**1. Introduction**

*Suaeda aralocaspica* is an annual halophyte (Amaranthaceae), with succulent leaves and grape-like fruits, distributed in the saline deserts of central Asia. In China, this plant is mainly distributed in the southern margin of the Junggar Basin in Xinjiang [1–3]. The study of *S. aralocaspica* is mainly focused on the leaf morphology and anatomical structure [4,5], germination characteristics [6–9], and photosynthetic type [10]. *S. aralocaspica* is a single-cell C4 photosynthetic plant without Kranz anatomy and has two types of chloroplast, called 'Borszczowioid type' [10,11]. This plant can produce two distinct types of seeds on a single plant, which have obvious differences in seed coat color, seed size, dormancy and germination characteristics. The brown seeds have high salt tolerance and are not dormant, while the black seeds have low salt tolerance and non-deep physiological dormancy [12–14]. However, there is no significant difference in growth, mineral nutrient content, and salt tolerance at middle and late growth stages [12]. Besides, short time pre-soaking, with a low concentration of abscisic acid (ABA), promotes the germination and seedling growth of dimorphic seeds of *S. aralocaspica* [15].

**Citation:** Si, Y.; Haxim, Y.; Wang, L. Optimum Sterilization Method for In Vitro Cultivation of Dimorphic Seeds of the Succulent Halophyte *Suaeda aralocaspica*. *Horticulturae* **2022**, *8*, 289. https://doi.org/10.3390/ horticulturae8040289

Academic Editor: Sergio Ruffo Roberto

Received: 18 February 2022 Accepted: 28 March 2022 Published: 29 March 2022

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There are few molecular studies about *S. aralocaspica*. Compared with four traditionally used reference genes, *GAPDH* and *β-TUB* are stable internal reference genes and more suitable for the subsequent gene research of *S. aralocaspica* under different experimental conditions [16]. The results of KEGG enrichment and gene expression analysis reveal that specific genes and miRNAs are regulated differently between black and brown seeds during germination, which may contribute to the different germination behaviors of dimorphic seeds of *S. aralocaspica* in unpredictable environments [17]. The sequencing and assembly of the *S. aralocaspica* whole genome are finished and the length is 425 Mb. In addition, a complete chloroplast genome is also assembled. *S. aralocaspica* is the first sequenced halophyte and single-cell C4 plant [18].

*Suaeda. aralocaspica* can grow normally in typical saline soils (salt content of the topsoil exceeds 10%). Brown seeds can germinate at high salinity (over 1000 mmol/L NaCl). This plant is valuable for the study of salt tolerance, C4 photosynthesis without Kranz anatomy, and seed heteromorphism. However, this plant is limited to saline deserts in central Asia, and the field sampling is also restricted by the season and the low density of persistent soil seed bank. These factors influence the supplement of *S. aralocaspica* material for experimental research. Thus, an efficient seed sterilization and culture method is essential for the effective supply of this research material.

Obtaining high-quality sterile seeds and seedlings is affected by various factors, such as the type of disinfectant, the time duration of sterilization, the pH of the medium, etc. [19]. Common surface disinfectants include ethanol, sodium hypochlorite, hydrogen peroxide, and mercury chloride [20–22]. As a commonly used medical disinfectant, 70–75% ethanol inactivates some bacteria by infiltrating through their cell membranes to denature various proteins. The bactericidal effect of 75% ethanol, when used together with other disinfectants, is better than that of using only ethanol as a disinfectant [22]. Mung bean seeds are disinfected with 75% ethanol for 30 s and then disinfected with 1.0% NaClO for 10 min [23]. *Salicornia europaea* seeds are treated with different concentrations of sodium hypochlorite (NaClO), mercuric chloride (HgCl2), and hydrogen peroxide (H2O2) on a Murashige and Skoog (MS) medium, with different concentrations of the hormone. The optimal sterilization effect of *S. europaea* seeds is after they have been treated with mercuric chloride, with a quality fraction of 0.1% for 10–20 min [24].

Seed sterilization and aseptic seedling cultivation have played a crucial role in subsequent research. Sterile seedlings are the source of explants in the tissue culture system. However, the sterilization methods of *S. aralocaspica* seeds have not been reported. We hypothesized that dimorphic seeds had distinct responses to disinfectants, sterilization time, and pH value of the mediums. Therefore, the present study was conducted to compare different sterilizing protocols, employed for different types of seeds in vitro culture, and to find out the best and most efficient sterilization procedure, based on germination, contamination, and seedling survival, which can be used for the rapid propagation system of *S. aralocaspica*.
