*3.2. In Vitro Germination Steps of Untreated Black Cardamom Seeds*

In the in vitro germination of untreated black cardamom seed (control), the radicle emerges from the hilum of the seed and forms the primary root at around the 75th day of culture (Figure 3a). After that, the coleoptile emerges, grows to approximately 0.2–0.3 cm, then stops growing and will be pierced by the first leaf of the plumule (Figure 3c,d). The first leaf will grow, expand, and be supplemented by additional leaves (Figure 3e). At the other end of the embryonic axis, the primary root soon dies while adventitious roots (roots that arise directly from the shoot system) emerge, develop, and will ultimately produce a fibrous root system (Figure 3b).

**Figure 3.** In vitro germination steps of untreated black cardamom seed: (**a**,**b**) radicle emerges (at 75th day of culture): op—operculum, pr—primary root, sbr—shoot-borne root; (**c**) coleoptile emerges and is pierced by the primary leaf (at 85th day of culture): cp—coleoptile, hc—hypocotyl, pl—primary leaf; (**d**,**e**) the first leaf grows, expands, and is supplemented by additional leaves (at 85th and 110th day of culture, respectively): lb—leaf blade, ls—leaf sheath. Scale bars = 0.5 cm.

*3.3. Effect of Seed Treatments on the Germination and Seedling Growth of Black Cardamom under In Vitro Conditions*

Efficiency of seed surface disinfection by 0.1% HgCl2 for 10 min was significantly different between seed treatments after 10 days of culture (Table 2).

**Table 2.** Different seed treatment effects on seed germination and seedling growth of black cardamom cultured in vitro.


Treatments: control; ME—soak in water for 24 h + scarify by scalpel; HW2m and HW4m—soak in hot water at 100 ◦C for 2 and 4 min, respectively; CW—soak in cold water for 24 h; NAS10m and NAS15m—soak in 50% HNO3 for 10 and 15 min, respectively; HAS10m and HAS15m—soak in 25% HCl for 10 and 15 min, respectively; GA324h—soak in 200 ppm GA3 for 24 h; NAA24h—soak in 200 ppm NAA for 24 h. Means followed by a different letter are significantly different at an alpha level of 0.05 according to the Duncan's multiple range test. Percentage values were arcsin <sup>√</sup><sup>X</sup> transformed prior to statistical analysis. <sup>1</sup> Mean <sup>±</sup> standard error. <sup>2</sup> GP (%) = germination percentage. <sup>3</sup> MGT (days) = mean germination time. <sup>4</sup> GRI = germination rate index.

Acid seed scarification had a significantly higher percentage of contamination-free seed compared with other treatments. Among these, seeds were dipped into 25% HCl for 15 min and disinfected with 0.1% HgCl2 for 10 min in the next step and gave the highest percentage of contamination-free seeds, reaching 86.7%. In the other seed treatments, there was not a statistically significant difference in the percentage of contamination free seed compared to the control.

Results also showed that there was a significant difference in germination of black cardamom seeds under different seed treatments at α = 0.05 (Table 2). Applying mechanical scarification (ME) provided the highest mean germination percentage (reaching 68.0%) and the lowest mean germination time (reaching 53.7 days) after 90 days of culture. In this treatment, it took only approximately 50 days for 50% seed germination, while all other treatments had a lower germination percentage of 50% (Figure 4). Treatment of seed immersion in cold water (CW) or PGRs (GA324h and NAA24h) also showed significantly higher mean germination percentages than the control. Acid seed scarification methods (NAS10m, NAS15m, HAS10m, and HAS15m) did not show a significant effect on the germination percentage of black cardamom seeds, but they significantly reduced the seed germination time compared to the control. Hot-water-soaking treatments (HW2m and HW4m) appeared to have a negative effect on seed germination (Table 2). In addition, after the 90th day of culture, seed germination was not observed in any of the treatments.

**Figure 4.** In vitro black cardamom seed germination patterns under different treatments.

Generally, the ME, GA324h, and NAA24h treatments significantly improved in vitro seedling length and number of leaves after 20 days of subsequent culture from the 90th day. In the other seed treatments, there were not statistically significant differences in mean seedling length and mean number of leaves compared to the control (Table 2, Figure 5).

**Figure 5.** Seedling growth of black cardamom at the 110th day under different seed treatments: T0—Control; T1—ME (soak in water for 24 h + scarify by scalpel); T2—HW2m (soak in hot water at 100 ◦C for 2 min); T3—HW4m (soak in hot water at 100 ◦C for 4 min); T4—CW (soak in cold water for 24 h); T5—NAS10m (soak in 50% HNO3 for 10 min); T6—NAS15m (soak in 50% HNO3 for 15 min); T7—HAS10m (soak in 25% HCl for 10 min); T8—HAS15m (soak in 25% HCl for 15 min); T9—GA324h (soak in 200 ppm GA3 for 24 h); T10—NAA24h (soak in 200 ppm NAA for 24 h). Scale bars = 1 cm.

#### *3.4. Effect of Different PGRs on Shoot Multiplication, Elongation, and Rooting in Black Cardamom*

After 7 weeks of culture on the MS medium supplemented with various concentrations of PGRs (auxin and cytokinin), the growth responses of the explants (shoot tip explants derived from in vitro grown seedlings) were different (Table 3).

**Table 3.** Effect of different PGRs in MS culture medium on shoot multiplication, elongation, and rooting in black cardamom.


Means followed by the same letter are not significantly different at α = 0.05 according to the Duncan's multiple range test. Values of callus induction were arcsin <sup>√</sup><sup>X</sup> transformed prior to statistical analysis. The data have been recorded on a per-explant basis and recorded after 7 weeks of culture. +: small shoots, thin and light green leaves, and thin and few root-hairs roots; ++: medium shoots, thin and light green leaves, and medium and a few root-hairs roots; +++: fat shoots, thick and dark green leaves, and fat and many root-hairs roots.

Results indicated that the combination of BAP and NAA showed a high response for both shoot, leaf, and root induction after seven weeks of culture. MS medium supplemented with 4.0 mg/L BAP and 0.5 mg/L NAA exhibited the overall best response (mean shoot number: 5.4 ± 0.3, mean shoot length: 6.8 ± 0.3 cm, and mean root number: 16.2 ± 0.8) (Table 3). Shoots developed in clusters, stout pseudo-stems, thick and dark green leaves, and fat and many root-hairs roots (Figure 6c,e–g). The second-highest response was obtained using MS culture medium supplemented with 4.0 mg/L BAP and 1.0 mg/L NAA (mean shoot number: 4.92 ± 0.22, mean shoot length: 5.8 ± 0.2 cm, and mean root number: 8.9 ± 0.7) (Table 3). The in vitro response of seedling shoot tips on medium without PGRs (control) was low (mean shoot number: 0.6 ± 0.1, mean shoot length: 3.0 ± 0.1 cm, and mean root number: 2.7 ± 0.2) (Table 3). It can be seen that MS medium supplemented with a combination of BAP at high concentration and NAA is very suitable not only for shoot multiplication but also for root formation, and, hence, a separate constitution for root initiation is not required, as is often the case.

On the other hand, the combination of high-concentration auxin (including NAA and 2,4-D) with lower-concentration cytokinin (BAP) also showed different effects on callus induction of explants. The treatments related to 2,4-D produced a high callus induction rate (Table 3). However, produced callus were white and did not initiate shoot organogenesis in any of the explants when they were subjected to callogenesis induction treatments (Figure 6d). At the combination of 4.0 mg/L NAA with 1.0 mg/L BAP, callus induction was not observed; the explants were increasingly dark brown to blackish and, finally, rotten.

**Figure 6.** Different stages of in vitro micropropagation of black cardamom through seedling shoot tip culture on MS medium supplemented with BAP (4 mg/L) and NAA (0.5 mg/L) (except Figure (**d**)): (**a**) in vitro seedling, the source of explant; (**b**) explant (seedling shoot tip) after 1 week; (**c**,**e**,**g**) cluster of multiple shoots after 3, 5, and 7 weeks; (**f**) rooting after 7 weeks; (**d**) callus induction on MS medium supplemented with BAP (1 mg/L) and 2,4-D (1 mg/L) after 7 weeks. Scale bars = 1 cm.
