Assessment of Genetic Stability During Cryopreservation of Vanilla (V. planifolia) Shoot-tips Using ISSR Markers

Maria Teresa Gonzalez-Arnao, Pedro Isidro-Adolfo, Maria F. NetzahualcoyotlMata, Lourdes G. Iglesias Andreu, Jaime Martínez-Castillo, University of Veracruz, INBIOTECA, CICY, 

In this study we used six selected ISSR primers (T05, T06, C07, UBC823, UBC825, UBC848) to evaluate the genetic stability during cryopreservation of vanilla (V. planifolia) shoot-tips subjected to a Droplet-Vitrification (D-V) protocol using PVS2 solution for dehydration. We studied the impact of tissues culture and cryopreservation, assessing the effect of multiplication cycles of donor plantlets, with few (less than 4) and multiples (more than 12) subcultures, from which were isolated the shoot-tips to be cryopreserved. It was also assessed the effect of treatments, which allowed growth recovery after immersion in liquid nitrogen. To apply D-V procedure, shoot-tips were dissected of motherplantlets with few or multiple subcultures, and then preconditioned for 1 day on MS semisolid medium supplemented with 0.3M trehalose, loaded in a solution of 0.4M sucrose + 2M Glycerol and exposed to PVS2 solution for 30 min at room temperature prior to the ultra-rapid cooling in liquid nitrogen placed on droplets of PVS solution over aluminium cryoplates. The analysis of electrophoretic profiles of in vivo plants and in vitro plantlets with different subculture numbers revealed a total of 153 bands and 21.2% of polymorphism. UPGMA dendrogram based on Nei’s genetic distance demonstrated that plants with multiple subcultures induced the greatest genetic variation but kept a high (0.90) similarity index. The electrophoretic profiles of all successive steps of D-V protocol revealed a total of 199 bands and 34.2% polymorphism. Treatment with PVS2 promoted the greatest variation and continued declining the level (0.816) of similarity compared to that of the plantlets with multiples subcultures. Plantlets regenerated after cryopreservation kept the same level (0.819) of those treated with PVS2, indicating that liquid nitrogen immersion did not induce a further genetic variation. Our studies revealed that both, subculturing number and the composition of vitrification solution influenced genetic variability.