**1. Introduction**

*Aconitum violaceum* Jacq. ex Stapf is a biennial herbaceous medicinal plant of the Ranunculaceae family and is endemic to the north-western Himalayan region of India, Pakistan, and Nepal [1–3]. In India, it is primarily found in the alpine and subalpine regions of the north-western Himalayas, at an elevation range of 3000–4000 m asl and shares its position with vulnerable plant species [4,5]. The toxic alkaloids of *A. violaceum* can be easily converted into less harmful alkaloids by heating or by using an alkaline

**Citation:** Hadi, A.; Singh, S.; Rafiq, S.; Nawchoo, I.A.; Wagay, N.A.; Mahmoud, E.A.; El-Ansary, D.O.; Sharma, H.; Casini, R.; Yessoufou, K.; et al. In Vitro Propagation of *Aconitum violaceum* Jacq. ex Stapf through Seed Culture and Somatic Embryogenesis. *Horticulturae* **2022**, *8*, 599. https://doi.org/10.3390/ horticulturae8070599

Academic Editor: Sergio Ruffo Roberto

Received: 9 June 2022 Accepted: 28 June 2022 Published: 4 July 2022

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treatment. They can then be employed in Ayurvedic and Unani medicines after they have been detoxified [6]. Traditionally, it is used to treat boils [7–9], asthma, high fever [7–12], gastric troubles [8–12], sciatic pains [8,11,13], intestinal worms [7,12,13], renal pain [7,11,12], and snake and scorpion bites [6]. This is due to its wide spectrum of biological activities, such as antioxidant [14], antimicrobial [14], anti-inflammatory [15–17], anti-malarial [15–18], anti-proliferative [16–20], analgesic, and antipyretic properties [14,15,17].

*A. violaceum* faces a great threat of extinction in its natural habitat due to various factors, such as physiological seed dormancy of >5 months [4,21,22], and long-term burying of seeds beneath snow (more than five months). Due to its specific ecological requirement, seed germination and seedling establishment in *A. violaceum* are quite challenging. *A. violaceum* grows along the margins of irrigation canals and at the edges of alpine streams; consequently, the majority of the seeds are susceptible to being carried away from their natural environment by running water, heavy rainfall, and floods, exposing them to adverse environmental conditions [4,21]. The plant maintains its spatial continuity through its rhizome. Therefore, uprooting whole plants due to collection, overgrazing, and premature harvesting, alongside construction of high-altitude roads, dams, cemented water channels, and human settlements, also contribute to the decline of this species from its natural habitat. Beetles and aphids are another potential hazard to this species as they consume the flowers and other reproductive portions of the plant, thereby reducing the species' sexual potential [22]. In addition, the species has a unique niche, which may limit its area of occupancy and dispersion [4].

When properly applied, mass propagation methods may help to reduce the extinction risk of vulnerable species by using procedures such as in vitro propagation [22]. In vitro regeneration techniques have been established for some *Aconitum* species such as *A. chasmanthum* Stapf ex Holmes [22], *A. ferox* [23], *A. heterophyllum* [24], *A. nagarum* [25], and *A. vilmorinianum* [26], but no such attempts have been made for *A. violaceum.* Giri et al. [24] regenerated complete plantlets from the somatic embryos of leaf and petiole explants in *Aconitum heterophyllum*. Hatano et al. [27] regenerated complete plantlets from the somatic embryo of anther explant in *Aconitum carmichaeli*. High concentrations of NAA (10 mg L<sup>−</sup>1) and darkness promote somatic embryo development in *Ranunculus sceleratus* [28]. After surveying extensive literature from various databases (DOAJ, Google Scholar, PubMed, Scihub, ScienceDirect, etc.) only a few studies have reported on the in vitro propagation of *A. violaceum* to date. Rawat et al. [29] attempted in vitro propagation from the nodal explant. Given the aforementioned issues, the current research on *A. violaceum* was carried out to develop: (1) direct in vitro seed germination protocols; (2) embryogenic callus production; and (3) complete plant regenerations from somatic embryos.

Due to its immense medicinal and economic value, establishing in vitro propagation protocols for *A. violaceum* would not only provide elite clones for pharmaceutical uses and facilitate rapid propagation and germplasm conservation, but it would also help to domesticate the plant and preserve wild populations.

#### **2. Materials and Methods**

#### *2.1. Collection of Plant Material*

The seeds of *A. violaceum* Jacq. ex Stapf were collected in the month of August (10–28) as soon as the follicle burst for seed dispersal, from the villages of Khawous and Numsuru (3220 m asl to 3340 m asl; 34◦15.905 N to 34◦12.705 N; 75◦96.040 E to 75◦96.720 E) in Ladakh, India. The plant specimen was identified and authenticated by the Center for Biodiversity and Taxonomy (CBT), Department of Botany, University of Kashmir, Hazratbal-Srinagar. A voucher specimen (accession number 3738-KASH) was deposited at the Kashmir University Herbarium (KASH). Seeds were collected in dry plastic and glass bottles and brought to the plant tissue culture laboratory, Department of Botany, University of Kashmir, where they were stored between 4 ◦C and 6 ◦C with 55 to 65% relative humidity for experimental purposes.
