Evaluation of in vitro shoot multiplication and ISSR marker based assessment of somaclonal variants at different subcultures of vanilla (Vanilla planifolia Jacks)

The effect of subculture cycles on somaclonal variation of V. planifolia using intersimple sequence repeat (ISSR) markers was analyzed. Nodal segments of 2cm in length were established in vitro and multiplied by 10 subculture cycles in Murashige and Skoog (MS) medium supplemented with 8.86M BAP (benzylaminopurine). After 45days in each culture, the length and number of shoots per explant were evaluated. For ISSR markers, ten shoots per each subculture and the mother plant were used. Ten ISSR primers were used and a total of 118 bands were obtained. The polymorphism (%) was calculated and a dendrogram based on Jaccard's genetic distance between the subcultures and the donor plant was obtained. These results show that the multiplication rate tends to increase until subculture five, whereas shoot length decreases as the number of subcultures increases. The ISSR markers revealed an increase in the polymorphism percentage after the fifth culture cycle. The dendrogram showed the formation of two groups. The first group, with less genetic variability, is the donor plant and subcultures 1-5; the second group has greater genetic distance and is formed by subcultures 6-10. The results revealed that the number of subcultures with 8.86M BAP is a factor that affects the somaclonal variation during in vitro regeneration of V. planifolia. In conclusion, the subculture number affects somaclonal variation and in vitro development of V. planifolia.