**6. References**


Cryopreserving Vegetatively Propagated Tropical Crops

Cooling Factors *CryoLetters* 23, 283-290

*Sciences* 83, pp. 205-215.

*Cryoletters* 15, pp. 47-52.

*Plant Physiology* 92, pp. 666-671.

simple freezing process. *Cryo-Letters* 17, 171-176.

tobacco tissue cultures. *Physiologia Plantarum* 15, 473 – 497.

by vitrification. *Cryoletters* 19, pp. 27-36.

cryostorage. Cryoletters 20, pp 77-82.

vitrification. *Cryoletters* 21 pp. 349 – 356.

JH Dodds, Chapman and Hall, London, pp 11-39.

Genetic Resources Institute, Rome, Italy, pp 136-139.

*Ekerbergia capensis*, Sparrm. *Cryoletters* 27 (1), pp. 5-16.

Durban, South Africa.

*Physioplogy* 96, 1093-1098.

346.

– The Case of *Dioscorea* Species and *Solenostemon rotundifolius* 503

[22] Gnanaprasam, S and Vasil IK (1992). Cryopreservation of immature embryos,

[23] González-Benito ME, Prieto RM, Herradón E & Martín C (2002) Cryopreservation Of

[24] Grospietsch M, Stadulkora E & Jiri Z (1999) Effect of osmotic stress on the dehydration

[25] Hatanaka T, Yasuda T, Yamaguchi T & Sakai A (1994). Direct regrowth of encapsulated

[26] Langis P, & Steponkus PL (1990). Cryopreservation of rye protoplast by vitrification.

[27] Matinez-Montero ME, Gonzalez-Arnao MT, Borroso-Nordelo C, Puentes-Diez C &

[28] Matsumoto T, Sakai A & Nako Y (1998). A novel preculturing for enhancing the

[29] Murashige T & Skoog E (1962) A revised medium for rapid growth and bioassays with

[30] Mycock D (1999). Addition of Calcium and Magnesium to a Glycerol and Sucrose

[31] Naidoo, S, (2006). *Investigations into the Post-harvest behavior and germplasm conservation of* 

[32] Niino T, Hettiarachchi A, Takahashi J &. Samarajeewa PK, (2000). Cryopreservation of

[33] Nishizawa, S, Sakai A, Amano Y & Matsumoto T (1993), Cryopreservation of

[34] Ng SYC & Ng NQ (1991) in *Tissue Culture for Conservation of Plant Genetic Resources,* (ed)

[35] Ng NQ & Daniel IO (2000) in *Cryopreservation of Tropical Germplasm, Current Research* 

[36] Pammenter NW , Vertucci CW & Berjak P (1991). Homoiohydrous (recalcitrant) seeds:

[37] Panis B, Strosse H, Van Den Hende S & Swennen R (2002) Sucrose preculture to simplify cryopreservation of banana meristem cultures. *CryoLetters* 23, 375-384 (2002) [38] Perán R, Berjak P, Pammenter NW & Kioko JI (2006). Cryopreservation, encapsulation

regeneration by simple freezing method. *CryoLetters* 13, 379-388.

embryogenic callus and cell suspension cultures of gramineous species. *Plant* 

*Quercus Suber* And *Quercus Ilex* Embryonic Axes: In Vitro Culture, Desiccation And

tolerance and cryopreservation of *Solanum Tuberosum* shoot tips. *Cryoletters* 20, 339-

somatic embryos of coffee (*Coffee canephora*) after cooling in liquid nitrogen.

Engelmann F (1998) Cryopreservation of sugarcane embryogenic callus using a

survival of *in vitro* – grown meristems of wasabi (*wasabi japonica*) cooled to -196oC

cryoprotectant solution improves the quality of plant embryo recovery from

*the seed of selected* Amaryllid *species*. MSc Thesis University of Kwa-Zulu Natal

lateral buds of *in vitro* grown innala plants (*Solenostemon rotundifolius*) by

Asparagus (*Asparagus officinalis* L.) embryogenic cells and subsequent plant

*Progress and Application,* (eds) F Engelmann and H Takagi, Japan International Research Centre for Agricultural Sciences, Tsukuba, Japan / International Plant

dehydration, the state of water and viability characteristics in *Landolphia kirkii*. *Plant* 

and promotion of shoot production of embryonic axes of a recalcitrant species


[6] Chandel, KPS, Chaudhury R & Radhamani J (1994), Biological mechanisms determining the recalcitrance in seeds of tea, cocoa and jackfruit. IBPGR-NBPGR Report. [7] Charoensub R, Phansiri S, Sakai A & Yongmanotchai W (1999). Cryopreservation of

[8] Cho EG, Noor NM, Kim HH, Rao VR & Engelmann F (2002) Cryopreservation of *Citrus* 

[9] Dereuddre J, Bland S. & Hassen N (1991). Resistance of alginate-coated somatic embryos

[10] Dumet D, Engelmann F, Chardrillange N, Duval Y, & Dereuddre J (1993). Importance of

[11] Egnin M, Mora A & Prakash, C.S. (1998). Factors Enhancing *Agrobacterium tumefaciens*-

[12] Engelmann F & Engels JMM, (2002), Technologies and strategies for existing

[13] Engelmann F, Dambier D & Ollitraut P (1994), cryopreservation of cell suspension and

[14] González-Arnao MT, Urra C, Engelmann F, Ortiz R, & Delafe C (1999).

[15] Gonzales-Benito ME & Perez C (1994). Cryopreservation of embryonic axes of two

[16] Harding K & Benson EE (1994). A study of growth, flowering, and tuberisation in plants

[17] Hatanaka T, Yasuda T, Yamaguchi T & Sakai A (1994). Direct regrowth of encapsulated

[18] Jitsuyama Y, Suzuki T, Harada T & Fujikawa S (2002). Sucrose incubation increases

[19] Kay DE (1973) TPI Crop and Product Digest No. 2 Root CROPS. London: Tropical

[20] Kioko J, Berjak P, Pammenter NW, Watt MP & Wesley-Smith J, (1998). Desiccation and

[21] Kioko, J, Berjak P, Pritchard H & Daws M (2000). Seeds of African pepper bark

cultivars of hazelnut (*Corylus avellana* L.). *Cryoletters*, 15, pp. 41-46

pp. 89-94.

309-316.

pp 89-104.

58.

of Preculture. Cryoletters 12 pp.125-134.

*Developmental Biology-Plants* 34, 310-318.

palm somatic embryos. *Cryo-Letters* 14: pp. 243-250.

and storage duration. *CryoLetters* 20, pp. 347-352.

collections. *Cryo Letters* 15, 59-66.

suspensions. *Cryoletters* 23, pp. 103-112.

*Research Progress and Application.* IPGRI: 371-377

*Cryoletters* 15, pp. 47-52.

Products Inst.

15-26

cassava *in vitro*-grown shoot tips cooled to –196oC by vitrification*. CryoLetters* 20,

*aurantifolia* seeds and embryonic axes using a desiccation protocol. *CryoLetters* 23,

of carrot (*Daucus carota* L.) to desiccation and freezing in Liquid nitrogen: 1. Effect

sucrose for the acquisition of tolerance to desiccation and cryopreservation of oil

Mediated Gene Transfer in Peanut (*Arachis Hypogaea* L.). In Vitro *Cellular and* 

conservation. In JMM Engels, VR Rao, AHD BROWN and MT Jackson (eds), Managing plant Genetic Diversity, CAB International Wallingfield/IPGRI, Rome,

embryogenic callus of citrus using a simple freezing process. *Cryo-Letters* 15: pp. 53-

Cryopreservation of encapsulated sugarcane apices - effect of storage-temperature

derived from cryopreserved potato shoot-tips: implications for *in vitro* germplasm

somatic embryos of coffee (*Coffee canephora*) after cooling in liquid nitrogen.

freezing tolerance of Asparagus (*Asparagus officianalis* L) embryonic cell

cryopreservation of embryonic axes of *Trichilia dregeana* SOND. *Cryo-Letters* 19: pp.

(Wurdurgia *salutaris*) can be cryopreserved after rapid dehydration in silica gel. In F. Engelmann and H. Takagi (eds.) *Cryopreservation of Tropical Germplasm, Current* 


**Part 8** 

**Equipment and Assays** 


**Part 8** 

**Equipment and Assays** 

504 Current Frontiers in Cryobiology

[39] Plessis P, Leddet C, Collas A & Dereuddre J. (1993). Cryopreservation of *Vitis Vinefera*

[41] Quain, MD, Berjak, P, Acheampong, E, and Kioko, JI, (2009) Sucrose Treatment And

[42] Reinhoud PJ, Schrijnemakers WM, van Iren F & Kijne JW (1995) vitrification and heat

[44] Santos IRI & Stushnoff C (2003) Desiccation and freezing tolerance of embryonic axes from *Citrus sinensis* (L) OSB. pretreated with sucrose. *CryoLetters* 24, 281-292. [45] Schippers, R.R. (2000). African Indigenous Vegetables. An Overview of the Cultivated

[46] Shibli RA, Moges AD, and Karam NS (2004). Cryopreservation Of African Violet

[47] Spurr A R (1969) A low-viscosity epoxy resin embedding medium for

[48] Tetteh JP & Guo JI (1993) *Problems of Frafra Potato Production in Ghana*. A dissertation.

[49] Thammasiri K. (1999). Cryopreservation of embryonic axes of jackfruit. *Cryo-letters* 20,

[50] Turner SR, Touchell DH, Senarata T, Bunn E, Tan B & Dixon KW (2001) Effect of Plant

[51] Veisseire P, Guerrier J & Coudret A. (1993). Cryopreservation of embryonic suspension

[52] Wang J-H, Bian R-W, Zhang Y-W & Cheng H-P (2001) The dual effect of antifreeze

[53] Wang Q, Li P, Batuman Ö, Gafny R & Mawassi M., (2003). Effect of benzyladenine on

[54] Walter C, DH. Touchell, P Power, J Wesley-Smith and MF. Antolin (2002), A

[55] Wesley-Smith J, Vertucci CW, Berjak P, Pammenter NW & Crane J (1992)

[56] Withers, LA (1985). Cryopreservation of cultured cells and protoplasts. In: K.K. Kartha (ed) *Cryopreservation of plant cells and organs*. CRC press Inc., Boca Raton. pp 243-267.

electromicroscopy. *Journal of Ultrastructure Research* 26, 31-43

two-step freezing. *Plant Cell Tissue and Organ Culture* 42: pp. 261-267. [43] Sakai A, Kobayashi S & Oiyama I (1990) Cryopreservation of nuceller cells of navel orange

18, pp.185-190.

*rotundata* (Yam). *CryoLetters*, 30 (3), 212-223

Species. Chatham. UK: NRI/ACP-EU Technical

of *Hevea brasiliensis*. *CryoLetters* 14, 295-302.

*plant* 40, 4, pp. 389-395(7)

*Cryoletters* 22, 163-174.

*CryoLetters* 22, 175-182.

*CryoLetters* 23, 291-298.

*Plant Physiology* 140, 596-604.

Cape Coast.

pp. 21-28.

293-302.

L. Cv Chardonnay shoots tips by encapsulation-dehydration: effects of pretreatment, cooling and postculture conditions. *Cryoletters* 14, pp. 309-320. [40] Potts SE & Lumpkin TA (1997). Cryopreservation of Wasabi species seeds. Cryoletters.

Explant Water Content: Critical Factors to Consider in Development of Successful Cryopreservation Protocols for Shoot Tip Explants of the Tropical Species *Dioscorea* 

shock treatment improve cryopreservation of tobacco cell suspensions compared to

(*Citrus sinensis* Obs, var. *brasiliensis* Tanaka) by vitrification. *Plant Cell Report* 9, 30-33.

(*Saintpaulia Ionantha* Wendl.) Shoot Tips. In vitro *Cellular and Develoment Biology -* 

School Centre for Agricultural and Rural Cooperation of Agriculture, University of

Growth Regulators on Survival and Recovery Growth Following Cryopreservation.

protein on cryopreservation of rice (*Oryza sativa* L.) embryogenic suspension cells.

recovery of cryopreserved shoot tips of grapevine and citrus *in vitro*. *CryoLetters* 24,

Cryopreservation protocol for embryos of the endangered species *Zizania texana.*

Cryopreservation of desiccation-sensitive axes of *Camellia sinensis* in relation to dehydration, freezing rate and the thermal properties of tissue water*. Journal of* 

**18** 

*UK* 

**Precision in Cryopreservation –** 

**1.1 Different samples may have differing cryopreservation requirements** 

What properties are the retrieved samples required to possess?

and logistics of the freezing and storing process.

 How many batches are to be stored? What is the expected duration of storage?

cryopreservation process?

What is to be stored?

For any cryopreservation protocol there are five key questions that govern the methodology

Are there packaging requirements in addition to those dictated by the

Reduction of temperature results in the retardation of metabolic processes and this can, in some circumstances, provide sufficient stability for the required period of storage. However, at temperatures below 0 °C the biological effects of cooling are dominated by the crystallization of ice: typically, water constitutes around 80 % of tissue mass. Freezing is the conversion of liquid water to crystalline ice but the term is commonly misused in circumstances where samples are cooled below their expected freezing point but without the formation of ice, for example by supercooling or by vitrification. The result of the freezing of water in a complex solution is that the concentration of the solutes in the remaining liquid phase increases and some solutes may precipitate if their concentration exceeds their solubility limits. This realisation provides two potential mechanisms of damage: direct mechanical effects of the formation of ice, and the rise in concentration of

In 1948 a method was discovered that permitted the freezing of many types of animal cells with good post-thaw recovery of living cells: Polge, Smith, and Parkes (1948) showed in a landmark paper that adding 10-20 % of glycerol enabled avian spermatozoa to survive freezing at -80 °C. Theories of freezing injury that were current at the time envisaged ice crystals damaging the cells and intracellular structures, and because glycerol increased the total solute concentration in the system, the amount of ice that formed was reduced. A little later, in the 1950s, Lovelock (1952) showed that the increase in concentration of salts as the volume of the suspending solution decreased was in fact the dominant damaging mechanism: salt concentration, rather than ice formation, was a major cause of freezing

**1. Introduction** 

dissolved solutes.

**Equipment and Control** 

Stephen Butler and David Pegg

*Planer plc, University of York* 
