1	Cryopreservation using Slow Cooling with PGD Barbara M. Reed USDA-ARS National Clonal Germplasm Repository Corvallis, Oregon CRIS 5358-21000-033-00D
2	Slow Cooling/Two-Step Freezing There are many variations of the slow cooling technique that cool the specimens at less than 1 degree per minute to -35 or -40°C and then plunge them in liquid nitrogen This form uses the cryoprotectant PGD developed by Finkle and Ulrich in 1979 This is the standard technique used for slow cooling at NCGR (Reed, et al.) Many samples can be processed at one time Recipes are given at the end of the presentation
3	Basic Steps In-vitro cultured plants (3 weeks from last transfer) Cold-acclimate plantlets for 2-4 weeks with alternating temperatures (8 hr 22°C and 16 hr -1°C) Dissect 1 mm shoot tips and plate on 5% DMSO pretreatment medium for 48 hr in cold acclimation Add PGD cryoprotectant to shoot tips in a cryo tube over 30 minutes (on ice) Hold at 0°C for 30 minutes Cool at 0.1°C to -40°C with exotherm at -9°C Plunge in LN Thaw 1 min in 45 °C water then 2 min 25°C water Rinse with liquid MS medium and plate on recovery medium
4	In-vitro cultured plants (3 weeks from last transfer) are placed in cold acclimation for 2-4 weeks
5	Dissect 0.8 mm shoot tips and culture on medium with 5% DMSO and 1 g/L more agar than normal medium
6	Place cryotubes in an ice tray and add 2 drops of liquid MS medium. Add shoot tips and gradually add PGD up to 1.2 ml over 30 min. Hold in the freezer for 30 additional minutes. Drain down to 1 ml PGD and close caps.
7	Cool with the following program to -40 °C then plunge in LN 1) 0.1 °C/min to sample temperature -9 °C 2) Initiate exotherm by taking chamber temperature to - 50 °C at 99.9 °C /min, then warm chamber to -15 °C at 20 °C/min 3) Resume 0.1 °C/min to sample temperature -40 °C
8	Thaw quickly in 45°C water for 1 min then move to 25°C water for 2 min
9	Drain off PGD and add liquid MS medium. Drain on filter paper and plate on recovery medium in multi-cell plates or petri dishes
10	Recovering Rubus shoot tips Controls are in the column on the right. All others were exposed to liquid nitrogen
11	Protocols for slow cooling developed at NCGR Fragaria (Reed, B.M. and K. Hummer. 1995. Conservation of germplasm of strawberry (Fragaria species), p. 354-370. In: Y.P.S. Bajaj (ed.). Biotechnology in Agriculture and Forestry, Cryopreservation of Plant Germplasm I, Vol. 32. Springer-Verlag, Berlin.) 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) Lolium (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.) Pyrus (Reed, B.M. 1990. Survival of in vitro-grown apical meristems of Pyrus following cryopreservation. HortScience 25:111-113;Chang Y. and B. M. Reed. Extended Alternating-Temperature Cold Acclimation and Culture Duration Improve Pear Shoot Cryopreservation Cryobiology 40:311-322. 2000; Chang Y. and B.M. Reed. Preculture Conditions Influence Cold Hardiness and Regrowth of Pyrus cordata Shoot Tips after Cryopreservation. HortScience. 36: 1329-1333. 2001. Ribes (Reed, B.M. and X. Yu. 1995. Cryopreservation of in vitro-grown gooseberry and currant meristems. CryoLett. 16:131-136; 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.) Rubus (Reed, B.M. and H.B. Lagerstedt. 1987. Freeze preservation of apical meristems of Rubus in liquid nitrogen. HortScience 22:302-303; Reed, B.M. 1988. Cold acclimation as a method to improve survival of cryopreserved Rubus meristems. Cryo-Lett. 9:166-171; Reed, B.M. 1993. Responses to ABA and cold acclimation are genotype dependent for cryopreserved blackberry and raspberry meristems. Cryobiology 30:179-184; Chang, C. and B.M. Reed. 1999. Extended cold acclimation and recovery medium alteration improve regrowth of Rubus shoot tips following cryopreservation. CryoLett 20:371-376.) Vaccinium (Reed, B.M. 1989. The effect of cold hardening and cooling rate on the survival of apical meristems of Vaccinium species frozen in liquid nitrogen. Cryo-Lett. 10:315-322.)
12	Conclusions This method is easy to do and successful for many types of temperate plants Be sure to include controls for the dissection and cryoprotectant steps PGD is relatively nonphytotoxic so cryoprotectant controls can be held in the refrigerator, then rinsed and plated either at the exotherm or at the end of the freezing cycle. Shoots will begin to develop into plantlets in 3-4 weeks
13	NCGR Storage Dewars
14	Basic Steps In vitro cultured plants (3 weeks from last transfer) Cold-acclimate plantlets for 2-4 weeks with alternating temperatures (8 hr 22°C and 16 hr -1°C) Dissect 1 mm shoot tips and plate on 5% DMSO pretreatment medium for 48 hr in cold acclimation (Appendix A) Add PGD cryoprotectant to shoot tips in a cryo tube over 30 minutes (on ice) (Appendix B) Hold at 0°C for 30 minutes Cool at 0.1°C/min to -40°C with exotherm at -9°C Plunge in LN Thaw at 45 °C for 1 min and 25°C for 2 min Rinse with liquid MS medium and plate
15	Appendix A DMSO Pretreatment Medium
16	Appendix B Preparation of PGD cryoprotectant In a 50 ml graduated cylinder add 30 ml of liquid MS medium (no hormones) and small stir bar. Add 4.9 ml DMSO (reagent grade, do not use DMSO older than 1 year). Wear gloves Slowly add 5 g glucose (dextrose) while stirring. Very slowly add 5 g polyethylene glycol mw10,000 (pgd) while stirring and let stir until dissolved. Bring to volume (50 ml) with liquid ms (not water), cover with parafilm to mix. Filter-sterilize cryoprotectant through a filter flask and place the flask in the freezer until ready for use (at least 30 minutes but not much longer or it will freeze). Wash out the cylinder and stir bar immediately yourself, do not leave it for others. Wear gloves. While the cryoprotectant is cooling you have 30 min to transfer the shoot tips to the cryo tubes.