Genome Engineering for Crop Improvement. Группа авторов

52 T₀ lines obtained by Agrobacterium‐mediated transformation, seven lines were reported to carry the psy1 knockout furthermore, the sequencing analysis undertaken to evaluate the variations generated and mutation efficiency. The results showed that no off‐target sites were edited and stable psy1 mutants were obtained. Feng et al. (Feng et al. 2016) have demonstrated the utility of the CRISPR/Cas9 system in maize by targeting the albino marker gene, Zmzb7 through protoplast system. Knockout of Zmzb7 results in albino plant, with the sgRNA designed to target a region in the eighth exon of Zmzb7 and maize U3 promoter was used for expression. Following Agrobacterium‐mediated transformation of maize embryos, T₀ lines were found to show a 31% mutation efficiency. Gene editing tools that can affect multiple gene knockouts are of immense importance to accelerate and achieve efficient crop breeding. For the first time, multiplex genome editing in maize was demonstrated by Qi et al. (Qi et al. 2016) using a tRNA‐RNA processing system. A multiplex editing vector can incorporate a cluster of gRNAs separated by spacers in a polycistron, producing multiple gRNAs from one primary transcript. The study targeted three transcription factor genes (MADS, MYBR, and AP2) for simplex editing and three other genes (RPL, PPR, and IncRNA) for multiplex editing. Increased editing efficiency (up to 100%) was observed for t‐RNA processing based multiplex editing. Current high yielding maize varieties are the result of hybrid maize seed production and the production of hybrid maize requires sterilization to avoid self‐fertilization. Maize thermosensitive genic male‐sterile 5 (ZmTMS5), known to cause male sterility was targeted for genome editing by CRISPR/Cas9 approach (Li et al. 2017). Three gRNAs were used to knockout the gene, with one sgRNA targeting the first exon and the other two sgRNAs targeting the second exon. Mutation efficiency was examined in maize protoplasts using PCR/restriction enzyme assays. Analysis of mutational efficiency revealed that the sgRNA targeting the first exon had no off‐targets whereas the other two sgRNAs had off‐targets in the maize genome. The AUXIN REGULATED GENE INVOLVED IN ORGAN SIZE (ARGOS) gene family are negative regulators of the ethylene response and modulate ethylene signal transduction. Overexpression of ARGOS genes in transgenic maize plants enhances drought tolerance and identification of new allelic variants would be of immense importance in maize breeding programs. Shi et al. (Shi et al. 2017)utilized CRISPR/Cas9 genome editing to create new allelic variants of ARGOS8. Two genome‐edited variants (ARGOS8‐v1 and ARGOS8‐v2) were used for the production of hybrids and evaluated in the field in multi‐location trials. Improved yield under stress condition was observed for the mutant than wild‐type. This study demonstrated the use of CRISPR/Cas9 genome editing method for creating new variants and their application in maize crop improvement.

3.4.1.6 Cotton

      Cotton is an important crop for the production of fiber, oil and biofuel. In addition, cotton serves as a cash crop for more than 20 million farmers in Asia and Africa. Despite the availability of synthetic alternatives, cotton remains an important source of fiber because of the advantages related to cost of production and unique features offered by cotton lint. Consumption of cotton products in the world is increasing day by day in a lot of places, but world cotton production is stagnant because of biotic and abiotic stresses. To meet the demands of the masses, production of cotton needs to be very high, with good quality. Cotton is also affected by diseases, causing significant losses to industry. Therefore, it became evident to utilize plant‐breeding approaches to tackle threats caused by both biotic and abiotic factors ultimately reducing fiber quality.

The genus Gossypium includes approximately 45 diploid (2n = 2x = 26) and five tetraploid (2n = 4x = 52) species, all exhibiting disomic patterns of inheritance. Diploid species (2n = 26) fall into eight genomic groups (A–G, and K). The African clade, comprising the A, B, E, and F genomes (Wendel and Cronn 2003), occurs naturally in Africa and Asia, while the D genome clade is indigenous to the Americas. A third diploid clade, including C, G, and K, is found in Australia. All 52 chromosome species, including Gossypium hirsutum and Gossypium barbadense, are classic natural allotetraploids that arose in the New World from interspecific hybridization between an A genome‐like ancestral African species and a D genome‐like American species. More than 95% of the annual cotton crop worldwide is G. hirsutum, Upland or American cotton, and the extra‐long staple or Pima cotton (G. barbadense) accounts for less than 2% (National Cotton Council, http://www.cotton.org, 2006).The genome of both sea‐island and upland cotton were sequenced in 2015, paving the way for the use of tools such as genome editing in genetic improvement programs (Zhang et al. 2015). Modern biotechnology and molecular approaches have been applied to plant breeding to promote plant‐genome manipulation and enhance selection of desired traits and performance of crops. The recent availability of genome‐editing technologies provides a vast opportunity to introduce targeted modifications in the genome efficiently to study the functional aspects of various components of plants.

3.4.1.7 Application of CRISPR/Cas9 for Cotton Quality Improvement

      Cotton fiber quality is directly related to boost the economy determining the income of almost 100 million families from more than 100 countries (Guan et al. 2014). Tetraploid cotton retains special features such as larger fiber length and fiber strength to facilitate more spinnable cotton. Cotton fibers contain single‐celled trichomes originating from outer integument cells of the ovular surface. Fiber developmental mechanisms comprising four levels, fiber‐cell initiation, elongation, secondary cell wall biosynthesis and maturation (Manik and Ravikesavan 2009). The overlapping developmental stages have some special features differentiating cellular and physiology. This is due to complexity of cotton fiber transcriptome involving ~18 000 and 36 000 genes in diploid and allotetraploid cotton genomes, respectively (Arpat et al. 2004). Several key fiber‐related genes have been identified creating interest to study their functions and subsequent improvement of enhance fiber quality. Some key genes including E6 (John and Crow 1992), GhExp1 (Harmer et al. 2002), GhSusA1 (Jiang et al. 2012), PIP2s (Li et al. 2013) and GA20ox (Bai et al. 2014) were reported predominantly expressed during fiber initiation, secondary cell wall biosynthesis (Brill et al. 2011), and fiber elongation (Yang et al. 2014). Taking an example, protodermal factor1 gene (GbPDF1) 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