International Journal of

Molecular Sciences

Review
Development of Drought-Tolerant Transgenic Wheat:
Achievements and Limitations
Shahbaz Khan 1 , Sumera Anwar 2 , Shaobo Yu 1 , Min Sun 1 , Zhenping Yang 1 and
Zhi-qiang Gao 1, *
1 College of Agriculture, Shanxi Agricultural University, Taigu 030801, China
2 State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China
* Correspondence: [email protected]; Tel.: +86-354-6287226




Received: 13 June 2019; Accepted: 5 July 2019; Published: 8 July 2019


Abstract: Crop yield improvement is necessary to keep pace with increasing demand for food. Due
to climatic variability, the incidence of drought stress at crop growth stages is becoming a major
hindering factor to yield improvement. New techniques are required to increase drought tolerance
along with improved yield. Genetic modification for increasing drought tolerance is highly desirable,
and genetic engineering for drought tolerance requires the expression of certain stress-related genes.
Genes have been identified which confer drought tolerance and improve plant growth and survival
in transgenic wheat. However, less research has been conducted for the development of transgenic
wheat as compared to rice, maize, and other staple food. Furthermore, enhanced tolerance to drought
without any yield penalty is a major task of genetic engineering. In this review, we have focused on
the progress in the development of transgenic wheat cultivars for improving drought tolerance and
discussed the physiological mechanisms and testing of their tolerance in response to inserted genes
under control or field conditions.

Keywords: genetic modification; Triticum aestivum L.; water stress; transcription factors

1. Introduction
Bread wheat (Triticum aestivum L.) is one of the most important cereal and staple food crops
worldwide, mainly grown in semiarid and arid regions of the world where water scarcity is causing
severe yield losses [1]. The constantly increasing population and land degradation have increased the
desire for per acre yield increase [2]. It is estimated that the global wheat production should increase
approximately 60% from the current 3.3 t ha−1 to 5 t ha−1 by 2050 to feed a population of 9 billion [3,4].
However, wheat production is severely affecting and reducing by 29% due to various abiotic stresses,
especially drought stress [5,6].
Therefore, improving the tolerance of wheat to drought stress through adaptive strategies is
important to ensure food security. To achieve this goal without increasing the area of cultivated land,
which is simply not available, emphasis must be concentrated on key traits related to plant productivity
and adaptation to environmental challenges. Genetic improvement and developing drought-tolerant
wheat cultivars is critically important and a main aim for wheat breeders [7]. Different techniques
such as marker-assisted breeding, quantitative trait locus mapping, and introgression from the wild
gene pool are being employed to improve drought tolerance [8–10]. Various genes of interest could be
inserted by genetic transformation. In contrast to conventional breeding, transgenic technique seems
to be a more attractive approach which allows the direct induction of a single or group of desired
genes [11].
Drought tolerance is a complex quantitative polygenic trait controlled by a large number of genes
and thus, it is difficult to understand the molecular and physiological mechanisms [1,12]. Until now,

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genetically-modified Glycine max (soybean) and Zea mays (maize) for drought tolerance have been
developed and approved. However, despite continuous research, less progress has been made for the
development of transgenic wheat as compared to other staple crops like rice and maize which might
be ascribed to the complex genetic characteristics of wheat [13].
Although transgenic plants have a potential to transform agriculture, limited progress has been
achieved, particularly for wheat where no drought-tolerant transgenic wheat has been approved for
commercialization [13]. The first fertile herbicide-resistant transgenic wheat plant was obtained by
microprojectile bombardment of regenerable embryonic callus [14]. The first approved transgenic
wheat for glyphosate herbicide tolerance was developed by Monsanto. It is an important step forward
in an area where progress is urgently needed, though it is too early to claim that transgenic wheat will
form the backbone of a second Green Revolution [15].
The task of the successful generation of transgenic plants is not only limited to the success in the
transformation process but also to properly incorporate the stress tolerance. The proper understanding
of physiological effects of the inserted gene and evaluation of transgenic plants under stress are
major challenges. After controlling and inserting ‘single-action” genes, the regulatory mechanism of
transcription factors has emerged as a new tool for controlling the expression of many stress-responsive
genes. González et al. [16] reported that transgenic wheat transformed with mutated transcription
factor (HaHB4) from Helianthus annuus (sunflower), which belongs to homeodomain-leucine zipper
family (HD-Zip I), and had increased water use efficiency and yield. Transgenic wheat carrying a
GmDREB1 gene from soybean, under a ubiquitin promoter, showed drought tolerance [17]. One main
interest is to improve WUE so plants could grow well under water stress. Water use efficiency could
be improved by genetically engineering in two ways. One way is inserting genes for compatible
osmolytes such as sugar and amino acids [18]. The other strategy is constitutive over-expression of the
late embryogenic abundant proteins which provide dehydration tolerance.
This review focuses mainly on the recent studies of genes involved in physiological or biochemical
processes which have been used successfully in the genetic engineering of wheat for improvement
of drought tolerance (summarized in Table 1). Furthermore, we will discuss recent successes and
limitations in the application of genetic manipulation to increase drought tolerance in wheat.

2. Drought-Induced Gene Expression/Single Action Gene

Numerous genes have been isolated from various plants and inserted in transgenic wheat to
induce stress resistance. These genes could be classified into two groups. The first group is genes
involved in cellular protection including osmoprotectants, membrane stabilization, detoxification,
and transport proteins. The genes in the second group are the transcription factors and signaling
molecules [19].

3. Osmoprotectants, Metabolites and Protective Genes

These osmoprotectants are the favorite targets for genetic engineering and many crops are
genetically engineered using osmoprotectants, like glycine betaine, mannitol, and trehalose to increase
tolerance by protecting important macromolecules. The higher accumulation of these compounds by
transferring genes in transgenic wheat plays a critical role in improving drought tolerance [20]. Higher
tolerance is by osmotic adjustment and chaperone-like activity in stabilizing membrane and protein
and detoxification by scavenging reactive oxygen species [21].

3.1. Proline
Proline is a well-known proteogenic amino acid which acts as a compatible osmoprotectant
and accumulates under osmotic stress to protect cellular structure and function [22,23]. Proline
under stress works as an antioxidant and scavenges reactive oxygen species, protects denaturation of
macromolecules, and regulates cytosolic activity [24]. The accumulation of proline contents in plants is
correlated with the tolerance to drought stress; therefore, the overexpression of proline in transgenic
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plants increased stress tolerance in wheat [25], potato (Solanum tuberosum L.) [23] and tobacco (Nicotiana
tabacum) [26]. In plants, proline is synthesized from glutamate through the glutamate pathway. The
reduction of glutamate to its semialdehyde is catalyzed by P5CS (∆1 -pyrroline-5-carboxylate synthase)
enzyme, which then reduces to proline. P5CS is a rate-limiting enzyme for the synthesis of proline by
feedback inhibition of P5CS [27].
Vendruscolo et al. [19] used the biolistics transformation method to transform cDNA of the P5CS
gene of Vigna aconitifolia under stress-inducible AIPC promotor. Transgenic wheat plants were exposed
to 14 days of water withholding at the booting stage. Transgenic plants showed 50% intact cellular
membrane as compared to wild type with 13% intact membrane. The enhanced tolerance to water
deficit was mainly due to improved antioxidants also manifested with less MDA contents, rather than
the enhanced osmotic adjustment. Pavei et al. [28] evaluated seven transgenic lines in T2 generation
containing the p5cs gene, and transgenic plants produced 1.85 times more proline after 8 days of
water stress content than non-transgenic showing the overexpression of the p5cs gene. However,
MDA contents were not decreased in transgenic plants which indicated that despite higher proline
accumulation, cell membrane protection against oxidative stress was not increased.

3.2. Glycine Betaine

Glycine betaine is a quaternary ammonium compound and is known to have a protective
role against drought stress by maintaining osmotic balance and protecting quaternary structures of
proteins [29]. Various genes (BADH, COD, CDH, and betaA) involved in the synthesis of glycine
betaine, have been inserted into transgenic plants [30–32]. The gene encoding betaine aldehyde
dehydrogenase enzyme (BADH) synthesizes glycine betaine by catalyzing betaine aldehyde into
glycine betaine. However, transgenic plants with glycine betaine synthesizing genes could accumulate
lower levels of glycine betaine than natural accumulators, still enhancing the tolerance to various
abiotic stresses [33,34]. Transgenic wheat lines overexpressing BADH showed enhanced tolerance to
salt stress especially by protecting the thylakoid membrane [34].
The wheat plants naturally accumulate glycine betaine, but the in-vivo level is too low to maintain
the osmotic balance under drought stress [29,33]. Transgenic wheat transformed and cloned containing
cDNA of BADH from Atriplex hortensis showed higher betaine aldehyde dehydrogenase activity [35].
Wang et al. [36] generated T6 transgenic wheat line overexpressing the BADH gene cloned from
Atriplex hortensis under maize ubiquitin promoter through the microprojectile bombardment method.
Transgenic and wild type (Shi 4185) were exposed to osmotic stress at −1.88 MPa using 30% PEG and
results indicated that transgenic plants overexpressing glycine betaine led to increased photosynthesis
by improving water status and antioxidant activity. In the second study, T6 lines of transgenic
wheat plants were exposed to drought (78–82% relative water content) by controlling irrigation [31].
Transgenic plants increased glycine betaine production and enhanced plant tolerance to drought by
improving photosynthesis. In another study, transgenic wheat was produced by introducing betA
gene-encoding choline dehydrogenase under maize ubiquitin promoter [32]. Transgenic plants under
drought stress (12–14% relative water content) were less injured and showed higher root length and
biomass which indicated that glycine betaine reduced the extent of the injury and worked as an
important osmoprotectant in transgenic wheat under drought stress.

3.3. Mannitols
Mannitol is sugar-alcohol, which serves as a major carbon source and has a role in osmoregulation
as a coenzyme regulator and scavenging of reactive oxygen species. Mannitol is widely distributed
in various plant species [21]. Transgenic plants for mtlD gene from Escherichia coli in tobacco [37,38],
rice (Oryza indica) [39], arabidopsis (Arabidopsis thaliana) [40] and peanut (Arachis hypogaea L.) [41]
demonstrated improved photosynthesis and ROS scavenging activity and enhanced the tolerance
to abiotic stresses [21,42]. Wheat plants naturally do not accumulate mannitol, although the higher
expression of mtlD gene transformed from Escherichia coli caused the accumulation of mannitol in
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transgenic wheat [18]. Moderate accumulation of mannitol enhanced the tolerance to water stress,
however, several abnormalities were also observed in transgenic plants due to short and sterile plants,
twisted leaves and low sucrose contents [18]. Transgenic wheat plants with the accumulation of mannitol
showed higher biomass, plant height, and flag leaf length as compared to non-accumulating [18].

3.4. Heat/Cold Shock Protein Chaperons/Molecular Chaperons

Bacterial cold shock proteins (CSPS ) are important bacterial genes for bacterial acclimatization
to low temperature and drought stress. CSPS genes conferred resistance to drought stress and
improved grain yield in maize under drought conditions [43]. Yu et al. [44] (2017) conducted a field
experiment using transgenic wheat lines transformed with SeCspA and SeCspB genes from Escherichia
coli. Transgenic wheat lines with SeCspA showed enhanced tolerance with lower water loss, less
wilting and MDA content, and higher proline, chlorophyll content, 1000-grain weight, and grain yield
compared to non-transgenic under drought stress. Transgenic lines with SeCspB showed no such
improvement under field condition. Seven-day-old seedlings were exposed to severe water deficit
and the recovery rate of SeCspA transgenic plant after re-watering was significantly higher than the
non-transgenic plants [44].

3.5. Late Embryogenesis Abundant (LEA) Proteins

Late embryogenesis-abundant (LEA) proteins are hydrophilic and are induced by drought, salt,
and ABA. LEA acts as a molecular chaperone protein by stabilizing the protein or membrane structure.
HVA1 is an ABA-inducible LEA protein which is naturally accumulated in the aleurone layer of barley
under seed desiccation [45]. Transgenic plants overexpressing HVA1 gene in rice [46] OsLEA3-1 in
rice [47], and HVA1 in spring wheat [48] enhanced abiotic stress tolerance.
Transgenic wheat over-expressing HVA1 gene, which is a group-3 LEA protein, indicated the
potential of the HVA1 gene to enhance the tolerance to water deficit stress [48,49]. The barley HVA1
gene transformation in transgenic wheat and its constitutive expression much improved the drought
tolerance in terms of higher dry mass accumulation, WUE and higher cell integrity under moderate
water deficit conditions in greenhouse tests [48]. After this, Bahieldin et al. [49] conducted nine field
experiments to test the drought tolerance of HVA1 transgenic wheat using T4 progeny of six transgenic
lines and two new lines. Substantial variability was found for transgenic lines and coincided with the
expression rate of HVA1 transgene. Line 111/1 showed higher yield and biomass than the wild type,
and in sixth season, the yield of new transgenic lines (765 and 1201) increased more than in line 111/1
and wild type. Higher relative water content was observed in lines with higher expression of the HVA1
gene which shows the potential of the HVA1 gene to confer drought tolerance.

4. Transporters Genes
Transporters genes play an important role in restoring ionic homeostasis under stress. Ferritins
are iron-storage proteins which sequester and release iron when needed. Ferritins are highly conserved
in plants with two genes in hexaploid wheat on chromosomes 4 and 5 [50]. Previous studies showed
that the over-expression of ferritin gene induced tolerance to various abiotic stresses like cold, heat and
drought stresses [50,51]. Zang et al. [51] studied the response of transgenic wheat over-expressing
TaFER-5B under various abiotic stresses. TaFER-5B from thermo-tolerant wheat ‘TAM1070 was
transformed into cultivar ‘JM52650 under maize ubiquitin promoter via the particle bombardment
method. TaFER-5B transgenic wheat lines had higher total root length, whereas GR enzyme activity
and H2 O2 contents were lower under 10% PEG.

5. Carbon Metabolism
Transforming C4 enzymes genes is also a promising approach to increasing the yield and
photosynthesis rate of C3 plants like wheat. Transgenic wheat plants over-expressing C4 enzymes,
PEPC (phosphoenolpyruvate carboxylase) and PPDK (pyruvate orthophosphate dikinase), showed a
Int. J. Mol. Sci. 2019, 20, 3350 5 of 18

significant positive effect on photosynthetic traits and yield [52]. Later, transformed wheat-expressing
PEPC gene from maize was studied for drought tolerance [53]. Transgenic plants had higher yield,
photosynthesis, soluble sugar, and proline contents and improved water use efficiency with more roots
under drought stress. The increased photosynthesis in transgenic wheat was attributed to higher PEPC
responsive proteins such as pyruvate, chlorophyll binding proteins, and phosphate dikinase enzymes.

6. Transcription Factors
Plants induce the expression of various transcription factors to cope with environmental stresses.
Then the transcription factors up or down-regulate the expression of series of genes by binding with
the enhancer or promoter region of the gene with DNA-binding domains [54–56]. Several transcription
factors families like WRKY, DREB, MYB, NAC, and ERF are involved in the plants in response to
drought stress by controlling the expression of stress-related genes [9,56–58]. Engineering transcription
factor is an effective and practical approach to improve abiotic stress tolerance by genetic engineering.
Previous studies indicated that transgenic plants overexpressing transcription factor-encoding genes
could increase tolerance to abiotic stresses [59–61]. Transcription factors involved in drought tolerance
could be utilized to develop drought-tolerant wheat cultivars [56].

6.1. DREB
Dehydration-responsive element binding (DREB) transcription factors have been reported to
enhance drought tolerance in transgenic wheat [62,63]. In wheat, the TaDREB1 is a DREB2-type
transcription factor induced by drought and other abiotic stresses [64]. Likewise, a DREB-related
gene, TdDRF1, has been isolated from durum wheat and is characterized to be involved in drought
tolerance [65].
Wheat transgenic plants with the DREB1A gene were produced at CIMMYT and were tested in
small pots under greenhouse [62]. DREB1A gene of Arabidopsis thaliana was transferred into bread
wheat under the control of the stress-inducible rd29A promoter. Transgenic wheat plants expressing
the DREB1A gene demonstrated substantial resistance to water stress as compared to wild type under
greenhouse conditions, as manifested by delayed wilting and leaf bleaching after withholding water.
On the other hand, the non-transgenic plants were dyed after 15 days of holding water [62]. The
higher drought tolerance of the AtDREB1A transgenic plant is achieved by increasing the relative water
content, chlorophyll, sugar, and proline contents as compared to non-transgenic plants [66].
Shiqing et al. [17] transformed the GmDREB gene of soybean into wheat by bombardment.
Transgenic wheat plants using ubiquitin and rd29A promoters showed normal growth and higher
drought resistance than the wild type. Furthermore, some transgenic seeds germinate at 18% PEG6000
solution where all seeds of wild type could not even germinate. Pierre et al. [63] conducted a field trial
to test the performance of 14 transgenic wheat lines (variety Bobwhite) overexpressing the DREB1A
gene as described by Pellegrineschi et al. [62]. Results indicated that the transgenic lines showed no
unwanted pleiotropic effect as compared to control lines and the DREB1A gene increased the survival
rate and water use efficiency. Therefore, it might be possibly assumed that high-yielding transgenic
wheat lines could be achieved by adequate screening and transformation protocols.
Jiang et al. [61] investigated miRNAs in seeds of non-transgenic varieties and GmDREB1 transgenic
wheat line using deep sequencing and bioinformatic techniques. They found that 23 miRNAs were
found differentially expressed in transgenic wheat seeds. The variations between transgenic and
non-transgenic wheat lines were higher than which was induced by transgene.

6.2. WRKY2
The WRKY proteins form one of the largest families of transcription factors in plants, characterized
by their WRKY domain of about 60 amino acids. WRKY acts as a repressor or activator and regulate
various plant growth processes as well as abiotic and biotic stress responses [67]. Various transgenic
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plants, arabidopsis [68] and tobacco [69] overexpressing WRKY genes from wheat conferred abiotic
stress tolerance.
Okay et al. [55] investigated the expression patterns of TaWRKY proteins in the leaf and root of
drought-tolerant and susceptible bread wheat cultivars under drought. Through in-silico searches,
35 WRKY transcripts belonging to 10 TaWKRY genes were detected and the expression of all the
quantified TaWRKY transcripts was found to be up-regulated in the roots of drought-tolerant wheat
under drought. Gao et al. [70] cloned the promoter of the TaWRKY2 gene from a drought-tolerant
wheat ‘Xifeng200 into a spring wheat cultivar ‘Fielder’. The over-expression of the promoter of the
TaWRKY2 gene contributes to drought tolerance as indicated by lesser water loss and higher survival
rate as compared to wild type and also of spring wheat. In addition, the transgenic TaWRKY2 wheat
had a higher yield and higher chlorophyll, proline, and sugar contents [70].

6.3. HDG11
HDG11, a transcription factor belongs to the HDZip IV TF subfamily, was found to induce drought
tolerance by increasing the expression of numerous genes responsive to drought, including genes
involved in calcium signaling and ABA synthesis pathway [71,72]. Transgenic wheat overexpressing
the AtHDG11 gene were studied after 30 days of water stress conditions [60]. Transgenic lines
overexpressing AtHDG11 not only improved the physiological traits but also increased the yield and
exhibited lower water loss rate and stomatal density, while accumulating more proline contents. The
activities of antioxidative enzymes, net photosynthesis rate, water use efficiency, and grain yield were
higher in transgenic wheat than non-transgenic after drought stress.

6.4. TaSHN1
Cuticle protects plant from various abiotic and biotic stresses and from water loss. Cuticle
biosynthesis depends on various metabolic activities which are regulated by several TFs from different
families [73]. SHINE1/WAX INDUCER1 is TF in Arabidopsis belongs to the APETALA2/Ethylene
Response Factor family involved in the regulation of cuticle biosynthesis. Overexpression of SHINE1
increased the accumulation of wax by activating gene expression encoding enzymes involved in
wax biosynthesis [74]. Bi et al. [58] reported that the activation of the TaSHN1 promoter conferred
drought tolerance in transgenic wheat without any yield loss under controlled growth conditions.
The overexpression of the TaSHN1 gene in transgenic wheat plants enhanced drought tolerance by
changing cuticle composition, effecting cuticle biosynthetic genes and decreasing the leaf stomatal
density as compared to non-transgenic plants [58].

6.5. NAC
NAC constitutes one of the largest families of transcription factors and is characterized by a
highly-conserved DNA binding domain at the N-terminus (NAC domain). Several members of
the NAC family have been identified in many plant species and are known to be involved in the
regulation of plant response under abiotic and biotic stresses by constitutive expression [75,76]. Many
NAC transcription factors have been genetically engineered in crops to improve tolerance [77,78].
Xue et al. [79] identified four highly homologous NAC genes (TaNAC69) in wheat. The expression of
TaNAC69 genes was highly upregulated in the leaf and root of wheat under drought stress, however,
the expression of TaNAC69 gene in roots under normal conditions indicated the involvement of the
TaNAC69 gene in root cellular activities [79]. Later, Tang et al. [80] identified six NAC transcription
factors in wheat, which are grouped into development-related, stress-related and membrane-associated
transcription factors. All these NAC were induced by dehydration and other abiotic stresses. The
over-expression of TaNAC69 was studied in transgenic wheat under drought-inducible barley HvDhn4s
promoter [77]. The constitutive over-expression of TaNAC69 in transgenic wheat lines induced the
enhanced expression of several stress-related genes. Transgenic lines produced more root biomass
under PEG-induced dehydration stress [77].
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6.6. bZIP2
The bZIP transcription factors, characterized by a highly conserved bZIP domain, are composed
of the leucine zipper and amino acids. bZIP transcription factors are involved in ABA signaling
and plant response to various stresses such as drought. Its overexpression increased tolerance to
drought stress [80]. Several group A bZIP transcription factors have been characterized in wheat
and their role in stress response of transgenic plants has been described [56,81]. bZIP transcription
factor, TabZIP2, was overexpressed in transgenic wheat lines under the drought-inducible ZmRab17
promoter. Under moderate water stress, transgenic plants were smaller with a lesser number of spikes
and seeds, but seed weight was increased compared to control plants. These changes were attributed
to the rearrangement of carbohydrates in plant parts under drought and wrong information about
the strength of drought by a higher number of TaZIP2 transcripts in transgenic plants, indicating the
possible role of TaZIP in the signaling pathway [82].

7. Post-Translational Modification
Post-translational modification of small ubiquitin-like modifiers (SUMOylation) is an important
mechanism and regulates plant growth and development under stress conditions [9,83]. SUMOylation
is reversible and SUMO-substrate linkage is cleaved by SUMO protease in the deSUMOylation process.
Overexpression of OVERLY TOLERANT TO SALT-1 (OTS1) protease has increased salt tolerance in
arabidopsis [84], and salt and drought tolerance of transgenic rice [85]. Le Roux et al. [86] performed
the first study on the SUMOylation effect in wheat by transferring the cysteine protease AtOTS1
from Arabidopsis thaliana to wheat. Overexpression of AtOTS1 in bread wheat improved the drought
tolerance of transgenic wheat. Transgenic wheat showed improved growth and delayed senescence
under drought by increasing photosynthesis and chlorophyll content.

8. Protein Kinase

8.1. Phosphoenolpyruvate Carboxylase Kinase Related Kinases

Protein kinases regulate key aspects of cellular function, including responses to external signals [87].
Phosphoenolpyruvate carboxylase kinase-related kinases (PEPKRs) belong to the CDPK-SnRK
superfamily of protein kinase [87]. Drought-tolerant wheat cultivar showed higher induction of
TaPEPKR2 under drought stress, while sensitive cultivar exhibited less induction of TaPEPKR2 [88].
Phosphoenolpyruvate carboxylase kinase-related kinase gene (TaPEPKR2) is a highly conserved
serine-threonine protein kinase gene and is reported to be induced by heat and drought. Transgenic
wheat lines transformed with the TaPEPKR2 gene exhibited enhanced heat and dehydration stress
tolerance and higher total root lengths in the presence of 10% PEG than wild type [89].

8.2. Signal Transduction Genes

Calcineurin B-like (CBLs) proteins are Ca2+ sensor proteins that under unfavorable conditions
and drought stress interact with target proteins to transduce signals. CBLs proteins interact
with CBL-interacting protein kinase (CIPKs) [90]. Cui et al. [91] reported that 21 TaCIPK genes
were overexpressed in wheat under drought, and TaCIPK23 was most responsive. Transgenic
wheat over-expressing TaCIPK23 genes showed a higher survival rate under drought stress with a
higher accumulation of osmoprotectants, and the expression of drought and ABA-responsive genes
was increased.

9. Nuclear Factor
Nuclear factor Y (NF-Y) gene family is involved in various regulatory functions for plant
development and performance under stress [92]. In heaxaploid wheat, 80 NF-Y genes have been
identified, but the exact number of NF-Y genes is still unknown [93].
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Nelson et al. [94] reported that transgenic maize-overexpressing ZmNF-YB2 showed improved
growth under drought stress. Yadav et al. [95] isolated NF-YB and NF-YC encoding genes from
drought-tolerant Triticum aestivum cultivar RAC875, and TaNF-YB4 was placed in wheat cultivar
Gladius under constitutive polyubiquitin maize promoter by using biolostic bombardment. The effect
of overexpression of TaNF-YB4 gene in T2 lines was investigated under mild water stress. T2 lines
overexpressing TaNF-YB4 indicated a greater number of spikes under water stress whereas other yield
components were not increased. Although under well-watered conditions, a significant increase in
yield was observed [95] (Table 1).
Int. J. Mol. Sci. 2019, 20, 3350 9 of 18

Table 1. Improving drought tolerance of wheat through engineering gene.

Transgene Transgenic Recipient Source Improved Traits References

TaWRKY2 Fielder, a spring Triticum aestivum cultivar Xifeng20, a drought tolerant wheat Higher survival rate, proline, soluble sugar and chlorophyll. [70]
calcineurin B-like protein
Higher survival rate, increased osmolytes, induction of
(CBL)-interacting protein kinase Fielder, a Triticum aestivum cultivar Triticum aestivum cultivar Xiaobaimai [91]
stomatal closure, enhanced ABA sensitivity.
CIPK23
Higher detoxification activity for the aldehyde substrate;
aldose reductase gene MsALR CY-45, a spring Triticum aestivum cultivar Alfalfa [96]
higher biomass and seed weight.
Hi-Line, a spring Triticum aestivum
HVA1 Barley Improved biomass and water use efficiency. [48]
cultivar
Hi-Line, a spring Triticum aestivum
HVA1 Barley Higher plant height, total biomass and grain yield. [49]
cultivar
Mannitol-1-phosphate
Bobwhite, Triticum aestivum cultivar Escherichia coli Improved biomass, mannitol accumulation. [18]
dehydrogenase mtlD
betA encoding choline dehydrogenase Jinan 17, Triticum aestivum cultivar Escherichia coli Accumulation of glycinebetaine. [32]
Betaine aldehyde dehydrogenase,
Triticum aestivum Atriplex hortensis Higher BADH activity, show normal growth. [35]
BADH
Betaine aldehyde dehydrogenase,
Line (T6), from Shi4185 line Atriplex hortensis Accumulation of glycinebetaine. [31]
BADH
Betaine aldehyde dehydrogenase,
Line (T6), from Shi4185 line Atriplex hortensis Decreased PSII photoinhibition. [36]
BADH
Improved drought tolerance with more leaves, roots and
GmDREB Lumai22, Triticum aestivum cultivar Glycine max cultivar Jinong27 [17]
high soluble sugar contents.
∆1 -pyrroline-5 carboxylate
CD200126, Triticum aestivum cultivar Vigna aconitifolia Proline biosynthesis. [19]
synthetase, P5CS
∆1 -pyrroline-5 carboxylate
Triticum aestivum Triticum aestivum Proline accumulation. [28]
synthetase, P5cs
More branched root phenotype higher total number of
DREB1A bread wheat Arabidopsis thaliana [62]
heads, enhance drought tolerance.
sedoheptulose-1, 7-bisphosphatase
Line (T2) from cultivar Cadenza Brachypodium distachyon SBPase promoter fully drive the GUS expression. [97]
SBPase
HaHB4 cv. Cadenza Sunflower Increased yield and water use efficiency. [16]
Higher biomass, photosynthesis, relative water content,
AtWRKY30 Sakha-61 genotype, Triticum aestivum Arabidopsis thaliana prolines, soluble proteins, soluble sugars, and antioxidant [98]
enzymes activities.
More yield, higher proline content and photosynthesis,
AtHDG11 Chinese Spring, Triticum aestivum Arabidopsis thaliana lower stomatal density, lower water loss rate, and increased [60]
activities of catalase and superoxide dismutase.
Higher proline, grain weight and grain yield, less reduction
cold shock protein gene SeCspA cultivar KN199, winter wheat Escherichia coli [44]
in chlorophyll, low MDA content.
Int. J. Mol. Sci. 2019, 20, 3350 10 of 18

Table 1. Cont.

Transgene Transgenic Recipient Source Improved Traits References

Improved leaf iron content and ROS, enhanced drought and
ferritin gene, TaFER-5B Jimai5265, wheat cultivar wheat cultivar, TAM107 [51]
temperature tolerance.
phosphoenolpyruvate carboxylase
Liaochun10, wheat cultivar wheat cultivar, TAM107 Enhanced drought tolerance, higher root length. [89]
kinase-related kinase gene, TaPEPKR2
Australian drought tolerant genotype Lower stomatal density and leaf water loss, and improved
TaSHN1 Triticum aestivum cultivar Gladius [58]
RAC875 recovery after severe drought.
TaNF-YB4 Triticum aestivum cultivar Gladius Triticum aestivum cultivar RAC875 More spikes. [95]
DREB/CBF gene TaRAP2.1Lmut Triticum aestivum cultivar Gladius Triticum aestivum cultivar RAC785 Enhanced ability to survive frost and drought. [99]
Delayed senescence, higher relative water content,
OTS1, overly tolerant to salt-1 Triticum aestivum Gamtoos-R Arabidopsis thaliana [86]
photosynthesis and antioxidants.
TaNAC69 Triticum aestivum cultivar Bobwhite Triticum aestivum More root biomass, longer roots. [77]
TabZIP2 Triticum aestivum cultivar Gladius Triticum aestivum cultivar RAC875 Fewer spikes and seeds, increased single seed weight. [82]
Improved survival, slow growth, delayed flowering, less
DREB Triticum aestivum cultivar Bobwhite Triticum durum L. cultivar Langdon [100]
grain yield.
Triticum aestivum cultivar 8901, 5–98, Still green after 15 d withholding water, high proline
DREB Arabidopsis thaliana [101]
99–92, Baofeng 104 contents.
PEPC Triticum aestivum cultivar Zhoumai19 Maize Higher proline, soluble sugar and water use efficiency. [53]
Lower water loss rate and MDA content, higher chlorophyll,
CspA and CspB Triticum aestivum cultivar KN199 Escherichia coli [44]
proline and yield.
Int. J. Mol. Sci. 2019, 20, 3350 11 of 18

10. Limitations
Progress for successful development of drought-resistant wheat depends on the knowledge of
functional genomics. Drought resistance is a complex trait and it is necessary to identify the function
and structure of key genes in the development of wheat plants for drought tolerance and finally for
higher grain yield. Plant biologists could use this knowledge to alter the structure and functions of
selected genes through genetic manipulation.
Less progress has been made for the development of transgenic wheat, and a number of reasons
could explain the lack of success. Wheat has a complex and large hexaploid genome, almost five times
greater than the human genome which contains almost 128,000 genes [102], with 80–85% of them being
repetitive sequences of DNA due to hexaploidy genome [103]. The genetic transformation efficiency of
wheat is low and dependent on genotypes. Most of the studies have focused on the survival rate of
plants under severe water stress which rarely has importance for wheat and other crops [104]. In some
cases, the survival rate is accompanied with undesirable phenotypic features such as reduced plant
size and yield [100,105,106].
The development of drought tolerance by transforming a single gene seems unsatisfactory. A single
gene is easier to manipulate however, it is less suitable to confer tolerance under various field conditions.
Thus, multiple genes should be considered to manipulate the built-in feedback control mechanism
and to evade the overaccumulation of intermediate products. Moreover, the wheat transformation
may generate completely new interactions between genes making them function differently from
what would be expected. There is a need to account for negative interactions between the drought
tolerance and other traits such as the photosynthetic rate in order to get a high yield. For instance,
transforming homeodomain-leucine zipper I (HD-Zip I) transcription factors into wheat, the plants
showed improved resistance to drought but also exhibited undesirable phenotypic characteristics such
as reduced biomass and yield [105,106].
In many previous studies, molecular biologists are screening transgenic plants in small pots under
severe water stress applied for few hours or days and injury or survival-related plant responses were
induced under the conditions of severe stress, which will be different from the response for a long-term
slowly-induced water deficit stress. However, under natural conditions, water deficit stress advances
slowly in a gradual manner with the drying of soil and plants under these conditions adjusting the
morphological and physiological traits accordingly, like the accumulation of osmolytes to maintain cell
hydration [15]. Thus, it is important that experiments must be under conditions similar to the field.
Furthermore, the intensity and duration of stress are key factors for screening which have been ignored.
Most of the studies which reported the yield increase of transgenic plants were conducted in the
greenhouse-controlled conditions [96] and the response of specific transgene could be reversed under
field [104]. Therefore, transgenic lines failed to sustain the benefits observed under control conditions
in field trials [104]. The 14 transgenic wheat lines with DREB1A gene, selected under control conditions
for high survival and water use efficiency, failed to show improved yield under water deficit in the
field [63]. Only some of the researches have confirmed the improved drought resistance of transgenic
wheat under field conditions [16].
Accurate phenotyping is most crucial for drought tolerance and should be selected under
suboptimal field conditions in order to draw correct assumptions about the role of the discovered genes
towards drought tolerance and their utilization in plant breeding. The selection of phenotypic traits
for water use efficiency, osmotic adjustment, root architecture, stomatal conductance, carbohydrates
remobilization, chlorophyll contents, and traits related to stay green and delayed leaf senescence will
be useful for the improvement of yield under water stress [56].
Furthermore, there are some significant issues related to the cascading effect of the regulatory
gene on different genes pathways, such as increased competition due to introgression of a transgene
into wild, and the impact of regulatory genes on the environment and human health, which need to be
properly addressed [15].
Int. J. Mol. Sci. 2019, 20, 3350 12 of 18

11. Future Directions in the Development of Drought-Tolerant Transgenic Wheat

Genetic modification is a promising approach which gives us an insight into the regulatory
mechanism by a genetic change of a single or few genes. Recently, some progress has been made by
transgenic approaches in identifying the key regulators of drought tolerance in wheat. Transgenic
wheat plants with inserted genes such as structural and protected genes of mannitol, proline, glycine
betaine, and LEA protein and regulatory genes of NAC and DREB transcription factors are being used
for drought tolerance. The first drought-tolerant genetically-modified wheat was developed by the
Argentine company, but needs approval for commercial release. After the approval for commercial
release, Argentina will be the first country to release genetically-modified wheat engineered for drought
tolerance [107].
It is of importance that the tolerance to a specific stress such as water stress be considered with
the tolerance to other stresses. Wheat has a complex hexaploid allopolyploidy genome structure.
Therefore, future breeding and genetic transformation efforts require the complete information of
functional genomics and identification of the functional and structural role of genes involved in
tolerance and determining higher grain yield. Some potential genes have been discovered which
have not yet been tested in wheat and could be targeted to improve drought tolerance and yield.
These potential genes are responsible for allocating sucrose during seed development, recovering
from drought stress at the vegetative stage, and signaling genes involved in plant growth regulations.
Research is being undergone in Mexico by the International Maize and Wheat Improvement Centre
in which genetically-modified wheat inserted with these genes will be studied, and the yield will be
determined under drought. Furthermore, genetic variations in wheat genotypes for sucrose allocation
will be focused. Hopefully, the new developed drought-tolerant varieties will soon be developed
and deployed through exchanging knowledge between research institutes and industries. Some
modern and advanced molecular techniques like CRISPR/Cas9 and RNA interference are emerging
as new end-products-based regulators in stress tolerance. These approaches could provide a more
favorable potential base for future wheat-breeding programs by effective collaboration with traditional
breeders. By collaboration and successful introgression of these genetic techniques, the outcome could
be translated from laboratory tools to fields.

Funding: This work was jointly supported by scientific research programs by Modern Agriculture Industry
Technology System Construction (CARS-03-01-24), the National Key Research and Development Program of
China (2018YFD020040105), Natural Science Foundation of China, (31771727), Sanjin Scholar Support Special
Funds Projects, Research Project Supported by Shanxi Scholarship Council of China (2017-068), Crop Ecology and
Dry Cultivation Physiology Key Laboratory of Shanxi Province (201705D111007), Shanxi Science and Technology
Innovation Team Project (201605D131041), and Shanxi Collaborative Innovation Center with Featured Crops
High-quality and Efficiency Production in Loess Plateau.
Conflicts of Interest: The authors declare no conflict of interest.

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