Establishment of Agrobacterium-mediated genetic transformation and application of CRISPR/Cas9 gene-editing system to Chinese cabbage (Brassica rapa L. ssp. pekinensis)

2020 ◽  
Author(s):  
Xiaonan Li ◽  
Haiyan Li ◽  
Yuzhu Zhao ◽  
Peixuan Zong ◽  
Zongxiang Zhan ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Brassica Rapa ◽  
Chinese Cabbage ◽  
Transformation Method ◽  
Sequencing Analysis ◽  
Related Gene ◽  
Transformation System ◽  
Regeneration System ◽  
Regeneration Rate ◽  
Functional Studies

Abstract Background: Chinese cabbage, belonging to Brassica rapa species, is an important vegetable in Eastern Asia. It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low. The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies.Results: In this study, we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors, with 93.50% regeneration rate. Based on this, a simple and efficient Agrobacterium-mediated genetic transformation method was established, without a pre-culture procedure and concentration adjustment of hormone and AgNO3 in co-cultivation and selection media. Using this system, transformants could be obtained within 3.5 to 4.0 months. Average transformation frequency is up to 10.83%. Furthermore, using this transformation system, the CRISPR/Cas9 technology was successfully applied in Chinese cabbage by knocking out a self-incompatibility-related gene SRK. Gene sequencing analysis in the positive transgenic lines revealed various mutations, including deletions, insertions, and substitutions. Conclusion: A simple, stable and efficient genetic transformation method was established for Chinese cabbage and successfully applied to the CRISPR/Cas9 system. The results of this study pave the way for further gene functional studies and genome editing in Chinese cabbage.

Establishment of regeneration system and transformation of Zm401 gene in Lilium longiflorum×L. formosanum

2008 ◽  
Vol 5 (2) ◽  
pp. 113-119 ◽  
Author(s):  
Li Qiu-Hua ◽  
Hong Bo ◽  
Tong Zheng ◽  
Ma Chao ◽  
Guan Ai-Nong ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Lilium Longiflorum ◽  
Bacterial Suspension ◽  
Specific Gene ◽  
Naphthaleneacetic Acid ◽  
Transformation System ◽  
Regeneration System ◽  
Regeneration Rate ◽  
Maize Pollen ◽  
Genetic Transformation System

AbstractIn vitro bulb scales of Lilium longiflorum×L. formosanum were used as explants to develop a highly efficient regeneration system. A high regeneration rate (100%) was reached through organogenesis on basal Murashige and Skoog (MS) medium supplemented with 1.0 mg/l 6-benzylaminopurine (6-BA) and 1.0 mg/l naphthaleneacetic acid (NAA). A genetic transformation system for the lily was developed using an Agrobacterium tumefaciens-mediated method. An improved genetic transformation rate (12‰) was obtained when the explants were pre-cultured for 3 days, immersed in bacterial suspension (OD600≈0.8) for 5 min, and co-cultivated for 5 days. The binary vector pBI121 containing Zm401, a maize pollen-specific gene, was introduced into the Agrobacterium strain LBA4404 and transformed into the explants using the genetic transformation system. Gene integration into the lily genome was confirmed by polymerase chain reaction (PCR) and PCR–Southern analysis. These results could lead to the production of new pollenless lily plants.

scholarly journals Establishment of a Genetic Transformation System in Guanophilic Fungus Amphichorda guana

2021 ◽  
Vol 7 (2) ◽  
pp. 138
Author(s):  
Min Liang ◽  
Wei Li ◽  
Landa Qi ◽  
Guocan Chen ◽  
Lei Cai ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Genetic Manipulation ◽  
Major Facilitator Superfamily ◽  
Protoplast Regeneration ◽  
Transformation System ◽  
Regeneration Rate ◽  
Bioactive Natural Products ◽  
Genetics Research ◽  
Genetic Transformation System ◽  
Major Facilitator

Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus Amphichorda guana LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene AG04914 encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi.

scholarly journals Optimization of Protoplast Isolation from Leaf Mesophylls of Chinese cabbage (Brassica rapa ssp. pekinensis) and Subsequent Transfection with a Binary Vector

Plants ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 2636
Author(s):  
Ganeshan Sivanandhan ◽  
Solhee Bae ◽  
Chaemin Sung ◽  
Su Ryun Choi ◽  
Geung-Joo Lee ◽  
...  
Keyword(s):  
Chinese Cabbage ◽  
Binary Vector ◽  
Transfection Efficiency ◽  
Genome Structure ◽  
Transformation Method ◽  
Protoplast Isolation ◽  
Breeding Cycle ◽  
Functional Studies ◽  
Gfp Reporter Gene ◽  
Protoplast Transfection

Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and the lack of an efficient plant transformation protocol. Thus, a protoplast transfection-based transformation method may be useful for cell-based breeding and functional studies involving Chinese cabbage plants. In this study, we established an effective method for isolating Chinese cabbage protoplasts, which were then transfected with the pCAMBIA1303 binary vector according to an optimized PEG-based method. More specifically, protoplasts were isolated following a 4 h incubation in a solution comprising 1.5% (v/v) cellulase, 0.25% (v/v) macerozyme, 0.25% (v/v) pectinase, 0.5 M mannitol, 15 mM CaCl2, 25 mM KCl, 0.1% BSA, and 20 mM MES buffer, pH 5.7. This method generated 7.1 × 106 protoplasts, 78% of which were viable. The gfp reporter gene in pCAMBIA1303 was used to determine the transfection efficiency. The Chinese cabbage protoplast transfection rate was highest (68%) when protoplasts were transfected with the 40 µg binary vector for 30 min in a solution containing 40% PEG. The presence of gusA and hptII in the protoplasts was confirmed by PCR. The methods developed in this study would be useful for DNA-free genome editing as well as functional and molecular investigations of Chinese cabbage.

scholarly journals Multiplex CRISPR/Cas9 Mutagenesis of BrVRN1 Delays Flowering Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Agriculture ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1286
Author(s):  
Joon Ki Hong ◽  
Eun Jung Suh ◽  
Sang Ryeol Park ◽  
Jihee Park ◽  
Yeon-Hee Lee
Keyword(s):  
Flowering Time ◽  
Brassica Rapa ◽  
Chinese Cabbage ◽  
Crop Improvement ◽  
Sequencing Analysis ◽  
Vrn1 Gene ◽  
Floral Repressor ◽  
Dna Sequencing Analysis ◽  
Delayed Flowering ◽  
Golden Gate Cloning

The VERNALIZATION1 (VRN1) gene is a crucial transcriptional repressor involved in triggering the transition to flowering in response to prolonged cold. To develop Chinese cabbage (Brassica rapa L. ssp. pekinensis) plants with delayed flowering time, we designed a multiplex CRISPR/Cas9 platform that allows the co-expression of four sgRNAs targeting different regions of the endogenous BrVRN1 gene delivered via a single binary vector built using the Golden Gate cloning system. DNA sequencing analysis revealed site-directed mutations at two target sites: gRNA1 and gRNA2. T1 mutant plants with a 1-bp insertion in BrVRN1 exhibited late flowering after the vernalization. Additionally, we identified ‘transgene-free’ BrVRN1 mutant plants without any transgenic elements from the GE1 (gene-editing 1) and GE2 generations. All GE2 mutant plants contained successful edits in two out of three BrVRN1 orthologs and displayed delayed flowering time. In GE2 mutant plants, the floral repressor gene FLC1 was expressed during vernalization; but the floral integrator gene FT was not expressed after vernalization. Taken together, our data indicate that the BrVRN1 genes act as negative regulators of FLC1 expression during vernalization in Chinese cabbage, raising the possibility that the ‘transgene-free’ mutants of BrVRN1 developed in this study may serve as useful genetic resources for crop improvement with respect to flowering time regulation.

scholarly journals Transcriptome and small RNA combined sequencing analysis of cold tolerance response in non-heading Chinese cabbage

2020 ◽  
Author(s):  
Jin Wang ◽  
Qinxue Zhang ◽  
Xiong You ◽  
Xilin Hou
Keyword(s):  
Gene Expression ◽  
Cold Tolerance ◽  
Brassica Rapa ◽  
Chinese Cabbage ◽  
Small Rna ◽  
Target Genes ◽  
Differentially Expressed ◽  
Sequencing Analysis ◽  
Heading Chinese Cabbage ◽  
Gene Expression Levels

Abstract Background: Non-heading Chinese cabbage ( Brassica rapa ssp. chinensis ), as an important leaf vegetable grown worldwide. However, there is currently no enough transcriptome and small RNA combined sequencing analysis of cold tolerance, which hinders further functional genomics research.Results: In this study, 63.43 Gb of clean data was obtained from the transcriptome analysis. The clean data of each sample reached 6.99 Gb, and the basic percentage of Q30 was 93.68% and above. The clean reads of each sample were sequence aligned with the designated reference genome ( Brassica rapa, IVFCAASv1 ), and the efficiency of the alignment varied from 81.54% to 87.24%. According to the comparison results, 1,860 new genes were discovered, of which 1,613 were functionally annotated. Among them, 13 common differentially expressed genes were detected in all materials, including 7 up-regulated and 6 down-regulated. At the same time, we used quantitative real-time PCR to confirm the changes of these gene expression levels. In addition, we sequenced miRNA of the same material. Our findings revealed a total of 34,182,333 small RNA reads, 88,604,604 kinds of small RNA, among which the most common size was 24 nt. In all materials, the number of common differential miRNAs is 8. According to the corresponding relationship between miRNA and its target genes, we carried out Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis on the set of target genes that each group of differentially expressed miRNAs. Through the analysis, it is found that the distribution of candidate target genes in different materials is different. We not only use transcriptome sequencing and small RNA sequencing, but also use experiment to prove that the expression level of differentially expressed genes are obtained by sequencing. Sequencing combined with experiments proved the mechanism of some differential gene expression levels after low temperature treatment.Conclusions: In all, this study provides a resource for genetic and genomic research under abiotic stress in Pak-choi.

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