**5. Cryopreservation protocols for pineapple**

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 al., 2007).

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

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 several species (Engelmann, 2004).

#### **5.1 Apices**

380 Current Frontiers in Cryopreservation

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

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

calluses

Stem diameter (cm) 2,62 2,59 2,65 0,10 Stem length (m) 1,93 1,98 2,04 0,16 Stem fresh weight (kg) 1,50 1,51 1,54 0,21 Stem number per meter 9,77 9,65 10,09 2,20 Agriculture recovery (t/ha) 122,10 121,42 129,48 5,12 Juice brix (°Brix) 23,22 23,02 22,98 1,79

w/w) 20,45 20,07 19,95 1,85

w/w) 17,81 17,62 17,45 1,80

(t/ha) 21,75 21,35 22,59 1,51

Fibre percentage (%, w/w) 12,9 12,17 12,5 0,35

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* 

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

Origin of stools Typical

Buds isolated

Cryopreserved Error

Control calluses

and growth regulators in the medium.

in our experiments.

Parameter measured

Pol percentage in juice (%,

Pol percentage in cane (%,

Agro-industrial recovery

*(ANOVA).*

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 formation.

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

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 383

Fig. 9. Dissected pineapple (*Ananas comosus* L. Merrill cv. MD2) shoot tips as viewed by stereo-microscope (A, B); and by light microscopy 10x (C, D). Dissected apices type I (A, C) with apical dome (dm) and 3-4 primordial leaves (pf) and mechanical damaged apices type

In the case of pineapple, it is believe as symbol in the province of Ciego de Avila (Cuba) due to great cultivated areas dedicated to this crop. In our Institution (Bioplantas Centre) is located the unique field collection of pineapple germplasm in the country. However, this field genebank is prone to disease, or damage through natural disaster and need very high maintenance. For this reason, the cryopreservation of apices obtained from vitroplants could constitutes the most relevant strategy for long-term conservation of pineapple germplasm, since true to type and virus-free plants can be regenerated directly from cryopreserved

The successful application of the vitrification protocol for nine accessions of the *in vitro* collection at Bioplantas Centre was accomplished with the following conditions: type of shoot tip (consisted in meristematic dome area and 3-4 primordial leaves with 2,5 – 3 mm in size); 0,3 mol.L-1 sucrose preculture during 2 days; application of the loading solution (0,4 mol.L-1 sucrose + 2 mol.L-1 glycerol) during 25 min at 25°C; dehydration with plant vitrification solution number three (PVS3: 50% w/v glycerol + 50% w/v sucrose) during 7 hours at 0°C. The results per accessions expressed as percentage of recovery before (-LN) and after (+LN) cryopreservation for six apices per replicate, four replicates per treatment

and each experiment was repeated three times are showed in Table 7.

II (B, D) with one primordial leaf used as controls.

**5.1.1 Extension of vitrification protocol** 

apices (Martinez-Montero et al., 2005).

remaining freezable water upon freezing. This detrimental crystallization might be avoided by slowly freezing of the encapsulated apices, which would result in removing the remaining freezable water by means of freeze-induced dehydration. Several plant materials cryopreserved by encapsulation-dehydration technique have required slow freezing regime to achieved optimal survival (Engelmann, 2010).

In another set of experiments, our group obtained positive results again only with vitrification (Martinez-Montero et al., 2005). The best protocol comprised a 2-d preculture on semi-solid MS medium supplemented with 0.3 M sucrose, a loading treatment in liquid medium containing 0.4 M sucrose + 2 M glycerol for 25 min, and dehydration for 7h at 0°C with PVS3 before immersion into liquid nitrogen. The highest survivals of apices were: Smooth Cayenne (45%), Cabezona (33%) and Red Spanish (25%).

Fig. 8. Cryopreservation protocol established for pineapple apices using vitrification technique.

However, contrary to most vitrification reports, pineapple apices required a prolonged exposure (7 h) to the vitrification solutions (Engelmann, 2010). This result is probably due to the large size, and compact structure of the pineapple apices employed in our experiments with success (Fig. 9): the apices were around 3mm long, and comprised the apical dome tightly covered by 2-3 leaf primordial with a very thick cuticle. Long treatment durations were needed for the vitrification solution to sufficiently dehydrate these very compact structures.

Fig. 9. Dissected pineapple (*Ananas comosus* L. Merrill cv. MD2) shoot tips as viewed by stereo-microscope (A, B); and by light microscopy 10x (C, D). Dissected apices type I (A, C) with apical dome (dm) and 3-4 primordial leaves (pf) and mechanical damaged apices type II (B, D) with one primordial leaf used as controls.

### **5.1.1 Extension of vitrification protocol**

382 Current Frontiers in Cryopreservation

remaining freezable water upon freezing. This detrimental crystallization might be avoided by slowly freezing of the encapsulated apices, which would result in removing the remaining freezable water by means of freeze-induced dehydration. Several plant materials cryopreserved by encapsulation-dehydration technique have required slow freezing regime

In another set of experiments, our group obtained positive results again only with vitrification (Martinez-Montero et al., 2005). The best protocol comprised a 2-d preculture on semi-solid MS medium supplemented with 0.3 M sucrose, a loading treatment in liquid medium containing 0.4 M sucrose + 2 M glycerol for 25 min, and dehydration for 7h at 0°C with PVS3 before immersion into liquid nitrogen. The highest survivals of apices were:

Fig. 8. Cryopreservation protocol established for pineapple apices using vitrification

However, contrary to most vitrification reports, pineapple apices required a prolonged exposure (7 h) to the vitrification solutions (Engelmann, 2010). This result is probably due to the large size, and compact structure of the pineapple apices employed in our experiments with success (Fig. 9): the apices were around 3mm long, and comprised the apical dome tightly covered by 2-3 leaf primordial with a very thick cuticle. Long treatment durations were needed for the vitrification solution to sufficiently dehydrate these very compact

technique.

structures.

to achieved optimal survival (Engelmann, 2010).

Smooth Cayenne (45%), Cabezona (33%) and Red Spanish (25%).

In the case of pineapple, it is believe as symbol in the province of Ciego de Avila (Cuba) due to great cultivated areas dedicated to this crop. In our Institution (Bioplantas Centre) is located the unique field collection of pineapple germplasm in the country. However, this field genebank is prone to disease, or damage through natural disaster and need very high maintenance. For this reason, the cryopreservation of apices obtained from vitroplants could constitutes the most relevant strategy for long-term conservation of pineapple germplasm, since true to type and virus-free plants can be regenerated directly from cryopreserved apices (Martinez-Montero et al., 2005).

The successful application of the vitrification protocol for nine accessions of the *in vitro* collection at Bioplantas Centre was accomplished with the following conditions: type of shoot tip (consisted in meristematic dome area and 3-4 primordial leaves with 2,5 – 3 mm in size); 0,3 mol.L-1 sucrose preculture during 2 days; application of the loading solution (0,4 mol.L-1 sucrose + 2 mol.L-1 glycerol) during 25 min at 25°C; dehydration with plant vitrification solution number three (PVS3: 50% w/v glycerol + 50% w/v sucrose) during 7 hours at 0°C. The results per accessions expressed as percentage of recovery before (-LN) and after (+LN) cryopreservation for six apices per replicate, four replicates per treatment and each experiment was repeated three times are showed in Table 7.

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

2000).

(Gonzalez-Arnao et al., 2003).

**5.2 Embryogenic callus** 

before rapid immersion into liquid nitrogen.

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 385

tolerance to dehydration and deep cooling than a preculture in sucrose alone (Sakai et al,

Dehydration at 0°C instead of 25°C for both vitrification solutions gave better results, as previously reported for pineapple apices by our group. This low temperature reduces the toxicity of the vitrification solutions and increases the potential period of exposure (Withers & Engelmann, 1997). In all our cryopreservation experiments, dehydration with PVS3 at 0°C gave higher recovery rates compared with PVS2, even for diverse genotypes. The encapsulation-vitrification method using PVS3 gave greater survival than the vitrification procedure. Additionally, the manipulation of encapsulated apices permits handling large quantities of material that from the practical point of view is more convenient. These results corroborated that encapsulation-vitrification may also be very useful for cryopreserving desiccation-sensitive germplasm such as pineapple, that could not be successfully cryopreserved using an encapsulation-dehydration approach

The cryopreservation protocol presented here improved survival and shortened the process compared with previous protocols from our group (table 8). The optimal conditions involved the encapsulation of pineapple apices in calcium alginate (3%) beads, followed by a 2-d progressive preculture in liquid medium with 0.16 M sucrose + 0.3 M proline for 24 h, then 0.3 M sucrose + 0.3 M proline for 24h, a loading treatment for 25 min in 0.75 M sucrose + 1M glycerol solution at room temperature and dehydration for 60 min with PVS3 at 0°C

Table 8. Comparison of the encapsulation-vitrification and vitrification procedures on regrowth (%) of pineapple apices after dehydration with PVS2 or PVS3 solutions at 0°C.

The results of some studies indicated *Fusarium subglutinans* isolates cause fusariose which constitutes the most serious pineapple disease and causes losses as high as 80% of marketable pineapple fruit. It produces phytotoxins in culture that were phytotoxic on


Table 7. Effect of vitrification protocol on survival of apices from eight pineapple accessions and one related specie (*Bromelia* sp.) before (-LN) and after cryopreservation (+LN).

#### **5.1.2 Optimization of methodology for pineapple apices**

Further modifications to the procedures might be useful in order to reduce the exposure duration to PVS and achieve higher survival after cooling. Therefore, the objective was to develop a more effective cryopreservation protocol using both vitrification and encapsulation/vitrification. As previously we reported (Gonzalez-Arnao et al., 1998b; Martinez-Montero et al., 2005), pineapple apices are sensitive to sucrose and dehydration exposures. A progressive treatment increasing sucrose concentrations was effective at enhancing their tolerance to dehydration and cooling.

Proline has been shown to have a beneficial effect in several cryopreservation protocols (Luo & Reed, 1997; Rasmunsen et al., 1997; Rudolf & Crowe, 1985; Thierry et al., 1999). In our experiments we confirmed that a 2-d progressive preculture in a mixture of sucrose and proline improved the results obtained after cooling in comparison with sucrose alone. This modification in pregrowth also considerably reduced the required dehydration time in PVS. As previously reported, apices of Puerto Rico variety treated for 2 d in 0.3 M sucrose needed an extended exposure (7 h) to PVS2 at 0°C to achieve high levels of survival (65 %) after cryopreservation. However, following the same vitrification approach, higher survival (72%) was obtained using the best pretreatment in sucrose-proline and only 30 min of exposure to PVS2. As regards PVS3, we also demonstrated that 30 min or 1 h of dehydration were also enough to obtain higher survival (76 %) after cryopreservation.

The role of proline has been associated with its ability to act as source of nitrogen and carbon for reparative post-stress processes (Rabbe & Lova, 1984), to increase the nonfreezable fraction of water (Rasmussen et al, 1997), to inhibit membrane mixing and to stabilize proteins during dehydration and freezing (Rudolf & Crowe, 1985). On the other hand, the combination of chemical cryoprotectants may also improve the response of tissues to cryopreservation in comparison with the application of one chemical alone. As reported for wasabi apices, a mixture of glycerol with sucrose was more effective at enhancing their tolerance to dehydration and deep cooling than a preculture in sucrose alone (Sakai et al, 2000).

Dehydration at 0°C instead of 25°C for both vitrification solutions gave better results, as previously reported for pineapple apices by our group. This low temperature reduces the toxicity of the vitrification solutions and increases the potential period of exposure (Withers & Engelmann, 1997). In all our cryopreservation experiments, dehydration with PVS3 at 0°C gave higher recovery rates compared with PVS2, even for diverse genotypes. The encapsulation-vitrification method using PVS3 gave greater survival than the vitrification procedure. Additionally, the manipulation of encapsulated apices permits handling large quantities of material that from the practical point of view is more convenient. These results corroborated that encapsulation-vitrification may also be very useful for cryopreserving desiccation-sensitive germplasm such as pineapple, that could not be successfully cryopreserved using an encapsulation-dehydration approach (Gonzalez-Arnao et al., 2003).

The cryopreservation protocol presented here improved survival and shortened the process compared with previous protocols from our group (table 8). The optimal conditions involved the encapsulation of pineapple apices in calcium alginate (3%) beads, followed by a 2-d progressive preculture in liquid medium with 0.16 M sucrose + 0.3 M proline for 24 h, then 0.3 M sucrose + 0.3 M proline for 24h, a loading treatment for 25 min in 0.75 M sucrose + 1M glycerol solution at room temperature and dehydration for 60 min with PVS3 at 0°C before rapid immersion into liquid nitrogen.


Table 8. Comparison of the encapsulation-vitrification and vitrification procedures on regrowth (%) of pineapple apices after dehydration with PVS2 or PVS3 solutions at 0°C.

#### **5.2 Embryogenic callus**

384 Current Frontiers in Cryopreservation

Cayenne of Puerto Rico 80.2 65.5

Smooth Cayenne of Serrana 50.3 25.3

Red Spanish of Caney 45.5 12.1

Table 7. Effect of vitrification protocol on survival of apices from eight pineapple accessions

Further modifications to the procedures might be useful in order to reduce the exposure duration to PVS and achieve higher survival after cooling. Therefore, the objective was to develop a more effective cryopreservation protocol using both vitrification and encapsulation/vitrification. As previously we reported (Gonzalez-Arnao et al., 1998b; Martinez-Montero et al., 2005), pineapple apices are sensitive to sucrose and dehydration exposures. A progressive treatment increasing sucrose concentrations was effective at

Proline has been shown to have a beneficial effect in several cryopreservation protocols (Luo & Reed, 1997; Rasmunsen et al., 1997; Rudolf & Crowe, 1985; Thierry et al., 1999). In our experiments we confirmed that a 2-d progressive preculture in a mixture of sucrose and proline improved the results obtained after cooling in comparison with sucrose alone. This modification in pregrowth also considerably reduced the required dehydration time in PVS. As previously reported, apices of Puerto Rico variety treated for 2 d in 0.3 M sucrose needed an extended exposure (7 h) to PVS2 at 0°C to achieve high levels of survival (65 %) after cryopreservation. However, following the same vitrification approach, higher survival (72%) was obtained using the best pretreatment in sucrose-proline and only 30 min of exposure to PVS2. As regards PVS3, we also demonstrated that 30 min or 1 h of dehydration

The role of proline has been associated with its ability to act as source of nitrogen and carbon for reparative post-stress processes (Rabbe & Lova, 1984), to increase the nonfreezable fraction of water (Rasmussen et al, 1997), to inhibit membrane mixing and to stabilize proteins during dehydration and freezing (Rudolf & Crowe, 1985). On the other hand, the combination of chemical cryoprotectants may also improve the response of tissues to cryopreservation in comparison with the application of one chemical alone. As reported for wasabi apices, a mixture of glycerol with sucrose was more effective at enhancing their

were also enough to obtain higher survival (76 %) after cryopreservation.

and one related specie (*Bromelia* sp.) before (-LN) and after cryopreservation (+LN).

**5.1.2 Optimization of methodology for pineapple apices** 

enhancing their tolerance to dehydration and cooling.

Perolera 49.9 33.8

Cabezona 61.5 27.9 Piña Blanca of Caney 57.9 24.7

P3R5 53.1 20.0

MD2 80.1 60.2 *Bromelia* sp. 33.1 6.3

Survival (%)


The results of some studies indicated *Fusarium subglutinans* isolates cause fusariose which constitutes the most serious pineapple disease and causes losses as high as 80% of marketable pineapple fruit. It produces phytotoxins in culture that were phytotoxic on

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

the tissue culture protocol is sufficiently operational for this species.

The authors thank the Food and Agriculture Organization of the United Nations (FAO), the International Plant Genetic Resources Institute (IPGRI, actually Bioversity International) and the International Foundation for Science (IFS) for partly funding the research programme.

Anchordoguy, T.J.; Cecchini, C.A.; Crowe, J.H.; Crowe, L.M. (1991) Insights into the

Arakawa, T.; Carpenter, J.F.; Kita, Y.A.; Crowe, J.H. (1990) The basis for toxicity of certain

Aronen, T.S.; Krajnakova, J.; Haggman, H.M., Ryynannen, L.A. (1999) Genetic stability of

*Cryobiology* 28(5), (October 1991), 467-473. ISSN: 0011-2240

cryoprotective mechanism of dimethyl sulphoxide for phospholipid bilayers.

cryoprotectants: An hypothesis. *Cryobiology* 27(4), (August 1990), 401-15. ISSN:

cryopreserved embryogenic clones of white spruce (*Picea glauca*). *Plant Cell Rep.* 18,

**7. Acknowledgment** 

0011-2240

948-953. ISSN: 0721-7714

**8. References** 

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 387

terms of numbers of genotypes/varieties within a given species. With a few exceptions, vitrification-based protocols have been employed. It is also interesting to note that in many cases, different protocols can be employed for a given species and produce comparable results. Survival is generally high to very high. Regeneration is rapid and direct, and callusing is observed only in cases where the technique is not optimized. Different reasons can be mentioned to explain these positive results. The meristematic zone of apices, from which organised growth originates, is composed of a relatively homogenous population of small, actively dividing cells, with little vacuoles and a high nucleocytoplasmic ratio. These characteristics make them more susceptible to withstand desiccation than highly vacuolated and differentiated cells. As mentioned earlier, no ice formation takes place in vitrificationbased procedures, thus avoiding the extensive damage caused by ice crystals which are formed during classical procedures. The whole meristem is generally preserved when vitrification-based techniques are employed, thus allowing direct, organised regrowth. By contrast, classical procedures often lead to destruction of large zones of the meristems, and callusing only or transitory callusing is often observed before organised regrowth starts. Other reasons for the good results obtained are linked with tissue culture protocols. Many vegetatively propagated species successfully cryopreserved until now are cultivated crops, often of great commercial importance, for which cultural practices, including *in vitro* micropropagation, are well established. In addition, *in vitro* material is "synchronized" by the tissue culture, and pregrowth procedures and relatively homogenous samples in terms of size, cellular composition, physiological state and growth response are employed for freezing, thus increasing the chances of positive and uniform response to treatments. Finally, vitrification-based procedures allow using samples of relatively large size (shoot tips of 0.5 to 2–3 mm), which can regrow directly without any difficulty. Cryopreservation techniques are now operational for large-scale experimentation in an increasing number of cases. In view of the wide range of efficient and operationally simple techniques available, any vegetatively propagated species should be amenable to cryopreservation, provided that

calluses (Jin et al., 1996; Kaur et al., 1987) and the correlation between pineapple variety and the toxicity of culture filtrates suggests that filtrates could be used to screen in vitro for disease resistance (Borras et al., 2001). Therefore, the cryopreservation of pineapple calluses can provide a means of effective source when in vitro screening of germplasm for fusariose disease would be attempted. Storing calluses in liquid nitrogen could preserve their regeneration capacity and limits the risk of somaclonal variation, which increases with culture duration.

A simplified freezing protocol mentioned before for sugarcane embryogenic calluses (Martinez-Montero et al., 1998) was used for pineapple calluses of the genotypes "Smooth Cayenne" and "Perolera" (Martinez-Montero et al., 2005). For cryopreservation experiments, 15 to 20 day-old calluses, about 3 to 6 mm in diameter, were employed. Calluses were pretreated with a cryoprotective solution containing 5 to 20 % (v/v) dimethylsulfoxide (DMSO) and 0.5M sucrose for 1h at 0°C. After freezing calluses were transferred directly to recovery medium (MS medium supplemented with dicamba:BAP (2.5:0.5 mg.L-1) and citric acid (0.1 mg.L-1)). The survival, evaluated 45 days after thawing, corresponded to the percentage of calluses which had increased in size during the recovery period.

As results, the survival of calluses after pretreatment was high and similar for the two genotypes studied (Table 8). It decreased only with 20% of DMSO. After freezing in liquid nitrogen, survival was achieved with 10 and 15% DMSO only and was highest with 15% DMSO (57-67%). Re-growth of successfully cryopreserved calluses was very rapid and they increased in size during the recovery period. With this work, the application of a simplified freezing protocol achieved survival from used pineapple genotypes and this confirm that our previously simplified freezing protocol for sugarcane (Martinez-Montero et al., 1998) can be used wider to others species.


Table 9. Effect of cryoprotective solution (DMSO + sucrose) on the survival rate (%) before (- LN) and after (+LN) application of cryopreservation protocol for calluses of two pineapple genotypes. *Values represent means of 50 samples from three replicate experiments, ± SE. 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) for percentage of survival.* 
