Indian Journal of Biotechnology

Vol. 17, July 2018, pp 459-465

Genetic improvement in tomato (Solanum lycopersicum) against salt stress

Sohail Ahmad Jan1,2*, Ghulam Muhammad Ali1,2, *Shaukat Ali1, 2, Sabir Hussain Shah3, Nazir Ahmad1, 2
1
PARC Institute of Advanced Studies in Agriculture (PIASA), National Agricultural Research Centre (NARC),
Park Road, Islamabad 45500, Pakistan
2
National Institute for Genomics and Advanced Biotechnology (NIGAB), National Agricultural Research Centre (NARC),
Park Road, Islamabad 45500, Pakistan
3
Department of Agricultural Sciences, Allama Iqbal Open University, Islamabad, Pakistan

Received 5 November 2014; revised 8 July 2016; accepted 24 December 2016

Salinity and drought are the two major abiotic stresses that affect the quality and quantity of tomato. The dehydration
response transcription element binding factor (DREB) gene enhances tolerance in plants against salinity. The present study
was conducted to transformed DREB1A gene through Agrobacterium in tomato cultivar Roma against salt stresses. A binary
vector containing DREB1A gene under the control of inducible promoter Lip 9 along with hygromycin screening marker
was used. The few major factors which directly affect transformation efficiency were optimized such as acetosyringone
levels, cefotaxime concentrations, pre-selection time period and hygromycin levels. Four different concentrations (50, 100,
150 and 200 mM) of NaCl were applied on both transgenic and non transgenic plants. The addition of 200 µM
acetosyringone in co-cultivation media, 200 mg/L cefotaxime in pre-culturing media and 25 mg/L hygromycin
concentration significantly increased transformation efficiency. Maximum transformation efficiency 16.6% was obtained in
tomato cultivar Roma. The transgenic plants were confirmed with PCR and the expected band size ~630 bp was amplified.
The transgene express in transgenic tomatoes was confirmed by reverse transcriptase- polymerase chain reaction (RT-PCR).
The resulted transgenic plants showed enhanced tolerance against high salt concentration (200 mM) while the non
transgenic plants showed the symptoms of wilting and eventually die at that concentration. The DREB1A gene was
successfully transformed into local tomato cultivar Roma. The resulted transgenic tomato cultivar Roma will be
recommended against extreme salt stresses.

Keywords: Agrobacterium, DREB1A, tomato, salinity stress

Introduction stress related genes and thus enhanced tolerance in

Tomato (Solanum lycopersicum) belongs to a
commercially important family Solanaceae. It is
cultivated in tropical, subtropical and temperate
areas1. Biotic and abiotic factors are the two major
constrains that affect its quality and quantity2-3.
Abiotic stresses such as drought, cold and salinity
affect plant severely. Plants are more susceptible to
these extreme environmental stresses as compare to
any other living organisms. When plants are subjected
to these extreme environmental stress conditions they
cannot perform normal physiological function so
affect their growth and productivity. Therefore for
continuous improvement in agriculture it is important
to develop resistance varieties through genetic
approach4. Transcription factors provide resistant to
plant against abiotic stresses by activation of multiple
——————
*Author for correspondence:
Tel: +923459118133
drgmails@gmail.com; shaukat_parc@yahoo.co.in
plants. Under abiotic stress condition the plants
produce some signals, as result by these signal
perception through series of transcription factors
starts there function and thus regulate different genes
by attaching with specific sequence at promoter
region and provide protection to plants and allow
plants for its normal physiological functions5.
The dehydration responsive element binding
factors (DREB) belong to AP2/ERF (apetala2 and
ethylene responsive factors) class of transcription
factors distinct to plants. The two subclass DREB1
and DREB2 transcription factors activate under
drought and salt stress condition and activate
other stress related genes which leads enhanced
tolerance to plants against these environmental
extreme condition6. Transgenic tobacco plant
expressing the Brassica junica DREB1B gene shows
high level of proline content which leads enhanced
tolerance to drought and salt stresses7. DREB gene
460 INDIAN J BIOTECHNOL, JULY 2018
have important role in signal transduction in abiotic
stress condition and activate many stress related
genes. They expression analysis of cloned rice DREB
gene revealed that DREB gene enhanced the
expression of multiple genes under drought, salt
and cold stress condition but have no function under
biotic stress8.
Agrobacterium-mediated transformation is quick
and easy method of gene transfer to plant.
The stability and integration of DNA into host
genome is excellent9. Kasuga et al (2004)10 studied
that transformed DREB1A gene in tobacco increase
tolerance against drought and cold. Shen et al
(2003)11 conducted an experiment by transformation
of DREB1A gene into different cultivars of wheat. To
address the salt stress problem in tomato the present
study was conducted to transform the DREB1A gene
in tomato for salt tolerance.
Materials and Methods
Plant Materials
The genetic transformation experiment was
conducted at National Institute for Genomics and
Advanced Biotechnology (NIGAB), National
Agricultural Research Centre (NARC), Islamabad,
Pakistan. Mature seeds of important tomato cultivar
Roma were taken from Horticultural Research
Institute (HRI), National Agricultural Research
Centre (NARC), Islamabad, Pakistan.
Cloning Vector and Agrobacterium Strain
A binary cloning vector having AtDREB1A gene
was used. The binary vector express in the
presence of inducible promoter Lip 9 (Fig. 1). The
Agrobacterium tumefaciens strain EHA105 was used,
due to its quick and stable transformation efficiency.
Seed Sterilization and Germination
The seeds were treated with 80% alcohol for
3 minutes. The seeds were then gentle shake with
20% Clorox for two times for 15 minutes each. The
Fig. 1 — Diagram for vector containing DREB1A gene under
lip9 promoter. LB, left border; T-G7, terminator; hpt (hygromycin
phosphotransferase gene), P-NOS promoter of nopaline
synthase gene, Lip-9 promoter, DREB1A gene, T-NOS terminator
of nopaline synthase gene, RB right border.
seeds were dried and transferred to Murashig &
Skoog (MS) media12 and the pH of media was
maintained 5.7 to 5.8. The tubes were placed in
growth room at optimum growth condition for
7-10 days in order to achieve maximum germination.
Agrobacterium Mediated Transformation
The viable strain EHA105 of Agrobacterium
tumefaciens was taken and streaked with sterilized
loop on Luria-Bartani (LB) media (Peptone 10 gm/L,
yeast extract 10 gm/L, NaCl 5 gm/L, agar 6 gm/L,
pH 7) along with kanamycin sulphate (100 mg/L).
The plates were then kept in incubator for 24 hours at
28°C. The fresh single colony was selected and
transfers to LB media in falcon tube and kept in
incubator for 20 hours at 28°C. Once the bacterial
growth was observed after 20 hours the culture was
then centrifuged at 8000 rpm for 8 minutes to get a
clear pellet. The supernatant was discarded and
the pellet was re-dissolved in fresh LB media.
The optimum Agrobacterium optical density (OD600)
0.4 was maintained.
Infection and Co-cultivation
The nodes were cuts from the freshly regenerated
plantlets and were placed in plates. The Agrobacterium
culture having optimum optical density (OD600)
0.4 was added to plates containing explants for 1, 3
and 5 minutes. After infection the explants were
carefully removed and transfer to co-cultivation media
plates containing three different concentrations of
acetosyringone (100, 200 and 300 µM). The plates
were shifted to growth room and were placed for three
days for efficient transformation of targeted gene. The
morphology of explants was also checked regularly at
different bacterial concentrations.
Washing of Infected Explants and Pre-selection
After three days infection period the explants were
single washed with distilled water for 5 minutes
followed by two times washed with MS liquid media
having three different levels of cefotaxime antibiotic
(200, 400 and 600 mg/L) for 10 minutes each. The
explants were then transferred to pre-selection media
having three different levels of cefotaxime (200, 400
and 600 mg/L) for 2, 4, 6 and 8 days.
Selection
The explants were changed into callus stage on pre-
selection media and were transferred to selection
media containing three different levels of selectable
marker hygromycin (25, 50 and 75 mg/L) and were
 AHMAD JAN et al: GENETIC TRANSFORMATION IN TOMATO
kept in light/dark (16/8 hours) at 28°C for three days.
The transformed green calli showed resistance to
hygromycin. And transformation efficiency was
measured from these resistant calli.
Regeneration of Transformed Calli
The transformed calli were transferred into
regeneration media containing 0.5 mg/L of indole
3-acetic acid and 3 mg/L of benzylaminopurine. After
three weeks of regenerated plants were carefully
transferred into pots and were shifted into glass house
with supply of water (Fig. 3).
Molecular Analysis
The transgenic plants were screened with
polymerase chain reaction (PCR) for transgene
integration. The young green leaves were removed
from the transgenic tomato plants for genomic
DNA isolation through modified CTAB method13.
PCR analysis was performed using set of DREB1A
primers 5’-CTACGCGTACGTACGATACA-3’F
and 3’-TCGGCTTGGAGACTCCGAAT-5’R. The
amplified PCR product were then loaded on 1% gel
electrophoresis containing TAE buffer and the
amplified PCR product band were visualized by
UV illuminator. The target PCR bands size approx.
630 bp were than confirmed with the 1 Kb ladder.
Gene Expression Analysis
The mRNA was extracted from transgenic and
non transgenic tomato plants after 2 weeks of salt
stress by using RNA extraction kit PureLinkTM
RNA Mini Kit (Invitrogen). The mRNA was used
for the construction of cDNA by using specialized
AmbionTM Reverse Transcriptase Kit (Fermentas).
The PCR condition was set for 10 minutes at 95oC,
followed by 40 cycles at 95oC for 30 sec, 64oC for
30 sec and last cycle at 74oC for 1 minute.
Salt Stress Treatments
The salt stress was subjected to both transgenic and
non transgenic plants. Both the transgenic and non
transgenic plants were grown in pots containing
compost and soil (1:1). The four different salt
concentrations (50, 100, 150 and 200 mM) were given
to both transgenic and non transgenic plants after two
weeks of regeneration. The salt stress was given for
1-2 weeks and various morphological changes that
occur during this period were noted.
Data Analysis
All the experiments were arranged in completely
randomized design (CRD) and were repeated three
461
times. The data was analysed by using ANOVA P ≤
0.05. The significant mean differences among
genotypes were studied with least significant
difference test (LSD)25.
Results
DREB1A gene play important role in plants by
providing tolerance against environmental stresses.
The DREB1A gene was successfully transformed and
various factors that affect transformation process were
optimized for important tomato cultivar Roma.
Effect of Acetosyringone on Transformation
Acetosyringone is a phenolic substance which
helps in transformation of T-DNA region into
host plants. In present study three different levels
(100, 200 and 300 µM) of acetosyringone were used.
The ANOVA results showed that acetosyringone
have significant effect on transformation efficiency at
p < 0.05. The 100 µM acetosyringone concentration
ion MS media was found optimum for efficient and
transformation efficiency was highest 45% at that
level. The high amounts of acetosyringone (200,
300 µM) in MS media decrease the transformation
frequency up to several folds (Table 1). The node
explants were placed on co-cultivation media are
shown in Figure 2a.
Influence of Pre-selection Time Period on Transformation
Efficiency
Pre-selection time and transformation efficiency
both are correlated. Four different events of pre-
selection were tested in present study. Our results
showed that 6 and 8 days pre-selection period
increased the transformation efficiency up to 34%.
From ANOVA results we noted that pre-selection
period shows significant effects on transformation
frequency. The lower pre-selection period showed
inverse effect on transformation efficiency (Table 2).
Table 1 — Effect of acetosyringone (AS) on transformation
efficiency in tomato cultivar Roma
AS (µM) Calli selected on Transformation
hygromycin efficiency (%)
100 16.66a ± 0.69a 40a
200 10.00b ± 0.38 25b
300 04.66c ± 0.24 10c
LSD 3.20
Mean values followed by the same letter within a column shows no
significant differences (p < 0.05). Transformation efficiency was
calculated by using following formula. Transformation efficiency =
(Number of hygromycin resistant plants regenerated/total number of
explants co-cultivated with Agrobacterium) × 100
462 INDIAN J BIOTECHNOL, JULY 2018

Fig. 3 — Regeneration of transformed plants (a) Regeneration of

tomato cultivar Roma and (b) Transgenic plants of tomato cultivar
Roma in glass house.

Table 3—Transformation efficiency of tomato cultivar Roma

Fig. 2 — Factors effecting on transformation of tomato cultivars
Roma: (a) Primary leaves explants of Roma on co-cultivation
media; (b) Explants of Roma on pre-selection media; (c) Effect of
cefotaxime on explants; and (d) Selection of transformed calli
with hygromycin.
Table 2 — Effect of days of pre-selection on transformation
efficiency in tomato cultivar Roma
Pre-selection Calli selected on Transformation
(days) hygromycin efficiency (%)
2 02.66d ± 0.71 03
4 05.00c ± 0.38 06
6 18.66a ± 0.24 34
8 14.00b ± 0.58 34
LSD 0.57
Mean values followed by the same letter within a column shows no
significant differences (p < 0.05). Transformation efficiency was
calculated by using following formula. Transformation efficiency =
(Number of hygromycin resistant plants regenerated/total number of
explants co-cultivated with Agrobacterium) ×100
The nodes explants of tomato cultivar Roma on
pre-selection media were shown in (Fig. 2b).
Influence of Cefotaxime on Transformation Efficiency
Cefotaxime is one of the broad spectrum antibiotic
commonly used in plant transformation experiments
to control the excess growth of bacteria. In present
study three different concentrations (200, 400 and
600 mg/L) were used. The low dose 200 mg/L of
cefotaxime was found effective for explants
survivability and for control of bacterial overgrowth.
The high dose of cefotaxime (400 and 600 mg/L)
caused sclerosis of explants and followed by the death
of all the explants (Fig. 2c).
Influence of Hygromycin Selectable Marker on
Transformation Efficiency
Hygromycin is one of the commonly used
screening markers in genetic transformation
using hygromycin as selectable marker
Hygromycin Calli selected on Transformation
(mg/l) hygromycin efficiency (%)
25 14.66a ± 0.88 20.00a
50 08.00b ± 1.23 16.60b
75 02.33c ± 0.33 00.00c
LSD 2.13
Mean values followed by the same letter within a column shows
no significant differences (p < 0.05). Transformation efficiency was
calculated by using following formula. Transformation efficiency =
(Number of hygromycin resistant plants regenerated/total number of
explants co-cultivated with Agrobacterium) ×100
experiments for selection of transgenic plants.
Three different concentration (25, 50 and 75 mg/L) of
hygromycin were used. The ANOVA results revealed
that different concentrations of hygromycin have
significant effect on transformation frequency and
selection of transgenic and non-transgenic calli at
p < 0.05. The results revealed that transformation
efficiency was highest 20% at 25 mg/L hygromycin
level while it was lower 16.6% on 50 mg/L (Table 3).
The high concentration 75 mg/L showed negative
effect on transformation frequency (Fig. 3).
Molecular Analysis
The PCR results revealed that the transgenic plants
showed positive results. The target PCR bands size
approx. 630 bp were than confirmed with the 1 kb ladder
from the transgenic plants while the non-transgenic
plants showed no bands pattern (Fig. 4a).
Expression Analysis
The foreign DREB1A gene expression was
confirmed by RT-PCR. The transgenic plants after
different salt events show stable expression of foreign
gene while the wild non-transgenic plants showed no
expression DREB1A gene (Fig. 4b). The presence of
 AHMAD JAN et al: GENETIC TRANSFORMATION IN TOMATO
Fig. 4 — (A) Molecular detection of alien gene through PCR: (B)
Molecular detection of alien gene at different salt concentration
through RT-PCR. [(A) M = Marker 1 kb, PC = Positive control, T
1 = Transgenic plant 1, T2 = Transgenic plant 2, T4 = Transgenic
plant 4, NC = Negative control NT1 = Non-transgenic plant 1,
NT2 = Non-transgenic plant 2, NT3 = Non-transgenic plant 3 and
(B) T1: Trangenic plants (50 mmol), T2: Trangenic plants
(100 mmol), T3: Trangenic plants (150 mmol), T4: Trangenic
plants (200 mmol), C: Controled plants (0 mmol), NT1: Trangenic
plant (50 mmol), NT2: Trangenic plant (100 mmol), NT3:
Trangenic plant (150 mmol), NT4: Trangenic plant (200 mmol)].
Fig. 5 — Transgenic and non transgenic plants after salt stress:
(a)Transgenic plants after 2- weeks of salt stress; transgenic
plants remain green and healthy, and (b) Non transgenic plants
after 2-weeks of salt stress; the non transgenic plants shows
yellowish and necrotic symptoms.
transgene in transgenic tomato genotypes gives
more tolerance to salt stress events as compared to
non-transgenic plants.
The Effect of Salt Stress on Transgenic and Non Transgenic
Plants
The effect of salinity was determined by exposing
both transgenic and non-transgenic plant to various
salt concentrations (50, 100, 150 and 100 mM) for
1-2 weeks after two weeks of regeneration. Our
results revealed the transgenic plants showed
resistance to low (50 mM) and even very high salt
concentration (200 mM) and remain green after two
weeks of salt trials. While the non transgenic plant
showed the symptoms of sclerosis and wilting at
moderate salt concentration (100 mM) and eventually
died at high salt concentration (200 mM) after one
week of salt stress (Fig. 5).
463
Discussion
Plant transformation through Agrobacterium is a
quick, efficient and easy method of transformation.
However genetic transformation process is affected
by various factors which directly affect the efficiency
of transformation. In present study different
factors such as acetosyringone levels, co-cultivation
time, cefotaxime levels, pre-selection time period
and screening marker gene concentrations were
optimized. Optimum acetosyringone concentration is
important for the activation of virulence region of
Ti-plasmid. Three different concentrations of
acetosyringone were tested. The results showed
that 200 mg/L concentration was optimum for
maximum transformation (Table 1). Our results are
not in commitment with van Wordragen
and Dons (1992)14 who reported that transformation
efficiency increased in dicot with the increase
concentration of acetosyringone. Our findings
show agreement with Raj et al (2005)15 who achieved
maximum transformation frequency with 100 µM
acetosyringone. Our results are in accord with
Moghaieb et al (2004)16 who used 100 µM
acetosyringone in co-cultivation media.
Cefotaxime is one the broad spectrum commonly
used in transformation to control the overgrowth of
Agrobacterium. From results we noted that with low
cefotaxime concentration (200 mg/L) maximum
explants survive and remain healthy but high
cefotaxime concentration (400 - 600 mg/L) leads death
of explants (Fig. 2c). Paramesh et al (2010)17 used
250 mg/L concentration of cefotaxime and observed
no toxic effect on explants but when the concentration
was increase up to 500 mg/L the explants become
necrotic and eventually died after 7 days of
pre-selection. However, our findings are not similar
with the findings of Huang et al (2001)18 who used
300 mg/L cefotaxime concentration for controlling
bacterial overgrowth in rice and all the calli were
healthy at that concentration but high concentration
inhibited the germination.
Pre-selection is important step before the selection.
The pre-selection period provide strength to explants
and the entire explants change into calli in this
period. In present study four different pre-selection
periods were provided to explants. The 6 and
8 days pre-selection period enhanced efficiency of
transformation up to 34% in tomato cultivar Roma.
The transformation efficiency was low with small
pre-selection period for 2 - 4 days (Table 2). Our
results are contradictory with other workers with
464 INDIAN J BIOTECHNOL, JULY 2018
Ahsan et al (2007)19 that obtained maximum
transformation frequency in tomato without providing
pre-selection period. Out results are supported by Gao
et al (2009)20, who obtained best transformation
efficiency in tomato with longer pre-selection period.
Sensitivity of calli to screening marker hygromycin
was also studied in present work. Three different
concentration of hygromycin (25, 50 and 75 mg/L)
was used for best selection of transgenic calli
from non-transgenic calli. The results revealed that
25 mg/L hygromycin concentration was found
effective and maximum transformation efficiency
20% was obtained at this concentration. The high
concentration 75 mg/L decreased both the selection
pressure and transformation efficiency (Table 3). Our
findings show contradiction with Roy et al (2006)21
who used 40 mg/L hygromycin concentration for
transgenic calli selection of tomato cultivar Pusa
Ruby. This deviation might be due to different
genotype used. Other worker also used other types of
selection marker in tomato such as kanamycin was
used by Moghaieb et al (2004)16 and Raj et al
(2005)15. It is reported that the concentrations of
hygromycin vary with the genotype22.
Salt stress is a persistent and economically
damaging environmental factor that affects both the
qualitative and quantitative characters of tomato plant
at different developmental stages. At the flowering
and fruiting stages, tomato plants are able to survive
NaCl concentrations which are sufficient to kill them
at the seedling and early developmental stages. In
present study four different salt concentration (50,
100, 150 and 200 mM) were tested and from our
finding we noted that the transgenic plants showed
resistant event at very high salt concentration while
the non-transgenic plant showed the yellowish
symptoms at early salt stress and finally died after one
week of salt trial (Fig. 5). Our findings show
agreement with other co-workers such as,
Yamaguchi-Shinozaki and Shinozaki (2007)23 who
reported that the transgenic Arabidopsis plant
produced tolerance against drought, cold and salinity
by over expression of the DREB1A gene. The stable
integration of DREB2A gene in Populus euphratica
provides tolerance against salt stress24. The transgenic
Arabidopsis and rice plants having OsDREB1F gene
in their genome showed enhanced tolerance to both
extreme salt stress8. The transgenic tomato shows the
presence of DREB1A gene and expressed in all stress
levels as compared to non-transgenic tomatoes
(Fig. 4a & b). The transgenic tomatoes expressing
DREB1A gene shows maximum tolerance to frost
stress as compared to non-transgenic tomatoes. The
transgenic plants shows long term expression of
DREB1A gene in frost stress conditions26, 27.
Acknowledgements
This work was financially support by the Project
National Institute for Genomics and Advanced
Biotechnology (NIGAB), National Agricultural Research
Centre (NARC), Islamabad, Pakistan. The authors are
thankful to the Director of Horticultural Research Institute
(HRI), National Agricultural Research Centre (NARC),
Islamabad Pakistan for providing seeds.
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