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Hilda E. Lee-Espinosa, Joaquín Murguía-González, Benjamín García-Rosas, Ana L. Córdova-Contreras, Antonio Laguna-Cerda, Javier O. Mijangos-Cortés, Luis F. Barahona-Pérez, Lourdes G. Iglesias-Andreu, and Nancy Santana-Buzzy

A complete and efficient regeneration protocol was developed for Vanilla planifolia ‘Andrews’, an endangered orchid species that represents an important crop in several tropical countries. Axillary buds excised from the first to the eighth node, considering the first to fourth nodes as “young” (zone 1) and the fifth to eighth as “mature” (zone 2), were cultured on Murashige and Skoog (MS) medium supplemented with 5.73, 7.64, 9.55, or 11.46 μm 6-benzylaminopurine (BA) for shoot induction and in combination with 4.45 μm naphthalene acetic acid (NAA) to induce multiple shoot proliferation. Cytokinin concentration and bud position in the stem had a significant effect on the number of shoots regenerated. The greatest shoot formation per explant, for the two tested zones, was obtained with 9.55 μm BA on MS medium supplemented with 100 mg·L−1 myoinositol, 150 mg·L−1 citric acid, 100 mg·L−1 ascorbic acid, and 20 g·L−1 sucrose. Young buds from zone 1 were able to form an average of 18.5 ± 2.4 shoots per explant, whereas buds from zone 2 induced a maximum of 11.0 ± 1.0 shoots per explant. Plants of 2 to 3 cm height developed a root system in half-strength MS medium supplemented with 0.44 μm NAA and, once they reached 5 cm height on average, were transferred to greenhouse conditions for their acclimatization where a 100% rate survival was achieved. The optimal use of both young and mature buds from each mother plant to induce adventitious shoots permitted a marked increase in the number of shoots per explant. By using all buds from the upper stem part (zone 1 + zone 2) and subculturing every 90 d, the multiplication rate was 1.1 to 1.86 × 105 shoots per bud per year.