**2. Materials and Methods**

#### *2.1. Plant Material*

The mother plant was collected from a commercial plantation in Veracruz, Mexico (19.39◦81 00" N, −96.76◦22 62" W). The accession was identified by the National Agro-Alimentary Health, Safety and Quality Service (SENASICA, certificate No. LAB 30044001/2018) and handling authorization was approved by the Mexico program of the Ministry of the Environment and Natural Resources (permission No. SGPA/DGVS/2868/19). The accession has been deposited in a public in vitro germplasm bank at the Plant Tissue Culture Laboratory of Colegio de Postgraduados, Veracruz, Mexico. In this study, all procedures performed were in compliance with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

#### *2.2. In Vitro Establishment and Multiplication*

Young, 20–30 cm long vanilla mother seedling stems containing three buds were used for in vitro establishment under greenhouse conditions. Leaves were removed from the stems and 2 cm long nodal segments containing one bud were cut. The nodes were used as explants and washed with running water and two drops of Tween 20 (Sigma-Aldrich, Chemical Company, St. Louis, MO, USA) for 30 min. Subsequently, the explants were immersed in a 0.1 mg·L−<sup>1</sup> fungicide solution of 50 WP Captan-ultra (Arysta Life Science México S.A. de C.V. Coah., MX) for 30 min. The explants were submerged in a 5% (*w*/*v*) solution of NaClO (Cloralex, Industrias Alen, S.A. de C.V, NL, MX) (6% a.i.). Finally, they were immersed in a 1.3% (*w*/*v*) mercuric chloride (HgCl2) solution for 15 min and rinsed five times with sterile distilled water. The explants were cultured individually in <sup>22</sup> × 150 mm test tubes containing 10 mL MS [32] medium, supplemented with 2 mg·L−<sup>1</sup> 6-benzylaminopurine (BAP) (Sigma-Aldrich, St. Louis, MO, USA) and 30 g·L−<sup>1</sup> sucrose. The Murashige and Skoog (MS) medium was adjusted to a pH of ±5.8 with 1 N sodium hydroxide (NaOH), and 0.25% (*w*/*v*) phytagel (Sigma-Aldrich) was used. The material was sterilized in an autoclave at 120 ◦C for 20 min. Cultures were incubated at 24 ◦C and the photoperiod was 16 h with a white LED light (460 and 560 nm) at an irradiance of <sup>45</sup> ± <sup>5</sup> <sup>μ</sup>mol m−<sup>2</sup> <sup>s</sup><sup>−</sup>1. After two weeks of culture, the explants were transferred to 500 mL jars with 30 mL MS multiplication medium supplemented with 2 mg·L−<sup>1</sup> BAP under the aforementioned light and temperature conditions. Three subcultures were performed in 60-day periods prior to the 60Co irradiation treatments.
