genes influencing grain quality to be studied, while new genetic and molecular influences on wheat quality can be revealed by considering variations in the whole genome and relating all the differences to explain the basis in fluctuations in wheat grain quality.
3.4.1.3 Application of CRISPR/Cas9 for Wheat Quality Improvement
Most of the studies provided insights on starch composition and its biosynthesis pathways based on the knowledge gained, several biotechnologies approaches have been utilized to enhance the quality of starch related properties. Weichert et al. (Weichert et al. 2010) over expressed the barley sucrose transporter gene (HvSUT1) that uplifts sucrose uptake along with protein content of wheat grains with less compromise on starch content. In another attempt, expression of an optimized maize ADP‐glucose pyrophosphorylase (ZmAGPase) resulted in elevated yield and enhanced photosynthetic rates in the transgenic wheat lines (Smidansky et al. 2007).
Subsequently, down regulation of the transcription factor TaRSR1 (a wheat homolog of Rice Starch Regulator; OsRSR1), showed a negative regulation of some starch synthesis‐related enzymes in wheat grains (Kang et al. 2013),resulting in a significant 30% increase in starch content, and also a ~ 20% increase in yield in terms of 1000‐kernel weight (Liu et al. 2016). The amylose to amylopectin ratio influences the starch quality. The higher amylose content contributes more toward resistant starch (RS) in food with potential benefits to human health. There is evidence that RS can provide protection from several health conditions such as diabetes, obesity, and cardiovascular diseases (Meenu and Xu 2018). Many experiments have been conducted on down regulation of starch‐branching enzymes, SBEIIa, and SBEIIb, which led to substantially increased amylose levels in wheat (Regina et al. 2006). The starch with higher concentration of AC demonstrated a health impact in rats (Regina et al. 2006), and a similar study showed obesity in humans (Sestili et al. 2010). Glutelin makes the major portion of Wheat SSPs, determining the dough viscoelastic properties (Anjum et al. 2007). Glutelin contribute 70–80% of crude protein and are composed of gliadins and glutenins. Therefore, it is essential to understand and manipulate the genes controlling/improving nutritional and dough‐ related properties. The application of transgenic approaches enhances the understanding for underlying attributes and their genomic architecture controlling quality traits. The genes controlling high molecular glutelin subunits (HMW‐GS) were introgressed in Wheat to improve dough‐related properties (Altpeter et al. 1996). The transformation of subunits 1Ax1 and 1Dx5 in common wheat cultivars modify dough properties to various extent (Alvarez et al. 2001). The genes influencing HMW‐GS were introduced into Wheat cultivars Bobwhite through a transgenic approach. Further, the transgenic lines were crossed with elite varieties to improve grain qualities which demonstrated the feasibility of transgenic wheat breeding (Li et al. 2007). In comparison to HMW‐GS, the gliadins contribute to dough viscosity and extensibility (Payne 1987). The gliadins are further grouped into α‐, γ‐, and ω‐gliadins, multigenic in nature. The gliadins possess research interest owing to its contribution to influence dough quality, immunogenic epitopes linked with immune condition, e.g. wheat‐dependent exercise‐induced anaphylaxis (WDEIA) and celiac disease (Scherf et al. 2016).
Iron and zinc are essential micronutrients contributing to nutrition. As per the WHO reports, approximately one billion in the human population are suffering from several diseases directly linked with nutrition deficiency. Moreover, the annual death rate is approximately half a million below the age of five. The cultivable Wheat lacks in micronutrients (Cakmak et al. 2000), and one possible reason is that the micronutrients accumulate in the outer husk, aleurone and embryo, lost during the milling and polishing process (Welch and Graham 1999). The anti‐nutritional factor, i.e. phytic acid also deposited in the aleurone storage vacuoles with impact on iron and zinc in the human digestive tract. The micronutrient deficiency can easily be manipulated through biofortification mechanism. The transgenic approaches have been successfully carried out to make micronutrients especially iron contents in Wheat. The plants store iron in ferritin structures deposited into no‐green plastids, etioplasts and amyloplasts. The expression of gene controlling Aspergillus niger phytase, phytic acid degrading enzyme in Wheat aleurone and endogenous (Borg et al. 2012) or Soybean (Sui et al. 2012) ferritin genes in wheat endosperm, were the first successful attempts to fortify wheat grains with iron through transgenic approaches. Recently, Connorton et al. (Connorton et al. 2017) have isolated, characterized, and overexpressed two wheat Vacuolar Iron Transporter (TaVIT) genes under the control of an endosperm‐specific promoter in wheat and barley. The introduction of TaVIT2 gene through the transgenic approach enhances, almost two‐fold of iron content in Wheat grains.
In wheat, the principal applicability of CRISPR/Cas9 was demonstrated in protoplasts and suspension cultures, where multiple genes were successfully targeted and achieved in the year following the publication of the original CRISPR/Cas9 principle (Upadhyay et al. 2013). Many agriculturally important traits of wheat have been targeted by genome editing among which include; (i) resistance/tolerance to biotic and abiotic stresses, (ii) yield and grain quality, and (iii) male sterility. The first experiment was conducted by Shan et al. (Shan et al. 2014) and successfully revealed the application of CRISPR/Cas9 system in wheat protoplasts for TaMLO gene (Mildew resistance locus O). The CRISPR TaMLO knockout has shown to confer resistance to powdery mildew disease caused by Blumeriagraminis f. sp. Tritici (Btg). The lipoxygenase genes, TaLpx1 and TaLox2, attracted attention as potential subjects for gene editing in relation to resistance to Fusarium, one of the most devastating fungal diseases in wheat. Lipoxygenases hydrolyze polyunsaturated fatty acids and initiate biosynthesis of oxylipins, playing a role in the activation of jasmonic acid‐mediated defense responses in plants. Silencing of the TaLpx‐1 gene has resulted in resistance to Fusarium graminearum in wheat (Nalam et al. 2015). TaLpx1 and TaLox2 genes were edited in protoplasts with a mutation frequency of 9 and 45%, respectively (Shan et al. 2014; Wang et al. 2018). Wheat plants with mutated TaLOX2 were obtained with a frequency of 9.5%, of which homozygous mutants accounted for 44.7% (Zhang et al. 2016). With the aim of enhancing grain size and yield, several genes have been edited by the CRISPR/Cas9 system: TaGASR7 (Liang et al. 2017), TaGW2 (Li et al. 2017), and TaDEP1 (Zhang et al. 2016). TaGASR7, a member of the Snakin/GASA gene family, has been associated with grain length in wheat. A CRISPR/Cas9 vector designed to target TaGASR7 was delivered by particle bombardment into shoot apical meristems. Eleven (5.2%) of the 210 bombarded plants carried mutant alleles, and the mutations of three (1.4%) of these were inherited in the next generation (Wang et al. 2018). Transiently expressing the CRISPR/Cas9 DNA and using the CRISPR/Cas9 RNP mediated method were also highly effective for TaGASR7 editing (Liang et al. 2017). The TaGW2 gene encodes a previously unknown RING‐type E3 ubiquitin ligase that was reported to be a negative regulator of grain size and thousand grain weight in wheat (Li et al. 2017). Recent studies, detailing the functionality of the allelic TaGW2 genes through genome specific knockouts, showed that the TaGW2 gene in wheat acts by regulating the gibberellin hormone biosynthesis pathway (Li et al. 2017), principally confirming the parallel functions of these genes in rice and wheat. T1 plants carrying knock‐out mutations in all three copies of the TaGW2 gene (genotype aabbdd) showed significantly increased thousand grain weight (27.7%), grain area (17.0%), grain width (10.9%), and grain length (6.1%) compared to the wild‐type cultivar (Wang et al. 2018). CRISPR/Cas9 technology was also successful in obtaining low immunogenic wheat. Sanchez‐Le'on et al. (Sánchez‐León et al. 2018) have shown that CRISPR/Cas9 technology can be used to efficiently reduce the amount of alpha‐gliadins in the seeds, providing bread and durum wheat lines with reduced immune reactivity for consumers with celiac disease. Twenty‐one mutant lines were generated (15 bread wheat and 6 durum wheat), all showing a strong reduction in alpha‐gliadins. Up to 35 of the 45 different genes identified in the wild‐type were mutated in one of the lines, and immunoreactivity, as measured by competitive ELISA assays using two monoclonal antibodies, was reduced by 85%. These examples provide an insight into the many ways in which modern genome modification technologies are being used to mine the core research findings from model plant transgenesis, and finally harness that understanding to drive essential crop. The ability to enact targeted changes to the genome
