ASHS 2015 Annual Conference
Cryopreservation Techniques of Plumules of Brazilian Green Dwarf Coconut Accession ( BGD)
Cryopreservation Techniques of Plumules of Brazilian Green Dwarf Coconut Accession ( BGD)
Friday, August 7, 2015
Napoleon Expo Hall (Sheraton Hotel New Orleans)
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.