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Plant Methods

, 5:16

First Online: 24 November 2009Received: 07 October 2009Accepted: 24 November 2009

Abstract

BackgroundProtoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using Arabidopsis mesophyll protoplasts TEAMP, is currently commonly used for studies of subcellular protein localization, promoter activity, and in vivo protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments.

ResultsIn this report, we present a new procedure that we call the Tape-Arabidopsis Sandwich. This is a simple and fast mesophyll protoplast isolation method. Two kinds of tape Time tape adhered to the upper epidermis and 3 M Magic tape to the lower epidermis are used to make a -Tape-Arabidopsis Sandwich-. The Time tape supports the top side of the leaf during manipulation, while tearing off the 3 M Magic tape allows easy removal of the lower epidermal layer and exposes mesophyll cells to cell wall digesting enzymes when the leaf is later incubated in an enzyme solution. The protoplasts released into solution are collected and washed for further use. For TEAMP, plasmids carrying a gene expression cassette for a fluorescent protein can be successfully delivered into protoplasts isolated from mature leaves grown under optimal conditions. Alternatively, these protoplasts may be used for bimolecular fluorescence complementation BiFC to investigate protein-protein interactions in vivo, or for Western blot analysis. A significant advantage of this protocol over the current method is that it allows the generation of protoplasts in less than 1 hr, and allows TEAMP transfection to be carried out within 2 hr.

ConclusionThe protoplasts generated by this new Tape-Arabidopsis Sandwich method are suitable for the same range of research applications as those that use the current method, but require less operator skill, equipment and time.

Electronic supplementary materialThe online version of this article doi:10.1186-1746-4811-5-16 contains supplementary material, which is available to authorized users.

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Autor: Fu-Hui Wu - Shu-Chen Shen - Lan-Ying Lee - Shu-Hong Lee - Ming-Tsar Chan - Choun-Sea Lin

Fuente: https://link.springer.com/



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