Abstract
The protocol outlined in this chapter describes a detailed step by step procedure for the stable transformation of tobacco protoplasts by microinjection. Microinjection is greatly facilitated by immobilization of the protoplasts in a thin layer of alginate, thereby eliminating the need of a holding capillary. The injection process can be visually controlled by co-injecting the DNA with a fluorochrome (FITC-dextrane) and observing the injection under fluorescent illumination. Injected cells are directly cultured and selected in the alginate, omitting complicated single cell cultures. Using this protocol 50 to 100 cells can be injected in 1 h while achieving a transformation efficiency of up to 20%. The protocols for protoplast isolation and plant regeneration, as well as the protocols for alginate embedding, microinjection, and selection of transgenic clones, are done, with some modifications, according to Potrykus and Shillito (1986) and Schnorf et al. (1991), respectively.
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© 1995 Springer-Verlag Berlin Heidelberg
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Schnorf, M., Kost, B., Galli, A., Neuhaus, G. (1995). Microinjection into Tobacco Protoplasts and Regeneration of Transgenic Plants. In: Potrykus, I., Spangenberg, G. (eds) Gene Transfer to Plants. Springer Lab Manual. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-79247-2_21
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DOI: https://doi.org/10.1007/978-3-642-79247-2_21
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-48967-9
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