*2.4. Embryogenic and Non-Embryogenic Callus Production*

Mature and immature seeds of *A. violaceum,* taken from the wild plants, were cultured on the MS basal medium augmented with various concentrations of PGRs, such as Kn (0.05 mg L−<sup>1</sup> to 3.0 mg L−1), IAA (0.1 mg L−<sup>1</sup> to 1.5 mg L−1), and 2,4-D (0.05–2.5 mg L−1) with sucrose 3% (*w*/*v*) and 0.8 % agar for production of embryogenic and non-embryogenic calluses. The pH of the medium was adjusted to 5.8 ± 0.02 before autoclaving at 121 ◦C. Immature and mature seeds were surface sterilized with 1% sodium hypochlorite (NaClO) (*v*/*v*) for 7 min with occasional stirring, then rinsed 3–5 times with sterile water before being immersed in 70% ethanol for about 30 s and washed 3–4 times with sterile (autoclaved, double-distilled) water. The remaining moisture was removed by putting the seeds in the folds of sterile filter paper. Cultures were incubated at 10 ± 2 ◦C in a 16/8-h light/dark cycle or in completely dark conditions on racks fitted with cool fluorescent tube lights of 60.0 μmol m−<sup>2</sup> s−<sup>1</sup> illuminance and RH 55%. The frequency of embryogenic callus induction was studied after 6 to 8 weeks (wk) of incubation. Consequently, embryogenic

and non-embryogenic calluses sub-cultured within 1 culture were used for whole plant regeneration. The experiment was conducted at least 3 times with a total of 10 replicates for each treatment (1 vial was considered as 1 replicate).
