Fig. 4

Transformation and gene expression analysis of leaf mesophyll protoplasts of Phaseolus vulgaris. a-b Laser-scanning confocal microscope showing protoplasts that were transformed with the PvSnRK1-RNAi vector expressing red fluorescence. c Quantitative RT-PCR analysis showing the downregulation of the PvSnRK1 transcript in protoplasts that were transformed with the PvSnRK1-RNAi vector. d-e Protoplasts that were transformed with the PvSnRK1-OE vector expressing green fluorescence under a laser scanning confocal microscope. f Quantitative RT-PCR analysis showing the overexpression of the SnRK1 transcript in protoplasts that were transformed with the PvSnRK1-OE vector. For RT-qPCR analysis, the total RNA was isolated from transformed protoplasts after 20 h of incubation at room temperature in the presence of light. Transcript accumulation was normalized to the expression of Ef1α and IDE, which were used as reference genes. The data are the averages of three biological replicates (n > 9). The statistical significance of the differences between the control (non-transformed and vector control) and transformed protoplasts was determined using a one-way ANOVA Newman-Keuls Multiple Comparison Test (**, P < 0.01). The error bars represent means ± SD