**Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation – Review of Sugarcane (***Saccharum* **spp.) and Pineapple (***Ananas comusus* **(L.) Merrill) Cases**

Marcos Edel Martinez-Montero1, Maria Teresa Gonzalez Arnao2 and Florent Engelmann3,4 *1University of Ciego de Avila/Bioplantas Centre 2Universidad Veracruzana, 3IRD, UMR DIAPC 4Bioversity International 1Cuba 2Mexico 3France 4Italy* 

#### **1. Introduction**

358 Current Frontiers in Cryopreservation

Zamecnik, J.; Faltus, M.; Kotkova, R. & Hejnak, V. (2011). Glass transition determination in

Zhao, Y.H.; Wu, Y.J.; Engelmann, F. & Zhou, M.D. (2001). Cryopreservation of axillary buds

Zhao, Y.H.; Wu, Y.J.; Engelmann, F.; Zhou, M.D.; Zhang, D.M. & Chen, S.Y. (1999).

Vol.20, No.2, (March 1999), pp. 103-108, ISSN0143-2044

ISSN0567-7572

pp. 321-328, ISSN0143-2044

Allium shoot tips after dehydration. Acta Hort. (ISHS) 908 (April 2009), pp. 33-38,

of grape (*Vitis vinifera*) in vitro plantlets. *Cryoletters*, Vol.22, No.5, (September 2001),

Cryopreservation of apple shoot tips by encapsulation-dehydration: Effect of preculture, dehydration and freezing procedure on shoot regeneration. *Cryo-Letters*,

> 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 using classical macropropagation techniques.

> *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

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

were recovered from the cryopreserved cell suspension.

a limited number of albino plantlets from cryopreserved calluses.

(1992) was successfully applied to calluses of 10 varieties.

**2.1 Cell suspensions** 

**2.2 Embryogenic callus** 

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 361

The first experimental research on sugarcane cell suspension was accomplished at the end of 1970 and at the beginning of 1980 decades (Finkle & Ulrich, 1979; Finkle & Ulrich, 1982; Ulrich et al., 1979; Ulrich et al., 1984). These authors demonstrated that resistance of cells to freezing to -23°C and -40°C was possible with little decrease in survival by using mixtures of glucose, dimethylsulfoxide and polyethylene glycol as cryoprotectants. However, no plants

Later on, Gnanapragasam & Vasil (1990) reported that efficient plant regeneration was obtained from a cryopreserved embryogenic cell suspension of one commercial sugarcane hybrid established from leaf derived callus. They observed pregrowing the cells for three days in Murashige & Skoog (1962) basal medium supplemented with 0.33 M sorbitol was essential to the process. A regeneration efficiency of 92% was obtained and plants regenerated from cryopreserved cells, and grown to maturity in the greenhouse, were morphologically identical to regenerated control plants. Later, there were not detected differences at molecular level using RFLP technique comparing plants regenerated from cryopreserved and control cells for three sugarcane hybrids (Chowdhury & Vasil, 1993).

The first success for cryopreservation of sugarcane embryogenic callus was obtained by Ulrich et al., (1979) for the hybrid H50-7209. It was a pretreatment using a combination of 10% polyethylene glycol, 8% glucose and 10% DMSO, freezing rate of 2°C.min-1 until a first transfer temperature of -40°C and freezing rate of 5°C.min-1 until second transfer temperature of -80°C. However, the recovery of cryopreserved callus was achieved only with root regeneration. Ulrich et al., (1984) obtained after modifications of the same protocol

Later on, high survival rates (ca. 90%) and recovery of whole plants were obtained by Jian et al., (1987), Eksomtramage et al., (1992) and Gnanapragasam & Vasil (1992). The conditions defined were different from that used by Ulrich et al., (1979, 1984). For cryoprotective treatment, a mixture of sorbitol and DMSO was used by Jian et al., (1987) and Gnanapragasam & Vasil (1992); Eksomtramage et al., (1992) employed a mixture of sucrose and DMSO. Freezing conditions were also different: 1°C.min-1 from 0°C to -10°C, and kept for 15min at the same freezing rate from -10°C down to -40°C and kept for 1-5 h, and finally immersed into liquid nitrogen (Jian et al., 1987); or 0.5°C.min-1 down to -40°C or -45°C with no plateau at the end of the controlled freezing sequence (Eksomtramage et al., 1992 and Gnanapragasam & Vasil, 1992). Moreover, the technique developed by Eksomtramage et al.,

These authors have followed the strategy known as dehydration by extracellular freezing, which uses a controlled freezing regime (Withers & King, 1980). However, this procedure requires expensive and sometimes complex programmable freezing devices, limiting its use to laboratories specializing in cryopreservation (Ashmore, 1997; Reed, 2001). Furthermore, their research has been focused on the cryopreservation of sugarcane calli obtained from segments of immature leaves belonging to *in vitro* cultured plants; however, such explants are known to have a limited morphogenetic capacity (Krishnaraj & Vasil, 1995) and it is widely acknowledged that immature embryos, as well as young inflorescences, are

greatly facilitate the international exchange of germplasm as the size of the samples is drastically diminished and they can be shipped in sterile conditions. Different *in vitro* conservation methods are employed, depending on the storage duration requested. For short- and medium-term storage, the aim is to reduce growth and to increase the intervals between subcultures. For long-term storage, cryopreservation, i.e. storage at ultra-low temperature, usually that of liquid nitrogen (−196°C), is the only current method ensuring long-term storage of germplasm from vegetatively propagated species. At this temperature, all cellular divisions and metabolic processes are stopped; therefore, plant material can thus be maintained without alteration or modification. Moreover, cultures are stored in a small volume, protected from contamination, requiring very limited maintenance.

This Chapter comprises two main sections focusing on the establishment, optimization and application of cryopreservation techniques to different tissues of *in vitro* sugarcane and pineapple cultures. The first part presents the cryopreservation protocols developed for sugarcane apices isolated from *in vitro* grown plants, embryogenic calluses and somatic embryos, as well as some analytical techniques (electrolyte leakage, protein content and lipid peroxidation products), used to describe the impact of the successive steps of the protocol on the physiological state of the cultures, which are also useful to refine the cryopreservation protocol. The effect of cryopreservation on the phenotypical development, both *in vitro* and in the field, of sugarcane plants regenerated material will be also presented. The second section presents the studies performed to set up and refine a cryopreservation protocol for apices of pineapple *in vitro* plantlets. The protocol established following the vitrification approach was successfully applied for the first time to shoot tips of three pineapple varieties, and then extended to nine pineapple accessions belonging to the *in vitro* collection of Bioplantas Centre in Cuba. In addition, we present the preliminary assays developed using callus of two pineapple cultivars. In the conclusion, we discuss the possibilities and prospects of utilisation of cryopreservation techniques for the long-term storage of other vegetatively propagated tropical plant species.

### **2. Cryopreservation protocols for sugarcane**

Several review papers have been published, which provide lists of species which have been successfully cryopreserved (Cyr, 2000; Engelmann, 1997; Engelmann & Takagi, 2000; Sakai et al., 2002). For vegetatively propagated species, cryopreservation has a wide applicability in terms of species coverage, since protocols have been successfully established for roots and tubers, fruit trees, ornamentals, forestry species and plantation crops from both temperate and tropical origin (Engelmann, 2004; Kaczmarczyk et al., 2008; Engelmann, 2010; Engelmann, 2011).

In the case of sugarcane, cryopreservation protocols have been developed for various materials: apices of *in vitro* plantlets using the encapsulation-dehydration technique (Gonzalez-Arnao et al., 1993; Paulet et al., 1993); cell suspensions (Finkle & Ulrich, 1979; Gnanapragasam & Vasil, 1990) and embryogenic callus using classical freezing protocols (Eksomtramage et al., 1992; Gnanapragasam & Vasil, 1992; Jian et al., 1987) and simplified cryopreservation protocols (Martinez-Montero et al., 1998). Recently, it was published the cryopreservation procedure based on vitrification techniques for somatic embryos (Martinez-Montero et al., 2008).

### **2.1 Cell suspensions**

360 Current Frontiers in Cryopreservation

greatly facilitate the international exchange of germplasm as the size of the samples is drastically diminished and they can be shipped in sterile conditions. Different *in vitro* conservation methods are employed, depending on the storage duration requested. For short- and medium-term storage, the aim is to reduce growth and to increase the intervals between subcultures. For long-term storage, cryopreservation, i.e. storage at ultra-low temperature, usually that of liquid nitrogen (−196°C), is the only current method ensuring long-term storage of germplasm from vegetatively propagated species. At this temperature, all cellular divisions and metabolic processes are stopped; therefore, plant material can thus be maintained without alteration or modification. Moreover, cultures are stored in a small

This Chapter comprises two main sections focusing on the establishment, optimization and application of cryopreservation techniques to different tissues of *in vitro* sugarcane and pineapple cultures. The first part presents the cryopreservation protocols developed for sugarcane apices isolated from *in vitro* grown plants, embryogenic calluses and somatic embryos, as well as some analytical techniques (electrolyte leakage, protein content and lipid peroxidation products), used to describe the impact of the successive steps of the protocol on the physiological state of the cultures, which are also useful to refine the cryopreservation protocol. The effect of cryopreservation on the phenotypical development, both *in vitro* and in the field, of sugarcane plants regenerated material will be also presented. The second section presents the studies performed to set up and refine a cryopreservation protocol for apices of pineapple *in vitro* plantlets. The protocol established following the vitrification approach was successfully applied for the first time to shoot tips of three pineapple varieties, and then extended to nine pineapple accessions belonging to the *in vitro* collection of Bioplantas Centre in Cuba. In addition, we present the preliminary assays developed using callus of two pineapple cultivars. In the conclusion, we discuss the possibilities and prospects of utilisation of cryopreservation techniques for the long-term

Several review papers have been published, which provide lists of species which have been successfully cryopreserved (Cyr, 2000; Engelmann, 1997; Engelmann & Takagi, 2000; Sakai et al., 2002). For vegetatively propagated species, cryopreservation has a wide applicability in terms of species coverage, since protocols have been successfully established for roots and tubers, fruit trees, ornamentals, forestry species and plantation crops from both temperate and tropical origin (Engelmann, 2004; Kaczmarczyk et al., 2008; Engelmann, 2010;

In the case of sugarcane, cryopreservation protocols have been developed for various materials: apices of *in vitro* plantlets using the encapsulation-dehydration technique (Gonzalez-Arnao et al., 1993; Paulet et al., 1993); cell suspensions (Finkle & Ulrich, 1979; Gnanapragasam & Vasil, 1990) and embryogenic callus using classical freezing protocols (Eksomtramage et al., 1992; Gnanapragasam & Vasil, 1992; Jian et al., 1987) and simplified cryopreservation protocols (Martinez-Montero et al., 1998). Recently, it was published the cryopreservation procedure based on vitrification techniques for somatic embryos

volume, protected from contamination, requiring very limited maintenance.

storage of other vegetatively propagated tropical plant species.

**2. Cryopreservation protocols for sugarcane** 

Engelmann, 2011).

(Martinez-Montero et al., 2008).

The first experimental research on sugarcane cell suspension was accomplished at the end of 1970 and at the beginning of 1980 decades (Finkle & Ulrich, 1979; Finkle & Ulrich, 1982; Ulrich et al., 1979; Ulrich et al., 1984). These authors demonstrated that resistance of cells to freezing to -23°C and -40°C was possible with little decrease in survival by using mixtures of glucose, dimethylsulfoxide and polyethylene glycol as cryoprotectants. However, no plants were recovered from the cryopreserved cell suspension.

Later on, Gnanapragasam & Vasil (1990) reported that efficient plant regeneration was obtained from a cryopreserved embryogenic cell suspension of one commercial sugarcane hybrid established from leaf derived callus. They observed pregrowing the cells for three days in Murashige & Skoog (1962) basal medium supplemented with 0.33 M sorbitol was essential to the process. A regeneration efficiency of 92% was obtained and plants regenerated from cryopreserved cells, and grown to maturity in the greenhouse, were morphologically identical to regenerated control plants. Later, there were not detected differences at molecular level using RFLP technique comparing plants regenerated from cryopreserved and control cells for three sugarcane hybrids (Chowdhury & Vasil, 1993).

### **2.2 Embryogenic callus**

The first success for cryopreservation of sugarcane embryogenic callus was obtained by Ulrich et al., (1979) for the hybrid H50-7209. It was a pretreatment using a combination of 10% polyethylene glycol, 8% glucose and 10% DMSO, freezing rate of 2°C.min-1 until a first transfer temperature of -40°C and freezing rate of 5°C.min-1 until second transfer temperature of -80°C. However, the recovery of cryopreserved callus was achieved only with root regeneration. Ulrich et al., (1984) obtained after modifications of the same protocol a limited number of albino plantlets from cryopreserved calluses.

Later on, high survival rates (ca. 90%) and recovery of whole plants were obtained by Jian et al., (1987), Eksomtramage et al., (1992) and Gnanapragasam & Vasil (1992). The conditions defined were different from that used by Ulrich et al., (1979, 1984). For cryoprotective treatment, a mixture of sorbitol and DMSO was used by Jian et al., (1987) and Gnanapragasam & Vasil (1992); Eksomtramage et al., (1992) employed a mixture of sucrose and DMSO. Freezing conditions were also different: 1°C.min-1 from 0°C to -10°C, and kept for 15min at the same freezing rate from -10°C down to -40°C and kept for 1-5 h, and finally immersed into liquid nitrogen (Jian et al., 1987); or 0.5°C.min-1 down to -40°C or -45°C with no plateau at the end of the controlled freezing sequence (Eksomtramage et al., 1992 and Gnanapragasam & Vasil, 1992). Moreover, the technique developed by Eksomtramage et al., (1992) was successfully applied to calluses of 10 varieties.

These authors have followed the strategy known as dehydration by extracellular freezing, which uses a controlled freezing regime (Withers & King, 1980). However, this procedure requires expensive and sometimes complex programmable freezing devices, limiting its use to laboratories specializing in cryopreservation (Ashmore, 1997; Reed, 2001). Furthermore, their research has been focused on the cryopreservation of sugarcane calli obtained from segments of immature leaves belonging to *in vitro* cultured plants; however, such explants are known to have a limited morphogenetic capacity (Krishnaraj & Vasil, 1995) and it is widely acknowledged that immature embryos, as well as young inflorescences, are

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

procedure proposed by Maddox et al., (1983).

(C) for the establishment of cryopreservation procedures.

freeze sugarcane calli, provide little detail on its implementation.

et al., 1994; Withers, 1985).

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 363

a practical point of view, seeding ice crystals is much more difficult when using the

a b c Fig. 2. Devices used by Maddox et al*.* (1983) (A), Withers (1985) (B) and our research group

In general, the "classical" cryopreservation protocols provide insufficient detail on seeding ice crystals step (Martinez-Montero et al*.,* 2006). For instance, although the analysis of this parameter must have been unavoidable for the development of the protocols of Jian et al., (1987) and Gnanapragasam & Vasil (1992), these analyses are not described in their articles; and even Eksomtramage et al., (1992), who first mentioned the need for this step when

Optimizing the effect of post-subculture time on the survival and regeneration of plants from cryopreserved calli the results reported by Jian et al., (1987) and Martinez-Montero et al*.,* (2006) coincided. We founded that survival after cryopreservation is associated with the selection step during the post-subculture period, reaching a maximum at 15 days postsubculture. Moreover, we correlated this finding to the physiological state of the calli before cryopreservation and measured the growth of the calli. These results are the basis for a rational selection of the material to be cryopreserved, since several authors have shown that there is a correlation for different species between the phase of active growth of the calli and its performance upon cryopreservation (Reinhoud et al., 2000; Withers, 1985; Yoshida et al., 1993). It has been proven that the morphology of the cultured cells has a marked influence on cryotolerance. In most species, only small cells with a highly dense cytoplasm, usually found in small cellular aggregates in the periphery of the callus, survive after cryopreservation; whereas large, vacuolated cells are damaged during freezing (Kristensen

We also founded the decrease in survival and regeneration when using sucrose concentrations higher than 0.3 M. The importance of sucrose tolerance within this setting is determined by the role of this disaccharide in the regulation of the hydric potential of the cells (Tetteroo, 1996); sucrose has also occasionally been considered an inducer of cellular division and differentiation (Feher, 2003). Furthermore, there is evidence suggesting that sucrose functions as a genetic regulatory signal for genes coding for enzymes and proteins involved in transport and storage (Lunn & MacRae, 2003). Additionally, Ausborn et al., (1994) and Turner et al., (2001) detected that sucrose stabilizes the lipid bilayers on the membranes by forming disaccharide-lipid hydrogen bonds, whereas Niu et al., (1997)

physiologically better explants for calli production because they retain their embryogenic capacities (Merkle et al., 1995).

#### **2.2.1 Optimization of methodology for sugarcane callus**

Our research team (Martinez-Montero et al*.*, 1998, 2006), using the cryo-research for sugarcane callus described above as starting point, published the results for establishing step by step a methodology for the cryopreservation of sugarcane calli with embryogenic structures obtained from immature inflorescence (Figure 1). We optimized the following aspects according to the *in vitro* survival and regeneration (plants per 500 mg of calli) percentages for: Selection of the cooling procedure, the effect of the cooling procedure and of the type of alcohol, the effect of the induction time of extracellular ice crystals, the effect of post-subculture time, the effect of sucrose and dimethylsulfoxide concentration in the cryoprotective medium, and the effect of the pre-freezing time.

Fig. 1. Optimized methodology for the cryopreservation of sugarcane calli with embryogenic structures.

Firstly, we based on the results carried out by Maddox et al*.* (1983) and Withers (1985) who successfully used uncomplicated freezing procedures for cellular suspensions of *Nicotiana* and *Musa*, respectively (Figure 2). We evaluated the application of one of these devices as an alternative to establish techniques for the cryopreservation of sugarcane calli with embryogenic structures (i.e., cooling rate controlled by a computer-coupled programmable freezer).

As results was detected a survival after storage in liquid nitrogen for both cooling procedures implying the existence of a protective dehydration process that allows the vitrification of some cells without the formation of intracellular ice crystals. However, from

physiologically better explants for calli production because they retain their embryogenic

Our research team (Martinez-Montero et al*.*, 1998, 2006), using the cryo-research for sugarcane callus described above as starting point, published the results for establishing step by step a methodology for the cryopreservation of sugarcane calli with embryogenic structures obtained from immature inflorescence (Figure 1). We optimized the following aspects according to the *in vitro* survival and regeneration (plants per 500 mg of calli) percentages for: Selection of the cooling procedure, the effect of the cooling procedure and of the type of alcohol, the effect of the induction time of extracellular ice crystals, the effect of post-subculture time, the effect of sucrose and dimethylsulfoxide concentration in the

capacities (Merkle et al., 1995).

embryogenic structures.

freezer).

**2.2.1 Optimization of methodology for sugarcane callus** 

cryoprotective medium, and the effect of the pre-freezing time.

Fig. 1. Optimized methodology for the cryopreservation of sugarcane calli with

Firstly, we based on the results carried out by Maddox et al*.* (1983) and Withers (1985) who successfully used uncomplicated freezing procedures for cellular suspensions of *Nicotiana* and *Musa*, respectively (Figure 2). We evaluated the application of one of these devices as an alternative to establish techniques for the cryopreservation of sugarcane calli with embryogenic structures (i.e., cooling rate controlled by a computer-coupled programmable

As results was detected a survival after storage in liquid nitrogen for both cooling procedures implying the existence of a protective dehydration process that allows the vitrification of some cells without the formation of intracellular ice crystals. However, from a practical point of view, seeding ice crystals is much more difficult when using the procedure proposed by Maddox et al., (1983).

Fig. 2. Devices used by Maddox et al*.* (1983) (A), Withers (1985) (B) and our research group (C) for the establishment of cryopreservation procedures.

In general, the "classical" cryopreservation protocols provide insufficient detail on seeding ice crystals step (Martinez-Montero et al*.,* 2006). For instance, although the analysis of this parameter must have been unavoidable for the development of the protocols of Jian et al., (1987) and Gnanapragasam & Vasil (1992), these analyses are not described in their articles; and even Eksomtramage et al., (1992), who first mentioned the need for this step when freeze sugarcane calli, provide little detail on its implementation.

Optimizing the effect of post-subculture time on the survival and regeneration of plants from cryopreserved calli the results reported by Jian et al., (1987) and Martinez-Montero et al*.,* (2006) coincided. We founded that survival after cryopreservation is associated with the selection step during the post-subculture period, reaching a maximum at 15 days postsubculture. Moreover, we correlated this finding to the physiological state of the calli before cryopreservation and measured the growth of the calli. These results are the basis for a rational selection of the material to be cryopreserved, since several authors have shown that there is a correlation for different species between the phase of active growth of the calli and its performance upon cryopreservation (Reinhoud et al., 2000; Withers, 1985; Yoshida et al., 1993). It has been proven that the morphology of the cultured cells has a marked influence on cryotolerance. In most species, only small cells with a highly dense cytoplasm, usually found in small cellular aggregates in the periphery of the callus, survive after cryopreservation; whereas large, vacuolated cells are damaged during freezing (Kristensen et al., 1994; Withers, 1985).

We also founded the decrease in survival and regeneration when using sucrose concentrations higher than 0.3 M. The importance of sucrose tolerance within this setting is determined by the role of this disaccharide in the regulation of the hydric potential of the cells (Tetteroo, 1996); sucrose has also occasionally been considered an inducer of cellular division and differentiation (Feher, 2003). Furthermore, there is evidence suggesting that sucrose functions as a genetic regulatory signal for genes coding for enzymes and proteins involved in transport and storage (Lunn & MacRae, 2003). Additionally, Ausborn et al., (1994) and Turner et al., (2001) detected that sucrose stabilizes the lipid bilayers on the membranes by forming disaccharide-lipid hydrogen bonds, whereas Niu et al., (1997)

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

therefore, be involved in dimethylsulfoxide-mediated toxicity.

result in a better performance of the cryopreserved material after thawing.

(%)

+LN CP52-43 89,0 b 150 b

CP52-43 98,8 a 230 a C91-301 69,5 c 72 c C1051-73 44,1 d 55 d

C91-301 38,8 d 42 e C1051-73 22,2 e 25 f Typical Error 0,190 1,421

Table 1. Effects of optimized cryopreservation protocol on survival and plant regeneration produced from control (-LN) and cryopreserved (+LN) sugarcane embryogenic calluses (varieties CP52-43, C91-301 and C1051-73). *Means within columns followed by the same letter are not significantly different (ANOVA p < 0,05 Tukey,). Data were transformed for statistical analysis in accordance with x'= 2 arcsine ((x/100)0,5) and with x'= (0,5 + x)0,5 for percentage of survival and* 

Variety Survival


*plant regeneration, respectively.* 

toxicity of dimethylsulfoxide.

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 365

al., (1999) in embryogenic cultures of *Abies cephalonica*. We later founded that the storage in liquid nitrogen eliminates a high proportion of cells which had been previously damaged by dimethylsulfoxide, since only small, meristematic cells survive this treatment. Furthermore, the use of cryoprotective mixtures containing other agents greatly minimizes the inherent

On the other hand, it is recognized that the process of apoptosis (programmed cell death) is not circumscribed to animals, but also occurs in plants, where it is used for the selective elimination and suicide of unwanted cells (Krishnamurthy et al., 2000). According to Joyce et al., (2003), among the cells undergoing this process are those which have sustained high levels of *in vitro* stress, which can compromise their physiology. Such a mechanism might,

After using the simple freezing procedure proposed by Martinez-Montero et al*.* (1998), it was determined that, apparently, the best dehydration levels are reached by the sugarcane calli when kept for 2 or 3 hours at -40 °C. It should be noted that the survival percentages achieved in this study were comparable to the best values obtained by Jian et al., (1987) and Eksomtramage et al*.,* (1992). Survival rates did not increase with longer pre-freezing times, probably due to excessive dehydration of the material. Studies based on the use of nuclear magnetic resonance spectroscopy in *Catharanthus roseu*s cells (Chen et al., 1984), vegetative apple buds (Tyler et al., 1988) and different tissues from *Rhododendron japonicum* (Ishikawa et al., 2000) have determined that the optimum pre-freezing time for a specified pre-freezing temperature depends on the amount of water still remaining inside the cells. Tyler et al*.*, (1988) proved the need for the pre-freezing step when they showed that the incubation of samples at an intermediate negative temperature before immersion in liquid nitrogen would

Finally, the optimized protocol carried out by our team took into account the *in vitro* survival and regeneration (plants per 500 mg of sugarcane calli) percentages and was validated for: a) three varieties (CP52-43, C1051-73, C91-301) (Table 1) ; b) explants obtained either from immature inflorescences or immature leaves from *in vitro* plants; c) calli stored for up to 16 months under liquid nitrogen, belonging to the CP52-43 variety (Table 2).

> Regeneration (plants per 500mg of calluses)

founded that the right amount of intracellular sucrose can protect a number of enzymes from ion-mediated toxicity.

According to our results, the sugarcane calli did not survive the cryopreservation procedure when dimethylsulfoxide was omitted from the cryoprotective mixture; and both the survival and plant regeneration percentages rose steadily with increasing dimethylsulfoxide concentrations, up to 10%. However, in clear contrast with the results obtained when testing different amounts of sucrose, there are no differences in survival between the cryopreserved and non-cryopreserved samples at concentrations higher than the optimum (10% v/v in this case), and the contrast is even starker when comparing plant regeneration rates, where the cryopreserved material performs even better than the non-cryopreserved calli. These results agree with those of Finkle et al., (1985) for rice cells, who concluded that the effects achieved by using dimethylsulfoxide are paradoxical, since although this substance is clearly toxic, but inhibits the growth of ice crystals during cryopreservation.

Our data obtained during the experiment for dimethylsulfoxide tolerance are coherent with the reports for other biological systems, associated to the high degree of toxicity of dimethylsulfoxide (Arakawa et al., 1990; Fahy et al., 1990). Kartha et al., (1988) detected that dimethylsulfoxide produces an inhibition of 35 to 42% on the growth of embryogenic cultures of white spruce when used at a concentration higher than 5% (v/v), and Klimaszewska et al., (1992) reported a 28% reduction in the growth of embryogenic tissues from black spruce when treated with 15% (v/v) dimethylsulfoxide; this effect, according to the microscopic observations of these authors, is due to the induction by this substance of a strong plasmolytic effect at the cellular level.

However, and in spite of these findings, dimethylsulfoxide has been, and still is used as a cryoprotectant during storage at ultra-low temperatures. According to Engelmann, (2000) this apparent paradox is due to the fact that dimethylsulfoxide is always used as part of a cryoprotective mixture, rather than individually. Arakawa et al., (19990) have provided evidence that the toxicity of dimethylsulfoxide in isolated proteins is mediated by hydrophobic interactions, which are favored at increasing temperatures; in this context, this effect is minimized by the use, during preculture, of sucrose at 0°C, which induces the biosynthesis of proteins that neutralize the toxic effects of this agent (caused by its interaction mainly with lysine residues) (Anchordoguy et al., 1991; Klimazewska et al., 1990; Swan et al., 1999).

Although the exact cryoprotective mechanism of dimethylsulfoxide at ultra-low temperatures remains unknown, it is widely acknowledged that it depends on the colligative properties of this penetrating compound; that is, dimethylsulfoxide affects the formation of ice crystals by decreasing the equilibrium freezing point of the solution, in direct dependence on its molar concentration (Kinoshita et al., 2001; McGann, L.E. & Walterson, 1987). Dimethylsulfoxide, as a cell-penetrating agent, also decreases the intracellular concentration of toxic electrolytes on unfrozen cells (Finkle et al., 1985).

Anchordoguy et al., (1991) suggested, furthermore, that there is another, not colligative mechanism for dimethylsulfoxide-mediated cryoprotection, which involves ionic interactions between the oxygen atom from this molecule and phospholipid bilayers. Such a mechanism would stabilize the cell membranes during the freeze-thaw cycle. The findings by us related wit better plant regeneration percentages from cryopreserved calli as compared to calli which had not been cryopreserved could be similar to those of Aronen et

founded that the right amount of intracellular sucrose can protect a number of enzymes

According to our results, the sugarcane calli did not survive the cryopreservation procedure when dimethylsulfoxide was omitted from the cryoprotective mixture; and both the survival and plant regeneration percentages rose steadily with increasing dimethylsulfoxide concentrations, up to 10%. However, in clear contrast with the results obtained when testing different amounts of sucrose, there are no differences in survival between the cryopreserved and non-cryopreserved samples at concentrations higher than the optimum (10% v/v in this case), and the contrast is even starker when comparing plant regeneration rates, where the cryopreserved material performs even better than the non-cryopreserved calli. These results agree with those of Finkle et al., (1985) for rice cells, who concluded that the effects achieved by using dimethylsulfoxide are paradoxical, since although this substance is clearly toxic,

Our data obtained during the experiment for dimethylsulfoxide tolerance are coherent with the reports for other biological systems, associated to the high degree of toxicity of dimethylsulfoxide (Arakawa et al., 1990; Fahy et al., 1990). Kartha et al., (1988) detected that dimethylsulfoxide produces an inhibition of 35 to 42% on the growth of embryogenic cultures of white spruce when used at a concentration higher than 5% (v/v), and Klimaszewska et al., (1992) reported a 28% reduction in the growth of embryogenic tissues from black spruce when treated with 15% (v/v) dimethylsulfoxide; this effect, according to the microscopic observations of these authors, is due to the induction by this substance of a

However, and in spite of these findings, dimethylsulfoxide has been, and still is used as a cryoprotectant during storage at ultra-low temperatures. According to Engelmann, (2000) this apparent paradox is due to the fact that dimethylsulfoxide is always used as part of a cryoprotective mixture, rather than individually. Arakawa et al., (19990) have provided evidence that the toxicity of dimethylsulfoxide in isolated proteins is mediated by hydrophobic interactions, which are favored at increasing temperatures; in this context, this effect is minimized by the use, during preculture, of sucrose at 0°C, which induces the biosynthesis of proteins that neutralize the toxic effects of this agent (caused by its interaction mainly with lysine residues) (Anchordoguy et al., 1991; Klimazewska et al., 1990;

Although the exact cryoprotective mechanism of dimethylsulfoxide at ultra-low temperatures remains unknown, it is widely acknowledged that it depends on the colligative properties of this penetrating compound; that is, dimethylsulfoxide affects the formation of ice crystals by decreasing the equilibrium freezing point of the solution, in direct dependence on its molar concentration (Kinoshita et al., 2001; McGann, L.E. & Walterson, 1987). Dimethylsulfoxide, as a cell-penetrating agent, also decreases the

Anchordoguy et al., (1991) suggested, furthermore, that there is another, not colligative mechanism for dimethylsulfoxide-mediated cryoprotection, which involves ionic interactions between the oxygen atom from this molecule and phospholipid bilayers. Such a mechanism would stabilize the cell membranes during the freeze-thaw cycle. The findings by us related wit better plant regeneration percentages from cryopreserved calli as compared to calli which had not been cryopreserved could be similar to those of Aronen et

intracellular concentration of toxic electrolytes on unfrozen cells (Finkle et al., 1985).

but inhibits the growth of ice crystals during cryopreservation.

strong plasmolytic effect at the cellular level.

Swan et al., 1999).

from ion-mediated toxicity.

al., (1999) in embryogenic cultures of *Abies cephalonica*. We later founded that the storage in liquid nitrogen eliminates a high proportion of cells which had been previously damaged by dimethylsulfoxide, since only small, meristematic cells survive this treatment. Furthermore, the use of cryoprotective mixtures containing other agents greatly minimizes the inherent toxicity of dimethylsulfoxide.

On the other hand, it is recognized that the process of apoptosis (programmed cell death) is not circumscribed to animals, but also occurs in plants, where it is used for the selective elimination and suicide of unwanted cells (Krishnamurthy et al., 2000). According to Joyce et al., (2003), among the cells undergoing this process are those which have sustained high levels of *in vitro* stress, which can compromise their physiology. Such a mechanism might, therefore, be involved in dimethylsulfoxide-mediated toxicity.

After using the simple freezing procedure proposed by Martinez-Montero et al*.* (1998), it was determined that, apparently, the best dehydration levels are reached by the sugarcane calli when kept for 2 or 3 hours at -40 °C. It should be noted that the survival percentages achieved in this study were comparable to the best values obtained by Jian et al., (1987) and Eksomtramage et al*.,* (1992). Survival rates did not increase with longer pre-freezing times, probably due to excessive dehydration of the material. Studies based on the use of nuclear magnetic resonance spectroscopy in *Catharanthus roseu*s cells (Chen et al., 1984), vegetative apple buds (Tyler et al., 1988) and different tissues from *Rhododendron japonicum* (Ishikawa et al., 2000) have determined that the optimum pre-freezing time for a specified pre-freezing temperature depends on the amount of water still remaining inside the cells. Tyler et al*.*, (1988) proved the need for the pre-freezing step when they showed that the incubation of samples at an intermediate negative temperature before immersion in liquid nitrogen would result in a better performance of the cryopreserved material after thawing.

Finally, the optimized protocol carried out by our team took into account the *in vitro* survival and regeneration (plants per 500 mg of sugarcane calli) percentages and was validated for: a) three varieties (CP52-43, C1051-73, C91-301) (Table 1) ; b) explants obtained either from immature inflorescences or immature leaves from *in vitro* plants; c) calli stored for up to 16 months under liquid nitrogen, belonging to the CP52-43 variety (Table 2).


Table 1. Effects of optimized cryopreservation protocol on survival and plant regeneration produced from control (-LN) and cryopreserved (+LN) sugarcane embryogenic calluses (varieties CP52-43, C91-301 and C1051-73). *Means within columns followed by the same letter are not significantly different (ANOVA p < 0,05 Tukey,). Data were transformed for statistical analysis in accordance with x'= 2 arcsine ((x/100)0,5) and with x'= (0,5 + x)0,5 for percentage of survival and plant regeneration, respectively.* 

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

nitrogen ranged between 38 and 91% for the 5 varieties experimented.

had been only slightly harmed, as revealed by histological examination.

duration tested.

always rapid and direct.

varieties without any need for further adaptation.

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 367

medium with 3% alginate. Optimal conditions comprised preculture for 2d in liquid medium with 0.75M sucrose, desiccation for 6h under the laminar flow or for 10–11 hours with silicagel followed by rapid freezing and slow thawing. Survival after freezing in liquid

Later on, Gonzalez-Arnao et al. (1993) in CNIC, Havana Cuba investigated the effect of sucrose concentration during the pregrowth treatment and of freezing procedure on the survival of encapsulated apices of six sugarcane varieties. The optimal sucrose concentration was 0.75 M during 24h. We showed that encapsulated apices of sugarcane could withstand freezing in liquid nitrogen using various freezing procedures. Growth recovery of apices after thawing was very rapid and direct, due to the fact that most cells of the apical region

Moreover, our group studied apices sampled on *in vitro* plantlets of different varieties and could be cryopreserved using the encapsulation-dehydration technique and stored for one year at the temperature of liquid nitrogen without modification in their recovery percentage (Gonzalez-Arnao et al., 1999). By contrast, apices placed at -70°C or -25°C lost viability very rapidly. There are several explanations for this result: even though vitrification of internal solutes has been observed during freezing of these materials, including sugarcane devitrification and recrystallization processes, which are detrimental to 'cellular integrity, take place at these temperatures (Gonzalez-Arnao et al., 1996). These contrasting results might be linked to the presence of higher levels of residual free water in the latter systems, which would recrystallize rapidly at these temperatures and result in the death of the explants. At lower temperatures comprised between -135 and -196"C, no differences were noted in the regrowth capacity of all materials mentioned above whatever the storage

It is interesting to note that, even though the two protocols set up were slightly different, the average results obtained on a total of 15 sugarcane varieties (8 frozen with the CNIC protocol, 7 with the CIRAD protocol) were similar (Table 3). It should also be noted that different varieties showed different sensitivities to preculture and desiccation, and to preculture, desiccation and freezing. However, there was only one case (Ja 60-5) where the difference between control and cryopreserved samples was very high, 70% and 24% survival, respectively. Both protocols are thus potentially applicable to a large range of

Recently, *in vitro* shoot tips of two clones were successfully cryopreserved using encapsulation-dehydration according to Gonzalez-Arnao et al., (1993) and dropletvitrification with two vitrification solutions, PVS2 and PVS3 (Barraco et al., 2011). For both clones, encapsulation-dehydration induced significantly higher recovery, reaching 60% for clone H70-144 and 53% for clone CP68-1026, compared with droplet-vitrification in which recovery was 33–37% for clone H70-144 and 20–27% for clone CP68-1026. Optimal conditions included preculture of encapsulated shoot apices for 24 h in liquid medium with 0.75 M sucrose and dehydration with silica gel to 20% moisture content (fresh weight basis) before direct immersion in liquid nitrogen. With both protocols employed, regrowth of cryopreserved samples, as followed by visual observation, was


Table 2. Effect of extended storage duration on the survival and plantlet produced from control (-LN) and cryopreserved (+LN) sugarcane embryogenic calluses (variety CP52-43). *Means within columns followed by the same letter are not significantly different (ANOVA p < 0,05 Tukey,). Data were transformed for statistical analysis in accordance with x' = 2 arcsine ((x/100)0,5) and with x' = (0,5 + x)0,5 for percentage of survival and plant regeneration, respectively.*

#### **2.3 Apices**

The first attempt to freeze sugarcane apices were carried out by the group of Bajaj et al., (1987) using apices from *in vivo* plants. This material was pretreated with a mixture of 5% (v/v) of each DMSO, sucrose and glycerol during 45min. Then the freezing was accomplished by rapid immersion in liquid nitrogen of samples. However, the apices recovery was very scarce and with only small callus formation without plant regeneration.

Later on, research for the development of a cryopreservation protocol for sugarcane apices was carried out in the framework of collaborative program involving IRD (Institut de recherche pour le développement, Montpellier, France), CIRAD (Centre de coopération internationale en recherche agronomique pour le développement, Montpellier, France), CNIC (Centro Nacional de Investigaciones Cientificas, Havana, Cuba), IPGRI (International Plant Genetic Resources Institute, Rome, Italy) and FAO (Food and Agriculture Organisation of the United Nations, Rome, Italy).

A cryopreservation process using encapsulation/dehydration was set up for apices sampled on *in vitro* plantlets of sugarcane by Paulet et al., (1993) in CIRAD, Montpellier, France. After dissection, apices were cultured for one day on standard medium and then encapsulated in

plants (%)

1 100 a 100,0 a 225,2 a

4 98,6 a 100,0 a 224,3 a

8 96,7 ab 66,7 b 77,0 c

12 97,5 ab 7,1 c 19,1 d

16 11,5 c 0,0 d 0,0 e

1 90,6 b 96,7 a 149,3 b

4 87,6 b 97,8 a 142,9 b

8 88,0 b 93,3 a 135,1 b

12 86,4 b 97,0 a 141,7 b

16 90,0 b 97,5 a 140,3 b

Typical Error 0,189 0,253 1,312

Table 2. Effect of extended storage duration on the survival and plantlet produced from control (-LN) and cryopreserved (+LN) sugarcane embryogenic calluses (variety CP52-43). *Means within columns followed by the same letter are not significantly different (ANOVA p < 0,05* 

*= (0,5 + x)0,5 for percentage of survival and plant regeneration, respectively.*

The first attempt to freeze sugarcane apices were carried out by the group of Bajaj et al., (1987) using apices from *in vivo* plants. This material was pretreated with a mixture of 5% (v/v) of each DMSO, sucrose and glycerol during 45min. Then the freezing was accomplished by rapid immersion in liquid nitrogen of samples. However, the apices recovery was very scarce and with only small callus formation without plant regeneration. Later on, research for the development of a cryopreservation protocol for sugarcane apices was carried out in the framework of collaborative program involving IRD (Institut de recherche pour le développement, Montpellier, France), CIRAD (Centre de coopération internationale en recherche agronomique pour le développement, Montpellier, France), CNIC (Centro Nacional de Investigaciones Cientificas, Havana, Cuba), IPGRI (International Plant Genetic Resources Institute, Rome, Italy) and FAO (Food and Agriculture

A cryopreservation process using encapsulation/dehydration was set up for apices sampled on *in vitro* plantlets of sugarcane by Paulet et al., (1993) in CIRAD, Montpellier, France. After dissection, apices were cultured for one day on standard medium and then encapsulated in

*Tukey,). Data were transformed for statistical analysis in accordance with x'*

Organisation of the United Nations, Rome, Italy).

Regeneration (plants per 500mg of calluses)

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

(months) Survival (%) Calluses that regenerated

Time


+NL

*and with x'*

**2.3 Apices** 

medium with 3% alginate. Optimal conditions comprised preculture for 2d in liquid medium with 0.75M sucrose, desiccation for 6h under the laminar flow or for 10–11 hours with silicagel followed by rapid freezing and slow thawing. Survival after freezing in liquid nitrogen ranged between 38 and 91% for the 5 varieties experimented.

Later on, Gonzalez-Arnao et al. (1993) in CNIC, Havana Cuba investigated the effect of sucrose concentration during the pregrowth treatment and of freezing procedure on the survival of encapsulated apices of six sugarcane varieties. The optimal sucrose concentration was 0.75 M during 24h. We showed that encapsulated apices of sugarcane could withstand freezing in liquid nitrogen using various freezing procedures. Growth recovery of apices after thawing was very rapid and direct, due to the fact that most cells of the apical region had been only slightly harmed, as revealed by histological examination.

Moreover, our group studied apices sampled on *in vitro* plantlets of different varieties and could be cryopreserved using the encapsulation-dehydration technique and stored for one year at the temperature of liquid nitrogen without modification in their recovery percentage (Gonzalez-Arnao et al., 1999). By contrast, apices placed at -70°C or -25°C lost viability very rapidly. There are several explanations for this result: even though vitrification of internal solutes has been observed during freezing of these materials, including sugarcane devitrification and recrystallization processes, which are detrimental to 'cellular integrity, take place at these temperatures (Gonzalez-Arnao et al., 1996). These contrasting results might be linked to the presence of higher levels of residual free water in the latter systems, which would recrystallize rapidly at these temperatures and result in the death of the explants. At lower temperatures comprised between -135 and -196"C, no differences were noted in the regrowth capacity of all materials mentioned above whatever the storage duration tested.

It is interesting to note that, even though the two protocols set up were slightly different, the average results obtained on a total of 15 sugarcane varieties (8 frozen with the CNIC protocol, 7 with the CIRAD protocol) were similar (Table 3). It should also be noted that different varieties showed different sensitivities to preculture and desiccation, and to preculture, desiccation and freezing. However, there was only one case (Ja 60-5) where the difference between control and cryopreserved samples was very high, 70% and 24% survival, respectively. Both protocols are thus potentially applicable to a large range of varieties without any need for further adaptation.

Recently, *in vitro* shoot tips of two clones were successfully cryopreserved using encapsulation-dehydration according to Gonzalez-Arnao et al., (1993) and dropletvitrification with two vitrification solutions, PVS2 and PVS3 (Barraco et al., 2011). For both clones, encapsulation-dehydration induced significantly higher recovery, reaching 60% for clone H70-144 and 53% for clone CP68-1026, compared with droplet-vitrification in which recovery was 33–37% for clone H70-144 and 20–27% for clone CP68-1026. Optimal conditions included preculture of encapsulated shoot apices for 24 h in liquid medium with 0.75 M sucrose and dehydration with silica gel to 20% moisture content (fresh weight basis) before direct immersion in liquid nitrogen. With both protocols employed, regrowth of cryopreserved samples, as followed by visual observation, was always rapid and direct.

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

embryos (SE).

vitrification (Fig. 3D).

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 369

Table 4. Effect of the cryopreservation protocol on the recovery pattern of sugarcane somatic

Untreated embryos were white (Fig. 3A & B). Cryopreserved embryos were white to yellow when they were placed on recovery medium; viable embryos turned yellow to green after about 2 weeks; they converted to plants within an additional 2 week period and produced green shoots and roots (Fig. 3 C & D). Callus formation was not observed in germinated embryos and no secondary embryos were produced after the droplet-vitrification procedure (Fig. 3 C). However, callus appeared together with germinated embryos after encapsulation-

Fig. 3. Initial embryogenic sugarcane callus (A); clumps of somatic embryos selected for cryopreservation experiments (dashed line) (B); recovered clumps of somatic embryos after cryopreservation and 4 weeks after transfer to MS medium under light conditions (C, using droplet-vitrification procedure; D, using encapsulation-vitrification procedure) (bar = 1mm).


Table 3. Survival of control (-LN) and cryopreserved (+LN) apices using the encapsulationdehydration technique according to the protocol described by Gonzalez-Arnao et al. (1993, \*) and Paulet et al. (1993, \*\*).
