Cryopreservation Techniques of Plumules of Brazilian Green Dwarf Coconut Accession ( BGD)

The objective was to evaluate the effect of cryoprotectant solutions and immersion times on the survival and regeneration of Brazilian green dwarf (BGD) accession. Encapsulation-dehydration and the droplet-vitrification techniques were tested in plumules excised from mature zygotic embryos of Coconut fruits  (10-11 months) of Active Germplasm Bank of Embrapa Coastal Tablelands, Sergipe, Brazil. The plumules, after the desinfection,  were initially precultured for 72 hours in Y3 medium with 0.12 M sucrose.  For the  encapsulation, the plumules were suspended in 3% (w/v) sodium alginate in a growth regulator- and calcium-free liquid MS medium supplemented with 0.1 M sucrose. The plumules with some alginate solution were dispensed into a liquid MS medium containing 100 mM calcium chloride and 0.1 M sucrose to form beads. Thereafter the beads were transferred to cryoprotectant solutions composed by Y3 medium supplemented with 0.5 or 1.0 M sucrose and incubated on rotary shaker providing gentle shaking for 48 hours at 25 ± 2°C in the absence of light. Half of the beads, from each treatment, was then transferred to a recovery medium (-LN), while the other half of the beads was placed in 2 mL sterile cryogenic vials and rapidly cooled by direct immersion into liquid nitrogen (+LN) by 24h. For the droplet-vitrification technique the explants were immersed in the PVS2 vitrification solution ( 30% DMSO plus 1 M sucrose, or 15% DMSO plus 1.0 M sucrose in a hormone-free liquid MS medium) for 30, 45 and 60 minutes. After dehydrating the plumules were transferred to aluminum foil containing drops of 10 µL PVS2/foil and introduced into sterile polypropylene cryotubes and quickly immersed in liquid nitrogen by 24 h. After cryopreservation, cryogenic vials containing beads or aluminum foil were thawed in a water bath at 38 ± 2°C for 2 to 3 minutes and then cultured on regeneration medium. After three months, the explants (-LN and +LN) were evaluated for the percentage of survival and induction of somatic embryogenesis. The regenerative capacity of BGD plumules (control) was 80% through somatic embryogenesis. The cryoprotectant solutions composed through Y3 medium supplemented with 0.5 or 1 M sucrose provide 86.67 and 66.67%, respectively,  of survival in plumule encapsulated. The immersion in PVS2 solution for 30 minutes promoted 100% survival. Both methods and can be recommended for future BGD accession coconut cryopreservation protocols.