Ziyao Zhao1,2, Yaguang Qi1, Zhimin Yang1, Liyu Cheng1, Rahat Sharif1,3, Ali Raza4, Peng Chen5, Dong Hou6, Yuhong Li7. 1. College of Horticulture, Northwest A&F University, Yangling, 712100, Shaanxi, China. 2. College of Horticulture, Shanxi Agricultural University, Taigu, 030801, Shanxi, China. 3. College of Horticulture and Plant Protection, Yangzhou University, Yangzhou, 225009, Jiangsu, China. 4. Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Center of Legume Crop Genetics and Systems Biology/College of Agriculture, Oil Crops Research Institute, Fujian Agriculture and Forestry University (FAFU), Fuzhou, China. 5. College of Life Science, Northwest A&F University, Yangling, 712100, Shaanxi, China. 6. Vegetable Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou, 730070, Gansu, China. 7. College of Horticulture, Northwest A&F University, Yangling, 712100, Shaanxi, China. liyuhong73@nwsuaf.edu.cn.
Abstract
BACKGROUNDS: The narrow genetic basis of cucumber makes breeding of this species difficult. CRISPR/Cas9 system is characteristic of simple design, low cost and high efficiency, which has opened a new path for cucumber functional genetics and the development of cucumber mocular breeding. However, the immature genetic transformation system is the main limiting factor for applying this technology in cucumber. METHODS AND RESULTS: In this study, a Histochemical β-glucuronidase (GUS) assay was used to analyze the effect of various parameters, including slight scratch of explants, pre-culture time, acetosyringone (AS) concentration, infection time in Agrobacterium solution, and co-culture period on the transformation efficiency. The results showed that the explants slightly scratched after cutting, pre-cultured for 1 day, Agrobacterium bacterial solution containing AS, and 20 min length of infection could significantly increase the GUS staining rate of explants. On this basis, two sequences with high specificity (sgRNA-1 and sgRNA-2) targeted different loci of gene CsGCN5 were designed. The corresponding vectors Cas9-sgRNA-1 and Cas9-sgRNA-2 were constructed and transformed using the above-optimized cucumber genetic transformation system, and three and two PCR positive lines were obtained from 210 and 207 explants, respectively. No sequence mutation at target loci of CsGCN5 was detected in the Cas9-sgRNA-1 transformed three PCR positive lines. However, one mutant line with targeted homozygous change was recognized from the Cas9-sgRNA-2 transformed two PCR positive lines. CONCLUSION: In this study, 2.4‰ of total explants had directed mutation in the CsGCN5 gene. The results in the present study would be beneficial to further optimize and improve the efficiency of the genetic transformation of cucumber.
BACKGROUNDS: The narrow genetic basis of cucumber makes breeding of this species difficult. CRISPR/Cas9 system is characteristic of simple design, low cost and high efficiency, which has opened a new path for cucumber functional genetics and the development of cucumber mocular breeding. However, the immature genetic transformation system is the main limiting factor for applying this technology in cucumber. METHODS AND RESULTS: In this study, a Histochemical β-glucuronidase (GUS) assay was used to analyze the effect of various parameters, including slight scratch of explants, pre-culture time, acetosyringone (AS) concentration, infection time in Agrobacterium solution, and co-culture period on the transformation efficiency. The results showed that the explants slightly scratched after cutting, pre-cultured for 1 day, Agrobacterium bacterial solution containing AS, and 20 min length of infection could significantly increase the GUS staining rate of explants. On this basis, two sequences with high specificity (sgRNA-1 and sgRNA-2) targeted different loci of gene CsGCN5 were designed. The corresponding vectors Cas9-sgRNA-1 and Cas9-sgRNA-2 were constructed and transformed using the above-optimized cucumber genetic transformation system, and three and two PCR positive lines were obtained from 210 and 207 explants, respectively. No sequence mutation at target loci of CsGCN5 was detected in the Cas9-sgRNA-1 transformed three PCR positive lines. However, one mutant line with targeted homozygous change was recognized from the Cas9-sgRNA-2 transformed two PCR positive lines. CONCLUSION: In this study, 2.4‰ of total explants had directed mutation in the CsGCN5 gene. The results in the present study would be beneficial to further optimize and improve the efficiency of the genetic transformation of cucumber.
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Authors: Ricardo Oliva; Chonghui Ji; Genelou Atienza-Grande; José C Huguet-Tapia; Alvaro Perez-Quintero; Ting Li; Joon-Seob Eom; Chenhao Li; Hanna Nguyen; Bo Liu; Florence Auguy; Coline Sciallano; Van T Luu; Gerbert S Dossa; Sébastien Cunnac; Sarah M Schmidt; Inez H Slamet-Loedin; Casiana Vera Cruz; Boris Szurek; Wolf B Frommer; Frank F White; Bing Yang Journal: Nat Biotechnol Date: 2019-10-28 Impact factor: 68.164