Direct introduction of neomycin phosphotransferase II protein into apple leaves to confer kanamycin resistance

Numata, Keiji; Horii, Yoko; Motoda, Yoko; Hirai, Narumi; Nishitani, Chikako; Watanabe, Satoru; Kigawa, T.; Kodama, Yutaka

doi:10.5511/plantbiotechnology.16.0929a

Cited by 13 publications

(12 citation statements)

References 20 publications

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“…electroporated a stress-related protein ERD14 into the cell cytoplasm of tobacco BY-2 cells 29 , although the microscopic observation of a fluorescent probe does not unequivocally prove that intracellular delivery was successful, as the proteins fused with fluorescent proteins do not often reflect their original localization 33 . In addition, if the protein of interest can exert its function outside of the cell nucleus or cytoplasm, such as the endosome or intercellular gap, the protein may appear but not actually localize to the cell nucleus or cytoplasm 34 . Thus, one must pay special attention to proving the intracellular delivery of biomaterials.…”

Section: Resultsmentioning

confidence: 99%

A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall

Furuhata

Sakai

Murakami

et al. 2019

Sci Rep

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Genome engineering in plants is highly dependent on the availability of effective molecular techniques. Despite vast quantities of research, genome engineering in plants is still limited in terms of gene delivery, which requires the use of infectious bacteria or harsh conditions owing to the difficulty delivering biomaterial into plant cells through the cell wall. Here, we describe a method that uses electroporation-mediated protein delivery into cultured Arabidopsis thaliana cells possessing an intact cell wall, and demonstrate Cre-mediated site-specific recombination. By optimizing conditions for the electric pulse, protein concentration, and electroporation buffer, we were able to achieve efficient and less-toxic protein delivery into Arabidopsis thaliana cells with 83% efficiency despite the cell wall. To the best of our knowledge, this is the first report demonstrating the electroporation-mediated protein delivery of Cre recombinase to achieve nucleic acid-free genome engineering in plant cells possessing an intact cell wall.

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Section: Resultsmentioning

confidence: 99%

A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall

Furuhata

Sakai

Murakami

et al. 2019

Sci Rep

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“…One example of CPP applications in plant biotechnology is transient modifications of plants using chemicals and enzymes. We previously introduced neomycin phosphotransferase II (NPTII) into apple leaf cells to add transient kanamycin resistance using the fusion peptide system 37 . Moreover, gene delivery systems for plant cells have been developed using CPP as a part of carrier molecules, indicating that CPP can be combined with the other functional molecules and will become versatile tools for gene delivery and transformation of plants without requiring special equipment.…”

Section: Resultsmentioning

confidence: 99%

Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus

Numata

Horii

Oikawa

et al. 2018

Sci Rep

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Cell-penetrating peptides (CPPs) are used for various applications, especially in the biomedical field. Recently, CPPs have been used as a part of carrier to deliver proteins and/or genes into plant cells and tissues; hence, these peptides are attractive tools for plant biotechnological and agricultural applications, but require more efficient delivery rates and optimization by species before wide-scale use can be achieved. Here, we developed a library containing 55 CPPs to determine the optimal CPP characteristics for penetration of BY-2 cells and leaves of Nicotiana benthamiana, Arabidopsis thaliana, tomato (Solanum lycopersicum), poplar (hybrid aspen Populus tremula × tremuloides line T89), and rice (Oryza sativa). By investigating the cell penetration efficiency of CPPs in the library, we identified several efficient CPPs for all the plants studied except rice leaf. In the case of rice, several CPPs showed efficient penetration into rice callus. Furthermore, we examined the relationship between cell penetration efficiency and CPP secondary structural characteristics. The cell penetration efficiency of Lys-containing CPPs was relatively greater in plant than in animal cells, which could be due to differences in lipid composition and surface charge of the cell membranes. The variation in optimal CPPs across the plants studied here suggests that CPPs must be optimized for each plant species and target tissues of interest.

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“…In the future, nanoparticles may become new tools for the delivery of RNPs into plants for genetic engineering regardless of plant species. Other delivery methods demonstrated in plants include the modification of proteins with cell-penetrating materials, such as cell-penetrating peptides ( Numata et al., 2016 ; Guo et al., 2019 ; Midorikawa et al., 2019 ). These could potentially be used for RNP delivery into plant cells, as has been demonstrated previously for human cells ( Ramakrishna et al., 2014 ).…”

Section: Challenges and Future Perspectivesmentioning

confidence: 99%

CRISPR ribonucleoprotein-mediated genetic engineering in plants

Zhang

Iaffaldano

2021

Plant Communications

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CRISPR-derived biotechnologies have revolutionized the genetic engineering field and have been widely applied in basic plant research and crop improvement. Commonly used Agrobacterium - or particle bombardment-mediated transformation approaches for the delivery of plasmid-encoded CRISPR reagents can result in the integration of exogenous recombinant DNA and potential off-target mutagenesis. Editing efficiency is also highly dependent on the design of the expression cassette and its genomic insertion site. Genetic engineering using CRISPR ribonucleoproteins (RNPs) has become an attractive approach with many advantages: DNA/transgene-free editing, minimal off-target effects, and reduced toxicity due to the rapid degradation of RNPs and the ability to titrate their dosage while maintaining high editing efficiency. Although RNP-mediated genetic engineering has been demonstrated in many plant species, its editing efficiency remains modest, and its application in many species is limited by difficulties in plant regeneration and selection. In this review, we summarize current developments and challenges in RNP-mediated genetic engineering of plants and provide future research directions to broaden the use of this technology.

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Direct introduction of neomycin phosphotransferase II protein into apple leaves to confer kanamycin resistance

Cited by 13 publications

References 20 publications

A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall

A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall

Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus

CRISPR ribonucleoprotein-mediated genetic engineering in plants

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