**1. Introduction**

*Amomum tsao-ko* Crevost & Lemarié, belonging to the ginger family (Zingiberaceae), is a valuable medicinal plant in Vietnam and is commonly known as "black cardamom", "do ho", or "sa nhan coc" [1,2]. It is a particularly shade-loving and moisture-loving species, so it can only be grown under the forest canopy, at an altitude of 1300–2200 m, with an average annual temperature of 13–15.3 ◦C, usually frequent fog, annual rainfall of 3500–3800 mm, and more than 90% humidity [1,3–5]. Therefore, it is distributed and cultivated in high ranges with damp soil rich in humus and under the partial shade of evergreen forests

**Citation:** Khuat, Q.V.;

Kalashnikova, E.A.; Kirakosyan, R.N.; Nguyen, H.T.; Baranova, E.N.; Khaliluev, M.R. Improvement of In Vitro Seed Germination and Micropropagation of *Amomum tsao-ko* (Zingiberaceae Lindl.). *Horticulturae* **2022**, *8*, 640. https://doi.org/ 10.3390/horticulturae8070640

Academic Editor: Sergio Ruffo Roberto

Received: 15 June 2022 Accepted: 11 July 2022 Published: 15 July 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

belonging to the Northern Midland and Mountainous region of Vietnam (Lai Chau, Lao Cai, Yen Bai, Ha Giang, Tuyen Quang, and Cao Bang provinces). Additionally, black cardamom is also grown in China (Yunnan province) and Laos (Phongsaly province) [1,2,6]. In Vietnamese traditional medicine, black cardamom fruit is utilized to treat dyspepsia, nausea, malaria, bad breath, infections, etc. [1,7,8]. Black cardamom is also a spice used in many traditional Vietnamese dishes, e.g., Vietnamese beef noodle, chicken herbal dish, hotpot, etc.

For the above-mentioned reasons, black cardamom has become one of the main medicine crops for local people in the Northern Midland and Mountainous region of Vietnam. The area of black cardamom cultivation is constantly expanding. In Vietnam, local people use mainly seeds and rhizome segments to cultivate black cardamom. Because plants grown from rhizome segments are susceptible to diseases caused by viruses, fungi, or bacteria, they often provide a lower yield and fruit quality than plants grown from seeds. Additionally, the destructive harvesting of the rhizomes for vegetative propagation appears not to be workable because there is always the possibility of losing the mother plant during this process. However, the practice of seed propagation of local people illustrates that cardamom seeds germinate slowly and unevenly, and seedlings are slow growth. Studies on other species from the ginger family such as *Alpinia malaccensis* Roscoe [9], *Alpinia galanga* Willd. [10], korarima (*Aframomum corrorima* P.C.M. Jansen) [11,12], large cardamom (*Amomum subulatum* Roxb.) [13], and green cardamom (*Elettaria cardamomum* Maton) [14] have all shown that seed germination of these species was not fast and/or that many seeds do not germinate due to the presence of some kind of dormancy, possibly associated with the hard and impermeable nature of the seed coat. Additionally, in the above-mentioned articles, various seed treatments (including mechanical scarification, soaking in hot or cold water, acid scarification, and soaking in plant growth regulators) were applied to break seed dormancy. Presently, similar studies in black cardamom are lacking. Quyet et al. (2021) [15] found that surface disinfection with 0.1% HgCl2 for 10 min showed the best seed disinfection effect, and MS medium diluted to 1/16 concentration was the best for in vitro germination of black cardamom seeds. Additionally, the authors applied a mechanical scarification treatment to improve the germination rate of dried seeds. However, the germination rate was reported to be quite low (33.3%). Determining the cause and applying more seed treatments to establish the best seed treatments in order to further improve the germination rate of these seeds is necessary.

Currently, the technique involving propagation using plant tissue promises to produce many disease-free and uniform-quality crops in a short time compared with traditional methods. In vitro propagation has been reported in various species of the genus *Amomum*, e.g., green cardamom [16–18], large cardamom [19–21], siam cardamom (*Amomum krevanh* Pierre) [22], white amomum (*Amomum villosum* Lour.) [23,24], purple amomum (*Amomum longiligulare* T.L. Wu) [25], and *Amomum* sp. [26]. Rhizome buds are used by most researchers as a source of explants when carrying out in vitro propagation of these species. Regarding black cardamom, Quyet et al. (2021) [27] carried out clonal micropropagation and also used rhizome buds as an explant source. In this study, the authors evaluated the separate effects of two cytokinins, 6-benzylaminopurine (BAP) and kinetin, in the fast multiplication phase and two auxins, indole-3-butyric acid (IBA) and 1-naphtaleneacetic acid (NAA), for root induction. Eyob S. (2009) [11], when carrying out the micropropagation of korarima, a species of the ginger family, used two different explant sources including shoot tip derived from in vitro grown seedlings and rhizome buds. The author found that the highest survival and number of shoots were obtained from the shoot tips of in vitro seedlings compared with rhizome buds. Reghunath (1989) [28] applied the shoot tips of green cardamom to perform micropropagation experiments through callus culture. The results of this study showed that Murashige and Skoog (MS) medium supplemented with the combination of 4.0 mg/L NAA or 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg/L BAP provided the highest callus induction rate after 28 days of culture. On the other hand, the combined effect of cytokines and auxins in micropropagation has been reported to be effective in many species of the genus *Amomum*, including green cardamom, large cardamom, and purple amomum. These were not reported in the previous study [27]. Therefore, similar studies on black cardamom are needed to determine the most effective micropropagation protocol for black cardamom.

Therefore, the overall goal of the present study was to investigate the effects of different seed treatment methods on the seed germination and seedling growth of black cardamom under in vitro conditions and determine the effect of different PGRs on in vitro shoot multiplication and root formation of seedling shoot tips in order to develop a protocol that produces a constant supply of viable and clean clonally propagated plant materials for crop production.
