**1. Introduction**

The *Plectranthus* genus is among the most prominent members of the Lamiaceae family. *Plectranthus amboinicus* (Lour.) Spreng, native to eastern and southern Africa, is an important medicinal succulent perennial herb which tends to be creeping or climbing; it has highly aromatic leaves with short erect hairs [1]. In folk medicine of different countries, it is used for the treatment of skin diseases, constipation, asthma, flu, fever, cough, and headache [2–4]. The high concentration of active compounds that are found in *P. amboinicus* is predominantly responsible for its varied therapeutic potential as well as its culinary uses. The plant's raw leaves are eaten or added as a flavoring ingredient in traditional drinks, food, and meat stuffing [1,5]. Biochemical studies revealed that the essential oil extracted from this plant has high amounts of thymol [6], carvacrol [7], α-humulene, α-terpineol, β-caryophyllene, β-selinene, γ-terpinene, and p-cymene [4,8]. Previous literature revealed that this plant has several pharmacological effects, including antimicrobial [9], antibacterial [10,11], antifungal [8,12], and antiviral [13–16] activities. In addition to its antiepileptic [17], antitumorigenic [18,19], anti-inflammatory [20,21], and antioxidant [22,23] effects, *P. amboinicus* shows antagonistic activities against respiratory [24], cardiovascular [25], oral [26], digestive [27], and genitourinary [28] disorders.

**Citation:** Faisal, M.; Alatar, A.A. Establishment of an Efficient In Vitro Propagation Method for a Sustainable Supply of *Plectranthus amboinicus* (Lour.) and Genetic Homogeneity Using Flow Cytometry and SPAR Markers. *Horticulturae* **2022**, *8*, 693. https://doi.org/ 10.3390/horticulturae8080693

Academic Editor: Sergio Ruffo Roberto

Received: 23 June 2022 Accepted: 27 July 2022 Published: 1 August 2022

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Furthermore, the leaf paste of this plant is effective in the treatment of wounds [29] and skin diseases [30,31].

Since 1960, tissue culture has been used as an alternative method for conventional propagation in many plant species, where a large number of plants can be produced in a relatively short time and small space from a small piece of plant tissue [32]. Culturing of plant cells and tissues in vitro has become a reliable and necessary technique for propagation, improvement, and mass-multiplication of several plant species [33]. Micropropagation has been used to propagate many species of *Plectranthus*, such as *P. bourneae* [34–36], *P. edulis* [37,38], *P. amboinicus* [39,40], *P. barbatus* [41], *P. zeylanicus* [42], and *P. esculentus* [43].

One of the major problems facing the micropropagation system is the occurrence of morphological, physiological, and molecular changes in in vitro regenerated plants [44]. Therefore, it is necessary to evaluate the genetic stability of the micropropagated plants to ensure their conformity with the donor plants. Polymerase chain reaction (PCR) based on SPAR (single primer amplification reaction) markers is one of the techniques used to analyze the genetic stability of micropropagated plants. Among SPAR markers, inter-simple sequence repeat (ISSR) and directed amplification of minisatellite-region DNA (DAMD) are successfully used to reveal the genetic homogeneity between micropropagated and donor plants in many plant species [33,45–51]. ISSR and DAMD techniques are cost-effective, quick, simple, avoid the use of radioactivity and DNA blotting, and are susceptible to automation [33,51,52]. In addition, genetic changes in regenerated plants can be detected by flow cytometry, which has been shown to be an efficient and reliable tool for estimation of ploidy level and genome size by calculating the nuclear DNA content [53]. The flow cytometry system has been used to evaluate the genetic fidelity of regenerated plants in many medicinal plants, including *Bacopa monnieri* [44], *Ficus carica* [54], *Salix lapponum* [55], and *Brassica juncea* [53].

The objective of this research was to develop an efficient and reproducible method for in vitro propagation of *P. amboinicus* from nodal explants for a sustainable large-scale production. Genetic homogeneity of micropropagated plantlets was assessed for the first time through DNA-based SPAR markers and flow cytometry to ensure the propagation and supply of true-to-type plantlets.

## **2. Materials and Methods**

#### *2.1. Shoot Materials and Explants Preparation*

Shoots of the *Plectranthus amboinicus* were harvested during March–April 2021 from a plant maintained in the growth chamber at the Botany & Microbiology Department, King Saud University, which was full of healthy growth. The plant samples were identified with the help of a taxonomist in the department and verified at the department's herbarium, where vouchers were deposited. The excised shoots were thoroughly cleaned in water with Tween-20 (2-3 drops) for a period of twenty minutes in order to eliminate any dust and dirt. After that, they were cut into pieces that were 2–3 cm in size and placed under running tap water for a period of 30 min. All the materials were moved into a biosafety laminar-air flow hood, where they were sterilized for three minutes with a freshly prepared 0.1 percent (*w*/*v*) mercury chloride (HgCl2) solution (Riedel-de Haan AG, Seelze, Germany) while being gently stirred. The sterilized shoots (nodal segments) were given a thorough washing in deionized ultra-pure water to eliminate any residues of the sterilant before being cut into pieces measuring 0.5–0.7 cm in length.
