Fig. 4: Parameters affecting the probability of protoplast release.
Probability of protoplast release from the nanosurgery-produced incision, superimposed on the corresponding histogram bin of parameter values. a Distance of the laser incision from the hyphal protoplast during nanosurgery: distribution and dependence of the probability of protoplast release. b Hyphal width at the site of nanosurgery: distribution and dependence of the probability of protoplast release. c Length of surgical incision: distribution and dependence of the probability of protoplast release. d In a separate series of experiments, the effect of increased calcium concentration on the probability of protoplast release was measured as a function of incision length in the nanosurgical phase of the procedure, without subsequent steps of deplasmolysis. High [Ca2+]: 30 mM, as used in the surgical protocol. The hyperosmotic solution used was 600-617 mOsm throughout the protoplast release period. Standard [Ca2+]: 3 mM and 1 mM (pooled data). The hyperosmotic solution used was 555-560 mOsm throughout the protoplast release time. For each 3 µm incision size bin in the histogram, the corresponding number of protoplasts obtained divided by the number of surgical sections performed is shown above the probability curve. In all graphs, the abscissa represents the upper limit of the bin. For a–c: ntot(incisions) = 203 (a); 209 (b); 208 (c). ntot(protoplast) = 71(a), 81(b), 112 (c). For d: ntot(incisions) = 272 (Standard Ca2+); 148 (High Ca2+). ntot(protoplast) = 43 (Standard Ca2+); 55 (High Ca2+)
