1	Cryopreservation using Encapsulation-Dehydration Barbara M. Reed USDA-ARS National Clonal Germplasm Repository Corvallis, Oregon CRIS 5358-21000-033-00D
2	Encapsulation-Dehydration The E-D technique was originally developed in the laboratory of Jean Dereuddre in Paris using a cold-acclimation pretreatment This adaptation using a sucrose pretreatment was developed by Dominique Dumet for use with somatic embryos The technique was later modified for Ribes meristems in a collaboration of Dr. Dumet with Erica Benson and Barbara Reed Recipes are given at the end of the presentation
3	Basic Steps In vitro cultured plants (3 weeks from last transfer) Remove 1-2 cm shoot tips and plant on 0.75 M sucrose growth medium for 7 days. (If using cold-acclimated plants, skip this step) Dissect 1 mm shoot tips and encapsulate in alginate beads Shake beads in 0.75 M sucrose solution for 20 hrs Dry under laminar flow or over silica gel to 20% moisture content Enclose in a cryo tube and plunge in liquid nitrogen Thaw at room temperature for 20 minutes Rehydrate in liquid MS medium for 10 minutes and plate on recovery medium with 1 g/L agar less than the normal multiplication medium
4	In vitro cultured plants (3 weeks from last transfer) can either be cold acclimated or pretreated on 0.75 M sucrose medium
5	Sucrose Pretreatment Remove 1-2 cm shoot tips and grow on 0.75 M sucrose medium for 7 days
6	Dissect 1 mm shoot tips and encapsulate in alginate beads. To encapsulate, place tips in the alginate medium in a small sterile beaker or dish, then draw them up into a sterile pipette and drop them one by one into the calcium chloride solution. Hold the pipette vertically and avoid air bubbles in the beads. Let them stand in the calcium chloride for 20 minutes.
7	Shake beads in 0.75 M sucrose solution for 20 hrs at 22-25° C
8	Briefly blot beads dry on sterile filter paper, then arrange them so they do not touch, and dry them under laminar flow (4-6 hrs) or over silica gel to 20% moisture content. You must calibrate the drying time to fit your laboratory conditions.
9	This is an example of the change in moisture content of alginate-encapsulated meristems during air dehydration under the laminar flow
10	Moisture content determination of alginate beads
11	Enclose in cryo tube and plunge into liquid nitrogen. Thaw 20 min at room temperature
12	Rehydrate in liquid MS medium for 10 minutes and plate on recovery medium with 1 g/L agar less than normal
13	Step 1: Dissection only. These shoots are 3 weeks old
14	Step 2: Encapsulation and 20 hr Sucrose Step 3: Dried to 20% Moisture Content These shoots are 3 weeks old
15	Step 4: Liquid Nitrogen Exposure These shoots are 3 weeks old
16	Conclusions This method is easy to do and successful for many types of plants When starting to use this method be sure to test for each control step Control 1 and 2 should be 80-100% before you continue to Control 3 Control 3 should be high if you expect to have recovery after liquid nitrogen exposure Shoots will easily grow out of the rehydrated beads and develop into plantlets in 1-2 weeks
17	Genera cryopreserved at NCGR using E-D Cynodon (Bermudagrass) Reed, B.M., N. Wang, J. D’Achino, and R.E. Barker. Cryopreservation of Bermudagrass Germplasm. In press. Humulus Reed, B.M., N. Okut, J. D’Achino, L. Narver, and J. DeNoma. Cold storage and cryopreservation of hops (Humulus L.) shoot cultures through application of standard protocols. CryoLetters. 24:389-396. 2003. Ribes Reed, B.M., D. Dumet, J. M. DeNoma and E.E. Benson. 2001. Validation of cryopreservation protocols for plant germplasm conservation: A pilot study using Ribes L. Biodiversity and Conservation 10(6): 939-949; Dumet D., Y. Chang, B.M Reed, and E.E. Benson. Replacement of cold acclimatization with high sucrose pretreatment in black currant cryopreservation. In Cryopreservation of Tropical Germplasm. Current research progress and application, Engelmann F. and Takagi H., eds., Japan International Research Center for Agricultural Sciences and International Plant Genetic Resources Institute, Rome, Italy, 385-387. 2000; Reed, B.M., L. Schumacher, D. Dumet, and E.E. Benson. Evaluation of a modified encapsulation-dehydration cryopreservation procedure for storing Ribes germplasm. (in press). Zoyzia grass Chang Y., R. Barker, and B. M. Reed. 2000. Cold acclimation improves recovery of cryopreserved grass (Zoysia and Lolium sp.). Cryo-Letters 21:107-116.
18	Basic Steps for Sucrose Pretreatment and Encapsulation-Dehydration In vitro cultured plants (3 weeks from last transfer) Remove 1-2 cm shoot tips and plant on 0.75 M sucrose growth medium for 7 days (Appendix A) Dissect 1 mm shoot tips and encapsulate in alginate beads (Appendix B) formed in Calcium Chloride (Appendix C) Shake beads in 0.75 M sucrose solution for 20 hrs (Appendix C) Dry under laminar flow or over silica gel to 20% moisture content Enclose in a cryo tube and plunge in liquid nitrogen Thaw at room temperature for 20 minutes Rehydrate in liquid MS medium for 10 minutes and plate on recovery medium with 1 g/L agar less than the normal multiplication medium (Appendix D)
19	Appendix A 0.75 M Sucrose Pretreatment Medium Plant 1 cm shoot tips on this medium for 7 days before taking 1 mm shoot tips for E-D MS base medium with 0.75 M sucrose Sucrose 256.73 g/L = 0.75 M 0.8% agar Pour into petri dishes
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21	Appendix C 0.75 M Sucrose Medium for shaking Alginate Beads and CaCl₂ Medium for forming beads
22	Appendix D Standard MS medium for Rehydrating Beads Use liquid Murashige and Skoog 1962 medium for rehydrating beads (30 g/L sucrose) Add enough to cover the beads in the cryo tube Hold for 10 minutes to rehydrate the beads Drain off liquid MS and plate beads on soft recovery medium (1 g/L less agar than the multiplication medium you normally use)