Protoplast Isolation, Culture and Plant Formation of Psycomitrella

1983 ◽  
pp. 42-43
Author(s):  
A. Batra ◽  
W. O. Abel
Keyword(s):  
Protoplast Isolation

Protoplast Isolation and Fusion Induced by PEG with Leaves and Roots of Tea Plant (Camellia sinensis L. O. Kuntze)

2018 ◽  
Vol 44 (3) ◽  
pp. 463 ◽  
Author(s):  
Zhang PENG ◽  
Hua-Rong TONG ◽  
Guo-Lu LIANG ◽  
Yi-Qi SHI ◽  
Lian-Yu YUAN
Keyword(s):  
Camellia Sinensis ◽  
Protoplast Isolation ◽  
Tea Plant ◽  
Leaves And Roots

Callus induction, suspension culture and protoplast isolation in Camellia oleifera

2021 ◽  
Vol 286 ◽  
pp. 110193
Author(s):  
Su-Fang Li ◽  
Tian-Wen Ye ◽  
Xin Xu ◽  
De-Yi Yuan ◽  
Shi-Xin Xiao
Keyword(s):  
Suspension Culture ◽  
Callus Induction ◽  
Protoplast Isolation ◽  
Camellia Oleifera

scholarly journals Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2

2022 ◽  
Vol 23 (2) ◽  
pp. 837
Author(s):  
Sudip Biswas ◽  
Nancy J. Wahl ◽  
Michael J. Thomson ◽  
John M. Cason ◽  
Bill F. McCutchen ◽  
...  
Keyword(s):  
Genome Editing ◽  
New Technologies ◽  
Fluorescent Protein ◽  
Peanut Butter ◽  
Processing System ◽  
Protoplast Isolation ◽  
Sequencing Analysis ◽  
Ara H 2

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

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.

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