International Journal of

Molecular Sciences

Review
Transgenic Breeding Approaches for Improving
Abiotic Stress Tolerance: Recent Progress and
Future Perspectives
Ali Anwar and Ju-Kon Kim *
Graduate School of International Agricultural Technology and Crop Biotechnology Institute/GreenBio Science &
Technology, Seoul National University, Pyeongchang 25354, Korea; [email protected]
* Correspondence: [email protected]




Received: 8 February 2020; Accepted: 9 April 2020; Published: 13 April 2020


Abstract: The recent rapid climate changes and increasing global population have led to an increased
incidence of abiotic stress and decreased crop productivity. Environmental stresses, such as
temperature, drought, nutrient deficiency, salinity, and heavy metal stresses, are major challenges for
agriculture, and they lead to a significant reduction in crop growth and productivity. Abiotic stress
is a very complex phenomenon, involving a variety of physiological and biochemical changes in
plant cells. Plants exposed to abiotic stress exhibit enhanced levels of ROS (reactive oxygen species),
which are highly reactive and toxic and affect the biosynthesis of chlorophyll, photosynthetic capacity,
and carbohydrate, protein, lipid, and antioxidant enzyme activities. Transgenic breeding offers
a suitable alternative to conventional breeding to achieve plant genetic improvements. Over the
last two decades, genetic engineering/transgenic breeding techniques demonstrated remarkable
developments in manipulations of the genes for the induction of desired characteristics into transgenic
plants. Transgenic approaches provide us with access to identify the candidate genes, miRNAs,
and transcription factors (TFs) that are involved in specific plant processes, thus enabling an integrated
knowledge of the molecular and physiological mechanisms influencing the plant tolerance and
productivity. The accuracy and precision of this phenomenon assures great success in the future
of plant improvements. Hence, transgenic breeding has proven to be a promising tool for abiotic
stress improvement in crops. This review focuses on the potential and successful applications, recent
progress, and future perspectives of transgenic breeding for improving abiotic stress tolerance and
productivity in plants.

Keywords: plant biotechnology; transgenic breeding; abiotic stress; CRISPR/Cas9; QTL; miRNA

1. Introduction
The world’s population is very intensively increasing every day; it is estimated that it will increase
to 9.7 billion by 2050 [1]. The intensive increase in biotic and other environmental stresses (high
and low temperature, salinity, drought, and heavy metal stresses) due to climate changes can pose
a severe threat to tropical crop production [2]. Although conventional breeding techniques have
significantly improved crop production and yields, new methodologies and techniques are needed for
crop production improvement to fulfill the food demand [3]. Additionally, environmental stresses
are defined as the adverse effects of non-living factors on living organisms (plants) in a specific
environment. Abiotic stresses include drought, heat, cold, and heavy metals [2]. Excess of salts or toxic
metals, such as aluminum, arsenate, and cadmium, in the soil are major environmental factors that
significantly influence the growth of plants and lead to a decline in plant productivity [4].
To improve plant survival and production efficiency, different approaches have been established
to improve plant resistance [3,5]. The exposure of plants to these environmental stresses leads to

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Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 28
Int. J. Mol. Sci. 2020, 21, 2695 2 of 29
leads to reactive oxygen species (ROS) overproduction, which negatively affects the activities of
enzymes, the biosynthesis of carbohydrates, DNA, proteins, and other biochemical activities, thus
reactive oxygen species (ROS) overproduction, which negatively affects the activities of enzymes,
leading to oxidative stress (Figure 1) [6,7]. ROS also influence the expression of a number of genes
the biosynthesis of carbohydrates, DNA, proteins, and other biochemical activities, thus leading to
involved in many processes, including growth, the cell cycle, programmed cell death (PCD), abiotic
oxidative stress (Figure 1) [6,7]. ROS also influence the expression of a number of genes involved in many
stress responses, pathogen response, systemic signaling, and development [8]. The defense system
processes, including growth, the cell cycle, programmed cell death (PCD), abiotic stress responses,
(superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione reductase (GR),
pathogen response, systemic signaling, and development [8]. The defense system (superoxide
ascorbate peroxide (APX) enzymes, etc.) plays a key role in the regulation of the production of ROS
dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione reductase (GR), ascorbate peroxide
and hence protects plants from abiotic stresses [4].
(APX) enzymes, etc.) plays a key role in the regulation of the production of ROS and hence protects
To meet the growing demand for food and to contrast the harmful effects of abiotic stress on
plants from abiotic stresses [4].
plant production, it is indispensable to produce transgenic lines that have improved resistance to a
To meet the growing demand for food and to contrast the harmful effects of abiotic stress on
wide range of abiotic stresses [6,9,10]. The traditional breeding techniques were used for genetic
plant production, it is indispensable to produce transgenic lines that have improved resistance to
variations arising from varietal germplasms and intergeneric or interspecific hybridizations to induce
a wide range of abiotic stresses [6,9,10]. The traditional breeding techniques were used for genetic
mutations at a cell and tissue culture level to increase the plant’s resistance to environmental stresses.
variations arising from varietal germplasms and intergeneric or interspecific hybridizations to induce
However, these techniques have many limitations [11]. A long time is required to introduce new
mutations at a cell and tissue culture level to increase the plant’s resistance to environmental stresses.
plants and numerous undesirable genes can be transferred along with desirable genes, there is a low
However, these techniques have many limitations [11]. A long time is required to introduce new plants
yield improvement under stress conditions due to the complexity of the stress response and its
and numerous undesirable genes can be transferred along with desirable genes, there is a low yield
mechanisms, and there is no guarantee of obtaining a particular gene combination over millions of
improvement under stress conditions due to the complexity of the stress response and its mechanisms,
crosses [12].
and there is no guarantee of obtaining a particular gene combination over millions of crosses [12].
The recent advances in biotechnology have dramatically changed the capability of gene
The recent advances in biotechnology have dramatically changed the capability of gene discovery
discovery and functional genomics in plants to regulate a specific character [10,13]. Biotechnology
and functional genomics in plants to regulate a specific character [10,13]. Biotechnology techniques
techniques make protein and metabolite profiling easy and allow us to understand the
make protein and metabolite profiling easy and allow us to understand the physiologically complex
physiologically complex processes and cellular functions [11]. Current efforts to improve the abiotic
processes and cellular functions [11]. Current efforts to improve the abiotic stress tolerance of plants
stress tolerance of plants resulted in significant achievements [14]. Plant biotechnology and genetic
resulted in significant achievements [14]. Plant biotechnology and genetic engineering approaches
engineering approaches for abiotic stress tolerance [15] are based on the gene expression that is
for abiotic stress tolerance [15] are based on the gene expression that is involved in regulatory and
involved in regulatory and signaling pathways that control genes encoding stress-resistant proteins
signaling pathways that control genes encoding stress-resistant proteins and enzymes for the synthesis
and enzymes for the synthesis of functional metabolites [10,16–18].
of functional metabolites [10,16–18].

Figure 1. A simplified scheme of mechanism for abiotic stress tolerance in plants. Abiotic stress is
Figure 1. in
involved A the
simplified scheme of reactive
overproduction mechanism for species
oxygen abiotic stress
(ROS),tolerance in plants.
which causes Abiotic
cell death. stress is
A complex
involved
plant in the
defense overproduction
system of reactive
stabilizes the oxygen species
ROS production (ROS),the
and protects which causes
plant cells. cell death. A complex
plant defense system stabilizes the ROS production and protects the plant cells.
2. Physiological and Biochemical Mechanisms of Abiotic Stress
2. Physiological and Biochemical
Plants’ exposure Mechanisms
to abiotic stress of Abiotic
leads to diverse Stress and biochemical changes, such as
physiological
overproduction of reactive
Plants’ exposure oxygen
to abiotic species
stress leads(ROS:
to diverse O2 − , OH) [5].and
H2 O2 ,physiological Plants possess achanges,
biochemical very efficient
such
defense system (including SOD, POD, CAT, GR, antioxidant enzymes, etc.), which is
as overproduction of reactive oxygen species (ROS: H2O2, O2 , OH) [5]. Plants possess a very
• − involved in
efficient
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 28
Int. J. Mol. Sci. 2020, 21, 2695 3 of 29
defense system (including SOD, POD, CAT, GR, antioxidant enzymes, etc.), which is involved in
plant protection during stress conditions [8]. The overproduction of ROS influences the expression
plant
of protection
numerous during
genes stress conditions
involved [8]. Thephotosynthesis,
in chlorophylls, overproduction of ROS influences
enzymes, cells, andthe expression
the defense of
numerous genes involved in chlorophylls, photosynthesis, enzymes, cells,
response [2]. The hydrogen peroxide signaling pathway (H2O2) plays dual roles: at lower and the defense response [2].
The hydrogen peroxide
concentrations, signaling
it activates pathway
the plant’s (H2 Osystem
defense 2 ) plays todual roles:
reduce theatharmful
lower concentrations,
effects of abiotic it activates
stress,
the plant’s defense system to reduce the harmful effects of abiotic stress, while it
while it is highly toxic at higher concentrations, causing negative effects on the plant’s defense system is highly toxic at
higher
[4,8]. concentrations, causing negative effects on the plant’s defense system [4,8].
Inthe
In thepast
pastdecade,
decade,several
severalcandidate
candidategenes/transcription
genes/transcriptionfactorsfactors(TFs)
(TFs)have
havebeen
beenidentified
identifiedto tobebe
involved in plant defense systems. MYB transcription factors have been identified
involved in plant defense systems. MYB transcription factors have been identified as being involved as being involved in
a wide
in a widerange of regulatory
range networks,
of regulatory including
networks, metabolism
including and biotic
metabolism andand abiotic
biotic andstresses
abiotic [19]. Similarly,
stresses [19].
the ZmPP2C-A (protein phosphatase type 2C clade A) gene was identified to facilitate
Similarly, the ZmPP2C-A (protein phosphatase type 2C clade A) gene was identified to facilitate drought drought tolerance
in maize in
tolerance [20]. The[20].
maize author
Thereported that PP2C
author reported (protein
that phosphatase
PP2C (protein type 2C)type
phosphatase was2C)involved in ABA
was involved
biosynthesis
in and defense-related
ABA biosynthesis genes to reduce
and defense-related genes to thereduce
harmful theeffects of drought
harmful effects stress.
of droughtAs displayed
stress. As in
Figure 2, plant defense-related enzymes (SOD, CAT, POD, GR, etc.) are involved
displayed in Figure 2, plant defense-related enzymes (SOD, CAT, POD, GR, etc.) are involved in in decreasing the
overproduction
decreasing of ROS [8].
the overproduction of ROS [8].

Figure 2. The mechanisms of plant defense systems. Plant antioxidants (superoxide dismutase (SOD),
Figure 2. The mechanisms of plant defense systems. Plant antioxidants (superoxide dismutase (SOD),
peroxidase (POD), catalase (CAT), glutathione reductase (GR), ascorbate peroxide (APX), etc.) act as
peroxidase (POD), catalase (CAT), glutathione reductase (GR), ascorbate peroxide (APX), etc.) act as
the plant defense system, which reduces the toxic effects of abiotic stress, as reported previously [8].
the plant defense system, which reduces the toxic effects of abiotic stress, as reported previously [8].
3. Biotechnology Applied to the Breeding of Abiotic Stress Tolerance
3. Biotechnology Applied to the Breeding of Abiotic Stress Tolerance
Plant biotechnological approaches, such as molecular breeding and genetic engineering, offer the
Plant biotechnological
possibility to obtain improved approaches,
and genome suchedited
as molecular
crops inbreeding and[3,10].
a short time geneticGenetic
engineering, offer
engineering
the
maypossibility
also allowtous
obtain improved
to overcome theand genome edited
reproductive crops
barrier amongin a different
short time [3,10].
plant Genetic
species engineering
[10]. Transgenic
may also allow us to overcome the reproductive barrier among different
breeding efficiently improved crop production through genetic modification and improvements, plant species [10].
Transgenic
with a shortbreeding
breedingefficiently
phase [21].improved crop production
Genome sequences are nowthrough
availablegenetic modification
for different and
plant species
improvements, with a short breeding phase [21]. Genome sequences are now available
(Arabidopsis, cucumber, tomato, rice, etc.), including species with large and complex genomes [22,23]. for different
plant species
Moreover, the(Arabidopsis, cucumber,
advent of so-called tomato, rice, sequencing’
‘next-generation etc.), including species with
technologies large
offers theand complex
possibility to
genomes
sequence,[22,23]. Moreover,
relatively quickly the
andadvent
cheaply,ofnew
so-called
crops ‘next-generation sequencing’
and different varieties technologies
of the same offers
crop to develop
the
newpossibility
markers to tobe
sequence, relatively breeding
used in molecular quickly and cheaply, new crops and different varieties of the
[24,25].
same As
cropimprovements
to develop newin markers to be used in molecular
plant physiology enhance our breeding [24,25]. on the complexity of
knowledge
As improvements
drought-tolerant in plantand
mechanisms physiology enhance
its relation our knowledge
to different on the
traits, selection complexity
efficiency usingof molecular
drought-
tolerant mechanisms and its relation to different traits, selection efficiency using
and genomic approaches will result in the identification of quantitative trait loci (QTL) and genes molecular and
Int. J. Mol. Sci. 2020, 21, 2695 4 of 29

linked with traits [26]. The identification of the candidate genes responsible for plant tolerance
under different abiotic stresses is essential for developing transgenic crops with enhanced drought
stress tolerance [12]. Once the genes controlling drought tolerance have been identified through QTL
mapping, these genes can then be incorporated into the genetic background of any desirable cultivar
using genetic engineering and hybridization with marker-assisted selection [10,21,23].
Plant engineering strategies for abiotic stress tolerance were intensively studied over the last two
decades. Scientists investigated the gene expression that was potentially involved in plant defense
signaling pathways that encoded proteins conferring abiotic stress tolerance. The current efforts to
improve plant stress tolerance through genetic transformation have resulted in several important
achievements [10]; however, the genetically complex mechanisms of abiotic stress tolerance make the
task extremely difficult [3]. Therefore, plant biotechnology should be fully combined with conventional
breeding and physiology. The molecular mechanisms of abiotic stress tolerance, the number of stress
responsive genes, the transcription factors, and the signaling pathways are reported in several extensive
reviews and research that was recently published.

4. Quantitative Trait Loci (QTL)

Quantitative trait loci (QTL) mapping is an emerging technique in plant breeding [26] and is
a perfectly adequate method for investigating genetically complex systems for the tolerance of abiotic
stresses [27]. The enhancement of crop production under environmental/abiotic stress conditions was
achieved through QTL manipulation, which controlled specific agronomic traits and physiological
mechanisms to enhance crop production. Quantitative trait loci can be categorized as “adaptive and
constitutive” according to their stability and response to abiotic stresses [28]. The constitutive QTL
are frequently discovered in most environments, but the adaptive QTL are detected under specific
environmental conditions, such as QTL that are expressed under increasing or decreasing temperature,
thus indicating that QTL are responsible for controlling temperature stresses [27].
Studies identified specific resistance loci through QTL mapping and assessed the race-specificity
of resistance and interactive genes involved in the plant developmental process under specific
environmental conditions [26,29]. These studies provide insights into the number of QTL involved
in complex disease resistance, epistatic and environmental interactions, race-specificity of partial
resistance loci, interactions between pathogen biology, plant development and biochemistry, and the
relationship between qualitative and quantitative loci [30]. QTL mapping also provides a framework
for the marker-assisted selection of complex disease resistance characters and the positional cloning of
partial resistance genes [29].
A number of important QTL were identified to control key traits in plants. Recently, 25 QTL
were identified for timing of flowering (TOF), 17 QTL for spring yield, and 6 QTL for cumulative
summer biomass in Medicago sativa L [30]. Three TOF-related QTL were stable, and four TOF-QTL were
detected in the corresponding genomic locations of the flowering QTL of M. truncatula, an indication of
possible evolutionarily conserved regions [29]. The potential candidate genes for the SNP sequences of
QTL regions were identified for all three traits, and these genes would be potential targets for further
molecular studies [30].
These QTL, markers and the potential candidate genes associated with spring flowering time
and the biomass yield of alfalfa constitute valuable genomic resources for improving these traits via
marker-assisted selection (MAS). Another study reported five major QTL, qHII-1-1, qHII-1-2, qHII-1-3,
qHII-2-1, and qCC-1-5 (qREC-1-3), in tomatoes involved in high temperature stress tolerance [30].
The authors reported that qHII-1-1, qHII-1-2, and qHII-1-3 were located, respectively, in the intervals
of 1.43, 1.17, and 1.19 Mb on chromosome 1, while qHII-2-1 was located in the interval of 1.87 Mb on
chromosome 2.
The locations observed with conventional QTL mapping and QTL-seq were consistent. qCC-1-5
and qREC-1-3 for CC (chlorophyll contents) and REC (relative electrical conductivity), respectively,
were located at the same position observed by conventional QTL mapping [31]. These findings were also
Int. J. Mol. Sci. 2020, 21, 2695 5 of 29

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 28

confirmed by RNA-seq, and four candidate genes (SlCathB2, SlGST, SlUBC5, and SlARG1) associated
with heat tolerance
associated were
with heat finally detected
tolerance within
were finally the major
detected QTLthe
within by DEG
majoranalysis,
QTL byqRT-PCR screening
DEG analysis, qRT-
and biological function analysis. The findings concluded that the combination of conventional
PCR screening and biological function analysis. The findings concluded that the combination of QTL
mapping, QTL-seq,
conventional QTLand RNA-seq
mapping, analysisand
QTL-seq, can rapidly
RNA-seq identify candidate
analysis genes,identify
can rapidly thus greatly shortening
candidate genes,
the
thus greatly shortening the breeding process and improving the breeding efficiency [31].excellent
breeding process and improving the breeding efficiency [31]. QTL can work as an QTL can
tool foras
work translational
an excellentgenomics and the breeding
tool for translational of quantitative
genomics traits of
and the breeding inquantitative
a variety of traits
plantsinthrough
a variety
comparative genomics approaches [26,27,29,30]. These studies clearly demonstrated
of plants through comparative genomics approaches [26,27,29,30]. These studies the importance of
clearly
QTL identification
demonstrated theand hence offered
importance of QTL newidentification
and unique markers
and hence (Figure
offered3) with
new great capabilities
and unique to
markers
contribute to and improve abiotic stress tolerance and yields.
(Figure 3) with great capabilities to contribute to and improve abiotic stress tolerance and yields.

Figure 3. A schematic model for quantitative trait loci (QTL) mapping and the discovery of functional
Figure
genes for3.a A schematic
specific trait.model for quantitative
To discover candidate trait
genesloci
for(QTL) mapping
specific traits, aand the discovery
number of functional
of plant populations
genes for a specific trait. To discover candidate genes for specific traits, a number of
were used as a genetic resource. These resources were QTL mapping, GWAS (genome-wide associationplant populations
were used as a genetic resource. These resources were QTL mapping, GWAS (genome-wide
study), and transcriptomic analysis to allocate candidate genes for control of a specific character.
association study), and transcriptomic analysis to allocate candidate genes for control of a specific
5. miRNAs in Abiotic Stress Tolerance
character.
MicroRNAs (miRNA) are a class of single-stand RNA molecules that are 21 to 24 nucleotides in
5. miRNAs
length in Abiotic
[25]. Plant miRNAs Stress
are Tolerance
frequently investigated as key regulators of specific gene expression
duringMicroRNAs
the plant developmental
(miRNA) are a process, mediating aRNA
class of single-stand defense response
molecules thatagainst
are 21 biotic and abioticin
to 24 nucleotides
stresses 0 0 end of
length [32]. A phosphate
[25]. Plant miRNAsgroup is attached
are frequently at the 5 end
investigated andregulators
as key a hydroxyl
of group
specificatgene
the 3expression
during the plant developmental process, mediating a defense response against biotic and abiotic
stresses [32]. A phosphate group is attached at the 5′ end and a hydroxyl group at the 3′ end of mature
miRNA. miRNA targets the 3′ UTR region of specific mRNA, thereby provoking the silencing of post-
Int. J. Mol. Sci. 2020, 21, 2695 6 of 29

mature miRNA. miRNA targets the 30 UTR region of specific mRNA, thereby provoking the silencing
of post-transcription, causing the degradation of mRNA and/or inhibition of its translation [33–35].
In the past decade, researchers mainly aimed to identify miRNAs with a great capability to induce
abiotic (chilling, drought, and heavy metal) stress tolerance. A previous study found miRNAs that are
involved in a wide variety of processes in plant cells, including development, transcription, protein
degradation, and detoxification (Table 1) [36–38].
Here, we discuss a current overview of miRNA identification, functions, specific roles, and the
mechanisms in major crops for abiotic stress tolerance, as well as the recent case studies for
improving abiotic stress tolerance through overexpressing selected miRNAs (Figure 4) [37]. In brassica
napus, farnesyltransferase (alpha-subunit) confers resistance to seed abortion during flowering,
which is thought to be influenced by water deficiency during drought stress [38]. miR393 was
observed to be strongly up-regulated by dehydration, cold, high salinity, and ABA treatments
in various plant species [36,37]. In Arabidopsis, miR169 was down-regulated by drought stress,
and its target nuclear factor YA5 (NF-YA5) was significantly induced upon exposure to drought
stress [34,39]. The miRNA169a-overexpressing plants were enhanced by leaf water loss and were
highly sensitive to drought stress, when compared to wild type plants, while the miRNA169a-targeted
NF-YA5-overexpression increased drought stress tolerance [40].
Likewise, the characterized GmNF-YA3 gene, a target of miR169, led to significantly improved
tolerance of drought stress in Arabidopsis thaliana (Figure 4) [41]. The researchers also reported
that the over-expression of GmNF-YA3 in Arabidopsis resulted in increased sensitivity to salinity
and ABA stresses [40]. Tomato miR169 overexpression lines induced drought stress through its
four targets NF-YA1/2/3 and multidrug resistance-associated protein gene 1 (MRP1), which were
all down-regulated [42]. Constitutive over-expression of tomato miR169c improved drought stress
tolerance as compared to wild-type plants through reduction of leaf water loss, the stomatal opening,
and transpiration rate [42]. The Agrostis stolonifera plants overexpressing a miR319 rice gene Osa-miR319a,
increased their wax contents in leaves for the retention of water and reduced their Na+ ion uptake.
Thus, they induced salinity and drought stress tolerance [43].
Gene expression analysis indicated that the enhanced abiotic stress tolerance can be attributed
to a significant down-regulation of at least four putative turf miR319 target genes, teosinte
branched/cycloidea/proliferating factors (TCP) AsPCF5, AsPCF6, AsPCF8, AsTCP14, and a homolog of
a rice NAC (NAM, ATAF, and CUC) domain gene AsNAC60 [42–44]. Additionally, overexpression
of Osa-miR319 enhanced leaf size, enlarged stem size, and reduced the number of tillers of Agrostis
stolonifera [43]. Numerous conserved miRNAs have been identified in Medicage trunctula plants under
water-deficit conditions [45]. Among these, miR169 was down-regulated in the roots, while miR408
and miR398a/b were highly expressed in the shoots as well as the roots. The miR171 family induced
drought stress tolerance in potatoes [46].
The overexpression of the Osa-miR319 gene led to increased cold stress tolerance (4 ◦ C) after chilling
acclimation (12 ◦ C) of plants as compared to wild-type plants [43,47,48]. The miR319 overexpression
lines showed more tolerance to cold stress than the OsPCF5 and OsTCP21 RNAi lines in rice [49]. miR398
is involved in plant thermo-tolerance mechanisms, especially for the protection of the reproductive
organs. Researchers reported that miR398 down-regulated its target CSD (copper/zinc superoxide
dismutase) genes [50], CSD1 and CSD2, as well as CCS, a gene encoding a copper chaperone for both
CSD1 and CSD2 [32]. The csd1, csd2, and ccs mutants displayed a higher heat stress tolerance than the
wild-type plants associated with the increased accumulation of heat stress transcription factors and
heat shock proteins and reduced damage to flowers [50]. Thus, it can be concluded that miR398 and its
target gene can be useful for crop improvements to enhance heat stress tolerance.
 Int. J. Mol. Sci. 2020, 21, 2695 7 of 29
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 28

Figure 4. The
Figure regulatory
4. The mechanisms
regulatory mechanismsof miRNAs under
of miRNAs abiotic
under stress
abiotic conditions
stress in Arabidopsis
conditions [32].[32].
in Arabidopsis
TheThe
proposed network defines the molecular mechanism of miRNA response to a variety
proposed network defines the molecular mechanism of miRNA response to a variety of biotic of biotic
andand
abiotic stresses.
abiotic The The
stresses. network is based
network on expression
is based profiles
on expression and and
profiles targeted genegene
targeted transcripts under
transcripts under
stress condition.
stress condition.

TheThe
overexpression
overexpressionof miR398-resistance
of miR398-resistance from CSD2
from is involved
CSD2 in the
is involved enhanced
in the enhancedtolerance of of
tolerance
various kinds of abiotic stresses
various kinds of abiotic stresses [51]. The investigation of interactions between miR398 and
The investigation of interactions between miR398 and its targeted its
targeted
CSD1CSD1 and CSD2
and CSD2 genes genes provided
provided a possible
a possible description
description to previous
to previous studies studies [33,34,45].
[33,34,45]. The
The transgenic
transgenic
lines of lines
SOD of SOD
with with miR398
miR398 targetedtargeted sites negatively
sites negatively impacted impacted the miR398-mediated
the miR398-mediated gene
gene regulation.
regulation.
Based on Based on the abovementioned
the abovementioned facts, wefacts, we concluded
concluded that miRNAsthat miRNAs
played aplayed a fundamental
fundamental role in the
roleimprovements
in the improvements of stress.
of abiotic abiotic stress.
Int. J. Mol. Sci. 2020, 21, 2695 8 of 29

Table 1. List of miRNAs involved in plant growth and developments.

miRNA Species Targeted Genes Function References

miR172a Soybean SSAC1 Salt tolerance [52]
miR528 Maize ZmLAC3, ZmLAC5 Lignin biosynthesis [53]
miR319 Arabidopsis TCP4 Leaf development and hormone biosynthesis and signaling [54]
miR171c Arabidopsis SCL6-II, SCL6-III, SCL6-IV Shoot and branching [55]
TCP21, JA biosynthesis and signaling-related genes
miR319 Rice Rice ragged stunt virus resistance [56]
(PLDα1, LOX5, LOX11, CORI1b and CORI2)
miR528 Rice OsSPL9 Rice stripe virus resistance [57]
miR528 Rice OsRFI2 Flowering time [58]
miR156 Brassica BrpSPL9-2 Control heading time [59]
miR157b SPL Control of various developmental process [60]
Involved in GA signaling pathway and control
miR159 Arabidopsis AtMYB [61]
programmed cell death and flowering
miR393 Rice OsAFB2, OsTIR1 Early flowering, salt and drought stress tolerance [62]
miR169 Arabidopsis NY-YA2 Promote flowering and stress tolerance [63]
miR171 Arabidopsis SPL Control plant growth and flowering time [64]
miR159 Rice OsGAMYBLs Regulate heading [65]
miR172 Maize ZmTOE1 Regulate flowering time [66]
miR169 Arabidopsis PH2 Abiotic stress response [67]
miR159 Tobacco GAMYB Pathogen defense response [68]
Int. J. Mol. Sci. 2020, 21, 2695 9 of 29

6. The Development of Abiotic Stress-Tolerant Crops by CRISPR (Lustered Regularly

Interspaced Short Palindromic Repeats)/Cas9
Plants are challenged by different kinds of abiotic stresses, such as salinity, drought, temperature,
and heavy metals, which significantly limit crop production around the world [6]. Conventional
breeding techniques have been used for crop improvements, which are now becoming constrained
by the declining genetic resources of plants and time consuming [21]. There is a crucial need for
efficient crop improvement strategies with novel genome editing techniques, such as CRISPR-Cas9 [69],
which is easy, fast, efficient, and accurate in obtaining new genome edited crops (Figure 5). Global
warming is leading to increases in several kinds of abiotic stress, including temperature and drought,
which are major abiotic stresses that limit crop production [70]. Scientists are screening to determine
candidate genes using CRISPR-Cas9 technology to overcome these environmental problems.
The KUP (K+ uptake permease) gene family is the largest family of K+ transporters in
Arabidopsis [71]. Recently, researchers discovered that differences in cassava genotypes influenced
drought resistance through the differential expression of KUP genes [72]. A genome-wide study
revealed that MAPKKK (mitogen-activated protein kinase kinase kinase) genes played important roles
in the tissue development of cassava and its resistance to drought stress [73]. When considering
tropical climates, Elaeis guineensis is the main source of edible oil in Africa and is very sensitive to
low temperature, but resists drought and salinity well [23]. WRKY genes demonstrated tissue-specific
expression under low temperature stress in oil palms [70].
Almost all the EgWRKY genes were upregulated under abiotic stresses [74], which suggested that
the expression of EgWRKY genes in African oil palms play an important role in the responses to abiotic
stresses [49]. The CRISPR/Cas9 technology has not been fully applied for crop improvements; thus,
the opportunity exists to apply the technology for a variety of crop genetic modifications for enhanced
yield and biotic/abiotic stress tolerance.
Recently, a plant multi-genome editing toolkit was developed using a CRISPR/Cas9 binary
vector set and a gRNA module vector set [23,69,75]. This will make it easier to use CRISPR/Cas9 in
a variety of plant systems and is especially useful for high-efficiency multiplex plant genome editing.
Therefore, the direct introduction of cas9 and sgRNA into host cells by genetic transformation is the
only requirement for plant genome editing with regard to abiotic stresses [76]. The previous study
demonstrated that gemini virus replicons (gvrs) could be used to transfer cas9/sgRNA to plant cells
with enhanced mutations when the replication initiation protein gene (rep) was co-transformed with
Cas9/sgRNA structures [71]. In addition, to develop the practicability of CRISPR-cas9 technology,
more efforts are required to make targeted genome editing in plants easier and faster [7,71].
Two recent reports using direct delivery of the tobacco rattlesnake virus (Trv) [75] and cabbage
leaf virus (Calcv) [77] clearly demonstrated the feasibility of different virus-mediated Cas9/sgRNA
delivery for efficient plant genome editing. CRISPRi (CRISPR interference) in plants was proven to
regulate the transcription of target genes in plants stably and effectively under the guidance of RNA
by fusing inactivated dCas9 into the effect domain [76,78]. dCas9 was used in functional genetics to
regulate gene expression and new synthetic biological applications [79]. gRNAs were used to recruit
them into specific DNA sequences as fusion proteins with transcription factor activation or inhibition
domains [80,81].
The transcription of the reported constructs and endogenous PDS (phytoene desaturase) genes in
tobacco [81] was regulated by fusing dCas9 C terminal into the EDLL domain as a transcriptional
activator and into the SRDX domain as a repressor. The DCAS9/SGRNA/effector recognition complex
interfered with effective SGRNA-dependent induction and the reversible suppression of gene expression
transcriptional regulation [82,83]. The target site of effective CRISPRi should be between -50 BP and
+300 BP relative to the transcription initiation site (TSS). The CRISPR activator (crispr a) system
regulates the gene expression in the range of 1000 times the expression of a single sgRNA at a binding
site [84]. The development of genome-scale crispri and crispra libraries will provide significant tools
for the functional genomic study of stress response signaling pathways [85]. The availability of online
 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 28
Int. J. Mol. Sci. 2020, 21, 2695 10 of 29

availability of online resources for CRISPR/Cas system materials and tools ensures that the wide
adoptionfor
resources and applicationsystem
CRISPR/Cas of this materials
technologyandremains very simple.
tools ensures This
that the includes
wide weband
adoption resources for the
application
CRISPR/CAS system and software tools of sgRNA design.
of this technology remains very simple. This includes web resources for the CRISPR/CAS system and
software tools of sgRNA design.

Figure 5. Overview of plant genome editing using the CRISPR (lustered Regularly Interspaced Short
Figure 5. Overview of plant genome editing using the CRISPR (lustered Regularly Interspaced Short
Palindromic Repeats)-Cas9 system.
Palindromic Repeats)-Cas9 system.
7. Transgenic Breeding to Improve Abiotic Stress Tolerance
7. Transgenic Breeding to Improve Abiotic Stress Tolerance
7.1. Drought Stress
7.1.Drought
Drought Stress
is a key threat for plant production around the world, due to insufficient rainfall or
a lack Drought
of availability
is a key of irrigation waterproduction
threat for plant [86]. Globally, one-third
around of total
the world, dueagricultural
to insufficient landrainfall
is arid oror a
semi-arid, due to insufficient
lack of availability water
of irrigation [87]. [86].
water Thus,Globally,
drought one-third
stress, together with
of total other climatic
agricultural land changes,
is arid or
causes a significant loss of crop yield [88]. According to previous
semi-arid, due to insufficient water [87]. Thus, drought stress, together with other climatic reports, the global temperature
changes,
increased
causes aby 1.2 ◦ C in loss
significant the last century
of crop and[88].
yield wasAccording
expected totohave increased
previous an additional
reports,
◦ C by 2010.
the global3 temperature
The development
increased by 1.2 of°Ccrop
in the plants with improved
last century and wasperformance
expected to have underincreased
drought an stress is therefore
additional 3 °Caby major
2010.
breeding objective for scientists. Transgenic breeding or biotechnology
The development of crop plants with improved performance under drought stress is therefore a approaches play a fundamental
role in mining
major breedingcandidate
objective genes forpotentially
scientists.involved
Transgenicin drought
breeding stress
ortolerance [3,10]. Various
biotechnology approaches molecular
play a
techniques
fundamental (marker-assisted
role in miningselection candidate (MAS)genes and genomic selection
potentially involved (GS) and QTL-mapping
in drought stress tolerance are used
[3,10].
toVarious
identifymolecular
candidatetechniques
gene (that’s can control a specific
(marker-assisted selection character),
(MAS) and andgenomic
CRISPR-Cas9
selection is being
(GS) andused to
QTL-
make
mappingtransgenic linestofor
are used the drought
identify stressgene
candidate tolerance
(that’sofcan crops (Supplementary
control Table S1) and
a specific character), [34]. CRISPR-
Plant hormones are involved in the response to drought stress,
Cas9 is being used to make transgenic lines for the drought stress tolerance of crops (Supplementary and they play a crucial role in
drought stress
Table S1) [34]. tolerance by regulating multiple processes, such as stomatal closure, root growth, and the
production
Plant of protective
hormones aremetabolites
involved in [5].theInresponse
the modeltoplant droughtArabidopsis,
stress, and sucrose
they non-ferment
play a crucial 1 related
role in
kinase 2 protein kinases (subgroup III SnRK2) are reported to function
drought stress tolerance by regulating multiple processes, such as stomatal closure, root growth,as serine/threonine protein kinasesand
and as central and positive regulators of the ABA signaling pathway [88].
the production of protective metabolites [5]. In the model plant Arabidopsis, sucrose non-ferment 1 Upon the perception of ABA,
the ABA kinase
related receptor 2 PYR
protein (pyrabactin resistance 1)/PYL
kinases (subgroup (PYR1-like
III SnRK2) protein)/RCAR
are reported (regulatory
to function components
as serine/threonine
ofprotein
the ABA kinases and as central and positive regulators of the ABA signaling pathway [88]. Uponthe
receptor) proteins inhibit the activity of clade A PP2C phosphatases, thus releasing the
SnRK2s to phosphorylate the downstream proteins [89].
perception of ABA, the ABA receptor PYR (pyrabactin resistance 1)/PYL (PYR1-like protein)/RCAR
Under drought
(regulatory components stress,ofSnRK2s
the ABA phosphorylate (especially
receptor) proteins RK2.6)
inhibit thethe key ion
activity clade AKAT1
of channel PP2C
(inward rectifying potassium channel 1) SLAC1
phosphatases, thus releasing the SnRK2s to phosphorylate the downstream proteins [89]. stomatal
and (guard call slow ion channel 1) to facilitate
opening [90]. drought
Under The activated stress, SnRK2
SnRK2s can also phosphorylate
phosphorylate AREB/ABFs
(especially RK2.6) (ABAtheresponsive
key ion channelprotein) KAT1and
bZIP transcription factors and upregulate the expression ABA responsive
(inward rectifying potassium channel 1) and SLAC1 (guard call slow ion channel 1) to facilitate stomatal genes, thus activating the
ABA signaling pathway to induce drought stress tolerance [91]. The
opening [90]. The activated SnRK2 can also phosphorylate AREB/ABFs (ABA responsive protein) and ABA independent transcription
factor
bZIP(DREB1A)
transcription transgenic
factors and plants improve the
upregulate water use efficiency
expression ABA compared
responsivetogenes,wild typethus plants
activating[84].the
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 28
Int. J. Mol. Sci. 2020, 21, 2695 11 of 29
ABA signaling pathway to induce drought stress tolerance [91]. The ABA independent transcription
factor (DREB1A) transgenic plants improve water use efficiency compared to wild type plants [84].
DREB1Adistinctly
DREB1A distinctlystimulated
stimulatedthe theresponse
responseofof groundnut
groundnut roots
roots when
when exposed
exposed toto water
water scarcity,
scarcity,
wherein the stimulated roots grew significantly longer, especially in the
wherein the stimulated roots grew significantly longer, especially in the deeper layers of soil [21]. deeper layers of soil [21].
TransgenicDREB1A
Transgenic DREB1Agroundnut
groundnut(DREB1A
(DREB1Agenetically
geneticallytransformed
transformedby rd29promoter),
byrd29 promoter),led ledtotoaltering
altering
the root system in a consistent manner throughout the soil profile, thus
the root system in a consistent manner throughout the soil profile, thus increasing the root length increasing the root length
density to facilitate the water extraction frequency [92]. Thereby, the transgenic groundnut resultedin
density to facilitate the water extraction frequency [92]. Thereby, the transgenic groundnut resulted
inenhanced
enhancedpod podyield,
yield,enhanced
enhancedyieldyieldcomponents,
components,and andenhanced
enhancedharvest
harvestindex.
index.
Most drought inducible genes are activated by drought
Most drought inducible genes are activated by drought responsive transcription responsive transcriptionfactors factors(TF),
(TF),
suchas
such asNAC,
NAC, MYB, MYB, etc.,
etc.,which
whichregulate
regulate drought
droughtstress tolerance
stress [93,94].
tolerance NACNAC
[93,94]. is a domain name derived
is a domain name
from the first letters of three different genes, including NAM (no apical
derived from the first letters of three different genes, including NAM (no apical meristem), ATAFmeristem), ATAF (Arabidopsis
transcriptiontranscription
(Arabidopsis activation factor), andfactor),
activation CUC and (cup-shaped cotyledon),
CUC (cup-shaped which are
cotyledon), involved
which in plant
are involved
indevelopmental
plant developmental and abiotic
and stress
abioticresponses [95–98].[95–98].
stress responses A previous study reported
A previous that OsNAC6
study reported mediated
that OsNAC6
root structural adaptations, enhanced the number and diameters of roots,
mediated root structural adaptations, enhanced the number and diameters of roots, and increased and increased drought stress
tolerance in rice [87].
drought stress tolerance in rice [87].
Moreover,OsNAC10
Moreover, OsNAC10isisanother
anothermember
memberofofthe theNAC
NACfamily,
family,involved
involvedinindrought
droughtstressstresstolerance
tolerance
in field conditions and also increases the grain yield in rice, as presented in Figure
in field conditions and also increases the grain yield in rice, as presented in Figure 6 [96,99]. Similarly, 6 [96,99]. Similarly,
theoverexpression
the overexpression of of OsNAC5
OsNAC5 enlarges
enlarges the root
the root diameter
diameter in riceinand
riceleads
and toleads to increased
increased droughtdrought
stress
stress tolerance and grain yield in the field [99,100]. The overexpression
tolerance and grain yield in the field [99,100]. The overexpression of AtNAC2 demonstrated an of AtNAC2 demonstrated
an improved
improved tolerance
tolerance under under limited
limited water
water conditions,
conditions, providing
providing a candidate
a candidate genegene forforwater
waterstress
stress
toleranceinincrops
tolerance crops[101–103].
[101–103].The Theoverexpressed
overexpressedMuNAC4 MuNAC4gene geneperformed
performeda asubstantial
substantialpart partinin
successful water stress tolerance by reducing any injury to membrane
successful water stress tolerance by reducing any injury to membrane structures and enhancing the structures and enhancing the
osmoticand
osmotic andantioxidative
antioxidativeenzyme enzymeregulation
regulationininhorse
horsegram
gram[104].
[104].

Figure
Figure6.6.The
Theregulatory
regulatory mechanism of ofGBF3
GBF3inina combined
a combined drought
drought andand Pseudomonas
Pseudomonas syringae
syringae stress
tolerance
stress [105].[105].
tolerance Root-Specific expression
Root-Specific of OsNAC10
expression increased
of OsNAC10 drought
increased stress
drought tolerance
stress and and
tolerance grain
yieldyield
grain in rice
in [96].
rice [96].

The G-BoxBinding
TheG-Box BindingFactor
Factor3 3(GBF3)
(GBF3)TF TF(transcription
(transcriptionfactor)
factor)gene
genereported Arabidopsisthaliana
reportedininArabidopsis thaliana
showeda asignificant
showed significantresistance
resistancetotodrought
droughtand andheat
heatstress. AtGBF3isisa amember
stress.AtGBF3 memberofofthe thebasic
basicleucine
leucine
zipper (bZIP) superfamily and binds to the palindromic, hexameric “G-box” element (CACGTG) inin
zipper (bZIP) superfamily and binds to the palindromic, hexameric “G-box” element (CACGTG)
thepromoters
the promotersofofmany
manystress-responsive
stress-responsivegenes genes[105].
[105].The AtGBF3protein
TheAtGBF3 proteincomprises
comprisesa aproline-rich
proline-rich
domain at the N-terminal and a basic leucine zipper domain at the C-terminal, conferring
domain at the N-terminal and a basic leucine zipper domain at the C-terminal, conferring tolerance tolerance
to individuals and combined drought and pathogen stress responses [106]. AtGBF3-overexpressing
to individuals and combined drought and pathogen stress responses [106]. AtGBF3-overexpressing
plantswere
plants weretolerant
tolerantunder
underindividual
individualand andcombined
combineddrought
droughtand Pseudomonassyringae
andPseudomonas syringaepv.pv.tomato
tomato
infection stresses, when compared to control plants, as presented in Figure
infection stresses, when compared to control plants, as presented in Figure 6. The global6. The global transcriptome
of Atgbf3 mutants
transcriptome under
of Atgbf3 combined
mutants under stresses
combinedled to the identification
stresses of its downstream
led to the identification targets, PYL,
of its downstream
PIL, and
targets, GLR
PYL, [92].
PIL, andResearchers confirmed that
GLR [92]. Researchers AtGBF3that
confirmed regulated
AtGBF3the combined
regulated thestress tolerance
combined stressby
Int. J. Mol. Sci.
Sci. 2019, 21, x2695
2020, 20, FOR PEER REVIEW 4 of 28
12 29

tolerance by activating ABA-mediated signaling. They concluded that AtGBF3 significantly regulated
activating ABA-mediated
combined drought signaling. syringae
and Pseudomonas They concluded that AtGBF3 significantly regulated combined
stresses [21,105].
drought and Pseudomonas syringae stresses [21,105].
MicroRNAs played important roles in plant responses to environmental stresses, including
MicroRNAs
many developmental played important
processes androles in plant
yields [26,38].responses to environmental
Plants respond to droughtstresses,
stress byincluding many
up-regulating
developmental processes and yields [26,38]. Plants respond to
or down-regulating the expression of certain miRNAs or synthesizing new miRNAs [36,107]. Some drought stress by up-regulating
or down-regulating
drought stress response themiRNAs
expression haveofbeencertain miRNAs
reported or synthesizing
in numerous plants, such newasmiRNAs
A. thaliana,[36,107].
Oryza
Some drought stress response miRNAs have been reported in numerous
sativa, barley, wheat, and soybeans, using high-throughput sequencing technology [34–36]. For plants, such as A. thaliana,
Oryza
example, sativa,
the barley,
miR169wheat,family and is a soybeans,
conserved using high-throughput
and large-scale miRNA sequencing
gene familytechnology [34–36].
in plants, which is
For example, the miR169 family
involved in abiotic stress tolerance [40]. is a conserved and large-scale miRNA gene family in plants, which is
involved in abiotic stress tolerance [40].
To date, about 400 miR169 gene family members have been identified in 35 plant species,
To date,
including about 400dicots,
monocots, miR169and genesome
familyancient
members have been identified
gymnosperms in 35 The
[40,41,107]. plantmiR169
species, family
including in
monocots, dicots, and some ancient gymnosperms [40,41,107]. The miR169
Arabidopsis, rice, and Populus trichocarpa consists of 14, 18, and 33 members, respectively. Previous family in Arabidopsis,
rice, andhave
studies Populusshowntrichocarpa
that miR169consists of 14, are
targets 18, and 33 members,
usually respectively.
NF-YA (Nuclear FactorPrevious
Y, subunitstudies have
A) family
shown that miR169 targets are usually NF-YA (Nuclear Factor
members, widely involved in abiotic stress responses, such as drought, high salt, extremeY, subunit A) family members, widely
involved
temperature,in abiotic
and Nstress responses,
deficiency, such asplant
in different drought, high[98,108].
species salt, extreme
miR169 temperature, and Nregulatory
exerts a negative deficiency,
in different plant species [98,108]. miR169 exerts a negative regulatory role
role in the response to drought stress by inhibiting the expression of its target gene, nuclear factor in the response to droughtYA
stress
(NF-YA) inhibiting the expression of its target gene, nuclear factor YA (NF-YA) [35,40,41].
by [35,40,41].
Histochemical ß-glucuronidase (GUS)
Histochemical ß-glucuronidase (GUS) staining
staining showed
showed that that the
the gma-miR169c
gma-miR169c promoter
promoter drives
drives
GUS
GUS reporter gene expression in various transgenic Arabidopsis tissues, and the stress-induced
reporter gene expression in various transgenic Arabidopsis tissues, and the stress-induced
pattern
pattern waswasconfirmed
confirmed in transgenic
in transgenic Arabidopsis and transgenic
Arabidopsis soybean hairy
and transgenic soybean rootshairy
[35]. Arabidopsis
roots [35].
overexpressing gma-miR169c is more sensitive to drought stress,
Arabidopsis overexpressing gma-miR169c is more sensitive to drought stress, with reduced with reduced survival, accelerated leaf
survival,
water loss and
accelerated leafshorter
waterrootlosslengths than wild-type
and shorter root lengths plants,
thanas wild-type
shown in Figure
plants,7.asMoreover,
shown inresearchers
Figure 7.
identified a precise cleavage site for 10 gma-miR169c targets
Moreover, researchers identified a precise cleavage site for 10 gma-miR169c targets and and found reduced transcript levels
foundof
the AtNF-YA1 and AtNF-YA5 transcription factors in gma-miR169c-overexpressing
reduced transcript levels of the AtNF-YA1 and AtNF-YA5 transcription factors in gma-miR169c- Arabidopsis and
reduced expression
overexpressing of the stress
Arabidopsis andresponse
reducedgenes, AtRD29A,
expression of theAtRD22, AtGSTU25,
stress response genes, AtCOR15A
andAtRD29A, [34,35].
AtRD22,
These results indicated that gma-miR169c played a negative regulatory
AtGSTU25, and AtCOR15A [34,35]. These results indicated that gma-miR169c played a negative role in drought stress and is
aregulatory
candidaterole miRNA for improving plant drought adaptation.
in drought stress and is a candidate miRNA for improving plant drought adaptation.

Figure 7.
Figure 7. Arabidopsis
Arabidopsis plants
plants overexpressing
overexpressing the
the 35S:gma-MIR169c
35S:gma-MIR169c gene gene show
show an
an increased
increased sensitivity
sensitivity
to the
to drought stress compared to the wild type ** pp <
wild type < 0.05;** p << 0.01
0.01 [35].
[35].
Int. J. Mol. Sci. 2020, 21, 2695 13 of 29

7.2. Salinity Stress

Salinity is one of the most serious factors limiting the productivity of agricultural crops. Saline
soil has a high concentration of soluble salts [2]. Salinity affected soils contain excessive soluble
salts and exchangeable sodium on the surface, which affects plant root systems. According to the
FAOs (Food and Agriculture Organization) 2008 report, approximately 800 million hectares of land
around the world are affected by salinity [109]. High salinity affects plants in several ways: the
alteration of metabolic processes, membrane disorders, irregular cell division and expansion, decreased
photosynthetic activity and protein synthesis, increased ion toxicity, and enzymatic disorders [110].
Plants under salinity stress uptake excess amounts of Na+ and Cl− ions, thus increasing their
accumulation in different tissues of the plants, leading to oxidative stress [8]. However, the accumulation
of these ions in plant tissues may also have direct toxic effects by inhibiting protein synthesis,
photosynthesis, and susceptible enzymes [8]. Plants respond to salt stress through a series of
mechanisms, such as Na+ /K+ homeostasis and Na+ exclusion. Additionally, the excess amount of
these Na+ and Cl− ions in plant cells leads to the overproduction of ROS, which is highly toxic and
causes oxidative stress [103,110].
Salt/salinity stress caused numerous physiological and biochemical changes in plants, including
osmatic stress, ionic imbalances, and secondary stress [111]. High salinity disturbs the osmatic balance,
causing biochemical and enzymatic variation, thus leading to a significant reduction in water and
nutrient uptake [79,102]. Researchers suggested that salinity can disturb the overall plant growth
by influencing the complex interactions in nutrient uptake and accumulation, hormonal imbalance,
and oxidative stress (Supplementary Table S2).
Generally, glycophytes are salt-sensitive plants, and they showed resistance through accumulating
osmoprotectants; however, halophytes can survive and grow well under salinity stress (about 200 mM
NaCl) [112,113] by employing specific mechanisms to reduce the uptake and accumulation of Na+ and
Cl- ions, thus reducing the harmful effects [114]. Some species of halophytes have specialized glands
to excrete Na+ and Cl- ions at the leaves surfaces, while in the case of plant regulation of Na+ ions out
of the cell, some cells have large vacuoles that can act as a sink for the accumulation of excess Na+
through transport into vacuoles [113,115].
During the last two decades, researchers searched for the genes responsible for salinity tolerance.
Using biotechnological tools, numerous genes, transcription factors, and miRNAs were identified with
effects on salinity stress. For example, high-affinity potassium transporter (HKT) genes, belonging to
the Trk/Ktr (K+ transporter)/HKT transporter family, are known to be responsible for regulating the
transportation of Na+ and K+ in higher plants [116]. The first plant HKT gene was found in wheat,
TaHKT2;1 [117]. A number of studies reported that HKT genes should be involved in the exclusion of
Na+ from leaves in crops [118–120].
SKC1 is a key QTL (encode OsHKT1;5) that controls salt tolerance in Oryza sativa [121,122].
The previous study presented that Nax1 and Nax2 loci can carry TmHKT1;4-A2 and TmHKT1;5-A
(sodium transporter) and have great capability to restrict the Na+ accumulation in wheat leaf tissues,
thus leading to enhanced salt tolerance [123,124]. Previous studies reported that Na+ /K+ transport is
mainly controlled by HvHKT1 and HvHKT2 in barley and SbHKT1;4 in Sorghum bicolor and concluded
that HKTs significantly control salt tolerance [125,126].
Knockout plants demonstrated more salt sensitivity, and the sodium ion uptake and accumulation
in shoots suggested that OsHKT1;1 is potentially involved in recovering Na+ from the leaf blade.
It is expressed in the vascular tissues of roots and leaves [127], but its exact role in salt tolerance is
unknown. OsHKT1;4 is mainly expressed in the leaf sheath and encodes a plasma membrane-localized
protein [128]. Thus, the HKT genes family has great potential in Na+ /K+ homeostasis and is predicted
to show broad importance in salinity tolerance. Recently, another study also explored the role of the
HKT gene in salt tolerance. The researchers reported that the overexpression of zmKHT1;5 enhanced
salt tolerance by activating antioxidant enzyme activities (SOD, POD, and CAT) and reduced the
accumulation of MDA and H2 O2 [121–123].
Int. J. Mol. Sci. 2020, 21, 2695 14 of 29

The salt overly sensitive (SOS) signaling pathway plays an important role in ion homeostasis
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 28
and salinity stress tolerance [129]. It consists of SOS1, 2, and 3, and SOS1 is particularly important
for+ Na + and H+ transport in the plasma membrane [112]. Moreover, phosphorylation of the SOS3
Na and H + transport in the plasma membrane [112]. Moreover, phosphorylation of the SOS3 and

SOS2 complexes leadsleads

and SOS2 complexes to theto the activation
activation of SOS1.
of SOS1. AsAs mentioned
mentioned in in Figure8,8,the
Figure thenewly
newlyactivated
activated
pathway of SOS3-SOS2 stimulates the transcription of SOS1 and stabilizes
pathway of SOS3-SOS2 stimulates the transcription of SOS1 and stabilizes the cellular levels of the cellular levels ofSOS1
SOS1
mRNA[130–132].
mRNA [130–132].
The phosphorylatedform
The phosphorylated formof SOS1 plays
of SOS1 plays anan essential
essential role
role in Na++efflux
in Na effluxand
and contributes
contributesto to the
the
reduction of of Na +
Na+ toxicity.
toxicity. Overall,
Overall,SOS1 SOS1functions
functionsas asanan Na +
Na+ transporter
transporterand andmainly
mainlyexists
existsinin the
the
reduction
cytosolic compartment of the cell along with the Na + sensor. Previous studies reported that OsSOS1 of
cytosolic compartment of the cell along with the Na sensor. Previous studies reported that OsSOS1
+

Oryza
of Oryzasativa
sativawas
wasisolated
isolatedbased
basedon onthe
thehomology
homologywith withAtSOS1
AtSOS1 of of Arabidopsis
Arabidopsis [114,133]. Similar to
[114,133]. Similar to
AtSOS1, OsSOS1
OsSOS1 encodes
encodes aa plasma
plasma membrane-localized
membrane-localized Na + +
/H+ antiporter,
antiporter,which
whichmediates
mediatesthetheactive
active
AtSOS1, Na+/H
Na + extrusion in roots under salt stress [133].
Na extrusion in roots under salt stress [133].
+

The OsSOS1knockout
TheOsSOS1 knockoutline’s line’sresponse
responseto tosalt
saltstress
stressisisunknown;
unknown;however, OsSOS1had
however,OsSOS1 hadaa great
great
abilityto
ability toresist
resistthe
thesalinity
salinitystress
stressofofthethe atsos1
atsos1 mutant,
mutant, which
which suggested
suggested thatthat OsSOS1
OsSOS1 cancan
alsoalso
playplay
an
an important role in the salt tolerance of rice [111]. In rice, OsCIPK24 (CBL
important role in the salt tolerance of rice [111]. In rice, OsCIPK24 (CBL interacting protein kinases)interacting protein kinases)
and
and OsCBL4 facilitated OsSOS1 transport activity to reduce + ion accumulation in the cell and
OsCBL4 facilitated OsSOS1 transport activity to reduce thetheNaNa+ ion accumulation in the cell and
reduce the
reduce the harmful
harmfuleffects of of
effects salinity stress
salinity [130][130]
stress (Supplementary
(Supplementary Table S2).
TableThe SOS
S2). signaling
The pathway
SOS signaling
played an important role in salinity stress tolerance in mono- and dicot
pathway played an important role in salinity stress tolerance in mono- and dicot plants. Thus, we plants. Thus, we concluded
that plant biotechnology
concluded plays an important
that plant biotechnology role in identifying
plays an important candidate genes
role in identifying that genes
candidate are potentially
that are
involved in salinity stress tolerance.
potentially involved in salinity stress tolerance.

Figure8.
Figure Theproposed
8. The proposedmodel
modelof
ofthe
thesalt
saltoverly
overlysensitive
sensitive(SOS)
(SOS)pathway
pathwayfor
forsalinity
salinitystress
stresstolerance
tolerance
in plants.
in plants.
7.3. Temperature Stresses
7.3. Temperature Stresses
Among various environmental factors, “temperature” is considered to be a vital factor that
Among various environmental factors, “temperature” is considered to be a vital factor that
facilitates the ecological distribution of plant species and productivity in various parts of the world.
facilitates the ecological distribution of plant species and productivity in various parts of the world.
Extreme heat and cold temperatures have a hostile influence on all phases of plant development,
Extreme heat and cold temperatures have a hostile influence on all phases of plant development,
growth, reproduction, and yield. Within the plant life cycle, the reproductive stage is the most sensitive
growth, reproduction, and yield. Within the plant life cycle, the reproductive stage is the most
sensitive to high or low temperature stresses. As reported previously, a very small change in
temperature stress (mostly at the flowering stage) can influence a huge amount of yield loss [5].
According to the Intergovernmental Panel on Climate Change (IPCC), plant growth will be
challenged with warmer environments as the average surface temperature will increase 2.0–4.5 °C by
the end of this century [134]. A previous study reported that changes of 1 °C in temperature will
Int. J. Mol. Sci. 2020, 21, 2695 15 of 29

to high or low temperature stresses. As reported previously, a very small change in temperature stress
(mostly at the flowering stage) can influence a huge amount of yield loss [5].
According to the Intergovernmental Panel on Climate Change (IPCC), plant growth will be
challenged with warmer environments as the average surface temperature will increase 2.0–4.5 ◦ C
by the end of this century [134]. A previous study reported that changes of 1 ◦ C in temperature will
cause great influences on plant physiological and biochemical activities [2]. As temperature stresses
become more frequent, there is an urgent need to identify the genes associated with tolerance to
temperature stresses and understand their regulatory mechanisms in order to develop crops with
enhanced temperature stress tolerance through genetic manipulation (Supplementary Table S3) [134].
Here, we will describe some examples of transgenic breeding for temperature stress. Genetic
engineering is defined here as using genes encoding temperature tolerance proteins and metabolites in
different crops [135–137]. The omega-3 fatty acid desaturase gene (FAD7) induces chilling stress tolerance
in tobacco, due to a strong correlation with the cold responsive gene (COR) [138,139].
A number of candidate genes involved in low temperature adaptation are regulated by C-repeat
binding factor/dehydration-responsive element binding (CBF/DREB1) transcription factors [140–142].
Genes encoding a family of cold-regulated (COR) proteins were used to identify a family of
Arabidopsis transcription factors known as either C-repeat binding factors (CBF) (CBF1, 2, and 3)
or dehydration-responsive element binding factors (DREB) (DREB1B, DREB1C, and DREB1A) [142,143].
Three CBF/DREB1 genes (CBF3/DREB1a, CBF1/DREB1b, and CBF2/DREB1c) belonging to the AP2/DREBP
family of DNA-binding proteins were identified in Arabidopsis [144].
In various plants, CBF1/DREB1b and CBF1/DREB1b overexpression lines increased the
chilling stress tolerance, including increased COR gene expression levels and proline and sugar
accumulation. The overexpression of CBF1/DREB1b in Arabidopsis, activated COR homologous genes at
non-acclimating temperatures [145]. In another study, the Arabidopsis and rice CBF/DREB1-dependent
cold response pathway was shown to play a predominant role in the freezing tolerance through the
process of cold acclimation [144]. These transcription factors activated the C-repeat (CRT)/dehydration
responsive element (DRE) binding factor (CBF/DREB1) TFs, which are the key facilitator of COR and
hence induce chilling stress tolerance [146].
Thus, the CBF/DREB1 genes are thought to be activators that integrate several components of the
cold acclimation response, by which plants increase their tolerance to low temperatures after exposure
to nonfreezing conditions [144]. NAC transcriptional factors belong to a large family. Currently,
approximately 150 members of NAC have been reported in rice, and they play a crucial role in the
abiotic stress tolerance in the plants [100]. In a recent study, researchers reported the characterization of
a rice stress-responsive ONAC095 gene for drought and cold stress tolerance [130]. The overexpression
of ONAC095 (ONAC095-OE) and the dominant chimeric repressor-mediated suppression of ONAC095
(ONAC095-SRDX) plants showed comparable phenotypes to wild-type plants under drought and cold
stress conditions [147]. These findings indicate that ONAC095 plays opposing roles in low temperature
and drought stress and acts as a positive regulator for low temperature and a negative regulator for
drought stress in Oryza sativa.
As mentioned above, miRNAs play essential roles in growth, development, and the responses to
environmental stress. Many cold stress-responsive miRNAs, including miR396, miR397, and miR319,
have been identified in various plant species, such as wheat, rice, Arabidopsis, and tomatoes.
Recent studies demonstrated that miR396b positively regulated cold tolerance by repressing ethylene
synthesis through reducing 1-aminocyclopropane-1carboxylic acid oxidase (ACO) transcript levels in
Arabidopsis [148,149].
The overexpression of miR319 in rice led to enhanced cold tolerance likely via reducing the
expression level of the OsPCF5 and OsPCF8, two TCP family transcription factors [134]. Similarly,
the overexpression of miR397 significantly improved plant tolerances to chilling and freezing stresses
in Arabidopsis by enhancing the expression of cold-regulated C-repeat binding factors (CBFs) and the
Int. J. Mol. Sci. 2020, 21, 2695 16 of 29

related downstream genes [33]. In brief, the role of miRNA has been well established as performing
a vital role in the cold stress response and adaptation.
Similarly, high temperature or heat stress is also a key barrier for crop production. The exposure of
Arabidopsis plants to warm temperatures caused negative effects on the physiological and biochemical
activities and growth [2]. During the last two decades, data from genetic engineering on high
temperature stress has been accumulating and includes several putative heat sensors, HSF and HSP
(heat shock factors and proteins) response pathways and the network of phytohormones, chaperones,
and secondary metabolites [132].
A recent study explored the central role of the basic helix–loop–helix (bHLH) transcription
factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) in warmth-mediated morphological
acclimation and the acceleration of flowering [150]. Additionally, warm temperatures promoted
auxin accumulation and activated the gibberellin (GA) and brassinosteroids (BRs) pathway resulting
in hypocotyl elongation [151]. PIF4 played a central positive role in the acclimation to increased
ambient temperature. Previously, PIF4 was demonstrated to control the morphological acclimation
to warm temperatures via auxin. PIF4 bound to the promoters of the key auxin biosynthesis genes
in a temperature-dependent manner [152,153]. Moreover, PIF4 directly or indirectly stimulated the
expression of auxin target SMALL AUXIN UP RNA (SAUR) 19–24 genes, which drive warmth-induced
hypocotyl elongation and likely petiole elongation and leaf hyponasty [152]. Finally, many miRNAs
and candidate genes have been identified to reduce the harmful effects of temperature stresses.

7.4. Heavy Metal Stress

At the present time, the world contains fast growing technology and industrialization, and the
toxicity of heavy metals (Iron, Fe; arsenate, As; cadmium, Cd; chromium, Cr; lead, Pb; copper,
Cu; mercury, Hg; and aluminum, Al) has developed as a global threat for all human beings.
The accumulation of heavy metals causes devastation to the fertility of agricultural lands. Additionally,
heavy metals are important to life only when they are present in a trace amount [154]. The presence of
heavy metals in an excess amount is toxic to plant cells [155]. The excess amount of these heavy metals
not only disturbs the plant kingdom, but also affects the animal kingdom. Their damaging impact on
our agriculture has also been very well-documented [156,157].
At a cellular level, the elevated quantity of heavy metals imposes damage by a wide number
of mechanisms. The most common mechanism is the production of reactive oxygen species (ROS)
inducing oxidative stress, while others include the inactivation of biomolecules by the displacement of
essential metal ions or by blocking essential functional groups [8]. Previous studies suggested that
heavy metals acted as exchangers of essential metal ions or by blocking functional groups and caused
oxidative damage at the cellular level. Metals like Fe and Cu, which are redox active, generated ROS
directly through redox reactions; in contrast, other metals like Pb, Cd, Ni, Al, Mn, and Zn generated
ROS through indirect mechanisms (Figure 9) [2].
Previous studies suggested that the toxicity of heavy metals disturbs redox homeostasis due
to the overproduction of ROS such as singlet oxygen (1 O2 ), superoxide radicals (O2 − ), hydrogen
peroxide (H2 O2 ), and hydroxyl radicals (• OH) [8]. The plant’s molecular response to heavy metal
stress is characterized by the synthesis of stress-related amino acids, protein, genes, and signaling
molecules [140]. A higher proline (Pro) level was found in the Cd-hyperaccumulator plant Solanum
nigrum more than in the non-accumulator plant (Solanum melongena L), indicating its role in heavy
metal detoxification [158,159].
The different kinetics of mitogen-activated protein kinase (MAPK) cascades in response to metal
stress have also been reported [160]. The seedlings of Medicago sativa being exposed to Cd/Cu stress
resulted in the activation of MAPK genes, including SAMK, MMK2, MMK3, and SIMK. Importantly,
Cu stress rapidly activated SIMK, MMK2, MMK3, and SAMK, while Cd showed a similar but delayed
MAPK activation [161,162].
Int. J. Mol. Sci. 2020, 21, 2695 17 of 29

Similarly, calcium signaling, hormone signaling, and MAPK signaling networks also demonstrated
a positive response to the toxic effect of heavy metal stresses [161]. Calcium signaling employs
a multitude of calcium sensing proteins like calmodulins (CaMs), CaM-like proteins (CMLs), calcineurin
B-like proteins (CBLs), and Ca2+ -dependent protein kinases (CDPKs) that bind to Ca2+ and trigger
different downstream signaling pathways [162].
Another mechanism, cation diffusion facilitators (CDFs), also known as the metal tolerance
protein (MTP) family, is reported in a diverse group of organisms such as bacteria, fungi, animals,
and plants [163]. Twelve and ten MTP genes have been recognized so far in Arabidopsis and rice,
respectively [164]. In Arabidopsis, the first CDF gene was characterized as the Zinc Transporter 1 gene
(ZAT1) and later renamed as METAL TOLERANCE PROTEIN 1 (AtMTP1) [165]. The AtMTP1 gene is
expressed constitutively in the roots as well as in the shoots, and when overexpressed in Arabidopsis,
it enhances Zn tolerance (Supplementary Table S4) [166–168].
In A. halleri, a Zn hyperaccumulator plant, the AhMTP1 gene is believed to have a role in Zn
hyper-tolerance [167]. Unlike the AtMTP1 gene, AtMTP3 is expressed predominantly in the roots
and is reported to be engaged in the maintenance of Zn homeostasis by excluding Zn under Zn
oversupply [168–170]. Another member of the MTP family, AtMTP11, has been reported to transport
and to provide Mn tolerance [171]. In rice, an ortholog of MTPs, OsMTP1, was characterized and is
thought to be located on chromosome 5; it is highly expressed in the mature leaves and stem [172,173].
Recently, it was reported that, in response to metal stress, plants regulate the location
and accumulation of auxin by the differential and dynamic expression of auxin-related genes,
like phosphoribosyl anthranilate transferase 1 (PAT1), CYP79B2 and CYP79B3, YUCCA (YUC), Gretchen
Hagen (GH3) genes (TIR1), the PIN family, and the ABCB family [156]. Cu2+ toxicity in Arabidopsis
led to changes in auxin and cytokinin accumulations and mitotic activity within the primary and
secondary root tips [174]. Moreover, aux1-7 and pin2 mutants showed a high tolerance to Fe stress as
compared to wild-type plants, which suggested that AUX and PIN proteins are potentially involved in
the protection of lateral root formation under Fe stress [175].
Apart from this, Cd disrupts the maintenance of auxin homeostasis in Arabidopsis seedlings
by increasing the IAA (indole-3-acetic acid) oxidase activity and altering the expression of several
auxin biosynthetic and catabolic genes [176,177]. The Cd-mediated up-regulation of biosynthesis
gene NITRILASE (NIT) resulted in increasing the IAA concentration in Arabidopsis roots promoting
lateral root growth, thus protecting the roots from Cd [178]. Moreover, a recent report revealed the
inhibition of root meristem growth through Cd-induced NO accumulation, which in turn repressed
auxin transport and stabilized AUX/IAA proteins to repress auxin signaling [179]. A positive role for
auxin transport through AUX1 on plant tolerance to As stress via ROS-mediated signaling was also
disclosed in a study [180].
root growth, thus protecting the roots from Cd [178]. Moreover, a recent report revealed the inhibition
of root meristem growth through Cd-induced NO accumulation, which in turn repressed auxin
transport and stabilized AUX/IAA proteins to repress auxin signaling [179]. A positive role for auxin
transport through AUX1 on plant tolerance to As stress via ROS-mediated signaling was also
disclosed in 2020,
Int. J. Mol. Sci. a study [180].
21, 2695 18 of 29

Figure 9. The mechanism of metal stress and oxidative damage in plant cells. Plants uptake an excess
Figure 9. The mechanism of metal stress and oxidative damage in plant cells. Plants uptake an
amount of heavy metals, which leads to the overproduction of ROS. These ROS cause an oxidative
excess amount of heavy metals, which leads to the overproduction of ROS. These ROS cause an
burst and affect a variety of plant biochemical activities in the plant cell.
oxidative burst and affect a variety of plant biochemical activities in the plant cell.
8. Recent Progresses in Genome Editing for Crop Improvement
8. Recent Progresses in Genome Editing for Crop Improvement
Genome editing has become a very useful tool for crop improvement, and the advancements
Genome editing
of CRISPR/Cas9 havehas become a very
significantly speduseful toolbreeding
up crop for crop improvement, andathe
[11,12]. Recently, advancements
great improvementof
CRISPR/Cas9 have significantly sped up crop breeding [11,12]. Recently, a great
in plants genome editing has been reported. Here, we will discuss some important progress for improvement in
plants
genome genome editing
editing. Singlehasgene
beentraits
reported. Here, we will
are considered to discuss some important
be phenotypes progress
controlled for genome
by a single gene.
During the mutation process, these genes typically affect a specific trait without compromising other
agronomic characteristics, making genome-editing tools especially suitable. For example, AtNAC2
demonstrated an improved tolerance under limited water conditions without affecting the yield and
other traits [95,102].
On the other hand, conventional breeding is unable to control/work on a single gene and is also
time consuming. It is difficult to reduce the accumulation of cadmium in rice grains using traditional
breeding methods, which can have serious health consequences for consumers. CRISPR/Cas9 has
recently been used to knock out the metal transporter gene OsNramp5 [52], which significantly reduced
the concentration of Cd in seeds without significantly affecting yield. In Arabidopsis, AhMATP1
and AtMATP1 overexpression lines significantly enhanced the Zn tolerance [166,167]. The Fe stress
tolerance in Arabidopsis was enhanced in aux1-7 and pin2 mutants [173–178].
Similarly, the other traits, such as flowering, height, and yield, were able to be controlled by
a single gene. The FT2a in soybean can delay flowering time under both short-day and long-day
conditions [179], thereby adapting the transgenic mutant plants to a larger geographic extent of the
growing region [52]. Some traits, such as yield, are controlled by a complex network of genes or family.
To overcome abiotic stress tolerance, a number of micro RNAs (miR319, miR169, miR398, miR169c,
miR408, miR398a/b, etc.) have been identified as involved in a wide range of abiotic (cold, drought,
and salinity) stress tolerances using CRISPR-Cas9 [47,49,50].
Drought is considered a key hurdle for crop production. Genome editing technology and plant
biotechnology played a leading role in identifying candidate genes not only to enhance stress tolerance
but also to improve crop production. SnRK2 phosphorylates KATs and SLAC1 to facilitate the stomatal
opening to induce drought stress tolerance [90–92]. Similarly, the NAC transcription factor is involved
in plant growth, development, and defense mechanisms. Recently, an OsNAC6 overexpression line was
reported to regulate the root architecture and increase drought stress tolerance [95], while OsNAC10 and
OsNAC5 not only induced drought stress tolerance but also facilitated the grain yield in rice [96,99–101].
High affinity potassium transporter (HKT) genes, such as OsHKT1;1, OsHKT1;4, and OsHKT1;5,
facilitated salinity stress tolerance in rice [127,128]. Additionally, TaHKT2;1, AtSOS1, OsCIPK24,
Int. J. Mol. Sci. 2020, 21, 2695 19 of 29

and OsCBL4 were reported in different plant species in the regulation of salinity stress tolerance.
Likewise, several transcription factors and micro-RNAs have been reported related to temperature
stress tolerance. In rice, miR319, miR535, miR396b, and ONAC095 were reported to increase cold stress
tolerance [47–49,147]. Additionally, PIF4 is a key facilitator for auxin biosynthesis and is also required
for hypocotyl elongation and high temperature stress tolerance [131].
Xing and Zhang (2010) demonstrated that rice grain yield can be controlled by many quantitative
trait loci (QTL) [178] and reported that independent or multiplex editing of these QTL can result in
an improved yield [76]. The editing of the same yield-related QTL in different elite rice varieties
can have inconsistent or even negative effects under field conditions [27,179]. Thus, the ability to
incorporate some complex traits, which cannot be controlled using conventional breeding techniques,
can be controlled by genome editing technology.
Recently, two innovative rapid-breeding approaches, IMGE (haploid-inducer mediated
genome editing) and Hi-Edit (haploid induction-edit), which combine haploid induction with
CRISPR/Cas9-mediated genome editing was used to introduce desirable traits into elite inbred
lines within two generations, avoiding the time-consuming crossing and back-crossing processes [180].
The MiMe phenotype in rice can be reproduced by the simultaneous editing of OsSPO11-1, OsREC8,
and OsOSD1, suggesting that different sets of genes involved in meiosis can be manipulated to create the
same phenotype [181,182]. Thus, an important benefit of genome editing tools is the ability to integrate
complex features that cannot be introduced through traditional enhancement techniques. Moreover,
plant biotechnology plays an important role in the genome editing of crops toward improvement for
the benefit of humanity.

9. Conclusions
In the last two decades, numerous achievements have been reported in different plant species
(rice, maize, tomato, tobacco, Arabidopsis, etc.) using CRISPR/Cas9 for genome editing. To have
a successful impact on agricultural production under environmental stresses, more efforts are required
to enhance and improve the CRIPSR/Cas9 technology to produce easy, approachable, and accessible
methods for researchers.
Plant abiotic stresses are a major threat to crop production around the world, and these effects
are also predicted to increase in the future. As mentioned above, plant responses to abiotic stresses
are through very complex signaling pathways, and determining the genes involved and untangling
the responses for practical application requires a multi-pronged approach. During the last two
decades, plant abiotic stress has become a key issue for researchers to identify candidate genes and
transcriptional factors. Several transcription factors (NAC, ARF, MYB, SOC, MAPK, CBFs, etc.) have
been identified to overcome abiotic stresses, including low and high temperatures, drought stress,
and heavy metal stress.
Transgenic breeding can create new and significant sources of resistance with rapid multiplication
potentials, for instance, CRISPR/Cas9. The genome-edited crops through CRISPR/Cas9 should be
considered as BE (bioengineered) crops for the quick application and acceptance of the technology
in the field. We predict that CRISPR/Cas9 technology application in numerous plant species could
revolutionize agriculture in a second green revolution, which could ensure the food demands and
nutritional security are met for the increasing populations. As mentioned above, the global population
is increasing rapidly, and this leads to enhanced food consumption. By using genome editing
technologies, we can offer improved crop production to meet the demand for food. We should grasp
this opportunity to increase crop production and the potential to save the lives of millions of people
who are facing food shortages around the globe, especially in developing nations.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/8/2695/s1.

Funding: This research was supported by the Rural Development Administration under the Next-Generation
BioGreen 21 Program (Project No. PJ013666 to J.-K.K.) and by the Basic Science Research Program through the
National Research Foundation of Korea, Ministry of Education (NRF-2017R1A2B4007457 to J.-K.K.).
Int. J. Mol. Sci. 2020, 21, 2695 20 of 29

Conflicts of Interest: The authors declare no conflicts of interest.

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