*2.3. Explant Selection, Culture Conditions, and Establishment of In Vitro Cultures*

*A. violaceum* seeds were used as explants to start in vitro cultures. The seeds were immersed in tap water for 96 h (h) before inoculation on the MS medium. Prior to culture, seeds were rinsed for about 2 h under running tap water, surface sterilized with 1% NaClO (*v*/*v*) for 7 min with occasional agitation, washed 4–5 times with doubledistilled autoclaved water, and then dipped in 70% ethanol for about 30–45 s. They were then washed with aseptic (autoclaved, double-distilled) water 3–4 times. The seeds were placed in the folds of sterile filter paper to absorb the leftover moisture. Seeds were then cultured in 30 mL borosilicate culture vials on the MS basal medium containing 0.8% (*w*/*v*) agar and sucrose (3% *w*/*v*), fortified with different PGRs at different concentrations (0.05–2.0 mg L−1), either individually or in combination. They were then incubated at <sup>10</sup> ± <sup>2</sup> ◦C with 50–55% humidity for a 12–12 h photoperiod (42–60 <sup>μ</sup>mol m−<sup>2</sup> <sup>s</sup><sup>−</sup>1) in a plant growth chamber. Under the laminar air flow hood, all studies, from surface sterilization to inoculation, were carried out successfully. Data were represented as a mean of 50 replicates per repetition. Observations were recorded from each non-contaminated vial (experimental unit). Mean germination time (MGT) in days was calculated by following the methods of Darrudi et al. [30] using the following equation:

$$\text{MGT} = \frac{\sum (n \times D)}{N}$$

where

'*n*' = number of newly germinated seeds after each incubation period

'*D*' = number of days since the experiment began, and

'*N*' = total number of seeds germinated at the end of the experiment.

Seed germination rate was calculated using the following equation:

% seed germination = (nx/Na) × 100

where

'nx' = total germinated seeds

'Na' = number of seeds used at the beginning of the experiment.
