Callus formation and plant regeneration
After 3 weeks, growth of the protoplast derived microcolonies on semi-solid medium containing the same components of those used for the first culture (except that mannitol was eliminated), resulted in the initiation of microcalli 2–5 mm in diameter. KM8p medium supplemented with 2.7 or 5.4 µM NAA plus 2.2 µM BA resulted in the highest number of microcallus formation from microcolonies (Supplementary Table 2). The callus formed from both types of protoplast derived
microcolonies was transferred to semi-solid MSB5 medium (MS minerals with Gamborg B5 organics) or WPM containing different concentrations of NAA in combination with 4.4 µM BA (Supplementary Table 3) for further growth and proliferation. MSB5 medium with 10.8 µM NAA plus 4.4 µM BA was best for proliferation of microcalli from both leaf- and callus-derived protoplasts (Supplementary Table 3). In addition, MSB5 medium supplemented with 8.1 µM NAA plus 4.4 µM BA, and WPM with 10.8 µM NAA plus 4.4 µM BA were also suitable media for proliferation of microcalli from callus-derived protoplasts. WPM with 5.4 µM NAA plus 4.4 µM BA produced the least amount of callus proliferation from the leaf (35%) and callus (27.5%) protoplast-derived microcalli after 4 weeks of culture.