Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method

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.

SND1 Transcription Factor-Directed Quantitative Functional Hierarchical Genetic Regulatory Network in Wood Formation in Populus trichocarpa

“…Our optimized system (using stem dipping) requires a very short total processing time of ;25 min (from tissue harvest to protoplast recovery) and gives a high yield (;2.5 3 10 7 protoplasts/g SDX), making it one of the most efficient protoplast isolation systems reported (Wu et al, 2009;Tan et al, 2013). Furthermore, our system allows protoplast isolations from milligrams to tens of grams of SDX tissue at a similar efficiency and high protoplast yield.…”

PSEUDO RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day length

“…For FRET in A. thaliana cells, protoplasts from adult leaves of plants grown under 12‐h light/12‐h dark were isolated by previously described methods (Yoo et al , 2007; Wu et al , 2009). In these experiments, 9 μg of plasmids carrying 35S:: PRR:CFP or 35S:: CO:YFP was used for transfection to ~100 000 protoplasts.…”

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

“…Protoplasts were isolated from the leaves of 2-4 weekold plants using the Tape-Sandwich method (Wu et al, 2009), and transfected with fluorescent reporter constructs. Following transfection, protoplasts were incubated at RT under constant darkness in W5 solution (154 mM NaCl, 125 mM CaCl 2 , 5 mM KCl, 5 mM glucose, and 2 mM MES pH 5.7) for 12-24 h prior to examination with an Olympus Fluoview 1000 laser scanning confocal microscope.…”