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Scientia Horticulturae 130 (2011) 320–324

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l homepage: www.e lsev ier .com/ locate /sc ihor t i

omparing encapsulation-dehydration and droplet-vitrification forryopreservation of sugarcane (Saccharum spp.) shoot tips

iuseppe Barracoa,b,∗, Isabelle Sylvestrea, Florent Engelmanna,c

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

r t i c l e i n f o

rticle history:eceived 4 May 2011eceived in revised form 29 June 2011ccepted 1 July 2011

a b s t r a c t

In this study, in vitro shoot tips of two sugarcane clones were successfully cryopreserved usingencapsulation-dehydration and droplet-vitrification with two vitrification solutions, PVS2 and PVS3. Forboth clones, encapsulation-dehydration induced significantly higher recovery, reaching 60% for clone

eywords:ugarcaneryopreservationncapsulation-dehydrationroplet-vitrification

H70-144 and 53% for clone CP68-1026, compared with droplet-vitrification in which recovery was33–37% for clone H70-144 and 20–27% for clone CP68-1026. Optimal conditions included precultureof encapsulated shoot apices for 24 h in liquid medium with 0.75 M sucrose and dehydration with silicagel to 20% moisture content (fresh weight basis) before direct immersion in liquid nitrogen. With bothprotocols employed, regrowth of cryopreserved samples, as followed by visual observation, was alwaysrapid and direct.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Cryopreservation [liquid nitrogen (LN), −196 ◦C] is the onlyechnique currently available for safe and cost-effective long-termonservation of genetic resources of vegetatively propagated plantpecies such as sugarcane, which cannot be stored in the form ofehydrated seeds in seedbanks. At such ultra-low temperature, alletabolic activities are virtually stopped, thereby enabling theo-

etically unlimited storage durations (Engelmann, 2004).Cryopreserved material is sheltered from biotic and abiotic

tresses that can affect field collections and cause serious dam-ge, as illustrated with the US sugarcane germplasm collection, inhich 61% of the clones were lost between 1957 and 1977, mostlyue to pests and diseases (Berding and Roach, 1987). Furthermore,N storage requires limited maintenance and reduced space. Com-ared to traditional in vitro conservation (normal or slow growthulture), it suppresses the risk of occurrence of somaclonal varia-ions and of contamination during transfers (Ashmore, 1997).

In the case of sugarcane, cryopreservation protocols havelready been developed for cell suspensions (Ulrich et al.,984; Gnanapragasam and Vasil, 1990), embryogenic calluses

∗ Corresponding author at: IRD, UMR DIADE, 911 avenue Agropolis, BP 64501,4394 Montpellier Cedex 5, France. Tel.: +33 0467416224; fax: +33 0467416222.

E-mail address: giu.barra 82@alice.it (G. Barraco).

304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi: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 shoottips, encapsulation-dehydration (E-D), a technique first developedby Dereuddre et al. (1990) was successfully applied to 15 sugar-cane varieties (Gonzalez-Arnao, 1996). Storage of apices sampledfrom in vitro plantlets appears as the ideal procedure for long-termconservation of sugarcane genetic resources due to the ability ofsuch explants to guarantee genetic stability of regenerated plantmaterial (Gonzalez-Arnao et al., 2008).

In recent years, new cryopreservation protocols based on vit-rification of intracellular solutes have been developed, includingencapsulation-vitrification and droplet-vitrification (D-V) (Paniset al., 2005). The D-V protocol, which has proved its efficiencywith various plant species, has not been tested yet with sugar-cane. We therefore decided to compare the E-D protocol alreadydeveloped for cryopreservation of sugarcane shoot tips (Gonzalez-Arnao 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 ofVS placed on aluminium strips, which are rapidly immersed in LN

(Sakai and Engelmann, 2007). Rewarming is performed by plung-ing the aluminium foils in liquid medium containing 0.8–1.2 Msucrose. Samples are retrieved and placed on recovery medium. Themain advantage of this technique is the possibility of achieving very

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igh cooling/warming rates, due to the very small volume of VS inhich explants are placed and the direct contact between explants

nd LN during cooling and between explants and the unloadingolution during rewarming, thus allowing higher recovery com-ared with “standard” vitrification protocols (Kim et al., 2006). Inur experiments, we compared E-D and D-V using shoot apices ofwo commercial clones of sugarcane. In the D-V protocol, osmoticehydration was performed with the two most generally used VSs,VS2 (Sakai et al., 1990) and PVS3 (Nishizawa et al., 1993).

. Materials and methods

.1. Plant materials

In vitro plantlets of two sugarcane US commercial clones weresed: H70-144, from Hawaii, and CP68-1026, from Canal PointFlorida). Mother-plants were cultivated on semi-solid MS basal

edium (Murashige and Skoog, 1962) with 60 g L−1 sucrose andg L−1 agar at pH 5.6. Plantlets were placed at 27 ± 1 ◦C, under12 h d−1 photoperiod and a light intensity of 50 � mol m−2 s−1.other plants were subcultured monthly. For cryopreservation

xperiments, we used explants of a size of 0.5–1.0 mm, consisting ofhe apical dome, one or two leaf primordia and a basal part (Fig. 1a).pices were dissected under the binocular microscope from in vitrolantlets, 30–40 days after their last transfer. After dissection,hoot tips were maintained for 16 h in the dark on the mediumsed for culture of mother-plants to minimize the dissectiontress.

.2. Encapsulation-dehydration

Encapsulation-dehydration was performed according toonzalez-Arnao et al. (1993). Excised apices were encapsulated in% calcium alginate beads (diameter 4–5 mm) and precultured for4 h in liquid MS medium 0.75 M sucrose with constant shaking90 rpm). Encapsulated apices were dehydrated to moisture con-ents (MC) between 35 and 20% (fresh weight basis) by placinghem in hermetically closed containers (10 beads per container)lled with 80 g silica gel. Beads were then placed in 2 mL polypropy-

ene cryovials (10 beads per vial) and directly plunged in LN wherehey were kept for a minimum of 15 min. Cryopreserved beadsere rewarmed by placing them directly on recovery medium

n Petri dishes. This procedure ensured rapid rewarming due theirect contact between the beads and the ambient air at roomemperature.

Recovery medium consisted of semi-solid MS medium with0 g L−1 sucrose, 0.2 mg L−1 6-benzylaminopurine (BAP), 0.1 mg L−1

inetin (KIN), 7 g L−1 agar and 1 g L−1 Plant Preservative Mix-ure (PPM, Plant Cell Technology, Washington, USA) to avoidroliferation of endophytic bacteria. After cryopreservation,hoot tips were kept in the dark for 7 days to avoid photo-xidation damage, and then transferred under standard cultureonditions.

.3. Droplet-vitrification

Excised apices were treated for 20 min with a loading solutionontaining 2 M glycerol and 0.4 M sucrose (Nishizawa et al., 1993),hen dehydrated with PVS2 (30% glycerol, 15% DMSO, 15% EG and3.7% sucrose, w/v; Sakai et al., 1990) at 0 ◦C for 20–80 min or withVS3 (50% glycerol and 50% sucrose, w/v; Nishizawa et al., 1993)t room temperature for 20–100 min. Five min before the end of

ehydration with the VSs employed, apices were transferred to0 �L drops of VS (10 explants per drop) placed on aluminiumtrips and directly plunged in LN. After 15 min storage in LN, apicesere quickly rewarmed by plunging the aluminium strips in 10 mL

lturae 130 (2011) 320–324 321

unloading solution containing 1.2 M sucrose at room temperature(Panis et al., 2005). Explants were kept in the unloading solutionfor 20 min, then transferred on recovery medium. Recovery wasperformed as in the case of E-D described above.

2.4. Assessment of survival and recovery percentage andstatistical analyses

The effects of the different treatments were evaluated bymeasuring survival and recovery percentages of control and cry-opreserved shoot tips. Survival, corresponding to the presence ofliving tissues and to the observation of any regrowth pattern, wasrecorded 10 days after cryopreservation; after this duration deadexplants normally turned white. Recovery (Fig. 1b), correspond-ing to the production of normal shoots from treated explants,was observed after 40 days. Measuring survival was important asit allowed rapid evaluation of the experiments performed. Com-paring survival with recovery percentages was also useful as itshowed the range of potential improvement of the cryopreser-vation procedures. Explants were observed every 10 days using abinocular microscope to assess recovery features and the absenceof callus production. Each treatment was performed with threereplicates of 10 explants. The results, presented as percentage ofsurviving/recovering samples over the total number of explantstreated per experimental condition, were analyzed using analysis ofvariance, following arcsin transformation, with Duncan’s multiplerange test (DMRT), using the SPSS 14.0 software.

3. Results

3.1. Encapsulation-dehydration

With E-D, survival and recovery of non-cryopreserved shoot tipsof clone H70-144 decreased progressively, in line with decreasingbead MCs, while for CP68-1026 survival did not change significantlywith changes in MC (Table 1). No large differences were recordedbetween survival and recovery percentages since almost all surviv-ing explants were able to recover growth ability.

After cryopreservation, survival and recovery of shoot tips ofboth clones gradually increased, in line with decreasing bead MC,reaching a maximum at 20–22% MC; at such MC, recovery was40–60% 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 bothclones dehydrated with PVS2 decreased gradually, in line withincreasing durations of exposure to the VS (Table 2). Most surviv-ing apices were able to produce shoots after transfer to recoverymedium.

After cryopreservation, no survival was achieved without PVS2treatment. When dehydrating apices with PVS2, the best resultswere obtained after 20 and 40 min for clone H70-144; these treat-ments enabled recovery between 33 and 37%. For clone CP68-1026survival was not significantly different after all treatment dura-tions tested. The highest recovery, occurred between 10 and 20%,was obtained after 40–80 min dehydration. Treating samples withPVS2, survival and recovery of CP68-1026 apices were generallylower compared with H70-144.

After treatment with PVS3, survival and recovery of apicesof both clones decreased progressively in line with increasingtreatment durations (Table 3). After cryopreservation, the highest

survival and recovery percentages of apices of clone H70-144 wereobtained after 20–40 min treatment durations, reaching 17–33%.Longer exposures to VS induced lower recovery percentages andslower shoot production.

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

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 cryopreservedby 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 1Effect 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 treatedwith the E-D protocol.

Moisture content (%) H70-144 CP68-1026

−LN +LN −LN +LN

Survival (%) Recovery (%) Survival (%) Recovery (%) Survival (%) Recovery (%) Survival (%) Recovery (%)

75 94 a 94 a 0 c 0 c 79 a 79 a 0 c 0 c35 93 a 90 ab 0 c 0 c 87 a 87 a 0 c 0 c30 87 b 80 b 10 c 10 c 86 a 86 a 6 b 6 b25 77 bc 73 bc 27 b 23 b 86 a 80 a 10 b 7 b22 77 bc 60 cd 47 ab 40 ab 90 a 80 a 43 a 37 a

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alues followed by the same letter in the same column are not significantly differen

For CP68-1026, the highest survival percentages were obtainedfter treatment for 20–60 min. Recovery was not significantly dif-erent whatever the treatment duration tested; it varied betweenand 27%.

.3. Comparison between encapsulation-dehydration androplet-vitrification

The best results obtained with the D-V and E-D protocolsmployed in our experiments are presented in Table 4. For bothlones, E-D enabled the highest recovery percentages after cryop-eservation (60% for H70-144 and 53% for CP68-1026).

able 2ffect of duration of PVS2 dehydration on survival and recovery (%) of control (−LN) and cith the D-V protocol.

PVS2 dehydration (min) H70-144

−LN +LN

Survival (%) Recovery (%) Survival (%) Rec

0 87 a 80 a 0 c 0 c20 77 ab 67 ab 43 a 37 a40 60 bc 50 bc 47 a 33 a60 40 cd 33 cd 27 b 23 b80 23 d 17 d 23 b 13 c

alues followed by the same letter in the same column are not significantly different at t

83 a 83 a 53 a 53 a

he 0.05 probability level.

With clone H70-144, survival and recovery of non-cryopreserved apices were significantly higher using D-V,compared with E-D. By contrast, survival of cryopreservedapices was not significantly different between the three protocolsbut E-D led to significantly higher recovery. With clone CP68-1026,survival and recovery of non-cryopreserved controls were higherwith E-D and PVS3 D-V, compared with PVS2 D-V. After cryop-reservation, the highest survival (53%) was obtained with E-D,

even though survival using PVS3 D-V (40%) was not significantlydifferent. E-D also induced significantly higher recovery comparedwith D-V. With E-D, all surviving apices resumed growth aftercryopreservation and this was also the case for most apices treated

ryopreserved (+LN) shoot tips of sugarcane clones H70-144 and CP68-1026 treated

CP68-1026

−LN +LN

overy (%) Survival (%) Recovery (%) Survival (%) Recovery (%)

77 a 73 a 0 b 0 c63 ab 53 b 17 a 7 b50 bc 50 b 23 a 20 a33 cd 27 c 17 a 10 ab27 d 23 c 17 a 10 ab

he 0.05 probability level.

G. Barraco et al. / Scientia Horticulturae 130 (2011) 320–324 323

Table 3Effect 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 treatedwith the D-V protocol.

PVS3 dehydration (min) H70-144 CP68-1026

−LN +LN −LN +LN

Survival (%) Recovery (%) Survival (%) Recovery (%) Survival (%) Recovery (%) Survival (%) Recovery (%)

0 80 a 63 a 0 c 0 c 97 a 93 a 0 d 0 b20 83 a 73 a 53 a 33 a 87 ab 87 a 40 a 27 a40 50 b 30 b 37 ab 17 ab 60 bc 37 b 43 a 27 a60 53 b 37 b 27 b 10 bc 53 bc 47 b 30 ab 17 a80 43 bc 30 b 20 b 10 ac 43 c 37 b 20 b 17 a

100 22 c 9 c 3 c 3 bc 27 c 20 b 7 c 7 ab

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

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

Protocol H70-144 CP68-1026

−LN +LN −LN +LN

Survival (%) Recovery (%) Survival (%) Recovery (%) Survival (%) Recovery (%) Survival (%) Recovery (%)

D-V PVS2 77 a 67 ab 43 a 37 b 50 b 50 b 23 b 20 bD-V PVS3 83 a 73 a 53 a 33 b 87 a 87 a 40 ab 27 b

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ith PVS2 D-V. By contrast, only a part of the apices cryopreservedsing the PVS3 D-V resumed shoot production as shown by theecrease between survival and recovery percentages.

In all experimental conditions tested, shoot regrowth, assessedy observation under a binocular microscope, was direct, withoutallus formation.

. Discussion

In vitrification-based cryopreservation protocols, the highlyoncentrated intracellular solutes solidify during the rapid coolingrocedure, thus reaching an amorphous glassy state that pro-ects cells against structural damage. Vitrification is obtained byncreasing viscosity of intracellular solutes through physical and/orsmotic dehydration before cryopreservation and by rapid immer-ion of samples in LN (Gonzalez-Arnao et al., 2008).

In this paper, we investigated the efficiency of two vitrification-ased protocols, E-D and D-V for cryopreserving apices of twougarcane clones. Recovery of cryopreserved material was obtainedith both protocols, demonstrating the possibility of using bothethods for LN storage of sugarcane apices. The differences

etween survival and recovery data were small, indicating the use-ulness of this parameter for the early evaluation of the efficiencyf cryopreservation protocols for sugarcane apices.

E-D is a technique based on the synthetic seed technology,hich was experimented with plant material by Dereuddre et al.

1990), then successfully applied to numerous temperate andropical species (Gonzalez-Arnao and Engelmann, 2006) includ-ng sugarcane (Gonzalez-Arnao et al., 1993, 1999; Paulet et al.,993). E-D offers many advantages in terms protocol simplifica-ion and manipulation of explants (Engelmann et al., 2008). In ouresearch the highest recovery percentage using the E-D protocolas 53–60%, which was comparable to the 62% recovery obtained

y Gonzalez-Arnao et al. (1993), Paulet et al. (1993), Gonzalez-rnao (1996) and Gonzalez-Arnao et al. (1999) using 15 sugarcane

ccessions. The optimization of the pretreatment procedure and ofample residual MC are key factors for successful application of E-(Gonzalez-Benito et al., 1998). In our study, the highest recoveryas obtained when dehydrating the shoot tips to 20–22% MC. These

83 a 83 a 53 a 53 a

e 0.05 probability level.

results are in accordance with those of Gonzalez-Arnao (1996) whoshowed that dehydration to 22% MC ensured the highest survival(57%) after cryopreservation of shoot tips of five sugarcane com-mercial clones.

Our experiments showed for the first time that sugarcane apicescould be cryopreserved using D-V. We compared the most widelyused VSs, PVS2 and PVS3, which were employed for different dura-tions. PVS2 is characterized by its high chemical toxicity, becauseit includes the permeating cryoprotectants DMSO and ethyleneglycol, while PVS3, which includes the non-penetrating cryopro-tectants sucrose and glycerol, can be toxic because of the highosmotic pressure it exerts on plant cells (Kim et al., 2009). In ourexperiments, apices of the two clones tested showed relatively hightolerance to dehydration with both VSs (PVS2 and PVS3); however,recovery of cryopreserved samples was low, reaching a maximumof 20–37%. This is in contrast with what is observed with numeroustemperate and tropical plants species, for which D-V produces veryhigh recovery (Sakai and Engelmann, 2007). In experiments carriedout by Panis et al. (2005) on 56 accessions of banana shoot tips cry-opreserved using D-V, recovery was between 50 and 95%. Whenapplying D-V to shoot tips of 18 taro cultivars, recovery was 89%(Sant et al., 2008); it reached 73% with Chrysanthemum shoot tipsand 95% with garlic bulbil primordia (Kim et al., 2009). This clearlyshows the necessity of additional studies aiming at optimizing thedifferent 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, whichis 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 cryop-reserving sugarcane apices using E-D, which had already beensuccessfully used by various researchers (Gonzalez-Arnao et al.,

1993, 1999; Paulet et al., 1993) and D-V, which was applied to sug-arcane for the first time. In our experiments, the already optimizedE-D was clearly superior, leading to higher recovery percentages.However, additional studies aiming at optimizing the different

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teps of the D-V protocol may increase survival and recovery ofhoot tips cryopreserved with this technique. Moreover, our studyas performed with two clones only. Our results should thus be

onfirmed using additional clones of sugarcane.

cknowledgements

The assistance of Marie-Jo Darroussat and Jean-Claude GirardCIRAD Baillarguet, France) for providing the experimental mate-ial is gratefully acknowledged. This work was partly supported byRCAD, a flagship program of Agropolis Fondation (I. Sylvestre).

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