**2.4 Somatic embryos**

In Cuba, a micropropagation protocol based on the artificial seed technology has been established for sugarcane, which uses somatic embryos produced on semi-solid medium, an alternative which allows mass multiplication of plants from elite varieties (Nieves et al., 2001; Tapia et al., 1999). However, this protocol has an important limiting factor, which lies with the necessity of safely storing somatic embryos for the long-term (Benson, 2008). Establishing a cryopreservation protocol for somatic embryos would allow solving this problem.

In this sense our group (Martinez-Montero et al., 2008) compared three vitrification-based cryopreservation techniques, viz. vitrification, encapsulation-vitrification and dropletvitrification for cryopreserving sugarcane somatic embryos. No viability was achieved using the vitrification procedure. The comparison of the recovery pattern of cryopreserved somatic embryos showed that droplet-vitrification procedure was more efficient than encapsulation-vitrification (Table 1) based on the presence of green colour in somatic embryos and on the percentage of clumps with embryos converted into plants. Moreover, the presence of callus together with converted plants was observed with the encapsulationvitrification procedure.

C 87-51 (\*) 90 70 C 266-70 (\*) 90 86 B 34104 (\*) 60 67 B 4362 (\*) 88 74 Ja 60-5 (\*) 70 24 IAC 5448 (\*) 50 38 POJ 2878 (\*) 56 60 CP 70-1133 (\*) 80 60 CP 68-1026 (\*\*) 100 64 B 69566 (\*\*) 100 91 Co 6415 (\*\*) 80 64 Co 740 (\*\*) 80 38 IAC 5118 (\*\*) 50 38 My 5514 (\*\*) 83 75 Q 90 (\*\*) 100 82


Variety Survival

Table 3. Survival of control (-LN) and cryopreserved (+LN) apices using the

cryopreservation protocol for somatic embryos would allow solving this problem.

Arnao et al. (1993, \*) and Paulet et al. (1993, \*\*).

**2.4 Somatic embryos** 

vitrification procedure.

encapsulationdehydration technique according to the protocol described by Gonzalez-

In Cuba, a micropropagation protocol based on the artificial seed technology has been established for sugarcane, which uses somatic embryos produced on semi-solid medium, an alternative which allows mass multiplication of plants from elite varieties (Nieves et al., 2001; Tapia et al., 1999). However, this protocol has an important limiting factor, which lies with the necessity of safely storing somatic embryos for the long-term (Benson, 2008). Establishing a

In this sense our group (Martinez-Montero et al., 2008) compared three vitrification-based cryopreservation techniques, viz. vitrification, encapsulation-vitrification and dropletvitrification for cryopreserving sugarcane somatic embryos. No viability was achieved using the vitrification procedure. The comparison of the recovery pattern of cryopreserved somatic embryos showed that droplet-vitrification procedure was more efficient than encapsulation-vitrification (Table 1) based on the presence of green colour in somatic embryos and on the percentage of clumps with embryos converted into plants. Moreover, the presence of callus together with converted plants was observed with the encapsulation-


Table 4. Effect of the cryopreservation protocol on the recovery pattern of sugarcane somatic embryos (SE).

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 encapsulationvitrification (Fig. 3D).

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).

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

techniques yet.

(Guy, 1999).

after freezing.

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 371

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

**3.1 Effect of cryopreservation on the structural and functional integrity of cell** 

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

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

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

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

**membranes of sugarcane embryogenic callus** 

stressing events (Harding, 1999; Verleysen et al., 2004).

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 (Engelmann, 2011).

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 impossible and alternative options had to be sought.

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 encapsulationvitrification.

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

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 become vitrified on direct exposure to liquid nitrogen.
