**8. References**

386 Current Frontiers in Cryopreservation

calluses (Jin et al., 1996; Kaur et al., 1987) and the correlation between pineapple variety and the toxicity of culture filtrates suggests that filtrates could be used to screen in vitro for disease resistance (Borras et al., 2001). Therefore, the cryopreservation of pineapple calluses can provide a means of effective source when in vitro screening of germplasm for fusariose disease would be attempted. Storing calluses in liquid nitrogen could preserve their regeneration capacity and limits the risk of somaclonal variation, which increases with

A simplified freezing protocol mentioned before for sugarcane embryogenic calluses (Martinez-Montero et al., 1998) was used for pineapple calluses of the genotypes "Smooth Cayenne" and "Perolera" (Martinez-Montero et al., 2005). For cryopreservation experiments, 15 to 20 day-old calluses, about 3 to 6 mm in diameter, were employed. Calluses were pretreated with a cryoprotective solution containing 5 to 20 % (v/v) dimethylsulfoxide (DMSO) and 0.5M sucrose for 1h at 0°C. After freezing calluses were transferred directly to recovery medium (MS medium supplemented with dicamba:BAP (2.5:0.5 mg.L-1) and citric acid (0.1 mg.L-1)). The survival, evaluated 45 days after thawing, corresponded to the

As results, the survival of calluses after pretreatment was high and similar for the two genotypes studied (Table 8). It decreased only with 20% of DMSO. After freezing in liquid nitrogen, survival was achieved with 10 and 15% DMSO only and was highest with 15% DMSO (57-67%). Re-growth of successfully cryopreserved calluses was very rapid and they increased in size during the recovery period. With this work, the application of a simplified freezing protocol achieved survival from used pineapple genotypes and this confirm that our previously simplified freezing protocol for sugarcane (Martinez-Montero et al., 1998)

(%) Sucrose (M) -LN +LN -LN +LN

10 818 a 253 b 856 a 304 b 15 7512 a 577 a 806 a 656 a 20 556 b 0 c 604 b 0 c Table 9. Effect of cryoprotective solution (DMSO + sucrose) on the survival rate (%) before (- LN) and after (+LN) application of cryopreservation protocol for calluses of two pineapple genotypes. *Values represent means of 50 samples from three replicate experiments, ± SE. Means within columns followed by the same letter are not significantly different (ANOVA p<0,05 Tukey,).* 

For vegetatively propagated species, cryopreservation has a wide applicability both in terms of species coverage, since protocols have been successfully established for root and tubers, fruit trees, ornamentals and plantation crops, both from temperate and tropical origin and in

Survival (%) Smooth Cayenne Red Spanish

8511 a 0 c 876 a 0 c

*= 2 arcsine ((x/100)0,5) for* 

percentage of calluses which had increased in size during the recovery period.

culture duration.

can be used wider to others species.

0.5

*Data were transformed for statistical analysis in accordance with x'*

DMSO

5

*percentage of survival.* 

**6. Conclusions** 


Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

varieties. *Cryo-Letters* 13, 239-52. ISSN: 0143-2044.

pp. 119-161. ISBN: 0851991424, CABI, Wallingford.

*Cryobiology* 27(3), (June 1990), 247-68. ISSN: 0011-2240

IPGRI; ISBN 92-9043-428-7, Rome, Italy.

*Plant* 40, 427–433. ISSN 1054-5476

Science+Business Media, LLC 2011.

7, Rome, Italy.

1020-3362

0011-2240

Montpellier, pp. 15-17.

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 389

Eksomtramage T.; Paulet F.; Guiderdoni E.; Glaszmann J.C.; Engelmann F. (1992)

Engelmann, F. & Takagi, H. Eds. (2000) *Cryopreservation of tropical plant germplasm - Current* 

Engelmann, F. (1997) Importance of desiccation for the cryopreservation of recalcitrant seed

Engelmann, F. (1997) *In vitro* conservation methods. In: *Biotechnology and Plant Genetic* 

Engelmann, F. (2000) Importance of cryopreservation for the conservation of plant genetic

Engelmann, F. (2004) Plant cryopreservation: progress and prospects. *In Vitro Cell Dev Biol* 

Engelmann, F. (2010) Use of biotechnologies for the conservation of plant biodiversity. *In Vitro Cell Dev Biol Plant* DOI 10.1007/s11627-010-9327-2, ISSN 1054-5476 Engelmann, F. (2011) Cryopreservation of Embryos: An Overview. In: *Plant Embryo Culture:* 

Esterbauer, H.; Zollner, H.; Schauer, R.J. (1988) Hydroxyalkenals: Citotoxics products of

Fabre, J. & Dereuddre, J. (1990) Encapsulation-dehydration: A new approach to cryopreservation of *Solanum* shoot tips. *Cryo-Letters* 11, 413 – 426. ISSN: 0143-2044 Fahy, G.M.; Lilley, T.H.; Linsdell, H.; Douglas, M.S.J.; Meryman, H.T. (1990) Cryoprotectant

Fahy, G.M.; McFarlane, D.R.; Angell, C.A.; Meryman, H.T. (1984) Vitrification as an

Feher, A.; Pasternak, T.P.; Dudits, D. (2003) Transition of somatic cells to an embryogenic state. *Plant Cell, Tissue and Organ Culture* 74, 201-228. ISSN: 0167-6857 Feldmann, P.; Sapotille, J.; Gredoire, P.; Rott, P. (1994) Micropropagation of sugarcane. In: *In* 

Finkle, B.J. & Ulrich, J.M. (1979) Effect of cryoprotectants in combination on the survival of

Finkle, B.J. & Ulrich, J.M. (1982) Cryoprotectant removal as a factor in the survival of frozen rice and sugarcane cells. *Cryobiology* 19(3), (June 1982) 329-335. ISSN: 0011-2240

frozen sugarcane cells. *Plant Physiology* 63, 598-604. ISSN: 0032-0889

Development of a cryopreservation process for embryogenic calluses of a commercial hybrid of sugarcane *(Saccharum* sp.) and application to different

*research progress and applications*. Tsukuba: JIRCAS; Rome: IPGRI; ISBN 92-9043-428-

and vegetatively propagated species. *Plant Genet. Res. Newsletter* 112, 9-18. ISSN:

*Resources Conservation and Use*. BV Ford-Lloyd, JH Newburry & JA Callow (Eds).

resources. In: *Cryopreservation of tropical plant germplasm - Current research progress and applications*. Engelmann, F.; Takagi, H., Eds. (8-20) Tsukuba: JIRCAS; Rome:

*Methods and Protocols, Methods in Molecular Biology.* Trevor, A.T. & Yeung, E.C. Eds. Vol. 710, DOI 10.1007/978-1-61737-988-8\_13, ISBN 978-1-61737-987-1, Springer

lipid peroxidtion. In: *ISI Atlas of Sciences: Biochemistry* 311-317. ISBN. 0-941708-00-4

toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms.

approach to cryopreservation. *Cryobiology*, 21(4), (August 1984), 407-426. ISSN:

*vitro culture of tropical plants*. C Teisson (ed). CIRAD, ISBN 2-87614-162-0,


Ashmore, S.E. (1997) *Status reports on the development and application of in vitro techniques for* 

Ausborn, M.; Schreier, H.; Brezesinski, G.; Fabian, H.; Meyer, H.W.; Nuhn, P. (1994) The

Barraco, G.; Sylvestre, I.; Engelmann, F. (2011) Comparing encapsulation-dehydration and

Benson, E.E., Lynch, P.T., Jones, J. (1992) Variation in free radical damage in rice cell

Borras, O.; Santos, R.; Matos, A.; Cabral, R.; Arzola, M. (2001) A first attempt to use a

Côte, F.X.; Goué, O.; Domergue, R.; Panis, B.; Jenny, C. (2000) In-field behavior of banana

Cyr, D.R. (2000) Cryopreservation: roles in clonal propagation and germplasm conservation

Duan, X.Q.; Hall, J.A.; Nikaido, H.; Quicho, F.A. (2001) Crystal structures of the

Dumet, D.; Block,W.; Worland, R.; Reed, B.M.; Benson, E.E. 2000. Profiling cryopreservation

resistant to fusariose disease. *Plant Breed.* 120: 435-438. ISSN: 0179-9541 Chen, T.H.H.; Kartha, K.K.; Constable, F.; Gusta, L.V. (1984) Freezing characteristics of

*Scientia Horticulturae*, 130(1), (August 2011), 320-324. ISSN: 0304-4238 Benson, E.E. (2008a) Cryopreservation of phytodiversity: A critical appraisal of theory and practice. *Critical Reviews in Plant Sciences* 27(3), 141-219. ISSN: 0735-2689 Benson, E.E. (2008b) Cryopreservation theory. In: *Plant Cryopreservation: A Practical Guide.*

freeze-drying of liposomes. *J Control Release* 30, 105-116. ISSN: 0168-3659 Baier, S.K. & McClements, D.J. (2005) Influence of cosolvent systems on the gelation

Resources Institute, ISBN 92-9043-339-6, Rome, Italy.

43-54. ISSN: 1541-4337

*Genet* 86, 181-188. ISSN: 0040-5752

IPGRI. ISBN 92-9043-428-7, Rome, Italy.

(9 March 2001), 1115-1126. ISSN: 0022-2836

367–378. ISSN: 0143-2044

suspensions. *Cryo-Letters* 21, 19-24. ISSN: 0143-2044

USA.

0935

*the conservation and use of plant genetic resources.* International Plant Genetic

protective effect of free and membrane bound cryoprotectants during freezing and

mechanism of globular protein: Thermodynamic, kinetic, and structural aspects of globular protein gelation. *Comprehensive Reviews in Food Science and Food Safety* 4,

droplet-vitrification for cryopreservation of sugarcane (*Saccharum* spp.) shoot tips.

Reed, B.M. Ed., pp. 15–32. Chapt. 2. Springer, ISBN 978-0-387-72275-7, New York,

suspensions with different embryogenic potentials. *Planta* 188: 296–305. ISSN: 0032-

*Fusarium subglutinans* culture filtrate for the selection of pineapple cultivars

cultured *Catharanthus roseus* (L.) G. Don cells trated with dimethylsulfoxide and sorbitol in relation to cryopreservation. *Plant Physiol.* 75, 720-725. ISSN: 0032-0889 Chowdhury, M.K.U. & Vasil, I.K. (1993) Molecular analysis of plant regenerated from

embryogenic cultures of hybrid sugarcane cultivars (*Saccharum* spp.). *Theor. Appl.* 

plants (*Musa* spp.) obtained after regeneration of cryopreserved embryogenic cell

of conifers. In: *Cryopreservation of tropical plant germplasm - Current research progress and applications.* Engelmann, F.; Takagi, H., Eds. (261-268) Tsukuba: JIRCAS; Rome:

maltodextrin/maltose-binding protein complexed with reduced oligosaccharides: flexibility of tertiary structure and ligand binding. *Journal of Molecular Biology* 306:5,

protocols for *Ribes ciliatum* using differential scanning calorimetry. *Cryo-Letters* 21:


Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

9043-428-7, Rome, Italy.

*Dis.* 80: 922-927. ISSN: 0191-2917

7923-2493-5, Dordrecht.

0011-3891

ISSN 1991-8178

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 391

Harding, K.; Johnston, J.; Benson, E. E. (2005) Plant and algal cell cryopreservation: issues in

Jackson, W.; Wagnespack, H.; Richard, C.; Garrison, D.; Lester, W. (1990) CP65-357 Kleentek

Jian, L.C.; Sun, D.L.; Sun, L.H. (1987) Sugarcane callus cryopreservation. In: *Plant cold hardiness.* Li, P.H. Ed., 323-337, Alan R. Liss, Inc., ISBN 0-8451-1084-8, New York. Jiménez, E.; Pérez, J.; Gil, V.; Herrera, J.; García, Y.; Alfonso, E. (1995) Sistemas para la

Riego E, Limonta E, Tellez P, Fuentes J (Eds).. Elfos Scientiae. Cuba 3:11.2 Jin, H.; Hartman, G.L.; Nickell, C.D.; Widholm, J.M. (1996) Phytotoxicity of culture filtrate

Joyce, S.M.; Cassells A.C.; Jain, M.S. (2003) Stressand aberrant phenotypes *in vitro* culture.

Kaczmarczyk, A.; Shvachko, N.; Lupysheva, Y.; Hajirezaei, M.R.; Keller, E.R.J. (2008)

Kartha, K.K. & Engelmann F. (1994) Cryopreservation and germplasm storage. In: *Plant cell* 

Kartha, K.K.; Fowke, L.C.; Leung, N.L.; Caswell, K.L.; Hakman I. (1988) Induction of somatic

Kaur, G.; Singh, U.S.; Garg, G.K. (1987) Mode of action of toxin isolated from *Fusarium* 

Kinoshita, K.; Li, S.J.; Yamazaki, M. (2001) The mechanism of the stabilization of the

Klimazewska, K.; Ward, C.; Cheliak, W.M. (1992) Cryopreservation and plant regeneration

Krishnamurthy, K.V.; Kishnaraj, R.; Chozavendam, R.; Samuel, C.F. (2000) The programme

Krishnaraj, S. & Vasil, I.K. (1995) Somatic embryogenesis in herbaceous monocots. In: *In* 

*oxysporum* f. sp. ciceri. *Indian Phytopathol* 40: 76-84. ISSN 0367-973X

dimethyl sulfoxide. *Eur Biophysics J* 30, 207-220. ISSN 0175-7571

*Plant Cell Tissue and Organ Culture* 74, 103-21. ISSN:0167-6857

shoot tips. *Plant Cell Rep.* 27: 1551-1558. ISSN: 0721-7714

*glauca*). *J. Plant Physiol* 132, 529-39. ISSN: 0176-1617

*mariana*). *J Exp Bot* 43, 73-79. ISSN: 0022-0957

genetic integrity, concepts in 'Cryobionomics' and current European applications. In: *Contributing to a Sustainable Future*. Benett, I.J.; Bunn, E.; Clarke, H.; McComb, J. A., Eds., (112–119) Proc. Australian Branch IAPTC & B, Perth, Western Australia. Ishikawa, M.; Ide H.; Price, W.S.; Arata, Y.; Kitashima, T. (2000) Freezing behaviors in plant

tissues as visualized by NMR microscopy and their regulatory mechanisms. In: *Cryopreservation of tropical plant germplasm - Current research progress and applications.* Engelmann, F.; Takagi, H., Eds. (22-35) Tsukuba: JIRCAS; Rome: IPGRI. ISBN 92-

test in Lousiana. *Journal of the American Society of Sugar Cane Technologist* 21, 10-13.

propagación de caña de azúcar. In: *Avances de la Biotecnología Moderna* Estrada M,

from *Fusarium solani*, the causal agent of Soybean Sudden Death Syndrome. *Plant* 

Influence of altering temperature preculture on cryopreservation results for potato

*and tissue culture.* Vasil, I.K. & Thorpe, T.A. Eds. (195–230) Kluwer, ISBN 978-0-

embryos and plantlets from cryopreserved cell cultures of white spruce (*Picea* 

hexagonal II (HII) phase in PE membranes in the presence of low concentrations of

from embryogenic cultures of larch (*Larix* x *eurolepis*) and black spruce (*Picea* 

of cell death in plants and animals. A comparison. *Curr Sci* 79, 1169-1181. ISSN:

*vitro embryogenesis in plants. Current Plant Science and Biotechnology in Agriculture,*


Finkle, B.J. ; Zavala, M.E.; Ulrich, J.M. (1985) Cryoprotective compounds in the viable

Fleck, R.A.; Day, J.G.; Clarke, K.J.; Benson, E.E. (1999) Elucidation of the metabolic and

Flynn, L. & Anderlini, T. (1990) Disease incedence and yield performance of tissue culture-

Gnanapragasam, S. & Vasil, I.K. (1990) Plant regeneration from a cryopreserved

Gnanapragasam, S. & Vasil, I.K. (1992) Cryopreservation of immature embryos,

Gonzalez-Arnao, M.T.; Engelmann F.; Huet C.; Urra C (1993) Cryopreservation of

Gonzalez-Arnao, M.T.; Engelmann. F.; Urra-Villavicencio. C.; Morenza, M.; Rios, A. (1998a)

Gonzalez-Arnao, M.T.; Juarez, J.; Ortega, C.; Navarro, L.; Duran-Vila, N. (2003)

Gonzalez-Arnao, M.T.; Moreira, T.; Urra, C. (1996) Importance of pregrowth with sucrose

Gonzalez-Arnao, M.T.; Ravelo, M.M.; Urra-Villavicencio, C.; Martinez-Montero, M.M.;

Gonzalez-Arnao, M.T.; Urra, C.; Engelmann, F.; Ortiz, R.; de la Fe, C. (1999)

Grune, T.; Michel, P.; Sitte, N.; Eggert, W.; Albrecht-Nebe, H.; Esterbauer, H.; Siems, W.G.

Guy, C. (1999) Molecular responses of plants to cold shock and cold acclimation. *J Mol* 

Harding, K. (1999) Stability assessments of conserved plant germplasm. In: *Plant* 

Harding, K. (2004) Genetic integrity of cryopreserved plant cells: A review. *Cryo-Letters 25,* 

dehydration technique. *Cryo-Letters* 24 (2), 85-94. ISSN: 0143-2044

and storage duration. *Cryo-Letters* 20, 347-352. ISSN: 0143-2044

dehydration. *Cryo-Letters* 27, 141-148. ISSN: 0143-2044

Ed., 243-267, ISBN 0-8493-6102-8, CRC Press, Boca Raton. USA.

*Xanthophyceae*. *Cryo-Letters* 20, 271–282. ISSN: 0143-2044

*Sugar Cane Technologists* 10, 113. ISSN 1991-8178

*Plant Cell Rep.* 9, 419-423. ISSN: 0721-7714

*Science* 83, 205-215. ISSN: 0168-9452

*Letters* 14, 303-308. ISSN: 0143-2044

*Cryo-Letters* 19,177-182. ISSN: 0143-2044

*Cryo-Letters* 19, 375-382. ISSN: 0143-2044

(November 1996), 357-360. ISSN: 0891-5849

*Microbiol Biotechnol.* 1(2), 231-242. ISSN: 1464-1801

Francis Ltd., ISBN 0-7484-0746-4, London, UK.

*3-22.* ISSN: 0143-2044

freezing of plant tissues. In: *Cryopreservation of plant cells and organs. K*artha, K.K.

structural basis for the cryopreservation recalcitrance of *Vaucheria sessilis*,

generated seed cane over the crop cycle in Lousiana. *Journal of American Society of* 

embryogenic cell suspension of a commercial sugarcane hybrid (*Saccharum* sp.)

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

encapsulated apices of sugarcane: Effect of freezing procedure and histology. *Cryo-*

Cryopreservation of citrus apices using the encapsulation-dehydration technique.

Cryopreservation of ovules and somatic embryos of citrus using the encapsulation-

and vitrification for the cryopreservation of sugarcane apices using encapsulation-

Engelmann, F. (1998b) Cryopreservation of pineapple (*Ananas comosus*) apices.

Cryopreservation of encapsulated sugarcane apices: Effect of storage temperature

(1997) Increased levels of 4-hydroxynonenal modified proteins in plasma of children with autoimmune diseases. *Free Radical Biology and Medicine* 23 (3),

*Conservation & Biotechnology*. Chapt. 7. Benson, E.E. Ed., pp. 97–107, Taylor and


Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

method. CryoLetters. 16: 189-196. ISSN: 0143-2044

method. *Cryo-Letters* 16, 189-196. ISSN: 0143-2044

7923-3149-4, Dordrecht, Netherlands.

28, 283-287. ISSN: 0114-0671

161-169. ISSN: 0001-5342

0167-6857

Rome, Italy.

0168-9452

IPGRI, Rome, Italy.

Press Inc., New York.

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 393

Matsumoto T, Sakai A, Takahashi C y Yamada K (1995) Cryopreservation of *in vitro*-grown

Matsumoto, T.; Sakai, A.; Takahashi, C.; Yamada, K. (1995) Cryopreservation of *in vitro*-

McGann, L.E. & Walterson, M.L. (1987) Cryoprotection by dimehyl sulfoxide and dimethyl

Merkle, S.A.; Parrot, W.A.; Flinn, B.S. (1995) Morphogenicaspects of somatic embryo-

Mittler, R.; Vanderauwera, S.; Gollery, M.; Van Breusegem, F. (2004) Reactive oxygen gene

Mix-Wagner, G.; Conner, A.J.; Cross, R.J. (2000) Survival and recovery of asparagus shoot

Murashige, T.; Skoog, F. (1962) A revised medium for rapid growth and bioassays with

Nieves, N.; Martinez-Montero, M.E.; Castillo, R.; Blanco, M.A.; González-Olmedo, J.L. (2001)

Niu, D.K.; Wang, M.G.; Wang, Y.F. (1997) Plant cellular osmotica. *Acta Biotheoretica* 45(2),

Panis, B. & Lambardi, M. (2006) Status of cryopreservation technologies in plants (crops and

Panis, B. & Thinh, N.T. (2001) *Cryopreservation of Musa germplasm*, INIBAP Technical

Panis, B.; Piette, B.; Swennen, R. (2005) Droplet vitrification of apical meristems: a

Parsegian, V.A.; Rand, R.P.; Rau, D.C. (1995) Macromolecules and water: Probing with

Parsegian, V.A.; Rand, R.P.; Rau, D.C. (2000) Osmotic stress, crowding, preferential

*Academy of Sciences USA*, 3987-3992. ISSN-0027-8424

sulfone. *Cryobiology* 24(1), (February 1987), 11-16. ISSN: 0011-2240

network of plants. *Trends Plant Sci.* 9, 490–498. ISSN: 1360-1385

tobacco tissue cultures. *Physiol. Plant.* 15: 473-497. ISSN: 0031-9317

apical meristems of wasabi (Wasabia japonica) by encapsulation-vitrification

grown apical meristems of wasabi (*Wasabia japonica*) by encapsulation-vitrification

genesis. In: *In vitro embryogenesis in plants. Current Plant Science and Biotechnology in Agriculture,* Thorpe, T.A. Ed. Kluwer Academic Publishers; 20, 155-204. ISBN: 0-

tips after cryopreservation using the ¨droplet¨ method. *New Zeal. J. Crop & Hort Sci.,*

Effect of abscisic acid and jasmonic acid on partial desiccation of encapsulated somatic embryos of sugarcane. *Plant Cell Tissue and Organ Culture* 65, 15-21. ISSN:

forest trees). In: *The role of Biotechnology in Exploring and Protecting Agricultural Genetic Resources*, Ruane, J. & Sonnino, A. Eds. (61-78) FAO, ISBN 92-5-105480-0,

Guideline 5. Escalant, J.V. & Sharrock, S. Eds., International Network for the Improvement of Banana and Plantain, ISBN: 2-910810-45-3, Montpellier, France.

cryopreservation protocol applicable to all *Musaceae*. *Plant Sci.* 168, 45–55. ISSN:

osmotic stress. In: *Methods in Enzymology*: *Energetics of Biological Macromolecules.* Johnson, M.L. & Ackers, G.K. Eds. 259, (43-94), ISBN: 978-0-12-182160-9, Academic

hydration, and binding: A comparison of perspectives. *Proceedings of the National* 

Thorpe, T.A. Ed. Kluwer Academic Publishers; 20, 417-469. ISBN: 0-7923-3149-4, Dordrecht , Netherlands.


Kristensen, M.M.H.; Find, J.I.; Floto, F.; Moller, J.D.; Norgaard, J.V.N.; Krogstrup, P. (1994)

Leprince, O.; Hendry, G.A.F.; McKersie, B.D. (1993) The mechanisms of desiccation tolerance in developing seeds. *Seed Science Research*, 3: 231-246. ISSN: 0960-2585 Lorenzo, J.C.; Ojeda, E.; Espinosa, A.; Borroto, C. (2001) Field performance of temporary

Lourens, A.G. & Martin, F.A. (1987) Evaluation of *in vitro* propagated sugarcane hybrids for

Lunn, J.E. & MacRae, E. (2003) New complexities in the synthesis of sucrose. *Current Opinion* 

Luo, J. & Reed, B.M. (1997) Abscisic acid-responsive protein bovine serum albumin and

Maddox, A.D.; Gonsalves, F.; Shields, R. (1983) Successful preservation of suspension

Martinez-Montero, M.E.; Gonzalez-Arnao, M.T.; Borroto-Nordelo, C.; Puentes-Diaz, C.;

Martínez-Montero, M.E.; Martínez, J.; Castillo, R.; Engelmann, F.; González-Arnao, M.T.;

Martinez-Montero, M.E.; Martinez, J.; Engelmann, F. (2008) Cryopreservation of sugarcane

Martinez-Montero, M.E.; Mora, N.; Quiñones, J.; Gonzalez-Arnao, M.T.; Engelmann, F.;

Martinez-Montero, M.E.; Ojeda, E.; Espinosa, A.; Sánchez, M.; Castillo, R.; Gonzalez-Arnao,

callus-derived sugarcane plants. *Cryo-Letters* 23(1), 21-26. ISSN: 0143-2044

calluses. *Acta Hort. (ISHS)* 666, 127-131. (ISSN 0567-7572

somatic embryos. *Cryo-Letters*, 29(3), 229-242. ISSN: 0143-2044

simplified freezing process. *Cryo-Letters*, 19, 171-176. ISSN: 0143-2044 Martinez-Montero, M.E.; Lorenzo, J.C.; Ojeda, E.; Quiñones, J.; Mora, N.; Sánchez, M.;

cryopreserved by vitrification. *Cryobiology* 34:240-250. ISSN: 0011-2240 Lynch, P.T.; Benson, E.E.; Harding, K. (2007) Climate change: the role of ex situ and cryo-

somaclonal variation. *Crops Science* 27, 793-796. ISSN: 0011-183X

*in Plant Biology* 6, 208-224. ISSN: 1369-5266

*Science Letters* 28, 157-162. ISSN: 0304-4211

23(4), 360-375. ISSN: 1027-2852

244. ISSN: 0143-2044

Dordrecht , Netherlands.

0033-183X

1054-5476

ISSN: 1462-0316

Thorpe, T.A. Ed. Kluwer Academic Publishers; 20, 417-469. ISBN: 0-7923-3149-4,

The origin and development of somatic embryos following cryopreservation of an embryogenic suspension culture of *Picea sitchensis*. *Protoplasma* 182, 65-70. ISSN:

bioreactor-derived sugarcane plants. *In Vitro Cell Dev. Biol.-Plant*. 37, 803-806. ISSN

proline pretreatments improve recovery of *in vitro* currant shoot tips and callus

conservation in the future security of economically important, vegetatively propagated plants. *Journal of Horticultural Science & Biotechnology* 82 (2), 157–160.

cultures of three *Nicotiana* species at the temperature of liquid nitrogen. *Plant* 

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

Iglesias, A.; Martínez, J.; Castillo, R. (2006) Methodology for the cryopreservation of calli with embryogenic structures for the culture of sugarcane. *Biotecnología Aplicada*

(2005) Cryopreservation of pineapple (*Ananas comosus* (L.) Merr) apices and

Lorenzo, J.C. (2002a) Effect of cryopreservation on the structural and functional integrity of cell membranes of sugarcane embryogenic calluses. *Cryo-Letters* 23, 237-

M.T.; Engelmann, F.; Lorenzo, J.C. (2002b) Field performance of cryopreserved


Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

*tuberosus* L.). *Cryo-Letters* 20, 325-336. ISSN: 0143-2044

CP5243. *Biotecnología Aplicada* 16, 20-23. ISSN: 1027-2852

Agricultural University, The Nederlands. ISBN 90-5485-517-7

9780792308195, Dordrecht, Netherlands.

*Physiol. Biochem.* 37, 145-154. ISSN: 0981-9428

ISSN: 0006-3495

2044

0168-9452

*SugarCane 3*, 11-14.

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 395

Stanley, C.; Krueger, S.; Parsegian, V.A.; Rau, D.C. (2008) Protein structure and hydration

Sun, W.Q. (1999) State and phase transition behaviors of *Quercus rubra* seed axes and

of recalcitrant seeds. *Cryobiology*, 38(4), (June 1999), 372-385. ISSN: 0011-2240 Swan, T.W.; O´Hare, D.; Gill, R.A.; Lynch, P.T. (1999) Influence of preculture conditions on

Swartz, H.J. (1991) Post culture behaviour: genetic and epigenetic effects and related

Tapia, R.; Castillo, R.; Blanco, M.A.; González, J.L.; Sánchez, M.; Rodríguez, Y. (1999)

Taylor, P.; Ko, H.; Adkins, S.; Rathus, C.; Birch, R. (1992) Establishment of embriogenic

spp. hybrids) *Plant Cell Tissue and Organ Culture* 28: 69-78. ISSN: 0167-6857 Tetteroo, F.A.A. (1996) *Desiccation tolerance of somatic embryoids.* PhD Thesis, Wageningen

Thierry, C.; Florin, B.; Petiard, V. (1999) Changes in protein metabolism during the

Thinh, N.T.; Takagi, H.; Yashima, S. (1999) Cryopreservation of in vitro-grown shoot tips of

Thomashow, M.F. (1999) Plant cold acclimation: Freezing tolerance genes and regulatory mechanisms. *Annu. Rev. Plant Physiol. Plant Mol. Biol* 50, 571-599. ISSN:1040-2519 Timasheff, S.N. (1993) The Control of Protein Stability and Association by Weak Interactions

Turner, S.; Senaratna, T.; Touchell, D.; Bunn, E.; Dixon, K.; Tan, B. (2001) Stereochemical

Turner, S.R.; Krauss, S.L.; Bunn, E. Senaratna, T.; Dixon, K.W.; Tan, B.; Touchell, D.H. (2001)

storage and cryopreservation. *Plant Science* 161, 1099-1106. ISSN: 0168-9452 Tyler, N.; Stushnoff, C.; Gusta, L.V. (1988) Freezing of water in dormant vegetative apple buds in relation to cryopreservation. *Plant Physiol* 87, 201-205. ISSN: 0032-0889 Ulrich, J.M.; Finkle, B.J.; Moore, P.H. (1984) Frozen preservation of cultured sugarcane cells.

*and Biomolecular Structure* 22, 67-97, (June 1993), ISSN: 1056-8700

probed by SANS and osmotic stress. *Biophysical Journal* 94, 2777–2789, (April 2008),

cotyledonary tissues: relevance to the desiccation sensitivity and cryopreservation

the post-thaw recovery of suspension cultures of Jerusalem artichoke (*Helianthus* 

problems. In*: Micropropagation. Technology and Application.* PC Debergh & RH Zimmermann (eds.). pp. 95-121, Kluwer Academic Publishers, ISBN:

Induction, maduration and encapsulation of sugarcane somatic (*Saccharum* spp) cv

callus and high protoplast yielding suspension cultures of sugarcane (*Saccharum*

acquisition of tolerance to cryopreservation of carrot somatic embryos. *Plant* 

banana (*Musa* spp.) by vitrification method. *Cryo-Letters* 20(3), 163-174. ISSN: 0143-

with Water: How Do Solvents Affect These Processes? *Annual Review of Biophysical* 

arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation. *Plant Science* 160, 489-497. ISSN:

Genetic fidelity and viability of *Anigozanthos viridis* following tissue culture, cold


Paulet, F.; Engelmann, F.; Glaszmann, J.C. (1993) Cryopreservation of apices of *in vitro*

Pena, M. & Stay, M. (1997) Ventajas de la propagacion in vitro. *Canaveral* 3(24), 473–497.

Pérez, J.N.; Jiménez, E.; Gómez, R. (1999) Mejora genética de la caña de azúcar mediante la

Rabe, E. and C. J. Lovatt (1984). De novo arginine biosynthesis in leaves of phosphorus-

Rasmussen, P.H.; Jorgensen, B. & Nielsen, J. (1997) Cryoprotective properties of proline in

Reed, B.M. (2001) Implementing cryogenic storage of clonally propagated plants. *Cryo-*

Reinhoud, P.J.; Van Iren, F.; Kijne, J.W. (2000) Cryopreservation of undifferentiated plant

Ruan, K.; Xu, Ch.; Li, T.; Li, J.; Lange, R.; Balny, C. (2003) The thermodynamic analysis of

Rudolph, A.S. & Crowe, J.H. (1985) Membrane stabilization during freezing: the role of two

Sakai, A. & Engelmann, F. (2007) Vitrification, encapsulation-vitrification and droplet-

Sakai, A.; Kobayashi, S.; Oiyama, I. (1990) Cryopreservation of nucellar cells of navel orange

Sakai, A.; Matsumoto, T.; Hirai, D.; Charoensub, R. (2002) Survival of tropical apices cooled

Schäfer-Menuhr, A.; Schumacher, H.M.; Mix-Wagner, G. (1997) Long-term storage of old

Semenova, M.G.; Antipova, A.S.; Belyakova, L.E. (2002) Food protein interactions in sugar solutions. *Current Opinions in Colloid Interface Science* 7, 438–444. ISSN: 1359-0294 Spiteller, G. (1996) Enzymic lipid peroxidation-α consequence of cell injury? *Free Radical* 

vitrification: a review. *Cryo-Letters* 28, 151–172. ISSN: 0143-2044

*Plant Cell Rep.* 12, 525-529. ISSN: 0721-7714

ISSN: 1026-0781

ISSN: 0143-2044

*Letters* 22, 97-104. ISSN: 0143-2044

IPGRI. ISBN 92-9043-428-7, Rome, Italy.

Publishers, ISBN: 0306472864, New York.

*Res. Newsletter* 111, 19-24. ISSN: 1020-3362

*Biology and Medicine* 21(7), 1003-1009. ISSN: 0891-5849

*Journal of Biochemistry* 270, 1654–1661. ISSN: 0014-2956

0889

2240

ISSN: 0721-7714

Elfos Scientiae, Cuba.

plantlets of sugarcane (*Saccharum* sp. hybrids) using encapsulation-dehydration.

inducción de mutaciones y la selección in vitro. In: *Biodiversidad y Biotecnología de la Caña de Azúcar.* A Arencibia & MT Cornide (eds.). pp. 79-92, ISBN: 959-235-015-9,

deficient Citrus and Poncirus species. *Plant Physiology* 76(3): 747-752. ISSN: 0032-

cod muscle studied by differential scanning calorimetry. *Cryo-Letters* 18, 293-300.

cells. In: *Cryopreservation of tropical plant germplasm - Current research progress and applications.* Engelmann, F.; Takagi, H., Eds. (91-102) Tsukuba: JIRCAS; Rome:

protein stabilization by sucrose and glycerol against pressure-induced unfolding: The typical example of the 33-kDa protein from spinach photosystem II. *European* 

natural cryoprotectants, trehalose and proline. *Cryobiology* 22, 367-377. ISSN: 0011-

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

to -196°C by vitrification. In: *Plant cold hardiness, gene regulation and genetic engineering.* Li, P.H.; Palva, E.T. Eds., 109-119, Kluwer Academic/Plenum

potato varieties by cryopreservation of shoot-tips in liquid nitrogen. *Plant Genet.* 


**19**

*Portugal* 

Conceição Santos

**Somatic Embryogenesis and Cryopreservation** 

It is widely accepted that propagation and conservation strategies are crucial in any forest breeding program and that plant biotechnology has powerful tools to propagate and preserve selected genotypes (Jain, 1999; Park et al., 1988; Park, 2002). In particular, in vitro techniques have demonstrated to be essential in a large number of agricultural and forest breeding programs, allowing large scale clonal propagation of elite genotypes (Pinto et al., 2008) or of endangered forest species (Brito et al., 2009). In vitro techniques are also essential in breeding programs involving genetic improvement (e.g. using techniques such as somatic hybridization or genetic transformation) or involving long term germplasm conservation by

Somatic embryogenesis offers advantages in improving forest species over other in vitro propagation methods (Park, 2002), namely: a) somatic embryos simultaneously possess both the shoot and root meristems; so a distinct rooting stage, which usually involves stressing procedures, is not necessary; b) during somatic embryogenesis, embryos/clusters are often formed faster and potentially at extremely high numbers per explant; c) somatic embryogenesis is amenable to automation, which means it may become cheaper than other clonal propagation techniques (Pinto et al., 2002; 2008); d) finally, a robust and efficient protocol of somatic embryogenesis will allow that embryogenic clonal lines can be preserved for long periods (e.g., in liquid N2) while corresponding plants are transferred to

The combination of somatic embryogenesis and conservation strategies in breeding programs allows that one may develop high-value forest clonal varieties merely by recovering from N2 those genotypes/clones that showed best characteristics in the field. These selected genotypes can later be used for advanced breeding programs and

From the exposed advantages, it is generally accepted among breeders that breeding programs using in vitro clonal strategy require as a pre-requisite that a cloning technique (preferably somatic embryogenesis) is well established. Then it also requires the optimization of long-term genetic testing methodologies of clonal lines and long term

**1. Introduction** 

**1.1 General concepts** 

cryopreservation (Benson, 2008; Fernandes et al., 2008).

field conditions and are monitored for their characteristics.

commercial forestry (Park, 2002).

**in Forest Species: The Cork Oak Case Study** 

*CESAM & Department of Biology, University of Aveiro, Aveiro* 

