was shown through both complementation and CRISPR/Cas9‐mediated gene knockout experiments (Tang et al. 2016). CRISPR was used to disrupt the pathogen virulence gene (Avr4/6) in Phytophtho rasojae (Fang and Tyler 2016). Homologous genes placement of Avr4/6 by a marker gene (NPT II) stimulated by the CRISPR/Cas9 system emphasized the contribution made by the virulence gene in recognition of the pathogen by plants containing the soybean R gene loci, Rps4 and Rps6. CRISPR knockout of the soybean flowering time gene, GmFT2, was stably heritable in the subsequent T2 generation, with homozygous GmFT2a mutant inhibiting late flowering under both long‐day and short‐day conditions (Cai et al. 2018). The first soybean product, produced by genome editing, was announced in early 2019 and will be available on the market soon. By using the transcription activator‐like effector nuclease (TALEN) technique, a new high‐oleic‐acid soybean cultivar has been made without introducing foreign DNA into the soybean genome (Calyxt Inc2019). Vegetable oil, produced with this new soybean cultivar, contains 80% oleic acid and has 20% less saturated fat, which makes the oil healthier for human consumption, and the shelf life of the oil is also extended (Splitter 2019). It is expected that the high‐oleic‐acid soybean oil will be the first gene‐edited plant product to be commercialized (Calyxt Inc 2019; Splitter 2019). Most of the genome editing studies were focused on disease resistance and photoperiod sensitivity in soybean, however, the nutrition aspects are less studied which need further attention. So far, the genomics of soybean nutrition is a concern and several studies have been undertaken to highlight the genomic architecture soybean nutrition.
To improve the oxidative stability and quality of soybean oil, breeding programs have mainly focused on reducing the saturated fatty acid and linolenic acid contents and increasing oleic acid in the oil. Hence, delta‐12 fatty‐acid desaturase 2 (FAD2), which converts oleic acid (18 : 1) to linolenic acid (18 : 2), becomes the target for modification via molecular breeding. There are two FAD2 loci, FAD2–1A (Glyma10g42470) and FAD2–1B (Glyma20g24530), in the soybean genome. To develop high‐oleic‐acid lines (> 80% of total oil content vs. an average of 20–50% among existing soybean cultivars), efforts were made to combine a recessive mutant allele at the FAD2–1A locus (Glyma10g42470) and a recessive mutant allele at the FAD2–1B locus (Glyma20g24530) using molecular marker assays (Pham et al. 2011). In a later study, a non‐synonymous SNP on FAD2–1A (S117N) from 17D, an EMS mutant of Wm82, and another one on FAD2–1B (P137R), a mutant allele from PI 283327, were identified to be highly associated with the high‐oleic‐acid phenotype (Kulkarni et al. 2018). Similarly, an EMS mutant, PE1690, derived from the cultivar, Pungsannamul, was found to be low in linolenic acid. The phenotype was the result of a single‐base mutation (W128*) in the GmFAD3A gene on the locus Glyma14g37350, which rendered the desaturase enzyme non‐functional (Chahal and Gosal 2002). A derived cleaved amplified polymorphic sequence (dCAPs) assay specific for this artificial polymorphism was thus developed for future breeding activities (Kim et al. 2015). These studies have provided insights and, based on these observations, future studies can be designed utilizing modern technologies to improve soybean quality to fulfill the consumer requirements.
3.5 Regulatory Measures for Genome Engineering Crops
The classical breeding methods largely depend on selection and crossing to develop homologous recombinant. These techniques are utilized from decades ago to make selection of novel traits and introgression into elite cultivars to obtain desirable phenotype. The traditional breeding methods have helped to develop many improved cultivars in several crops important for food security, however, it requires much time and successive generations for selection (Chahal and Gosal 2002). Moreover, the unavailability of genetic variability in major crops have further hindered the classical breeding approach (Pacher and Puchta 2017). The modern breeding technologies have potential to overcome these challenges and are successfully utilized to a variety of crops. Mutation breeding for the development of transgenic and non‐transgenic plants are worthy strategies utilized for crop improvement programs (Scheben et al. 2017). The traditional mutagenesis approaches have helped to develop genetic variations in traits of interest to improve yield and quality. However, the traditional mutagenesis approaches require, labor oriented screening of a large number of mutant population (Sikora et al. 2011), less precise and leaves breeding programs, that unable to meet the requirements (Scheben et al. 2017). The transgenic breeding efforts have been utilized to introduce important genes in cultivars with promising attributes. The transgenic breeding approach has potential to overcome crossing barriers to enhance genetic variability. The major concern with transgenic approaches is the public concern regarding the impact on human health and the environment, therefore, commercialization of genetically modified crops (GMOs) is under strict scrutiny (Hartung and Schiemann 2014). In European countries, the GM crops are still not allowed for commercial purposes and are prohibited in the consumer market along with, the GE non‐transgenic crops also going through strict regulatory measures. The new breeding techniques, i.e. ZFNs, TALENs, and CRISPR do not fall under the category of GMOs due to no residues of foreign particles being in targeted genome. The US Department of Agriculture had exempted CRISPR edit crops from GMOs and are allowed to be cultivated and sold to consumers (Waltz 2018). The CRISPR technique will help to reduce the cost required for field trail and data collection of GMOs along with overcoming public concerns. However, there is an urgent need for intensive uniform of regulatory policy for GE tools around the globe.
3.6 Conclusion
The utilization of genome engineering for quality improvement in crops essential for food security requires substantial expression of genes without influencing other regulatory pathways essential for plant growth and development. The lack of understanding of biosynthesis pathways and their complex interaction for quality attributes in the majority of crops is a limiting factor. The availability of genomic data of several important crops helped in identification, characterization and regulation of genes controlling quality traits but, requires further research. The modern GETs have revolutionized the crops quality improvement programs but still requires further investigation. These breeding techniques possess the ability to make precise and quick targeted modifications in genes of interest compared to conventional breeding techniques. CRISPR/Cas proved to be a fundamental breakthrough in genome editing which is successfully utilized for yield enhancement, quality improvement, disease resistance and other traits of interest. In the past few years, targeted mutagenesis has been vigorously utilized in plant‐based research for functional studies. Moreover, the CRISPR/Cas genome editing system has potential to be adopted as a viable approach to achieve zero hunger and nutritional imbalance for growing human population.
Acknowledgement
The authors are thankful to the scientific insights of Dr. Aamir Riaz from China National Rice Research Institute, Hangzhou, Zhejinag, P.R. China.
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