Culture media and culture conditions
For explant development, disinfected explants were incubated in a culture room at 25 ± 1°C in light conditions (under 16 h photoperiod under cool white fluorescent tubes (F40 tubes Gro-lux, Sylvania) with 45 µmol m− 2 s− 1 (400–700 nm) Photosynthetic Active Radiation). on MS salts (Murashige and Skoog, 1962) supplemented with 100 mg l− 1 i-inositol, 30 g l− 1 sucrose and 8 g l− 1 of Agar A-1296 (Sigma), and after the aseptic establishment and due to the null growth of explants this medium was supplemented with different concentrations of plant growth regulators.
Different concentrations of cytokinins and auxins were tested: KIN (0, 0.1, 0.3, 0.4, 0.5, 0.6, 1.0) mg l− 1; BAP (0, 0.1, 0.3, 0.5, 1.0) mg l− 1; Adenine hemisulfate (0, 10, 20, 30, 40) mg l− 1 and various levels of NAA (0, 0,1, 0.4, 05, 0.6) mg l− 1; IBA, IAA, Picloram, pCPA (0, 0.1, 0.5) mg l− 1; multiple combinations cytokinins and auxins to select the best combination possible and later, when the optimal combination of auxins plus cytokinins was stablished, gibberellic acid (1, 5, 10 mgl− 1 during 3 days) was tested. After the selection of the best combination of PGRs, different levels of sucrose (0, 10, 20, 30, 40, 50, 60) g l− 1 as well as the growth retardant Ancymidol (0, 1.0, 2.0, 3.0, 4.0, 5.0) mg l− 1 were tested to evaluate growth, rooting rate and callus proliferation.
In all the experiments 50 explants were used per treatment and the experiments were repeated twice.
For micropropagation three different media have been developed MSM-1 (Mammillaria Snowcap Medium-1, for explant and offshoots growth and maintenance), MSM-2 (Mammillaria Snowcap Medium-2, for offshoots induction and prolifereation) and MSM-3 (Mammillaria Snowcap Medium-3, for rooting). These media consisting on basal Murashige Skoog medium (Murashige and Skoog, 1962) supplemented as follows:
MSM-1: MS plus 0.5 mg l− 1 NAA + 0.3 mg l− 1 KIN + 20 g l− 1 sucrose.
MSM-2: MS plus 0.5 mg l− 1 NAA + 0.3 mg l− 1 KIN + 5.0 mg l− 1 GA3 + 20 g l− 1 sucrose.
MSM-3: MS plus 0.5 mg l− 1 NAA + 0.3 mg l− 1 KIN + 5 mg l− 1 ANC + 10g l− 1 sucrose.
The pH of all the culture media was adjusted to 5.7 before autoclaving. For culture initiation, rooting and multiplication experiments, 75 ml of medium per jar were aliquoted into 120 mL culture jars covered with polypropylene caps (Magenta Corp., Chicago, IL) and autoclaved for 20 min at 121°C and 1.05 Kg cm− 2. All cultures were incubated at 25 ± 1°C under a 16/8 photoperiod under cool white fluorescent tubes (F40 tubes Gro-lux, Sylvania) with 45 µmol m− 2 s− 1 (400–700 nm) Photosynthetic Active Radiation.