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Öğe CRISPR-Cas9-Mediated Reduced Expression of Potato Apoplastic Invertase Inhibitor Gene and Analysis of Transformation Efficiency Parameters(Springer, 2024) Das Dangol, Sarbesh; Caliskan, Mehmet Emin; Bakhsh, AllahPathogen infections that affect potato yield cause severe economic losses every year. Many studies point to the role of apoplastic (cell wall) invertase (CWIN) enzyme in plant defence mechanisms. Apoplastic invertase inhibitor (INVINH1) post-translationally regulates the CWIN enzyme activity. Nevertheless, the role of INVINH1 needs to be elucidated for several effects in plant transformation parameters and its gene expression which we sought to explore using CRISPR/Cas9 technology. In this study, we sequenced the first exon of INVINH1 gene in cv. Desiree and Solanum chacoense M6. We identified in the first exon two alleles for StINVINH1 gene in cv. Desiree and one allele for ScINVINH1 gene in S. chacoense M6. We designed two single-guided RNAs (sgRNAs) to target INVINH1 gene from diploid S. chacoense M6 and tetraploid S. tuberosum cv. Desiree using CRISPR/Cas9-based technology. In our earlier study, we have already optimised the transformation protocol for M6 and cv. Desiree using Agrobacterium strains, based on which Agrobacterium strain AGL1 was chosen for the CRISPR/Cas9 experiment. Our experimentation showed that heat stress at 37 degrees C could increase the mutagenesis capability and CRISPR/Cas9 targeting affected the plant transformation parameters. It was found from the knockout experiment that the indels were present in the calli and the candidate regenerated plants showed reduced gene expression level conducted via RT-qPCR. Our study demonstrated that INVINH1 targeting affected the calli induction and regeneration rates, was effective under heat stress and reduced its gene expression level. More studies are required to comprehend the function of the INVINH1 enzyme in potato stress response and defence mechanism.Öğe CRISPR/Cas9: A New Genome Editing Tool to Accelerate Cotton (Gossypium spp.) Breeding(Springer International Publishing Ag, 2019) Sattar, Muhammad N.; Iqbal, Zafar; Das Dangol, Sarbesh; Bakhsh, AllahCotton has a tremendous economic value worldwide due to its high-quality fiber, edible oil and protein contents. However, the intensifying scenario of human population expansion and global environmental changes demand a proportionate increase in cotton production. In the past, several successful attempts have been made by introgression of many quality- and yield-related traits into elite cotton cultivars through conventional breeding. However, those measures are time consuming due to the reliance on introgression of naturally-existing genetic variation through extensive backcrossing. Nonetheless, plant breeding can be accelerated through modern genome editing (GE) tools. Various GE techniques including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeats and CRISPR-associated proteins systems (CRISPR/Cas)-based approaches have been successfully employed for various crop plants. Among them, CRISPR/Cas-based approaches hold great GE potential due to their simplicity, competency and versatility. In cotton, this system can regulate the gene expression associated with quality traits, to circumscribe phytopathogens and/or to stack molecular traits at a desired locus. In gene stacking through site-specific endonucleases, the desired genes can be introduced in close proximity to a specific locus in the cotton genome with a low risk of segregation. However, such executions are tedious to achieve through classical breeding techniques. Moreover, through the CRISPR/Cas-based approaches, transgene-free cotton plants can easily be produced by selfing or backcrossing to meet the current genetically modified organisms (GMO) guidelines. In this chapter, we address the potential application of CRISPR/Cas-based approaches in available whole cotton genomes to sustain cotton productivity, and achieve genetic improvement, pathogen resistance and agronomic traits. Future prospects of GE applications in cotton breeding are also addressed.Öğe Genome editing of potato using CRISPR technologies: current development and future prospective(Springer, 2019) Das Dangol, Sarbesh; Barakate, Abdellah; Stephens, Jennifer; Caliskan, Mehmet Emin; Bakhsh, AllahPotato (Solanum tuberosum L.) has tremendous significance due to its nutritional quality. The mounting pressure of increasing population further reinforces its importance as potato is believed to be a vital crop to meet food needs for population growth. Although conventional approaches of breeding, irradiation/mutagens and introgression of quality and yield related traits have improved potato yield, biotic and abiotic stresses continue to impose crop damages. Modern tools such as CRISPR/Cas have assisted plant scientists in accelerating breeding processes by providing new, simple, versatile and robust technologies. These tools make it possible to eliminate traits that are involved in negative regulation of quality and yield parameters. Besides that, genes of interest can also be introduced in close proximity to specific loci that may remain linked throughout the generations. This review focuses on the endeavors, applications and prospects of CRISPR/Cas-based approaches in potato with the potential to increase sustainable crop productivity. Key message This manuscript focuses the endeavors, applications and prospects of CRISPR/Cas-based approaches in potato with the potential to increase sustainable crop productivity.Öğe Manipulating genome of diploid potato inbred line Solanum chacoense M6 using selectable marker gene(Tubitak Scientific & Technological Research Council Turkey, 2020) Das Dangol, Sarbesh; Yel, Ilknur; Caliskan, Mehmet Emin; Bakhsh, AllahThe development of transgenic potatoes is imperative to investigate various gene functions as well as to develop robust potato varieties resistant to different biotic and abiotic stresses. Directing a potato breeding program from cultivated tetraploids to self-compatible diploid lines will be highly helpful for potato breeders. However, diploid potatoes are considered recalcitrant to regeneration. We aimed to develop a protocol for the transformation of diploid Solanum chacoense M6 potatoes using leaf, internodes, and microtubers as the explants. Agrobacterium tumefaciens strain GV2260 harboring pIBIN19 expression vector containing gusA gene (interrupted by an intron to induce expression from eukaryotic cells) was used for this purpose. Different inoculation times (10, 20, 30 min) were applied in aforementioned explants for gene transfer. After cocultivation, explants were transferred to the media with various hormone concentrations [6-benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA), trans-zeatin, kinetin, and 2, 4-Dichlorophenoxyacetic acid (2,4-D)]. The calli generated were then transferred to shoot generating medium supplemented with thidiazuron (TDZ) and gibberellic acid (GA(3)). According to histochemical GUS analysis, we found a 20 min inoculation time to be optimal for gene transfer and the medium containing 2 mg L-1 BAP and 2 mg L-1 NAA was the most suitable medium for callus induction from 20 min inoculated explants (41% callus formation for internodes and 65% for leaf explants). Abundant transcripts levels of gusA gene was found in primary transfonnants when subjected to RTAPCR. GUS fluorometric assay further confirmed the primary transfonnants at protein level. The present study can serve as a gateway to transfer gene(s) of interest in diploid potatoes.
