Transient gene expression is a common approach for studying subcellular localisation, promoter activities or protein complexes in vivo. 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. Each method has certain merits and demerits. Leaf infiltration (usually tobacco) with Agrobacterium is easy to perform, but many plant species are recalcitrant to this type of transformation. Also, 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. For instance, Agrobacterium alters the activity of several plant proteins [1, 2]. Thus, their function cannot be studied in the native state of the plant, i.e. uncoupled from the pathogen effect. Cell bombardment causes severe tissue damage, requires expensive equipment and frequently yields relatively low transformation rates. 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. Transgene expression can usually be observed 16 to 48 hours post transformation. 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.
Genetic transformation of protoplasts has been reported for diverse plant species, including those of e.g. Brassicacea, Solanacea and some ornamental plant families (reviewed in ). However, protoplast isolation, transformation as well as downstream analyses are often hampered by a number of factors. For cell culture-derived protoplasts, the plant cell cultures need to be established, which is time-consuming, cost-intensive and requires specific laboratory equipment (e.g. sterile laminar hood, temperature-regulated shaker). In addition, there is a permanent risk of microbial contamination of the cell culture.
These problems are partially circumvented when using mesophyll-derived protoplasts. Many mesophyll protoplast isolation procedures involve the (not necessarily sterile) cutting of leaves, followed by enzymatic lysis of the cell wall and separation of released protoplasts from non-protoplasted tissue debris. However, there is significant tissue damage, often accompanied by a high proportion of broken protoplasts in the final isolates.
In a recent study, Wu et al.  reported isolation and transformation of Arabidopsis thaliana protoplasts using the so-called “tape-Arabidopsis-sandwich” method. In this, the protoplasts are isolated by pulling leaf layers apart using sticky tapes. The leaf layers attached to the tapes are then exposed to a suspension of cell wall-degrading enzymes. The protoplasts consequently released are harvested by centrifugation. Despite the undeniable break-through of the “tape-Arabidopsis-sandwich” method and its evident advantage for the study of well-characterised ecotypes and mutants, we see a number of technical and particularly biological limitations. We therefore sought an alternative protoplast system that would be complimentary to the Arabidopsis protoplast system and equally simple in its application.
Arabidopsis need to be sown and grown routinely to secure continuous supply of plant material. Since plant growth conditions (light intensity, day/night length and humidity) largely determine protoplast yield and transformation efficiencies , the availability of well-controlled climate chambers is a prerequisite.
The major draw-back of protoplasts derived from green leaves is their high content of chloroplasts and chlorophyll, which impedes certain microscopical applications and protein analyses: The strong autofluorescence of chlorophyll can mask the signal of fluorescent-tag-labelled proteins in UV microscopy [5, 6]. To some extent, this problem can be alleviated through the use of costly narrow-bandpass filters.
Another chloroplast-associated limitation is the high abundance of photosynthesis-related proteins, particularly ribulose bisphosphate carboxylase (RuBisCo) and light harvesting complex a/b protein (LHC) in mesophyll-derived protoplast protein extracts. In fact, Lhcb1 is the most abundant chlorophyll a/b-binding protein in eukaryotic phototrophs and is often coded by several genes. Due to their abundance, RuBisCo and LCH can impede immuno-detection of proteins of interest due to masking effects or non-specific cross-reactivity with antibodies (reviewed in ). Chloroplast-associated complications might be prevented using etiolated leaves. However, transformation efficiencies in Arabidopsis mesophyll protoplasts are much lower in low-light grown plants as compared to high-light-grown plants .
Our needs and expectations of an alternative and complementary protoplast isolation and transformation technique were:
Continuous supply of plant material for protoplast isolation.
Source plants should have minimal requirements for growth and plant care; no controlled environment chambers should be required.
Isolation and subsequent transformation of protoplasts should be simple, fast, efficient and reproducible.
Isolated protoplasts should be robust, minimizing cell damage during transformation and centrifugation steps.
Protoplasts should contain few or no chloroplasts in order to minimise the interference of chlorophyll autofluorescence in UV microscopy. For some (e.g. immunoblotting) applications, a lack of certain highly abundant protein species, e.g. chloroplastic proteins RuBisCo (small and large subunit) and LHC, potentially masking or causing nonspecific hybridisation signals at app. 55 kDa, 11 kDa and 26 kDa may also be desirable.
Here, we report an alternative versatile system, Poinsettia (Euphorbia pulccherrima), for transient expression studies. Poinsettia plants can be grown in the laboratory and special green house space is not required. The isolation and highly efficient transformation of mesophyll protoplasts largely devoid of chloroplasts is described. The method is very time-efficient, as large quantities of vital protoplasts can be obtained within one day. The transformation efficiency is high (>70%); and compared to many other transformation protocols requires only low amounts of plasmid DNA. In addition, this is the first study to describe protoplast isolation and transformation in Poinsettia. Transformed protoplasts are of fundamental value in basic research, e.g. anthocyan synthesis/degradation, as well as for the ornamental industry.