Transformation of Nicotiana Tabacum by Agrobacterium Tumefaciens Carrying Salt and Drought Tolerance Gene

Abstract

A RING-H2 zinc-finger motif with small protein increased tolerance to salt and drought after used the 35S promoter. In present work, expressed in Nicotiana tabacum plant by Agrobacterium tumefaciens leaf. Tissue culture technique was used to indicate the tolerance of plant was able to tolerate NaCl up to 100mM and mannitol up to 50 mM, same technique with transformed bacteria and the antibiotic kanamycin used as selectable marker to select the transformed explants. PCR tech to ensure that the foreign gene presented in transgenic Tobacco plantle concentration of NaCl and mannitol in MS medium and the transformed plants were able to grow in 150mM and 100mM of salt and mannitol respectively, these results indicate that concentrations of salt and drought. From this work we concluded that abiotic stresses can be overcomes by producing GM crops specific gene control.

Full text

Advances i
n
Advan
ces
Journal home page: http://www.aensiweb.com/AEB/
Copyright © 2016 by authors and
American
Transformation of
Agrobacterium
Drought Tolerance Gene
Ahmed Abdul-Jabbar Suleiman
Anbar University, Center of Desert Studies-
Iraq
Address For Correspondence:
Ahmed Abdul-Jabbar Suleiman,
Anbar University, Center of Desert Studies
E-mail: drvaccine@yahoo.com
This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/
Received 28 December 2015
; Accepted
ABSTRACT
A RING-H2 zinc-finger
motif with small protein
increased tolerance to salt and drought after used the 35S promoter. In present work,
expressed in Nicotiana tabacum plant by
Agrobacterium tumefaciens
leaf. Tissue culture technique was used to indicate the tolerance of
plant was able to tolerate NaCl up to 100mM and mannitol up to 50 mM, same technique
with transformed bacteria and the antibiotic kanamycin used as selectable marker to select the transformed explants. PCR tech
to ensure that the foreign gene presented in transgenic Tobacco plantle
concentration of NaCl and mannitol in MS medium and the transformed plants were able to grow in 150mM and 100mM of salt and
mannitol respectively, these results
indicate that
concentrations of salt and drought. From this work we concluded that abiotic stresses can be overcomes by producing GM crops
specific gene control.
KEYWORDS
:
salt tolerance,
drought tolerance, Tobacco, Agrobacterium.
Salinity, w
ater, and temperature
factors responsible for 50%
reduction
drought-affected
areas are expanding and this trend is expected to
has supplied
a better understanding of
proteins have been identified
which
temperature stress,
drought, salinity, etc
some
of these useful genes implicated in
encoding enzymes of the biosynthetic pathways of
betaine, pinitol, sorbitol, etc.
have
different plants
through genetic engineering
A putative RING-H2 factor
encoded by
manner. Under conditions of
white light,
status. This mechanism
suggested a mechanism
n
Environmental Biology, 10(1)
January 2016, Pages: 150-154
AENSI Journals
ces
in Environmental Biology
ISSN-1995-0756 EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
American
-Eurasian Network for Scientific Information
(AENSI Publication).
Transformation of
Nicotiana
Tabacum
Tumefaciens Carrying
Drought Tolerance Gene
Iraq
Anbar University, Center of Desert Studies
-Iraq
This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/
; Accepted
28 January 2016;
Available online 24 February 2016
motif with small protein
encodes by XERICO gene was previously expressed in
Arabidopsis
increased tolerance to salt and drought after used the 35S promoter. In present work,
XERICO
gene under control of 35S promoter was
Agrobacterium tumefaciens
and the transformation efficiency was
15% transformants of infected
leaf. Tissue culture technique was used to indicate the tolerance of
wild-type
Tobacco to salt and drought and the results indicated that the
plant was able to tolerate NaCl up to 100mM and mannitol up to 50 mM, same technique
was used to grow the spliced explants infected
with transformed bacteria and the antibiotic kanamycin used as selectable marker to select the transformed explants. PCR tech
to ensure that the foreign gene presented in transgenic Tobacco plantle
ts. The transformed Tobacco plants were exposed to a
concentration of NaCl and mannitol in MS medium and the transformed plants were able to grow in 150mM and 100mM of salt and
indicate that
XERICO gene under the control of 35S promoter increases
concentrations of salt and drought. From this work we concluded that abiotic stresses can be overcomes by producing GM crops
drought tolerance, Tobacco, Agrobacterium.
INTRODUCTION
ater, and temperature
now are most important factors that effect on
crop productivity,
reduction
in yields in world. Approximately 22% of
cultured
areas are expanding and this trend is expected to
increase [2].
Researches
a better understanding of
the molecular mechanism of stress responses in
plants.
which
get stimulated when plants exposed to various
abiotic stresses
drought, salinity, etc
[11]. In addition,
tools of biotechnology have been applied to
of these useful genes implicated in
tolerance of stresses to plants, also these stress-
induced proteins, genes
encoding enzymes of the biosynthetic pathways of
different osmolytes such as
glycine,
have
been expressed and evaluated in improving
abiotic
through genetic engineering
[6].
encoded by
XERICO promoting synthesis of
ABA in
white light,
the expression of XERICO mRNA increase
under low
suggested a mechanism
responsible for the increasing
(AENSI Publication).
Tabacum
by
Tumefaciens Carrying
Salt and
Available online 24 February 2016
Arabidopsis
thaliana and showed
gene under control of 35S promoter was
15% transformants of infected
Tobacco to salt and drought and the results indicated that the
was used to grow the spliced explants infected
with transformed bacteria and the antibiotic kanamycin used as selectable marker to select the transformed explants. PCR tech
nology used
ts. The transformed Tobacco plants were exposed to a
relatively high
concentration of NaCl and mannitol in MS medium and the transformed plants were able to grow in 150mM and 100mM of salt and
the plants tolerance to high
concentrations of salt and drought. From this work we concluded that abiotic stresses can be overcomes by producing GM crops
with
crop productivity,
these
cultured
lands is saline, and
Researches
in the last decades
plants.
Several genes and
abiotic stresses
low and high
tools of biotechnology have been applied to
transfer
induced proteins, genes
glycine,
proline, trehalose,
abiotic
stress-tolerance in
ABA in
non understanding
under low
gibrillins GA
of endogenous ABA

151
Ahmed Abdul-Jabbar Suleiman, 2016
Advances in Environmental Biology, 10(1) January 2016, Pages: 150-154
concentration after seed inhibition under low GA conditions [12]. Same strategy, involving GA was earlier
proposed in seedlings, in which GA bind XERICO promoter sequences and positively regulates its mRNA
accumulation. The XERICO expression of finger protein RING-H2 confers tolerance of drought through
increasing in ABA biosynthesis and E2 ubiquitin-conjugating enzyme (AtUBC8) interaction which is play a role
in ABA pathway signaling [10].
Plant adaptation to water stress is dependent on the activation of cascades of molecular networks involved
in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites [9].
The plant hormone abscisic acid(ABA), accumulated in plant cells exposed to water stress, is the central
regulator of water stress resistance in plants, and coordinates a complex regulatory network enabling plants to
cope with water stress conditions [8].
Abscisic acid plays important role in development of plant and responses to stress such as growth regulation
and dormancy, desiccation tolerance and leaf senescence. High ABA levels reach during maturation of seed and
dormancy onset, and regulate vegetative development as response to various stresses such as high-salinity and
drought conditions. Under conditions of drought the level of ABA increases so stimulate its complex signaling
cascade, and as a result stomata are closed to prevent water loss through transpiration [1]. When water available
for growth, the level of ABA decreases and as a result the process is reversed.
Transformation mediated by Agrobacterium has some advantages over other transformation methods, and
these advantages involve transfer of large segments of DNA with defined flanking sequence, integration of a
lower number of copies of gene into chromosomes, and no need for protoplast culture which may be hard work.
Furthermore, transformation mediated by Agrobacterium more efficient in producing transformants with lower
number of gene copy simultaneously and insertion patterns are more simpler than genes inserted by particle
bombardment through gene gun [4].
This study was designed to test the role of XERICO gene under the control of 35S Cauliflower mosaic virus
(CaMV) promoter in drought and salt tolerance in another model plant Nicotiana tabacum after its evaluation in
Arabidopsis thaliana and this study was done in Dr.song lab at Michigan State University (MSU) during
Summer of 2013.
MATERIALS AND METHODS
Plant materials: -
Seeds of Tobacco (Nicotiana tabacum L. cv. Xanthi) were germinated in vitro. Approximately 100 seeds
were put on a filter paper (No.5:11 cm diameter), and then small pack was made. Each pack of seeds was soaked
in 95% (v/v) ethanol for 5 sec, and using a sterile forceps the seeds were transferred to 50% (v/v) Clorox with a
drop of Tween-20 for 20 min with stirring, after that seeds were rinsed in sterile water 5 times and dried on
sterile Whatman filter papers (No.5:11 cm diameter). Ten seeds were planted per Petri dish on the T-media (per
L; 0.1 g myo-inositol, 0.4 mg thiamine⋅HCl, 30.0 g sucrose, pH 5.6, and ten grams of agar for solid medium and
4.4 g MS salts). Seeds were germinated and grown in the growth room under constant light period 16 hrs at
room temperature (Murashige and Skoog 1962). Plantlets were sub-cultured every two to three weeks. Stems
with one node and two leaves were cut out from grown plantlets and then transplanted to fresh T-medium.
Preparation and culture of explants:
For all the experiments including transformation 30 d old first pairs of fully expanded leaves were used.
The leaves were trimmed in 1 × 1 mm pieces and aseptically placed with the abaxial surface touching the
culture medium. MS with B5 vitamins, 2% (w/v) sucrose, 0.03% (w/v) phytagel was used in all the experiments.
The pH of the medium was adjusted to 5.8 by 1 M NaOH or 1 M HCl before autoclaving at 121°C for 20 min.
All plant growth regulators were added after autoclaving the medium. The cultures were incubated at 25 ± 2°C
in a culture room with 50 μmol m
−2
s
−1
irradiance provided by cool fluorescent lamps and were exposed to a
photoperiod of 16 h and 55% relative humidity.
Checking of drought and salt tolerant:
The ability of tobacco to tolerate salt was investigated with increased concentration of NaCl using
0,25,50,75,100,150,200, and 250 mM of NaCl in MS media complete media with hormone also the drought
tolerant were checked by investigation the leaf ability to still green in mannitol with different concentration
0,25,50,75,100,125, and 150 in sterile well.
Construction of vector:
The full-length cDNA of XERICO (At2g04240) was amplified by PCR using primers 5-
Tggatccgacaacatcatttctaccgaca-3 (forward) and 5- Ccctctagatagctgtacacaacaaacacactc-3 (reverse) designed to
contain BamHI and XbaI sites, respectively. The resulting product was digested withBamHI and XbaI, and
inserted between a 35S promoter of Cauliflower mosaic virus(CaMV) and a nopaline synthase terminator in the

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Ahmed Abdul-Jabbar Suleiman, 2016
Advances in Environmental Biology, 10(1) January 2016, Pages: 150-154
pCB302-3 binary vector, containing kanamycin resistance gene inside the T-DNA for selection of trans
formants [14].
Transformation protocol:
The vector was introduced into Agrobacterium tumefaciens strain C58, which was used to transform
Arabidopsis ecotype Columbia by the floral-dip method as described by Clough and Bent [5]. EighteenT3
progeny resulting from homozygous self crosses were used for phenotypic characterizations. Well matured leaf
let were used to carry out in-vitro transformation studies. Sensitivity to antibiotic kanamycin selection is a factor
that affects our ability to produce fertile transgenic tobacco.
RESULT AND DISCUSSION
After about 10 days germination was occurred on 1/2 strength MS medium. Tobacco seeds were obtained
from the store of MSU, USA. Results indicated that most of seeds were geminated in 1/2 strength medium
containing thymine and myo-inositol at pH 5.8 after one week of incubation at 25 °C under 16:8 hrs (dark :
light) photoperiod, plantlets then were successfully transferred to light box bottle and the resulted leaves were
used for tissue culture and transformation experiments (Fig 1).
Fig. 1: Germination of tobacco seeds on 1/2 strength MS media.
Leaves formed on seedling were dissected to small pieces of (5x5 mm), regeneration ability of tobacco
explants to form complete plant was tested on MS medium supplemented with NAA and BAP, indicates
regeneration occurred with shoot formation after 10 days of culture. Shoots were transferred to another Petri
dish for rooting and plantlets were obtained after 30 days, and thus plants were ready to be transferred to a green
house (figure 2).
Fig. 2: Tobacco regeneration from explants on MS media containing growth hormone after 3 weeks.
The ability of wild-type tobacco to tolerate salt were evaluated on MS media and it were tolerated until
100mM as shown in figure 3.
Also, wild type tobacco were able to tolerate mannitol as indicator to drought tolerance by use of mannitol
drop on leaves and see the yellowish appearance on leaves with time and the tolerance reached to 50mM as
shown in figure 4.
After cotransformation with Agrobacterium tumefaciens the transformation efficiency was measured on MS
media containing kanamycin as a selectable marker and the efficiency of transformation was 13% of total
cultured explants. The growing explants were then transferred to MS media with growth hormone contain salt to
check the ability of transformed plant to tolerate salt and the leaves were then put in mannitol to check the
ability of drought tolerance. The transformed plant showed 150mM salt tolerance and 100mM mannitol
tolerance which mean drought tolerance. The transformation then confirmed by PCR detection of XIRCO gene

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Ahmed Abdul-Jabbar Suleiman, 2016
Advances in Environmental Biology, 10(1) January 2016, Pages: 150-154
with CaMV promoter and the resulted band was 1230 bp indicate that gene was transferred to Tobacco plant as
shown in figure 5.
.
Fig. 3: Tolerance of Tobacco explants to salt with increased concentration from 25 to 200mM respectively.
Fig. 4: The drought tolerance test by mannitol drop on tobacco leaves.
Fig. 5: Agarose gel electrophoresis of PCR product of transformed tobacco plant. M: molecular marker, W.T:
wild type plant, 1 and 2 transformed plant showed the 1230bp XIRCO gene.
The resulted explants able to grow on kanamycin-containing media then checked to ability to tolerate salt
and drought on increased concentration than which seen with wild type, transformed explants showed ability to
tolerate 150mM and 100mM of salt and drought respectively figure 6
Fig. 6: Tissue culture of transformed tobacco on MS media containing growth hormone and 150mM NaCl after
15 days of explants culture.

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Advances in Environmental Biology, 10(1) January 2016, Pages: 150-154
The high-level expressions of ABA-biosynthetic and ABAresponsive genes in 35S: XERICO plants, even
in the absence of ABA or drought treatment, suggested that the transgenic plant might have elevated
endogenous ABA. The transcriptional upregulation of XERICO in Arabidopsis induced hypersensitivity to salt
and osmotic stress and to ABA treatments during germination and early seedling growth. The hypersensitivity to
ABA may have come from altered ABA metabolism or signaling.
The analyses of ZFP36-overexpressing and silenced transgenic rice plants showed that ZFP36 is involved in
ABA-induced up-regulation of the expression and the activities of superoxide dismutase (SOD) and ascorbate
peroxidase (APX). Overexpression of ZFP36 in rice plants was found to elevate the activities of antioxidant
enzymes and to enhance thetolerance of rice plants to water stress and oxidative stress. In contrast, an RNA
interference (RNAi) mutant of ZFP36had lower activities of antioxidant enzymes and was more sensitive to
water stress and oxidative stress. ABA-induced H2O2 production and ABA-activated mitogen-activated protein
kinases (MAPKs) were shown to regulate the expression of ZFP36 in ABA signalling. On the other hand,
ZFP36 also regulated the expression of NADPH oxidase genes,the production of H2O2, and the expression of
OsMPK genes in ABA signaling [16].
Abscisic acid is known to affect the expression of many genes involved in ABA metabolism that
upregulation of the XERICO gene substantially increased ABA biosynthesis. However, it should be noted that
the expression of transcriptional regulators of ABA signaling (e.g.ABI5, ABI3, AtMYB2, AtMYC) was not
changed in 35S:XERICO plants. Many RING-H2 proteins function as part of the E3 ubiquitin ligases [3].
Ubiquitin-mediated protein degradation plays key regulatory roles during several plant growth and
developmental events, and has been implicated in plant hormone signaling [7]. Yeast two-hybrid screening
analysis identified AtTLP9 and AtUBC8 as potential XERICO interacting partners. AtTLP9 (At3g06380) was
reported as an ASK1-interacting F-box protein, which is involved in the ABA signaling pathway [15]. ASK1,
one of the 21 Skp proteins in Arabidopsis, is involved in both vegetative growth and reproductive development.
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