*Article* **Gamma Radiation (60Co) Induces Mutation during In Vitro Multiplication of Vanilla (***Vanilla planifolia* **Jacks. ex Andrews)**

**María Karen Serrano-Fuentes 1, Fernando Carlos Gómez-Merino 1, Serafín Cruz-Izquierdo 2, José Luis Spinoso-Castillo <sup>1</sup> and Jericó Jabín Bello-Bello 3,\***


**Abstract:** In vitro mutagenesis is an alternative to induce genetic variation in vanilla (*Vanilla planifolia* Jacks. ex Andrews), which is characterized by low genetic diversity. The objective of this study was to induce somaclonal variation in *V. planifolia* by gamma radiation and detect it using inter-simple sequence repeat (ISSR) molecular markers. Shoots previously established in vitro were multiplied in Murashige and Skoog culture medium supplemented with 2 mg·L−<sup>1</sup> BAP (6-benzylaminopurine). Explants were irradiated with different doses (0, 20, 40, 60, 80 and 100 Gy) of 60Co gamma rays. Survival percentage, number of shoots per explant, shoot length, number of leaves per shoot, and lethal dose (LD50) were recorded after 60 d of culture. For molecular analysis, ten shoots were used for each dose and the donor plant as a control. Eight ISSR primers were selected, and 43 fragments were obtained. The percentage of polymorphism (% P) was estimated. A dendrogram based on Jaccard's coefficient and the neighbor joining clustering method was obtained. Results showed a hormetic effect on the explants, promoting development at low dose (20 Gy) and inhibition and death at high doses (60–100 Gy). The LD50 was observed at the 60 Gy. Primers UBC-808, UBC-836 and UBC-840 showed the highest % P, with 42.6%, 34.7% and 28.7%, respectively. Genetic distance analysis showed that treatments without irradiation and with irradiation presented somaclonal variation. The use of gamma rays during in vitro culture is an alternative to broaden genetic diversity for vanilla breeding.

**Keywords:** gamma rays; hormetic effect; ISSR markers; polymorphism; somaclonal variation
