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United States Department of Agriculture

Agricultural Research Service

Research Project: RESEARCH TO DEVELOP STRATEGIES AND TECHNOLOGIES FOR PRESERVING PLANT GENETIC DIVERSITY IN EX SITU GENEBANKS

Location: Plant Germplasm Preservation Research Unit

Title: Increased efficiency using the encapsulation-dehydration cryopreservation technique

Authors
item Bonnart, Remi
item Volk, Gayle

Submitted to: CryoLetters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 19, 2010
Publication Date: May 9, 2010
Citation: Bonnart, R.M., Volk, G.M. 2010. Increased efficiency using the encapsulation-dehydration cryopreservation technique. CryoLetters. 31:95-100.

Interpretive Summary: Arabidopsis thaliana shoot tips were successfully cryopreserved using encapsulation-dehydration cryopreservation methods. We sought to increase the processing efficiency of encapsulation-dehydration by placing more than 1 shoot tip in each 4 mm calcium alginate bead. The time required to make 10 beads, each containing 5 shoot tips (4 min), was less than the time required to make 50 beads containing 1 shoot tip (12 min) and shoot tip regrowth after liquid nitrogen exposure remained high (between 60 and 68%). Using five Arabidopsis shoot tips per bead, alginate beads were formed either in the presence of 2 M glycerol+0.4 M sucrose or 0.5 M sucrose. Beads formed in the presence of glycerol were immediately air-dried and alginate beads formed in 0.5 M sucrose were incubated in solutions of 0.5, 0.75, and 1 M sucrose for 1 day each prior to air-dehydration. Shoot tip regrowth after cryoexposure was between 42 and 65%, with no significant differences among treatments. For Arabidopsis, we successfully reduced the amount of time needed for shoot tip processing by encapsulating five shoot tips per alginate bead and by using an encapsulation method that employed glycerol, without a lowering shoot tip regrowth levels after cryopreservation.

Technical Abstract: Arabidopsis thaliana shoot tips were successfully cryopreserved using encapsulation-dehydration cryopreservation methods. Between one and seven shoot tips were encapsulated within 4 mm calcium-alginate beads. Beads were formed in the presence of 2 M glycerol+0.4M sucrose. The time required to make 10 beads, each containing 5 shoot tips (4 min), was less than the time required to make 50 beads containing 1 shoot tip (12 min). Shoot tip regrowth after cryoexposure was between 60 and 68%, with between 1 and 7 shoot tips per bead. Using five Arabidopsis shoot tips per bead, alginate beads were formed either in the presence of 2 M glycerol+0.4 M sucrose or 0.5 M sucrose. Beads formed in the presence of glycerol were immediately air-dried to moisture contents between 0.21 to 0.26 g H2O/g FW (0.27 to 0.38 g H2O/g DW). Alginate beads formed in 0.5 M sucrose were incubated in solutions of 0.5, 0.75, and 1 M sucrose for one day each prior to air-dehydration, achieving moisture contents of 0.19 to 0.21 g H2O/g FW (0.23 to 0.27 g H2O/g DW). Shoot tip regrowth after cryoexposure was between 42 and 65%, with no significant differences among treatments. For Arabidopsis, we successfully reduced the amount of time needed for shoot tip processing by encapsulating five shoot tips per alginate bead and by using a glycerol-encapsulation method, without a lowering shoot tip regrowth levels after cryopreservation.

Last Modified: 7/28/2014