Cryo Biotechnology for Somatic Embryos of Agave Tequilana Cv. Chato: An Endangered Genetic Resource

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.

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