Pi rnase propidium iodide rnase

The ploidy status of anther-derived calli was examined using the flow cytometry method as described by Galbraith [43 (link)]. A total of 3 samples of anther-derived callus were randomly chosen, along with a reference standard of diploid leaves of C. roseus with a known 2C DNA content of 1.51 pg [44 (link)]. Approximately 50 mg of callus was added to a Petri plate having 1.0 mL ice-cold Galbraith’s buffer (nuclei isolation buffer) and finely macerated with the help of a surgical blade. The homogenate was then filtered with a 100 µm nylon mesh to eliminate larger cellular remnants and was finally stained with 50 µg/mL PI RNase (propidium iodide RNase) (Sigma-Aldrich, St. Louis, MO, USA) for 8–10 min. The samples were incubated in the dark at 4 °C for about 40 min and eventually examined on a BD FACS(Calibur) flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The relative nuclear DNA of anther-derived callus of C. roseus was estimated using the below formula [45 (link)]: $$\mathrm{Nuclear}\mathrm{DNA}\mathrm{content}\mathrm{of}\mathrm{sample}\left(\mathrm{pg}\right)=2\mathrm{C}\mathrm{DNA}\mathrm{content}\mathrm{of}\mathrm{standard}\left(\mathrm{pg}\right)\times \frac{\mathrm{mean}\mathrm{position}\mathrm{of}\mathrm{G}0/\mathrm{G}1\mathrm{peak}\mathrm{of}\mathrm{sample}}{\mathrm{mean}\mathrm{position}\mathrm{of}\mathrm{G}0/\mathrm{G}1\mathrm{peak}\mathrm{of}\mathrm{standard}}$$

Flow cytometric analyses were conducted to determine the genetic stability and the genome size of in vitro regenerated plantlets according to the method described by Galbraith [29 (link)]. Fresh young leaves were randomly selected from field-grown Digitalis (control) and plants regenerated via somatic embryogenesis and indirect organogenesis for ploidy level analysis. Digitalis lanata Ehrh. was used as a reference standard with known 2C nuclear DNA content of 3.0 pg [30 (link)]. Approximately 100 mg of each leaf sample was finely chopped using a fresh surgical blade in a pre-chilled Petri-dish containing 1.5 mL ice-cold Galbraith’s buffer (45 mM MgCl₂, 30 mM sodium citrate, 20 mM MOPS and 0.1% Triton-X) for nuclei extraction. Afterwards, the suspensions were filtered using a double-layered nylon mesh of 50 μm pore size to remove larger cellular debris and were stained with 50 μg/mL of PI RNase (Propidium iodide RNase) (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. The samples were then put in dark conditions at 4 °C for about 1 h. Finally, the incubated samples were analyzed on a BD FACS (Calibur) flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The 2C DNA content of regenerated plants of D. purpurea was estimated by using the formula [31 (link)]: