**4. Discussion**

The objectives of the study reported in this paper were to find solutions to improve the germination rate of black cardamom seeds, thereby creating an explant source for micropropagation to create many disease-free crops with outstanding characteristics of the selected mother plant to provide for mass production.

Seed coat hardness is an important factor that affects germination in seeds [32]. Seed dormancy has been reported in several species of the Zingiberaceae, including *Alpinia malaccensis* Roscoe [9], *Alpinia galanga* Willd. [10], korarima [11,12], large cardamom [13], and green cardamom [14]. In black cardamom, the morphological and anatomical characteristics of seeds established in the study, including the very hard testa, small endosperm (i.e., possessing low food storage), and small embryo, can be responsible for the difficulty of seed germination and the slow growth of seedlings. The strong inhibitory effect of the seed coat on seed germination may be caused by several possible mechanisms, including mechanical constraint, prevention of water and oxygen uptake, and retention or production of chemical inhibitors [33–35]. The integument breaking or softening, for instance, is needed to remove dormancy imposed by seed coat hardness or impermeability. Several authors (Alamgir and Hossain 2005 [36], 2005 [37]; Azad et al., 2006 [38], 2006 [39], 2010 [40], 2010 [41], 2011 [42]) have discussed different methods of pre-sowing treatments for seed germination in order to break dormancy, enhance the rate of germination, and speed up the germination process. Our studies showed that the seed treatments had a significant effect on the germination and growth parameters of black cardamom seeds. Among the applied seed treatments, mechanical scarification produced the best effect on seed germination parameters and subsequent seedling development. The findings of our previous study [15] also displayed similar results, but the germination rate of seeds was significantly improved in this experiment. The difference in the type of seeds used and the time of sowing in our two experiments is probably the cause for that difference. While in the first report we used dried seeds that were collected in October and dried in the open sun for 3 days, in this experiment we used fresh seeds that were collected in December. According to local people's experience, the best time to sow black cardamom seeds is at the end of December, and they must be sown immediately after harvest because they will lose their ability to germinate when stored for a long time. In a study on the effect of moisture in seeds on the germination of green cardamom seeds, Sangakkara U. R. (1990) [43] found that reducing the moisture content of seeds significantly reduced and even destroyed the viability of them. Robert (1973) [44] termed them recalcitrant seeds. Bearing similar physiological characteristics to green cardamom seeds may be responsible for the difference in results in our two experiments. Further studies are needed to confirm this issue. Copeland (1995) [45], Hartmann (1997) [46], Missanjo E. (2014) [47], and Botseleng B. (2014) [48] also confirmed that mechanical scarification produced the best effect in breaking the dormancy of seeds. However, recent studies on breaking dormancy of some Zingiberaceous species have shown that the effect of chemical scarification treatments is better than mechanical scarification treatments. According to Radhamani et al. [49], the treatments of green cardamom seeds in India with 25% sulfuric acid (H2SO4) for ten minutes and 80% absolute alcohol for 30 min were the most effective treatments in breaking seed dormancy. Dahanayake [14] reported that soaking in 50%nitric acid (HNO3) for 15 min was the most effective for green cardamom seeds' dormancy in Sri Lanka. In our study, acid scarification treatments were not effective in increasing the germination rate of black cardamom seeds. Similarly, the report of Seid et al. [50] also showed that acid scarification treatments were not effective in breaking dormancy of green cardamom seeds. According to this report, soaking in 80% alcohol for 30 min was the most effective method for breaking the dormancy of green cardamom seed. Endogenous gibberellins have been widely studied in relation to the breaking of seed dormancy in various species. GA3 has been exogenously applied as a substitute for stratification and has increased germination in many plant species, e.g., *Fagus sylvatica* L. [51], *Crataegus pseudoheterophylla* Pojark. [52], and *Juniperus polycarpos* K. Koch [53]. In a previous study on korarima, combined treatment of 50% H2SO4 for 60 min and 250 mg/L GA3 for 24 h yielded the highest dormancy-breaking effect [11]. In this study, the positive effects of PGRs on the seed germination efficiency and growth of black cardamom seedlings were also noted. Hot water treatments have been reported to enhance germination of hard-coated seeds by elevating water and O2 permeability of the testa [54]. Rivai et al. [9] showed that treatment with hot water at 75 ◦C for 5 min gave the highest germination rate of *Alpinia malaccensis* Roscoe seeds. However, in our study, the hot water

treatment seemed to have a negative effect on the seed germination and growth of black cardamom seedlings. This result is similar to that of Okunlola et al. (2010) [55] regarding African locust bean (*Parkia biglobosa* Benth.). Cold water treatment of black cardamom gave a fair germination percentage and a reduced mean germination time when compared to hot water and control treatments. This also implies that soaking in cold water could also reduce the dormancy period in seeds of black cardamom when compared to control. This result concurs with earlier reports of Emerhi and Nwiisuator (2010) [56] and Falemara et al. (2014) [57] that show that soaking in cold water is a feature that enhances germination in seeds of tropical trees. The subsequent increase in the germination percentage, decrease in mean germination time, and increase in germination index when subjected to different treatment methods are indications that the hard seed coat is responsible for the dormancy in black cardamom.

The next step in our study was to apply shoot tip explants derived from in-vitrogrown seedlings to establish an efficient in vitro propagation procedure that could be used for commercial purposes. In a previous study on large cardamom [19], when rhizome segments were cultured on an MS medium supplemented with BAP or NAA alone or in a combination of BAP + IBA, BAP + NAA, or IBA + NAA, they produced shoots and roots simultaneously. These researchers reported 90% survival of plants after transfer to soil [19]. In the present investigation, the MS medium supplemented with 4.0 mg/L BAP and 0.5 mg/L NAA was found to be suitable for large-scale multiplication of black cardamom. The use of MS medium supplemented with BAP at high concentration and NAA significantly shortened the time of in vitro plantlet generation (7 weeks) compared with our previous study (14 weeks). Earlier reports on the clonal micropropagation of other Zingiberaceous species such as green cardamom, ginger, and turmeric, indicated similar results [16,58–62]. According our previous study [27], MS medium supplemented with 1 mg/L BAP without auxin was best for shoot multiplication of rhizome buds of black cardamom after six weeks of culture (mean shoot number and mean shoot length were 4.5 and 5.5 cm, respectively). However, in this study, the growth response of shoot tip explants derived from in vitro grown seedlings to this medium was moderate (mean shoot number: 3.4 ± 0.3, mean shoot length: 5.1 ± 0.1 cm, and mean root number: 5.6 ± 0.2) (Table 3).

"Fasco" and "Garden Star" are two commonly used soils in the Russian Federation because they have a composition suitable for most crops grown here. The main components of these two soils are peat and mineral fertilizers. However, in the composition of "Fasco" bio-soil, there is also bio-humus—an organic fertilizer containing many beneficial microorganisms for the soil, which helps to increase the fertility and aeration of the soil. This difference may explain the more positive effect of "Fasco" bio-soil on survival and subsequent plantlets growth compared with "Garden Star" soil.

#### **5. Conclusions**

This study is one of the first reports of the morphological, anatomical, and germinating characteristics of black cardamom seeds collected in Vietnam. Seed coat ultrastructural features of the black cardamom using SEM were first reported. Because of the consistency in seed morphological features, SEM is considered useful when it comes to phylogenetic information and a great potential source of taxonomy. Additionally, we identified black cardamom seeds as physical dormancy seeds and the hard seed coat as the major cause. Applying mechanical scarification treatment before sowing has been shown to be the most effective for improving seed germination rates. Methods of immersion in cold water or PGRs before sowing are also recommended, although the effect of dormancy breaking is not as high as that of the mechanical scarification method. Traditional methods of propagation of black cardamom (by seeds and rhizomes) do not currently meet the increasing demand for large-scale production of crops. Therefore, the developed in vitro propagation protocol can be an effective solution for rapid multiplication of high-yielding elite plants to meet the needs of expanding cultivation of this important crop, but it needs to be tested for largescale multiplication and field planting. It can be said that this investigation carries great commercial significance, as black cardamom is used as a spice in many Asian countries, as a flavoring agent, and for pharmaceutical purposes by other industries.

**Author Contributions:** Q.V.K. and R.N.K.—experiments on black cardamom culture in vitro. E.N.B.—preparation of plant material for SEM; Q.V.K. and H.T.N.—statistical analysis of experimental data; E.A.K.—manuscript conceptualization. Q.V.K., E.A.K. and M.R.K.—writing of the manuscript; Q.V.K. and M.R.K.—reviewing and editing of the manuscript; all authors approved the final manuscript for publication and agreed to be accountable for all aspects of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The results of Sections 3.1–3.3 were supported by assignments 122020300187-2 (Tsitsin Main Botanical Garden of Russian Academy of Sciences) and 0431-2022-0003 (All-Russia Research Institute of Agricultural Biotechnology) of the Ministry of Science and Higher Education of the Russian Federation, respectively. Additionally, Sections 3.4 and 3.5 were supported by the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement № 075-15-2022-746, dated 13 May 2022 (internal number MK-3084.2022.1.4) on providing a grant in the form of subsidies from the federal budget of the Russian Federation under grants from the President of the Russian Federation for state support of young Russian scientists—PhDs— and leading scientific schools of the Russian Federation.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing is not applicable to this article.

**Acknowledgments:** The authors are grateful to Ma A Chang for provided photos of black cardamom and Tam H. M. for botanical identification.

**Conflicts of Interest:** The authors declare no conflict of interest.
