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.

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.

scholarly journals L-Cysteine Increases the Transformation Efficiency of Chinese Cabbage (Brassica rapa ssp. pekinensis)

2021 ◽  
Vol 12 ◽  
Author(s):  
Ganeshan Sivanandhan ◽  
Jiae Moon ◽  
Chaemin Sung ◽  
Solhee Bae ◽  
Zhi Hong Yang ◽  
...  
Keyword(s):  
Chinese Cabbage ◽  
Transformation Efficiency ◽  
Transformation Method ◽  
Gus Expression ◽  
Chain Reaction ◽  
Functional Studies ◽  
Cotyledon Explants ◽  
Polymerase Chain ◽  
Subsequent Regeneration

Successful Agrobacterium-mediated transformations of Chinese cabbage have been limited owing to the plant’s recalcitrant nature, genomic background and explant necrosis upon infection, which hinders the transfer of T-DNA region into the Chinese cabbage. Consequently, in the current experiment, a stable Agrobacterium tumefaciens-mediated transformation method for Chinese cabbage cv. Kenshin established by employing important anti-oxidants in the co-cultivation and subsequent regeneration media. Four-day-old in vitro derived cotyledon explants were infected with A. tumefaciens strain GV3101 harboring the vector pCAMIBA1303. Cotyledon explants exposed to an Agrobacterium suspension (OD600 of approximately 0.6) for 10 min and then incubated for 3 days co-cultivation in Murashige and Skoog medium containing an L-cysteine + AgNO3 combination exhibited the highest β-glucuronidase (GUS) expression (94%) and explant regeneration efficiency (76%). After 3 days, the cotyledon explants were subjected to three selection cycles with gradually increasing hygromycin B concentrations (10 to 12 mg/L). The incorporation and expression of hptII in T0 transformed plants were verified by polymerase chain reaction and Southern blot analyses. These transgenic plants (T0) were fertile and morphologically normal. Using the present protocol, a successful transformation efficiency of 14% was achieved, and this protocol can be applied for genome editing and functional studies to improve Chinese cabbage traits.

Transformation of Sinorhizobium meliloti MTCC 100 and Mesorhizobium ciceri TAL 620 by CaCl2 method

2010 ◽  
Vol 56 (1) ◽  
pp. 74-76 ◽  
Author(s):  
Urmi Patel ◽  
Sarika Sinha
Keyword(s):  
Escherichia Coli ◽  
Optical Density ◽  
Sinorhizobium Meliloti ◽  
Transformation Efficiency ◽  
Binary Vector ◽  
Transformation Method ◽  
Mesorhizobium Ciceri ◽  
Competent Cells

The CaCl2 method, commonly used for transformation of Escherichia coli, was modified and used to develop a simpler and easier transformation method for Rhizobia sp. Two species of Rhizobia, Sinorhizobium meliloti MTCC 100 and Mesorhizobium ciceri TAL 620, were transformed with the 13.2 kb binary vector pGA482. At an optical density of 0.4, the transformation efficiencies in Sinorhizobium meliloti MTCC 100 and Mesorhizobium ciceri TAL 620 were 104 and 103, respectively. Competent cells of Sinorhizobium meliloti MTCC 100 were prepared at different growth intervals and transformed by the same vector. A maximum transformation efficiency of 104 was achieved at an optical density of 0.5.

scholarly journals Optimization of Cell-Based cDNA Microarray Conditions for Gene Functional Studies in HEK293 Cells

2017 ◽  
Vol 22 (8) ◽  
pp. 1053-1059
Author(s):  
Hi Chul Kim ◽  
Jin Yeong Heo ◽  
Tae-Kyu Lee ◽  
Ssang-Goo Cho ◽  
Yong-Jun Kwon
Keyword(s):  
Cdna Microarray ◽  
Time Course ◽  
Transfection Efficiency ◽  
Final Concentration ◽  
Hek293 Cells ◽  
Functional Studies ◽  
Transfection Reagent ◽  
Reverse Transfection ◽  
The Stability ◽  
Lipofectamine 2000

Since the cell-based cDNA microarray (CBCM) technique has been a useful tool for gain-of-function studies, many investigators have used CBCMs to identify interesting genes. However, this method requires better-established conditions to ensure high reverse transfection efficiency without cross-contamination. Therefore, we optimized CBCM techniques through various means. We determined that Lipofectamine 2000 was the most appropriate transfection reagent by evaluating eight commercialized reagents, and we determined that the most effective concentrations for printing solution constituents were 0.2 M sucrose (to yield a final concentration of 32 mM) and 0.2% gelatin (to yield a final concentration 0.075%). After examining various combinations, we also determined that the best concentrations of cDNA and transfection reagent for optimal reverse transfection efficiency were 1.5 µg/5 µL of cDNA and 5.5 µL of Lipofectamine 2000. Finally, via a time course, we determined that 72 h was the most effective reaction duration for reverse transfection, and we confirmed the stability of cDNA spot activity of CBCMs for various storage periods. In summary, the CBCM conditions that we have identified can provide more effective outcomes for cDNA reverse transfection on microarrays.

scholarly journals Design and Validation of a Process Based on Cationic Niosomes for Gene Delivery into Novel Urine-Derived Mesenchymal Stem Cells

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 696
Author(s):  
Yerai Vado ◽  
Gustavo Puras ◽  
Melania Rosique ◽  
Cesar Martin ◽  
Jose Luis Pedraz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Intracellular Localization ◽  
Cell Types ◽  
Functional Studies ◽  
Cellular Models ◽  
Cationic Niosomes

Background: Mesenchymal stem cells (MSCs) are stem cells present in adult tissues. They can be cultured, have great growth capacity, and can differentiate into several cell types. The isolation of urine-derived mesenchymal stem cells (hUSCs) was recently described. hUSCs present additional benefits in the fact that they can be easily obtained noninvasively. Regarding gene delivery, nonviral vectors based on cationic niosomes have been used and are more stable and have lower immunogenicity than viral vectors. However, their transfection efficiency is low and in need of improvement. Methods: We isolated hUSCs from urine, and the cell culture was tested and characterized. Different cationic niosomes were elaborated using reverse-phase evaporation, and they were physicochemically characterized. Then, they were screened into hUSCs for transfection efficiency, and their internalization was evaluated. Results: GPxT-CQ at a lipid/DNA ratio of 5:1 (w/w) had the best transfection efficiency. Intracellular localization studies confirmed that nioplexes entered mainly via caveolae-mediated endocytosis. Conclusions: In conclusion, we established a protocol for hUSC isolation and their transfection with cationic niosomes, which could have relevant clinical applications such as in gene therapy. This methodology could also be used for creating cellular models for studying and validating pathogenic genetic variants, and even for performing functional studies. Our study increases knowledge about the internalization of tested cationic niosomes in these previously unexplored cells.

scholarly journals Identification of biological characteristics and preliminary research on genetic transformation of the Aspergillus niger TL8 strain isolated in Vietnam

2017 ◽  
Vol 33 (2S) ◽  
Author(s):  
Do Thi Binh Xuan Loc ◽  
Tran Van Tuan
Keyword(s):  
Aspergillus Niger ◽  
Extracellular Enzymes ◽  
Carbon Sources ◽  
Morphological Characteristics ◽  
Transformation Method ◽  
Plant Materials ◽  
Rdna Its ◽  
Black Mold ◽  
Gfp Reporter Gene

Aspergillus niger is a mold commonly used in industrial production of many enzymes and organic acids. Because this fungus can produce different extracellular enzymes to degrade plant materials, it also causes the damages for some agricultural products at postharvest stages. In this study, we isolated a black mold strain named TL8 from a decayed dragon fruit. Based on morphological characteristics and the rDNA ITS (internal transcribed spacer) sequence, the TL8 strain was identified as A. niger. The A. niger TL8 strain is able to use different carbon sources for the growth and decay the peel of dragon fruits in vitro. In order to establish the basis for future studies on the mechanism of plant material decomposition of the fungus, we have successfully transferred and expressed the GFP reporter gene in this A. niger strain using the Agrobacterium tumefaciens-mediated transformation method and the hygromycin resistance marker.

scholarly journals Creating a novel petal regeneration system for function identification of colour gene of grape hyacinth

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Qian Lou ◽  
Hongli Liu ◽  
Wen Luo ◽  
Kaili Chen ◽  
Yali Liu
Keyword(s):  
Transformation Method ◽  
Breeding Cycle ◽  
Regeneration System ◽  
Flower Bud ◽  
Juvenile Period ◽  
Colour Gene ◽  
Function Identification ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
Grape Hyacinth

Abstract Background Grape hyacinth (Muscari spp.) is one of the most important ornamental bulbous plants. However, its lengthy juvenile period and time-consuming transformation approaches under the available protocols impedes the functional characterisation of its genes in flower tissues. In vitro flower organogenesis has long been used to hasten the breeding cycle of plants but has not been exploited for shortening the period of gene transformation and characterisation in flowers. Results A petal regeneration system was established for stable transformation and function identification of colour gene in grape hyacinth. By culturing on Murashige and Skoog medium (MS) with 0.45 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 8.88 μM 6-benzyladenine (6-BA), during the colour-changing period, the flower bud explants gave rise to regeneration petals in less than 3 months, instead of the 3 years required in field-grown plants. By combining this system with Agrobacterium-mediated transformation, a glucuronidase reporter gene (GUS) was delivered into grape hyacinth petals. Ultimately, 214 transgenic petals were regenerated from 24 resistant explants. PCR and GUS quantitative analyses confirmed that these putative transgenic petals have stably overexpressed GUS genes. Furthermore, an RNAi vector of the anthocyanidin 3-O-glucosyltransferase gene (MaGT) was integrated into grape hyacinth petals using the same strategy. Compared with the non-transgenic controls, reduced expression of the MaGT occurred in all transgenic petals, which caused pigmentation loss by repressing anthocyanin accumulation. Conclusion The Agrobacterium transformation method via petal organogenesis of grape hyacinth took only 3–4 months to implement, and was faster and easier to perform than other gene-overexpressing or -silencing techniques that are currently available.

scholarly journals Agrobacterium-Mediated Capsicum annuum Gene Editing in Two Cultivars, Hot Pepper CM334 and Bell Pepper Dempsey

2021 ◽  
Vol 22 (8) ◽  
pp. 3921
Author(s):  
Sung-il Park ◽  
Hyun-Bin Kim ◽  
Hyun-Ji Jeon ◽  
Hyeran Kim
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Capsicum Annuum ◽  
Gene Editing ◽  
Binary Vector ◽  
Transformation Method ◽  
Bell Pepper ◽  
Hot Pepper ◽  
Genome Sequences ◽  
Agrobacterium Strain ◽  
Cultivated Species

Peppers (Capsicum annuum L.) are the most widespread and cultivated species of Solanaceae in subtropical and temperate countries. These vegetables are economically attractive worldwide. Although whole-genome sequences of peppers and genome-editing tools are currently available, the precision editing of peppers is still in its infancy because of the lack of a stable pepper transformation method. Here, we employed three Agrobacterium tumefaciens strains—AGL1, EHA101, and GV3101—to investigate which Agrobacterium strain could be used for pepper transformation. Hot pepper CM334 and bell pepper Dempsey were chosen in this study. Agrobacterium tumefaciens GV3101 induced the highest number of calli in cv. Dempsey. All three strains generated similar numbers of calli for cv. CM334. We optimized a suitable concentration of phosphinothricin (PPT) to select a CRISPR/Cas9 binary vector (pBAtC) for both pepper types. Finally, we screened transformed calli for PPT resistance (1 and 5 mg/L PPT for cv. CM334 and Dempsey, respectively). These selected calli showed different indel frequencies from the non-transformed calli. However, the primary indel pattern was consistent with a 1-bp deletion at the target locus of the C. annuumMLO gene (CaMLO2). These results demonstrate the different sensitivity between cv. CM334 and Dempsey to A. tumefaciens-mediated callus induction, and a differential selection pressure of PPT via pBAtC binary vector.

scholarly journals Cell Penetrating Peptides Mediated DNA-Free Transfection in Arabidopsis and Chinese Cabbage

2021 ◽  
Author(s):  
Han Wu ◽  
Zhipeng Zhang ◽  
Kai Zhu ◽  
Fulai Ke ◽  
Fei Zhang ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Genome Editing ◽  
Chinese Cabbage ◽  
Dna Sequences ◽  
Room Temperature ◽  
Genetically Modified Organisms ◽  
Transfection Efficiency ◽  
Cell Penetrating Peptides ◽  
Root Tips ◽  
Cell Penetrating

Abstract Background: The third-generation genome editing system CRISPR/Cas had shown strong application prospects in crop genetic improvement. However, this technology largely depends on genetic transformation. Public concerns on GMO (genetically modified organisms) safety, as well as related regulations, have restricted the application. Therefore, establishing DNA-free transfection system is important to promote CRISPR/Cas genome editing in agriculture.Results：In this paper, cell penetrating peptides fusion protein (CPP-mCherry) was found to be effective on DNA-free transfection. DNA sequences of nine tandem arginine (R9), one cysteine (cys), reporter mCherry and histidine label were sequentially constructed into pET 45B+ expression vector and transformed into Escherichia coli BL21(DE3) strain. CPP-mCherry fusion protein can be induced by 1mM IPTG for at least 1 hour in 28 °C. CPP-mCherry fusion protein can be obtained by 200W ultrasonication, then purified by Ni column and MWCO dialysis. The Arabidopsis thaliana root tips and leaves, as well as Chinese cabbage microspores and 3-week-old microspore embryo can be used as transfected recipient. Concentration can be selected between 10-100μg/ml and incubated overnight at room temperature. R9-cys-mCherry protein can be translocated into the nucleus of microspore. The transfection efficiency of root tips reached 100% and of microspore and MDE was 8.13% and 94.79%, respectively. Conclusions: Here, a CPP mediated DNA-free transfection system was built in dicots. These results lay a technical foundation of DNA-free genome editing.

scholarly journals A simplified method to isolate rice mitochondria

2020 ◽  
Author(s):  
Yanghong Xu ◽  
Xiaoyi Li ◽  
Jishuai Huang ◽  
Leilei Peng ◽  
Dinghui Luo ◽  
...  
Keyword(s):  
Genome Structure ◽  
Plant Mitochondria ◽  
Extraction Methods ◽  
Protoplast Isolation ◽  
Physiological Status ◽  
Time Saving ◽  
Plant Mitochondrial Genome ◽  
Protein Levels ◽  
Modified Method ◽  
Simplified Method

Abstract Background: Mitochondria play critical roles in plant growth, development and stress tolerance. Numerous researchers have carried out studies on the plant mitochondrial genome structure, mitochondrial metabolism and nuclear-cytoplasmic interactions. However, classical plant mitochondria extraction methods are time-consuming and consist of a complicated ultracentrifugation procedure with expensive reagents. To develop a more rapid and convenient method for the isolation of plant mitochondria, in this study, we established a simplified method to isolate rice mitochondria efficiently for subsequent studies.Results: To isolate rice mitochondria, the cell wall was first disrupted by enzymolysis to obtain the protoplast, which is similar to animal mitochondria. Rice mitochondria were then isolated with a modified method based on the animal mitochondria isolation protocol. The extracted mitochondria were next assessed according to DNA and protein levels to rule out contamination by the nucleus and chloroplasts. Furthermore, we examined the physiological status and characteristics of the isolated mitochondria, including the integrity of mitochondria, the mitochondrial membrane potential, and the activity of inner membrane complexes. Our results demonstrated that the extracted mitochondria remained intact for use in subsequent studies.Conclusion: The combination of plant protoplast isolation and animal mitochondria extraction methods facilitates the extraction of plant mitochondria without ultracentrifugation. Consequently, this improved method is cheap and time-saving with good operability and can be broadly applied in studies on plant mitochondria.

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