Wheat Responses to Stress and Biotechnological Approaches for Improvement

Abstract

In the wheat crop, biotechnological approaches are yielding remarkable results in the area of molecular variability for pathogens, genome mapping and characterization, production of virus-free plants, transgenic plants with improved quality, and stress-tolerant traits plus breeding for elite traits up to micronutrient levels. Tissue culture is the prerequisite approach for almost all of the biotechnological applications. Tissue culture performs an important role in improving wheat for various traits including biotic and abiotic stress resistance. The most widely used approaches of tissue culture reviewed here include in vitro androgenesis, somatic embryogenesis, somaclonal variations, and double haploid production. Among its various applications, tissue culture is now being applied for generating heavy metal-tolerant wheat plants. Genetic engineering is the most promising technique that is currently being applied for resistance against particular fungal diseases and subsequently for increasing crop yield. Among fungal diseases, the main focus has been given to Fusarium head blight and powdery mildew diseases. Many resistant genes harbored in suitable constructs have been successfully integrated into the wheat genome via biolistic or Agrobacterium transformation approaches. Pathogen-derived resistance has also been observed for producing tolerant wheat cultivars against viral diseases. For managing insect/pest resistance in wheat, resistance genes (R-genes) have been identified in resistant varieties and are effectively incorporated into the wheat cultivars through breeding. They are being used in place of conventional and molecular breeding to develop resistant cultivars. The next-generation technology is the foremost technique for identification of not only the target genes but also for the transcription factors or families and miRNA responsible for regulating these genes. Most of the abiotic stress-related transcription factors and genes have been documented, for instance, drought factors and metal toxicity. Here the main focus is on the biotechnological approaches and next-generation sequencing technology used in wheat improvement along with certain factors that affect wheat yield by causing biotic and abiotic stress conditions.

1 Introduction

Among all the cereal crops, wheat (Triticum aestivum L.) is the most vital grain crop, and about 21% of the world’s food depends on wheat annually (Enghiad et al. 2017). Wheat crop supplies with more than 20% of the protein and 40% of the calories in the diet for the whole population globally (Braun et al. 2010; Kumar et al. 2015). Therefore, demand and value for intensification of wheat production is widely recognized. With this high demand, wheat still faces annual losses in its total production subjected to various abiotic and biotic stresses caused by virus, fungi, bacteria, nematodes, and insects (Agrios 2005).

Presently, the global wheat yield on an average is about 3.12 metric tonnes for crop year 2016/2017, but there are considerable differences among various areas of the world. The increase in yields is an intricate challenge, difficult to be realized by any one particular approach. Basically, there are three main challenges regarding crop yield: yield potential maximization, yield potential protection, and enhancing resource efficiency for sustainability. These challenges are interrelated, and there is healthy evidence that expansion in the yield potential will often also lead to greater yields even under stress states. The genetic improvement of wheat has received significant consideration over the past decades with the purpose of maximizing the grain yield, to reduce crop productivity due to harsh environmental states, plus damage caused by various pathogens and pests .

Crop losses not only affect the farmer on an individual level but also the whole countries’ economy due to biotic and abiotic stresses. Since crop production remains the same, the profit expected to be gained from the crop harvest gets significantly. Each year there is a 34.5% loss of cereals due to pests, and this loss also varies with the abiotic stresses (Agrios 2005).

At the beginning of the 1960s, the targets of genetic manipulation shifted to decrease the yield variability caused by several biotic and abiotic stresses and enhance input use efficiency (Pingali and Rajaram 1999). Subsequently, due to alterations in the world food policy during the last few years, biotechnology approaches offered a plausible solution initially, by reducing the costs of farm-level production by providing the plants tolerant to various biotic and abiotic stresses and furthermore by improving the product quality.

Thus, tissue culture techniques became established that could be routinely utilized for the maximization and improvement of many crop plants. In comparison with dicot plants, the monocotyledons respond poorly to tissue culture, and all the major cereal crops are monocots. It is well established that tissue culture of dicotyledons is simple as compared to monocot crop plants (Reinert and Bajaj 1976; Yildiz 2012). Therefore, extensive research was needed to establish the in vitro culture protocols for these crops. The potential significance of cells, tissue, and anther culture as a technique has been described for improvement of crop plants (Vasil 1987). Remarkably, the regeneration of cereal plant species into the whole plant has become possible today and included maize (Duncan et al. 1985), bread wheat (Redway et al. 1990), rice (Yamada et al. 1986), and barley (Luhrs and Lorz 1987).

Various biotechnological approaches used in wheat improvement like the use of next-generation sequencing in accelerating wheat improvement have been of great focus. For a holistic oversight, certain biotic factors, including bacteria, fungi, viruses, and insects, and abiotic factors, including drought, salinity , extreme temperatures, and heavy metals with a special focus on drought and heavy metals, affecting wheat yield have been discussed here.

2 Problems and Challenges

2.1 Physical or Abiotic Stresses

Abiotic stress is a situation based on certain environmental conditions when they deviate from the normal or optimal level. De Leonardis et al. (2007) demonstrated that salinity, temperature intensity, and most importantly drought are some major environmental abiotic stresses affecting plant growth to a large extent often resulting in decreased productivity of crops (Mott and Wang 2007). Mittler and Blumwald (2010) also demonstrated the role played by these abiotic factors in determining the growth of plants and their productivity. As a result of such severe environmental conditions, the plant copes by adapting to these stressed conditions through biochemical, physiological, and morphological changes undertaking certain metabolic strategies occurring as a result of altered gene expression. This involves an intricate regulatory system comprising of stress sensory elements, metabolites, and signaling pathways based on certain protein (Knight and Knight 2001).

Wheat being one of the most important staple crops feeding a large global populace, reduction in its productivity is a serious issue and needs to be handled very efficiently. To focus on its stress tolerance capabilities and to increase its resistance to certain stress conditions, thereby increasing its yield, certain research groups have studied and functionally characterized and analyzed the genes involved in response to stress, with drought and salinity being the major ones that limit the productivity of the crop.

2.1.1 Drought

Khakwani et al. (2012) demonstrated the adverse effects of drought on the metabolism of wheat varieties. This abiotic stress is affecting grain yield in bread wheat, an important cereal crop in Pakistan, which was significantly demonstrated recently in certain genotypes by Khavarinejad and Karimov (2012). Johari and Moharram (2011) evaluated different wheat cultivars under water-deficient conditions. Plaut et al. (2004) and Passioura (2007) also evaluated the conditions created in drought and extreme temperature and their adverse effects on the agricultural environment. Sial et al. (2012) reviewed crop yield lowered by approximately 20–30% globally as a result of the impact of drought, with salinity influencing on 20% irrigated land. This damage will have quite a greater impact on developing countries.

2.1.2 Metal Toxicity

Metals are present in the soil naturally and may be useful or harmful for plants. Metals are usually needed in low amounts for plant growth but when found in excess amounts in the soil cause toxicity and are harmful to plants’ growth and inhibit it. The main problem occurs when these ions and toxic metals accumulate in the plant cell. The accumulation of metals in the plant cells depends upon the plant species and their ability to absorb nutrients and the concentration of metals in the soil. The reason for the increase in toxicity in the soil is industrialization. There are some natural resources as well as anthropogenic resources that contribute to metal toxicity. Metal toxicity affects the soil properties. Metals such as iron, aluminum , cadmium , lead, etc. in excess amounts cause metal toxicity (Roy and Kumar 2009).

2.1.2.1 Aluminum Toxicity

Aluminum (Al) is the metal which is found most abundantly in the earth’s crust because according to weight if it comprises of an 8% level, its Al toxicity affects 40–70% arable land of the earth. It increases the plant growth when it is in low concentration. Al is found in all soils, but it is generally correlated with low pH (Roy and Kumar 2009). It is found in the form of primary and secondary minerals in the soil. When soil becomes slightly acidic, these primary and secondary minerals release Al in the soil and cause Al toxicity and increase soil acidity. The Al and Fe ions have a major contribution in increasing the soil acidity. In soil solutions, different species of Al occur. The pH and toxicity of various Al species are shown in Table 1.

Table 1 Different species of aluminum and their pH and toxicity

Al toxicity is a major factor that limits the growth of the plants. The initial response of the plant to Al toxicity stress occurs in roots progressing to affect the whole root system with the color of roots turning brown and root tips and lateral root becoming thick. Root branches remain underdeveloped; therefore no luxuriant root mass formed. These deformations affect the root function. They became inefficient in functioning to absorb nutrients and water that are essential for the plant growth and survival (Reynolds et al. 2001). In roots, Al toxicity blocks the cell-to-cell movement because of accumulation of the cellulose in the plasmodesmata of the cell wall of the root cells (Panda et al. 2009). It is abiotic stress that in some global locations (Brazil) is a wheat production constraint.

2.1.3 Nutrient Deficiency

Nutrients are essential for plants. These nutrients have many functions in plant growth and development. They have a role in physiological functions. They activate enzymes, regulate their functions, and are important parts of the metabolite complexes or the assembly of macromolecules. Many nutrients in ionic form maintain the osmotic potential of the cells. One nutrient has many functions in the cell, and thus one nutrient is involved in another nutrient’s metabolism. For example, in wheat, molybdenum is the part of an enzyme that reduces the amount of nitrate in the wheat plant, and due to deficiency of molybdenum, the amount of nitrate increases and causes toxicity. Deficiency symptoms of nitrogen, phosphorous, and potassium occur in older leaves first (Roy and Kumar 2009).

2.1.3.1 Potassium Deficiency

Potassium (K) is an important mineral for the wheat plant. It plays an important role in the plant growth, photosynthesis, and protein synthesis . Enzymes play a key role in plants, and K has a significant role in their activation. About 67% of the wheat fields are K deficient. The phosphorous and nitrogen are nearly balanced in the fields due to the use of fertilizers, but K often becomes a limiting factor for the wheat yield. It is necessary to improve the K efficiency to increase the yield of wheat (Ruan et al. 2013). K deficiency is difficult to diagnose because symptoms of K resemble that of the injury to the plant from the soil below. It is diagnosed by the sporting tissue test result (Stevens et al. 2002) where leaves become spindly and the growth of the plant is adversely affected before the symptoms appear. Necrosis of the older leaves starts on tips and leaf margins which spread inward on the leaves leaving green arrow-like streaks in the middle of the leaf. The leaves ultimately die out and appear to be drought affected.

2.2 Biological or Biotic Stresses

In common terms, a biotic stress is defined as the effect of fungi, bacteria, viruses, pests, and weeds on the plants which hinder its normal production and thus decrease their yield. More than 42% of cereal crop yield is decreasing due to these biotic and abiotic factors. The food production would have to be tripled by now to fulfill the needs of the population throughout the world due to demands of people for food with improved nutritional quality (Vasil 2003). For this purpose, yield potential of cereal crops needs to be increased.

Yield potential is defined as the yield of a crop or a cultivar which is grown in such environments that are fully adaptable to biotic and abiotic stresses, thus maximizing the potential yield of a particular cultivar (Evans and Fischer 1999). There are two methods to acquire yield that can fulfill the future demands, firstly by reducing the production losses due to biotic (pathogens, pests, and weeds) and abiotic losses (drought, salinity, etc.) and secondly by increasing the nutrient usage as well as the photosynthetic efficiency of plants (Sramkova et al. 2009). To attain such increases in yield, conventional breeding and marker-assisted breeding are being used. They have achieved considerable results in improving certain stresses, but due to its slow progress in cereal crop improvement, scientists are working on genetic engineering techniques to swiftly fulfill the rising food demands to integrate genes that can increase yield potential. This chapter discusses the factors associated with wheat biotic and abiotic stresses and how they can be controlled to maximize yield.

The above mentioned strategies have been applied in wheat to increase its yield potential. Methods by which abiotic stresses can be improved have already been mentioned. Some of the abiotic and biotic factors responsible for the decline in wheat crops are mentioned in Fig. 1. However, this part focuses on how biotic stresses in wheat can be controlled by the application of the genetic engineering that can ultimately increase yield. These biotic stresses of wheat include damages caused by fungi, bacteria, viruses, and pests.

Fig. 1

Abiotic and biotic stresses responsible for the decline in wheat yield

2.2.1 Fungal Diseases

There are more than 50 diseases of fungi that affect wheat varieties worldwide. These include diseases affecting spike and grain, diseases affecting leaves, and diseases affecting lower tiller portions and roots. Most common fungal diseases of wheat include Karnal bunt (causing agent: Neovossia indica), common root rot (causing agent: Bipolaris sorokiniana), leaf rust (causing agent: Puccinia recondita/Puccinia triticina), stem rust (causing agent: Puccinia graminis), stripe rust (causing agent: Puccinia striiformis), powdery mildew (causing agent: Erysiphe graminis/Blumeria graminis), fusarium head blight or scab (causing agent: Fusarium graminearum), and septoria leaf blotch or glume blotch (causing agent: Septoria tritici). Therefore, developing resistance to fungal stresses is the major challenge for wheat breeding.

2.2.2 Bacterial Diseases

Bacterial diseases have also been identified in wheat, and most common bacterial diseases include bacterial leaf blight and basal glume rot (Pseudomonas syringae) and black chaff or bacterial leaf streak (Xanthomonas campestris). However, no significant work on developing resistance against these bacterial diseases through genetic engineering has been reported.

2.2.3 Viral Diseases

Viruses have the ability to infect all the life forms. In plants, the intensity of the viral diseases is associated with the type of crop variety and its location that directly affects the crop by reducing its yield and quality. The range of viral infection also varies in plants from minor or mild to devastating effects (Agrios 2005).

Each year millions of crops are destroyed before reaching the harvesting stage. The main reason for the wheat disease is its susceptibility toward a number of viruses. Among various diseases the following viruses are of importance: Barley yellow dwarf virus (BYDV), Wheat dwarf virus, Soil-borne wheat mosaic virus (SBWMV), Wheat spindle streak mosaic virus, Wheat yellow mosaic virus, and Barley yellow striate mosaic virus (Agrios 2005).

2.2.3.1 Barley Yellow Striate Mosaic Virus

Barley yellow dwarf virus belongs to Luteoviridae and strictly causes diseases in Poaceae family. This virus causes 5–30% losses of wheat and is submitted by aphids including several species of Sitobion, notably S. avenae, Rhopalosiphum padi, and Metopolophium dirhodum (McIntosh 1998). It can be controlled in wheat by making the cultivars resistant. Breeding different varieties of cereals to provide tolerance against BYDV infection has importance (McIntosh 1998). It is transmitted by plant hopper Laodelphax striatellus (Signoret et al. 1977). About 11–12% loss in yield of wheat is observed by this virus in Morocco and Chile (El Yamani and Hill 1990; Ramirez et al. 1992).

2.2.3.2 Soil-Borne Wheat Mosaic Virus

It belongs to Furovirus and is transmitted by fungus Polymyxa graminis. This virus causes stunting growth and results in 50% wheat yield losses (Campbell and Choy 2005). Recombination breeding of resistant and susceptible wheat can be helpful in preventing SBWMV, but effective resistance is not available in wheat (McIntosh 1998).

2.2.3.3 Wheat Spindle Streak Mosaic Virus

It belongs to Bymovirus – soilborne. This virus causes 3–87% of loss in wheat; the intensity of disease depends on the wheat variety and climatic conditions, where reduced tillering is the main cause of yield losses. However, crossbreeding can improve the variety (Brunt et al. 1996).

2.2.3.4 Wheat Streak Mosaic Virus (WSMV)

This is one of those viruses that can cause 100% loss in wheat yields (McNeil et al. 1996). It belongs to the genus Rymovirus and is transmitted through eriophyid mite vector Aceria tosichella Keifer. Transgenic crops produced against this virus interrupt with the viral life cycle and maintenance of its regular functions (Sanford and Johnston 1985). Such type of resistance provided to the crop is derived from the expression of viral genes and is called pathogen-derived resistance (Sanford and Johnston 1985).

2.2.4 Insects/Pests

Insects are major pest in agriculture that were controlled using insecticides (Schoonhoven et al. 2005). But, due to the negative impact of insecticides both on the environment and beneficial insects, other methods for the resistance have been explored. Wheat resistance is important for insect pest management. More than 30 species of aphids are available that cause various damages to wheat (McIntosh 1998). Therefore, crop resistance against insects can be helpful in creating a defense in plants by determining the source of resistance (Schoonhoven et al. 2005).

Various insects coevolved with wheat include Hessian fly and Russian wheat aphid , while those that became pests were the result of cultivation practice. These include greenbug and wheat stem sawfly. Among all these, Hessian fly and wheat stem fly pose a threat to wheat quality and yield (Painter 1951). Synthetic insecticides were introduced in 1940, but to control insects, farmers have relied heavily on plant resistance. There are four types of insects that feed on wheat and cause yield loss each year. They have a short life cycle but are sources of transmitting viral diseases to the crop (Janick and Wiley 2003).

According to Painter (1951), host plant shows resistance to the crop plant by itself using one of the three mechanisms. Antixenosis mechanism of class modalities resists the insect to lay an egg or colonize the plant. The second mechanism is antibiosis that reduces the growth of the insect or causes the death of the insect. This, however, is not a reliable mechanism as if the host plant lacks the resistant genotype, the same insect will result in high damage to the crop. The last mechanism on which the breeders rely is tolerance where wheat grows despite the attack by insects (Janick and Wiley 2003).

2.2.4.1 Cephus cinctus

It is also known as wheat sawfly that feeds particularly on wheat among all cereal crops. It infects the solid stems of wheat that are less susceptible. Wheat sawfly causes yellowing and grain shriveling, a common problem in the Mediterranean basin (Prescott 1986). Therefore, for the control of this insect, wheat with solid stems is desirable.

2.2.4.2 Aphids

There are several varieties of aphids that cause damage to wheat including Rhopalosiphum padi (bird cherry-oat aphid), Schizaphis graminum (greenbug), R. maidis (corn leaf aphid), Metopolophium dirhodum (rose-grass aphid), Sitobion avenae (English grain aphid), and Diuraphis noxia (Russian wheat aphid). Among these, Russian wheat aphid and greenbug are the most important. They are found to cause damage in all the cereal crops and serve as an important vector for the transmission of BYDV. Russian wheat aphid produces leaf rolling, white strips, and sterile heads (Prescott 1986). Resistant wheat has been produced, with six genes responsible for resistance. Greenbug has many biotypes that affect the crop; however, different sources of resistance are also used for control. The greenbug causes necrotic areas along with leaves rolling, whereas other aphids cause yellowing and premature death of leaves (Prescott 1986).

2.2.4.3 Hessian Fly

It is also known as Mayetiola destructor. Hessian fly belongs to Diptera: Cecidomyiidae family of insects that produces galls in infected plants (Foster and Hein 2009). It feeds on a variety of cereals, but in wheat it’s highly damaging and presents a common problem in the USA and North Africa. Common symptoms include stunting, reduced yield, and lodging (Prescott 1986). The insect prefers laying eggs in young wheat plants during the fall or in early spring, where the larva feeds permanently and causes damage. The incidence of Hessian fly can be avoided by late plantations, controlling wheat responsible for the spread or using resistant wheat varieties to prevent the loss of yield. Insecticides on Hessian fly are less effective because of its sporadic nature . However, parasitic wasps are used as natural enemies of Hessian fly to prevent the crop from its attack (Foster and Hein 2009).

3 Applications and Approaches in Wheat Biotechnology

Wheat is among the most important cereals, as most of the efforts have been applied to increase the productivity of this crop using conventional methods. But, only minor improvement in productivity of this crop has been achieved (Gupta et al. 2008). The most widely and important applications and approaches employed to improve not only wheat grain quality but also its yield are in vitro tissue culture of wheat, genetic engineering technique to integrate the desired genes, and next-generation sequencing techniques to identify the target genes in the wheat genome accurately with precise timing (Fig. 2).

Fig. 2

Approaches and applications in wheat biotechnology

3.1 In Vitro Tissue Culture of Wheat

La Rue (1949) initiated the first successful in vitro tissue culture exploiting endosperm as explant in cereal crops. Gamborg and Eveleigh (1968) reported the protocol for generating suspension cultures of wheat by employing a defined medium supplemented with mineral salts, sucrose , vitamin B complex, and the auxin 2,4-D (2,4-dichlorophenoxyacetic acid). Shimada et al. (1969) successfully developed callus formation and single cell cultures in wheat crop. Ozgen et al. (1996) reported the study on in vitro culturing of mature and immature embryos from seven different varieties of wheat (winter durum) on MS medium provided with 2,4-D. On the basis of this finding, it was concluded that mature embryos had a lower efficacy of callus production than immature embryo but with high regeneration efficiency. Plant regeneration capacity via embryo culture is affected by regeneration media and the genetic factors that can be regulated and by environmental factors that cannot be controlled (Uppal et al. 1996). The concentrations of plant growth regulators are also a very sensitive factor in the control of explant regeneration and morphogenesis.

The high concentration ratio of auxins and low concentration of cytokinins usually promote profuse cell proliferation in the cultured medium with the formation of callus. Hormone-free medium is better for the shoot regeneration, or it can be supplemented with 2,4-D at low concentration as with BAP and IAA (Chawla and Wenzel 1987). The regeneration of explants can be established either indirectly by adventitious bud or somatic embryogenesis and subsequently shoot and root formation (Bhaskaran and Smith 1990), or sometimes it can be obtained directly through organogenesis (Li et al. 1992). Generally, low light intensity is required for the callus formation, which limits the plantlet regeneration efficiency, the ratio of green plantlets production (Ekiz and Konzak 1993).

These preliminary but important outcomes led to the utilization of in vitro cell and tissue culture for several applications including breeding (Wang and Hu 1984), anther and embryo culture (Letarte et al. 2006), the elimination of systemic pathogens (Hafeez et al. 2012) such as viruses, drought resistance (Bajji et al. 2004), and foremost genetic engineering (Vasil 2007) for wheat. The whole procedure and the most widely used application of tissue culture in wheat crops to get the improved and desired variety are demonstrated in Fig. 3.

Fig. 3

Wheat tissue culture protocol and its widely used applications

Pertinent to mention here is the role of embryo culture relative to the innovative aspects that have come on the wheat improvement scene around harnessing the genetic diversity of the wheat families’ wild relatives through wide hybridization which encompasses intraspecific, interspecific, and intergeneric hybridization to effect introgression of novel alleles from the related and distant wild species to enrich the wheat genomes. At the initial step when hybrids are produced, embryo culture is crucial since all F1 hybrids from such distant crosses are devoid of endosperm and require nutrient availability to differentiate seedlings from that becoming the stocks that permit alien gene transfers to occur and lead to wheat improvement. This importance and usage has become a standard practice in wheat wide hybridization programs globally and has been reviewed in recent publications of Ogbonnaya et al. 2013 and Mujeeb-Kazi et al. 2013.

3.1.1 In Vitro Androgenesis and Dihaploid Phenomenon in Wheat

For many important cereal crops, including cultivated wheat, specialized tissue culture methods for producing homozygous generations from microspores exploiting in vitro androgenesis have been efficiently used. With the utilization of anther culture in wheat and establishment of homozygous genotypes within a single generation, plant breeders are now proficient to reduce the time span prerequisite for the development of a uniform line from crossing. Moreover, the haploid chromosome numbers revealed that genetic recombination during meiosis and recessive gene effects are exhibited at the plant level. Haploid breeding is the word coined for this technologies’ emergence.

Microspore and anther cultures have been the most desired choice of explants to produce double haploids, because of a large number of available microspores within each anther that efficiently produces doubled-haploid plants (Zhou 1996). Procedures for anther culture in wheat are frequently being improved (Hu and Kasha 1997) and obtained a high yield of green plants in some genotypes (Bruins and Snijders 1995). Tuvesson et al. (2000) were successfully optimizing the method for production of wheat and triticale double haploids on a large scale by utilizing a single-anther culture technique. Generally, triticale responds poorly to anther culture (RyoÈppy 1997). The major limitation for the commercially exploitation of anther culture in breeding programs is the genotype effect. The production of double haploids from wheat and triticale was initiated in many breeding combinations but with limited success to raise green plants from anther culture in the F1 and F2 generations. For anther culture the substrate used was 190–2 (Wang and Hu 1984), provided with sucrose (9%), 2,4-D (1.5 mg/l), and kinetin (0.5 mg/l).

The key factors affecting the productivity of anther culture include donor genotype physiology, genotype, anther developmental stages, physical and chemical conditions, and pretreatment conditions for culture initiation and regeneration (Zheng et al. 2001). Liu et al. (2002) successfully improved green plant production through isolated anther culture in bread wheat. Their findings clearly illustrated that providing some nutrients to microspores at the stage when embryogenesis was initiated is a vital factor affecting in vitro regeneration and green plant production for a genotype with a comparatively high tendency for albinism.

Anther pretreatment was also one of the most significant steps. The pretreated techniques include maintaining the spikes in cold temperature about at 4 °C for a week or much longer periods or in high temperature nearly at 33 °C for 2–3 days (Zheng 2003). Other pretreatments have been involved like osmotic shock, microtubule disruption agents, and starvation. Sugar starvation for 4–5 days employing mannitol instead of sucrose is generally used in barley (Cistue et al. 1999). Cistue et al. (1999) were able to produce doubled-haploid plants from durum wheat through the induction of androgenesis employing mannitol as the pretreatment and cocultivated the treated anther with ovaries. With this system, they successfully increased the number of embryoids, and as a result, the figures of green plants regenerated were also improved.

On the other hand, Letarte et al. (2006) established the protocol to improve the induction of embryogenesis in anther culture of wheat with much higher regenerable embryos without the presence of ovaries. They tested arabinogalactan (AG) Larcoll and the arabinogalactan protein (AGP) from gum arabic on two wheat spring genotypes. Their finding showed that Larcoll significantly declined microspore mortality in both genotypes irrespective to ovary presence or absence in the culture. Similarly, gum arabic had a strong influence on the amount of embryos generated and regenerated green plants.

Regarding the androgenic response, the colchicine effects on embryogenesis and frequency of green plant production highly depended on the genotype and the culture methodology. Soriano et al. (2007) found interesting effects of colchicine application on the androgenic response in anther culture and chromosome doubling of different bread wheat genotypes. Colchicine was applied not only over the first hours of anther culture but also at the time of mannitol stress pretreatment . Similarly, Islam (2010) also experimented to compare the impact of colchicine by applying it to the anthers and directly on the isolated microspores . He found that the direct colchicine treatment reduced embryoid induction but significantly improved fertile plant production. Lantos et al. (2013) also demonstrated that anther culture was an important application for crop improvement. Lantos and Pauk (2016) determined the effect of different induction media and genotypes of winter wheat. They analyzed the data on the basis of genotype response, year, and genotype with year interaction employing the numerous embryo-like structures, regenerated plantlets, and albino besides green plantlets, proving that anther culture was an efficient method in winter wheat breeding programs with lower costs than other alternative technologies.

Double haploid production through anther culture facilitates the plant breeders to accelerate breeding cycles and release wheat cultivars with biotic and abiotic stress resistance and good quality flour. In vitro androgenesis is useful not only for accelerating conventional breeding but also helpful for mutation breeding, in vitro selection of elite genotype, and genetic transformation techniques. Furthermore, doubled-haploid lines generated from anther culture facilitate the decent transfer of alleles/genes from exotic stock or alien species into wheat elite varieties. Dihaploids are basically isogenic lines which can be efficiently exploited for gene mapping (Barnabás et al. 2001).

The popularity for dihaploid breeding has thus targeted anther culture as described above. Microspore culture also has tremendous potential as microspores from one wheat spike have the capacity to provide several thousand green plants. This is exactly what is a need in a wheat improvement program: large number of outputs. Unfortunately, neither anther culture nor microspore culture can combat the genotypic constraint that limits haploids to be obtained across all wheat genotypes. The most posing procedure thus so far with 100% success in haploid production for utilization, wheat breeding has been the wheat/maize protocol. Apart from breeding swiftness, the wheat/maize route has varied applications in wheat research, and the details have been elucidated by Mujeeb-Kazi 2006. It would be fair to mention here that so far a better option for haploid breeding in wheat other than wheat/maize has to become available that can provide 100% efficiency and is genotype independent.

3.1.2 In Vitro Callogenesis and Somaclonal Variation in Wheat

During in vitro callogenesis, some genetic and phenotypic variations termed as somaclonal variation in the clonally propagated plants from a single clone are observed (Kaeppler et al. 2000). These somaclonal variations can be established either via meiotically or somatically stable events. Somatically stable variants involve phenotypes such as culture adaptation where the variations physiologically induced are examined among initial clones. This form of variation is generally not transferred to the following generations and has an impact in conditions where the primary clones are the final product, for instance, in ornamental plants being amplified and trees for direct utilization. Meiotically inherited variation is essential where the end products of the tissue culture are proliferated and retailed as seed (Kaeppler et al. 2000). Such types of variations can be adapted to cope with biotic and abiotic stresses in the plant genome.

Some studies on in vivo osmoregulation were reported that it is the vital adaptation to salinity in plants since it involved maintenance of the cell volume and turgor pressure (Ashraf and Waheed 1993). Due to lack of osmotic regulation, osmotic stress causes inhibition in the uptake of water and results in physiological drought causing salt injury to plants. Therefore, water loss or dissolved solute enhancement or both may be observed in the condition of the reduced osmotic potential of plants during salt stress (Carvagel et al. 1998).

Javed (2002) presented the study describing an efficient method to investigate the effect of salt (sodium chloride) stress on in vitro callus tissues of wheat genotypes in relation to water. He concluded that callus tissue of genotypes with more salt tolerance had great potential for osmotic maintenance and water reduction. Similarly, Bajji et al. (2004) experimented to produce improved drought-resistant wheat cultivars by exploiting somaclonal variations as a source of variability and placed explants in regenerated media containing polyethylene glycol. They were selecting tissue culture lines in the following generations and found differences in chlorophyll fluorescence , electrolyte leakage, electrolyte conductance in stomata , and days to heading among parent lines and tissue-cultured cultivars. Hence, somaclonal variations are the source of a wide range of modifications among progeny of drought resistance methodology.

Arun et al. (2003) regenerated immature embryos of spring wheat varieties that generated somaclones resistant for spot blotch disease for up to R2, R3, and R4 generations. They have successfully improved wheat varieties employing somaclonal variation for resistance to spot blotch disease and earliness. The resulting progenies have enhanced yield over the parents. Some of the other osmotic agents include sucrose , sorbitol , and mannitol as they provide carbon and energy in the cereal cell culture media (Al-Khayri and Al-Bahrany 2002) and also act as osmotica in explant organogenesis (Huang and Liu 2002). These were accumulated in plant tissues influenced by environmental stress such as water deficit and perform a role of osmoregulation. Javed and Ikram (2008) researched the response of sucrose that induces osmotic stress on callus development of two wheat genotypes. They used the seed for callus induction and propagated the resulting calli in media containing varying amounts of sucrose (control, 3–5%, and 8%). They conferred that increased sucrose concentration in media resulted in osmotic stress, enhancement of free proline, and total soluble carbohydrates .

Rashid et al. (2009) established a rapid protocol for callus production and regeneration and achieved a maximum of 97% callus induction efficacy using 2,4-D and 87% of regeneration frequency by employing different concentrations of IAA , kinetin, and 2iP. Similarly, Mehmood et al. (2013) obtained the highest callus induction frequency of 90% using 2,4-D and maximum regeneration efficiency of 59% in media supplemented with various concentrations of BAP.

3.1.3 In Vitro Embryogenesis and Organogenesis in Wheat

Among the most important food, wheat is the extensively examined in vitro regeneration employing different explant sources including anthers (Cistué et al. 2006), inflorescences (Ozias-Akins and Vasil 1982), shoot tips (Ahmad et al. 2002), immature embryos (He et al. 1992), and isolated microspores (Liu et al. 2002). These explant tissues have shown variable responses to regeneration into the whole plant during tissue culture system.

Many studies reported in vitro embryogenesis in wheat were either direct in which no callus induction phase or indirect with callus formation stage. Delporte et al. (2001) reported the indirect system for wheat embryogenesis via callus induction phase from exploiting thin mature embryo. The sterilized mature embryos were placed on solid medium supplemented with 2,4-D, and 90% callus formation rate was achieved, followed by 47% embryogenic calli induction frequency with the suppressed amount of 2,4-D. However, Mendoza and Kaeppler (2002) established the protocol for the regeneration of mature embryo with modifying and altering the amount of auxin and sugar for callus induction media. They compared the effects of four auxins, viz., 2,4-D, dicamba, picloram, and 2-MCPP, and sucrose versus with maltose. They found that the effect of the sugar type depended upon the kind of auxin used. The 2,4-D and picloram enhanced callus regeneration on media with maltose. A similar study was performed to investigate the role of plant growth regulators and plant hormones on in vitro somatic embryogenesis (Jiménez 2005). They suggested that endogenous plant hormone and exogenously provided growth regulators could provide evidence of divergent patterns occurring during stages of somatic embryogenesis and in vitro tissue culture.

Reported an efficient plant regeneration method employing mature embryos of ten wheat genotypes that analyzed the influence of auxin type on callus development (Nasircilar et al. 2006). They illustrated that the callus obtained from mature embryos was a good source of somatic embryogenesis and later on organogenesis. A study revealed that in vitro regeneration of mature embryo on media supplied with BAP and TDZ increased shoot production per explant (Ganeshan et al. 2006). The shoot production frequency in winter wheat genotypes ranged between 11 and 25 shoots per explant.

More recently plant genetic engineering has provided several options for improvement of cereal crops, particularly wheat. For the genetic transformation of wheat, an efficient tissue culture protocol is required. Therefore, researchers have routinely attempted different protocols with variable inputs. One such attempt reported a vigorous callus formation and regeneration method using mature embryos as the explant source with various hormonal regimes (Raziuddin et al. 2010; Aydin et al. 2011).

The plant tissue culture technique can be opted to produce plantlet tolerant to stress. One of the earliest efforts was made to generate drought resistance. Farshadfar et al. (2012) provided the experiment to identify in vitro predictors of drought resistance in the wheat landraces employing mature embryo for callus induction on media containing 2,4-D and followed by providing drought stress condition. In one of their studies, they provided PEG and mannitol for callus germination for screening drought-resistant plantlets. PEG has high molecular weight and, therefore, is unable to cross membranes and does not change the osmotic potential of the cell. Hence, it activates water scarcity in in vitro cells.

In wheat, immature embryos are the most extensively used explants for culture induction, but sometimes this is problematic because of its temporary accessibility and culture requirements. On the other side, mature embryos are easily available and stored similar to a seed. However, there are some potential studies conducted to provide a protocol for in vitro culture of an immature embryo. Yasmin et al. (2009) established the protocol for initiation of callus and regeneration in the immature embryo supplied with culture media with auxins and cytokinin . Murín et al. (2012) compared the regeneration efficacy of mature and immature embryo of various wheat genotypes in media containing auxin and found no significant correlation between their growths, hence suggesting different protocols for each regeneration step on media.

Einkorn (Triticum monococcum L.) is a recalcitrant diploid wheat, comprised of A-genome, and has a potential to employ as a useful model for understanding the concept of biology and genomics in tribe Triticeae. Miroshnichenko et al. (2017) developed a protocol for direct or indirect somatic embryogenesis and organogenesis and found that daminozide together with auxin remarkably improved establishment of morphogenic structures. They also observed that the maximum shoots per initial explant were obtained on medium containing 0.25 mg/L TDZ, 3.0 mg/L dicamba, and 50.0 mg/L daminozide.

In vitro embryogenesis is also exploited to assess disease resistance in wheat genotypes. In the context, Hafeez et al. (2012) selected three wheat landraces that showed leaf rust resistance and regenerated immature embryos on in vitro culture media supplemented with 2,4-D. Maximum regeneration was observed in LLR-16. Similarly, Soliman and Hendawy (2013) exploited the in vitro embryogenesis for producing drought-tolerant plantlets employing immature embryos as the explant source and providing water-deficit conditions with various concentrations of PEG for durum wheat genotypes. RAPD-PCR diagnostics were carried out with four primer pairs to discriminate the plantlets produced from PEG resistant and control plantlets. They found that durum wheat genotypes were differentiated with amplified DNA capacity.

3.1.4 In Vitro Regeneration and Multiplication in Wheat

Much research has been conducted to establish the most rapid and cost-effective protocol in tissue culture employments, but still, the most efficient method is always obscure. In this context, many methodologies have been reported, and some are in progress of completion.

One of the major applications of tissue culture is the production of multiple plantlets to enhance the yield of elite genotypes and eliminate diseases such as viral infections from the plant genome. Besides these applications, one of the recent uses of tissue culture is a requirement of the most flourished in vitro regeneration protocol for the production of transformed plantlets.

Therefore, Ahmad et al. (2002) reported the study to generate multiple shoots and later on somatic embryo formation on media provided with 2,4-D and BA employing 7-days-old shoot apical meristem of four different wheat genotypes as explant source. They achieved multiple shoots that produced fertile plantlets with viable seeds. Another similar approach was the influence of one of the plant regulators where thidiazuron was investigated on in vitro tissue culture of barley and wheat (Shan et al. 2000). They found vigorous shoot formation from callus derived from immature embryos. The highest regeneration frequency of 87% was achieved in wheat. Similarly, Satyavathi et al. (2004) investigated the effect of picloram, 2,4-D, and dicamba on callus formation and regeneration of durum wheat. They ascertained dicamba as the best plant regulator among them. Haliloglu (2006) provided an efficient growth system of two wheat genotypes exploiting leaf base segments, also investigated factors involved in callus initiation and plantlet regeneration. Remarkably, the highest figure of the somatic embryo was observed on media containing NAA and 2,4-D.

Recently, there has been an increase of heavy metal presence because of rapid industrialization that limits plant growth and cereal grain development and affects the chemical composition (Hart et al. 1998). Likewise, a higher amount of cadmium (Cd) in grains are lethal to human being and animals. Ganeshan et al. (2012) proposed an effective study that provides the uptake of Cd in the developing wheat grain via in vitro culture of the immature wheat spike on medium with varying concentration of cadmium chloride as the source of Cd metal. They successfully provided the evidence of grain development in media supplemented with Cd, accumulation of Cd in grain, and expression analysis of the Cd-related genes such as metallothionein, glutathione reductase, and phytochelatin synthase that have been activated in the tissue-cultured grains. AE Saeed et al. (2015) established an Agrobacterium-mediated transformation protocol for two wheat cultivars employing mature embryos as explant. They regenerated putative explants in MS medium with some modifications, e.g., for shoot initiation, used 2,4-D (4.0 mg/l); for shoot elongation, also added zeatin (1.0 mg/l); and for plantlet maintenance, used 2,4-D, zeatin, and GA₃ each of 1.0 mg/l in MS medium. The resulting transformation efficiency was 20–23% in both cultivars, and further molecular assays verified successful transformation.

3.2 Post-Sequencing Next-Generation Sequencing Technology

Next-generation sequencing (NGS) tends to be an emerging high-throughput technique for certain post-sequencing approaches and development due to its cost-effective and efficient nature. More recently, the wheat community (IWGSC 2014) has generated complete hexaploid wheat genome (T. aestivum) after sequencing diploid donors of A (T. urartu) and D (Aegilops tauschii) genomes by Ling et al. (2013) and Jia et al. (2013), respectively. Jia et al. (2017) reviewed some of the progress achieved on wheat genomics with assistance of NGS approach which included genomic polymorphism , sequencing of hexaploid wheat whole genome and its donor species, cloning of agronomical significant genes, development of wheat SNP microarrays, dynamics in wheat transcriptomes , and genotyping by sequencing (genome-wide association studies). In this chapter, the use of NGS in identifying certain microsatellite markers and miRNA from wheat has been reviewed.

3.2.1 Microsatellite Markers

Certain conventional methods currently in use for the identification of microsatellites are not so efficient according to the current research pace in the field, and these methods tend to be costly and slow, taking much longer for the identification process. These methods mainly include expressed sequence tag (EST) sequencing and mining of microsatellite libraries. However, Imelfort et al. (2009) demonstrated the promising nature of NGS technology for the purpose of marker development mainly due to being much economical, less time-consuming, impartial, and high throughput in nature. A large amount of work has been done on microsatellite markers in wheat due to their importance and high yield demand. However, no microsatellite markers specific to a single arm of chromosome were developed with the use of next-generation sequencing (NGS) technology until the foremost study regarding identification of microsatellite marker on specific chromosome arms based on the new and advanced technology of NGS was carried out by Nie et al. (2012) on the long arm of chromosome 7D (7DL) of wheat generating Illumina paired-read survey sequence of 7DL. Nie et al. (2012) demonstrated the efficacy of the newly identified chromosome arm-specific markers in various mapping and cloning outcomes such as their assistance in genetic mapping and saturation and positional cloning. On this chromosome, they assembled a sum of 1,61,061 contigs within which they identified 16,315 microsatellites and selected 33 markers, on a random basis, for validation in 20 cultivars of wheat with diversified genomes. For further validation regarding the specific nature of these markers developed by the research group, among the stock, they also screened two nulli-tetrasomic stocks.

In detecting the amplification of microsatellite markers, the primers used by Nie et al. (2012) exhibited a success rate of 79% which is quite high in contrast to 32% in a study carried out by Bryan et al. (1997), 36% in the case of Röder et al. (1995), and 68% with the experiments conducted by Nicot et al. (2004) suggesting detection of higher polymorphic activity using NGS technology than the conventional technologies. Thus the use of NGS technology in survey sequencing proves itself in all aspects to be an optimal approach for microsatellite marker development.

3.2.2 MicroRNA

MicroRNAs (miRNAs) are short, single-stranded, and noncoding RNAs made up of nearly 21 nucleotides. These are a highly conserved entity within the cell of all plant species and play a diverse role in many biochemical processes such as signal transduction, biological and physical stresses, growth and development, environmental adaptation, and protein processing along with their biogenesis. More recently miRNAs have been predicted within wheat genome on chromosomes 1, 6, and 5 of genomes A (long arm), B, and D, respectively (Lucas and Budak 2012; Tanaka et al. 2013; Kurtoglu et al. 2013).

3.2.3 MicroRNAs on Wheat Chromosome 5D

According to one study, out of 48 miRNAs, 36 are predicted to be located on long arm of chromosome 5D and 24 of 42 in short arm of chromosome 5D similarly present in the chromosome 4A of wheat (Kantar et al. 2012). Kurtoglu et al. (2013) used NGS to discover conserved miRNAs systematically for the very first time in wheat chromosome 5 of genome D and identified 55 putative miRNAs inclusive of 14 novels once reported for the first time in wheat. Here 13 miRNA of 55 are located on long arm of chromosome 5D, while 7 are present on the short arm of chromosome 5D. However, the remaining is present on both arms. It is notable that of the total read, long arm of wheat chromosome 5D has shown higher variety than that of the short arm of 5D.

Of these 55 miRNAs, 6 resulted in EST hits via in silico expression analysis after prediction of potential targets. They found three 5D-specific miRNAs in the process of verifying five miRNAs for their presence on chromosome 5D. This study also detailed the expression of one miRNA, miR2118, based on experimental assays. These findings of Kurtoglu and his colleagues are major contributions in wheat miRNA research. The novel 14 putative miRNAs on 5 chromosomes of D genome have been identified, are predicted to be involved in experimentally confirmed targets, and have role in many biological or metabolic functions with different percentages as a whole including binding activity of transcription factors with nucleic acid (miR5205; TC413453), hydrolase enzyme activity (miR3700; TC412324), oxidoreductase enzyme activity (miR482; CO348589), transferase enzyme activity (miR5568; TC446402, TC395950), and binding activity to metal ion (miR6197; AL821953) and respond to stress stimuli such as drought (miR5387; BE637541). Kurtoglu with his colleagues also identified that miR5387, miR6197/miR1118, miR1117/miR437, and miR1133 located on chromosome 5D targeted certain drought-responsive proteins: late embryogenesis abundant (LEA) protein, HVA22, aquaporin, and calmodulin-like protein, respectively.

Similarly, Kantar et al. (2011) identified the presence of certain miRNAs on wheat chromosome 5D including miR160, miR167, miR169, miR1125, and miR398 which were earlier demonstrated to be involved in responding to the drought stress independently in Arabidopsis and rice by Liu et al. (2008) and Zhou et al. (2010). Such finding will efficiently contribute to diverse miRNA functions located on wheat chromosomes, and many of similar research are on their way to recognition.

3.3 Identification of Genes and Transcription Factors for Physical Stresses

Purification and characterization of some genes that respond to drought, heat stress, and saline conditions in wheat carried out previously include TNHX1, TVP1 (Brini et al. 2005), TaERF3 (Zhang et al. 2007), SNF1-type S/T protein kinase (Mao et al. 2010), and TaAIDFa (Xu et al. 2008). However, the abiotic stress response of MYB3R proteins was first cloned in wheat by Cai et al. (2011) who isolated TaMYB3R1, a novel transcription factor of the family from bread wheat, performed sequence analysis by constructing alignments, and further performed phylogenetic studies through tree construction. They also performed a transient expression assay in the epidermal cells of onion for elucidating the localization of TaMYB3R1 at the subcellular level, DNA-binding activity, and trans-activation activity via yeast two-hybrid (Y2H). Cai et al. (2011) primarily focused on demonstrating the response of this MYB3R protein to certain stresses under various abiotic conditions including drought, salinity, and low temperature.

Rahaie et al. (2010) performed expression profiling of ten MYB genes in bread wheat against salinity to observe their response in recombinant inbred lines comprising of different levels of resistance to drought and salinity. Among the MYB genes involved in salinity and drought stress characterized by the research group, only one gene TaMYBsdu1 was evaluated to have been upregulated and expressed to a great extent in saline and drought conditions in a salt-tolerant line. Bartels and Sunkar (2005) demonstrated the involvement of certain genes in cell damage protection including the genes that code for reactive oxygen species (ROS) removal, osmolyte synthesis, and dehydrins.

In 2015, Kumar et al. sequenced the whole transcriptome under controlled (22° ± 3 °C) and heat stress (42 °C for 2 h) conditions at the flowering stage of wheat using two platforms, Illumina HiSeq and Roche GS-FLX 454. Their study revealed that under heat-treated condition, out of 1525 transcripts, 27 of them showed very high (>ten-fold) upregulation of gene expression. Moreover, metabolic processes, such as oxidation-reduction, protein phosphorylation, and others, were greatly influenced by heat stress.

Transcriptional regulation plays an important role in the expression of these genes as a coping mechanism against stressful environments created as a result of high salt and low water levels (Zhu 2002). Major transcription factor families that have been reported to respond to abiotic stress conditions include WRKY (Mare et al. 2004), C2H2 zinc finger (Kam et al. 2008), AP2 (Sakuma et al. 2006), basic domain-leucine zipper (bZIP; Uno et al. 2000), NAC (Xue et al. 2006), ethylene-responsive element-binding factors (ERF), and MYB (Jin and Martin 1999; Stracke et al. 2001). Riechmann et al. (2000) and Stracke et al. (2001) characterized the MYB transcription factor (TF) family constituting the MYB DNA-binding domain (MYB domain) of approximately 51–53 amino acids as being among the largest TF families in plants. Jin and Martin (1999) and Rosinski and Atchley (1998) demonstrated that this TF family could be divided into three subfamilies on the basis of the presence of a number of adjacent repeats in the characterizing domain: those with a single repeat in the MYB domain as MYB-1R, two repeat proteins as R2R3MYB, and three repeat proteins as MYB3R (Chen et al. 2005) have similar findings. Dubos et al. (2010) also identified some four repeats containing MYB proteins (MYB4R) in plants, but their detailed functionality in plants is yet to be demonstrated. The largest subfamily in MYB proteins is the R2R3MYB with a major role in responding to biotic and abiotic stress studied extensively in various studies by Abe et al. (2003), Agarwal et al. (2006), Cominelli et al. (2005), Maeda et al. (2005), Mengiste et al. (2003), Miyake et al. (2003), Seo et al. (2009), and Vannini et al. (2004). Although this family comprises of a large number of members in plants with quite divergent functions, drought- and salinity-related stress responsiveness among them has been studied to a very limited extent (Abe et al. 2003).

3.3.1 Drought-Related Genes

Sial et al. (2009) demonstrated the improvement in crop yields under water-deficient conditions, which can be attained by improved germplasm of crops developed to be well suited for conditions of lower water availability.

Sial et al. (2012) screened out the capabilities of a variety of wheat genotypes to tolerate drought stress by reviewing the grain yield in accordance with water availability and development of certain genotypes more capable of higher yield in the more stressed environment. These genotypes can be compared, keeping in view their yield, and can be utilized for developing drought-resistant wheat varieties. The higher the allele number and its stability of expression in the evolved genotype, the better the wheat variety gets in tolerance to drought conditions and performs normally to increase comparative yield. Zhang et al. (2012) reported MYB gene TaPIMP1 in wheat expressed through overexpression and underexpression and regulated by ABA (abscisic acid) and salicylic acid (SA) .

Budak et al. (2013) recently reviewed the drought stress-related genes and QTLs in cultivated (bread and durum) and wild (emmer) wheat over the last 3 years. These include TaPIMP1, TaSRG, TaMYB3R1, TaNAC (NAM/ATAF/CUC), TaMYB33, TaWRKY2, TaWRKY19, TdicDRF1, TaABC1, TaSnRK2.4, TaSnRK2.7, TdTMKP1, TaCHP, TaCP, TaEXPR23, TaL5, TdPIP1;1, TdPIP1;2, TdicATG8, TdicTMPIT1, Era1, and Sal1 (Fig. 4). The role of dehydrin genes (dhn, wcor, and dreb) in two bread wheat cultivars for tolerance in response to drought stress was observed by Hassan et al. (2015). They showed a striking finding of high induction expression of these genes in the leaves of one of the cultivars under water-deficit condition.

Fig. 4

Different genes and miRNAs activated under drought condition in wheat

3.3.2 Drought-Related miRNA Targets by NGS

MicroRNAs miR169, miR5085, miR6220, and miR2118 also get expressed in wheat tissues in certain environmental stress conditions at different phases during development. The drought- and frost-resistant proteins targeted by certain miRNAs have been represented in Table 2. Han et al. (2013) demonstrated various miRNAs in bread wheat and their target proteins by EST analysis. Some of those involved in certain stress responses are shown in Table 3. miRNA microarray approach has been utilized in order to screen root and leaf of bread wheat cultivar for identification of drought stress-related miRNAs (Akdogan et al. 2015). The results revealed 285 and 244 miRNAs in leaf and root tissues, respectively, among them the expression of miR169, miR172, miR159, miR160, miR166, miR395, miR1858, miR2118, miR396, miR408, miR472, miR477, miR482, and miR5049, differentiated significantly in bread wheat. Moreover, Bakhshi et al. (2017) identified 1813 miRNA belonging to 106 families, 104 of which predicted to be similar to 212 novel miRNA precursors.

Table 2 The miRNAs and their target proteins involved in drought and frost resistance

Table 3 The miRNA and their targeted proteins with various functions (Han et al. 2013)

3.3.3 Drought-Tolerant Cultivars in Pakistan

Raj is a wheat cultivar tolerant to certain biotic (leaf and yellow rust) and abiotic stresses (mainly drought tolerance) released to be cultivated in rainfed areas of KPK Pakistan (Khan and Khan 2010). Tijaban-10, a wheat variety, tolerant to biotic (yellow rust) and abiotic stresses (mainly drought but also low temperature), developed mainly in the rainfed areas of Baluchistan, Pakistan (Khan et al. 2013). These two major drought-tolerant varieties that have gone through several phases of testing and their main characteristics are summarized in Table 4.

Table 4 Characteristics of drought-tolerant wheat varieties

3.3.4 Transcription Factors in Wheat Plant Response to Aluminum Metal Toxicity

In the acidic soil, Al metal toxicity is one of the major constraints affecting plant yield. Signal transduction pathways conduct the signals within the cell and throughout the plant, and then as a result, gene expression and different cellular functions occur. Al stress leads to cascade pathways. The concentration of calcium ions Ca²⁺ may be affected by the Al toxicity. It increases the Ca ion concentration in the root apex.

Protein phosphorylation has a vital role in the pathway of signal transduction and in the regulation of many biological activities for the mediation of external stimuli into the cells. The major pathway for the signal transmission is the mitogen-activated protein kinase (MAPK) cascade. These pathway signals transduct the signals for light, temperature, nutrient deficiency, and all stress, etc. in yeast, MAP kinase plays a role in Al tolerance. In wheat, it is estimated that the MAP kinase plays a role in Al resistance because in root apex MAP kinase transduces the Al signal and its expressions (Mossor-Pietraszewska 2001).

Al tolerance genes are studied extensively in cereal crops, and the major emphasis is on the wheat. For the manipulation of genes, it is known in which plant and on which chromosome Al-tolerant genes are present (Reynolds et al. 2001). Most of the Al-tolerant genes are the general stress tolerance genes which increase Al tolerance. Al tolerance genetic variation is not only found between the species but also within the species. Major genes are present on the short arm of the 5A chromosome and long arms of 2D and 4D chromosomes in hexaploid wheat. In the wheat plant, root apex is the main site for the accumulation of Al. Ca uptake is affected by the Al toxicity. With the increase in Al, Ca concentration decreases in root and shoot of the plant (Mossor-Pietraszewska 2001).

Al resistance is not controlled by a single gene but is a multigenic trait. On chromosome , 4DL is a locus which controls most of the phenotypic variation as well as the release of malate which is the major Al-activated anion from the root apex. By chelating the Al and making it nontoxic, malate anion protects the root apex. The TaALMT1 gene encodes an anion channel called Al-activated malate transporter . In the promoter region, large tandem repeat sequences are present which increase the expression of TaALMT1. It has been proposed that these tandem repeats enhance the expression of the gene because these sequences have enhancer elements. Thus, by increasing the TaALMT1 expression, Al resistance can be increased (Pereira et al. 2010).

3.3.5 Transcription Factors in Wheat Response to Low Potassium

To improve the wheat tolerance to low-K stress in the transgenic plant, an Elymus dahuricus H⁺-PPase (EdVP1) gene is used. It is known that indole acetic acid (IAA) plays a key role in the root development. EdVP1 gene increases the IAA concentration in the root under low-K stress condition. EdVP1 also increases the concentration of H⁺ and root cation exchange capacity, which increases the ability of K accumulation in EdVP1 transgenic wheat. Transgenic wheat also releases more organic acids which help in the activation of more soil K, under low-K condition. The EdVP1 gene increases the ability of wheat to absorb and utilize K; thus this enhances the grain yield of the wheat (Ruan et al. 2013). The transcription factors and families responsible for regulating the metal toxicity as compared to drought in the wheat crop are listed in Fig. 5.

Fig. 5

The transcription factors and families responsible for regulating genes under the state of metal toxicity and drought

3.4 Genetic Engineering Approaches to Biological Stresses

To overcome these challenges, many approaches have been used including molecular breeding and developing tolerance by use of the D genome wheat synthetic hexaploids and genetic engineering approaches. Wheat is among the last major cereal crops that have been genetically engineered due to its reduced transformation efficiency. However successful wheat transformation studies have been published in the 1990s. Since then scientists have been using Agrobacterium as well as biolistic transformation protocols to develop transgenic wheat.

3.4.1 Wheat Transformation with Genes Resistant to Fungal Diseases

Earlier studies were conducted on transgenic wheat varieties against a devastating fungal disease powdery mildew caused by Erysiphe graminis. In this study RIP or β-1,3-glucanase genes under the regulation of rice actin 1 promoter and a barley seed chitinase gene II under the regulation of maize ubiquitin 1 promoter were transferred into wheat. The transgene was found to be stably expressing in the transgenic lines and showed increased resistance to powdery mildew infection (Bliffeld et al. 1999). The first study conducted to introduce resistance against scab or Fusarium head blight (FHB) by using genetic engineering techniques was published in 1999 in which the possibilities of engineering resistance were explored by expression of pathogen-related (PR) genes. In this study thaumatin-like protein (tlp) gene and chitinase gene (chi II) from rice were introduced in Bobwhite wheat cultivar by biolistic transformation protocol. The expression and integration of transgenes were analyzed by various techniques. However, no significant expression was observed. The inoculation of conidia of Fusarium graminearum done on the wheat variety showed that the infection rate was significantly slower in transgenic plants as compared to non-transgenic ones (Chen et al. 1999).

KP4, an antifungal protein from Ustilago maydis (corn smut), was genetically engineered to wheat to develop resistance against the stinking smut (Tilletia tritici). The transgene regulated by ubiquitin promoter was found to be stable over generations. Out of seven, three transgenic lines showed endogenous resistance against stinking smut disease (Clausen et al. 2000). Slight resistance against powdery mildew was achieved by biolistic transformation of stilbene synthase gene (vst 1) obtained from grapevine in T₃ generation of spring wheat Jinghong 5 (Hui et al. 2000). Moderate resistance against powdery mildew was developed by transforming wheat with barley seed ribosome-inactivating protein (RIP) regulated by cauliflower mosaic virus 35S promoter. It was found to be stable over four generations. A signal peptide derived from barley seed, that is, β-1,3-glucanase, is also engineered which has increased the antifungal properties of RIP (Bieri et al. 2000).

Another study of biolistic transformation was done in which resistance against powdery mildew and leaf rust (Puccinia recondita) was developed by transferring antifungal protein Ag-AFP from Aspergillus giganteus, chitinase gene from barley class II, and RIPs from barley type I under the control of ubiquitin 1 promoter. A significant decrease of about 40–50% in powdery mildew and leaf rust colonies was seen from afp and chitinase II at an inoculum density of 80–100 spores per cm³. However, disease resistance by the activity of RIP-I expression was not observed (Oldach et al. 2001). A study to develop scab (FHB)-resistant wheat has been done to reduce the effect of DON (deoxynivalenol) that poses a health hazard to humans and animals. An FsTR1101 gene from Fusarium sporotrichioides has been integrated in Bobwhite cultivar of wheat using biolistic transformation technique. FSTR1101 encodes trichothecene acetyltransferase enzyme that limits FHB pathogen’s hyphal spread in wheat. The expression of FSTR1101 was mostly in endosperm and glume. Greenhouse analysis of transgenics showed an accumulation of acetyltransferase in wheat that gives partial resistance against scab disease (Okubara et al. 2002).

Fusarium graminearum resistance in susceptible spring wheat Bobwhite has been developed by using biolistic transformation method. Pathogen-related (PR) proteins were isolated from Fusarium graminearum-resistant cultivar Sumai 3. Transgene expression was observed in four wheat lines. Among them, one wheat line showed co-expression of chitinase and β-1,3-glucanase genes. When assayed against scab infection under optimum greenhouse conditions, a delay in the spread of infection was observed. A second transgenic wheat line expressing rice thaumatin-like protein gene (tlp) showed moderated resistance to scab infection in greenhouse conditions. However, none of the lines were resistant to scab infection under field conditions (Anand et al. 2003). Another study has been done to increase the resistance of a fungal pathogen Blumeria graminis that causes powdery mildew diseases. Three gene constructs were designed for this experiment: plasmid A containing barley seed β-1,3-glucanase and chitinase coding sequences, plasmid B containing ribosome-inactivating protein (RIP) expressed with β-1,3-glucanase signal peptide, and plasmid C including signal peptides barnase and barstar. Most significant resistance was obtained from RIP expression. Barnase and barstar were less efficient, while lines containing chitinase and β-1,3-glucanase varied from highly resistant to highly susceptible. Combination of all three constructs did not show any resistance against powdery mildew (Bieri et al. 2003).

Resistance against fungal disease Puccinia recondita was developed by the expression of phytoalexins vst1 and vst2 obtained from grapevine (Vitis vinifera) involved in resveratrol synthase and pss gene from pine (Pinus sylvestris) involved in pinosylvan synthase were transformed into the bread wheat. This resulted in the accumulation of stilbene derivative when exposed to UV light and showed a significant reduction of disease ranging from 19 ± 9% to 27 ± 8% as compared to wild types (Serazetdinova et al. 2005). In another study published in 2005, enhanced resistance against powdery mildew disease has been achieved by integrating two defense-related genes oxalate-oxidase 9f-2.8 and TaPERO peroxidase under the control of Gst AI promoter in combination with WIR1a intron. Biolistic bombardment was used for developing transgenic wheat. The expression of defense-related genes is epidermis-specific. The results showed that TaPERO peroxidase gene had enhanced resistance against powdery mildew, while oxalate-oxidase overexpression did not have any effect (Altpeter et al. 2005).

In another study resistance against Fusarium head blight or scab has been developed by Agrobacterium-mediated transformation of Chinese cv. Sumai 3 wheat with Arabidopsis thaliana AtNPR-1 gene which develops a type II resistance to FHB by elevating levels of salicylic acid (SA) pathogenesis-related (PR) genes that cause the expression of antimicrobial proteins, thus regulating systemic acquired resistance in a FHB-susceptible wheat cultivar (Makandar et al. 2006). In 2006 the hexaploid wheat genome was genetically engineered to express the antimicrobial protein Ace-ANP1 obtained from Allium cepa (onion) against wheat fungal diseases like Karnal bunt (Neovossia indica) and powdery mildew. The transgene was controlled by the maize ubiquitin promoter and Bar gene on the medium of phosphinothricin (PPT) as a selectable marker. The expression of Ace-AMP1 was confirmed over two generations. About 50% increase in resistance was observed against powdery mildew disease, while the increase in expression of defense-related genes was observed in Karnal bunt-infected wheat plants (Roy-Barman et al. 2006).

Agrobacterium-mediated transformation of the basal portion of wheat seedlings was done with β-1,-3-glucanase gene to develop resistance against powdery mildew. A transformation efficiency of 9.82% was observed. The transgene was stable in the following generations. Study of T₂ generation showed high resistance against powdery mildew as compared to controls showing that the transgene could be efficiently expressed in wheat seedlings when transformed with Agrobacterium tumefaciens (Zhao et al. 2006). FHB resistance in wheat was developed by integrating a maize gene b-32 that encodes an RIP (ribosome-inactivating protein) by biolistic transformation and regulated by 35S CaMV promoter with the bar gene as a selectable marker. The transgene was stably expressed, and in all transgenic lines, reduction in FHB symptoms was observed from about 20% to 30% as compared to non-transgenic lines (Balconi et al. 2006).

Most of the work using genetic engineering techniques in wheat fungal diseases has been done to develop resistance against scab or FHB. In this study, defense-related genes have been overexpressed, and their effect on FHB-infected wheat under greenhouse and field conditions has been studied. These defense-related genes include α-1-purothionin, thaumatin-like protein 1 (tlp 1), and β-1,-3-glucanase. The wheat variety Bobwhite has been used for checking the expression. Results of this study showed that defense-related genes particularly β-1,-3-glucanase had enhanced resistance against FHB in both greenhouse and field conditions (Mackintosh et al. 2007). A similar study was conducted by developing resistance against FHB by transforming Bobwhite wheat variety with barley class II chitinase gene using biolistic bombardment, which showed that barley class II chitinase exhibited resistance against FHB in greenhouse and field conditions (Shin et al. 2008).

Studies on plant’s natural defense-related mechanism have also been done. One of these mechanisms includes plant’s glycoproteins PGIP (polygalacturonase-inhibiting proteins) that inhibit the activity of fungal endopolygalacturonases (endo-PGs). To access their effectiveness, transgenic wheat lines expressing bean’s PGIP genes have been developed. Results showed that transgenic wheat having PvPGIP2 accumulation has new PG recognition capabilities and showed increased resistance against Bipolaris sorokiniana. About 46–50% reductions in symptoms of fungal pathogens were observed (Janni et al. 2008). Transgenic tetraploid wheat (AABB genome) resistant against leaf rust has been developed. In this study puroindoline gene a (pin) that encodes puroindoline protein A (PINA) under the control of maize ubiquitin promoter, along with nptII gene (neomycin phosphotransferase II) used as a selectable marker, was transformed by biolistic bombardment using gold particles. These proteins located on chromosome 5D are known to have antibacterial and antifungal properties as well as control the grain hardness of wheat. Comparative study of transgenic and non-transgenic wheat varieties was done which showed that PINA protein significantly lowered the growth of P. triticina in vivo (Luo et al. 2008).

In another study an antibody fusion protein consisting of a Fusarium-specific recombinant antibody obtained from chicken and an antifungal peptide obtained from Aspergillus giganteus was transferred in wheat via biolistic bombardment. A significant resistance against types I and II scab diseases (FHB) was observed in T₂ and T₃ generations. This also significantly increased the yield of wheat cultivars demonstrating antibody fusion proteins were effective for scab disease (Li et al. 2008). Resistance against sharp eyespot (Rhizoctonia cerealis) is achieved by integrating an ethylene response factor (ERF) gene TiERF1 obtained from Thinopyrum intermedium via biolistic bombardment. TiERF1 was found to be stably expressed even in T₄ generation. TiEFR1 was found to be increasing the expression of PR (pathogenesis-related) genes such as chitinase and β-1,-3-glucanase which enhanced the resistance against sharp eyespot in transgenic wheat variety (Chen et al. 2008). A similar study was conducted for developing resistance against common root rot (B. sorokiniana) in which an ERF gene, TaPIEPI, was integrated in wheat variety using Yangmai 12 biolistic bombardment. The transgenic wheat was found to be stably expressing, conferring enhanced resistance, and was showing accumulation of defense-related genes, e.g., ethylene (ET) and jasmonic acid (JA), which may be useful in crop improvement (Dong et al. 2010).

Transgenic lines resistant to wheat fungal pathogens B. sorokiniana (spot blotch) and F. graminearum (FHB) were developed by modifying the cell wall pectin methyl esterification. PMEI gene which controls pectin methyl esterification has been obtained from Actinidia chinensis (i.e., AcPMEI). High expression of pectin methyl esterases (PME) makes a plant’s cell wall less susceptible to hydrolysis of fungal endopolygalacturonases (PG). The transgenic lines showed significant increase in PME activity as a result of which high resistance against spot blotch and FHB is seen (Volpi et al. 2011). Resistance to scab (FHB) disease and sharp eyespot (Rhizoctonia cerealis) is done by inserting a defensin gene RsAFP2 obtained from Radish (Raphanus sativus). RsAFP2 is a small cysteine-rich protein gene that exhibits antifungal properties.

Chinese wheat cultivar Yangmai 12 with RsAFP2 gene was transformed by biolistic bombardment. Four RsAFP2 gene integrated lines showed enhanced resistance to FHB, while two lines showed resistance to Rhizoctonia cerealis (Li et al. 2011). TaWRKY 45 is a wheat transcription factor that shows antifungal properties, so resistance against powdery mildew and leaf rust (Puccinia triticina) was obtained by transforming wheat with TaWRKY45 gene. Its constitutive overexpression was found to be enhancing resistance against both fungal pathogens but different from the expression of Pm 3 and Lr34 defense-related genes (Bahrini et al. 2011). Snakin-1 (SN1) gene obtained from Solanum chacoense is known to show antifungal properties. SN1 gene showed high properties. SN1 gene showed high resistance against wheat powdery mildew disease when transformed in a wild-type variety ProINTA Federal (Faccio et al. 2011). R genes specifically Pm 3 resistance locus was engineered in wheat lines to develop resistance against powdery mildew. Pm3 wheat multilines including Pm3a, Pm3c, Pm3d, Pm3f, or Pm3g were developed and found to be more resistant than non-transformed wheat lines (Brunner et al. 2011).

Multiple studies were done on scab (FHB) in 2012. Scab disease-resistant transgenic wheat lines have been developed by incorporating PvPGIP2 (polygalacturonase-inhibiting proteins) gene in wheat’s floral tissues (Ferrari et al. 2012). A bovine lactoferrin cDNA known to have antimicrobial properties in wheat was transferred in Bobwhite cultivar which showed high resistance against scab disease (Han et al. 2012). Similarly, resistance to FHB and Fusarium seedling blight (FSB) has been achieved by developing transgenic lines expressing two antifungal peptides (AFP) in Yangami 11 wheat cultivar (Liu et al. 2012). Puroindolines PINA and PINB (PINs) have been studied to provide enhanced resistance against Penicillium seed rot in wheat. Transgenic wheat seeds having high of PINA showed significant fungal growth reduction, while expression of both PINs didn’t have any effect on pathogen (Kim et al. 2012). Lr34 naturally shows resistance against wheat leaf rust (Puccinia triticina). Transgenic wheat lines with Lr34 gene were inserted to develop enhanced resistance. The results showed that Lr34 transgene expression did not regulate PR genes (pathogenesis-related genes). Only one Lr34-based transgenic line showed leaf rust resistance in the seedling stage (Risk et al. 2012).

Resistance against Cochliobolus sativus and F. graminearum was achieved by transferring lipid protein gene TaLTP5 by particle bombardment in wheat variety Yangami 18 (Zhu et al. 2012). Five winter wheat cultivars were particle bombarded with rice class I chitinase gene RC24 to develop resistance against stripe rust (P. striiformis). The transgene was stably integrated in T2 and T3 generations and found to be resistant to stripe rust (Huang et al. 2013). Lastly, resistance against taking all diseases (Gaeumannomyces graminis) was established by biolistic transformation. Integration of snakin-1 (SN1) gene was obtained from Solanum tuberosum. All five transgenic lines showed resistance against all mentioned diseases (Rong et al. 2013). Genes associated to confer resistance against fungal diseases like Fusarium head blight , eyespot, powdery mildew, and leaf rust are figured out for better understanding (Fig. 6). Eissa et al. (2017) successfully developed transgenic wheat to combat rust and powdery mildew fungal diseases by harboring barley chitinase (chi26) gene using biolistic bombardment.

Fig. 6

Some of the transgenes responsible for conferring resistance against fungal diseases in wheat; RsAFP2 and Ag-AFP genes are transformed in wheat for resistance to eyespot and fusarium head blight and leaf rust and powdery mildew, respectively

Hence, many studies have been done to develop resistance against fungal diseases by genetic engineering. These techniques can be successfully applied in improving wheat yield and productivity. In vitro growth of F. graminearum has also been reduced by seed defensin MsDef1 obtained from Medicago sativa (alfalfa). Hence transgenic wheat, with integrated MsDef1, can be used to obtain resistance against scab (FHB) disease (Spelbrink et al. 2004).

3.4.2 Wheat Crop Protection Against Viruses

Transformation of a plant with foreign genes does not disturb the plant genome but makes the plant accept the foreign sequence and express it. This technique has helped to make plants more immune to a variety of plant diseases that cause devastating effects on crops and prevent the virus from spreading throughout the plant by interfering with its replication (Agrios 2005). To minimize crop losses, transgenics are playing an important role in making tolerant varieties within high-temperature areas (Fahim et al. 2010).

Many practices have been explored to create virus-resistant plants that include: (1) Creating plants with natural resistant genes that help in virus resistance are extracted and transferred to another plant and (2) Pathogen-derived resistant (genes from viral sequences) (Sanford and Johnston 1985) involve blockage of molecules or interaction between infecting viruses and the plant. Pathogen-derived resistance involves two basic methods: protein-based protection, where viral gene product/coat protein interferes with the virus in providing resistance to plants or referred to as “coat protein-mediated resistance,” as they have reserved sequences that help to prevent diseases against a variety of strains of the same species, and nucleic acid-based protection, where the interference exists either at a transcriptional stage called transcriptional gene silencing (PGS) or at a posttranscriptional stage called posttranscriptional gene silencing (PTGS).

3.4.2.1 Pathogen-Derived Resistance

It is the most promising technique for providing resistance to crops against viruses and has proven to be successful in crop protection. Crops can be transformed by the virus to resist against virus vectors. Insect vectors play an important role in the transmission of viruses; thus controlling insect vectors can also broaden the ways of controlling some viral diseases. Serological testing can also be helpful to make sure the mother-plant is virus-free. Another way of improving the crop immunity against viral diseases is by crossing it with crops possessing resistance against viruses.

Scientists have adopted the method of cross-protection which allows the inoculation of crop plants with a mild strain of the same virus that helps in protection by severe strains of the same virus. A recent method for protection involves viral control using coat protein or another segment of the genome. This method is called pathogen-derived resistance that blocks the virus activity and its interaction with the plant. Viral gene silencing is an approach used in pathogen-derived resistance, where a homology inhibitory RNA sequence silences the virus gene and prevents it from causing disease. It is also considered that the plant defense system comprises of gene silencing against foreign material; therefore, to cause infection, virus needs to overcome this barrier (Agrios 2005).

3.4.2.1.1 Resistance Against Wheat Streak Mosaic Virus (WSMV)

Coat protein of WSMV is also used to make wheat resistant against the virus, by making CP gene constructs for transformation: pESCP45, pESCP35, and pRQ105 that showed resistance against Potyviruses (Smith et al. 1994) with 0.3–0.7% efficiency rates. Two viral transgenes from “Conrad-MT” isolate of WSMV were WSMV-CP and WSMV-NIb genes which are involved in providing resistance. But, resistance due to CP gene provides longer protection than replicase gene resistance (Jones et al. 2005). RNAi plays an important role in inhibiting the virus to cause a disease (Waterhouse et al. 1998), where dsRNA, or hpRNA, is sliced into siRNA by an enzyme Dicer (Tougou et al. 2006) and further incorporated into RNA-induced silencing complex (RISC) to find its complementary strand for degradation and to provide resistance to the crop (Campbell and Choy 2005).

Transformed wheat showed immunity against the WSMV and segregates in a Mendelian manner. WSMV can transmit through seed (Jones et al. 2005) or by the vector Aceria tosichella – the wheat curl mite – but the use of RNAi construct helps to achieve immunity in wheat even against vector transmission. Recently, a new strategy is adapted to create immunity in wheat against WSMV using applicable intron of hpRNA (Fahim et al. 2010) that targets nuclear inclusion protein a (NIa) gene of WSMV. The genome of WSMV undergoes co- and post-translation modification by three genes that cleave the protein including P1, HC-Pro, and NIa. Sivamani et al. (2002) reported that wheat resistance against WSMV with the inoculation of NIb and CP sense transgene, in comparison to dsRNA, provides more resistance against the virus (Waterhouse et al. 1998).

For wheat transformation hpRNA was created using an amplified clone of NIa gene of WSMV in vector pSTARGATE with a dehydrogenase kinase (pdk) intron and a polyubiquitin promoter. This method showed 3.5% efficiency when 16 independent T₀ transgenic wheat plants were transformed with hairpin. Furthermore, the transgenic cultivar showed Mendelian segregation, when resistance provided against WSMV “Conrad, MT” through isolated gene of WSMV-NIb gene by posttranscriptional gene silencing or by the expression of CP gene from “Conrad, MT” (Sivamani et al. 2000). Growth of transgenic wheat transformed against WSMV with WSMV-NIb gene showed disease-free symptoms (Sivamani et al. 2000) where CP gene of Potyviruses also showed the same results (Smith et al. 1994).

In some cases, wheat can be protected by the pre-resistance state by inoculation and degradation of viral RNA (Sivamani et al. 2000). F2 generations with intensive RNA expression from WSMV were intensively resistant. Thus, RNA degradation helps in resistance by activating the suppression mechanism (Waterhouse et al. 1998). As a result, transgenic crop with the transgene RNA has shown high resistance because of a large number of copies that resulted in transcriptional gene silencing.

3.4.2.1.2 Resistance Against Barley Yellow Striate Mosaic Virus (BYDV)

Resistant wheat lines against BYDV have been successfully produced. The resistant gene source in the host plant is located in Bdv1 gene which is linked to Lr34/Yr18 in the same chromosome; however, this resistant gene was considered ineffective in China (McIntosh 1998). Though a number of translocation wheat lines were produced, using Agropyron species was considered as more desirable for resistance to BYDV (Sharma et al. 1995) involving 7Ag chromosome exchanges with wheat chromosome 7D. Another resistant wheat line with 2Ag and 7Ag chromosomes was also produced (Sharma et al. 1995). The benefit of producing such resistant lines was to monitor virus attack serologically.

Resistance against BYDV in wheat exists in Thinopyrum intermedium that has been used for transformation of wheat with BYDV-PAV isolate and genotyping by a marker that reduced the infection against the particular strain. To create resistance against BYDV, antisense coat protein sequences are inserted using biolistic methods that showed positive results (Ma 2009). A wheat progenitor grass species, Th. intermedium, harbors genes for resistance against BYDV infection (Sharma et al. 1995). In wheat/Th. intermedium BYDV resistance genes were present in the long and short arm of a chromosome . In the wheat line PP9-1, a single gene showed resistance against BYDV that was crossed with different varieties to produce stable resistance. The wheat lines Yw642, Yw443, and Yw243 WITH resistant gene present in chromosome 7 of Th. intermedium resistant against BYDV serotypes are GPV and GAV. This wheat species in next-generation co-segregated with BYDV resistance (Xin et al. 2001). Spliceable intron in hpRNA has been shown to induce RNAi efficiently (Smith et al. 2000), against Barley yellow dwarf virus in wheat (Wang et al. 2000).

3.4.2.2 Wheat Crop Protection Using Marker-Assisted Selection

Wheat diseases caused by the virus are controlled by establishing tolerant or resistant cultivars. Genes for tolerance or resistance are taken from the infected ones; some cultivars are engineered with the resistant gene against the diseases to show resistance (http://www.apsnet.org/edcenter/intropp/lessons/viruses/Pages/BarleyYelDwarf.aspx). Disease leading to crop losses is a challenge for breeders; therefore, crop highly affected by the disease, the methods for control, and the source of resistance availability and durability is taken under consideration for disease resistance. Marker-assisted selection (MAS) helps in selecting desired makers for molecular breeding (Miedaner and Korzun 2012). So far markers for the resistance of diseases have been available for various types of biotic stresses (Koebner 2003). MAS uses about 50 genes in wheat, because of its high precision and its application in the seedling stage and cost-effectiveness. MAS helps in implementing the resistance into breeding crops because of its feasibility to linked markers (Miedaner and Korzun 2012).

Marker-assisted selection (MAS) is used widely in developed countries to help in the conventional breeding of wheat (Gupta et al. 2009). MAS is used for simple traits instead of complex polygenic traits, for instance, to increase the quality of wheat and to improve crop yields. But for wheat yield maximization, marker-assisted recurrent selection (MARS) and genome-wide selection (GWS) can be used (Bernardo and Yu 2007; Heffner et al. 2009). MAS targets varieties with useful traits, but it is important to consider the MAS nature, as it is preferred for traits that have low heritability, are difficult to phenotype, are recessive, and for disease resistance require gene pyramiding (Gupta et al. 2009). Therefore, for improvisation of wheat, simple traits for disease resistance are targeted. Once resistance against a variety of diseases has been achieved, it is easy to improve the grain including protein content, color, texture, hardness, and gluten strength. In contrast, MAS is also targeted to achieve resistance against abiotic stress protection such as metal toxicity, tolerance to salinity, heat, drought, and waterlogging (Gupta et al. 2009).

3.4.3 Wheat Crop Defense Against Insect/Pest Using Resistant Genes

Resistant wheat with 26 genes for resistance located in 5A chromosome (Ohm et al. 1995) was produced. But the emergence of new biotypes of Hessian fly still poses a threat. In 1955, first three wheat genes resistant to Hessian fly were bred into wheat varieties that had been gaining combat to this pest; but by the following years in 2000, the flies had overcome the resistant wheat varieties due to variations, introduced successfully tolerant against Hessian fly since 1986. Williams et al. (2003) mapped the new wheat genes: H31 and H32. As a result of high variability in the fly genome, more resistant genes are now created to defend the cultivars (USDA/Agricultural Research Service 2007).

The primary defense of wheat is through antibiosis genes that prevent the survival of larva, although some cultivars will allow the survival of insects that can develop to overcome host resistance (Foster and Hein 2009). However, Hessian fly is the most successful pest that overcame antibiosis genes H3, H5, and H6 of the host plant. The host plant resistance gene and virulence gene of the insect coevolved; as the plant resistance increases, insect virulence also increases. An elicitor is a resistant gene product of avirulent insect that triggers defense in plants. Whereas, virulent insects do not produce this product and are not recognized by the host; this strategy is used to make the host resistant (Janick and Wiley 2003). To make plants resistant, an avirulent protein (R-gene) from the insect is inserted to trigger plant defenses against particular insects (Dodds and Rathjen 2010). This wheat/Hessian fly interaction is responsible for provoking a response in wheat, resulting in programmed cell death of the fly (Klingler et al. 2009).

For resistance against Hessian fly, cuticle presence in wheat is vital (Kosma et al. 2010). William et al. (2011) found that the larva of Hessian fly produces saliva that acts on the plant and causes permeability in susceptible plants. However, the resistant plant variety starts producing surface waxes with the cuticle maintaining the integrity of its cell wall. The amount of gene expressing waxes and cuticle was verified using PCR giving evidence that the amount of mRNA helped to determine the gene expression responsible for a wax tissue. The plants were further confirmed by using a red dye that produced slight red spots as compared to the susceptible ones that absorbed the dye since no cuticles were present (Kosma et al. 2010). Recently, certain wheat lines have been produced against Hessian flies that act on the gut of the fly, thus decreasing its ability to absorb nutrients and leading to its death. Such wheat produces large amounts of lectin protein by gene Hfr1 and lectin-like protein from gene Hfr-3. Lectin produced is considered to be toxic for the fly (Shukle et al. 2010) that keeps the crop resistant.

R-gene (resistance gene)-mediated resistance is preferred over backcrossing. Insects have the ability to overcome this type of resistance, as it has been seen that introgression of h-genes in wheat against Hessian fly has failed within 10 years (Cambron et al. 2010). The effectiveness of R-gene can be increased using gene pyramiding (Yencho et al. 2000). Integration of multiple R-genes into cultivars proved useful toward resistance against insect pests. However, plants also consist of susceptible genes that can be switched on by the insects to prevent defense against them (Walling 2008; Giordanengo et al. 2010). Plant susceptibility can be removed, if certain genes are knocked down to increase its resistance against pathogens (Lorang et al. 2007; Pavan et al. 2010). Combination based on biological control and host resistance can be complicated but holds a strong future in preventing crop losses (Allmann and Baldwin 2010). Therefore, for long-lasting resistance, direct resistance can be combined with indirect defense traits that slow direct resistance (Mitchell et al. 2010).

Another method includes insertion of barley trypsin inhibitor CMe (BTI-CMe) in immature embryos of wheat by the biolistic method. Proteinase inhibitors were considered in plant defense against pests that protected the transgenic seeds of wheat from Angoumois grain moth of Sitotroga cerealella, Lepidoptera: Gelechiidae.

3.4.3.1 Wheat Crop Breeding for Protection Against Insects/Pests

A resistant crop is bred against a particular pest by keeping its life cycle and heritability of resistance in mind. The source of resistance used for this purpose should have particular resistant genes that can lower crop loss. Some cultivars have resistant genes against pests, but some may require the transfer of resistance from other sources. However, resistance genes transferred to distant cultivars provide very limited resistance and success as a new pest from outside has the potential to develop new biotypes against the resistance variety due to changes in agronomic practices (McIntosh 1998).

Producing insect-resistant varieties paves paths for crop protection instead of using expensive, hazardous pesticides. Insect-resistant varieties are rarely used by the breeders who prefer increasing the quality, quantity, and stability of desired abiotic traits and neglect the importance of insect resistance (William and Bonjean 2011). Insects are becoming virulent because of crop domestication with increased nutrients for insects that have reduced defenses against them. Under such circumstances, resistant varieties against the pests are important especially against Russian wheat aphid and Hessian fly. Although there is a concern of developing resistance against the resistant wheat by these insects, in the future, more than one gene may be required to protect wheat. To create resistant wheat varieties with high expression of the resistant gene, constitutive resistance is enough for protection. The drawback of such cultivars, however, is that they will show resistance against only one insect. This makes the cultivars susceptible to other insects (William and Bonjean 2011).

4 Conclusion and Future Perspectives: Biotechnology, Output Practicality, Vision 2050, and Food Security

Once wheat has been protected by various biotic and abiotic stresses, the yield of wheat increases automatically. Wheat being the source of protein and nutrition is utilized when it is refined into various products, so its grain and texture determine the quality. Wheat yield is directly linked with a number of grains per unit area (Peltonen-Sainio et al. 2007); improvement of grain number and quality increases the yield of wheat.

Controlling viral diseases in wheat can help to improvise its yield by using various methods including genetic modification in wheat at the cellular level to improve its photosynthetic capacity. Among viruses, BYDV- and WSMV-resistant cultivars have been produced using various methodologies from conventional breeding to the use of genetic engineering. Insect control is significantly important in making wheat resistant and improving its yield and quality. Hessian fly causes drastic damage to the crop that can be prevented by making resistant cultivars. By preventing the disease incidence, crop yield can be improved (McIntosh 1998). Once the disease is under control, various efforts can be made to improve the nutrition level to fulfill the requirement (Foulkes et al. 2011).

For maximization of a crop, factors involved in increasing the productivity of a crop are considered. Crop’s response to light duration and the growth inputs also provide high yield (Reynolds et al. 2009). Spike growth and stem elongation in wheat take place in the pre-anthesis period; increasing this duration improves spike dry matter. In the post-anthesis period, photosynthesis increases the strength and high grain numbers (Reynolds et al. 2005; Acreche and Slafer 2009). Improvement in photosynthetic rate increases crop productivity to accommodate grain numbers by developing partitioned spikes (Foulkes et al. 2011). Grain number can be increased by increasing spike development and increasing lodging resistance to improve water productivity and nutrient productivity and by increasing grain size. The quantitative approach in breeding can help to withstand the climatic change and to make faster progress in wheat yield returns (Foulkes et al. 2011).

Tissue culture being a biotechnological approach is very efficient in its applications. A wide range of tissue culture techniques are now worked out, and many more are in place. The scale of tissue culture revolution has initiated to impact over the survival in many ways. Hence civilization is now moving forward into the era of biotechnology. In the beginning, numerous protocols are devised for maximization and accomplishment of elite genotypes in a short period. Other major reasons for tissue culture enhancement are double haploid and somatic embryogenesis during breeding of different parental lines and genotypes. Tissue culture response of the wheat crop can be enhanced by achieving more success in improving and modifying elite genotypes for various agronomic traits. Wheat yield can be achieved, if the crop is protected against various biotic and abiotic environmental factors, but tissue culture and transformation difficulties in wheat pose a hurdle and are still in progress to control the yield. Further research efforts require reducing deleterious somaclonal variations during clonal production and transgenic plant propagation to get higher wheat yields.

Extensive work has been reported in this context. During this channel, some researchers have tried to produce in vitro plantlets with resistance to biotic and abiotic stresses. In this regard, plantlet’s ability to cope with abiotic stresses such as drought, salinity, heavy metal, etc. has been remarkably achieved. Similarly, regenerated plants withstand the biological stress condition, for instance, diseases, pathogen , etc., that has been accomplished. Fungal diseases such as scab (FHB), powdery mildew, rusts, smuts, and many others have been improved through genetic engineering. Many in vitro studies have been conducted, and outputs have emerged on achieving fungal stress resistance in fields. The confirmation of resistance in putative tissue culture plantlet is achieved through the molecular marker and target gene-specific primers.

Use of genetic engineering helps in resistance to disease incidence, increasing yield and making the crop more nutritious for use. The use of hpRNA helps to reduce amylose production by changing amylase amylopectin ratio in wheat that can help to reduce heart diseases and colon cancer incidences (Regina et al. 2006), type 2 diabetes, and other types of cancer. These benefits are lost as milling removes fibers, minerals, and phytochemicals present in the embryo aleurone layer and in salts of phytic acid. To make nutritionally improvised wheat, identification of composition source is required along with the development of a high portion of bran and germ. To overcome the problem of nutrition, GE uses the expression of fungal phytase enzyme in grain to reduce the amount of phytates (Brinch-Pedersen et al. 2003).

Genetic engineering is meaningless without the application of tissue culture technique. Therefore, in vitro tissue culture is an important technique and the hour of need that should be routinely practiced to uncover its more usage in across a wide array. However, genetic modification in wheat domestication entails knowledge about the genome of domestic wheat (Avni et al. 2017), particularly wild emmer (T. turgidum ssp. dicoccoides). This is the reference assemblage and would serve as a huge resource for genome-wide improvement of modern wheat varieties.