**4. Stability assessment**

376 Current Frontiers in Cryopreservation

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

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

Fig. 6. Effect of the total number of OH groups of glycerol and sucrose on the cell viability of

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

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

the cryopreserved sugarcane somatic embryos by droplet-vitrification procedure.

(Parsegian et al., 2000).

function through minimizing bilayer disruption and damages (Benson, 2007).

electrolyte efflux test for cryopreserved sugarcane embryos.

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 assessment of agronomic characteristics.

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

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 macropropagation.

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

*letter are not statistically different (ANOVA, Tukey test, p < 0,05).*

after 15 months of field growth of the first ratoon.

Parameter measured

Parameter measured

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 379

Stem diameter (cm) 1,51 b 1,45 b 1,82 a 0,12 Stem length (m) 0,57 b 0,49 b 0,85 a 0,10 Number of stems per meter 5,20 a 5,45 a 4,22 b 0,35 Table 4. Evaluation of several agronomic parameters after six months of field growth of sugarcane stools originating from cryopreserved calluses, control (non-cryopreserved) calluses and buds isolated from macropropagated plants. *Data in rows followed by the same* 

No significant difference between treatments was observed for any of the parameters studied after 12 months of stool field growth and 15 months of field growth of the first ratoon (Tables 5 and 6). This study has demonstrated that the differences observed for several agronomic characters between stools originating from cryopreserved and control calluses, and macropropagated material after 6 months of field growth disappeared progressively with time, as no differences could be uncovered in stools after 12 months nor

Only very few published reports on this topic deal with comparable number and duration components. No differences have been noted in the vegetative and floral development of several hundreds of palms regenerated from control and cryopreserved oil palm embryogenic cultures, but no detailed account of the observations made has been published (Engelmann, 1997). The most comprehensive study is the comparison of the field behaviour of banana plants regenerated from control and cryopreserved cell suspensions (Côte et al., 2000), which showed that two out of the eleven descriptors analyzed differed between control and cryopreserved material during the first culture cycle but that, similarly to our

calluses

Pol percentage in juice (%, w/w) 19,17 18,37 19,35 1,93 Fibre percentage (%, w/w) 11,55 11,23 11,52 0,43 Pol percentage in cane (%, w/w) 16,95 16,30 17,12 1,90 Agro-industrial recovery (t/ha) 18,96 19,06 19,81 1,51

sugarcane stools originating from cryopreserved calluses, control (non-cryopreserved) calluses and buds isolated from macropropagated plants. *No statistical differences were found (ANOVA).* 

Table 5. Evaluation of several agronomic parameters after 12 months of field growth of

Stem diameter (cm) 2,58 2,51 2,66 0,09 Stem length (m) 1,85 1,90 1,70 0,11 Stem fresh weight (kg) 1,37 1,33 1,49 0,09 Stem number per meter 9,80 10,55 9,32 1,50 Agriculture recovery (t/ha) 111,88 116,93 115,72 2,62 Juice brix (°Brix) 22,07 21,22 24,69 1,87

observations, these differences disappeared during the second culture cycle.

Origin of stools Typical

Origin of stools Typical

Buds isolated

Cryopreserved Error

Control calluses

Error Cryopreserved calluses Control calluses Buds isolated

Fig. 7. Regenerated plants from cryopreserved embryogenic sugarcane callus a) during acclimatization (42 days); b, c) after 12 months in the field of the stool; d) after 15 months in the field of the first ratoon.

Treatments were distributed following a randomised block experimental design including four repetitions per treatment. Experimental plots were 7.5 m long with 5 rows each. Intrarow spacing was 0.5 m. The commercial hybrid C266-70 (Co281 x POJ2878, INICA, Cuba) was planted as experiment border. Fertilization at the time of planting included 75 kg/ha urea; 50 kg/ha P2O5 and 50 kg/ha K2O. Additionally, 75 kg/ha urea were supplied 3 months after planting. The following measurements were performed on 100 plants originating from cryopreserved calluses, control calluses and macropropagated plants:


The results of the evaluation of field grown sugarcane plants after different periods showed significant differences between treatments only during the first six months of field growth of sugarcane stools (Table 4). Stems produced from *in vitro* cultured materials, irrespective of their cryopreservation status, had a smaller diameter and a shorter height than those produced from macropropagated buds. These differences disappeared during the course of the experiment as they were not observed anymore after 12 months of stool field growth.

Fig. 7. Regenerated plants from cryopreserved embryogenic sugarcane callus a) during acclimatization (42 days); b, c) after 12 months in the field of the stool; d) after 15 months in

Treatments were distributed following a randomised block experimental design including four repetitions per treatment. Experimental plots were 7.5 m long with 5 rows each. Intrarow spacing was 0.5 m. The commercial hybrid C266-70 (Co281 x POJ2878, INICA, Cuba) was planted as experiment border. Fertilization at the time of planting included 75 kg/ha urea; 50 kg/ha P2O5 and 50 kg/ha K2O. Additionally, 75 kg/ha urea were supplied 3 months after planting. The following measurements were performed on 100 plants originating from cryopreserved calluses, control calluses and macropropagated plants:

After 6 months of stool field growth: number of stems per clump (i.e. number of suckers

 After 12 months of stool field growth: number of stems per clump; stem diameter (cm); stem length (m); fibre percentage (w/w); juice brix (i.e. mass of sugar (g dry matter) per 100 g of juice, expressed in Brix degree) ; pol (total sugar content) percentage in juice;

 After cutting of stools and 15 months of field growth of the first ratoon: number of stems per clump; stem diameter (cm); stem length (m); single stem fresh mass (kg); fibre percentage (w/w); juice brix; pol percentage in juice; pol percentage in cane; juice

The results of the evaluation of field grown sugarcane plants after different periods showed significant differences between treatments only during the first six months of field growth of sugarcane stools (Table 4). Stems produced from *in vitro* cultured materials, irrespective of their cryopreservation status, had a smaller diameter and a shorter height than those produced from macropropagated buds. These differences disappeared during the course of the experiment as they were not observed anymore after 12 months of stool field growth.

produced from the original plant); stem diameter (cm); stem length (m).

pol percentage in cane; juice apparent purity tons of pol/ha.

apparent purity; tons of pol/ha.

the field of the first ratoon.


Table 4. Evaluation of several agronomic parameters after six months of field growth of sugarcane stools originating from cryopreserved calluses, control (non-cryopreserved) calluses and buds isolated from macropropagated plants. *Data in rows followed by the same letter are not statistically different (ANOVA, Tukey test, p < 0,05).*

No significant difference between treatments was observed for any of the parameters studied after 12 months of stool field growth and 15 months of field growth of the first ratoon (Tables 5 and 6). This study has demonstrated that the differences observed for several agronomic characters between stools originating from cryopreserved and control calluses, and macropropagated material after 6 months of field growth disappeared progressively with time, as no differences could be uncovered in stools after 12 months nor after 15 months of field growth of the first ratoon.

Only very few published reports on this topic deal with comparable number and duration components. No differences have been noted in the vegetative and floral development of several hundreds of palms regenerated from control and cryopreserved oil palm embryogenic cultures, but no detailed account of the observations made has been published (Engelmann, 1997). The most comprehensive study is the comparison of the field behaviour of banana plants regenerated from control and cryopreserved cell suspensions (Côte et al., 2000), which showed that two out of the eleven descriptors analyzed differed between control and cryopreserved material during the first culture cycle but that, similarly to our observations, these differences disappeared during the second culture cycle.


Table 5. Evaluation of several agronomic parameters after 12 months of field growth of sugarcane stools originating from cryopreserved calluses, control (non-cryopreserved) calluses and buds isolated from macropropagated plants. *No statistical differences were found (ANOVA).* 

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

**5. Cryopreservation protocols for pineapple** 

usually not longer than 2 h (Thinh et al., 1999).

several species (Engelmann, 2004).

embryogenesis.

al., 2007).

**5.1 Apices** 

formation.

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 381

Bioplantas for mass production of *in vitro* sugarcane plants by means of somatic

Pineapple is vegetatively propagated and crosses between varieties produce botanical seeds. However, these seeds are highly heterozygous and therefore of limited interest for the conservation of specific gene combinations. Cryopreservation of apices is the most relevant strategy for long-term conservation of vegetatively propagated crops, since true to type; virus free plants can be regenerated directly from cryopreserved apices (Lynch et

Vitrification and encapsulation-dehydration techniques have been widely applied for successfully cryopreserve apices of a large number of different crops which do not require sophisticated equipment for freezing and produce high recovery rates with a wide range of materials (Engelmann, 2010, 2011). Most vitrification protocols use a loading treatment and a stringently timed dehydration with PVS (Benson, 2008b). This two-step procedure has allowed tissues to be more tolerant to osmotic stress and to resist the chemical toxicity induced by the highly concentrated cryoprotective solutions. Exposure duration to PVS is

The encapsulation-dehydration method for cryopreservation is based on the fact that encapsulation protects the explants and preculture in medium enriched with osmoticum makes them tolerant to air drying (Fabre & Dereuddre, 1990). Preculture in 0.75 M sucrose and desiccation to about 25% water content in beads (fresh weight basis) are the most common conditions used (Gonzalez-Arnao et al., 1996). The application of vitrification solutions to dehydrate encapsulated cells or shoot tips has also been successfully applied to

The first successful result related with the cryopreservation of pineapple (*Ananas comosus*) apices was reported by our group in Cuba (Gonzalez-Arnao et al., 1998b). The encapsulation and vitrification techniques were experimented for freezing apices of pineapple *in vitro* plantlets. Positive results were achieved using vitrification only (Fig. 8). Optimal conditions included a 2 day preculture of apices on medium supplemented with 0.3M sucrose, loading treatment for 25 min in medium with 0.75M sucrose + 1M glycerol and dehydration with PVS2 vitrification solution at 0°C for 7 h before rapid immersion in liquid nitrogen. This method resulted in ranged survival (25-65%) depending of the genotypes. Recovery of cryopreserved apices took place directly, without transitory callus

The negative results after cryopreservation of pineapple apices by encapsulationdehydration technique can be related to the high sensitivity of pineapple apices to sucrose and dehydration. Pregrowth in media with sucrose concentrations higher than 0.5M was detrimental to survival and a prolonged treatment in 0.5M sucrose was required to improve survival after desiccation, but it was not sufficiently to obtain survival of apices after freezing. The viability loss observed after freezing may be due to the crystallization of

An interesting result in our study is that plants regenerated from control and cryopreserved calluses displayed the same differences in comparison with those originating from macropropagated material. These differences are therefore not induced by cryopreservation but are due to the fact that both groups of plants originate from in vitro cultured material. It is indeed a well known phenomenon that tissue culture induces temporary changes in the behaviour of *in vitro* cultured plants during their early in vivo growth phase (Swartz 1991). Such changes can be induced by *in vitro* culture conditions including notably low light intensity, high humidity, limited gas exchanges, presence of high sucrose concentrations and growth regulators in the medium.

In the case of sugarcane, changes in the field behaviour of plants have been frequently observed after *in vitro* culture. Several authors have reported an increase in the number of new stems per clump (Flynn & Anderlini, 1990; Jackson, 1990; Perez et al., 1999), which generally induces a reduction in the stem diameter and mass. Peña & Stay (1997) stated that, with sugarcane *in vitro* culture stimulated growth and vigour, induced rejuvenation and generally improved agricultural performance. Many authors (Jimenez et al., 1991; Lorenzo et al., 2001; Lourens. & Martin 1987; Taylor et al., 1992) indicate that such differences disappear during the course of field growth and the first clonal multiplication, as observed in our experiments.


Table 6. Evaluation of several agronomic parameters after 15 months of field growth of the first sugarcane ratoon originating from cryopreserved calluses, control (non-cryopreserved) calluses and buds isolated from macropropagated plants. *No statistical differences were found (ANOVA).*

In conclusion, the results obtained in our study validate the cryopreservation protocol developed by Martinez-Montero et al. (1998) for embryogenic calluses. Cryopreservation of embryogenic calluses will thus be incorporated in the scheme established by the Centro de Bioplantas for mass production of *in vitro* sugarcane plants by means of somatic embryogenesis.
