Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies

Andrea Pitzschke,Helene Persak

doi:10.1186/1746-4811-8-14

Abstract

BackgroundTransient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to few plant species often requiring substantial equipment and facilities. High chloroplast and chlorophyll content may further impede downstream applications of transformed cells from green plant tissue.ResultsHere, we describe a fast and simple technique for the high-yield isolation and efficient transformation (>70%) of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima). In this method no particular growth facilities or expensive equipments are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in a variety of commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence) or promoter activity studies. Due to low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence) are alleviated. Furthermore, the transgene expression is detectable within 90 minutes of transformation and lasts for several days.ConclusionsThe simplicity of the isolation and transformation procedure renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multi-colour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications.

Highlights

Transient gene expression systems are indispensable tools in molecular biology
Its red leaves are virtually devoid of chloroplasts
We found that cell layers of red, but not green Poinsettia leaves, can be pulled apart

Summary

Introduction

Transient gene expression systems are indispensable tools in molecular biology. Their routine application is limited to few plant species often requiring substantial equipment and facilities. The most frequently used transient transformation strategies involve: i) Agrobacterium tumefaciens-mediated transformation of leaves ii) biolistic approach, i.e. the bombardment of plant tissue (e.g. onion peel) with gold particle-loaded DNA and iii) protoplast transfection. The presence of Agrobacterium may alter the activity of several plant proteins This aspect should be considered while interpreting the data obtained from studies of stress signalling components involving Agrobacterium-mediated transformations. The third strategy, protoplast transfection, involves protoplast isolation from plant tissue by enzymatic removal of the cell wall and subsequent transfer of plasmid DNA carrying genes of interest. For plant species recalcitrant to Agrobacterium-mediated transformation, protoplast transformation and the subsequent attempt to generate entire plants may be a valuable alternative approach for obtaining stable transgenic plant lines

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Conclusion