**1. Introduction**

Vanilla (*Vanilla planifolia* Jacks. ex Andrews), of the family Orquidaceae, is cultivated for its fruits for the extraction of vanillin, one of the most valuable spices in the food, cosmetic and pharmaceutical industries [1,2]. Despite its economic importance, *V. planifolia* is classified in the category B2ab (iii, v) "Endangered" in The International Union for Conservation of Nature (IUCN) Red List (http://www.iucnredlist.org accessed on 22 June 2021) version 3.1. In Mexico, *V. planifolia* is a classified species with special protection due to severe fragmentation of its habitat [3].

Vanilla is propagated asexually by cuttings and is manually pollinated, obtaining pods that contain seeds with low or no germination [1,4]. Commercial propagation by cuttings limits the genetic diversity of this species, causing susceptibility to pests and diseases and loss of tolerance to abiotic factors [5], leading to premature fruit drop. Therefore, expanding the genetic diversity of *V. planifolia* is important in breeding programs [6].

An alternative to induce genetic variability is by in vitro mutagenesis techniques [7–9]. Genetic variations obtained from in vitro culture are also called somaclonal variations [10].

**Citation:** Serrano-Fuentes, M.K.; Gómez-Merino, F.C.; Cruz-Izquierdo, S.; Spinoso-Castillo, J.L.; Bello-Bello, J.J. Gamma Radiation (60Co) Induces Mutation during In Vitro Multiplication of Vanilla (*Vanilla planifolia* Jacks. ex Andrews). *Horticulturae* **2022**, *8*, 503. https://doi.org/10.3390/ horticulturae8060503

Academic Editor: Sergio Ruffo Roberto

Received: 11 May 2022 Accepted: 2 June 2022 Published: 5 June 2022

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Somaclonal variations obtained by in vitro mutagenesis with gamma radiation are free of regulatory constraints and allow the regeneration of genetic variations in a short period of time at low cost, added to which this system provides easy manipulation of explants in confined and controlled spaces under aseptic conditions [11–13]. Mutagenesis with cobalt 60 (60Co) has high penetration potential, poses no risk to the environment and can be used to irradiate cells, tissues, organs and whole plants [14,15]. In vitro mutagenesis with 60Co has been used in the breeding of San Francisco lily (*Laelia autumnalis*) [16], rice (*Oryza sativa* L.) [17], ginger (*Zingiber officinale* Rosc.) [8], potato (*Solanum tuberosum* L.) [18], and tomato (*Lycopersicon esculentum* L.) [19].

Molecular marker analysis is an important tool to estimate somaclonal variation [20–22]. Among molecular markers, inter simple sequence repeats (ISSRs) are characterized by being dominant, reproducible, inexpensive and they do not require prior knowledge of the genome [23]. Recently, ISSRs have been studied to analyze somaclonal variation in broadleaf plantain (*Plantago major*) [24], sugarcane (*Saccharum* spp.) [25], tulip (*Tulipa suaveolens*) [26], arracacha (*Arracacia xanthorrhiza*) [27] and *Disanthus cercidifolius* Maxim., an ornamental shrub [28]. In *V. planifolia*, ISSRs have been previously used by Bello-Bello et al. [29], Ramírez-Mosqueda et al. [30] and Pastelín-Solano et al. [31]. The aim of this study was to induce somaclonal variation in *V. planifolia* by means of gamma irradiation with 60Co and detect it using ISSR molecular markers.
