MANUAL DE PROCESOS

4.2.3.1 E. coli

The E. coli strains DH5α and NM552 were transformed with use of the CaCl₂/Heatshock- method (Cohen et al., 1972). Competent cells preparation was based on the Rubidiumchloride-method according to Hanahan (Hanahan, 1983). The strain JM110 was transformed by electroporation as described by Sambrook et al. (Sambrook et al., 1989). After transformation bacteria were plated on LB-medium supplemented with the appropriate antibiotic and incubated over night at 37°C.

4.2.3.2 Petunia hybrida

Isolation of protoplasts from petunia mesophyll

The entire procedure for protoplast isolation and transfection was accomplished under steril conditions. From totally 20-25 plants (3-4 weeks old) approximately 3 to 4 young leaves were harvested from each plant and transferred to a steril Petri dish. They were individually coarsely incised with a scalpell with 8-10 cuts perpendicular to the main vein. They were plasmolysed over night at room temperature in the dark in 30 ml steril filtered K3 medium containing 0.45 g Cellulase and 0.15 g Macerozyme. After digestion, protoplasts were swirled every 10 min for totally 30 min. By use of steril 10 ml wide tip plastic pipettes (such pipettes were used also in the next steps, every time protoplasts were

washed, resuspended, collected or transferred) protoplasts were collected and transferred to a sieve. By two consecutive filtrations through a 0.5 µm and a 0.25 µm stainless steel sieve, previously autoclaved, protoplasts were separated from undigested tissue. Protoplasts were then collected in aliquots of 10 ml suspension in totally 3×12 ml steril tissue culture tubes and centrifuged 10 min at 1000 rpm at room temperature with a Varifuge 3.0 R (Heraeus Sepatech) centrifuge. Sedimented cell remnants and the supernatant clarified K3 medium were sucked up with a glass capillary tube. Floating protoplasts collected in the bottom of the tube were washed with 16 ml W5-medium shared in 2×12 ml steril tissue culture tubes totally. After 10 min centrifugation at 600 rpm at room temperature, the sedimented protoplasts were resuspended in totally 10 ml W5 medium and stored in the dark for 90 min at 4°C in 1×12 ml steril tissue culture tube. Before storage step at 4°C, a 200 µl aliquot of the 10 ml protoplast suspension was added to 800 µl of W5-medium in 1.5 ml eppendorf tube and used to determine the number of total isolated protoplasts.

PEG-transfection of petunia protoplasts

After incubation at 4°C, protoplasts were collected by 10 min centrifugation at 600 rpm at room temperature, and resuspended in Ma-Mg transfection solution at a concentration of 1×106 _{protoplasts/300 µl solution. Aliquots of 300 µl of the protoplast suspension were}

transfected according to the Ca(NO₃)₂-PEG-method described by Negrutiu et al. (Negrutiu et al., 1987).

Unless stated otherwise, when protoplasts were cotransfected with a Ds reporter plasmid and the TPase expression vector, 10 µg of each plasmid type were added to 40 µg of sonified CT-DNA (calf thymus DNA). Steril bidest water was added to a final volume of 60 µl. Plasmid DNA employed for protoplast transfection was propagated either in the E.

coli strain DH5α (when dam-methylated DNA was needed) or in JM110 (when

unmethylated DNA was needed). Plasmid DNA was isolated by use of the Plasmid Maxi- Kit of Qiagen and further purified by CsCl-gradient centrifugation and resuspended in 1× TE buffer (pH 5.6).

The DNA solution was pipetted to the 300 µl protoplast suspension already aliquoted in a 12 ml steril tissue culture tube. After the addition of 300 µl of 40% PEG6000-solution and

transfection. Protoplasts were collected by 10 min centrifugation at 600 rpm at room temperature, carefully resuspended in 5 ml K3 medium and incubated at 26°C in the dark in Petri dishes.

Histochemical glucuronidase assay

The histochemical assay was performed essentially as described by Jefferson and coworkers (Jefferson et al., 1987) for tissue sections. The method allows the detection of the ß-glucuronidase expression in each transfected petunia cell. The hydrolase activity of the ß-glucuronidase can be recognized by incubation with the substrate X-Gluc: through oxidative dimerization of the reaction product (a indolyl derivative), an unsoluble, dark blue dye is generated that precipitates in correspondence of the enzymatic activity. Single ß-glucuronidase-expressing cells can be easily identified as blue spots through fixation on Nitrate Cellulose Membrane Filters (NC-filters) and incubation with the substrate X-Gluc. Steps of the protocol: NC-Filters (0.45 µm, ∅=4 cm) were wetted in protoplast fixation solution and dried on Glass Fibre Prefilters. 200 µl of each 5 ml K3 medium/protoplast suspension were collected on a nitrocellulose filter and fixed for 30 min at room temperature. The protoplast-coated surface of each NC-filter was then incubated 30 min in fixation solution. The filters were washed twice for 20 min in 50 mM-Na-Phosphate buffer (pH 7.0); afterwards they were wetted with 1 ml of the same buffer containing the indigogenic substrate X-Gluc (2 mM) and incubated at 37°C in sealed Petri dishes. The stained protoplasts were routinely counted after overnight incubation, with help of a dissecting microscope.

Normally two to three 200 µl-aliquots of protoplast suspension per 5 ml transfection sample were fixed on NC-filter. The remaining protoplasts were collected and frozen on liquid nitrogen and stored at -80°C for further DNA analysis.