High efficiency transient and stable transformation by optimized DNA microinjection into<i>Nicotiana tabacum</i>protoplasts

Kost, Benedikt; Galli, Alessandro; Potrykus, Ingo; Neuhaus, Gunther

doi:10.1093/jxb/46.9.1157

Cited by 14 publications

(6 citation statements)

References 29 publications

(41 reference statements)

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“…Protoplasts are the method of choice for DNA introduction, although whole plants or tissues have been reported to be transfectable by various methods (Fromm et al., ; Sparkes et al., ; Ueki et al., ). DNA can be delivered into plant protoplasts using various methods such as electroporation, PEG‐mediated transformation, and microinjection (Kost et al., ; Miao and Jiang, ; Yoo et al., ). Up to now, protoplast transient expression systems have been established in parsley ( Petroselinum crispum ; Boston et al., ; Lipphardt et al., ), maize ( Zea mays ; Sheen, ), carrot ( Daucus carota ; Boston et al., ; Rasmussen and Rasmussen, ), rapeseed ( Brassica napus ; Rasmussen and Rasmussen, ), switchgrass ( Panicum virgatum ; Mitra Mazarei et al., ), soybean ( Glycine max ; Rasmussen and Rasmussen, ), alfalfa ( Medicago sativa ; Harrison et al., ), Arabidopsis (Sheen, ; Yoo et al., ), and tobacco ( Nicotiana tabacum ; Miao and Jiang, ).…”

Section: Commentarymentioning

confidence: 99%

Isolation, Culture, and Transient Transformation of Plant Protoplasts

Shen

et al. 2014

CP Cell Biology

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Transient gene expression in protoplasts, which has been used in several plant species, is an important and versatile tool for rapid functional gene analysis, protein subcellular localization, and biochemical manipulations. This unit describes transient gene expression by electroporation of DNA into protoplasts of Arabidopsis or tobacco suspension‐cultured cells and by polyethylene glycol (PEG)‐mediated DNA transformation into protoplasts derived from rice leaf sheaths. PEG‐mediated DNA transformation for transient gene expression in rice protoplasts in suspension culture is also described as an alternative technique. Methods for collecting intracellular and secreted proteins are also provided. Curr. Protoc. Cell Biol. 63:2.8.1‐2.8.17. © 2014 by John Wiley & Sons, Inc.

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Section: Commentarymentioning

confidence: 99%

Isolation, Culture, and Transient Transformation of Plant Protoplasts

Shen

et al. 2014

CP Cell Biology

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“…Microinjection of fluorochromes 1 , antibodies 2-4 , proteins [5][6][7] , and genetic material [8][9][10] is widely practiced by biologists despite disadvantages associated with insertion of microcapillaries. In small cells, sealing of the plasma membrane around the tip (diameter, ∼1 µm) is often incomplete.…”

Section: Researchmentioning

confidence: 99%

A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes

et al. 1999

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A galinstan expansion femtosyringe enables femtoliter to attoliter samples to be introduced into prokaryotes and subcellular compartments of eukaryotes. The method uses heat-induced expansion of galinstan (a liquid metal alloy of gallium, indium, and tin) within a glass syringe to expel samples through a tip diameter of about 0.1 microm. The narrow tip inflicts less damage than conventional capillaries, and the heat-induced expansion of the galinstan allows fine control over the rate of injection. We demonstrate injection of Lucifer Yellow and Lucifer Yellow-dextran conjugates into cyanobacteria, and into nuclei and chloroplasts of higher organisms. Injection of a plasmid containing the bla gene into the cyanobacterium Phormidium laminosum resulted in transformed ampicillin-resistant cultures. Green fluorescent protein was expressed in attached leaves of tobacco and Vicia faba following injection of DNA containing its gene into individual chloroplasts.

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“…Nevertheless, further improvements are required before this approach becomes a standard technique in oil palm transformation programs. For example, the current transformation efficiency of 14% is relatively low compared to the frequencies achieved in other species, such as 26% in alfalfa [28] and 20–53% in tobacco [27] , [29] .…”

Section: Discussionmentioning

confidence: 90%

Efficient Transformation of Oil Palm Protoplasts by PEG-Mediated Transfection and DNA Microinjection

et al. 2014

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BackgroundGenetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants.Methodology/Principal FindingsWe recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation.Conclusions/SignificanceWe have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.

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High efficiency transient and stable transformation by optimized DNA microinjection intoNicotiana tabacumprotoplasts

Cited by 14 publications

References 29 publications

Isolation, Culture, and Transient Transformation of Plant Protoplasts

Isolation, Culture, and Transient Transformation of Plant Protoplasts

A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes

Efficient Transformation of Oil Palm Protoplasts by PEG-Mediated Transfection and DNA Microinjection

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