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Stable transformation of cotton by using Agrobacterium and the shoot apex. (A to E) represent the process of obtaining stable transgenic cotton plants. (A) Sterile cotton seeds were germinated on moistened filter paper in sterile glass plate. (B) Seeds with 1–2 cm length root were moved to glass vessels (5 cm in diameter, 8 cm in height) with MS medium (pH 5.8–6.0) and then grew at 28 °C in darkness. (C) One of each seedling's cotyledons was removed and the naked shoot apex was injured with a sterilized scalpel. A small sterile absorbent cotton ball with Agrobacterium suspension was placed on the injured shoot apex and vacuum-infiltrated at 0.5 MPa for 10 min at room temperature. (D) Infected seedlings were co-cultivated in an incubator at 28 °C for 2 d in darkness and 2 d under light. (E) Seedlings were transferred to soil and grew in the greenhouse.
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The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the g...
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... particle delivery system and Agrobacterium-mediated genetic transformation. Among them, Agrobacterium-mediated genetic transformation is the most widely used in cotton genetic transformation 16 . In our study, we delivered the CRISPR/Cas9 system into the cotton genome using the Agrobacterium-mediated method with the shoot apexes as receptors (Fig. 4). Unlike stem apexes as the explant, the injured shoot apexes can continue to grow on their original plants. Then, the transgenic lines can be tested with genomic DNA from the true leaves growing from the shoot apexes after 3-4 weeks. Although this method is time-saving, it is difficult to obtain homozygotes in the first generation (T 1 ...
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... transformation of cotton. The stable transformation of cotton was per- formed using the protocol of Lv et al. [20][21][22] (Fig. 4). Briefly, seeds of cotton cultivar Zhong 9807 were surface sterilized and germinated on moistened filter paper in glass plate at 28 °C in darkness. When the length of the seed root was 1-2 cm, we moved the seeds into sterilized glass vessels (5 cm in diameter, 8 cm in height) with MS medium. After incubation in an incubator at 28 °C in ...
Citations
... The CRISPR/Cas system has transformed the field of plant GE with its simple design and high efficiency, which uses short RNA molecules called single guide RNAs (sgRNAs). This system has overshadowed the complexity and limitations associated with ZFNs and TALENs in designing and cloning desired gene constructs [207,208]. Its cost-effectiveness and ease of use have made CRISPR/Cas the preferred choice for GE in animals and plants, transforming the landscape of genetic research. ...
... The genome editing approach has been successfully employed in cotton to induce targeted mutations with improved fiber quality traits or also to validate the functional relevance of genes involved in various fiber quality traits [78]. For instance, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) based genome editing was used to knock out the GhACTIN-1 gene and create null mutants with shorter and weaker fibers. ...
Natural fibers have garnered considerable attention owing to their desirable textile properties and advantageous effects on human health. Nevertheless, natural fibers lag behind synthetic fibers in terms of both quality and yield, as these attributes are largely genetically determined. In this article, a comprehensive overview of the natural and synthetic fiber production landscape over the last 10 years is presented, with a particular focus on the role of scientific breeding techniques in improving fiber quality traits in key crops like cotton, hemp, ramie, and flax. Additionally, the article delves into cutting-edge genomics-assisted breeding techniques, including QTL mapping, genome-wide association studies, transgenesis, and genome editing, and their potential role in enhancing fiber quality traits in these crops. A user-friendly compendium of 11226 available QTLs and significant marker-trait associations derived from 136 studies, associated with diverse fiber quality traits in these crops is furnished. Furthermore, the potential applications of transcriptomics in these pivotal crops, elucidating the distinct genes implicated in augmenting fiber quality attributes are investigated. Additionally, information on 11257 candidate/characterized or cloned genes sourced from various studies, emphasizing their key role in the development of high-quality fiber crops is collated. Additionally, the review sheds light on the current progress of marker-assisted selection for fiber quality traits in each crop, providing detailed insights into improved cultivars released for different fiber crops. In conclusion, it is asserted that the application of modern breeding tools holds tremendous potential in catalyzing a transformative shift in the textile industry. KEY POINTS Natural fibers possess desirable properties, but they often lag behind synthetic fibers in terms of both quality and quantity. Genomic-assisted breeding has the potential to improve fiber quality traits in cotton, hemp, ramie, and flax. Utilizing available QTLs, marker-trait associations, and candidate genes can contribute to the development of superior fiber crops, underscoring the significance of advanced breeding tools.
... Mostly, CRISPR application is focused mainly on insect and pest resistance whereas the abiotic stress tolerance is rather limited. However, CRISPR/Cas9 system employed for abiotic stress (continued) Ning et al. (2011) improvement is demonstrated in some major crops such as cotton (Chen et al. 2017), maize (Char et al. 2017;Svitashev et al. 2016), rice (Miao et al. 2013;Shan et al. 2014) and wheat (Shan et al. 2014). However, apart from other cereals like rice, wheat and maize, genetic transformation in millets falls behind. ...
Barnyard millet (Echinochloa species) is a versatile crop with a rich nutritional profile known for its innate ability to tolerate most abiotic stresses, including drought and high temperatures. Although potential yield losses are inevitable, further improvement is needed to make them more resilient to unprecedented effects of climate change and associated environmental stresses. Hence, strenuous efforts are required to characterize the germplasm resources and identify the specific traits, QTLs, genes and pathways associated with stress tolerance. In addition, understanding the mechanism underlying stress response in Echinochloa species may not only be useful to develop superior cultivars but also serve as the reservoir of unique alleles which assist in improving tolerance in major cereal crops. The release of genome and transcriptome sequence of both wild and cultivated barnyard millets will undoubtedly facilitate the dissection of genetic architecture behind stress tolerance. However, a major downside is the lack of large-scale genomic resources, research efforts and funding compared to other major millets like sorghum, pearl millet, foxtail millet and finger millet. Thus, with available knowledge on genetic and genomic resources in other major millets and cereals, attempts should be made to improve the stress tolerance of barnyard millet.
... Similar substitution mutations in rice have also been reported by Sony et al. [63] and Sun et al. [64]. Protoplast transformation in cotton (Gossypium hirsutum L.) using two gRNAs results in mutations that were majorly substitutions [65]. Cai et al. [66] induced single base pair substitutions in soybean (Glycine max (L.) Merr.) at target sites using CRISPR/Cas9 system. ...
Citrus reticulata Blanco also known as kinnow mandarin is a widely grown horticultural crop in Punjab. CRISPR/Cas9 technology is being widely used for generation of varieties with increased resilience towards abiotic and biotic stresses as well as improved horticultural traits. Xanthomonas citri subsp. citri (Xcc)-mediated Agroinfiltration offers a fast and transgene-free method for the delivery of CRISPR/Cas9 constructs for systemic introduction into plants for functional genomics and expression studies. The technology is currently unexplored in kinnow mandarin. This study is aimed at establishing an efficient method of Cas9 delivery for transient knockout of PDS (phytoene desaturase) gene in kinnow mandarin. The construct pKO-119-PDS N-Cas9/sgRNA:PDS1 carrying sgRNA and Cas9 enzyme was delivered into the dorsal surface of young leaves of kinnow mandarin. The leaves showed albino patches at the point of injection within 60 h. Two surfactants (Triton-X and Silwet™) were used to ease the Agroinfiltration process which resulted in variation in the expression of vector. The Sanger’s analysis of the treated plants showed a substitution within the sgRNA region which resulted in change in amino acid from proline to serine. The protocol provides a feasible and an efficient method for genome editing in C. reticulata which could be helpful in future studies aimed at genome editing as well as genetic transformation.
... Meanwhile, Kiryushkin et al. [9] included roots as a part of the in planta transformation concept. To the best of our knowledge, CRISPR/Cas9 components delivered via in planta transformation targeting non-root meristematic cells have only been achieved in upland cotton (Gossypium hirsutum L.) and wheat (Triticum aestivum L.) [10,11]. According to these studies, the cotton shoot apexes of young cotton seedlings were efficiently transformed using Agrobacterium. ...
... In contrast, the transformation of wheat relied on biolistic-based delivery that targeted the shoot apical meristem (SAM) of imbibed seeds. Chen et al. [10] achieved stable transformations in cotton, whereas Hamada et al. [11] achieved inheritable mutations in wheat through the transient introduction of the CRISPR/Cas9 system into the SAM. Agrobacterium-mediated in planta transformation for CRISPR/Cas9 delivery into sorghum (Sorghum bicolor L.) was reported by Sharma et al. [12]. ...
Agrobacterium-mediated transformation and particle bombardment are the two common approaches for genome editing in plant species using CRISPR/Cas9 system. Both methods require careful manipulations of undifferentiated cells and tissue culture to regenerate the potentially edited plants. However, tissue culture techniques are laborious and time-consuming.
In this study, we have developed a simplified, tissue culture-independent protocol to deliver the CRISPR/Cas9 system through in planta transformation in Malaysian rice (Oryza sativa L. subsp. indica cv. MR 219). Sprouting seeds with cut coleoptile were used as the target for the infiltration by Agrobacterium tumefaciens and we achieved 9% transformation efficiency. In brief, the dehusked seeds were surface-sterilised and imbibed, and the coleoptile was cut to expose the apical meristem. Subsequently, the cut coleoptile was inoculated with A. tumefaciens strain EHA105 harbouring CRISPR/Cas9 expression vector. The co-cultivation was conducted for five to six days in a dark room (25 ± 2 °C) followed by rooting, acclimatisation, and growing phases. Two-month-old plant leaves were then subjected to a hygromycin selection, and hygromycin-resistant plants were identified as putative transformants. Further validation through the polymerase chain reaction verified the integration of the Cas9 gene in four putative T0 lines. During the fruiting stage, it was confirmed that the Cas9 gene was still present in three randomly selected tillers from two 4-month-old transformed plants.
This protocol provides a rapid method for editing the rice genome, bypassing the need for tissue culture. This article is the first to report the delivery of the CRISPR/Cas9 system for in planta transformation in rice.
... Similar findings on the high level of substitutions mediated by CRISPR/Cas9 have been made by other studies. For example, in cotton (Gossypium hirsutum L.) protoplasts, only substitutions were found (Chen et al., 2017); in melon protoplasts and plants, 91% of mutations were substitutions (Hooghvorst et al., 2019); in cassava plants, substitutions occurred more frequently than InDel mutations (Odipio et al., 2017); in soybean (Glycine max L.) protoplasts and plants, a high level of substitutions were reported in the Cas9-edited events (Sun et al., 2015); and in rice (Oryza sativa L.), 25%-45% of the examined mutations were substitutions (Macovei et al., 2018). Observation of nucleotide substitution downstream of the target region implies activation of the homologous recombination (HR) pathway to repair double-strand breaks created in AcPDS. ...
Introduction
Clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) is a precise genome editing tool used to introduce genetic modifications in a wide range of crop species. Thus far, there is no report of CRISPR/Cas9-mediated genome editing in onions (Allium cepa L.).
Methods
In the present study, we targeted two exons of the gene coding for Phytoene desaturase (AcPDS) in onion cv. Bhima Super. The sgRNA-carrying constructs were co-cultivated with 8-week-old embryogenic calli using an Agrobacterium-mediated transformation protocol and incubated on the media without hygromycin B selection.
Results and discussion
Out of the total 617 co-cultivated calli, 21 (3.4%) regenerated shoots exhibited three distinct phenotypes: albino, chimeric, and pale green; in comparison to the wild-type non-transformed regenerated shoots. Total chlorophyll content was drastically reduced in albino shoots and significantly decreased in chimeric shoots. Out of the six Cas9 gene PCR-confirmed regenerated shoots, two exhibited the albino phenotype due to insertions/deletions (InDels) and substitution-based mutations in and around the AcPDS target sites. Deep amplicon sequencing revealed a significantly variable InDel frequency between two sgRNAs, ranging from 1.2% to 63.4%, along with a 53.4% substitution frequency. The mutation of the AcPDS gene generated a visually detectable albino phenotype, thus confirming the successful editing of the AcPDS gene. This is the first time a CRISPR/Cas9-mediated genome editing protocol has been successfully established in onion, with the AcPDS gene serving as an example. This study will provide the necessary momentum for researchers to further basic and applied research on onions.
... In vivo delivery of editing reagents in meristems, which results in edits that are transmittable to progeny, has been performed in only a few species [75][76][77][78]. Efficient in planta transformation No f [81] (continued on next page) ...
The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.
... Since 2017, several teams have utilized this system for tar- geting both exogenous and endogenous genes in cotton . Chen et al. (2017) analyzed the targeted genome editing system using the CRISPR/Cas9 system with one nucleotide insertion in the Cloroplastos alterados 1 (GhCLA1) gene and one nucleotide deletion in the vacuolar H+-pyrophosphatase (GhVP) gene and achieved the targeted editing frequencies up to 47.6%-81.8% in Upland cotton. The knocking out of the exogenous GFP gene through CRISPR/Cas9 targeted genome editing has been reported in cotton as well (Janga et al., 2017). ...
Cotton is an irreplaceable economic crop currently domesticated in the human world for its extremely elongated fiber cells specialized in seed epidermis, which makes it of high research and application value. To date, numerous research on cotton has navigated various aspects, from multi-genome assembly, genome editing, mechanism of fiber development, metabolite biosynthesis, and analysis to genetic breeding. Genomic and 3D genomic studies reveal the origin of cotton species and the spatiotemporal asymmetric chromatin structure in fibers. Mature multiple genome editing systems, such as CRISPR/Cas9, Cas12 (Cpf1) and cytidine base editing (CBE), have been widely used in the study of candidate genes affecting fiber development. Based on this, the cotton fiber cell development network has been preliminarily drawn. Among them, the MYB-bHLH-WDR (MBW) transcription factor complex and IAA and BR signaling pathway regulate the initiation; various plant hormones, including ethylene, mediated regulatory network and membrane protein overlap fine-regulate elongation. Multistage transcription factors targeting CesA 4, 7, and 8 specifically dominate the whole process of secondary cell wall thickening. And fluorescently labeled cytoskeletal proteins can observe real-time dynamic changes in fiber development. Furthermore, research on the synthesis of cotton secondary metabolite gossypol, resistance to diseases and insect pests, plant architecture regulation, and seed oil utilization are all conducive to finding more high-quality breeding-related genes and subsequently facilitating the cultivation of better cotton varieties. This review summarizes the paramount research achievements in cotton molecular biology over the last few decades from the above aspects, thereby enabling us to conduct a status review on the current studies of cotton and provide strong theoretical support for the future direction.
... Utilizing GenEd technologies, a number of organisms have already been genetically created for selective genetic modification e.g. Arabidopsis thaliana (Cermak et al. 2011), tomato (Brooks et al. 2014;Li et al. 2018; Bari et al. 2019), grapes (Ren et al. 2016), potato (Butler et al. 2016;Clasen et al. 2016), banana (Kaur et al. 2017;Kaur et al. 2018;Kaur et al. 2020), sorghum (Jiang et al. 2013b), soybean (Curtin et al. 2011;Jacobs et al. 2015), maize (Liang et al. 2014;Char et al. 2017), cassava (Odipio et al. 2017;Gomez et al. 2019), Citrus sinesis (Wang et al. 2019), Kiwi fruit (Wang et al. 2018a, b), wheat (Wang et al. 2014;Kim et al. 2018), rice (Li et al. 2012;Hu et al. 2016;Shufen et al. 2019), tobacco (Mahfouz et al. 2011;Gao et al. 2015), cotton (D'Halluin et al. 2013;Iqbal et al. 2016;Chen et al. 2017;Gao et al. 2017a;Wang et al. 2018a, b), bacteria (Jiang et al. 2013b, a), fungi (Liu et al. 2017), yeast (Saccharomyces cerevisiae) ), viruses (Ali et al. 2015Ji et al. 2015;Khan et al. 2018Khan et al. , 2019, drosophila (Gratz et al. 2013), mouse (Nelson et al. 2016), insects (Watanabe et al. 2017a), Caenorhabditis elegans (Cheng et al. 2013), zebrafish , rats (Tesson et al. 2011), sheep (Zhao et al. 2016), cattle (Gao et al. 2017b), goat (Zhou et al. 2017), pigs (Watanabe et al. 2017b), human cell lines (Miller et al. 2011). Advancement in gene editing technology, like base editing and prime editing which are more promising with high precision, has minimized the chance of off-targets (Mishra et al. 2020). ...
Cotton has enormous economic potential providing high-quality protein, oil, and fibre. A large increase in cotton output is necessary due to the world's changing climate and constantly expanding human population. In the past, conventional breeding techniques were used to introduce genes into superior cotton cultivars to increase production and to improve quality. The disadvantages of traditional breeding techniques are their time-consuming, reliance on genetic differences that are already present, and considerable backcrossing. To accomplish goals in a short amount of time, contemporary plant breeding techniques, in particular modern genome editing technologies (GETs), can be used. Numerous crop improvement initiatives have made use of GETs, such as zinc-finger nucleases, transcription-activator-like effector nucleases, clustered regularly interspaced palindromic repeats (CRISPR), and CRISPR-associated proteins systems (CRISPR/Cas)-based technologies. The CRISPR/Cas system has a lot of potential because it combines three qualities that other GETs lack: simplicity, competence, and adaptability. The CRISPR/Cas mechanism can be used to improve cotton tolerance to biotic and abiotic stresses, alter gene expression, and stack genes for critical features with little possibility of segregation. The transgene clean strategy improves CRISPR acceptability addressing regulatory issues associated with the genetically modified organisms (GMOs). The research opportunities for using the CRISPR/Cas system to address biotic and abiotic stresses, fibre quality, plant architecture and blooming, epigenetic changes, and gene stacking for commercially significant traits are highlighted in this article. Furthermore, challenges to use of CRISPR technology in cotton and its potential for the future are covered in detail.
... During cold acclimation, a large chromosomal deletion is attributed to the dispensable functionality of tandem arrayed CBF genes (C-repeat binding factor) in model plants . It was also reported that the tetraploid cotton genome could be edited Chen et al. 2017). In rice genomes, CRISPR/Cas9 has been extensively used for functional analysis. ...
Medicinal plants produce a heterogeneous group of natural metabolic products having distinct compounds conferring adaptive roles like in defense, attract pollinators, act as signaling agents for seed dispersal, produce molecules for abiotic stress resistance, and overall maintain an ecological balance. With the advancement of molecular tools, genetic manipulation of plants has proved to be a revolution for creation of plants with tremendous properties having traits conferring biotic and abiotic stress resistance. Metabolic engineering has made an innovative possibility to enhance the concentration of desired composites and introduction of new biotransformation pathways to a range of species that enhance the nutritive and economical value of crops and horticultural plants. To enhance research avenues in metabolic engineering, new genetic modification strategies have been developed for silencing of genes and enzyme precursors. Genome editing investigates the deep knowledge of biological systems. During the past decade, remarkable genome editing tools have been advanced utilizing nucleases like zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), and most widely clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). This chapter aims to summarize the status of research on metabolic engineering and genome editing tools in major classes of bioactive secondary metabolites with examples which have manifested as very efficacious and excellent tools for the pertinent advancement in agriculture.
