Table 2.
Effect of various treatments on TB phenotype in infected protoplasts
| Type of treatment | Treatment | Effect on TB |
| Hormones | Abscisic acid [5 μM] | – |
| Jasmonate [40 μM] | – | |
| Auxin [5 μM] | – | |
| Salicylic acid [1 mM] | – | |
| Mechanical/physical stress | Compacting by sedimentation | Tub+|TB, mixed-networks |
| Electroporation | – | |
| Heat shock | Tub+|TB | |
| Light/dark cycle | – | |
| Membrane depolarization | – | |
| Membrane hyperpolarization | – | |
| Microwaves | – | |
| Music | – | |
| Ultrasonication | – | |
| Vortexing | – | |
| Elicitors | Arabinogalactan [1 mg/ml] | – |
| Chitosan [40 μg/ml] | – | |
| Cryptogein [1 μM] | – | |
| Others | CO2 | Tub+|TB, mixed networks |
| Sodium azide [0.02%] | Tub+|TB, mixed networks | |
| pH | – |
Infected protoplasts were treated/incubated under the conditions indicated, and the TB phenotype was then analyzed by immunofluorescence against P2 and α-tubulin. Protoplasts were incubated with hormones and elicitors, at the indicated final concentrations, for 60 min. Compaction of protoplasts by sedimentation was achieved by exposing them for 2 h at 9.81 m/s2 on a bench-top. Electroporation conditions were 400 Ω, 0.25 μFD and 0.5 or 1 kV. Heat shock was for 1 h at 37°C. Daylight/dark cycle was for 2 h each condition. Membrane depolarization and hyperpolarization were induced with 100 and 0.1 mM KCl in protoplast buffer, respectively. Microwave exposure was 3 s at 750 W. For the music treatment (inspired by Braam and Davis, 1990), Vanessa Paradis's ‘Joe le taxi’ song was played at moderate volume (∼60 db) for 3.5 min with protoplasts ‘listening’ from opened Eppendorf tubes. Ultrasonication consisted of a 2 s pulse at 80% power using a Bioblock Vibracell 72434 apparatus; vortexing was for 5 s at maximal power using a Vortex Genie 2 machine. Conditions for CO2 and sodium azide treatments are described in 'Materials and methods'. For pH treatment, cells were incubated for 5 min with 10 mM K2HPO4/KH2PO4 titrated to pH 3.0, 5.6, 6.9 or 8.2. Lower and higher pH values proved lethal to the cells and were not considered for analysis. In all cases, the survival of cells was verified as described by Widholm (1972) and only treatments sustaining viability of the cells were used for analysis.