**3. Use of analytical techniques for sugarcane cryopreservation protocols**

In the past, cryopreservation protocols have generally been developed using an empirical approach. However, considerable advances have been made in recent years in the use of analytical tools to enhance our current knowledge of the damages induced in biological tissues by cryopreservation (Engelmann, 2011). Various biophysical, biochemical and histocytological techniques are available for this purpose (Harding, 1999). Such analytical tools

The obtained results by Martinez-Montero et al., (2008) contrasted with what is generally observed in the literature, as vitrification is the most frequently employed vitrification-based procedure and it has been applied to a large number of species (Panis & Lambardi, 2006; Sakai & Engelmann, 2007). However, the number of successful reports of application of the droplet-freezing and encapsulation-vitrification techniques is increasing steadily

Sugarcane somatic embryos proved very sensitive to PVS2, even though the PVS2 treatment was performed at 0°C, which usually reduces the toxicity of the cryoprotectant solution (Benson, 2008). This high sensitivity rendered the utilization of the vitrification procedure

One of the options tested for cryopreservation of sugarcane somatic embryos was the encapsulation-vitrification technique, as developed by Matsumoto et al., (1995). These authors suggested that the toxicity of the PVS2 solution could be reduced by encapsulating the explants in alginate beads. Encapsulation also made the manipulation of the material easier. The positive effect of employing this technique was confirmed by the results, as some viability was achieved after cryopreserving sugarcane embryos using encapsulation-

We also tested the droplet-vitrification technique with sugarcane embryos (Martinez-Montero et al., 2008). Droplet vitrification combines the procedure called droplet-freezing, which has been established with cassava (Kartha & Engelmann, 1994) and applied notably to potato (Schäfer-Menuhr et al., 1997) and asparagus shoot tips (Mix-Wagner et al., 2000), in which explants are cooled in a droplet of cryoprotectant solution with the vitrification procedure (Sakai et al., 1990), since explants are cooled in a droplet of PVS2 solution. Droplet-vitrification is relatively easy to implement and generally ensures high recovery after cooling (Sakai & Engelmann, 2007). One of the advantages of this technique is the high cooling and warming rates achieved, compared with others procedures (Benson, 2007; Panis et al., 2005). These high cooling/warming rates ensure complete vitrification during cooling and reduce the risks of devitrification during warming of samples, which is important to

Moreover, Volk & Walters (2006) concluded that PVS2 imparts its effect in the previtrified solution, and at lower temperature the cryoprotectant restricts the mobility of water molecules, so that they are unable to nucleate and ice crystals are not allowed to growth. Benson (2008) empathized that cryoprotection using droplet-vitrification involves a somewhat different principle, due to the behavior of water molecules contained in microdroplets of vitrification solution. If the biophysical conditions are optimal the droplets can

**3. Use of analytical techniques for sugarcane cryopreservation protocols** 

In the past, cryopreservation protocols have generally been developed using an empirical approach. However, considerable advances have been made in recent years in the use of analytical tools to enhance our current knowledge of the damages induced in biological tissues by cryopreservation (Engelmann, 2011). Various biophysical, biochemical and histocytological techniques are available for this purpose (Harding, 1999). Such analytical tools

avoid the lethal effects of intracellular ice crystal formation (Benson, 2008).

become vitrified on direct exposure to liquid nitrogen.

(Engelmann, 2011).

vitrification.

impossible and alternative options had to be sought.

allow the detection of those components of a cryopreservation method which cause the most damage. Usually, these studies are correlated with survival responses and viability testing. However, the application of analytical tools for plant cryopreservation studies is still very scarce and in some cases they are costly to implement and complex to evaluate (Verleysen et al., 2004). Apart mention need the excellent review by Benson (2008) in which it is exposed that contemporary cryopreservation research is now supported by advanced biomolecular or 'omics' technologies, creating a new knowledge base which will hopefully help to solve some of the more difficult cryobiological challenges. However, it will become increasingly so as stakeholders invest in areas commonly interested in low temperature research. Therefore, our research experience is only limited to use non costly and complex analytical techniques yet.

#### **3.1 Effect of cryopreservation on the structural and functional integrity of cell membranes of sugarcane embryogenic callus**

Cell membranes are one of the main targets of numerous stressing events, including cryopreservation (Benson, 2007; Fahy et al., 1984; Engelmann, 2011). Various markers, including electrolyte efflux, lipid peroxidation products and cell membrane protein content, reflect the structural and functional integrity status of cell membranes after exposure to such stressing events (Harding, 1999; Verleysen et al., 2004).

Measurement of electrolyte leakage has been used notably for studying the desiccation and cryopreservation sensitivity of various recalcitrant seed species (Sun, 1999). Lipid peroxidation profiles have been used as markers of cell membrane damage during freezing of rice cell suspensions and of the coenocytic alga *Vaucheria sessilis* (Benson et al., 1992; Fleck et al., 1999). Watanabe et al., (1999) have shown that the acquisition of tolerance to cryopreservation of rice cells was related to changes in protein metabolism. An increasing number of proteins and peptides that might contribute to freezing tolerance by reducing the effects of dehydration associated with freezing have been identified (Thomashow, 1999). In the same way, Thierry et al., (1999) have observed in carrot somatic embryos the overaccumulation of boiling-stable proteins, which seems to be related to an increase in tolerance to cryopreservation. Besides, some enzymes, which are induced by low temperature, such as fatty acid desaturase and sucrose phosphate synthase, also contribute to freezing tolerance (Guy, 1999).

Our research group studied the effect of cryopreservation on the structural and functional integrity of cell membranes of sugarcane embryogenic calluses by measuring electrolyte leakage, lipid peroxidation products and membrane proteins (Fig. 4). Firstly, we showed (Martinez-Montero et al., 2002a) that survival and plantlet production were lower with cryopreserved sugarcane embryogenic calluses in comparison with unfrozen control calluses. However, the differences observed between control and cryopreserved calluses in the parameters studied to evaluate membrane structural and functional integrity, including electrolyte leakage, total cell membrane protein content, malondialdehyde and other aldehyde content were only transitory. Indeed, they had all disappeared within 3-4 days after freezing.

Electrolyte leakage, measured to evaluate the overall effect of cryopreservation on the semipermeability of plasma membranes, revealed a partial loss of membrane semipermeability

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

aldehydic products.

stage.

cryopreservation.

could damage the membranes.

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 373

unstable lipid peroxides. These compounds breakdown to form toxic secondary oxidation products (Esterbauer et al., 1988) such as aldehydes, including malondialdehyde and other

According to our results the main factors affecting sugarcane callus cell membrane damages and electrolyte efflux might thus be oxygen reactive species instead of malondialdehyde and aldehydes themselves, since the highest concentration of these compounds was reached later than the highest level of electrolyte leakage. However, it is also possible that the damages noted after cryopreservation could have been caused by the loss of cellular integrity due to the formation of ice crystals and to the cryoprotectants employed, which

We also showed that the content in malondialdehyde and other aldehydes in the microsomal fraction were higher for cryopreserved calluses than unfrozen controls, but only during the first three days after cryopreservation. Benson et al., (1992) have obtained similar results for malondialdehyde with cryopreserved rice cell suspensions. Therefore, they suggested that freezing stress could have caused disruption and uncoupling in some metabolic pathways as reported by Fleck *et a*l. (1999) and Dumet et al., (2000) with other biological systems. This could have led to the production of free radicals, thus promoting lipid peroxidation in the cellular membranes of calluses at a very early post-thaw recovery

Variations were also observed in control calluses, concerning mainly electrolyte leakage and lipid peroxidation. The significantly increased levels of malondialdehyde and aldehydes measured during the first 3 days in control calluses might be a result of mechanical membrane damage caused by cutting when preparing the starting material. Fleck et al., (1999) described an increase in lipid peroxidation products after cutting algae filaments into sections. In addition, transfer of material to fresh medium itself is another stress source that

The concentration of lipid peroxidation products decreased from the second day onwards and reached a constant value on the fourth day in both frozen and control calluses (Martinez-Montero et al., 2002a). This decrease must have been caused by the activation of antioxidant defense mechanisms. Plants produce antioxidant molecules and have scavenging systems (ß-carotenes, tocopherol isomers, ascorbic acid, glutathione) and enzymatic free radical processing systems (superoxide dismutase, catalase, glutathione reductase, ascorbate peroxidase and various other enzymes) as a protective response to stresses (Leprince et al., 1993). Those antioxidant systems are directly activated by oxidative stress and, consequently, diminish the levels of ROS and thiobarbituric acid reactive substances in cells. Martinez-Montero et al., (2002a) suggested additional experiments to be performed to measure the concentration of such antioxidant molecules and the activity level of the above-cited enzymes in sugarcane embryogenic calluses in relation to

An increase in cell membrane-related proteins has been described as a response to dehydration and freezing stress (Ausborn et al., 1994). Such proteins are produced as a protective mechanism to preserve membrane structure, ion sequestration and chaperon-like functions (Thierry et al., 1999). According to Martinez-Montero et al., (2002a) the total

may cause an increase in malondialdehyde and aldehydes (Benson, 2007).

in callus cells. The transitory character of the electrolyte efflux observed indicates that no dramatic mechanical cell membrane injuries were caused by cryopreservation; rather only reversible lesions were induced by this treatment. As part of this dynamic process, the electrolytes released by damaged cells may have been taken up by living cells.

Fig. 4. Effect of cryopreservation on the structural and functional integrity of cell membranes of sugarcane embryogenic calluses by measuring electrolyte leakage (A), total proteins (B), malondialdehyde content (C) and other aldehyde content (D). *Data followed by the same letter are not statistically different (ANOVA, Duncan, p<0.05). CC: cryopreserved callus. NCC: non-cryopreserved callus.* 

Freezing injury induces the production of free radicals, mainly reactive oxygen species (ROS) (Benson et al., 1992). The ROS signaling pathway is mainly controlled by the production of, and balance between, pro- and antioxidants and the perturbation of ROS homeostasis (Mittler et al., 2004). These changes are perceived by various proteins, enzymes and receptors which influence different developmental, metabolic and defense pathways. Free radicals then attack the lipid fraction of membranes, resulting in the formation of

in callus cells. The transitory character of the electrolyte efflux observed indicates that no dramatic mechanical cell membrane injuries were caused by cryopreservation; rather only reversible lesions were induced by this treatment. As part of this dynamic process, the

a b

c d

membranes of sugarcane embryogenic calluses by measuring electrolyte leakage (A), total proteins (B), malondialdehyde content (C) and other aldehyde content (D). *Data followed by the same letter are not statistically different (ANOVA, Duncan, p<0.05). CC: cryopreserved callus.* 

Freezing injury induces the production of free radicals, mainly reactive oxygen species (ROS) (Benson et al., 1992). The ROS signaling pathway is mainly controlled by the production of, and balance between, pro- and antioxidants and the perturbation of ROS homeostasis (Mittler et al., 2004). These changes are perceived by various proteins, enzymes and receptors which influence different developmental, metabolic and defense pathways. Free radicals then attack the lipid fraction of membranes, resulting in the formation of

Fig. 4. Effect of cryopreservation on the structural and functional integrity of cell

*NCC: non-cryopreserved callus.* 

electrolytes released by damaged cells may have been taken up by living cells.

unstable lipid peroxides. These compounds breakdown to form toxic secondary oxidation products (Esterbauer et al., 1988) such as aldehydes, including malondialdehyde and other aldehydic products.

According to our results the main factors affecting sugarcane callus cell membrane damages and electrolyte efflux might thus be oxygen reactive species instead of malondialdehyde and aldehydes themselves, since the highest concentration of these compounds was reached later than the highest level of electrolyte leakage. However, it is also possible that the damages noted after cryopreservation could have been caused by the loss of cellular integrity due to the formation of ice crystals and to the cryoprotectants employed, which could damage the membranes.

We also showed that the content in malondialdehyde and other aldehydes in the microsomal fraction were higher for cryopreserved calluses than unfrozen controls, but only during the first three days after cryopreservation. Benson et al., (1992) have obtained similar results for malondialdehyde with cryopreserved rice cell suspensions. Therefore, they suggested that freezing stress could have caused disruption and uncoupling in some metabolic pathways as reported by Fleck *et a*l. (1999) and Dumet et al., (2000) with other biological systems. This could have led to the production of free radicals, thus promoting lipid peroxidation in the cellular membranes of calluses at a very early post-thaw recovery stage.

Variations were also observed in control calluses, concerning mainly electrolyte leakage and lipid peroxidation. The significantly increased levels of malondialdehyde and aldehydes measured during the first 3 days in control calluses might be a result of mechanical membrane damage caused by cutting when preparing the starting material. Fleck et al., (1999) described an increase in lipid peroxidation products after cutting algae filaments into sections. In addition, transfer of material to fresh medium itself is another stress source that may cause an increase in malondialdehyde and aldehydes (Benson, 2007).

The concentration of lipid peroxidation products decreased from the second day onwards and reached a constant value on the fourth day in both frozen and control calluses (Martinez-Montero et al., 2002a). This decrease must have been caused by the activation of antioxidant defense mechanisms. Plants produce antioxidant molecules and have scavenging systems (ß-carotenes, tocopherol isomers, ascorbic acid, glutathione) and enzymatic free radical processing systems (superoxide dismutase, catalase, glutathione reductase, ascorbate peroxidase and various other enzymes) as a protective response to stresses (Leprince et al., 1993). Those antioxidant systems are directly activated by oxidative stress and, consequently, diminish the levels of ROS and thiobarbituric acid reactive substances in cells. Martinez-Montero et al., (2002a) suggested additional experiments to be performed to measure the concentration of such antioxidant molecules and the activity level of the above-cited enzymes in sugarcane embryogenic calluses in relation to cryopreservation.

An increase in cell membrane-related proteins has been described as a response to dehydration and freezing stress (Ausborn et al., 1994). Such proteins are produced as a protective mechanism to preserve membrane structure, ion sequestration and chaperon-like functions (Thierry et al., 1999). According to Martinez-Montero et al., (2002a) the total

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 375

Fig. 5. Effect of the imbibition duration in double-distilled water on the leakage of electrolytes in mixtures of cooled and non-cooled clumps by regression analysis to

**3.2.1 Optimization of methodology for somatic embryos** 

25 h.

determine the cell viability of sugarcane somatic embryos. A) after 5 h; B) after 15 h; C) after

The loading treatment is an essential step to achieve high post-rewarming survival of cryopreserved sugarcane somatic embryos because it induces or enhances tolerance of samples to PVS2 treatment (Panis & Thinh, 2001). A loading solution including 2 M glycerol

microsomal fraction protein content was higher in cryopreserved sugarcane calluses during the first 3 d after thawing. This increase was concomitant with an increase in malondialdehyde and aldehyde concentration. They hypothesized that some of the proteins induced by the freeze-thaw cycle may play a role in decreasing the malondialdehyde and aldehyde levels, in addition to the other functions mentioned above.

Even though electrolyte leakage, malondialdehyde, aldehyde and cell membrane protein contents became similar in control and cryopreserved samples 4 d after cryopreservation, cryopreservation consistently affected callus survival and plantlet regeneration (Martinez-Montero et al., 2002a). However, lipid peroxidation products (such as malondialdehyde and aldehydes) might have impaired various cell functions in the sugarcane embryogenic calluses by cross-linking to macromolecules such as DNA and proteins to form mutagenic compounds as reported by Yang & Scaich, (1996) for animal cells. Moreover, the free radicals induced by freezing stress are considered both cytotoxic and genotoxic because they are capable to modify protein structure, to form complexes with DNA and enzymes and to inhibit nucleic acid synthesis (Esterbauer et al., 1988; Grune et al., 1997, Spiteller, 1996). Such impairments might have affected the totipotency of these callus cells.

#### **3.2 Use of electrolyte leakage technique for sugarcane somatic embryos**

To allow a quick, reliable prognosis of the experiments performed and to refine the optimal conditions for cryopreservation of somatic embryos, viability was estimated using an electrolyte efflux test by our research group (Martinez-Montero et al., 2008). Firstly, four dissected clumps were either immediately incubated in 20 ml double-distilled water or immersed directly in liquid nitrogen before incubation. The percentage of living cells (cell viability) was calculated by making mixtures of cooled and non cooled clumps (ca. 20 mg total fresh weight). Conductivity of the water was measured before (C0) and after 5, 15 or 25 h of imbibition (Cx). Samples were then autoclaved (30 min at 112°C, 107 kPa) and cooled down to room temperature for 4 h to determine the total conductivity (Ctotal). The percentage of electrolyte leakage was calculated from the ratio: (Cx – C0)\*100/(Ctotal – C0). Lastly, a regression analysis was made between the results of the electrolyte leakage test and the cell viability (both were expressed in percentages). The best model that was suitable to represent the experimental data constitutes a standardized curve for analysis of cell viability during all experimentations.

The effect of the imbibitions duration in double-distilled water on the leakage of electrolytes in mixtures of cooled and non-cooled clumps by regression analysis demonstrated that there was a significant linear relationship (α = 0.05) between the electrolyte efflux and the viability of somatic embryos and high coefficients of determination (R2 > 0.88) were obtained (Fig. 5). It was clearly observed that a greater functional relationship existed in the lineal equation for imbibitions periods of clumps between 15 and 25 h. Our results confirmed that an equilibration period was necessary for accurate measurement of cell leakage and the electrolyte leakage was practically complete (85% of the electrolytes leaked in a sample with 100% of cooled clumps) after 15 h only. Moreover, the values of electrolyte leakage indicated that close to 10% of the somatic embryos were damaged after dissection for the cryopreservation experiments. Therefore, our test proved useful and precise as it was not only good for distinguishing between living and dead tissues but also for quantifying small differences in the amount of viable tissues.

microsomal fraction protein content was higher in cryopreserved sugarcane calluses during the first 3 d after thawing. This increase was concomitant with an increase in malondialdehyde and aldehyde concentration. They hypothesized that some of the proteins induced by the freeze-thaw cycle may play a role in decreasing the malondialdehyde and

Even though electrolyte leakage, malondialdehyde, aldehyde and cell membrane protein contents became similar in control and cryopreserved samples 4 d after cryopreservation, cryopreservation consistently affected callus survival and plantlet regeneration (Martinez-Montero et al., 2002a). However, lipid peroxidation products (such as malondialdehyde and aldehydes) might have impaired various cell functions in the sugarcane embryogenic calluses by cross-linking to macromolecules such as DNA and proteins to form mutagenic compounds as reported by Yang & Scaich, (1996) for animal cells. Moreover, the free radicals induced by freezing stress are considered both cytotoxic and genotoxic because they are capable to modify protein structure, to form complexes with DNA and enzymes and to inhibit nucleic acid synthesis (Esterbauer et al., 1988; Grune et al., 1997, Spiteller, 1996). Such

To allow a quick, reliable prognosis of the experiments performed and to refine the optimal conditions for cryopreservation of somatic embryos, viability was estimated using an electrolyte efflux test by our research group (Martinez-Montero et al., 2008). Firstly, four dissected clumps were either immediately incubated in 20 ml double-distilled water or immersed directly in liquid nitrogen before incubation. The percentage of living cells (cell viability) was calculated by making mixtures of cooled and non cooled clumps (ca. 20 mg total fresh weight). Conductivity of the water was measured before (C0) and after 5, 15 or 25 h of imbibition (Cx). Samples were then autoclaved (30 min at 112°C, 107 kPa) and cooled down to room temperature for 4 h to determine the total conductivity (Ctotal). The percentage of electrolyte leakage was calculated from the ratio: (Cx – C0)\*100/(Ctotal – C0). Lastly, a regression analysis was made between the results of the electrolyte leakage test and the cell viability (both were expressed in percentages). The best model that was suitable to represent the experimental data constitutes a standardized curve for analysis of cell viability

The effect of the imbibitions duration in double-distilled water on the leakage of electrolytes in mixtures of cooled and non-cooled clumps by regression analysis demonstrated that there was a significant linear relationship (α = 0.05) between the electrolyte efflux and the viability of somatic embryos and high coefficients of determination (R2 > 0.88) were obtained (Fig. 5). It was clearly observed that a greater functional relationship existed in the lineal equation for imbibitions periods of clumps between 15 and 25 h. Our results confirmed that an equilibration period was necessary for accurate measurement of cell leakage and the electrolyte leakage was practically complete (85% of the electrolytes leaked in a sample with 100% of cooled clumps) after 15 h only. Moreover, the values of electrolyte leakage indicated that close to 10% of the somatic embryos were damaged after dissection for the cryopreservation experiments. Therefore, our test proved useful and precise as it was not only good for distinguishing between living and dead tissues but also for quantifying small

aldehyde levels, in addition to the other functions mentioned above.

impairments might have affected the totipotency of these callus cells.

during all experimentations.

differences in the amount of viable tissues.

**3.2 Use of electrolyte leakage technique for sugarcane somatic embryos** 

Fig. 5. Effect of the imbibition duration in double-distilled water on the leakage of electrolytes in mixtures of cooled and non-cooled clumps by regression analysis to determine the cell viability of sugarcane somatic embryos. A) after 5 h; B) after 15 h; C) after 25 h.

#### **3.2.1 Optimization of methodology for somatic embryos**

The loading treatment is an essential step to achieve high post-rewarming survival of cryopreserved sugarcane somatic embryos because it induces or enhances tolerance of samples to PVS2 treatment (Panis & Thinh, 2001). A loading solution including 2 M glycerol

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

1995; Stanley et al., 2008).

favoured (Semenova et al., 2002).

assessment of agronomic characteristics.

macropropagation.

their early growth in vivo (Gonzalez-Arnao et al, 1999).

**4. Stability assessment** 

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 377

(Parsegian et al., 2000), dealing also with the movement of water molecules (Parsegian et al.,

Duan et al., (2001) observed that the hydroxyl groups present in the glucose units could contribute to protein-sugar interactions in aqueous solutions. These newly formed dipoledipole interactions could form a hydrophilic layer around the protein units and therefore increase the dispersability of the protein through protein hydration and/or alter the intramolecular interactions in such a way that folding and even dissociation may be

Before using cryopreservation as an additional tool in the overall conservation strategy for any plant material, it is essential to verify that the cryopreservation protocol developed does not have any destabilizing effect and that the plants produced from cryopreserved explants are true to type (Harding, 2004). This author firstly provided a definition for "Cryobionomics" - a novel term describing the remodeled concept of genetic stability and the re-introduction of cryopreserved plants into the environment. Later, Cryobionomics is proposed as an approach to explore links between cryoinjury and genetic instability during and after cryopreservation (Harding et al., 2005, 2008b). There are an increasing number of reports indicating that no changes are observed in the material regenerated from cryopreservation (Engelmann, 1997). However, most of these experiments have been performed very soon after cryopreservation on a small number of individuals, often using material still cultured *in vitro* or after a very short period of growth in vivo and they concern mainly in vitro growth characteristics, or a limited number of biochemical or molecular markers. Only in a limited number of cases (e.g. Côte et al., 2000; Engelmann, 1997; Schäfer-Menuhr et al., 1997) have plants been grown in the field for a long period allowing the

In the case of sugarcane, numerous experiments have been conducted to study the field behaviour of micropropagated plants (Feldmann et al., 1994; Flynn & Anderlini, (1990); Jackson et al., 1990; Lorenzo et al., 2001; Pena & Stay, 1997), uncovering the occurrence of rejuvenation phenomenons and of epigenetic changes. By contrast, only limited information is available concerning the stability of plants regenerated from cryopreserved material. RFLP analysis did not reveal any difference that could be attributed to cryopreservation between plants of one sugarcane variety produced from control and cryopreserved calluses (Eksomtramage et al., 1992) or cell suspensions (Chowdhury & Vasil 1993). Plants produced from control and cryopreserved shoot tips of one variety were similar as regards pattern of two isoenzymatic systems (Paulet et al., 1993) and six agronomic traits observed during

Moreover, we published data on the field performance of sugarcane plants originating from cryopreserved material (Martinez-Montero et al., 2002b). The field performance of plants produced from embryogenic calluses of one sugarcane commercial hybrid cv. CP52-43 (CP43-64 x CP38-34, Canal Point, USA) cryopreserved using the protocol developed by Martinez-Montero et al. (1998) was evaluated over a period of 27 months by observing several agronomic parameters (Fig. 7). Similar observations were carried out simultaneously for comparison on plants produced from the same callus cultures, but which were not cryopreserved and on plants of the same variety originating from classical

and 0.4 M sucrose is the most commonly employed in cryopreservation protocols (Sakai & Engelmann, 2007). The results obtained by Martinez-Montero et al., (2008) showed that modifying the composition of the loading solution improved viability according to electrolyte efflux test for cryopreserved sugarcane embryos.

This indicates the importance of carefully studying each step of the cryopreservation protocol to optimize survival. We hypothesized that increasing the number of OH groups present in the loading medium progressively decreased viability of control sugarcane somatic embryos, whereas there was an optimum in their number to achieve highest viability after cryopreservation (Fig. 6). It has been suggested that OH groups of sugars/polyalcohols replace water and interact with phospholipids forming hydrogen bonding with membrane phospholipids (Turner et al., 2001). This helps stabilizing cellular membranes during dehydration and cooling and helps maintaining membrane integrity and function through minimizing bilayer disruption and damages (Benson, 2007).

Fig. 6. Effect of the total number of OH groups of glycerol and sucrose on the cell viability of the cryopreserved sugarcane somatic embryos by droplet-vitrification procedure.

From a thermodynamic, kinetic, and structural point of view, the physico-chemical mechanism by which glycerol plus sucrose as co-solvent system can modulate the functionality of a given protein is very important (Baier & McClements, 2005; Ruan et al., 2003). However, the stabilization mechanism of these agents has been attributed to a protein preferential hydration mechanism, as proposed by Timasheff (1993) or to an osmotic stress (Parsegian et al., 1995) where, mathematically, the two mechanisms cannot be distinguished (Parsegian et al., 2000).

In a very well documented paper, Parsegian et al., (2000) indicated that there has been much confusion about the relative merits of different approaches, osmotic stress, preferential interaction (i.e. preferential hydration), and crowding, to describe the indirect effect of solutes on macromolecular conformations and reactions. The two first mechanisms (and crowding) cannot be distinguished as they are derived from the same solution theory. In the preferential hydration model proposed by Timasheff (1993), both the chemical nature and the size of the solute determine water exclusion from the protein surfaces. The osmotic stress emphasizes the role of the water that is necessarily included if solutes are excluded (Parsegian et al., 2000), dealing also with the movement of water molecules (Parsegian et al., 1995; Stanley et al., 2008).

Duan et al., (2001) observed that the hydroxyl groups present in the glucose units could contribute to protein-sugar interactions in aqueous solutions. These newly formed dipoledipole interactions could form a hydrophilic layer around the protein units and therefore increase the dispersability of the protein through protein hydration and/or alter the intramolecular interactions in such a way that folding and even dissociation may be favoured (Semenova et al., 2002).
