*2.3. Experimental Design and Treatments*

Germination of black cardamom seeds is erratic, delayed, and poor under the traditional type of sowing. Attempts made to improve germination percentage and also to achieve uniform germination include mechanical scarification, immersion in hot or cold water, acid scarification, and soaking in PGRs (Table 1). Seeds without damage to the uniform size and color were selected randomly to conduct the experiments.


**Table 1.** Summary of black cardamom seed treatments.

Control (without treatment). ME treatment are performed after disinfection seed step. Other treatments are performed before disinfection seed step. Each treatment was replicated three times with 25 seeds for each. To avoid contamination, only one seed was cultured per glass vessel.

## *2.4. Obtaining of Aseptic Donor Seeds and Culture Conditions*

Above-mentioned treated seeds (Table 1) were rinsed with tap water. After that, seeds were washed in liquid soap for 10 min and then rinsed directly under running tap water. In the next step, seeds were disinfected in 70% ethanol for 30 s, followed by immersion in 0.1% (*w/v*) aqueous mercuric chloride for 10 min [15]. After surface disinfection, seeds were rinsed 4–5 times with sterile distilled water and transferred to the culture vessels containing basal MS medium [29] with macronutrients diluted to 1/16 strength for germination and growth [15]. The pH of the medium was adjusted to 5.6–5.8 by 1N NaOH before being autoclaved at 121 ◦C and 1.1 atm for 20 min. The cultures were maintained in a culture room at 25 ± 2 ◦C during a long-day photoperiod (16 h of light: 8 h of dark) with cool white fluorescent light (2000–2500 lux).

## *2.5. Data Recording and Germination Assessment*

Seeds were considered germinated when the healthy, white radical had emerged through the integument. Data were scored from 30 to 90 days after culture. The contamination and germination seeds were counted every 10 days until the 90th day.

The following germination parameters were determined:

(1) Germination percentage (GP), the number of germinated seeds as a percentage of the total number of tested seeds, is given as:

GP = (germinated seeds/total tested seeds) × 100%;

(2) Mean germination time (MGT, days) is given according to Scott et al. [30] as:

$$\text{MGT} = \sum \text{Tk Nk/S}\_2$$

where Tk is the number of days since the beginning of the experiment, Nk the number of seeds germinated per day, and S is the total number of seeds germinated.

(3) Germination rate index (GRI) was calculated for each treatment using the following equation:

$$\text{GRI} = \text{(G}\_1/\text{1)} + \text{(G}\_2/\text{2)} + \dots \dots + \text{(G}\_{\text{i}}/\text{i})\_{\text{i}}$$

where G is the germination day 1, 2, ... , and i represents the corresponding day of germination [31].

Seedling length was measured using a ruler scale on the 110th day of culture.

#### *2.6. Culture Media for Clonal Propagation*

Seedlings (2–3 cm in length) were used as an explant source (shoot tips) for clonal propagation. They then were placed on agar-solidified MS medium supplemented with various concentrations of PGRs (auxin: 0.5 and 1.0 mg/L NAA, 1.0 mg/L and 2.0 mg/L 2,4-D); cytokinin: 1.0–4.0 mg/L BAP; see Table 3 for details). The experiment was arranged completely randomly and repeated three times, with 12 explants per treatment. Data were scored after 7 weeks. The length of shoots was measured using a ruler scale.
