Controlled Rate Cooling

  • Barbara M. Reed
  • Esther Uchendu

Controlled rate cooling is based on osmotic regulation of cell contents and freeze-induced dehydration. The samples are pretreated in cryoprotectant solutions and cooled at a standard rate to an intermediate temperature such as –35°C or –40°C, with ice nucleation initiated at about –9°C. At the freezing point of the cryoprotectant solution, ice nucleation is initiated, and ice forms in the cryoprotectant solution and the intercellular spaces. The cytoplasm remains unfrozen due to solute concentration and the cell wall protects the cell membrane from damaging ice crystals. As the temperature is further decreased to –35°C or –40°C, the extracellular solution becomes increasingly icy and the intracellular solutes become highly concentrated. The plant cells lose water to the exterior ice and the cytoplasm is further concentrated. The intracellular freezable water is safely reduced before samples are plunged into liquid nitrogen (LN). If the cells are optimally dehydrated, the cytoplasm vitrifies on contact with LN. If the samples are under dehydrated, leaving freezable water in the cytoplasm, ice will form. If over dehydrated, the cells may die from desiccation.

Controlled rate cooling is very efficient for storing suspension and callus cultures, embryogenic cultures, and in-vitro shoot tips from temperate and subtropical plants. The advantages of controlled rate cooling include the use of standardized procedures, programmed cooling rates, and large batch sizes, and the effective use of technician time.


Somatic Embryo Cold Acclimation Cold Hardiness Cryoprotectant Solution Plant Vitrification Solution 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Benson EE, Lynch PT, June J (1995) The use of iron chelating agent desferrioxamine in rice cell cryopreservation: A novel approach for improving recovery. Plant Sci 110: 249-258CrossRefGoogle Scholar
  2. Bravo LA, Zuniga GE, Alberdi M, Corcuera LJ (1998) The role of ABA in freezing tolerance and cold acclimation. Physiol Plant 103: 12-23CrossRefGoogle Scholar
  3. Brison M, de Boucaud MT, Dosba F (1995) Cryopreservation of in vitro grown shoot tips of two interspecific Prunus rootstocks. Plant Sci 105: 235-242CrossRefGoogle Scholar
  4. Chang Y, Barker RE, Reed BM (2000) Cold acclimation improves recovery of cryopreserved grass (Zoysia and Lolium sp.). CryoLetters 21: 107-116PubMedGoogle Scholar
  5. Chang Y, Reed BM (1995) Improved shoot formation of blackberry and raspberry meristems following cryopreservation. Cryobiology 32: 581Google Scholar
  6. Chang Y, Reed BM (1999) Extended cold acclimation and recovery medium alteration improve regrowth of Rubus shoot tips following cryopreservation. CryoLetters 20: 371-376Google Scholar
  7. Chang Y, Reed BM (2000a) Cold acclimation improves the cryopreservation of in vitro-grown Pyrus and Rubus meristems. In: Engelmann F, Takagi H (eds.) Cryopreservation of Tropical Germplasm. Current Research Progress and Application. Japan International Research Center for Agricultural Sciences and International Plant Genetic Resources Institute, Rome, Italy, pp 382-384Google Scholar
  8. Chang Y, Reed BM (2000b) Extended alternating-temperature cold acclimation and culture duration improve pear shoot cryopreservation. Cryobiology 40: 311-322CrossRefPubMedGoogle Scholar
  9. Chang Y, Reed BM (2001) Preculture conditions influence cold hardiness and regrowth of Pyrus cordata shoot tips after cryopreservation. HortScience 36: 1329-1333Google Scholar
  10. Chen THH, Kartha KK, Constabel F, Gusta LV (1984) Freezing characteristics of cultured Catharanthus-roseus (L.). Don, G. cells treated with dimethylsulfoxide and sorbitol in relation to cryopreservation. Plant Physiol 75: 720-725CrossRefPubMedGoogle Scholar
  11. Chen THH, Kartha KK, Gusta LV (1985) Cryopreservation of wheat suspension culture and regenerable callus. Plant Cell Tissue Organ Cult 4: 101-109CrossRefGoogle Scholar
  12. Dereuddre J, Fabre J, Bassaglia C (1988) Resistance to freezing in liquid nitrogen of carnation (Dianthus cryophyllus L. var Eolo) apical and axillary shoot tips excised from different-aged in vitro plantlets. Plant Cell Rep 7: 170-173CrossRefGoogle Scholar
  13. Dumet D, Chang Y, Reed BM, Benson EE (2000) Replacement of cold acclimatization with high sucrose pretreatment in black currant cryopreservation. In: Engelmann F, Takagi H (eds.) Cryopreservation of Tropical Germplasm. Current Research Progress and Application. Japan International Research Center for Agricultural Sciences and International Plant Genetic Resources Institute, Rome, Italy, pp 385-387Google Scholar
  14. Dumet D, Engelmann F, Chabrillange N, Duval Y, Dereuddre J (1993) Importance of sucrose for the acquisition of tolerance to desiccation and cryopreservation of oil palm somatic embryos. CryoLetters 14: 243-250Google Scholar
  15. Finkle BJ, Ulrich JM (1979) Effects of cryoprotectants in combination on the survival of frozen sugarcane cells. Plant Physiol 63: 598-604CrossRefPubMedGoogle Scholar
  16. Ford CS, Jones NB, van Staden J (2000) Cryopreservation and plant regeneration from somatic embryos of Pinus patula. Plant Cell Rep 19: 610-615CrossRefGoogle Scholar
  17. Forsline PL, Stushnoff C, Towill LE, Waddell J, Lamboy W (1993) Pilot project to cryopreserve dormant apple (Malus sp) buds. HortScience 28: 118Google Scholar
  18. Gonzalez-Benito ME, Fernandez-Llorente F, Perez-Garcia F (1998) Interaction between cryopreservation rewarming rate and seed humidification on the germination of two Spanish endemic species. Ann Bot 82: 683-686CrossRefGoogle Scholar
  19. Heino P, Sandeman G, Lang V, Nordin K, Palva ET (1990) Abscisic acid deficiency prevents development of freezing tolerance in Arabidopsis thaliena (L.) Heynh. Theor Appl Genet 79: 801-806CrossRefGoogle Scholar
  20. Heszky LE, Jekkel Z, Ali A-H (1990) Effect of cooling rate, cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture [Puccinellia distans (L.) Parl.] Plant Cell Tissue Organ Cult 21: 217-226CrossRefGoogle Scholar
  21. Hubalek Z (2003) Cryoprotectants used in the cryopreservation of microorganisms. Cryobiology 46: 205-229CrossRefPubMedGoogle Scholar
  22. Jain S, Jain RK, Wu R (1996) A simple and efficient procedure for cryopreservation of embryogenic cells of aromatic Indica rice varieties. Plant Cell Rep 15: 712-717CrossRefGoogle Scholar
  23. Kartha KK, Engelmann F (1994) Cryopreservation and germplasm storage. In: Vasil IK, Thorpe TA (eds.) Plant Cell and Tissue Culture. Kluwer, Dordrecht, pp 195-230Google Scholar
  24. Kartha KK, Leung NL, Gamborg OL (1979) Freeze-preservation of pea meristems in liquid nitrogen and subsequent plant regeneration. Plant Sci Lett 15: 7-15CrossRefGoogle Scholar
  25. Kartha KK, Leung NL, Pahl K (1980) Cryopreservation of strawberry meristems and mass propagation of plantlets. J Am Soc Hort Sci 105: 481-484Google Scholar
  26. Lee SP, Chen THH, Fuchigami LH (1991) Changes in the translatable RNA population during abscisic acid induced freezing tolerance in bromegrass suspension culture. Plant Cell Physiol 32: 45-56Google Scholar
  27. Lovelock JE, Bishop MWH (1959) Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature (London) 183: 1394-1395CrossRefGoogle Scholar
  28. Malabadi RB, Nataraja K (2006) Cryopreservation and plant regeneration via somatic embryogenesis using shoot apical domes of mature Pinus roxburghii Sarg. Trees. In Vitro Cell Dev Biol Plant 42: 152-159CrossRefGoogle Scholar
  29. Malaurie B, Trouslot MF, Engelmann F, Chabrillange N (1998) Effect of pretreatment conditions on the cryopreservation of in vitro-cultured yam (Dioscorea alata ‘Brazo Fuerte’ and D. bulbifera ‘Doumea Imboro’) shoot apices by encapsulation-dehydration. CryoLetters 19: 15-26Google Scholar
  30. Menges M, Murray JAH (2004) Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures. Plant J 37: 635-644CrossRefPubMedGoogle Scholar
  31. Meryman HT, Williams RJ (1985) Basic principles of freezing injury to plant cells: Natural tolerance and approaches to cryopreservation. In: Kartha KK (ed.) Cryopreservation of Plant Cells and Organs. CRC Press, Boca Raton, FL, pp 13-47Google Scholar
  32. Moriguchi T, Akihama T, Kozaki I (1985) Freeze-preservation of dormant pear shoot apices. Japan J Breed 35: 196-199Google Scholar
  33. Plessis P, Leddet C, Collas A, Dereuddre J (1993) Cryopreservation of Vitis vinifera L. cv Chardonnay shoot tips by encapsulation-dehydration: Effects of pretreatment, cooling and postculture conditions. CryoLetters 14: 309-320Google Scholar
  34. Quatrano RS (1968) Freeze-preservation of cultured flax cells utilizing DMSO. Plant Physiol 43: 2057-2061CrossRefPubMedGoogle Scholar
  35. Reed BM (1988) Cold acclimation as a method to improve survival of cryopreserved Rubus meristems. CryoLetters 9: 166-171Google Scholar
  36. Reed BM (1990) Survival of in vitro-grown apical meristems of Pyrus following cryopreservation. HortScience 25: 111-113Google Scholar
  37. Reed BM (1993) Responses to ABA and cold acclimation are genotype dependent for cryopreserved blackberry and raspberry meristems. Cryobiology 30: 179-184CrossRefGoogle Scholar
  38. Reed BM (1996) Pretreatment strategies for cryopreservation of plant tissues. In: Normah MN, Narimah MK, Clyde MM (eds.) In Vitro Conservation of Plant Genetic Resources. Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia, pp 73-87Google Scholar
  39. Reed BM (2001) Implementing cryogenic storage of clonally propagated plants. CryoLetters 22: 97-104PubMedGoogle Scholar
  40. Reed BM, Hummer K (1995) Conservation of germplasm of strawberry (Fragaria species). In: Bajaj YPS (ed.) Biotechnology in Agriculture and Forestry: Cryopreservation of Plant Germplasm I. Springer-Verlag, Berlin, pp 354-370Google Scholar
  41. Reed BM, Hummer KE (2002) Cryopreservation of Ribes. In: Towill LE, Bajaj YPS (eds.) Biotechnology in Agriculture and Forestry: Cryopreservation of Plant Germplasm II. Springer-Verlag, Berlin, pp 323-343Google Scholar
  42. Reed BM, Lagerstedt HB (1987) Freeze preservation of apical meristems of Rubus in liquid nitrogen. HortScience 22: 302-303Google Scholar
  43. Reed BM, Okut N, D’Achino J, Narver L, DeNoma J (2003) Cold storage and cryopreservation of hops (Humulus L.) shoot cultures through application of standard protocols. CryoLetters 24: 389-396PubMedGoogle Scholar
  44. Reed BM, Yu X (1995) Cryopreservation of in vitro-grown gooseberry and currant meristems. CryoLetters 16: 131-136Google Scholar
  45. Ryynanen LA (1998) Effect of abscisic acid, cold hardening and photoperiod on recovery of cryopreserved in vitro shoot tips of silver birch. Cryobiology 36: 32-39CrossRefPubMedGoogle Scholar
  46. Sakai A (1960) Survival of the twig of woody plants. Nature 185: 393-394CrossRefGoogle Scholar
  47. Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep 9: 30-33CrossRefGoogle Scholar
  48. Sakai A, Kobayashi S, Oiyama I (1991) Survival by vitrification of nucellar cells of navel orange (Citrus sinensis var. brasiliensis Tanaka) cooled to -196°C. Plant Physiol 137: 465-470Google Scholar
  49. Sakai A, Nishiyama Y (1978) Cryopreservation of winter vegetative buds of hardy fruit trees in liquid nitrogen. HortScience 13: 225-227Google Scholar
  50. Smith D (1983) Cryoprotectants and the cryopreservation of fungi. Trans Br Mycol Soc 80: 360-363CrossRefGoogle Scholar
  51. Suzuki M, Ishikawa M, Okuda H, Noda K, Kishimoto T, Nakamura T, Ogiwara I, Shimura I, Akihama T (2006) Physiological changes in Gentian axillary buds during two-step preculturing with sucrose that conferred high levels of tolerance to desiccation and cryopreservation. Ann Bot 97: 1073-1081CrossRefPubMedGoogle Scholar
  52. Tanino KK, Chen THH, Fuchigami LH, Weiser CJ (1990) Metabolic alterations associated with abscisic acid-induced frost hardiness in bromegrass suspension culture cells. Pl Cell Physiol 31: 505-511Google Scholar
  53. Tao D, Li PH (1986) Classification of plant cell cryoprotectants. J Theor Biol 123: 305-310CrossRefGoogle Scholar
  54. Thierry C, Tessereau H, Florin B, Meschine M, Petiard V (1997) Role of sucrose for the acquisition of tolerance to cryopreservation of carrot somatic embryos. CryoLetters 18: 283-292Google Scholar
  55. Towill LE (1988) Survival of shoot tips from mint species after short-term exposure to cryogenic conditions. HortScience 23: 839-841Google Scholar
  56. Tyler N, Stushnoff C (1988) Dehydration of dormant apple buds at different stages of cold acclimation to induce cryopreservability in different cultivars. Can J Plant Sci 68: 1169-1176Google Scholar
  57. Tyler N, Stushnoff C, Gusta LV (1988) Freezing of water in dormant vegetative apple buds in relation to cryopreservation. Plant Physiol 87: 201-205CrossRefPubMedGoogle Scholar
  58. Uchendu E, Reed BM (2007) A comparative study of three cryopreservation protocols for effective storage of mint (Mentha spp.) CryoLetters In PressGoogle Scholar
  59. Ulrich JM, Finkle BJ, Moore PH, Ginoza H (1979) Effect of a mixture of cryoprotectants in attaining liquid nitrogen survival of callus cultures of a tropical plant. Cryobiology 16: 550-556CrossRefPubMedGoogle Scholar
  60. Verleysen H, Samyn G, Van Bockstaele E, Debergh P (2004) Evaluation of analytical techniques to predict viability after cryopreservation. Plant Cell Tiss Organ Cult 77: 11-21CrossRefGoogle Scholar
  61. Weiser CJ (1970) Cold resistance and acclimation in woody plants. HortScience 5: 403-410Google Scholar
  62. Withers L (1979) Freeze preservation of somatic embryos and clonal plantlets of carrot (Daucus carota L.). Plant Physiol 63: 460-467CrossRefPubMedGoogle Scholar
  63. Withers LA (1979) Freeze-preservation of cultured cells and tissues. In: Thorpe TA (ed.) Frontiers of Plant Tissue Culture 1978 Proc 4th Int Congr Plant Tissue and Cell Culture. IAPTC/ Calgary University Press, AB, pp 297-306Google Scholar
  64. Withers LA (1985) Cryopreservation of cultured cells and protoplasts In: Kartha KK (ed.) Cryopreservation of Plant Cells and Organs CRC Press, Boca Raton, FL, pp 243-267Google Scholar
  65. Withers LA, King PJ (1979a) The freeze preservation of cultured plant cells. Experientia 35: 984Google Scholar
  66. Withers LA, King PJ (1979b) Proline: A novel cryoprotectant for the freeze preservation of cultured cells of Zea mays L. Plant Physiol 64: 675-678CrossRefPubMedGoogle Scholar
  67. Withers LA, King PJ (1980) A simple freezing unit and cryopreservation method for plant cell suspensions. CryoLetters 1: 213-220Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Barbara M. Reed
    • 1
  • Esther Uchendu
    • 2
  1. 1.US Department of Agriculture, Agricultural Research ServiceNational Clonal Germplasm RepositoryCorvallisUSA
  2. 2.Department of HorticultureOregon State UniversityCorvallisUSA

Personalised recommendations