specifically regulates fiber initiation by the HDZIP2‐ATATHB2 core cis element (Deng et al. 2012). Similarly, alpha‐expansins (GhExp1) overexpression regulate fiber elongation encoding cell wall loosening proteins (Harmer et al. 2002). In addition, several related genes are highly expressed during fiber elongation. Earlier, antisense suppression of sucrose synthase (SuSy) was revealed to suppress fiber elongation due to change in osmosis (Ruan et al.2007). In contrast, proline‐rich protein‐coding (GhPRP5) was found as a negative regulator of fiber development (Xu et al. 2013).
Cellulose synthesis is a principal event in fiber cells during the secondary cell wall biosynthesis. Previously, many efforts have been made to explore how the cotton fiber regulates and supports the strong irreversible carbon sink characterized by secondary wall cellulose synthesis (Brill et al. 2011). It has been shown that suppression of Sus gene expression affects the cellulose deposition (Brill et al. 2011), emphasizing the importance of this enzyme in cellulose synthesis. Subsequently, Brill et al. (Brill et al. 2011) identified and characterized a novel Sus isoform (SusC), which was up‐regulated during secondary wall cellulose synthesis in cotton fiber. Besides secondary wall cellulose synthesis, the maturation stage of fiber development begins. During fiber maturation, the majority of the expressed genes belong to cellular respiration (Kim et al. 2013). Many genes encoding transcription factors, that are MYB, C2H2, bHLH, WRKY, and HD‐ZIP families, were also expressed during cotton fiber development. Previously, various studies indicated that MYB‐related genes have high expression during fiber development in G. hirsutum (Machado et al. 2009). For example, expression studies of six MYB‐related genes in G. hirsutum indicated that GhMYB6 has high expression in cotton fiber (Loguercio et al. 1999), while R2R3 MYB‐like transcription factor‐encoding gene “GhMYB109” is particularly expressed in fiber initiation and elongation (Suo et al. 2003). The RAD‐like GbRL1 was also highly expressed in cotton ovules during fiber initiation (Zhang et al. 2011). TCP transcription factor has played a significant role in fiber and root hair development by controlling the jasmonic acid biosynthesis, ethylene signaling, calcium channel and reactive oxygen species (Hao et al. 2012). Though, GhHOX3 from class IV homeo‐domain‐leucine zipper (HD‐ZIP) family showed strong expression during early fiber elongation (Shan et al. 2014). Besides transcription factors, phytohormones such as ethylene, auxins and brassinosteroids (BR) also play a critical role during fiber development. Ethylene plays a vital function in fiber elongation by stimulating the pectin biosynthesis network (Qin and Zhu 2011), while gibberellins (GA) and indole‐3‐acetic acid (IAA) are required for fiber initiation and elongation in cotton (Xiao et al. 2010). In contrast, the persistent high concentration of jasmonic acid (JA) inhibits fiber elongation (Tan et al. 2012). Though several gene expression studies have been reported on cotton fiber development, some issues are illustrated here. First, most of the differentially expressed genes identified by the comparative analyses are associated with variations between species rather than related to fiber traits. Second, in some cases, the use of the protein‐coding gene sequences from Gossypium raimondii and Gossypium arboreum may not be accurate enough for gene annotation in tetraploid cotton. Third, it is unknown whether any of the expressed genes recognized from earlier reports had sequence variations between a cotton fiber mutant and its wild type because only the differentially expressed genes having sequence differences and co‐localization with target fiber traits are possible candidates for advanced cotton studies. These excellent contributions extremely facilitate vector construction for functional genes analyses and screening via the “genotype‐to‐phenotype” approach. Thus, the arsenal of cotton genomic manipulation urgently needs to be updated to meet the demand for rapid and precise dissecting gene functional analyses. Based on the presented facts and the well‐documented functional genomics of fiber quality traits, along with the availability of genetic resources and the high transformation efficiency, the employment of the CRISPR/Cas system is a better choice for cotton fiber quality improvement.
3.4.1.8 Soybean
Soybean (Glycine max L.) is an old polyploidy crop with immense economic benefits. The soybean genome is complex and probably arisen by two rounds of genome‐wide duplications or polyploidy events (Innes et al. 2008). Though several QTLs were mapped on the soybean genome before the publication of soybean genome sequence, the identification of underlying genes responsible for the quantitative trait of interest was rare (Watanabe et al. 2009). The availability of genome sequence of soybean has accelerated mapping and identification of genes responsible for various qualitative and quantitative traits (Schmutz et al. 2010).Soybean is an important crop owing to its potential for supplying protein, oil and animal forages. The world soybean production in the year 2019 was approximately 358.65 million tons. The US was the largest producer with 120.52million metric tons. Other major countries such as Brazil, Argentina, and China contributed 117, 55.30, and 15.90 million metric tons, respectively (http://www.sopa.org/statistics/world‐soybean‐production/).The classical breeding approaches are not affected due to limited genetic variability available in the germplasm. However, there are several ways through which the classical breeding approaches deals and is able to incorporate the traits of interest in elite cultivars. However, if cultivars show low genetic diversity, their existing genes/alleles may not be able to cope with new environmental stresses. The novel genes/alleles can be incorporated in existing germplasm from wild relatives and/or created by random mutagenesis. However, the incorporation of large genome from wild relatives may cause severe damage to the elite germplasm, i.e. low production making long breeding cycles essential to purify the background while reserving the introduced attribute. The utilization of Marker‐assisted breeding (MAB) which is marker‐assisted selection (MAS) and genomic selection (GS) can speed up the process and has been well documented to achieve the objective (Crossa et al. 2017; Zhang et al. 2018). In modern times, the targeted mutagenesis has been successfully exploited in many crops for several desirable phenotypes with no exception to soybean.
3.4.1.9 Application of CRISPR/Cas9 for Soybean Quality Improvement
The genetic transformation methods in plants have made tremendous progress especially in soybean breeding programs, to produce novel, and genetically variable, plant material. These transformation techniques have been utilized previously to study the functional aspects of soybean (Stewart et al. 1996). These transgenic plants represented a priceless tool for molecular, genetic, biochemical and physiological studies by gene overexpression or silencing, transposon‐based mutagenesis, protein sub‐cellular localization and/or promoter characterization. However, there are some disadvantages to this traditional method. The introduced genes may cause non‐targeted mutagenesis ultimately interrupting the endogenous or exogenous genes which may have negative results. The RNAi technique possesses the ability to silence a whole gene family hough, the silencing of only targeted gene is required. The genome modifications of soybean are complex due to the fact that genes are highly duplicated in nature. Thus, precise, efficient and straightforward methods for researching gene functions and genome engineering are required. Recently, CRISPR/Cas9 has emerged as a robust and effective technology for editing each member of a gene family without influencing other genes or simultaneously editing multiple genes of interest, thereby overcoming the shortcomings of the traditional plant‐breeding methods.
Genome editing has emerged as a better choice for molecular breeding than transgenesis and it is well accepted by society. Cai et al. (Cai et al. 2015) first successfully realized CRISPR/Cas9 mediated genome editing in soybean using a single sgRNA for a trans gene (bar) and six sgRNAs that targeted different sites of two endogenous soybean genes (GmFEI2 and GmSHR) and understood deficiency of the sgRNAs in a hairy root system. The targeted mutagenesis of two genomic sites in soybean chromosome 4 (DD20 and DD43) caused frame shift mutation (Li et al. 2015). The targeted gene integrations through HDR were detected by border‐specific polymerase chain reaction analysis at callus stage. Soybean GmU6–16‐1 promoter was found to be more efficient in simultaneous editing of multiple homoeoallelesrelativetotheArabidopsisAtU6‐26 promoter (Du et al. 2016). The role of a dominant nodulation restriction gene in soybean,
