**9. Conclusion**

352 Current Frontiers in Cryopreservation

tissue damage by chemical toxicity of the cryoprotectants. Otherwise, plant parts in solutes can be injured by the cryoprotectant, by strong osmotic stress during the cryoprotective solution treatment. The time for a high regeneration rate of plants after cryopreservation must be optimized. During this time the explants are dipped in the vitrification solutions, at the optimal temperature, which is involved in the procedure (Condello *et al*., 2011; Faltus &

The temperature-induced glasses-the point at which this occurs, is called the glass transition temperature (Tg) - molecular motion nearly ceases and the liquid becomes a glassy solid. Vitrification of cells and tissues is a physical process which avoids intracellular ice crystallization during ultra-rapid freezing by the transition of the aqueous solution of the symplast into an amorphous glassy state of the cells. As a consequence of the vitrification process, plant tissues are protected from the damage and remain viable during their long-

Experimental determination of glassy state in plants is complicated by the endothermic reaction overlapping with the glass transition. Ice crystallization as the first-order reaction has discontinuous change in heat capacity contrary to glass transition, which is characterized by heat capacity change. The main problem is to distinguish the endothermic reaction and the glass transition temperature during the measurement of the thermal events. Thermal analysis methods of glass transition temperature and temperature of ice crystals melting in plant tissues were determined. Standard Differential Scanning Calorimetry (DSC) method and Temperature Modulated Differential Scanning Calorimetry (TMDSC) were

Cryopreservation Method

Encapsulation -dehydration

Ultra-rapid freezing

Zamecnik, 2009; Sakai & Engelmann, 2007; Zámečník *et al*., 2007).

usually used (Condello *et al*., 2011; Zámečník & Faltus, 2009).

Droplet -vitrification (PVS3)

tested; NT- not tested; (§)-PVS2 was used.

solution at temperatures above the freezing point.

Garlic \*\* NT \* Potato \* \*\* \* Hop NT \*\* NT Apple tree \*(§) \* \*\*

Table 5. Cryopreservation methods used in this study. Results obtained by cryopreservation methods with a high regeneration rate were presented only: \*\* - high regeneration rate; \*-

The vitrification method, based on involvement of biological glass, requires a highly concentrated solution of cryoprotectant (from 5 to 8 M), at which the cells are osmotically dehydrated to a certain level. This level of dehydration is characterized by no frost-heaving of water and with a minimum, or no production of ice crystals. The cells treated by cryoprotectant are then vitrified before or during immersion into liquid nitrogen (Sakai *et al*., 1991). Vitrification belongs to new well-developed procedures supplying frost dehydration of cells pursued at a low temperature. Namely, it removes most of the freezable water through the exposure of the plant shoot tips to a highly concentrated vitrification

term storage at –196 °C.

Plant

The four main crops (garlic, potato, hop and apple tree) have been cryopreserved in the Czech Plant Cryobank. The methods of cryopreservation are based on cryoprotocol of the vitrification procedures (encapsulation-dehydration, dehydration by vitrification solution and a modified ultra–rapid freezing method based on preconditioning of the plant shoot tips on an osmotic solution). Cryopreservation is well advanced for vegetatively propagated species, and techniques are ready for large-scale experimentation in an increasing number of cases (Engelmann, 2011). We have started the routine cryopreservation with Czech potato genotypes in collaboration with Potato Research Institute Ltd., Havlíčkův Brod. The other three crops are supported by specialized companies (*Allium* genera by the Gene Bank Olomouc branch, the Research and Breeding Institute of Pomology at Holovousy and the Hop Research Institute). The Czech Plant Cryobank operates as a safe duplicate to repositories of germplasm kept in field or *in vitro* conditions. In this way the Czech Plant Cryobank in Prague joined the effort of the potato cryobank in Germany (Keller *et al*., 2008), and Korea (Kim *et al*., 2006). Currently three of the EU countries (The Czech Republic, Germany and Poland) involved in the maintenance of national vegetatively propagated *Allium* collections are developing the methodology for cryopreservation (project

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#### **10. Acknowledgment**

This work was partially supported by project 0002700604 MSMT and QH71228 MZe. The authors would like to thank M. Grospietsch, Jana Chittendenová, Aneta Babická and Adéla Hreňuková for their help and Dr. V. Skládal for critical review of this manuscript.

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**18**

*1Cuba 2Mexico 3France 4Italy* 

Florent Engelmann3,4

*2Universidad Veracruzana, 3IRD, UMR DIAPC 4Bioversity International* 

*1University of Ciego de Avila/Bioplantas Centre* 

**Cryopreservation of Tropical Plant Germplasm** 

**with Vegetative Propagation – Review of** 

**(***Ananas comusus* **(L.) Merrill) Cases** 

**Sugarcane (***Saccharum* **spp.) and Pineapple** 

Marcos Edel Martinez-Montero1, Maria Teresa Gonzalez Arnao2 and

Sugarcane (*Saccharum* sp. hybrids) is a crop of major importance, which is cultivated on a large scale in tropical and subtropical regions primarily for its high sucrose content. Cultivated pineapple (*Ananas comosus* (L.) Merrill, which is now called Ananas comosus var comosus) belongs to the family Bromeliaceae. It is economically the fourth most important crop worldwide in terms of tropical fruit production and follows banana, mangoes and citrus. One of the main drawbacks faced by sugarcane and pineapple agriculture worldwide is the vegetative (i.e. asexual) nature of its conventional propagation. The consequence is that plants in the field must be replaced at intervals ranging from 1 to 5 years, a process that is costly, tedious and time-consuming. Furthermore, if the planting material is of low quality, yields decrease and more tillage is needed. The crops are exposed to natural disasters, while the propagation system leads to systemic disease transmission, and natural selection and plagues also take their toll. Moreover, the industry is in dramatic need of planting material, which cannot be produced in sufficient quantities to meet the demand

*In vitro* culture techniques have been extensively developed and applied for several thousand plant species including sugarcane and pineapple. Their uses are of high interest for multiplication, conservation and transformation of plant germplasm. Indeed, they allow the multiplication of plant material with high multiplication rates in an aseptic environment, reduction of space requirements, genetic erosion is reduced under optimal storage conditions, and minimized of the expenses in labour costs. Moreover, tissue culture systems

**1. Introduction** 

using classical macropropagation techniques.

