Comparing encapsulation-dehydration and droplet-vitrification for cryopreservation of sugarcane (Saccharum spp.) shoot tips

Scientia Horticulturae 130 (2011) 320–324

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Comparing encapsulation-dehydration and droplet-vitrification for cryopreservation of sugarcane (Saccharum spp.) shoot tips Giuseppe Barraco a,b,∗ , Isabelle Sylvestre a , Florent Engelmann a,c a

IRD, UMR DIADE, 911 avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, France Università degli Studi di Palermo, Facoltà di Agraria, Viale delle Scienze ed. 4, 90128 Palermo, Italy c Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy b

a r t i c l e

i n f o

Article history: Received 4 May 2011 Received in revised form 29 June 2011 Accepted 1 July 2011 Keywords: Sugarcane Cryopreservation Encapsulation-dehydration Droplet-vitrification

a b s t r a c t In this study, in vitro shoot tips of two sugarcane clones were successfully cryopreserved using encapsulation-dehydration and droplet-vitrification with two vitrification solutions, PVS2 and PVS3. For both clones, encapsulation-dehydration induced significantly higher recovery, reaching 60% for clone H70-144 and 53% for clone CP68-1026, compared with droplet-vitrification in which recovery was 33–37% for clone H70-144 and 20–27% for clone CP68-1026. Optimal conditions included preculture of encapsulated shoot apices for 24 h in liquid medium with 0.75 M sucrose and dehydration with silica gel to 20% moisture content (fresh weight basis) before direct immersion in liquid nitrogen. With both protocols employed, regrowth of cryopreserved samples, as followed by visual observation, was always rapid and direct. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Cryopreservation [liquid nitrogen (LN), −196 ◦ C] is the only technique currently available for safe and cost-effective long-term conservation of genetic resources of vegetatively propagated plant species such as sugarcane, which cannot be stored in the form of dehydrated seeds in seedbanks. At such ultra-low temperature, all metabolic activities are virtually stopped, thereby enabling theoretically unlimited storage durations (Engelmann, 2004). Cryopreserved material is sheltered from biotic and abiotic stresses that can affect field collections and cause serious damage, as illustrated with the US sugarcane germplasm collection, in which 61% of the clones were lost between 1957 and 1977, mostly due to pests and diseases (Berding and Roach, 1987). Furthermore, LN storage requires limited maintenance and reduced space. Compared to traditional in vitro conservation (normal or slow growth culture), it suppresses the risk of occurrence of somaclonal variations and of contamination during transfers (Ashmore, 1997). In the case of sugarcane, cryopreservation protocols have already been developed for cell suspensions (Ulrich et al., 1984; Gnanapragasam and Vasil, 1990), embryogenic calluses

∗ Corresponding author at: IRD, UMR DIADE, 911 avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, France. Tel.: +33 0467416224; fax: +33 0467416222. E-mail address: giu.barra [email protected] (G. Barraco). 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.07.003

(Eksomtramage et al., 1992; Martinez-Montero et al., 1998), somatic embryos (Martinez-Montero et al., 2008) and shoot tips (Gonzalez-Arnao et al., 1993, 1999; Paulet et al., 1993). With shoot tips, encapsulation-dehydration (E-D), a technique first developed by Dereuddre et al. (1990) was successfully applied to 15 sugarcane varieties (Gonzalez-Arnao, 1996). Storage of apices sampled from in vitro plantlets appears as the ideal procedure for long-term conservation of sugarcane genetic resources due to the ability of such explants to guarantee genetic stability of regenerated plant material (Gonzalez-Arnao et al., 2008). In recent years, new cryopreservation protocols based on vitrification of intracellular solutes have been developed, including encapsulation-vitrification and droplet-vitrification (D-V) (Panis et al., 2005). The D-V protocol, which has proved its efficiency with various plant species, has not been tested yet with sugarcane. We therefore decided to compare the E-D protocol already developed for cryopreservation of sugarcane shoot tips (GonzalezArnao et al., 1993) with the D-V protocol established by Panis et al. (2005) for cryopreservation of banana shoot tips. In a D-V protocol, the explants are dehydrated osmotically with a loading solution, then with a vitrification solution (VS), put in 5–10 ␮L droplets of VS placed on aluminium strips, which are rapidly immersed in LN (Sakai and Engelmann, 2007). Rewarming is performed by plunging the aluminium foils in liquid medium containing 0.8–1.2 M sucrose. Samples are retrieved and placed on recovery medium. The main advantage of this technique is the possibility of achieving very

G. Barraco et al. / Scientia Horticulturae 130 (2011) 320–324

high cooling/warming rates, due to the very small volume of VS in which explants are placed and the direct contact between explants and LN during cooling and between explants and the unloading solution during rewarming, thus allowing higher recovery compared with “standard” vitrification protocols (Kim et al., 2006). In our experiments, we compared E-D and D-V using shoot apices of two commercial clones of sugarcane. In the D-V protocol, osmotic dehydration was performed with the two most generally used VSs, PVS2 (Sakai et al., 1990) and PVS3 (Nishizawa et al., 1993). 2. Materials and methods 2.1. Plant materials In vitro plantlets of two sugarcane US commercial clones were used: H70-144, from Hawaii, and CP68-1026, from Canal Point (Florida). Mother-plants were cultivated on semi-solid MS basal medium (Murashige and Skoog, 1962) with 60 g L−1 sucrose and 7 g L−1 agar at pH 5.6. Plantlets were placed at 27 ± 1 ◦ C, under a 12 h d−1 photoperiod and a light intensity of 50 ␮ mol m−2 s−1 . Mother plants were subcultured monthly. For cryopreservation experiments, we used explants of a size of 0.5–1.0 mm, consisting of the apical dome, one or two leaf primordia and a basal part (Fig. 1a). Apices were dissected under the binocular microscope from in vitro plantlets, 30–40 days after their last transfer. After dissection, shoot tips were maintained for 16 h in the dark on the medium used for culture of mother-plants to minimize the dissection stress.

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unloading solution containing 1.2 M sucrose at room temperature (Panis et al., 2005). Explants were kept in the unloading solution for 20 min, then transferred on recovery medium. Recovery was performed as in the case of E-D described above. 2.4. Assessment of survival and recovery percentage and statistical analyses The effects of the different treatments were evaluated by measuring survival and recovery percentages of control and cryopreserved shoot tips. Survival, corresponding to the presence of living tissues and to the observation of any regrowth pattern, was recorded 10 days after cryopreservation; after this duration dead explants normally turned white. Recovery (Fig. 1b), corresponding to the production of normal shoots from treated explants, was observed after 40 days. Measuring survival was important as it allowed rapid evaluation of the experiments performed. Comparing survival with recovery percentages was also useful as it showed the range of potential improvement of the cryopreservation procedures. Explants were observed every 10 days using a binocular microscope to assess recovery features and the absence of callus production. Each treatment was performed with three replicates of 10 explants. The results, presented as percentage of surviving/recovering samples over the total number of explants treated per experimental condition, were analyzed using analysis of variance, following arcsin transformation, with Duncan’s multiple range test (DMRT), using the SPSS 14.0 software. 3. Results

2.2. Encapsulation-dehydration 3.1. Encapsulation-dehydration Encapsulation-dehydration was performed according to Gonzalez-Arnao et al. (1993). Excised apices were encapsulated in 3% calcium alginate beads (diameter 4–5 mm) and precultured for 24 h in liquid MS medium 0.75 M sucrose with constant shaking (90 rpm). Encapsulated apices were dehydrated to moisture contents (MC) between 35 and 20% (fresh weight basis) by placing them in hermetically closed containers (10 beads per container) filled with 80 g silica gel. Beads were then placed in 2 mL polypropylene cryovials (10 beads per vial) and directly plunged in LN where they were kept for a minimum of 15 min. Cryopreserved beads were rewarmed by placing them directly on recovery medium in Petri dishes. This procedure ensured rapid rewarming due the direct contact between the beads and the ambient air at room temperature. Recovery medium consisted of semi-solid MS medium with 30 g L−1 sucrose, 0.2 mg L−1 6-benzylaminopurine (BAP), 0.1 mg L−1 kinetin (KIN), 7 g L−1 agar and 1 g L−1 Plant Preservative Mixture (PPM, Plant Cell Technology, Washington, USA) to avoid proliferation of endophytic bacteria. After cryopreservation, shoot tips were kept in the dark for 7 days to avoid photooxidation damage, and then transferred under standard culture conditions. 2.3. Droplet-vitrification Excised apices were treated for 20 min with a loading solution containing 2 M glycerol and 0.4 M sucrose (Nishizawa et al., 1993), then dehydrated with PVS2 (30% glycerol, 15% DMSO, 15% EG and 13.7% sucrose, w/v; Sakai et al., 1990) at 0 ◦ C for 20–80 min or with PVS3 (50% glycerol and 50% sucrose, w/v; Nishizawa et al., 1993) at room temperature for 20–100 min. Five min before the end of dehydration with the VSs employed, apices were transferred to 10 ␮L drops of VS (10 explants per drop) placed on aluminium strips and directly plunged in LN. After 15 min storage in LN, apices were quickly rewarmed by plunging the aluminium strips in 10 mL

With E-D, survival and recovery of non-cryopreserved shoot tips of clone H70-144 decreased progressively, in line with decreasing bead MCs, while for CP68-1026 survival did not change significantly with changes in MC (Table 1). No large differences were recorded between survival and recovery percentages since almost all surviving explants were able to recover growth ability. After cryopreservation, survival and recovery of shoot tips of both clones gradually increased, in line with decreasing bead MC, reaching a maximum at 20–22% MC; at such MC, recovery was 40–60% for H70-144 and 37–53% for CP68-1026 (Fig. 1b). 3.2. Droplet-vitrification With D-V, survival and recovery of control samples of both clones dehydrated with PVS2 decreased gradually, in line with increasing durations of exposure to the VS (Table 2). Most surviving apices were able to produce shoots after transfer to recovery medium. After cryopreservation, no survival was achieved without PVS2 treatment. When dehydrating apices with PVS2, the best results were obtained after 20 and 40 min for clone H70-144; these treatments enabled recovery between 33 and 37%. For clone CP68-1026 survival was not significantly different after all treatment durations tested. The highest recovery, occurred between 10 and 20%, was obtained after 40–80 min dehydration. Treating samples with PVS2, survival and recovery of CP68-1026 apices were generally lower compared with H70-144. After treatment with PVS3, survival and recovery of apices of both clones decreased progressively in line with increasing treatment durations (Table 3). After cryopreservation, the highest survival and recovery percentages of apices of clone H70-144 were obtained after 20–40 min treatment durations, reaching 17–33%. Longer exposures to VS induced lower recovery percentages and slower shoot production.

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Fig. 1. (a) Dissected shoot tip consisting of the meristematic dome, a leaf primordium and a basal part (bar 0.5 mm). (b) Recovery of shoot tip of clone CP68-1026 cryopreserved by E-D after dehydration to 20% MC (bar 10.0 mm). (c) Plants originated by cryopreserved shoot tips of CP68-1026 after 4 months cultivation (bar 20.0 mm). Table 1 Effect of bead MC (%, fresh weight basis) on survival and recovery (%) of control (−LN) and cryopreserved (+LN) shoot tips of sugarcane clones H70-144 and CP68-1026 treated with the E-D protocol. Moisture content (%)

H70-144

CP68-1026

−LN

75 35 30 25 22 20

−LN

+LN

+LN

Survival (%)

Recovery (%)

Survival (%)

Recovery (%)

Survival (%)

Recovery (%)

Survival (%)

Recovery (%)

94 a 93 a 87 b 77 bc 77 bc 50 c

94 a 90 ab 80 b 73 bc 60 cd 47 d

0c 0c 10 c 27 b 47 ab 60 a

0c 0c 10 c 23 b 40 ab 60 a

79 a 87 a 86 a 86 a 90 a 83 a

79 a 87 a 86 a 80 a 80 a 83 a

0c 0c 6b 10 b 43 a 53 a

0c 0c 6b 7b 37 a 53 a

Values followed by the same letter in the same column are not significantly different at the 0.05 probability level.

For CP68-1026, the highest survival percentages were obtained after treatment for 20–60 min. Recovery was not significantly different whatever the treatment duration tested; it varied between 7 and 27%. 3.3. Comparison between encapsulation-dehydration and droplet-vitrification The best results obtained with the D-V and E-D protocols employed in our experiments are presented in Table 4. For both clones, E-D enabled the highest recovery percentages after cryopreservation (60% for H70-144 and 53% for CP68-1026).

With clone H70-144, survival and recovery of noncryopreserved apices were significantly higher using D-V, compared with E-D. By contrast, survival of cryopreserved apices was not significantly different between the three protocols but E-D led to significantly higher recovery. With clone CP68-1026, survival and recovery of non-cryopreserved controls were higher with E-D and PVS3 D-V, compared with PVS2 D-V. After cryopreservation, the highest survival (53%) was obtained with E-D, even though survival using PVS3 D-V (40%) was not significantly different. E-D also induced significantly higher recovery compared with D-V. With E-D, all surviving apices resumed growth after cryopreservation and this was also the case for most apices treated

Table 2 Effect of duration of PVS2 dehydration on survival and recovery (%) of control (−LN) and cryopreserved (+LN) shoot tips of sugarcane clones H70-144 and CP68-1026 treated with the D-V protocol. PVS2 dehydration (min)

H70-144

CP68-1026

−LN

0 20 40 60 80

−LN

+LN

+LN

Survival (%)

Recovery (%)

Survival (%)

Recovery (%)

Survival (%)

Recovery (%)

Survival (%)

Recovery (%)

87 a 77 ab 60 bc 40 cd 23 d

80 a 67 ab 50 bc 33 cd 17 d

0c 43 a 47 a 27 b 23 b

0c 37 a 33 a 23 b 13 c

77 a 63 ab 50 bc 33 cd 27 d

73 a 53 b 50 b 27 c 23 c

0b 17 a 23 a 17 a 17 a

0c 7b 20 a 10 ab 10 ab

Values followed by the same letter in the same column are not significantly different at the 0.05 probability level.

G. Barraco et al. / Scientia Horticulturae 130 (2011) 320–324

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Table 3 Effect of duration of PVS3 dehydration on survival and recovery (%) of control (−LN) and cryopreserved (+LN) shoot tips of sugarcane clones H70-144 and CP68-1026 treated with the D-V protocol. PVS3 dehydration (min)

H70-144

CP68-1026

−LN

0 20 40 60 80 100

−LN

+LN

+LN

Survival (%)

Recovery (%)

Survival (%)

Recovery (%)

Survival (%)

Recovery (%)

Survival (%)

Recovery (%)

80 a 83 a 50 b 53 b 43 bc 22 c

63 a 73 a 30 b 37 b 30 b 9c

0c 53 a 37 ab 27 b 20 b 3c

0c 33 a 17 ab 10 bc 10 ac 3 bc

97 a 87 ab 60 bc 53 bc 43 c 27 c

93 a 87 a 37 b 47 b 37 b 20 b

0d 40 a 43 a 30 ab 20 b 7c

0b 27 a 27 a 17 a 17 a 7 ab

Values followed by the same letter in the same column are not significantly different at the 0.05 probability level.

Table 4 Optimal survival and recovery (%) of control (−LN) and cryopreserved (+LN) shoot tips of sugarcane clones H70-144 and CP68-1026 obtained using the three cryopreservation protocols tested, E-D, PVS2 D-V and PVS3 D-V. Protocol

H70-144

CP68-1026

−LN

D-V PVS2 D-V PVS3 E-D

−LN

+LN

+LN

Survival (%)

Recovery (%)

Survival (%)

Recovery (%)

Survival (%)

Recovery (%)

Survival (%)

Recovery (%)

77 a 83 a 50 b

67 ab 73 a 47 b

43 a 53 a 60 a

37 b 33 b 60 a

50 b 87 a 83 a

50 b 87 a 83 a

23 b 40 ab 53 a

20 b 27 b 53 a

Values followed by the same letter in the same column are not significantly different at the 0.05 probability level.

with PVS2 D-V. By contrast, only a part of the apices cryopreserved using the PVS3 D-V resumed shoot production as shown by the decrease between survival and recovery percentages. In all experimental conditions tested, shoot regrowth, assessed by observation under a binocular microscope, was direct, without callus formation. 4. Discussion In vitrification-based cryopreservation protocols, the highly concentrated intracellular solutes solidify during the rapid cooling procedure, thus reaching an amorphous glassy state that protects cells against structural damage. Vitrification is obtained by increasing viscosity of intracellular solutes through physical and/or osmotic dehydration before cryopreservation and by rapid immersion of samples in LN (Gonzalez-Arnao et al., 2008). In this paper, we investigated the efficiency of two vitrificationbased protocols, E-D and D-V for cryopreserving apices of two sugarcane clones. Recovery of cryopreserved material was obtained with both protocols, demonstrating the possibility of using both methods for LN storage of sugarcane apices. The differences between survival and recovery data were small, indicating the usefulness of this parameter for the early evaluation of the efficiency of cryopreservation protocols for sugarcane apices. E-D is a technique based on the synthetic seed technology, which was experimented with plant material by Dereuddre et al. (1990), then successfully applied to numerous temperate and tropical species (Gonzalez-Arnao and Engelmann, 2006) including sugarcane (Gonzalez-Arnao et al., 1993, 1999; Paulet et al., 1993). E-D offers many advantages in terms protocol simplification and manipulation of explants (Engelmann et al., 2008). In our research the highest recovery percentage using the E-D protocol was 53–60%, which was comparable to the 62% recovery obtained by Gonzalez-Arnao et al. (1993), Paulet et al. (1993), GonzalezArnao (1996) and Gonzalez-Arnao et al. (1999) using 15 sugarcane accessions. The optimization of the pretreatment procedure and of sample residual MC are key factors for successful application of ED (Gonzalez-Benito et al., 1998). In our study, the highest recovery was obtained when dehydrating the shoot tips to 20–22% MC. These

results are in accordance with those of Gonzalez-Arnao (1996) who showed that dehydration to 22% MC ensured the highest survival (57%) after cryopreservation of shoot tips of five sugarcane commercial clones. Our experiments showed for the first time that sugarcane apices could be cryopreserved using D-V. We compared the most widely used VSs, PVS2 and PVS3, which were employed for different durations. PVS2 is characterized by its high chemical toxicity, because it includes the permeating cryoprotectants DMSO and ethylene glycol, while PVS3, which includes the non-penetrating cryoprotectants sucrose and glycerol, can be toxic because of the high osmotic pressure it exerts on plant cells (Kim et al., 2009). In our experiments, apices of the two clones tested showed relatively high tolerance to dehydration with both VSs (PVS2 and PVS3); however, recovery of cryopreserved samples was low, reaching a maximum of 20–37%. This is in contrast with what is observed with numerous temperate and tropical plants species, for which D-V produces very high recovery (Sakai and Engelmann, 2007). In experiments carried out by Panis et al. (2005) on 56 accessions of banana shoot tips cryopreserved using D-V, recovery was between 50 and 95%. When applying D-V to shoot tips of 18 taro cultivars, recovery was 89% (Sant et al., 2008); it reached 73% with Chrysanthemum shoot tips and 95% with garlic bulbil primordia (Kim et al., 2009). This clearly shows the necessity of additional studies aiming at optimizing the different steps of the D-V protocol for sugarcane shoot tips. With both techniques, regrowth of cryopreserved apices, observed using a binocular microscope, was rapid and direct, which is a good indication of genetic stability of cryopreserved material, as observed with numerous plant species (Harding, 2004). 5. Conclusion In conclusion, this study showed the possibility of cryopreserving sugarcane apices using E-D, which had already been successfully used by various researchers (Gonzalez-Arnao et al., 1993, 1999; Paulet et al., 1993) and D-V, which was applied to sugarcane for the first time. In our experiments, the already optimized E-D was clearly superior, leading to higher recovery percentages. However, additional studies aiming at optimizing the different

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steps of the D-V protocol may increase survival and recovery of shoot tips cryopreserved with this technique. Moreover, our study was performed with two clones only. Our results should thus be confirmed using additional clones of sugarcane. Acknowledgements The assistance of Marie-Jo Darroussat and Jean-Claude Girard (CIRAD Baillarguet, France) for providing the experimental material is gratefully acknowledged. This work was partly supported by ARCAD, a flagship program of Agropolis Fondation (I. Sylvestre). References Ashmore, S.E., 1997. Status Report on the Development and Application of In vitro Techniques for the Conservation and Use of Plant Genetic Resources. IPGRI, Rome. Berding, N., Roach, B.T., 1987. Germplasm collection, maintenance and use. In: Heinz, D.J. (Ed.), Sugarcane Improvement Through Breeding. Elsevier, Amsterdam, pp. 143–210. Dereuddre, J., Scottez, C., Arnaud, Y., Duron, M., 1990. Resistance of alginate-coated axillary shoot tips of pear tree (Pyrus communis L. Cv Beurré Hardy) in vitro plantlets to dehydration and subsequent freezing in liquid nitrogen: effects of previous cold hardiness. C. R. Acad. Sci. III 310, 317–323. Eksomtramage, T., Paulet, F., Guiderdoni, E., Glaszmann, J.C., Engelmann, F., 1992. Development of cryopreservation for embryogenic calli of a commercial hybrid of sugarcane (Saccharum sp.) and application to different varieties. CryoLetters 13, 239–252. Engelmann, F., 2004. Plant cryopreservation: progress and prospects. In Vitro Cell Dev. Biol. Plant 40, 427–433. Engelmann, F., Gonzalez-Arnao, M.T., Wu, Y., Escobar, R., 2008. The development of encapsulation-dehydration. In: Reed, B.M. (Ed.), Plant Cryopreservation: A Practical Guide. Springer Science and Business Media LLC, New York, pp. 33–58. Gnanapragasam, S., Vasil, K.I., 1990. Plant regeneration from cryopreserved embryogenic cell suspension of commercial sugarcane hybrid (Saccharum sp.). Plant Cell Rep. 9, 419–423. Gonzalez-Arnao, M.T., Engelmann, F., Huet, C., Urra, C., 1993. Cryopreservation of encapsulated apices of sugarcane: effect of freezing procedure and histology. CryoLetters 14, 303–308. Gonzalez-Arnao, M.T., 1996. Desarollo de una técnica para la crioconservacion de ˜ azucar. Tesis de Doctorado. CNIC, Cuba. meristemos apicales de cana

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