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Advances in Environmental Biology, 6(10): 2646-2653, 2012
ISSN 1995-0756
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ORIGINAL ARTICLE
Overexpression of mtlD gene in potato (solanum tuberosum L.), cv. Arinda improves
salt tolerance
1,2
Aliakbar Askari, 2Astghik Pepoyan
1
2
Department of Biology, Islamic Azad University, BAM branch, Iran
Laboratory of Molecular Biology and Biotechnology, Armenian State Agrarian University, Yerevan, Armenia
Aliakbar Askari, Astghik Pepoyan: Overexpression of mtlD gene in potato (solanum tuberosum L.),
cv. Arinda improves salt tolerance
ABSTRACT
Potato as one of the world’s main tuber crops of the solanaceae family plays an important role in the
economics scene. Since one of the major reasons for yield decreasing in the world are abiotic stresses. The aim
of this research was producing transgenic potatoes with more tolerance to salinity stress and studying GMO
potato features. To produce GMO plants, new gene (mtlD, mannitol -1- phosphate dehydrogenase, E.C.1.1.1.17)
was transferred to a plant by using agrobacterium plasmid. For the production of transformed potato plant
resistant to salinity stress, mannitol, 1-phosphate dehydrogenase gene (mtlD, E.C.1.1.1.17) was used. At first
mtld gene was isolated from pCabmtld plasmid of E. Coli and then was cloned in PBI121 plasmid. The
promoters that have been most commonly used in the production of abiotic stress tolerant plants so far, include
the CaMV 35S (Bhatnagar-mathur et al., 2007). This vector was inserted into Agrobacterium tumefaciens which
was used for producing transformed potato plant and was stored in 28°c temperature. The freeze-thaw method
(Holsters et al., 1978) was used for replacement of recombinant binary vector PBI-VHH into Agrobacterium
tumefaciens strains LBA4404.. Existence of recombinant gene in transgenic plants was confirmed by PCR
technique. For final confirmation, the features of transgenic potato were studied. Briefly, the transgenic potatoes
and non- transgenic potatoes lines showed the different amounts of tolerance to salinity stress because the
transgenic lines produced mannitol which increased osmotic pressure in salinity stress. The salt tolerance of
transgenic potato (+mtlD) was recorded higher than that of non- transgenic potato (-mtlD). Growth analyses
include CGR (Crop Growth Rate) and RGR (Relative Growth Rate) in transgenic and non- transgenic potatoes
lines were showed that +mtlD potatoes lines grew better than –mtlD potatoes 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 (-mtlD) showed the more
tolerance of +mtlD to salinity stress than –mtlD potatoes. Osmotic pressure in this GMO plant was increased by
producing of mannitol and consequently producing mannitol certify that the mtlD gene was successfully
transferred to potato.
Key words: GMO potato, mtlD gene, tolerance, salinity stress
Introduction
Potato (solanum tuberosum L.) from the
solanaceae family is one of the important tuber crops
widely grown all over the world after rice, wheat and
corn [23]. Taking into account the significance of
potato as a food crop, the United Nation announced
the year 2008 as an International Year of the potato
in order to concentrate human attention on potato as
a main food resource in developing countries [21].
The abiotic stresses: drought, high temperature, soil
salinity and frost, are serious agricultural factors
influenced on production of potatoes. Besides world
population grows at an annual rate of about 1.5 %,
drought, high temperature, soil salinity and frost
causes crop loss and reduce average yield more than
50% all over the world [5]. (Phytophthora infestans)
is a potato’s fungal disease commonly known as
blight of potato (Irish: An Gorta Mor, The Bad Life)
caused the great Famine in Ireland. Because of the
disease, a million of the Irish population died and
about a million leaved the country [13]. Considering
the role of potato on human and animal nutrition, the
new biotechnologies are used for the introducing of
productive varieties of this crop. The use of GM
organisms offers the potential for increased
agricultural productivity and improved nutritional
significance contributing directly to enhance human
health and development [3,4,11]. GMOs were
mentioned as organisms in that the genetic material
(DNA) has been changed not in normal way [15].
Plant is one of the organisms that are mainly used for
Corresponding Author
Aliakbar Askari, Department of Biology, Islamic Azad University, BAM branch, Iran
E-mail:[email protected] 2647
Adv. Environ. Biol., 6(10): 2646-2653, 2012
genetic
modifications.
Genetically
modified
organism (GMO) is one of these viable organisms
that have been altered genetically by genetic
technology. Potato plants appear as a suitable model
plant in the area of genetic modifications for
different reasons [23]. By using the method of
agrobacterium gene transfer method, the first
transgenic crops were created in 1983[26]. Genetic
engineering was effectively used in developing
Crops varieties that today ended in planting genetic
modified crops on more than 109.2 million acres
over the world [17]. A famous genetically modified
potato known as Amflora (EH92-527-1) owned by
BASF Plant Science and it was approved for
industrial usage in the European Union market on
2007 by the European Commission. Amflora is
proved to be better than the natural starch recently
used [19].
When a plant is subjected to abiotic stress, some
genes that responsible for conferring a certain degree
of protection to these stresses are turn on. As a result
the levels of several metabolites and proteins will
increase. To produce better crops under stress
understanding the changes in cellular, biochemical
and molecular mechanism that happen in reaction to
stress, is necessary. Most crops are not able to
synthesis the certain osmoprotectants which are
naturally build up by stress tolerant organisms.
Researches proved that the best method for abiotic
stress tolerance is osmoregulation , especially when
osmoregulatory genes are trigged in reaction to
drought, salinity and high temperature. As a result, to
produce stress tolerant crops a method was suggested
in which special osmolytes were engineered and
these osmolytes were over expressed in plants.
Several methods are being followed to genetically
engineer osmoprotection in plants. The first process
for producing stress tolerant transgenic plants was to
engineer gens that encode enzymes for synthesis of
selected osmolytes [3].
The result was a lot of reports including
osmoprotectants like glycine-betaine and proline.
The various kinds of genes used for increasing. Also,
a number of ‘‘sugar alcohols’’ (mannitol, trehalose,
myo-inositol and sorbitol) have been intended for the
engineering of compatible-solute overproduction,
thereby protecting the membrane and protein
complexes during stress [1,20].
The various kinds of genes have been used for
increasing plants tolerance to water and salinity
stress involve 1- Detoxifying genes, In many aerobic
organisms, it is essential to effectively remove
reactive oxygen species (ROS) that are created by
environmental stresses. 2- Late embryogenesis
abundant (LEA) proteins, this kind of proteins show
a group of high molecular weight proteins that are in
large quantities during late embryogenesis and build
up during seed desiccation and as a reaction to water
stress. 3- Transporter genes, an important method to
gain more tolerance to abiotic stress is to help plants
to re-build homeostasis under stressful environments,
restoring both ionic and osmotic homeostasis. 4Multifunctional genes for lipid biosynthesis
Transgenic approaches by changes in the lipid
biochemistry of the membranes try to improve
photosynthesis under abiotic stress conditions. 5Regulatory genes, most genes that show reaction to
multiple stresses such as dehydration and low
temperature at the transcriptional level are also
induced by ABA which protects the cell from
dehydration [4].
The hypothesis of the investigation is the
possibility of increasing the salinity tolerance of
Arinda potato cultivar by formation of GMO potato.
Research questions to be ask: If the mtlD gene
introduction to potato’s callus cells increases the
salinity tolerance of potato plant? We planned to
produce a GMO potato by method of leaf disk
method [7] and then the features of this produced
were evaluated. The main aim of our investigation
was suggestion of ways for increasing the potato
plant tolerance to salinity and water stress by use of
mtlD gene.
Materials And Methods

Plant material:
Tubers of potato (solanum tuberosum L.) cv.
Arinda were obtained from Agricultural research
center of Bam (Iran).

Callus induction and disinfection methods:
Tubers of potato cv. Arinda were placed in
sodium hypochlorite (5%) solution for 15 minutes
then they were washed by distilled water three times
for 5 minutes. These disinfected tubers were planted
in pots which filled with sterilized soil by autoclaved
in 121◦C for 20 minutes. After 7 days the pieces of
terminal meristem(leaf buds) were isolated and were
cultured on prelature containing basal MS semi-solid
medium with 1.4% agar, supplemented with 2%
sucrose, and 2,4-D(2mg/l) for callus induction. The
cultures were maintained at 20-22°c under white
fluorescent lamps with 16h photoperiod.

Gene transformation method:
To produce a transgenic potato plant resistant to
salinity stress, mannitol, 1-phosphate dehydrogenase
gene (mtlD, E.C.1.1.1.17) was used. The mtlD gene
was isolated from pCabmtlD plasmid (E. coli) and
then was cloned in PBI121 plasmid (LBA4404). The
promoters that have been most commonly used in the
production of abiotic stress tolerant plants is the
CaMV 35S [18]. This vector was inserted into
(Agrobacterium tumefaciens) which was used to
produce transformed potato plant and was stored in
28°c temperature. The freeze-thaw method was used
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Adv. Environ. Biol., 6(10): 2646-2653, 2012
for replacement of recombinant binary vector
PBI121 into (Agrobacterium tumefaciens) strains
LBA4404. The (Agrobacterium tumefaciens) was
inserted in LB (Lysogeny broth) medium and was
centrifuged for 3 h in (4000-5000rpm) then lower
part of solution was inserted in ½ MS medium. The
most common growth media for bacteria
(microorganisms) are nutrient broths (liquid nutrient
medium) or LB medium (Lysogeny Broth). The
explants (leaf bud or terminal meristem) which had
inserted in preculture were incubated with
(Agrobacterium tumefaciens) containing the 35SmtlD gene for 5 min. After washing and cocultivation for 2-3 days, the explants were transferred
to liquid medium containing 250 mg/l cefotaxime
and 100mg/l kanamycin and washed several times to
select transformed cells and was checked by PCR
technique. These transformed explants were placed
in callus induction culture then were transferred to
MS medium, supplemented with shoot induction
medium (MS+2mg/l BAP+0.2mg/l NAA) then
shoots (5-6mm length) were transformed to root
induction medium (IAA, 1mg/l) for organogenesis.
For disinfecting all hormones and antibiotics syringe
filter (0.22µm) method was used. 2, 4-D hormone
was dissolved in methanol but other hormones and
antibiotics were dissolved in distilled water.

Study the features of transgenic potato:
Finally, the feature of this genetic modified
potato was studied. We produced the transformed
potato (20 plants) by transforming of mtlD gene to
potatoes plants. Existence of recombinant gene in
transgenic plants was confirmed by PCR technique.
Total DNA was extracted from plant tissues with
cetyl (hexadecyl) trimethylammonium bromide
(CTAB), essentially as described by Wulff and
Aljanabi [2,25]. Inserted DNA was detected by PCR.
The
forward
primer
was
5′GTCTAGATGAAAGCATTACATTTTGGCG-3′.
The reverse primer was
5′-CCGAGCTCCACCATT ATTGCATTGC-3′ .
PCR conditions were 30 cycles of 94 °C for 1 min,
55 °C for 1 min, and 72 °C for 1 min using PCR
Thermal Cycle. PCR products were run on a 1 %
agarose gel followed by staining with ethidium
bromide. For final confirmation, the transformed
plants were planted in pots and experiment was
conducted as a factorial arrangement using a
complete randomizes design with three replications
(factor A= salinity and factor B= mtlD, two levels:
+mtlD, -mtlD). The treatments were the different
amounts of salt (Nacl) solutions (0, 50, 100, 150 mM
Nacl). The Nacl was dissolved in a dilute (0.5% W/v)
commercial 20:20:20 fertilizer solution and plants
were irrigated at least one a day. The experiment
lasted 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.
Results:
As it is presented in table1, there are significant
differences between four salinity stress treatments (0,
50,150,200 mM Nacl) and also there were significant
differences between two mtlD treatments (+mtlD , mtlD ) in all growth indicators, by recorded
probability alpha= 0.010 . Considerably Interactions
recorded significant differences among all
treatments.
Data analysis of all growth indicators showed
the negative influence of salinity stress (different
Nacl concentration) on two potato lines (+mtlD and –
mtlD). Considerably, 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 40% in 50 mM Nacl , 65%
in 100 mM Nacl , 87% in 150 mM Nacl while in
+mtlD potatoes salt stress decreased fresh weight by
20% in 50 mM Nacl , 44% in 100 mM Nacl , 55% in
150 mM Nacl . In –mtlD plants, salt stress reduced
dry weight by 45% in 50 mM Nacl , 67% in 100
mM Nacl , 81% in 150 mM Nacl but in +mtlD
potatoes salt stress decreased dry weight by 19% in
50 mM Nacl , 37% in 100 mM Nacl , 45% in 150
mM Nacl (Table 1).
Table 1: The effect of salinity stress on transgenic and non-transgenic potatoes growth
Nacl *
Plant*
Fresh
Dry weight Height (cm) Numbers Total
weight (gr)
(gr)
of tubers
weights of
tubers (gr)
0
-mtlD
82.3 ± 0.7
17.50 ± 1.1
56.2 ± 0.64
5
45 ± 0.55
+mtlD
78.7 ± 1.83
16.83 ± 1.3
52.4 ± 1.40
5
40 ± 1.21
50
-mtlD
48.6 ±0.52
10.40 ± 0.7
24.7 ± 1.36
3
27 ± 0.87
+mtlD
62.9 ± 0.90
13.53 ± 1.1
43.6 ± 1.26
4
32 ± 1.32
100
-mtlD
28.7 ± 1.78
6.23 ± 0.9
17.8 ± 1.61
1
8 ± 1.27
+mtlD
43.8 ± 0.75
10.53 ± 0.9
28.2 ± 1.71
3
22 ± 1.34
150
-mtlD
10.4 ± 0.87
3.53 ± 1.1
12.9 ± 1.38
0
0
+mtlD
34.8 ± 1.61
9.36 ± 1.0
19.7 ± 0.36
2
16 ± 1.27
*(mol.cm-3) , Transgenic plant(+mtlD) and Non transgenic plant(-mtlD)
Harvest
Index
55 ± 0.55
51 ± 2.27
55 ± 2.24
50 ± 2.40
28 ± 3.02
50 ± 3.26
0
46 ± 4.58
Shoot
weight
(gr)
29 ± 0.5
31 ± 0.4
17.4± 0.5
25.1± 1.1
16.6± 1.1
19.8± 1.2
9.5± 0.45
17 ± 1.5
Root
weight
(gr)
7.3 ± 0.2
7.8 ± 2.8
4.2 ± 0.9
5.5 ± 2.4
3.9 ± 0.6
2.3 ± 0.6
1 ± 0.3
1.8± 1.83
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Table 2: The mannitol concentration in leaves, stems and roots of non – transgenic and transgenic potatoes
Mannitol concentration (µ mol.g-1 fresh weight)
Plant lines
Leaf A*
Leaf B *
Stem
+mtlD
1.86 ± 0.121
1.45 ± 0.604
1.01 ± 0.104
_mtlD
0.002 ± 0.001
0.001 ±0.0008
0.0
*Leaf A; leaves on the top of plant, Leaf B; leaves on the lower parts of plant
Root
2.85 ± 0.678
0.06
± 0.001
14
CGR(g.m‐2 ) 12
10
8
6
+mtlD
4
‐mtlD
2
0
5
10
15
20
25
30
Days after planting
Fig. 1: CGR (Crop Growth Rate) of +mtlD and –mtlD potatoes (irrigated with 150 mM Nacl)
Growth analysis is widely used analytical tool
for characterizing plant growth and in classical
growth analysis, CGR was applied to determine of
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 expressed in g per m2
per day W1 and W2 are crop dry weights at the
beginning 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 maximum values at the time of
flowering. The CGR (Crop Growth Rate) for +mtlD
is greater than –mtlD potatoes. The maximum CGR
for +mtlD was 13 gr per m2 and 10 gr per m2 for –
mtlD (Fig 1).
0.14
RGR(g.g‐1 .day‐1 )
0.12
0.10
0.08
0.06
‐mtlD
0.04
+mtlD
0.02
0.00
10
14
18
22
26
30
Day after planting
Fig. 2: RGR (Relative Growth Rate) of +mtlD and –mtlD potatoes (irrigated with 150 mM Nacl)
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Relative growth rate (RGR) is a measure used in
plant physiology to quantify the speed of plant
growth. It is measured as the mass increase per
aboveground biomass per day, for example as g g-1 d1
. RGR is calculated using the following equation:
RGR = (ln W2 - ln W1)/(T2-T1) where W1 and W2
are plant dry weights at times T1 and T2. In fact
many plants exhibit a declining RGR over time but
the RGR for –mtlD is fewer than transgenic potato
(Fig 2).
LAI 2.5
2
1.5
+mtlD
1
‐mtlD
0.5
0
10
15
20
25
30
Day after planting
Fig. 3: LAI (Leaf Area Index) of +mtlD and –mtlD potatoes (irrigated with 150 mM Nacl)
Leaf area index (LAI) is defined as leaf area per
unit soil area (cm2.m-2). The equation of LAI is ;
LAI= (A ×N)/10000 where A is leaf area (cm2) and
N is plants per m2. Also there is another equation for
LAI calculation: CGR=LAI ×NAR. It was showed
that LAI of GMO potatoes is much lower than that of
non-transgenic
potatoes
(Fig
3).
Chart Title
14
NAR (g.m‐2.d‐1)
12
10
8
6
+mtlD
4
‐mtlD
2
0
0
5
10
15
20
25
30
Day after planting
Fig. 4: NAR (Net Assimilation Rate) of +mtlD and –mtlD potatoes (irrigated with 150 mM Nacl)
A useful measure of the photosynthetic
efficiency of plants is ‘net assimilation rate’ which
was defined as the rate of increase of dry weight (W)
per unit of leaf area (L); that equation is: NAR=
(1/A) (dW/dt), whereA is leaf area and is the change
in plant dry matter per unit time.NAR values
decrease with crop growth due to mutual shading of
leaves and reduced photosynthetic efficiency of older
leaves. When comparing NAR values, it was
determined that transgenic potatoes lines have a
higher NAR than non- transgenic potatoes lines (Fig
4).
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Adv. Environ. Biol., 6(10): 2646-2653, 2012
The regression equation for salinity stress (Nacl
concentration) with dry weight of transgenic (+mtlD)
and non- transgenic potatoes lines (-mtlD) were
created by spss software. The linear regression
equations showed that the transgenic (+mtlD) has a
low negative gradient (slope) in compare with non-
transgenic (-mtlD). This expressed the low negative
effect of salinity on +mtlD potato due to production
of mannitol and osmotic pressure was increased in
transgenic potato. Then +mtlD has more tolerance to
salinity stress ( equation 1 and equation 2).
Equation 1(- mtlD).
Y = 16.327 – 0.92 X, Equation 2(+ mtlD).
Equation 1
Model Summary
R Square
F
0.955
42.926
.
.
.
.
Linear
Logarithmica
Powera
df1
1
.
.
Y = 16.374 – 0.051 X
df2
2
.
.
Model Summary
R Square
F
df1
df2
Linear
0.963
51.601
1
2
Logarithmica
.
.
.
.
Powera
.
.
.
.
The independent variable is Salinity and Dependent Variable is Dryweight
Sig.
0.023
.
.
Equation 2
Sig.
0.019
.
.
Parameter Estimates
Constant
b1
16.327
- 0.092
.000
.000
.000
.000
Parameter Estimates
Constant
b1
16.374
- 0.051
.000
.000
.000
.000
Fig. 5: Linear regression for salinity and dry weight of non- transgenic potato (- mtlD) and transgenic potato
(+ mtlD)
Discussion:
It is indicated that considerably, the more
tolerance to salinity stress of GMO potato (+mtlD)
was produced in compared with non- transgenic
potato (control treatment, - mtlD). Osmotic pressure
in this GMO plant was increased by producing of
mannitol and producing mannitol certify that the
mtlD( mannitol -1- phosphate dehydrogenase , EC
1.1.1.17) gene was transferred to potato plant
successfully and this gene expression in GMO
potatoes plants (+mtlD)( Table2). In the presented
research it has been observed that tolerance to
salinity increased with increase of mannitol content
and mannitol was significantly produced by
existence of mtlD gene in transgenic potatoes
(+mtlD)(Table1 and Table2). Similar results also had
been observed earlier [1]. Authors showed that
expression of mtlD gene for biosynthesis of mannitol
in wheat improves tolerance to salinity stress. They
also demonstrate that mannitol improves growth of
transgenic wheat under water stress and salinity both
at the callus and the plant.
Thomas and his coworkers transformed the mtlD
gene into Arabidopsis thaliana which encodes
mannitol 1- phosphate dehydrogenase. They
indicated that tolerance to salinity of seeds increased
due to accumulation of mannitol [22]. This result is
in an agreement with the research result. Huizhong
and his colleagues have also revealed that the salt
tolerance of transgenic rice (Oryza sativa L.), which
this transgenic plant was produced by expression
mtlD gene (E.C. 1.1.1.17), were much higher than of
their control [9]. The similar results to our research
results were observed by Tarczynski. They expressed
of a bacterial mtlD gene in transgenic tobacco leads
to production and accumulation of mannitol. Their
studies indicated the roles of sugar alcohols such as
mannitol, in stress tolerance in higher plants [20]. Hu
have studied on overexpression of mtlD gene in
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Adv. Environ. Biol., 6(10): 2646-2653, 2012
transgenic populous tomentosa improves salt
tolerance through accumulation of mannitol. They
achieved similar results to our results that relative
growth rate of transgenic plants were much less
influenced by salt stress than in non- transgenic
plants[8].
All growth analyses of transgenic and nontransgenic potatoes lines were exhibited that +mtlD
potatoes lines grew better than –mtlD potatoes lines
in salinity stress. This certified 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(mtlD) showed the more tolerance of +mtlD to
salinity stress than –mtlD potatoes( equation 1and
equation 2).
4.
5.
6.
7.
Conclusion:
As total achievement, we can conclude that, it
was produced GMO potato in this investigation. Also
it was certified that mtlD gene transfer to GMO
potatoes plants by PCR and measuring growth
indicators methods. The GMO potatoes (20 lines)
showed tolerance to salinity stress (even in150 mM
Nacl). This certified that mtlD gene in genetic
modified potatoes causes producing mannitol and
increase osmotic pressure (Table 2). Therefore
tolerances to salinity stress of GMO potatoes were
increased and this is in agreement with Pharr (1996),
Karakas (1997), Gilbert (2000), Wang (2003), Khare
(2010) and Moghaieb [6,10,12,14,16,24]. Transgenic
plants showed mannitol concentrations up to 0.5 – 2
µmol/gr of fresh weight, while mannitol
accumulation could not be detected in untransformed
potato.
8.
9.
10.
11.
Acknowledgment
I would like to thank Prof. Astghik Pepoyan
from Laboratory of Molecular Biology and
Biotechnology, Armenian State Agrarian University,
and Dr. Amin Baghizadeh from International center
for science, High Technology and environmental
science of Mahan (Iran) for their useful supervision.
12.
References
1.
2.
3.
Abebe, T., A.C. Guenzi, B. Martin, J.C.,
Cushman, 2003. Tolerance of mannitolaccumulating transgenic wheat to water stress
and salinity. Plant Physiology, 131: 1748-1755
Aljanabi S.M., I. Martinez, 1997. Universal and
rapid salt-extraction of high quality Nucleic
Acids Research, 25(22): 4692-4693
Bahieldin A., H. Eissa, A. Ramadan , Z.E.
Abdelsalam, 2007. Applications of genetic
engineering in addressing adverse environmental
13.
14.
conditions for agricultural production, journal of
Alstesmar zarai , 2007: 50- 58
Bhatnagar- Mathur P., V. Vadez , K. Sharma,
2007. Transgenic approaches for abiotic stress
tolerance in plants: retrospect and prospects,
Plant Cell Rep, 27: 411 - 424.
Burke E.J., S.J. Brown, N. Christidis, 2006.
Modeling the recent evolution of global drought
and projections for the twenty-first century with
the Hadley centre climate model, Journal of
Hydrometeor, 7: 1113–1125
Gilbert C., A.M. Rus, M. Carmen Bolarin, J.M.
Lopes- Coronado, I. Arrillaga, C. Montesinos,M.
Caro, R. Serrano, V. Moreno, 2000. The Yeast
HAL1 gene improves salt tolerance of transgenic
tomato, Plant physiology, 123: 393 - 402
Horsch R.B., J.E. Fry, N.L. Hoffmann, D.
Eichholtz, S.G. Rogers, R.T. fraley, 1985. A
Simple and General Method for transferring
Genes into Plants, Biological Sciences,
Monsanto Company, St. Louis, Missouri 63167
Hu L., H. Lu, Q. Liu, X. Chen, X. Jiang, 2005.
Overexpression of mtlD gene in transgenic
(Populous tomentosa) improves salt tolerance
through accumulation of mannitol, Tree
physiology, 25: 1273-1281
Huizhong W., D. Huang, L.U. Ruifang, L.I.U.
Junjun, Q. Qian, P. Xuexian, 2000. Salt
tolerance of transgenic rice (Oryza sativa L. )
with mtlD gene and gutD gene, Chinese science
bulletin, 45(18): 1685- 1690
Karakas B., P. Ozias-Akins, C. Stushnoff , M.
Suefferheld, M. Rieger, 1997. Salinity and
drought tolerance of mannitol- accumulating
transgenic tobacco, Plant Cell Environment, 20:
609- 616
Katiyar-Agarwal S., M. Agarwal, A. Grover,
1999. Emerging trends in agricultural
biotechnology research: use of abiotic stress
induced promoter to drive expression of a stress
resistance gene in the transgenic system leads to
high level stress tolerance associated with
minimal negative effects on growth, Curr
Science, 77: 1577-1579
Khare N., D. Goyary, N. Kumar Singh, P. Shah,
M. Rathore, S. Anandhan, D. Sharma, M. Arif,
Z. Ahmad, 2010. Transgenic tomato CV. Pusa
Uphar expressing a bacterial mannitol-1phosphate dehydrogenase gene confers abiotic
stress tolerance, plant cell tissue organ culture,
103: 267-277
Litton H., 2006. The Irish Famine: An Illustrated
Histor, Wolfhound Press. (2006 RP). ISBN 0
86327 912 0
Moghaieb-Reda E.A., A. Nakamura, H.
Saneoka, K. Fujita, 2011. Evaluation of salt
tolerance in ectoine-transgenic tomato plants
(Lycopersicon esculentum) in terms of
photosynthesis, osmotic adjustment and carbon
2653
Adv. Environ. Biol., 6(10): 2646-2653, 2012
15.
16.
17.
18.
19.
20.
partitioning, GM Crops, Landes Bioscience, 2:
58-65.
Morgan S., 2006. Genetic engineering: the facts.
Books Google.Com
Pharr D.M., J.M.H. Stoop, J.D. Williamson,
M.E. Studer Feusi, M.O. Massel, M.A.
Conkling, 1996. The dual role of mannitol as
osmoprotectant and photoassimilate in celery,
HortScience, 30: 1182-1188
Rashid A., 2009. Introduction To Genetic
Engineering
Of
Crop
Plants.
Aims
books.google.com
Romero C., J.M. Belles, J.L. Vaya, R. Serrano,
F.A. Culianez-Macia, 1997. Expression of the
yeast trehalose-6-phosphate synthase gene in
transgenic
tobacco
plants:
pleiotropic
phenotypes include drought tolerance, Planta,
201: 293-297.
Schmidt Raulf – Michael, 2009. Amflora Facts,
BASF Plant Science Agricultural Center
Tarczynski M. C., R.J. Jensen, H.J. Bohnert,
1992. Expression of a bacterial mtlD gene in
transgenic tobacco leads to production and
accumulation of mannitol, Proc. Natl. Academic
Science USA, Plant biology, 89: 2600-2604
21. Theisen K., 2008. World potato atlas: history
and overview. International Potato Center
22. Thomas J.C., M. Sepahi, B. Arendall, H.J.
Bohner, 1995. Transformed gene mtlD which
encodes mannitol 1- phosphate dehydrogenase,
into (Arabidopsis thaliana), Plant Cell and
Environment, (995) 18: 801-806
23. Vreugdenhil D., E. Bradshaw John, 2007. Potato
biology and biotechnology: advances and
perspectives. Book.google.com
24. Wang W., B. Vinocur, A. Altman, 2003. Plant
responses to drought, salinity and extreme
temperatures: towards genetic engineering for
stress tolerance, Planta, 218(1): 1- 14
25. Wulff, E.G., S. Torres, E. Gonzales Vigil, 2002.
Protocol for DNA extraction from potato tubers,
plant molecular biology reporter, 20(2): 187195.
26. Zupan J., T.R. Muth, O. Draper, P. Zambryski,
2000.
The
transfer
of
DNA
from
(Agrobacterium tumefaciens) into plants: a feast
of fundamental insights, Plant Journal, 23: 1128.