Salt tolerance of genetic modified potato (solanum tuberosum) cv. Agria by expression of a bacterial mtlD gene
ABSTRACT Water and salinity stresses are the major reasons to yield decreasing in the world. Potato is the world’s
main tuber crops of the Solanaceae family which is one of the most economically important annual vegetable crop.
The goal of this investigation was creation genetic modified potato cv.Agria with more tolerance to salinity stress
and evaluating GMO potato properties. To create transgenic potato plant, mtlD gene (mannitol -1- phosphate
dehydrogenase, E.C.22.214.171.124) was expressed to potato cv.Agria plant by using Agrobacterium tumefaciens.
Transgenic potato was produced by transforming of mtlD gene to potato plant cv.Agria. Existence of recombinant
gene in transgenic plants was approved by two ways 1. Polymerase Chain Reaction technique. 2. Measurement of
physiological parameters. The transgenic potatoes and non-transgenic potatoes lines exhibited the different
amounts of tolerance to salinity stress because in the transgenic lines mannitol accumulates that increased osmotic
pressure in salinity stress. The salt tolerance of transgenic potato cv.Agria (+mtlD) was recorded higher than that of
non-transgenic potato cv.Agria (-mtlD). Osmotic pressure in this transgenic potato plant was increased by
accumulating of mannitol and existence of mannitol in potato plant approved that the mtlD gene was successfully
expressed to potato cv.Agria.
- SourceAvailable from: indiana.edu[show abstract] [hide abstract]
ABSTRACT: The paper is focused on the potato production in Peru, that is the Latin America's biggestpotato producer, with a record harvest in 2007 of almost 3.39 million tones, followed by Brazil,Argentina, Colombia and Mexico. National potato production is enough for internal demand; the annualconsumption in is about 80 kg per capita. The cultivated area in Peru is about 270000 ha, which isproduced mainly by small farmers, at altitudes of from 2000 m to 4500 m in the Peruvian highlands.Today, in Peru, there are around 3000 cultivated varieties of potato, which include the eight speciessome of them are exclusive to the Andes. Unfortunately, potato biodiversity in Peru is vulnerable,partly due to the tradition of local knowledge being passed down through the generations by word-ofmouth,young people move to the cities, and also by abiotic and biotic threats. The study underlinesthe main constraints on potato productivity, but also the solutions the research activity may offer tosustain the efforts in crop improvement.Advances in Agriculture & Botanics. 01/2009;
- Journal of Hydrometeorology - J HYDROMETEOROL. 01/2006; 7(5).
- [show abstract] [hide abstract]
ABSTRACT: Previous work with model transgenic plants has demonstrated that cellular accumulation of mannitol can alleviate abiotic stress. Here, we show that ectopic expression of the mtlD gene for the biosynthesis of mannitol in wheat improves tolerance to water stress and salinity. Wheat (Triticum aestivum L. cv Bobwhite) was transformed with the mtlD gene of Escherichia coli. Tolerance to water stress and salinity was evaluated using calli and T(2) plants transformed with (+mtlD) or without (-mtlD) mtlD. Calli were exposed to -1.0 MPa of polyethylene glycol 8,000 or 100 mM NaCl. T(2) plants were stressed by withholding water or by adding 150 mM NaCl to the nutrient medium. Fresh weight of -mtlD calli was reduced by 40% in the presence of polyethylene glycol and 37% under NaCl stress. Growth of +mtlD calli was not affected by stress. In -mtlD plants, fresh weight, dry weight, plant height, and flag leaf length were reduced by 70%, 56%, 40%, and 45% compared with 40%, 8%, 18%, and 29%, respectively, in +mtlD plants. Salt stress reduced shoot fresh weight, dry weight, plant height, and flag leaf length by 77%, 73%, 25%, and 36% in -mtlD plants, respectively, compared with 50%, 30%, 12%, and 20% in +mtlD plants. However, the amount of mannitol accumulated in the callus and mature fifth leaf (1.7-3.7 micromol g(-1) fresh weight in the callus and 0.6-2.0 micromol g(-1) fresh weight in the leaf) was too small to protect against stress through osmotic adjustment. We conclude that the improved growth performance of mannitol-accumulating calli and mature leaves was due to other stress-protective functions of mannitol, although this study cannot rule out possible osmotic effects in growing regions of the plant.Plant physiology 05/2003; 131(4):1748-55. · 6.56 Impact Factor
Advances in Agriculture & Botanics
International Journal of the Bioflux Society
Volume 4 | Issue 1Page 10
Salt tolerance of genetic modified potato (Solanum
tuberosum) cv. Agria by expression of a bacterial
1,2Aliakbar Askari, 1Astghik Pepoyan, 3Ali Parsaeimehr
1 Laboratory of Molecular Biology and Biotechnology, Armenian State Agrarian University, Yerevan, Armenia; 2 Islamic Azad
University, BAM Branch, IRI; 3 Club of Young Researchers Organization of Azad University, Iran.
Potato (Solanum tuberosum) is a tuber plant from Solanaceae
family which is grown all over the world. The potato plant was
first grown and eaten in the Andes Mountains of South America
then it was brought to Europe and other countries (Blas & Petrescu
2009). It is the most important crop after rice, wheat and corn
(Vreugdenhil & Bradshaw 2007), and the year 2008 was named
as international year of potato by United Nation (Theisen 2008).
The potato tuber (of a medium size, 150 g) contains vitamin C
(27 mg, 45% of the daily value), 620 mg potassium (18% of
the daily value), 0.2 mg vitamin B6 (10% of the daily value),
thiamin, riboflavin, niacin, phosphorus, iron and zinc. It was
reported by the United Nations - FAO that the world produc-
tion of potatoes in 2009 was 330 million tones. Two thirds of
the global production is eaten directly by humans and the rest
is used to feed the animals or produce starch (Vreugdenhil &
Bradshaw 2007). One the major branch of genetic engineer-
ing is genetic modified organisms (GMO). Although this term
(GMO) is not the most representative for genetically engineered
organisms, GMO was defined as an organism which his genetic
material has been altered using genetic engineering techniques
(Bhatnagar et al 2007). These techniques were initially named
recombinant DNA technologies, and use DNA molecules from
different sources that are combined into one chromosome to
create a new organism with modified genetic material. Genetic
modification includes the insertion of a wanted gene or genes to
genetic material of a cell (Bahieldin et al 2007). GMO is used
in biological and medical research production of pharmaceuti-
cal drugs, gene therapy and agriculture. Many GMO organisms
that were created include plants, fishes, bollworms, mosquitoes,
fruit flies and microbes (Katiyar-Agarwal et al 1999).
Abiotic stresses have unfavorable effect on growth and produc-
tivity of plants and those cause a series of morphological, physi-
ological, biochemical and molecular changes in plants. Drought,
temperature extremes and saline soils are the most common
abiotic stresses. Approximately 22% of the agricultural lands
are saline, and regions under drought are increasing and it was
forecasted the areas under salinity and water stress will be in-
creased (Burk et al 2006). When a crop is exposed to abiotic
stress, a number of genes are turned on, resulting in increased
amount of some metabolites and proteins. Water and salinity
stresses are main abiotic stresses which limit plant productivity
and growth. One way of increasing yield in stress condition is
use of genetic engineering for improving plant tolerance to wa-
ter and salinity stresses (Abebe et al 2003). Horsch et al, in
1985, for first time introduced general method for transferring
Abstract. Water and salinity stresses are the major reasons to yield decreasing in the world. Potato is the world’s main tuber crops of the
Solanaceae family which is one of the most economically important annual vegetable crop. The goal of this investigation was creation genet-
ic modified potato cv. Agria with more tolerance to salinity stress and evaluating GMO potato properties. To create transgenic potato plant,
mtlD gene (mannitol-1-phosphate dehydrogenase, E.C.126.96.36.199) was expressed to potato cv. Agria plant by using Agrobacterium tumefaciens.
Transgenic potato was produced by transforming of mtlD gene to potato plant cv. Agria. Existence of recombinant gene in transgenic plants
was approved by two ways 1. Polymerase Chain Reaction technique. 2. Measurement of physiological parameters. The transgenic potatoes and
non-transgenic potatoes lines exhibited the different amounts of tolerance to salinity stress because in the transgenic lines mannitol accumu-
lates that increased osmotic pressure in salinity stress. The salt tolerance of transgenic potato cv. Agria (+mtlD) was recorded higher than that
of non-transgenic potato cv. Agria (-mtlD). Osmotic pressure in this transgenic potato plant was increased by accumulating of mannitol and
existence of mannitol in potato plant approved that the mtlD gene was successfully expressed to potato cv. Agria.
Key Words: transgenic potato, mtlD gene, salinity stress.
Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Corresponding Author: A. Askari, email@example.com
Askari et al 2012
Volume 4 | Issue 1Page 11
genes into plant. They have developed an approach for the trans-
formation that integrates the gene - transfer capability of A. tu-
mefaciens with the simple and general regeneration capability
of leaf explants. This method is more rapid and simple than
previous methods (Horsch et al 1985). Tarczynski et al, in 1992,
studied the expression of a bacterial mtlD gene in transgenic
tobacco that leads to production and accumulation of mannitol.
Mannitol concentration exceeded 6 µmol/g (fresh weight) in
the leaves and in the roots of some transformants whereas this
sugar alcohol was not detected in these organs of wild-type to-
bacco plants or untransformed tobacco plants (Tarczynski et al
1992). Tarczynski et al studied on the effect of mannitol which
has produced in transgenic tobacco, on stress protection of to-
bacco plant. In this research was detected that the accumulation
of sugar alcohols and other low molecular weight metabolites
such as proline and glycine – betaine is a response that may
protect against environmental stress. Growth of plant from con-
trol and mannitol-containing lines in the absence and presence
of added sodium chloride was analyzed. Plants containing man-
nitol had an increased ability to tolerate high salinity (Tarczynski
et al 1993). Phar & Stoop, in 1996, investigated the effect of
excess macronutrients in the root environment on mannitol and
sucrose metabolism in celery (Apium graveolens L. var dulce).
Celery is one of plants that normally produce mannitol in its
organs which increased ability to tolerate environmental stress
such as salinity and water stress (Stoop & Phar 1996). Thomas
et al transformed gene mtlD which encodes mannitol-1-phos-
phate dehydrogenase (E.C.188.8.131.52) into Arabidopsis thaliana.
MtlD-transformant accumulated mannitol, a sugar alcohol that
is not normally found in Arabidopsis thaliana. When mtlD-ex-
pressing seeds and control seeds were imbibed with solutions
containing NaCl (range 0–400 mol/m3), transgenic seeds con-
taining mannitol germinated in medium supplemented with up
to 400 mol/cm3 NaCl, while control seeds ceased germination
at 100 mol/m NaCl (Thomas et al 1995). Chakraborty et al
(2000) increased nutritive value of transgenic potato by express-
ing a non-allergenic seed albumin gene from Amaranthus hy-
pochondriacus. Improvement of nutritive value of crop plants,
in particular the amino acid composition, has been a major long-
term goal of plant breeding programs. They reported earlier the
cloning of the seed albumin gene ama1 from Amaranthus hy-
pochondriacus. The ama1 protein is non-allergenic in nature
and is rich in all essential amino acids, and the composition
corresponds well with the World Health Organization standards
for optimal human nutrition. In this research to improve the nu-
tritional value of potato, the ama1 coding sequence was suc-
cessfully introduced and expressed in tuber-specific and con-
stitutive manner. There was a noticeable increase in the growth
and production of tubers in transgenic populations and also of
the total protein content with an increase in most essential ami-
no acids. The expressed protein was localized in the cytoplasm
as well as in the vacuole of transgenic tubers. Thus they have
been able to use a seed albumin gene with a well-balanced ami-
no acid composition as a donor protein to develop a transgenic
crop plan (Chakraborty et al 2000). Kondrak and coworkers
(2011) studied on transcriptome analysis of potato leaves ex-
pressing the trehalose-6-phosphate synthase 1 gene of yeast.
The trehalose-6-phosphate synthase (TPS1) gene of yeast ex-
pressed to lines of the potato cultivar White Lady showed im-
proved drought tolerance. Transcriptome of wild-type
and TPS1-transgenic plants were compared by using the POCI
microarray containing 42,034 potato unigene probes to under-
stand the molecular basis of this phenomenon. One major way
to improve drought tolerance in plant species is to transfer genes
encoding metabolic enzymes or transcription factors that utilize
their effects through different mechanisms of action (Cattivelli
et al 2008). Genes of various sources involved in trehalose me-
tabolism have been used in some plant species to enhance their
drought tolerance. Trehalose, consisting of two glucose mole-
cules, is a very plentiful sugar in nature. In bacteria, yeast and
tolerant crops it accumulates under stress condition and plant
cells can survive by protecting membranes and proteins (Jain
& Roy 2009 and Gilbert et al 2000). In other plants trehalose
is synthesized at a different level. Trehalose is synthesized in
Escherichia coli, yeast and plants, in a two-step process. Initially,
trehalose-6-phosphate (T6P) is synthesized from glucose-6-phos-
phate (G6P) and UDP-glucose (UDPG) by trehalose phosphate
synthase (TPS) and then T6P is changed into trehalose by tre-
halose phosphatase (TPP). They expressed that water and pro-
tein amount in transgenic plants did not change compared to
the wild-type. Chlorophyll amount in transgenic leaves was a
little, but not significantly, higher than in the wild-type leaves.
But, shoot mass and leaf area of the TPS1-transgenic lines were
about 35 and 24% lower, respectively, than in the wild-type.
The 99 differentially transformed genes that we have identified
in the microarray experiments were exported into the Map Man
software for functional annotation. Of these 99 genes, 53 were
determined into different functional categories, while the bin
of “not determined” genes contains 46 genes of which 36 en-
code unknown, hypothetical proteins. Their microarray results
showed that a sucrose synthase gene (SUS3) and six other genes
associated with photosynthesis and carbon metabolism are reg-
ulated in TPS1 transgenic leaves. In 2007, Tang and coworkers
investigated to creation transgenic potato plant for increasing
tolerance to multiple environmental stresses by overexpressing
nucleoside diphosphate kinase 2 gene. Four lines for every
transgenic potato (Solanum tuberosum cv. Atlantic) plant (SN1,
SN19 and EN1, EN2) with high tolerance to methyl viologen
(MV, 10 LM) were used in that research. Plants were reproduced
under sterile conditions in Petri dishes containing MS medium
(Murashige & Skoog 1962) supplemented with 100 mg/L kana-
mycin. Then plants were transferred in pots and grown in a
growth chamber under 16-h photoperiod with light intensity
(100 Lmol m-2s-1), 60% (w/v) relative humidity at 25˚C. Two
vectors were used to express AtNDPK2 gene with SWPA2 pro-
moter or enhanced CaMV 35S promoter. For high temperature
stress, four-week-old potato plants growing at 25˚C growth
chamber were transferred to 42˚C for 20 h in the growth cham-
ber. These treated plants were transferred to normal conditions
(25˚C, 100 Lmol m-2s-1) for recovery from the stress. The toler-
ance of transgenic plants to high temperature stress was evalu-
ated as the photosynthesis activity (Fv/Fm) and the fresh weight
of the plants after treatment. To evaluate the salt tolerance, seed-
lings (NT, EN, and SN plants) were reproduced on MS medium
in vitro. Salt stress was accomplished by transferring shoots to
test tube containing MS medium (solid) supplemented with 80
mol/cm3 NaCl. Plants were cultured in a growth chamber under
a 16-h photoperiod with light intensity (100 Lmol m-2s-1) at
25˚C. Tolerance was evaluated by measuring the root length
and the root dry weight after 20 days of treatment. The root dry
Askari et al 2012
Volume 4 | Issue 1Page 12
weight was measured after drying the sample in the dry oven
at 70˚C for 48 h. To evaluate the tolerance of transgenic plants
to oxidative stress NT and transgenic plants (SN, EN) were es-
timated for visible damage 5 days after spraying with solutions
containing 0, 150, 200 or 250 LM methyl viologen (MV). MV
is a typical ROS generating redox active compound, which has
been used as non-selective herbicide. All plants showed a sig-
nificant symptom of leaf damage in correlation with MV con-
centration. However, SN and EN plants showed reduced symp-
toms of damage compared to NT plants. Especially, young leaves
from SN plants were unaffected even under high MV concen-
tration. They successfully created transgenic potato plants with
transferring AtNDPK2 gene with SWPA2 promoter or CaMV
35S promoter. Transgenic potato plants, especially SN plants
under SWPA2 promoter exhibited enhanced tolerance to envi-
ronmental stress including MV-induced oxidative stress, high
temperature and salt stress.
The hypothesis of the investigation is the possibility of increas-
ing the salinity tolerance of two potatoes cultivars (Agria) by
production of GMO potato. Research question to be asked: does
the mtlD gene introduction to potato’s callus cells increase the
salinity tolerance of potato plant (Agria cultivar)? We planned to
produce a GMO potato by method of leaf disk method (Horsch
et al 1985) and then to study the features of this product. The
main aim of our investigation was suggestion of ways for in-
creasing the potato plant tolerance to salinity and water stress
by use of mtlD gene.
Material and Methods
Tubers of potato (Solanum tuberosum L.) cv. Agria were achieved
from Agricultural Research Center of Bam (Iran). Tubers of
potato cv. Agria were sterilized by sodium hypochlorite (10%)
solution for 10 minutes then they were washed by distilled wa-
ter two times for 3 minutes. These tubers were planted in pots
which filled with sterilized (autoclaved) soil in 121°C for 20
minutes. The terminal meristem (leafs buds) were harvested after
10 days and were inserted on culture media of MS (Murashige
& Skoog 1962) supplemented with 3% sucrose, 8% agar and
2,4-D (3 mg/L) for callus induction. These samples were stored
at 20-25°C under white fluorescent lamps with 16h photoperiod.
Mannitol, 1-phosphate dehydrogenase gene (mtlD, E.C.184.108.40.206)
was used to create a transgenic potato plant, resistant to salin-
ity stress. This gene was isolated from pCabmtlD plasmid (E.
coli) and then was cloned in PBI121 plasmid. CaMV 35S is the
most commonly promoter which used in the creation of abiotic
stress tolerant plants (Romero et al 1997). Agrobacterium tu-
mefaciens (LBA4404) was used to create transgenic plant as a
vector (Holsters et al 1978) which the PBI121 plasmid transfers
to it and stored in 28°C temperature. The leaves buds of potato
plant were cut into discs then cultured in dark for 3 days on co-
cultivation medium (Murashige & Skoog) which supplemented
with 2.5 mg/L IBA (indol benzyl amino purine), 0.1 mg/L NAA
(naphthalene acetic acid), 30 g/L sucrose and 8 mg/L agar. Base
on leaf disc method, which initially was used by Horsch and
his colleagues in 1985, in this investigation, the leaf discs were
incubated in the diluted Agrobacterium culture for 20 minutes
(Horsch 1985). This culture should be co-cultivated in the dark
condition for 2 days. At next step, the leaf discs were trans-
ferred to Murashige and Skoog medium containing 300mg/L
cefotaxime and 150 mg/L kanamycin. The cultured was stored
in the light condition for 10 days. After this stage, samples were
transferred to MS medium containing 4 mg/L 2,4-D to callus
induction. To regenerate shoot, callus was incubated in culture
media supplement with 2.5 mg/L BA and 0.1 mg/L NAA. The
regenerated shoots were transferred to culture MS supplement-
ed with 1 mg/L IBA.
The properties of genetic modified potatoes which were created
in the previous stage were evaluated. To evaluate the features of
these transgenic potatoes two ways were applied that include:
1. Polymerase chain reaction and 2. Physiological assessment
of transgenic plants.
1. PCR (polymerase chain reaction)
The process of PCR include: 1.1 CTAB method for DNA ex-
traction: the protocol of this method is described as follows.
Cetyltrimethylammonium bromide (CTAB) extraction is a method
of extracting plant DNA that removes Polyphenolics from plant
cell walls (Aljanabi & Martinez 1997). Polyphenolics are com-
pounds with long chains that resemble DNA and precipitate in
similar ways. This process uses Chloroform:Isoamyl Alcohol,
which is both volatile and toxic. In the process, isopropanol is
also used. The CTAB method also uses liquid nitrogen as part
of the extraction process (Wulff et al 2002). The important ma-
terials used in CTAB method include: CTAB buffer, Microfuge
tubes, Mortar and Pestle, Liquid Nitrogen, Microfuge, Absolute
Ethanol (ice cold), 70 % Ethanol (ice cold), Ammonium Acetate
(7.5 M), 55˚C water bath, Chloroform:Isoamyl Alcohol (24:1).
1.2.Selection of primers; in this research the forward primer was
The reverse primer was
1.3.PCR cycle, 30 cycles of 94°C for 1 min, 55°C for 1 min,
and 72°C for 1 min using PCR Thermal Cycle.
1.4.PCR products were run on a 1% Agarose gel followed by
staining with Ethidium bromide.
2. Physiological assessment of transgenic potatoes
The transgenic and non-transgenic potatoes were subjected to
different NaCl treatments (0, 50, 100, 150, 200 mol/cm3) each
with three replications for 30 days.
The growth indicators include fresh weight, dry weight, height,
numbers of tubers, total weights of tubers; harvest index, shoot
weight and root weight were measured at the end of the stress
period. The obtained data were analyzed by SPSS software.
Table 1. The mannitol concentration in leaves, stems and roots
of non-transgenic and transgenic potatoes cv. Agria
Mannitol concentration (µ mol.g-1 fresh weight)
Leaf B *
0.5 ± 0.3
*Leaf A: leaves on the top of plant. Leaf B: leaves on the lower parts
Askari et al 2012
Volume 4 | Issue 1 Page 13
The results of this investigation were showed in Table 2. The
mean comparison showed that there are significant differences
between four salinity stress treatments (0, 50, 150, 200 mol/cm3
NaCl) and also there were significant differences between two
mtlD treatments (+mtlD, -mtlD) in all growth indicators, by
recorded probability alpha = 0.010. Data analysis of all growth
parameters indicated the negative effect of salinity stress (dif-
ferent NaCl concentration) on two potato lines (+mtlD and
-mtlD). Noticeably, the recorded growth indicators reduction in
+mtlD (GMO potato) line was recorded more than –mtlD lines.
In –mtlD plants, salt stress reduced fresh weight by 39% in 50
mol/cm3 NaCl, 61% in 100 mol/cm3 NaCl, 73% in 150 mol/
cm3 NaCl, while in +mtlD potatoes salt stress decreased fresh
weight by 28% in 50 mol/cm3 NaCl, 44% in 100 mol/cm3 NaCl,
56% in 150 mol/cm3 NaCl. In –mtlD plants, salt stress reduced
dry weight by 32% in 50 mol/cm3 NaCl, 55% in 100 mol/cm3
NaCl, 73% in 150 mol/cm3 NaCl but in +mtlD potatoes salt
stress decreased dry weight by 14% in 50 mol/cm3 NaCl, 30%
in 100 mol/cm3 NaCl, 35% in 150 mol/cm3 NaCl (see Table 2).
Growth analysis is used logical tool for evaluating plant growth
and in standard growth analysis, CGR was applied to determine
the plant growth. CGR is defined as dry matter accumulation
rate per unit land area. It has been calculated as follows: CGR =
(W2- W1)/SA (t2 – t1) where CGR is crop growth rate showed
in g per m2 per day, W1 and W2 are crop dry weights at the be-
ginning and end of intervals, t1 and t2 are corresponding days,
and SA is the land area occupied by plants at each sampling.
Values of CGR are normally low during early growth stages and
increase with time, reaching highest values at the time of flow-
ering. The CGR (Crop Growth Rate) for +mtlD is greater than
–mtlD potatoes. The highest CGR for +mtlD was 12 g per m2
and 8.5 g per m2 for –mtlD (Fig. 1). Relative growth rate (RGR)
is an evaluation used in plant physiology to measure the speed
of plant growth. It is evaluated as the mass increase per above-
ground biomass per day, for example as g g-1 d-1. RGR is cal-
culated using the following equation: RGR = (ln W2 - ln W1)/
(T2-T1) where W1 and W2 are plant dry weights at times T1
and T2. Many plants show a decreasing RGR over time but the
RGR for –mtlD is fewer than transgenic potato (Fig. 2). When
comparing RGR values, it was determined that transgenic pota-
toes lines have a higher RGR than non- transgenic potatoes lines.
The regression equation for salinity stress (NaCl concentra-
tion) with dry weight of transgenic (+mtlD) and non-transgenic
potatoes lines (-mtlD) were created by SPSS software. The lin-
ear regression equations exhibit that the transgenic (+mtlD) has
a low negative gradient (slope) in compare with non-transgenic
(-mtlD). This declared the low negative influence of salinity on
+mtlD potato due to creation of mannitol and osmotic pressure
was increased in transgenic potato cv. Agria. Then +mtlD has
more tolerance to salinity stress (equation 1 and equation 2).
GMO potato (+mtlD), Agria cultivar, showed the more toler-
ance to salinity stress compared with non-transgenic potato
(control treatment, -mtlD). It was observed that the osmotic
pressure in transgenic potato cv. Agria was increased by produc-
tion of mannitol which this approved the successfully expres-
sion of mtlD gene (mannitol-1-phosphate dehydrogenase, EC
220.127.116.11) to transgenic potato cv. Agria plant. The tolerance to
salinity stress increased with increase of mannitol content and
mannitol was significantly produced by existence of mtlD gene
in transgenic potato cv. Agria in this investigation (Table 1 and
Table 2). Similar results had also been reported by Thomas et
al (1995). Thomas and his colleagues described expression of
mtlD gene for biosynthesis of mannitol in Arabidopsis thaliana.
They showed that tolerance to salinity of seeds increased due to
accumulation of mannitol (Thomas et al 1995). Abebe and his
coworkers, in 2003, also declared the expression of mtlD gene
to wheat plant for increasing tolerance to salinity stress by pro-
duction of mannitol (Abebe et al 2003). Karakas et al (1997)
conducted an experiment to determine whether mannitol pro-
vides salt and/or drought stress protection through osmotic ad-
justment. In their study, Tobacco plants (Nicotiana tabacum L.)
were transformed with a mannitol-1-phosphate dehydrogenase
gene resulting in mannitol accumulation. Salt stress reduced dry
weight in wild-type plants by 44%, but did not reduce the dry
weight of transgenic plants. In 2007, Tang and coworkers in-
vestigated the creation of transgenic potato plant for increasing
tolerance to multiple environmental stresses by overexpressing
nucleoside diphosphate kinase 2 gene. They successfully cre-
ated transgenic potato plants with transferring AtNDPK2 gene
with CaMV 35S promoter. Transgenic potato plants, especially
SN plants under SWPA2 promoter exhibited enhanced tolerance
to environmental stress including MV-induced oxidative stress,
high temperature and salt stress.
Table 2. The effect of salinity stress on transgenic and non-transgenic potatoes cv. Agria growth
NaCl *Plant** Fresh weight (g)Dry weight (g) Height (cm)Numbers of
of tubers (g)
-mtlD 87.1 ± 0.418.40 ± 2.1 48.1±0.456 56±1.3364±1.11 25±0.346.1±0.7
+mtlD 83.3 ± 0.817.78 ± 0.845.4±1.226 48±1.23 58±1.429.4±0.55.9±0.3
-mtlD52.6 ±1.212.43 ± 1.220.6±1.12433±0.6863±1.2816±0.73.6±1.2
+mtlD 59.8 ± 0.615.23 ± 0.939.6±1.37335±1.2762±2.34 19.9±1.214.9±0.8
-mtlD34.1 ± 0.58.23 ± 1.216.8±1.362 17±0.8656±2.43 13.3±0.924.1±0.6
+mtlD46.2 ± 1.12 12.47 ± 0.825.7±1.18426±1.8563±2.11 16.6±0.893.6±0.5
-mtlD 23.2 ± 0.74.85 ± 0.610.2±0.431 10±0.26 76±1.7611±0.812.2±0.2
+mtlD 36.6 ± 0.9211.63 ± 1.318.8±1.42 214±0.56 79±1.4821.3±1.41.3±0.3
*mol.cm-3 ; **Transgenic plant (+mtlD) and non-transgenic plant (-mtlD)
Askari et al 2012
Volume 4 | Issue 1Page 14
Fig. 1. CGR (Crop Growth Rate) of +mtlD and –mtlD potatoes
cv.Agria (irrigated with 150 mol/cm3 NaCl)
Fig. 3. Linear regression for salinity and dry weight of non-
transgenic potato (-mtlD).
Moghaieb-Reda and coworkers, in 2011, from Department of
Genetics, Faculty of Agriculture, Cairo University, Giza, Egypt,
declared that they evaluated the salt tolerance in ectoine-trans-
genic tomato plants (Lycopersicum esculentum) in terms of
photosynthesis, osmotic adjustment and carbon partitioning.
In their study, the hypocotyl explants isolated from two tomato
cultivars were transformed with the Agrobacterium tumefaciens
LBA-4404 harboring the three genes involved in ecotin syn-
thesis. This was stimulated in the leaves and roots by salt ap-
plication, which improved water status by maintaining higher
activities of water uptake and transport to leaves. Furthermore
their results support hypothesis that ecotin alleviates inhibition
of root sink activity at a first response to salinity.
Growth analyses include, CGR (Crop Growth Rate) and RGR
(Relative Growth Rate) of transgenic and non-transgenic pota-
toes lines were showed that +mtlD potatoes cv. Agria lines grew
better than –mtlD potatoes cv. Agria lines in salinity stress. This
approved that +mtlD had more tolerance to salinity stress due
to production of mannitol.
The comparison of linear regression equations of transgenic
(+mtlD) and non- transgenic potatoes cv. Agria (-mtlD) showed
the more tolerance of +mtlD to salinity stress than –mtlD po-
Fig. 2. RGR (Relative Growth Rate) of +mtlD and –mtlD po-
tatoes cv.Agria (irrigated with 150 mol/cm3 NaCl)
Fig. 4. Linear regression for salinity and dry weight of trans-
genic potato (+mtlD)
tatoes cv. Agria (Table 3, Table 4, Figs 3-4).
On the whole, we can conclude that it was created GMO po-
tato cv. Agria in this study. Also it was approved that mtlD gene
transfer to GMO potatoes plants by Polymerase Chain Reaction
and physiological parameters assessment. The GMO potatoes
cv. Agria exhibited tolerance to salinity stress (even in 150 mol/
cm3 NaCl). This showed the expression of mtlD gene to genetic
modified potatoes plant which causes producing mannitol and
increase osmotic pressure (Table 2) and so tolerances to salin-
ity stress of GMO potatoes were increased and this is in agree-
ment with Karakas et al (1997), Huizhong et al (2000), Hu et
al (2005), Khare et al (2010)and Moghaieb-Reda et al (2011).
Transgenic plants exhibited mannitol concentrations up to 0.5–2
µmol/g of fresh weight, whereas mannitol accumulation could
not be seen in untransformed potato (Table 1).
I would like to thank Dr. Amin Baghizadeh from International
Center for Science, High Technology and Environmental
Science of Mahan (Iran) for his technical assistance.
Askari et al 2012
Volume 4 | Issue 1 Page 15
Table 3. The equation of -mtlD potato (non-transgenic potato)
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R SquareF df1 df2 Sig. Constantb1
0.983114.896 12 0.009 17.702-0.09
The independent variable is salinity and dependent variable is dry
0.959 46.965 1
df1 df2 Sig.
The independent variable is salinity and dependent variable is dry
Askari et al 2012
Volume 4 | Issue 1Page 16
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•Aliakbar Askari, Laboratory of Molecular Biology and
Biotechnology, Department of Biology, Armenian State Agrarian
University, 74 Teryan Str., 0009 Yerevan, Armenia, mazdakask@
•Astghik Pepoyan, Laboratory of Molecular Biology and
Biotechnology, Department of Biology, Armenian State Agrarian
University, 74 Teryan Str., 0009 Yerevan, Armenia, past@
•Ali Parsaeimehr, Club of Young Researcher Organization of
Azad University, Iran Ali.firstname.lastname@example.org
Askari, A., Pepoyan, A., Parsaeimehr, A., 2012. Salt tolerance of genetic
modified potato (Solanum tuberosum) cv. Agria by expression of a bacterial
mtlD gene. Advances in Agriculture & Botanics 4(1):10-16.
Editor I. Valentin Petrescu-Mag and Ştefan C. Vesa
Received 04 January 2012
Accepted 13 March 2012
Published Online 04 June 2012
Funding None Reported
Conflicts / Competing