, nopaline
synthase gene terminator.

J. Biosci. 34(3), September 2009

426 Manoj K Sharma et al.
AAGCATCCATCCAGGTA-3´. The LeMADS-RIN gene
is a conserved fruit-ripening regulator which has been
positionally cloned from tomato (Vrebalov et al. 2002). The
PCR product was digested with BamHI and SacI restriction
enzymes and cloned in antisense orientation into a modified
pCAMBIA vector (Jani et al. 2002), replacing the CTB
coding cassette to generate pRINAS. The 35S promoter
driving the LeMADS-RIN gene in the pRINAS vector was
replaced by a 35S promoter with a double enhancer and an
AMV leader sequence, as described earlier for construction
of the pCTBE2L vector, to generate pRINASE2L.
Agrobacterium cultures containing binary vectors were
grown in shaking culture for 72 h at 28°C and 200 rpm.
Cells were pelletted at room temperature and 5000 rpm,
followed by washing with washing medium (table 1).
The pellet was resuspended in 10 ml of washing medium.
Bacterial cell density was measured using a U-2001
spectrophotometer (Hitachi, Japan) and adjusted to the final
working concentration by diluting it with washing medium.
The optical density (OD)600 value of 1 corresponds to 108
cells/ml culture.
2.4 Plant transformation
Cotyledons from 10-day-old seedlings were cut at the tip
and base. Middle pieces (~0.7 cm × 1.0 cm) were pre-
cultured for 48 h at 28°C on pre-culture medium (table
1), with the adaxial surface in contact with the medium.
Healthy explants that responded to pre-culture, as evident
by swelling, were incubated in the bacterial suspension
for 30 min and inverted every 10 min during incubation.
The explants were then blotted on sterile tissue paper and
co-cultured on the same pre-culture medium for 72 h at
28°C with 50–80 explants in each 9 cm Petri plate. After
exposure for different experimental durations, co-cultured
explants were washed 4–5 times with washing medium
(table 1), blotted on sterile tissue paper and transferred to
a selection medium containing 1 mg/l trans-zeatin (table 1)
for regeneration. Each Petri plate (9 cm) had 20–25 explants
for regeneration. Plates were cultured under a 16 h light/8
h dark cycle at 28°C. Explants that showed regeneration
or callus formation were subcultured onto fresh selection
medium every 15 days. Regenerated shoots were excised
from the callus and transferred to a rooting medium (table
1). Shoots which did not produce roots after three weeks
of their transfer to the rooting medium were discarded.
Those plantlets which attained good shoot development (~8
cm in height) and produced roots were transferred to pots
containing 50% Soilrite (Kelperilite, Banglore, India; 1:1:1
ratio of vermiculite, perlite and Sphagnum moss) mixed with
soil for hardening. Pots were kept in a humidity chamber
for 3–5 days in the culture room under a 16 h light/8 h dark
cycle at 28°C and then in the greenhouse at 28 ± 2°C.
J. Biosci. 34(3), September 2009
The transformation efficiency was calculated as the
per cent co-cultivated explants producing independent
transformation events, leading to regeneration of a complete
plant on the selection medium. Multiple shoots generated
from a single callus were treated as a single transformation
event.
2.5 Analysis of putative transgenic tomato plants
Histochemical assay was performed to visualize GUS
activity as described elsewhere (Chaudhury et al. 1995).
Briefly, leaf tissues from randomly selected kanamycin-
resistant plantlets growing in the soil were incubated in
GUS histochemical buffer [50 mM sodium phosphate, pH
7.0; 50 mM EDTA, pH 8.0; 0.5 mM K3Fe(CN)6; 0.5 mM
K4Fe(CN)6; 0.1% Triton X-100; 1 mM X-gluc (5-bromo-4-
chloro-3-indolyl β-D-glucuronide)] at 37°C for up to 24 h.
Chlorophyll in leaf tissues was subsequently extracted by
incubation in acetone:ethanol (1:3) before assessment for
GUS activity.
For molecular analysis, plantlets grown on selection
medium were selected at random and genomic DNA was
isolated from leaf tissue as described by Dellaporta et
al. (1983). Briefly, 1 g of leaf tissue was frozen in liquid
nitrogen and ground to a fine powder using a pre-chilled
mortar and pestle. Powdered leaf tissue was added to 15 ml
extraction buffer (100 mM Tris-HCl, pH 8.0; 50 mM EDTA,
pH 8.0; 500 mM NaCl and 10 mM β-mercaptoethanol) in a
30 ml centrifuge tube. After lysis with 20% sodium dodecyl
sulphate (SDS), DNA was precipitated using isopropanol.
To remove RNA, samples were treated with RNaseA (30 μl
of 10 mg/ml stock per sample) for 1–2 h at 37°C. The sample
was extracted once with phenol:chloroform:isoamyl alcohol,
followed by another extraction with chloroform:isoamyl
alcohol only. DNA was precipitated with 0.1 volume of 3
M sodium acetate and 2.5 volumes of absolute ethanol for
1 h at –20°C. Presence of the transgene was confirmed by
Southern blot hybridization.
For Southern blot analysis, 15 μg of genomic DNA
from plants transformed with the LeMADS-RIN and ctxB
genes was digested with EcoRI and HindIII, respectively,
which was expected to generate a T-DNA fragment of at
least 4.5 kb for plants transformed with the LeMADS-RIN
gene and 3.5 kb for plants transformed with the ctxB gene.
Digested DNA samples were analysed on 1.2% w/v agarose
gel, blotted on nylon membrane (Hybond-N, Amersham
Biosciences UK Ltd.) by capillary transfer and hybridized
using a [32P]-labelled LeMADS-RIN gene or ctxB gene as
the probe following standard procedures (Sambrook et
al. 1989). The membrane was then autoradiographed by
exposing it to an X-ray film purchased from Kodak India
Ltd. Autoradiograms were recorded using a document
scanner.
 A simple and efficient Agrobacterium-mediated procedure for transformation of tomato 427

3. Results cotyledon explants responded to the pre-culture conditions

Tomato variety Pusa Ruby was used in the experiments to
determine optimum transformation conditions. Cotyledons
from 10-day-old seedlings (figure 2a) were used as explants
and cultured on pre-culture medium supplemented with
2 mg/l BAP (figure 2b). Cotyledons were cut at the base
and tip and the middle piece was used as described earlier
(Fillatti et al. 1987). These were placed with their adaxial
side in contact with the medium. More than 90% of the
by expanding in size within 48 h.
3.1 Optimization of various factors affecting plant
transformation
Agrobacterium carrying the pCTBE2L vector was used for
optimization of transformation parameters as this plasmid
carried the uidA reporter gene whose expression can be

Figure 2. Different stages of Solanum lycopersicum var. Pusa Ruby transformation. (a) Germinated seeds of tomato on MS-based
medium. (b) Cotyledonary explants on pre-culture medium. (c) Cotyledonary explants on selection medium without co-cultivation. (d)
Cotyledonary explants on selection medium after co-cultivation with Agrobacterium harbouring the pCTBE2L binary vector. (e) Callus
with regenerating shoot buds growing on selection medium. (f) Multiple shoots regenerating from a single callus. (g) Growing shoots on
callus. (h) Young plantlet with well developed roots on selection medium. (i) A soil acclimated plantlet. (j) Mature transgenic tomato plants
bearing fruit.

J. Biosci. 34(3), September 2009

428 Manoj K Sharma et al.
conveniently measured using histochemical GUS assay.
Bacterial densities of 0.5 × 108, 1.0 × 108, 2.0 × 108, 5.0 × 108
and 1.0 × 109 cells/ml culture were used for incubation and
co-culture of cotyledon explants for 72 h or 96 h (table 2).
A bacterial density of 1.0 × 108 cells/ml and co-cultivation
time of 72 h were found to be the most efficient for T-DNA
transfer. Cotyledon explants from Solanum lycopersicum
var. Pusa Ruby were used for optimization of transformation
conditions. Callus started developing from the co-cultivated
explants in the second week and shoots developed 4–6
weeks after co-culture (figure 2d, e) on selection medium
containing antibiotics and growth regulators (tables 1 and 3).
Pre-cultured control explants which did not receive co-
cultivation died on the selection medium after three weeks
of their transfer to the selection medium (figure 2c).
Lower bacterial density, i.e. 0.5 × 108 cells/ml and co-
cultivation for 72 h produced transformation efficiency
of up to 20%. Interestingly, an increase in transformation
efficiency was observed when the same bacterial density was
used to co-cultivate the explants for 96 h (table 2). However,
when a bacterial density of 1.0 × 109 cells/ml was used for 72
h of co-cultivation, transformation efficiency was reduced
to 12.9%. Similarly, an increase in co-cultivation time from
72 h to 96 h resulted in decreased transformation efficiency
from 24.3% to 17.9% (table 2). When a bacterial density
of 1.0 × 109 cells/ml was used to co-cultivate explants for
96 h, they could not be rescued (table 2) as all the explants
died due to overgrowth of Agrobacterium. Optimum
transformation efficiency of 41.4% was achieved when
explants were exposed to a bacterial density of 1.0 × 108
cells/ml for 72 h (table 2). The effect of zeatin or BAP alone
Table 2. Effects of bacterial density and co-cultivation period
on transformation of tomato cv. Pusa Ruby
Average per cent transformation
Bacterial efficiency ± SE
concentration No. of 72 h 96 h
(cells/ml) explants co-cultivation co-cultivation
0.5 × 108 70 20.7 ± 1.01 22.1 ± 1.01
1.0 × 108 70 41.4 ± 2.02 24.3 ± 2.02
8
2.0 × 10 70 38.6 ± 2.02 19.3 ± 3.03
5.0 × 108 70 36.4 ± 1.01 17.9 ± 1.01
1.0 × 109 70 12.9 ± 2.02 –
Transformation efficiency was calculated as the per cent co-
cultivated explants producing independent transformation
events, leading to regeneration of plantlets on antibiotic-
containing medium. Transformation efficiency values represent
the average ± SE of values from three independent experiments.
The binary vector used for transformation was pCTBE2L
carrying the ctxB gene coding for cholera toxin B subunit and
the uidA visual marker gene.
J. Biosci. 34(3), September 2009
and in combination with IAA on transformation of Pusa
Ruby was investigated (table 3). It was observed that zeatin
was more effective than BAP such that the use of the former
cytokinin (at 1 mg/l) resulted in a transformation efficiency
of 40.7% in this cultivar, whereas the use of BAP showed
only 30.3% efficiency. Supplementing the medium with
both BAP (2 mg/l) and IAA (0.1 mg/l) was found to improve
the transformation efficiency to 32.9% as compared with
30.3% when only BAP (2 mg/l) was used. No significant
improvement was observed on combining zeatin with IAA
as compared with zeatin alone (table 3). In fact, reduction
in the number of independent transformation events was
observed as the IAA concentration in the medium was
increased (table 3).
Approximately 10–15% of the calli produced leafy
structures or a large number of shoot buds (more than 4;
figure 2f), but none of these shoots grew sufficiently to allow
transfer to a rooting medium. When the leaves were removed,
the callus grew well and produced shoots which formed
plantlets on the rooting medium. When most of the shoots
from calli producing a large number of shoot buds were
removed leaving 2–3 shoots on each callus, shoots left on
the calli grew better and formed plantlets after transfer to the
rooting medium. In 5–10% of calli, either the development
of shoot buds was delayed or elongation of the shoots was
slow. In the regeneration medium supplemented with zeatin
as the growth regulator along with antibiotic selection, 2–4%
of calli which were otherwise green and healthy showed a
slowing in growth. Though the transgenic nature of these
slow-growing calli was confirmed by GUS expression, they
did not differentiate into shoots even after 6 months with
transfer to fresh medium after every 15 days. On transferring
slow-growing calli to fresh MS medium containing 1
mg/l zeatin and 0.2 mg/l IAA, these calli developed shoot
buds after 15–30 days of transfer. Shoots (figure 2g) were
excised from the calli and transferred to the rooting medium.
Transgenic shoots produced roots within 1–2 weeks of their
Table 3. Effects of different growth regulators and their
combinations on transformation of tomato cv. Pusa Ruby
Average
No. of transformation
Growth regulator (mg/l) explants efficiency (% ± SE)
BAP (2 mg/l) 50 30.29 ± 1.7
Zeatin (1 mg/l) 50 40.7 ± 1.2
BAP (2 mg/l), IAA (1mg/l) 50 32.9 ± 1.0
Zeatin (1 mg/l), IAA (0.1 50 41.6 ± 1.5
mg/l)
Zeatin (1 mg/l) IAA (1 mg/l) 50 37.3 ± 3.1
Transformation efficiency was calculated as described in
table 2. The vector used for transformation was pCTBE2L.
Standard error is mentioned for the values of transformation
efficiency. BAP, 6-benzylaminopurine
 A simple and efficient Agrobacterium-mediated procedure for transformation of tomato 429

transfer to the selection medium (figure 2h). These shoots
were transferred to fresh rooting medium after 15 days to
sustain root growth. Shoots which did not produce roots even
after 3–4 weeks of their transfer to the rooting medium were
discarded. The plantlets were transferred to soil (figure 2i).
Mature transgenic plants were morphologically similar to
wild-type plants and produced fruit (figure 2j).
To determine transgene expression, reporter protein
expression was assayed by GUS histochemical staining
(figure 3). Tissue from the youngest leaves of transgenic
plants grown in pots for 3–4 weeks was used. More than
90% of the plantlets selected on medium containing 100
mg/l kanamycin were found to exhibit GUS activity
(figure 3). The observed GUS expression level in different
transformation events was not uniform and ranged from
very low expression, showing blue colour after 10–24 h
incubation, to very high expression showing blue colour
within 1–2 h of incubation during GUS assay.
To determine the adaptability of the transformation
protocol optimized for Solanum lycopersicum var. Pusa
Ruby across other genotypes of tomato, Arka Vikas and
Sioux were used. Optimized parameters of bacterial density
(1.0 × 108 cells/ml), co-cultivation time (72 h) and hormone
(zeatin at 1 mg/l) in the regeneration medium were used for
transformation of Arka Vikas and Sioux cultivars of tomato
with the ctxB gene using the binary vector pCTBE2L or
LeMADS-RIN gene of tomato in antisense orientation using
the binary vector pRINASE2L. Whereas transformation
efficiency for the American cultivar Sioux was approximately
41%, it was approximately 22% for the other cultivar Arka
Vikas (table 4). In the case of Arka Vikas, calli grew slowly
and shoot regeneration was also delayed by 5–7 days
compared with Sioux or Pusa Ruby. Sioux and Pusa Ruby
did not show any major difference in callus formation. The
transgenic nature of the plants transformed with the ctxB or
LeMADS-RIN gene was checked by Southern hybridization
(figure 4). For Southern hybridization, genomic DNA
isolated from LeMADS-RIN transformed plants was digested
with the restriction enzyme EcoRI which cuts the T-DNA
between the LeMADS-RIN gene and nptII reporter gene, with
4.5 kb as the minimum size of the expected fragment. The
genomic DNA from ctxB-transformed plants was digested
with HindIII, which makes a cut in the T-DNA between the
ctxB and uidA gene, with 3.5 kb as the minimum size of the

Figure 3. Analysis of reporter gene expression in transgenic tomato var Pusa Ruby plants. Histochemical GUS staining was performed on
leaf tissue of transgenic tomato plants followed by removal of chlorophyll pigment. (a) Leaf from untransformed plant. (b–k) Leaves from
various transgenic plants showing GUS expression.

J. Biosci. 34(3), September 2009

430 Manoj K Sharma et al.

expected fragment. Transformants with single or multiple
copies of the transgene insertions were observed for the ctxB
gene (figure 4a) as well as LeMADS-RIN gene (figure 4b).
In more than 50% of the transformants, the transgene was
integrated as a single copy.
4. Discussion
The transformation efficiency of tomato depends upon many
factors such as the cultivar, type of explant and its age,
Agrobacterium strain and its density, co-cultivation time
and regeneration medium (Davis et al. 1991; Madhulatha et
al. 2007). As it is well established that cotyledon explants
show good regeneration in tomato (Fillati et al. 1987; Hamza
and Chupeau 1993; Frary and Earle 1996; Ellul et al. 2003),
these were used as explants for transformation. After 40–48
h of pre-culture, more than 90% of the cotyledon explants
expanded substantially. However, not all the pre-cultured
explants responded equally. It is important to select only
those explants for co-cultivation which show expansion in
response to the pre-culture medium. When placed with the
adaxial side towards the medium, the maximum surface

Table 4. Transformation efficiency using an optimized protocol for three tomato varieties
Vector used Tomato variety Average No. of explants co-cultivated Average transformation efficiency (% ± SE)
pCTBE2L Pusa Ruby 174 40.5 ± 0.80
pRINASE2L Pusa Ruby 163 41.1 ± 0.89
pCTBE2L Sioux 150 41.0 ± 1.00
pCTBE2L Arka Vikas 150 22.0 ± 1.50
Transformation efficiency was calculated as mentioned for table 1. Vector pCTBE2L was used for expression of the ctxB gene, coding
for cholera toxin B subunit and vector pRINASE2L was used for expression of the LeMADS-RIN gene coding for LeMADS-RIN
transcription factor in antisense orientation. Standard error is mentioned for the values of transformation efficiency.

of the cotyledon explant makes contact with the medium.

Figure 4. Detection of transgene integration by Southern blotting.
(a) ctxB and (b) LeMADS-RIN. Fifteen microgram of HindIII or
EcoRI digested genomic DNA was used for hybridization with [32P]
labelled probe for respective transgenes. Names of the transgenic
lines are mentioned at the top of the lanes. WT, wild-type plant
DNA; –, undigested DNA; +, EcoRI-digested DNA; transgenic
lines are numbered.
Therefore, this orientation was used in the present study.
Explants that did not respond to the pre-culture were not
used for co-cultivation with Agrobacterium. For the purpose
of calculating transformation efficiency, explants that did not
expand during pre-culture were not included. However, such
explants were less than 10% of the total explants used.
Previous reports on tomato transformations suggest the
use of a feeder layer during pre-culture and Agrobacterium
co-cultivation (McCormick et al. 1986; Fillatti et al. 1987;
Roekel et al. 1993; Frary and Earle 1996; Ling et al.
1998; Zhang and Blumwald 2001; Cortina and Culianez-
Macia 2004; Frary and Van Eck 2005), which makes the
transformation procedure tedious. In our study, pre-culture
and co-cultivation were done on a basal medium of MS
containing BAP at 2 mg/l, in the absence of a feeder layer or
acetosyringone in a manner similar to that reported by Qiu
et al. (2007) for cultivar Micro-Tom. The effects of bacterial
density and growth regulators on the transformation efficiency
and regeneration from explants infected with Agrobacterium
were studied. A bacterial density of 108 cells/ml and co-
cultivation time of 72 h resulted in the maximum number
of independent transformation events (41.4%; table 2). Most
published protocols for tomato transformation (McCormick
et al. 1986; Hamza and Chupeau 1993; Lipp-Joao and Brown
1993; Frary and Earle 1996; Oktem et al. 1999; Vidya et al.
2000; Pozueta-Romero et al. 2001; Jia et al. 2002; Ellul et
al. 2003) describe co-cultivation of the explants with various
Agrobacterium strains (LBA4404, C58C1, GV311SE or
A208) for 48 h with bacterial densities ranging from 108 to

J. Biosci. 34(3), September 2009

 A simple and efficient Agrobacterium-mediated procedure for transformation of tomato
5.0 × 108 cells/ml, giving variable transformation efficiencies
with different cultivars of tomato. In our study, we observed
that co-cultivation up to 72 h using bacterial strain AGL1 at a
density of 108 cells/ml enhanced the transformation efficiency
to 41.4% for variety Pusa Ruby compared with 14.2%
reported earlier (Raj et al. 2005). Using similar conditions,
we achieved transformation efficiency values of 22% and
41% for varieties Arka Vikas and Sioux, respectively. The
observed response can be attributed to the cumulative effect
of optimized parameters including bacterial strain and its
concentration, co-cultivation time and plant growth regulator
combination in the regeneration medium.
Further, increasing the bacterial concentration from 108
cells/ml or co-cultivation time from 72 h negatively affected
the health of explants. Affected areas in explants became
black, and overall, the explants became soft and lost their
regeneration capacity. In addition to this, removal of excess
bacterial solution from the explants before transfer to the
co-cultivation medium is also important. If the explants
were blotted dry on tissue paper, bacterial growth did not
affect the health of explants even after four days at higher
concentrations, but if a little bacterial solution was left
on the explants, bacterial growth affected the health of
explants even within 48 h. Therefore, it is important to
wipe off excess bacterial suspension before co-cultivation.
At a bacterial density of 0.5 × 108 cells/ml, the health of
the explants was not compromised even after 96 h of co-
culture but the improvement in transformation efficiency
was not significant (table 2). The same was not true when
Agrobacterium concentrations of 1.0 × 108 cells/ml or
higher were used. At bacterial densities of 1.0 × 108 cells/ml
or higher and co-cultivation for 96 h, significant decrease
in transformation efficiency was observed. Further, when
a bacterial density of 1.0 × 109 cells/ml was used, none
of the explants regenerated after 96 h of co-cultivation
(table 2). This indicates that there is a threshold range of a
combination of bacterial concentration and time of infection
required for efficient plant transformation. Transformation
efficiency obtained with zeatin alone at a concentration of 1
mg/l was found to be as good as that obtained with IAA and
zeatin, and was superior to that achieved by supplementing
the regeneration medium with BAP alone or BAP in
combination with IAA (table 3). This improvement in the
transformation efficiency using zeatin in the regeneration
medium rather than BAP was statistically significant using
t-distribution at 0.01. It has been reported earlier that zeatin
in combination with IAA improved the frequency of callus
formation and shoot regeneration in tomato (Park et al. 2003).
In our study, although the combined use of a cytokinin and
IAA in the regeneration medium showed a slight increase
in the number of regenerated transgenic plants, it was not
significantly better than the efficiency obtained using BAP
or zeatin alone in the regeneration medium.
431
Transgenic plants from independent transformation
events showed variable GUS expression. In some plants
it was barely detectable, while others expressed GUS at
high levels. This variation in transgene expression may
be attributed to position effect, copy number, silencing or
presence of regulatory sequences at the site of integration,
as reported earlier (Butaye et al. 2005; De Bolle et al. 2003;
Hobbs et al. 1993). Stable integration of the transgene was
shown by Southern analysis performed using genomic DNA
isolated from leaves of mature transgenic plants. Some of
the transgenic lines obtained had single copy insertions.
In summary, we report a highly improved transformation
efficiency of 41.4% for the tomato variety Pusa Ruby
compared with 14.2% reported earlier (Raj et al. 2005). In
addition, the improved protocol was shown to be effective in
transforming other tomato varieties, Arka Vikas and Sioux.
The optimized protocol is simple and reproducible, and may
be adapted for other tomato cultivars.
Acknowledgements
This research work is supported by financial assistance
from the Department of Biotechnology, New Delhi. Senior
Research Fellowship by University Grants Commission, New
Delhi to MKS and by Council of Scientific and Industrial
Research, New Delhi to AUS is acknowledged. We also
thank Dr V Siva Reddy, International Centre for Genetic
Engineering and Biotechnology, New Delhi, for providing the
pFF53-6 vector containing CaMV 35S promoter with double
enhancer and the AMV leader sequence.
References
Adhiguru P, Devi S V and Kanagaraj M 2004 Strengthening
economic and nutrition security: role of vegetables; in Impact of
vegetable research in India (eds) S Kumar, P K Joshi and S Pal
(New Delhi: NCAP) vol 13 , pp 190–200
Arumuganathan K and Earle E 1991 Estimation of nuclear DNA
content of plants by flow cytometry; Plant Mol. Biol. Rep. 9
208–218
Butaye K M J, Cammue B P A, Delaure S L and de Bolle M F C
2005 Approaches to minimize variation of transgene expression
in plants; Mol. Breed. 16 79–91
Chaudhury A, Maheshwari S C and Tyagi A K 1995 Transient
expression of gus gene in intact seed embryos of indica rice
after electroporation-mediated gene delivery; Plant Cell Rep.
14 215–220
Cortina C and Culianez-Macia F A 2004 Tomato transformation
and transgenic plant production; Plant Cell Tiss. Org. Cult. 76
269–275
Davis M E, Lineberger R D and Miller A R 1991 Effects of tomato
cultivar, leaf age, and bacterial strain on transformation by
Agrobacterium tumefaciens; Plant Cell Tiss. Org. Cult. 24
115–121
J. Biosci. 34(3), September 2009
432 Manoj K Sharma et al.
Davuluri G R, van Tuinen A, Fraser P D, Manfredonia A, Newman
R, Burgess D, Brummell D A, King S R, Palys J, Jhlig J,
Bramley P M, Pennings H M J and Bowler C 2005 Fruit-specific
RNAi-mediated suppression of DET1 enhances carotenoid and
flavonoid content in tomatoes; Nat. Biotechnol. 23 890–895
De Bolle M F C, Butaye K M J, Coucke W J W, Goderis I J W,
Wouters P F J, van Boxel N, Broekaert W F and Cammue B
P A 2003 Analysis of the influence of promoter elements and
a matrix attachment region on the inter-individual variation of
transgene expression in populations of Arabidopsis thaliana;
Plant Sci. 165 169–179
Dellaporta S L, Wood J and Hicks J B 1983 A plant DNA
minipreparation: version II; Plant Mol. Biol. Rep. 1 19–21
Ellul P, Garcia-Sogo B, Pineda B, Rios G, Roig L A and Moreno
V 2003 The ploidy level of transgenic plants in Agrobacterium-
mediated transformation of tomato cotyledons (Lycopersicon
esculentum Mill.) is genotype and procedure dependent; Theor.
Appl. Genet. 106 231–238
Fillati J J, Kiser J, Rose R and Comai L 1987 Efficient transfer
of a glyphosate tolerance gene into tomato using a binary
Agrobacterium tumefaciens vector; Bio/Technology 5 726–731
Frary A and Earle ED 1996 An examination of factors affecting
the efficiency of Agrobacterium-mediated transformation of
tomato; Plant Cell Rep. 16 235–240
Frary A and Van Eck J 2005 Organogenesis from transformed
tomato explants; Methods Mol. Biol. 286 141–150
Gamborg O L, Miller R A and Ojima K 1968 Nutrient requirements
of suspension culture of soybean root cells; Exp. Cell Res. 50
151–158
Hamza S and Chupeau Y 1993 Re-evaluation of conditions for
plant regeneration and Agrobacterium-mediated transformation
from tomato (Lycopersicon esculentum); J. Exp. Bot. 44
1837–1845
Hobbs S L A, Warketin T D and DeLong C M O 1993 Transgene
copy number can be positively or negatively associated with
transgene expression; Plant Mol. Biol. 21 17–26
Hu W and Phillips G C 2001 A combination of overgrowth-control
antibiotics improves Agrobacterium tumefaciens-mediated
transformation efficiency for cultivated tomato (L. esculentum);
In Vitro Cell Dev. Biol. Plant 37 12–18
Jani D, Meena L S, Rizwan-ul-Haq Q M, Singh Y, Sharma A K
and Tyagi A K 2002 Expression of cholera toxin B subunit in
transgenic tomato plants; Transgenic Res. 11 447–454
Janssen B, Lund L and Sinha N 1998 Overexpression of a
homeobox gene, LeT6, reveals indeterminate features in the
tomato compound leaf; Plant Physiol. 117 771–786
Jia G X, Zhu Z Q, Chang F Q and Li Y × 2002 Transformation
of tomato with the BADH gene from Atriplex improves salt
tolerance; Plant Cell Rep. 21 141–146
Lin W-C, Lu C-F, Wu J-W, Cheng M-L, Lin Y-M, Yang N-S, Black
L, Green S K, Wang J-F and Cheng C-P 2004 Transgenic tomato
plants expressing the Arabidopsis NPR1 gene display enhanced
resistance to a spectrum of fungal and bacterial diseases;
Transgenic Res. 13 567–581
Ling H-Q, Kriseleit D and Ganal M G 1998 Effects of ticarcillin/
potassium clavulanate on callus growth and shoot regeneration
in Agrobacterium-mediated transformation of tomato
(Lycopersicon esculentum Mill); Plant Cell Rep. 17 843–847
J. Biosci. 34(3), September 2009
Lipp-Joao K H and Brown T A 1993 Enhanced transformation
of tomato co-cultivated with Agrobacterium tumefaciens
C58C1Rifr::pCSFR1161 in the presence of acetosyringone;
Plant Cell Rep. 12 422–425
Lohar D P and Peat W E 1998 Floral characteristics of heat-tolerant
and heat-sensitive tomato (Lycopersicon esculentum Mill)
cultivars at high temperature; Sci. Horticul. 73 53–60
Lopez K, Libas E and Shanmugasundaram S 1996 Vegetable
research networking in South Asia; Savernet phase I final report
(Shanhua, Tainan, Taiwan: AVRDC) pp 76
Madhulatha P, Pandey R, Hazarika P and Rajam M V 2007 High
transformation frequency in Agrobacterium-mediated genetic
transformation of tomato by using polyamines and maltose
in shoot regeneration medium; Physiol. Mol. Biol. Plants 13
191–198
McCormick S, Niedermeyer J, Fry J, Barnason A, Horsh R and
Fraley R 1986 Leaf disc transformation of cultivated tomato
(L. esculentum) using Agrobacterium tumefaciens; Plant Cell
Rep. 5 81–84
Mueller L A, Tanskley S D, Giovannoni J J, van Eck J, Stack S,
Choi D, Kim D, Chen M, et al. 2005 The tomato sequencing
project, the first corner stone of the international Solanaceae
project (SOL); Comp. Funct. Genomics 6 153–158
Murashige T and Skoog F 1962 A revised medium for rapid growth
and bioassays with tobacco tissue culture; Plant Physiol. 15
473–497
Mythili J B D, Narasimha Murthy D K and Anand L 2005 Studies
on the influence of cytokinin independent gene (CKII) on
explants differentiation in tomato cv Arka Vikas through
Agrobacterium-mediated transformation; in Recent trends
in horticultural biotechnology (eds) R Keshavchandran, P A
Nazeem, D Girija, P S John and K V Peter (New Delhi: New
Delhi Publishing Agency) pp 703–710
Oktem H A, Bulbul Y, Oktem E and Yucel M 1999 Regeneration
and Agrobacterium-mediated transformation studies in tomato
(Lycopersicon esculentum Mill); Tr. J. Bot. 23 345–348
Otoni W C, Picoli E A, Costa M G, Nogueira F T and Zerbini F
M 2003 Transgenic tomato; in Plant genetic engineering – a
multivolume series: 1 vol. 5 (eds) R P Singh and P K Jaiwal
(Houston: Sci-Tech. Pub. Co) pp 41–131
Park S H, Morris J L, Park J E, Hirschi K D and Smith R H 2003
Efficient and genotype-independent Agrobacterium-mediated
tomato transformation; J. Plant Physiol. 160 1253–1257
Patil R S, Davey M R, Power J B and Cocking E C 2002 Effective
protocol for Agrobacterium-mediated leaf disc transformation
in tomato (Lycopersicon esculentum Mill); Indian J. Biotechnol.
1 339–343
Pozueta-Romero J, Houlne G, Canas L, Schantz R and Chamarro
J 2001 Enhanced regeneration of tomato and pepper seedling
explants for Agrobacterium-mediated transformation; Plant
Cell Tiss. Organ Cult. 67 173–180
Qiu D, Diretoo G, Tavarza R and Giuliano G 2007 Improved
protocol for Agrobacterium mediated transformation of
tomato and production of transgenic plants containing
carotenoid biosynthetic gene CsZCD; Scit. Horticul. 112
172–175
Raj S K, Singh R, Pandey S K and Singh B P 2005 Agrobacterium-
mediated tomato transformation and regeneration of transgenic
 A simple and efficient Agrobacterium-mediated procedure for transformation of tomato 433

lines expressing tomato leaf curl virus coat protein gene for
resistance against TLCV infection; Curr. Sci. 88 1674–1679
Roekel J S C, Damm B, Melchers L S and Hoekema A 1993 Factors
influencing transformation frequency of tomato (Lycopersicon
esculentum); Plant Cell Rep. 12 644–647
Sambrook J, Fritsch E F and Maniatis T 1989 Molecular cloning. A
laboratory manual 2nd edition (New York: Cold Spring Harbor
Laboratory Press)
Sun H-J, Uchii S, Watanabe S and Ezira H 2006 A highly efficient
transformation protocol for Micro-Tom, a model cultivar for
tomato functional genomics; Plant Cell Physiol. 47 426–431
Vidya C S S, Manoharan M, Kumar C T R, Savithri H S and Sita
G L 2000 Agrobacterium-mediated transformation of tomato
(Lycopersicon esculentum var. Pusa Ruby) with coat-protein gene
of physalis mottle tymovirus; J. Plant Physiol. 156 106–110
Vrebalov J, Ruezinsky D, Padmanabhan V, White R, Medrano D,
Drake R, Schuch W and Giovannoni J 2002 A MADS-box gene
necessary for fruit ripening at the tomato ripening-inhibitor
(Rin) locus; Science 296 343–346
Youm J W, Heung J, Jeon J H, Kim H, Kim Y H, Ko K, Joung
H and Kim H S 2008 Transgenic tomatoes expressing human
beta-amyloid for use as a vaccine against Alzheimer’s disease;
Biotechnol. Lett. 30 1839–1845
Zhang H and Blumwald W 2001 Transgenic salt-tolerant tomato
plants accumulate salt in foliage but not in fruit; Nat. Biotechnol.
19 765–768

MS received 5 March 2009; accepted 14 May 2009

ePublication: 18 June 2009

Corresponding editor: IMRAN SIDDIQI

J. Biosci. 34(3), September 2009