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Transient Expression Assay Using Arabidopsis Mesophyll Protoplasts (PDF reprint article)
A Transient Expression Assay Using Maize Mesophyll Protoplasts
Maxi-plasmid DNA Prep
A 10-min plasmid DNA miniprep method
PP2C assay

Arabidopsis plants for protoplast preparation


Figure 1. Viability of Arabidopsis mesophyll protoplasts. Arabidopsis leaves were digested with cellulase and macerozyme for 3 h atroom temperature. A homogeneous population of mesophyll protoplasts was released and observed under bright field (left) or with a FITC filter to show viable cells stained with a vital dye fluorescein diace-tate (right). The purity or viability of the mesophyll protoplasts is usually 95% without gradient purification. Scale bar 35 m.

Figure 2. High transfection efficiency of Arabidopsis and maize mesophyll protoplasts. Arabidopsis protoplasts were transiently transformed by the PEG method. Maize protoplasts were transiently transformed by electroporation. A cytosolic GFP marker was used to visualize the transformation efficiency. The mesophyll protoplasts showing only red chlorophyll autofluorescence are untransformed. The transformed cells appear yellow, orange, and/or green. The transformation efficiency is 90% for Arabidopsis protoplasts and 40% for maize protoplasts. Scale bar 35 m (Arabidopsis) and 25 m (maize).

Figure 3. Protoplast transient expression assays. Arabidopsis and maize mesophyll protoplasts are versatile systems for the elucidation of protein activities and functions in plant signal transduction pathways. Diverse signal responses have been detected in mesophyll protoplasts based on analyses of reporter gene expression. The nucleus of the Arabidopsis mesophyll protoplast is revealed by a nuclear GFP marker. Chloroplasts show red chlorophyll autofluorescence. Flg22, 22-amino acid peptide elicitor derived from pathogenic bacterial flagellin; PKs, protein kinases; PPs, protein phosphatases; TFs, transcription factors. UBQ, a constitutive ubi-quitin promoter.

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ASPB 2006 Protoplast Workshop

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