Lourdes Delgado-Aceves, Maria T. Gonzalez- Arnao, Liberato Portillo, Raquel Folgado, CUCBA-University of Guadalajara, University of Veracruz, The Huntington Library, Art Collections and Botanical Gardens

Agave tequilana cv. chato is an important resource widely used to produce a very emblematic and popular Mexican beverage called mezcal; however, its wild populations are currently severely degraded due to unsustainable management, mainly overexploitation. The application of tissue culture and cryopreservation would contribute to the propagation and conservation of this less studied cultivar. This work reports the in vitro multiplication via indirect somatic embryogenesis(ISE) and cryopreservation of regenerants. For ISE induction, segments (1 cm) of young leaves were cultivated for 40 days on MS basal solid medium with 10mg·L-1 4- amino-3,5,6-trichloro-2-pyridinecarboxylicacid (PIC) and 0.75 mg·L-1benzylaminopurine (BA). Generated calli were transferred to basal MS médium with reduced (76%) concentration of NH4NO3 and supplemented with 500 mg·L-1 glutamine, 250 mg·L- 1 casein hydrolysate and solidified with 6 g·L-1 phytagel for ISE conversion. After 50 days of culture, an average of 30±5 somatic embryos per explant were regenerated from calli mass. Cryopreservation experiments were performed following the V- cryoplate method and using somatic embryos of 1-3 mm in length, which were precultured on MS solid medium with 0.3 M sucrose for 1 d in dark, encapsulated over the cryoplate with calcium alginate (2%) containing 0.4 M sucrose, loaded in solution with 1 M sucrose and 2 M glycerol (15 min), and exposed to the vitrification solutions PVS2 and PVS3 for 30 min prior to direct immersion in liquid nitrogen. Rewarming took place in liquid medium with 1.2 M sucrose and samples were transferred to solid conversion medium in darkness for 7 days. More than 90% of somatic embryos shown post- cryopreservation recovery after 15 days of reculture, irrespective of the PVS used. Samples treated with PVS2 presented faster regeneration detected by the elongation of coleoptile and radicle. This is the first report on successful cryopreservation of somatic embryos from Agave genus.

Date Recorded: 
Thursday, July 25, 2019

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.

Date Recorded: 
Tuesday, July 23, 2019