Genetic

Engineering for
Abiotic Stress
Tolerance in
Agricultural Crops
Bidhan Roy, S.K. Noren, Asit B.
Mandal and Asit K. Basu

https://scialert.net/fulltext/?doi=biotech
.2011.1.22
Abiotic stresses have become an integral part of crop production. One or
other persist either in soil or in atmosphere. With the ultimate goal to raise
the crop plants with better suitability towards rapidly changing
environmental inputs, intense efforts are needed employing physiological,
biochemical and molecular tools to improve tolerance ability under abiotic
stresses. Attempts have been taken by plant breeders to develop tolerant
varieties of different crops for specific abiotic stress. Appreciable
improvement also has been done by the molecular biologists regarding to
perturbations in gene expression and protein during stress. Employing
transgenic technology, functional validation of various target genes involve
in diverse processes, such as signaling, transcription, ion homeostasis,
antioxidant defense etc. for enhanced abiotic stress tolerance has been
attempted in various model system and some of them have been extended
to crop plants. The information in the areas of gene and genetic engineering
for improvement of crop plants against abiotic stresses are lying
unorganized in different articles of journals and edited books. This
information has been compiled in this review article in organized way with
up-to-date citations, which will provide comprehensive literatures of recent
advances
Most crops growing under field conditions are often being exposed to various abiotic
stresses. The complex field environment with its heterogenic conditions and
global climate change are increasing day by day but a few of them challenges facing
modern agriculture (Mittler and Blumwald, 2010). A combination of plant breeding
approaches will likely be needed to improve significantly the abiotic stress tolerance of
crops in the field. A number of abnormal environmental parameters, such as drought,
salinity, cold, freezing, high temperature, waterlogging, high light intensity,
UV-radiation, nutrient imbalances, metal toxicities, nutrient deficiencies, climate
change etc. are collectively termed as abiotic stress. Only 10% of world's arable land
may be categorized as free from stress. The rapid change in environmental conditions
are likely to override the adaptive potential of plants, these environmental changes
mainly originated from anthropogenic activities mining, release of industrial waste,
smelting of As ore, incineration of fossil fuel,causing soil and air pollution, thus plants
are exposed to natural climatic. Among abiotic stresses drought is the main abiotic
factor as it affects 26% of arable areasuitable for growing crops.. Water stress is a
single most severe, limitation to the productivity of rice in the rainfed ecosystem
(Windawsky and O’Toole, 1990). Mineral toxicity/deficiencies are second in
importance. Among mineral toxicity, salinity is wide spread and is estimated to affect
10% of the world land surface (Richards, 1995). Increasing salinization of arable land is
expected to have devastating global effects, resulting in 30% land losses within the next
25 years and up to 50% by the year 2050 (Wang et al., 2003a). Soil acidity is another
major problem common to tropical regions, which constitutes about 3.95 billion ha of
land (FAO, 1991). Acid soils caused by combination of aluminium (Al) and manganese
(Mn) toxicity are major constraints to soil fertility and crop productivity. Al toxicity
problems are of enormous importance in production of rice, maize and sorghum. Low
temperature stress accounts about 15% crop area worldwide and such stress mainly
There is serious concern for food security of developing countries, which demands a conscious effort
to improve production from areas commonly exposed to abiotic stresses. As mindless urbanization and
industrialization swallow fertile lands and overuse of pesticides puts the environment jeopardy,
researchers are designing crops that could tolerate abiotic stresses. The limitations to increase crop
yield on existing cropped areas can be partially overcome with greater production inputs
manipulation, which are costly, laborious and beyond the reach of small and marginal farmers. Thus,
there is an urgent need to develop varieties that can not only withstand high levels of abiotic stresses
but can also maintain optimum yield levels. In conventional breeding programme, genetic markers are
being used by plant breeders, which are of morphological traits and controlled by single locus. The
morphological markers are not always useful for selection for abiotic stresses. Therefore, molecular
breeding (biochemical and/or DNA markers) is advancing as a new chapter for quick improvement in
crops suitable for problem soils. Molecular control mechanisms for abiotic stress tolerance are based
on the activation and regulation of specific stress-related genes. These genes are involved in the
whole sequence of stress response, such as signaling, transcriptional control, protection of
membranes and proteins and free-radical and toxic compound scavenging. The complex plant
response to abiotic stress, which involves many genes and biochemical-molecular mechanisms, is
schematically represented in Fig. 1. Furthermore, reproductive barriers limit transfer of favourable
alleles from interspecific and intergeneric sources. Transgenic development is another straight
forward technology to improve crop yield in abiotic stress affected land (Roy and Basu, 2009). The
development of tolerant crops by genetic engineering, on the other hand, requires the identification
of key genetic determinants underlying stress tolerance in plants and introducing these genes into
crops. Introduction of molecular change by genetic engineering takes less time compared to plant
breeding methods: only desired gene(s) can be transferred, whereas, in conventional breeding
approach is associated with simultaneous transfer of undesired gene(s). For re-cultivation of degraded
soils and reclamation of industrial sites, stress tolerant plants are required. The advent of plant
transformation may have placed within the grasp of the possibility of engineering greater abiotic
stress tolerance in plants.
 Plant response to abiotic stress to develop tolerance or
Fig. 1:
resistance
 SALT TOLERANCE
Salt tolerance is an important trait that requires overcoming salinity induced
reduction in plant productivity. The genetic response of plants to abiotic stresses is
complex involving simultaneous expression of a number of genes. Plant genetic
engineering techniques could be effectively utilized to exploit some of the
untapped potentials to increase the harvestable crop yield. It involves specific gene
manipulation either through over expression or silencing of alien/native genes. A
number of genes induced in response to salinity have been identified from a range
of organisms adapted to stressful environment. If a salt tolerant gene is identified
which can lead to betterment of the crops, it is possible to transfer that progress in
transgenic research for inclusion salinity stress tolerance, which has been presented
in Table 1.
Transgenic research has made significant progress in crop genetic improvement with
the advent of modern rDNA technologies. A large number of transgenics in diverse
crops are on large-scale cultivation. Moreover, multiple genes can be stacked or
transformed to a stock of interest through genetic transformation. There are a large
number of genes found to be instrumental and there are many functional targets for
engineering tolerance to salinity. Few of the genes of importance are briefed
below.
Ion transporter and Antiporter genes: A salt concentration of 200 mM is equivalent
to 40% of the salt concentration of sea water and will inhibit growth of almost all
crop plants.
Manitol gene: Osmotically shocked cells synthesize and accumulate massive
amount of osmoprotectory compounds. Such compounds possibly help the cells to
lower their osmotic potential and to draw water from the outside medium.
Manitol as an osmoprotectory compound is primarily found in microbes. By
introducing manitol-1-phosphate dehydrogenease gene (mt1D) isolated from E.
coli (Tarezynski et al., 1993) showed over-expression of manitol in tobacco
plants. These transgenic plants showed tolerance to high NaCl levels (250 mM).
Seeds of transgenic Arabidopsis transformed with mt1D gene under control of
CaMV 35 promoter over produced manitol and germinated in a medium
supplemented with high amount of NaCl (Thomas et al., 1995). Li et al.
(2004) introduced mt1D gene into upland rice (Oryza sativa var. japonica) by
microprojectile bombardment. Growth rate of transgenic plants was significantly
higher than the control on MS medium containing 1% NaCl. Non-transgenic plants
died after 35 days. They reported less membrane damage and low Na+/K+ ratio
than the control under salt stress.

Trehalose: Trehalose is a non-reducing disaccharide of glucose that functions as a

protectant in the stabilization of biological structure and enhances the tolerance
of organisms to abiotic stress. Zhang et al. (2005) transformed tobacco plants
with trehalose synthetase (Tsase) gene for manipulating abiotic stress tolerance.
They reported higher trehalose accumulation in transgenic plants as compared to
non-transgenics. The finding suggested that the transgenic plants transformed
with Tsase gene can accumulate higher levels of trehalose and have enhanced
tolerance to drought and salt stresses.
 H+-pyrophosphatase (H+-Ppase) gene: An H+-Ppase gene
named TsVP involved in basic biochemical and physiological mechanisms was
cloned from Thellungiella halophila. Transgenic tobacco
overexpressing TsVP had 60% greater dry weight than wild-type tobacco at
300 mM NaCl (Gao et al., 2006). Their findings suggested that over expression
of H+-Ppase causes the accumulation of Na+ in vacuoles instead of in the
cytoplasm and avoids the toxicity of excess Na+ in plant cells.
 WATERLOGGING TOLERANCE
The major changes in the waterlogged soils are physical, biological and
chemical changes. Upon flooding, the pore spaces (filled with air) in the soil
become saturated with water, as a result, the soil swells. Since, the exchange
of air between the atmosphere and the soil is impeded and since the water
particles are held by soil particles and prevent from percolating downward
and escaping. The absence of soil air in waterlogged condition causes a
change in the varieties of microbes, microscopic, organisms which live in the
soil. Anaerobic microbes tend to be much slower; less efficient decomposer
of organic matter than the aerobic microorganisms. Consequently, the rate
of decay of organic matter tends to be slow in flooded soils.
Chemical zones of flooded soil
Also the end products produced by anaerobic decomposition differ some are
toxic to crop plants; particularly those released during the first two weeks
after decomposition begins and the toxicity produced during decomposition
may stunt the growth of crop plants. Flooded soils develop two distinct
chemical zones (Fig. 2); oxidized and reduced zones. The upper oxidized
zone (1-10 mm) absorbs oxygen from the water, turns brown in colour and
reacts to nitrogen like an unfolded soil. The lower reduced zone, which
extends down as far as the water, is extremely low in available oxygen,
turns dark blue or gray in colour and takes on chemical properties quite
different from those of oxidized layer above.
Genetic engineering provides opportunities for germplasm improvement as
well as evaluating the impact of different mechanisms of tolerance to
waterlogging without confounding effects of complete changes in the
background. Two different approaches have been used to try and identify
limiting factors in the response to waterlogging. First is the
under-expression of single candidate genes, e.g., for ethanol synthesis,
using sense and anti-sense constructs. Second is the over-expression of
transcription factors (Dennis et al., 2000). It was anticipated that both
approaches may have a beneficial effect in switching on the longer term
adaptation response to low oxygen stress. Few achievements in transgenic
development have been presented in Table 2.
Transgenic cotton plants containing the ADH cDNA driven by a constitutive
35S promoter showed 10-30 fold increase in ADH activity and a significant
increase in the rate of ethanol fermentation (Dennis et al., 2000). Cotton
plants with rice pdc1 cDNA driven by a constitutive 35S promoter produce
more pdc protein but had only marginal more PDC activity. They also
observed that neither pdc or adh transgenic cotton plants, nor plants
containing both constructs showed increased tolerance of hypoxia stress.
 HEAT TOLERANCE
Heat shock proteins: It is important to maintain protein in their functional
conformations and preventing aggregation of non-native proteins under
stress.
Many stress responsive proteins, particularly Heat Shock Proteins (HSPs) have
been shown to act as molecular chaperones, which are responsible for
protein synthesis, targeting, maturation and degradation in a broad array of
normal cellular process.
Molecular chaperones function in the stabilization of proteins and
membranes and in assisting protein folding under stress conditions.
COLD TOLERANCE
Classical plant breeding has limited success in imparting cold hardiness to
crop plants.
Isolation of cold fighting genes help in the development of crop plants that
can withstand freezing temperature.
Table Gene encoding for molecular chaperones and transgenic
4: development
Table
Cold tolerance gens/transgenic plants
5:
cold acclimation is controlled by many genes and that cell membranes are
particularly vulnerable to cold damage.
Cold inducible alcohol dehydrogenase, β-amylase and many novel genes.
Dehydration responsive element: Many of the known cold regulated genes
were under control of a primary master regulator, CBF/DREB1. Dehydration
Response Element (DRE) plays an important role in the response to low
temperature.
Transcription factor DREB1A specifically interacts with DRE and induces the
expression of stress tolerance genes in plant.
Over expression of DREB1A in transgenic Arabidopsis plants activated the
expression of many stress tolerance genes and resulted in tolerance to
freezing
 ALUMINIUM TOXICITY
TOLERANCE
Aluminium is the third most abundant element in the earth’s crust. Al toxicity
mainly targets root apex, resulting in inhibited root growth and function.

Al toxicity leads to severe impairment in acquisition of water and nutrients from

the soil, which results in a significant reduction in crop yield on acid soils.

In plants with genetic resistance to Al toxicity, the Al-exclusion and uptake from
root tips have been found to be correlated to their increased capacity to
release organic acids such as citric acid, which chelates Al3+ outside the plasma
membrane.

There are two classes of physiological mechanisms that enable plants to withstand
toxic levels of Al in acid soils: exclusion of Al from the root apex and true tolerance
to Al in the root and shoot.
Table Gene/transgenic plants with gene conferring metal toxicity
6: tolerance
 IRON TOXICITY TOLERANCE

Fe-toxicity had been reported as one of the major soil constraints of

lowland acid soils, inland valley swamps, costal swamps and irrigated
lowlands in utisols and oxisols.

Fe is also abundantly found in heavy soils. In the humid forest and moist
Savana zones of Africa interflow of ferrous ion occurs from upper slopes
More than 50% lowland rice is being affected with Fe-toxicity in Siera
Leone, Liberia, Guinea, Nigeria, Ivory Coast and Senegal.

Transgenic development: The transgenic development study in case of

Fe-toxicity tolerance is very scanty.
Deak et al. (1999) developed transgenic rice with ferritin gene to
enhance high iron storage and they suggested that the enhanced Fe
storage ability can reduce reactive oxygen species. Ferritin gene has
been also transferred into rice, which enhanced iron level in the
endosperm.
 CADMIUM TOXICITY TOLERANCE
• Cadmium (Cd) is a strongly phytotoxic heavy metals in an
increasing environmental problem worldwide.
• It is one of the most dangerous metal due to its high mobility and
the small concentration at which its effects on the plants being to
appear.
• It is released into the environment by the power stations, heating
systems, metal-working industries or urban traffic. It is recognized
as an extremely significant pollutant due to its high toxicity and
large solubility in water.
• Soil solutions which have a Cd concentration ranging from 0.32 to
1.00 mM, can be regarded as polluted to a moderate level
(Sanita di Toppi and Garbrielli, 1999).
• Cd toxicity is highest in acidic environment and decrease as
the soil pH is increased (Przybulewska, 2004).
• Availability of Cd to plants is regulated by pH, redox potential and
other physicochemical parameters.
• The effect of Cd salt on the growth of seedlings was weaker in
loamy soil and stronger in sandy soil. The root system was more
sensitive to Cd ions than the cotyledons.
 Transgenic development

• Transgenic development is an alternative approach to improve cadmium

(Cd) tolerance and its accumulating capacity in crop plants.
• There are few successful reports on genetically engineered crop plants
tolerant to Cd-toxicity (Zhu et al., 1999; Creissen et al., 1999).
• Over expression of Escherichia coli gshI gene in Indian mustard
(Brassica juncia) increased γ-glutamylcysteine synthetase (γ-ECS)
activity leading increased Cd tolerance and accumulation.
 Conclusions
The load of abiotic stresses on crop production is being incremented gradually by
the directed demands of human beings for their food.
Abiotic stresses have become an integral part of crop production. The ground water
is depleting fast due to intensive and extensive cultivation during off-monsoon
periods as well as supplementation of water through irrigation during monsoon.
Poor quality irrigation water and depletion of ground water increased salinity in
arid and semi-arid zones.
Intensive cultivation and heavy feeder crops lead deficiency of some nutrients and
toxicity of others. The industrial growth and increase in vehicles created heavy
metal toxicity in many industrial areas.
In combination of industrial growth and improvement of transportation systems
facilitate aggressive air pollution, which subsequently forcing climate change
around the globe. All of these exert greater influence on plant growth and crop
productivity.