recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit...

Biotechnology Advances 31 (2013) 175–185

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Research review paper

Recent advances in the cryopreservation of shoot-derived germplasm ofeconomically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus,Pyrus and Vitis

Carla Benelli a,⁎, Anna De Carlo a, Florent Engelmann b

a IVALSA/Istituto per la Valorizzazione del Legno e delle Specie Arboree, CNR (National Research Council) 50019, via Madonna del Piano, 10, Sesto Fiorentino, Florence, Italyb IRD, UMR DIADE, 911 avenue Agropolis, BP 64501, 34032 Montpellier cedex 5, France

⁎ Corresponding author. Tel.: +39 055 5225698; fax:E-mail address: [email protected] (C. Benelli).

0734-9750/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.biotechadv.2012.09.004

a b s t r a c t
a r t i c l e i n f o
Article history:Received 18 April 2012Received in revised form 7 September 2012Accepted 22 September 2012Available online 27 September 2012

Keywords:BiotechnologyGenetic resourcesCryopreservationFruit treesShoot-tipsDormant budsCryotherapy

This paper presents the advances made over the last decade in cryopreservation of economically importantvegetatively propagated fruit trees. Cryopreservation protocols have been established using both dormantbuds sampled on field-grown plants and shoot tips sampled on in vitro plantlets. In the case of dormantbuds, scions are partially dehydrated by storage at −5 °C, and then cooled slowly to −30 °C using lowcooling rates (c.a. 1 °C/h) before immersion in liquid nitrogen. After slow rewarming and rehydration of sam-ples, regrowth takes place either through grafting of buds on rootstocks or excision of apices and inoculationin vitro. In the case of shoot tips of in vitro plantlets, the cryopreservation techniques employed are thefollowing: controlled rate cooling procedures involving slow prefreezing followed by immersion in liquid ni-trogen or vitrification-based procedures including encapsulation–dehydration, vitrification, encapsulation–vitrification and droplet-vitrification. The current status of cryopreservation for a series of fruit tree speciesincluding Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis is presented. Routine application of cryo-preservation for long-term germplasm storage in genebanks is currently limited to apple and pear, forwhich large cryopreserved collections have been established at NCGRP, Fort Collins (USA), using dormantbuds and in vitro shoot tips, respectively. However, there are a growing number of examples of pilot scaletesting experiments under way for different species in various countries. Progress in the further developmentand application of cryopreservation techniques will be made through a better understanding of the mecha-nisms involved in the induction of tolerance to dehydration and cryopreservation in frozen explants.

© 2012 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1762. Cryopreservation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

2.1. Cryopreservation of dormant buds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1772.2. Cryopreservation of in vitro shoot tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1772.3. Assessment of cryopreservation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

3. Cryopreservation of fruit tree species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1783.1. Actinidia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1783.2. Diospyros . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

3.2.1. Shoot tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1783.2.2. Dormant buds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

3.3. Malus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1803.3.1. Shoot-tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1803.3.2. Dormant buds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

3.4. Olea europaea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1813.5. Prunus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1813.6. Pyrus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

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176 C. Benelli et al. / Biotechnology Advances 31 (2013) 175–185

3.6.1. Shoot tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1823.6.2. Dormant buds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

3.7. Vitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1824. Concluding remarks and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

1. Introduction

Maintenance of plant genetic resources in situ and ex situ has twomain objectives, the conservation of genetic diversity at large and ofselected varieties with agronomic and economic value. Therefore,conservation plays a fundamental role in order to prevent the lossof plant species with economic and ecological importance.

Traditionally, the field genebank has been the ex situ storagemeth-od of choice for the aforementioned problem materials (Engelmannand Engels, 2002). In some ways, this method offers a satisfactory ap-proach to conservation. The genetic resources under conservation canbe readily accessed and observed, thus permitting detailed evaluation.However, there are certain drawbacks that limit its efficiency andthreaten its security (Engelmann, 1997; Withers and Engels, 1990).The genetic resources are exposed to pests, diseases and other naturalhazards such as drought, weather damage, human error and vandal-ism. In addition, they are not in a condition that is readily conduciveto germplasm exchange because of the great risks of disease transferthrough the exchange of vegetative material. Field genebanks arecostly to maintain, particularly when conserving multiple copies ofeach accession, and are thus prone to economic decisions that maylimit the level of replication of accessions, the quality of maintenanceand even their very survival in times of economic stringency. Evenunder the best circumstances, field genebanks require considerableinputs in the form of land, labour, management and materials, and,in addition, their capacity to ensure the maintenance of much diversi-ty is limited (Engelmann and Engels, 2002; Lambardi et al., 2001).

Cryopreservation in liquid nitrogen (LN,−196 °C) is the only tech-nique currently available to ensure the safe and cost-efficient long-term conservation of the germplasm of problem species, includingnon-orthodox seed species and vegetatively propagated plants(Engelmann, 2012). At this temperature, all cellular divisions andmetabolic processes are stopped. The plantmaterial can thus be storedwithout alteration or modification for a theoretically unlimited periodof time. Moreover, cultures are stored in a small volume, protectedfrom contamination, requiring very limited maintenance. Cryopreser-vation represents a valuable addition to the current conventionalmethods employed for the maintenance of clonally propagatedplants such as fruit tree species. Most fruit trees are vegetativelypropagated. They require the maintenance of large numbers of ac-cessions in clonal orchards in which periodic monitoring of theconserved trees is essential to maintain genetic diversity. The combi-nation of the traditional ex situ conservation approach with cryo-preservation has great potential to improve the conservation offruit tree germplasm.

Different types of tissues and organs can be cryopreserved, includingcell suspensions, pollen, embryogenic cultures, somatic and zygotic em-bryos, seeds, shoot tips and dormant buds. For vegetatively propagatedspecies, the organsmost commonly used in cryopreservation, are shoottips, excised from apical or axillary buds of in vitro-grown shoot cultures(Lambardi and De Carlo, 2003). The use of in vitro shoots in cryopreser-vation protocols has several advantages; they are easy to multiply andto manipulate, available throughout the year and cryoprotective treat-ments can be applied to the shoots or apices while cultured in vitro(Reed, 2004).

In fruit tree cultivars, for which the maintenance of genetic fidelityto the donor plants is fundamental, shoot tips are by far the most

utilized explants for cryopreservation (Engelmann, 2000; Takagi,2000; Towill, 2002; Zhao et al., 2008a). Indeed, shoot tips are differ-entiated organs composed of organized tissues and are thus lessprone to somaclonal variation than non-organized tissues like calliand cell suspensions (Panis and Lambardi, 2006). In some fruittrees, cryopreservation of dormant buds has also been developed,and recovery has been achieved by grafting (Forsline et al., 1998;Towill and Ellis, 2008).

Over the last 20 years, cryopreservation protocols have beenestablished for several hundreds of plant species, including numerousfruit tree species (Reed, 2008). In particular, the vitrification andencapsulation-dehydration methods have been continuously improvedand are thus the most frequently employed for cryopreservation ofclonally propagated plants (De Carlo et al., 2000; Gonzalez-Arnao andEngelmann, 2006; Gonzalez-Benito et al., 2004; Reed, 2008; Sakai andEngelmann, 2007). Nowadays, there are some examples of genebankswhich use cryopreservation (Sakai and Engelmann, 2007). In the caseof fruit trees, themost advanced genebank is theNational Centre for Ge-netic Resources Preservation (NCGRPP, Fort Collins, USA)where around1900 accessions of the national apple collection are conserved undercryopreserved storage (Towill et al., 2004). These accessions are storedusing dormant buds (Towill et al., 2004; Volk et al., 2008). More than100 pear cultivars and clones are also maintained under cryopreserva-tion at the NCGRPP (Reed, 2001; Trajkovski and Hjalmarsson, 2007).

The aim of this paper is to briefly summarize the cryopreservationtechniques developed for buds or shoot-tips, differentiated organswhich are used for cryopreservation of vegetatively propagated fruitplants, and to present the current state of development and applicationof cryopreservation in seven genera of fruit trees: Actinidia, Diospyros,Malus,Olea, Prunus, Pyrus and Vitis. These genera have been selected be-cause this paper focuses on top-fruit trees with great economic value inthe temperate region.

2. Cryopreservation techniques

Some materials such as orthodox seeds display natural dehydrationprocesses and can be cryopreservedwithout any pretreatment. Howev-er,most biologicalmaterials employed in cryopreservation (buds, shoottips, embryos, calli, cell suspensions) contain high amounts of cellularwater and are sensitive to freezing injury, sincemost of them are not in-herently freezing-tolerant. Cells have thus to be dehydrated artificiallyto protect them from the damages caused by the crystallization of intra-cellular water into ice (Mazur, 1984). The techniques employed andthe physical mechanisms, upon which they are based, are different incontrolled rate cooling and vitrification-based cryopreservation tech-niques (Withers and Engelmann, 1998). In controlled rate cooling tech-niques, dehydration of samples takes place both before and duringcooling (freeze-induced dehydration), whereas in vitrification-basedtechniques, dehydration takes place only before cooling. In optimalconditions, all freezable water is removed from the cells during dehy-dration and the highly concentrated internal solutes vitrify uponimmersion LN. Vitrification can be defined as the transition of water di-rectly from the liquid phase into an amorphous phase or glass, whileavoiding the formation of crystalline ice (Fahy et al., 1984).

Commercially important fruit tree species are propagated vegeta-tively, therefore dormant buds sampled from fieldmaterial after a suf-ficient duration of exposure to negative temperatures or shoot tips

177C. Benelli et al. / Biotechnology Advances 31 (2013) 175–185

from in vitro cultured plantlets are employed for cryopreservation. Inthe case of dormant buds, the methods used depend on the speciesas well as on the level of cold-hardiness of the collected material(Towill and Ellis, 2008). In some cases, dormant buds or scions holdingdormant buds are cryopreserved. After sample rewarming, regrowthcan be achieved through direct development of buds on scions,grafting of buds on rootstocks, in vitro culture of excised shoot tipsor micrografting of excised shoot tips on in vitro seedlings. In othercases, shoot tips sampled from in vitro plantlets are cryopreserved.Below we describe the techniques employed for cryopreserving dor-mant buds and in vitro shoot tips of fruit tree species. The informationprovided on cryopreservation of dormant buds is largely based on thereview chapter of Towill and Ellis (2008).

2.1. Cryopreservation of dormant buds

Branches or scions are generally harvested during mid-winter,after mother-plants have been exposed to a sufficient duration ofcold temperatures. The extent of cold-acclimation seems to be an im-portant factor to achieve high recovery after cryopreservation. Towillet al. (2004) showed that very cold-hardy Malus species displayedhigher recovery after cryopreservation than less hardy ones. Howev-er, Toldam-Andersen et al. (2007) indicated that the standard proto-col developed in North America for dormant apple buds (Forslineet al., 1998) proved to be applicable to less hardy cultivars grown inthe Danish mild, maritime winter climate. After collection, the dor-mant material can be stored in sealed plastic bags at temperatures be-tween −3 °C and −5 °C for several months without viability lossbefore its cryopreservation.

The samples used for cryopreservation can be of different sizes,ranging from small explants consisting of one axillary bud with5–10 mm vascular tissue (Niino et al., 1992a; Oka et al., 1991) to largerones consisting of 35 mm nodal sections with one bud (Forsline et al.,1998).

Another important parameter is the determination of sample opti-mal moisture content to achieve high recovery after cryopreservation.In most cases, explants are partly dehydrated to moisture contents(MCs) around 25–35% (freshweight, FW), before LN storage, dependingon the species, as recovery of dehydrated samples after cryopreserva-tion is usually higher than that of non-dehydrated samples (Volket al., 2008). Dehydration is usually performed by placing the samplesin a refrigerator at−5 °C for several days.

Samples are cooled slowly using controlled cooling rates. Explantsare either placed in the chamber of a programmable freezer and cooledat low rate, e.g. 1 °C/h from−5 °C to−30 °C followed by immersion inLN, as described by Towill et al. (2004) with Malus nodal sections, orplaced in a cold chamber, whose temperature is decreased progressive-ly at regular intervals, e.g. transfer for 24 h at −5 °C, −10 °C, −15 °C,−20 °C, −30 °C before immersion in LN, as performed by Oka et al.(1991)with pear dormant buds. Rewarming is generally slow, achievedby placing the cryotubes or bags containing the samples at 0 °C for 24 h.After rewarming, samples are allowed to rehydrate progressively in ahumid environment.

There are different procedures for growth recovery of cryopreservedsamples. They can be used directly, such as in the case of Salix, wherescions are planted in sterilized medium saturated with sterile waterand placed in controlled environmental conditions to achieve directregrowth of buds and rooting (Towill and Wildrlechner, 2004). Inother cases, buds are grafted on rootstocks using a chip bud (Forslineet al., 1998; Lambardi et al., 2011). Regrowth can also be achievedusing in vitro culture procedures. After rewarming, buds are sterilized,shoot tips are excised and inoculated in vitro for regrowth (Niinoet al., 1992a; Oka et al., 1991). Micrografting of cryopreserved buds onin vitro germinated seedlings is also feasible, as shown with pear(Suzuki et al., 1997).

2.2. Cryopreservation of in vitro shoot tips

In vitro shoot tips are generally used for cryopreservation when itis impossible to use dormant buds. This is the case with tropical andsubtropical species, or with cold-tender accessions of temperate spe-cies whose buds do not reach a sufficient level of dormancy, whichwould allow using them directly for cryopreservation. Shoot tips areeither sampled from already established in vitro plantlets or excisedfrom in vivo buds and immediately used for cryopreservation. Invitro shoot tips of fruit tree species have been cryopreserved usingboth controlled rate cooling and vitrification-based cryopreservationtechniques (Engelmann, 2012).

Controlled rate cooling techniques include the following succes-sive steps: pregrowth of shoot tips, treatment with cryoprotectants,slow cooling (0.5–2.0 °C/min) to a determined prefreezing temper-ature (usually around −40 °C), rapid immersion of samples in LN,storage, rapid rewarming and recovery. If the heterogeneous crystalli-zation temperature of a given cryoprotectant solution has a constantvalue, this is not the case for the ice nucleation temperature (i.e. thetemperature at which ice crystal formation begins to take place), aphenomenon which occurs randomly in different cryotubes of thesame batch and will result in uneven dehydration among samples. Inorder to control this parameter and to standardize the procedure, nu-cleation is induced manually by briefly pinching the cryotubes withforceps previously cooled in liquid nitrogen at a temperature interme-diate between the crystallization temperature and the average nucle-ation temperature of the cryoprotectant solution (Gonzalez-Arnaoet al., 2008). Controlled rate-cooling cryopreservation techniques aregenerally employed with apices of cold-tolerant species (Reed andUchendu, 2008). Cold acclimation of in vitro mother-plants has oftena positive effect on regrowth of cryopreserved shoot tips, as shownnotably with Rubus (Chang and Reed, 1999) and grape (Zhao et al.,2001).

Shoot tips of fruit tree species havemostly been cryopreserved usingfour of the seven existing vitrification-based cryopreservation tech-niques, viz. encapsulation-dehydration, vitrification, encapsulation-vitrification and droplet-vitrification.

The encapsulation–dehydration procedure is based on the technolo-gy developed for producing artificial seeds. Explants are encapsulated incalcium alginate beads (usually 3% w/v), pregrown in liquid mediumenriched with sucrose concentrations between 0.75 and 1.25 M for 1to 7 days, partially desiccated in the air current of a laminar airflowcab-inet or with silica gel down to MC around 20% FW, then cooled rapidly.Desiccation with silica gel is highly recommended, as it is more preciseand reproducible than air desiccation. Survival of cryopreserved sam-ples is generally high and growth recovery rapid and direct, withoutcallus formation. This technique has been applied to apices of numerousfruit tree species including notably apple, pear, Prunus and mulberry(Gonzalez-Arnao and Engelmann, 2006; Zamecnik et al., 2007). Asimplification of the encapsulation–dehydration technique has been re-cently proposed by Bonnart and Volk (2010), which involved encapsu-lating samples in alginate medium containing 2 M glycerol+0.5 Msucrose, immediately followed by air-dehydration.

Vitrification involves treatment of shoot tips with cryoprotectants,including exposure of samples to a loading solution with intermediateconcentration (usually 2 M glycerol+0.4 M sucrose, Matsumotoet al., 1994), followed by dehydration with highly concentrated vitri-fication solutions (total molarity around 5–7 M), rapid cooling andrewarming, removal of cryoprotectants in an unloading solution(containing 0.8–1.2 M sucrose) and recovery. The most commonlyemployed vitrification solutions are the so-called Plant VitrificationSolutions PVS2 (Sakai et al., 1990) and PVS3 (Nishizawa et al., 1993)developed by the group of Prof. Sakai in Japan. Vitrification has beenapplied for cryopreserving apices of numerous fruit tree species(Lambardi and De Carlo, 2003; Reed, 2001; Sakai and Engelmann,2007; Sakai et al., 2008).


Encapsulation–vitrification is a combination of encapsulation–dehydration and vitrification procedures, where shoot tips are encap-sulated in calcium alginate beads, then treated with vitrification solu-tions and cooled rapidly, as in a vitrification protocol. It has beenapplied to apices of an increasing number of species (Sakai andEngelmann, 2007; Sakai et al., 2008).

Droplet-vitrification is the latest technique developed (Panis et al.,2005). The number of species to which it has been successfully ap-plied is increasing steadily (Sakai and Engelmann, 2007). Apices arepretreated with a loading solution, then treated with a vitrificationsolution, placed on an aluminium foil in minute droplets of vitrifica-tion solution and cooled rapidly in LN. The good results obtainedwith this technique are due to the very high cooling and rewarmingrates achieved because of the direct contact between samples andLN during cooling, and between samples and the unloading solutionduring rewarming. Alternative loading and vitrification solutions,which proved to be more efficient and less toxic than the “classical”loading and vitrification solutions when applied to highly sensitivematerials, have been developed recently (Kim et al., 2009a,b).

2.3. Assessment of cryopreservation techniques

Cryopreservation of dormant buds is based on the natural cold accli-mation of mother-plants and on controlled dehydration of scions hold-ing one or several dormant buds. It requires mother-plants to be grownin a climate suitable for maximal cold acclimation, and the use of acontrolled rate freezer or programmable cold chamber (Reed, 2011).It is applicable only with moderately to very cold-hardy woody plants.Another limitation to cryopreservation of dormant buds lies with theregeneration procedure. Grafting of cryopreserved buds on appropriaterootstocks is feasible with some species such as apple or pear (Forslineet al., 1998; Lambardi et al., 2011). However in other cases, meristemshave to be excised from cryopreserved buds and introduced in vitrofor regrowth, which may prove difficult because of the structural dam-age caused to the explants by exposure to LN and possibly by theirnon-optimal physiological status.

Controlled rate cooling of in vitro shoot tips requires the use of aprogrammable freezer or of an alcohol-filled freezing containerplaced in a mechanical freezer (Reed, 2011). In this technique, cryo-protectants are employed at relatively low concentrations. Therefore,the precise control of the duration of exposure of samples to cryopro-tectants is not as critical as with some vitrification-based techniques.Another advantage of this technique is that it allows the simultaneousprocessing of many samples. However, this technique can be em-ployed only with shoot tips of temperate, cold-tolerant plant species,which restricts its spectrum of application (Engelmann, 2012; Reed,2011).

The encapsulation–dehydration technique is relatively labor inten-sive, as each bead is handled several times during the process (Reed,2011). It is also time consuming, as its implementation requires amin-imumof 2 days. However,many samples can be processed at the sametime. Moreover, encapsulation greatly facilitates the manipulation ofshoot tips, whereas naked apices are easily damaged during manipu-lation with forceps and some shoot tips are often lost during the im-plementation of a cryopreservation protocol, because of their smallsize (Engelmann et al., 2008).

In the vitrification technique, explants are exposed to highly concen-trated vitrification solutions, which are very effective in dehydratingsamples but which can be very toxic. A very precise control of the dura-tion of exposure of shoot tips to such vitrification solutions is of criticalimportance, which limits the number of samples which can beprocessed at the same time (Reed, 2011; Sakai and Engelmann, 2007).However, since the durations of treatmentwith loading and vitrificationsolutions are short, several batches of shoot tips can be processed overoneworking day. Encapsulation–vitrification can represent a good alter-native for plants, which are sensitive to direct contact with vitrification

solutions. Indeed, the alginate capsulemayprotect shoot tips fromdirectexposure to highly toxic vitrification solutions (Sakai and Engelmann,2007).

The high efficiency of droplet-vitrification is due to the high ratesof temperature changes achieved because of the direct contact be-tween samples and LN during cooling, and between samples and theunloading solution during rewarming. However, as with the vitrifica-tion technique, very precise timing of exposure of shoot tips to vitrifi-cation solutions is required. Moreover, this method is very laborintensive, requires careful handling and a sterile LN supply since theplant material is in direct contact with LN (Reed, 2011).

3. Cryopreservation of fruit tree species

The following section presents a brief overview of reports publishedfrom year 2000 onwards on cryopreservation of selected economicallyimportant fruit tree species in the temperate region (Tables 1–2).

3.1. Actinidia

Until now, encapsulation–dehydration has been the main tech-nique utilized for cryopreservation of Actinidia (Bachiri et al., 2001;Wu et al., 2001a; Zhai et al., 2003). Bachiri et al. (2001) applied encap-sulation–dehydration to a hybrid of A. arguta x A. deliciosa, then to sev-eral cultivars of A. deliciosa and A. chinensis. These authors preculturedthe beads in media with daily increasing sucrose concentrations (0.3,0.5 and 0.7 M), dehydrated them with silica gel to 20% MC andplunged them directly in LN. The range of survival obtained with thedifferent species employed was between 85% and 95%. Shoots re-grown from cryopreserved apices did not display any phenotypic ab-normality. Wu et al. (2001a) used encapsulated shoot tips of cvs.‘Tomuri M’ and ‘Tomuri F’ (A. deliciosa) and of A. chinensis preculturedin daily increasing sucrose concentrations (0.5, 0.7, 1.0 and 1.2 M) anddehydrated to 26%MC before LN exposure. Desiccated shoot tips werecryopreserved either by two-step freezing (prefreezing at 0.2 °C/minto temperatures between −20 and −40 °C followed by immersionin LN) or by direct immersion in LN. The former procedure providedbetter results, with regrowth ranging between 22% and 56%. By con-trast, low regrowth was obtained following rapid cooling. Zhai et al.(2003) evaluated the genetic stability of plantlets of cv. ‘Tomuri’ (A.deliciosa) recovered from cryopreserved shoot tips using the RAPDtechnique; RAPD fragment patterns of cryopreserved plantlets wereidentical to those of unfrozen controls. The only research related tothe application of the vitrificationmethod in Actinidia studied changesin cell ultrastructure during cryopreservation (Xu et al., 2006).

3.2. Diospyros

3.2.1. Shoot tipsIn the case of kaki, only few experiments were performed with in

vitro cultured material. Shoot tips excised from in vitro Diospyroskaki and D. lotus cold-hardened mother-plants were precultured onMS medium supplemented with sucrose (0.7 M) for 2 days, loadedin MS containing 2 M glycerol and 0.4 M sucrose at 20 °C for 20 minand dehydrated in PVS2 for 80 min at 0 °C prior to direct immersionin LN (Ai and Luo, 2003). After 6 weeks, shoot formation was over30%. Recently, Niu et al. (2012) developed a protocol combining theencapsulation and droplet-vitrification techniques. Shoot tips sam-pled from cold-acclimated mother-plants were encapsulated in algi-nate beads, transferred to droplets of PVS2 on aluminium foils, thenplunged in LN after 1.5 h exposure to PVS2. This protocol produced80% regeneration. Applying this method, the authors obtained thehighest regeneration (80%) without significantly different among ge-notypes, compared with vitrification and droplet-vitrification tech-niques (70–72%).

Table 1Reports on cryopreservation shoot tips of selected fruit tree species from year 2000 onwards.

Genus Plant species (no of cultivar/rootstock)a Cryopreservation techniqueb Maximum regrowth (%) Ref.

Actinidia A. chinensis (1) Vitrif 52 Xu et al. (2006)A. deliciosa (1) En-dehy 45 Zhai et al. (2003)Actinidia spp. (9) En-dehy 95 Bachiri et al. (2001)Actinidia spp. (3) En-dehy 56 Wu et al. (2001a)

Diospyros D. kaki (4) En-droplet-vitrif 80 Niu et al. (2012)Diospyros spp. (2) Vitrif 30 Ai and Luo (2003)

Malus M. domestica (5) En-dehy 88 Paul et al. (2000)M. domestica (4) Droplet 80 Wu et al. (2000)M. domestica (2) En-dehy 78 Kushnarenko et al. (2006)M. domestica (1) Vitrif 80 Liu et al. (2008)M. domestica (4) Vitrif or En-dehy 80 Kushnarenko et al. (2009)M. domestica (4) Droplet-vitrif 70 Halmagyi et al. (2010a)M. domestica (2) Droplet-vitrif 68 Halmagyi et al. (2010b)M. domestica (1) En-dehy 72 Forni et al. (2010)M. domestica (4) Droplet 70 Condello et al. (2011)M. pumila (3) En-dehy 87 Hao et al. (2001)M. pumila (1) Vitrif (PVSL)c 81 Liu et al. (2004)Malus spp. En-dehy 45 Zamecnik et al. (2007)

Olea O. europea Vitrif 15 Benelli et al. (2001)O. europea Vitrif 30 Nisi et al. (2006)O. europea Vitrif 38 Lynch et al. (2007)

Prunus P. avium Vitrif 78 Shatnawi et al. (2007)P. avium En-dehy 76 Shatnawi et al. (2007)P. cerasifera Droplet-vitrif 20 Vujovic et al. (2011)P. domestica Vitrif 57 De Carlo et al. (2000)P. dulcis Vitrif 88 Channuntapipat et al. (2000)P. dulcis En-dehy 62 Shatnawi et al. (2000)P. dulcis Droplet 58 De Boucaud et al. (2002)P. dulcis Vitrif 67 Al-Ababneh et al. (2003)P. dulcis En-dehy 60 Al-Ababneh et al. (2003)P. dulcis Vitrif 80 Wirthensohn et al. (2006)Prunus hybrids TSF 74 De Boucaud et al. (2002)P. persica Droplet 32 De Boucaud et al. (2002)Prunus rootstock Droplet-vitrif 52 De Boucaud et al. (2002)P. salicina Vitrif 60 Zhao et al. (2008b)Prunus spp. En-dehy 14 Zamecnik et al. (2007)

Pyrus P. cordata (1) TSF 75 Chang and Reed (2001)Pyrus spp. (8) TSF 83 Chang and Reed (2000)Pyrus spp. Vitrif 71 Wang et al. (2008)Pyrus spp. En-dehy 30 Zamecnik et al. (2007)P. pyraster (1) En-dehy 60 Condello et al. (2009)P. pyrifolia (1) En-dehy 82 Hao et al. (2005)

Vitis Vitis hybrid (1) En-dehy 63 Wang et al. (2003a)Vitis spp. (2) En-dehy 60 Wang et al. (2000)Vitis spp. (11) Vitrif 87 Matsumoto and Sakai (2003)V. vinifera (4) En-dehy 40 Zhao et al. (2001)V. vinifera (4) En-dehy 36 Zhai et al. (2003)

a Number of cultivars or rootstocks tested is given in parentheses, when reported in cited literature.b Vitrif = vitrification; En-dehy = encapsulation–dehydration; Droplet = droplet using DMSO; Droplet-vitrif = droplet vitrification using PVS2; En-droplet-vitrif = encapsulation–

droplet–vitrification; TSF = Two step-freezing, slow-cooling prior to storage in LN.c PVSL = Plant Vitrification Solution Liu.


3.2.2. Dormant budsDormant buds have been extensively used as explants for cryo-

preservation of Diospyros (Towill and Ellis, 2008). Matsumoto et al.(2001) reported for the first time successful cryopreservation ofDiospyros spp. dormant buds (Table 2). These authors tested vitrifica-tion on 16 cultivars of D. kaki and three tropical species (D. taitoensis,D. kuroiwai and D. morrisiana), using shoot tips excised from dormantaxillary buds. They were dehydrated with PVS2 for 20 min at 25 °C,then immersed in LN. The average shoot formation after cryopreser-vation was 89% for the cultivars, but it was lower for tropical species.A similar procedure, which comprised an extension of the PVS2 treat-ment duration up to 60–90 min, led to survival comprised between56% and 86% with the four D. kaki cultivars tested (‘Jiro’, ‘Triumph’and ‘Rojo Brillante’ and‘ Hiratanenashi’) (Benelli et al., 2009).

In another study, intact buds attached to shoot segments weresubjected to progressively decreasing temperatures down to −20 °C(12 h each at 0 °C, −5 °C, −10 °C and −20 °C), then plunged in LN(Ai and Luo, 2005). Shoot tips were excised from cryopreservedbuds after rewarming, sterilized and plated on modified MS medium.

Survival of shoot tips was over 60% with the five cultivars tested. Novariation in chromosome number or AFLP patterns was observed inplantlets regenerated from cryopreserved shoot tips.

Another promising protocol for shoot tips sampled from dormantaxillary buds is encapsulation–vitrification (Zhang and Luo, 2004).In this study, shoot tips of five D. kaki cultivars were encapsulated,precultured on MS medium supplemented with daily increasing su-crose concentrations (0.3, 0.5, 0.7 and 1 M), treated with loading solu-tion (Matsumoto et al., 1994) for 20 min and dehydrated with PVS2for 120 min at 25 °C before immersion in LN. Very high survival wasrecorded (near 100%) and no abnormalities in themorphological devel-opment of cryopreserved shoots were observed. The prefreezingmeth-od was tested on twigs bearing dormant buds of cv. ‘Saijo’ (Matsumotoet al., 2004). Explants were first cooled at 1 °C/min to−5 °C, then fur-ther cooled at the same rate to −10 °C, −15 °C, −20 °C, −30 °C andkept at each of these temperatures for one day, before their transfer ina deep-freezer at −150 °C in which they were held for 5 days. Afterrewarming at 25 °C, the percentage of in vitro shoot formation wasabout 70%.

Table 2Reports on cryopreservation dormant buds of selected fruit trees species from year 2000 onwards.

Genus Plant species (no cultivar/rootstock)a Cryopreservation techniqueb Maximum plant regrowth (%) Ref.

Diospyros D. kaki (5) En-vitrif 100 Zhang and Luo (2004)D. kaki (1) Slow freezingc 70 Matsumoto et al. (2004)D. kaki (5) Slow freezingd 76 Ai and Luo (2005)D. kaki (4) Vitrif 86 Benelli et al. (2009)D. kaki (6) En-droplet-vitrif 79 Niu et al. (2010)Diospyros spp. (19) Vitrif 100 Matsumoto et al. (2001)

Malus M. domestica (1) TSF 16 Wu et al. (2001b)M. domestica (3) Desi-TSF-Graft 100 Lambardi et al. (2009)M. domestica (3) Desi-TSF-Graft 85 Vogiatzi et al. (2011a, 2011b)M. domestica (3) Desi-TSF-Graft 80 Jenderek et al. (2011)Malus spp. (1915) Desi-TSF-Graft 100 Towill et al. (2004)Malus spp. (10) Desi-TSF-Graft 100 Towill and Bonnart (2005)Malus spp. (36) Desi-TSF-Graft 90 Toldam-Andersen et al. (2007)Malus spp. (10) Desi-TSF-Graft 77 Höfer (2007)Malus spp. (362) Desi-TSF-Graft 100 Volk et al. (2008)Malus spp. (15) Desi-TSF-Graft 78 Guyader et al. (2012)Malus spp. TSF 35 Zamecnik et al. (2007)

Pyrus Pyrus spp. (15) Desi-TSF-Graft 92 Guyader et al. (2012)

a Number of cultivars or rootstocks tested is given in parentheses, when reported in cited literature.b En-vitrif = encapsulation–vitrification; Vitrif = vitrification; En-droplet-vitrif = encapsulation–droplet–vitrification; TSF = Two step-freezing, slow-cooling prior to storage in

LN; Desi-TSF-Graft = desiccation-two step freezing–grafting.c Prefreezing at −5, −10, −15, −20 and −30 °C for 24 h intervals, prior to storage in LN.d Prefreezing at −20 °C for 12 h, prior to storage in LN.


A protocol combining droplet-vitrification and encapsulationwas applied to six cultivars of different D. kaki types (astringent andnon-astringent) (Niu et al., 2010). Shoot tips from dormant budswere encapsulated in alginate beads with 0.4 M sucrose and trans-ferred to droplets of PVS2 solution placed on aluminium foil strips at0 °C. After exposure to PVS2 for 90 min and immersion in LN, 79%regrowth was achieved. The addition of an appropriate concentrationof Pluronic F-68, a non-ionic detergent that protects cells from hydro-dynamic damage, in preculture and regrowth medium improvedshoot tip survival after cryopreservation.

3.3. Malus

3.3.1. Shoot-tipsApple is the fruit tree for which the largest number of reports on

cryopreservation has been published. Different protocols have beenreported over the years: encapsulation–dehydration (Zhao et al.,1999a), vitrification (Niino et al., 1990, 1992b; Wu et al., 1999; Zhaoet al., 1995), droplet-freezing (Zhao et al., 1999b), and two-step freez-ing (Chang et al., 1992). In the last 12 years, many papers reportedvery high survival of shoot tips, ranging from 68% to 88% with all pro-cedures involving direct immersion of explants in LN (Table 1).

Encapsulation–dehydration of shoot tips of three apple cultivars(‘Golden delicious’, ‘Mutsu’, ‘Reinette Clochard’) and two rootstocks(M26, M111) resulted in high regrowth percentages (Paul et al.,2000). The highest shoot regrowth (88% in cv. ‘Golden delicious’)was reached after 12 weeks cold-hardening of mother-plants andovernight preculture of encapsulated axillary shoot tips in modifiedMS medium containing 0.75 M sucrose followed by dehydration to21% MC (FW). In the same study, shoot tips of cv. ‘Reinette Clochard’treated with a cryoprotectant mixture containing sucrose and ethyl-ene glycol displayed 77% regrowth after cryopreservation usingencapsulation–vitrification. In these conditions, cold-hardening of invitro mother-plants could be omitted. Encapsulation–dehydrationwas also applied to shoot tips of the ancient apple cultivar ‘Annurca’.Seventy-two percent regrowth was obtained after preculture of en-capsulated explants in liquid medium containing 0.75 M sucrose for1 day followed by dehydration to 19% MC (FW) (Forni et al., 2010).This report also investigated changes in metabolism and protein ex-pression during dehydration. The concentration of two polyamines,putrescine and spermidine, decreased in shoot tips compared withuntreated controls. Sucrose preculture induced significant changes in

expression protein profiles, with 17 proteins being down-regulatedand nine up-regulated, among which six stress-related proteinswere identified.

For cryopreservation of shoot tips of cv. ‘Gala’ using vitrification,Liu et al. (2004) employed a new vitrification solution named PVSL(Plant Vitrification Solution Liu), which contained 40% (w/v) glycerol,45% (w/v) sucrose, 10% (w/v) ethylene glycol (EG) and 10% (w/v)DMSO in MS basal medium. In comparison with PVS2, in the PVSL so-lution, EG and DMSO concentrations were decreased from 15% to 10%,while glycerol and sucrose concentrations were increased from 30%to 40% for the former, and from 13.7% to 45% (w/v) for the latter.Regrowth of cryopreserved shoot tips was 77%. The PVSL solutionwas also effective with cv. ‘World’, producing 76% shoot formation(Liu et al., 2008).

Halmagyi et al. (2010a) compared the efficiency of two dropletprocedures applied for cryopreservation of apple shoot tips, droplet-vitrification (using PVS2) and droplet-freezing (using 10% DMSO).The highest regrowth after cryopreservation using droplet-vitrification (60–70%) was achieved after preculture of shoot tips inliquid medium with 0.5 M sucrose for 24 h, dehydration with PVS2at 24 °C for 30 or 40 min (depending on the cultivar). The effects ofpreculture conditions on survival of shoot tips of two apple cultivarsafter cryopreservation using the droplet-vitrification procedure wereanalyzed (Halmagyi et al., 2010b). A preculture treatment in liquidMS medium with 0.5 M sucrose, glucose, mannitol or sorbitol wasapplied to the explants for 24 or 48 h. Preculture with sucrose for24 h led to the highest regeneration. Various sucrose concentrations(0.1–1 M) were used during preculture and different vitrification so-lutions (PVS, PVS2 and PVS3) tested. The optimal protocol includedpreculture with 0.5 M sucrose for 24 h and dehydration with PVS2,resulting in 66% (cv. ‘Romus4’) and 62% (M106 rootstock) regrowth.

Droplet-vitrificationwas applied to two apple cultivars (‘Pinova’ and‘Jonagold’) and two rootstocks (M26 and Jork9) (Condello et al., 2011).Improved regrowth after cryopreservation was observed when axillarybuds were taken from 4-month-old mother-plants (46%), compared toaxillary buds excised from 1-month-old (17%). The highest regrowth ofcryopreserved shoot tips (70%) was obtained when recovery took placeon MS medium containing 4.5 μM benzyl adenine and 0.5 μM indolebutyric acid, devoid of glycine.

Four different methods (two-step freezing, vitrification,encapsulation–dehydration and droplet-freezing) were tested for cryo-preservation of four apple cultivars (Wu et al., 2000). Although the


highest regrowth (around 80%) was achieved with droplet-freezing,based on all quantitative and qualitative observations performed duringexperiments (callus formation and speed of apices regrowth) theencapsulation–dehydration technique was selected by the authors forroutine cryopreservation of apple germplasm.

The effect of cold-acclimation on cryopreservation of apple shoottips and their cellular ultrastructure was investigated by Kushnarenkoet al. (2006). Shootswere cold-acclimated for up to 6 weeks using alter-nating temperatures, 22 °C for 8 h (light) and −1 °C for 16 h (dark).An intense starch accumulation inside plastids was observed after3 weeks cold-acclimation in cv. ‘Grushovka Vernenskaya’, which dis-played low cold hardiness, while small starch grains and two typesof plastoglobules were formed in plastids of cv. ‘Voskhod’, whichdisplayed high cold hardiness. Kushnarenko et al. (2009) reported nosignificant differences, when using vitrification or encapsulation–dehydration, in regrowth of cryopreserved shoot tips with three Malusdomestica cultivars and onewild type ofM. sieversii, whatever the dura-tion of the cold-acclimation period. These authors recommended3 weeks of cold-acclimation as standard protocol, as it induced goodregrowth with the five genotypes tested, even though higher regrowth(around 80%) was observed with the three genotypes showing highercold hardiness.

The assessment of genetic stability after cryopreservation hasbeen performed using classical and innovative techniques, includingmorphological, cytological and molecular tools (Hao et al., 2001; Liuet al., 2004, 2008). No genetic changes were found in plants recoveredfrom cryopreserved shoot tips, using RAPDs, AFLPs and ISSR (Haoet al., 2005; Harding, 1991, 2004).

3.3.2. Dormant budsMalus spp. dormant buds have been cryopreserved to establish a

base collection at the NCGRPP (Fort Collins, USA) (Forsline et al.,1998). Dormant bud cryopreservation procedures can be species-dependent but they usually require the ability of buds to withstanddesiccation to b30% MC and slow cooling to−35 °C prior to LN expo-sure (Table 2).

Towill et al. (2004) applied cryopreservation to dormant buds of alarge number of Malus accessions (1195 Malus domestica and 720other Malus species). Buds were collected after temperature hadremained below 0 °C for at least 72 h. They were stored at −5 °C for1 to 8 weeks. Nodal sections with one bud (35 mm) were desiccatedto 30% MC in a −5 °C cold room (4–6 weeks), then cooled at 1 °C/hfrom −5 °C to −30 °C and held at this temperature for 24 h, beforetheir transfer in the vapor phase of a LN storage container. Rewarmingwas slow, achieved by placing the samples overnight at 4 °C. Afterrewarming, explants were placed in peat moss for rehydration at2 °C for 15 days before grafting buds using the chip budding tech-nique. According to NCGRP standards, an accession was consideredsuccessfully cryopreserved when at least 40% of the grafted budsformed shoots. Among 1915 accessions cryopreserved, less than 9%showed viability below 30% (Towill et al., 2004).

Towill and Bonnart (2005) assayed the possibility of cooling Maluswinter vegetative buds without any prior desiccation of nodal sections,in order and to simplify the cryopreservation technique and to improveits efficiency, since the desiccation step requires time, is labor intensiveand can damage materials if excessive. Desiccation to achieve thedesired MC takes between 2 and 6 weeks and some accessions requireeven longer durations. Most buds from several apple species,cryopreserved without drying, regrew after cooling at 5 °C/day until−30 °C and holding for 24 h at this temperature prior to transfer inthe vapor phase of a LN storage tank. Volk et al. (2008) studied the via-bility of dormant buds of a subsample of 362 Malus accessions after10 years of storage in the vapor phase of a LN container. These authorsshowed that all buds rewarmed maintained a least 40% viability aftercryostorage, as determined by grafting.

When the standard protocol developed by Forsline et al. (1998)was applied to 36 apple cultivars grown in a mild maritime winter cli-mate, two-third of the cultivars tested showed recovery above 70%(Toldam-Andersen et al., 2007). Moreover, collecting scions after ex-tended frost periods did not seem critical to achieve survival.

Dormant buds of three ancient Italian apple cultivars showed dif-ferences in tolerance to cryopreservation; the best result was achievedwith cv. ‘San Piero’, when budswere cryopreservedwithMCs between30% and 26%; in such conditions, 100% regrowth was achieved afterchip budding (Lambardi et al., 2009).

Lambardi et al. (2011) analyzed the cost-efficiency of cryopreser-vation using PVS2 vitrification of in vitro shoot tips compared to slowcooling of dormant buds for long-term storage of ancient apple culti-vars. The dormant-bud method was more effective in terms of timeand labor, since it required about 49% of the time and 50% of thelabor required to implement the shoot-tip vitrification approach.

Other examples of the application of cryopreservation to appledormant buds can be found in Wu et al. (2001b) and Höfer (2007).Investigations on the status of water after desiccation at −4 °C andduring the interval from−4 °C to−30 °C, together with the influenceof winter conditions on cryopreservation of apple dormant buds werecarried out by Vogiatzi et al. (2011a, 2011b, 2012). Most recently, aprotocol adapted from the one developed at NCGRP, USA (Towill andEllis, 2008; Towill et al., 2004) was tested in France using 15 differentcultivars (Guyader et al., 2012). Regeneration after cryopreservationand grafting ranged between 0% and 78%, with an average of 32%.

To study the effect of the intensity of cold-hardening of mother-plants in the field on survival of dormant buds after cryopreservation,Jenderek et al. (2011) cryopreserved dormant buds of three apple cul-tivars (Braeburn, Jonagold and Liberty) grown in different locations ofthe United States (Davis, CA; Corvallis, OR; Geneva, NY) following theprocedure by Forsline et al. (1998). No significant difference wasdetected in cryopreservability of dormant buds among the locations,even though, surprisingly, dormant buds sampled in Geneva, wherethe lowest pre-harvest temperatures were measured, displayed thelowest survival.

3.4. Olea europaea

In olive, different organs and tissues have been cryopreserved in-cluding embryos, embryogenic tissues, stones (complete seeds),seeds without endocarp and shoot tips (Revilla et al., 2002). Olive em-bryogenic lines appeared to be highly suitable materials for cryopres-ervation (Benelli et al., 2001; Lynch et al., 2011; Sanchez-Romero et al.,2009; Shibli and Al-Juboory, 2000). However, despite the difficultiesfaced with in vitro culture of olive shoots, reports on cryopreservationof shoot tips appear promising. Using vitrification, Benelli et al. (2001)and Nisi et al. (2006) obtained 15% and 30% post-rewarming shoot tipsurvival with cvs. ‘Canino’ and ‘Gentile di Larino’, respectively. Surviv-ing shoot tipswere green after rewarming, but showed poor regrowth.A regrowth of 38% was observed in cv. ‘Frantoio’ following two stepdehydration with PVS2 (50% PVS2 for 30 min, then 100% PVS2 for1 h), direct immersion in LN and post-rewarming culture on mediumcontaining a high concentration of zeatin (46 μM) (Lynch et al., 2007).Histological examination revealed that neither the PVS2 treatmentnor the immersion in LN did modify the apex structure; however,changes were observed in sub-apical cells after PVS2 treatment andimmersion in LN. These alterations, such as cell wall gelification, dehy-dration of external parenchyma cells and cellular starch accumulation,may contribute to explain the low regrowth percentage observed incryopreserved shoots.

3.5. Prunus

Before year 2000, Prunus cryopreservation had been performedusing embryonic axes, embryogenic tissues, dormant buds and in


vitro shoot tips (De Boucaud et al., 2002). Further research wasperformed only on in vitro shoot tips.

Using the vitrification technique, De Carlo et al. (2000) obtained 57%survival with shoot tips of P. domestica cv. ‘Regina Claudia’ preculturedat 4 °C for 2 days on medium with 0.09 M sucrose, loaded for 30 minwith a solution containing 2 M glycerol and 0.4 M sucrose, dehydratedwith PVS2 at 0 °C for 90 min and directly plunged in LN. With P. dulcis(cvs. ‘Non pareil’, ‘Nec Plus Ultra’ and the almond-peach hybrid‘Nemaguard’), shoot tips precultured on medium with 0.7 M sucrosefor 1 day, dehydrated with PVS2, for 45 min for the cultivar and60 min for the hybrid rootstock, showed no significant differences insurvival after 3 days (87–60% survival; Channuntapipat et al., 2000)and 2 years (80–54% survival; Wirthensohn et al., 2006) of LN storage.With P. dulcis shoot tips, 67% regrowth was obtained after 4 weeks ofcold-hardening ofmicroshoots onmediumwith 0.3 M sucrose followedby 120 min exposure to PVS2 (Al-Ababneh et al., 2003). Mother-plantsof P. salicinawere precultured at 5 °C for 21 days, shoot tips preculturedin medium with 0.7 M sucrose for 2 days, treated with PVS3 for100 min before direct immersion in LN. Under such conditions, survivalafter cryopreservation was above 60% (Zhao et al., 2008b).

De Boucaud et al. (2002) obtained good results using the dropletmethod with various Prunus genera. Regrowth after cryopreservationwas 52% with plum cv. ‘Ogden’, 47% with cv. ‘Torinel’, 32% with P.domestica, 40% with almond cv. ‘Chazanov’ and 58% with cv. ‘Samisch’.

Recently, the droplet-vitrification technique was successfully em-ployed for cryopreserving P. cerasifera shoot tips (Vujovic et al.,2011). Following treatment for 30 min with a loading solution com-prising 1.9 M glycerol and 0.5 M sucrose, shoot tips were dehydratedwith PVS2 or PVS3 for 10–30 min and 60–120 min, respectively.Cherry plum shoot tips were very sensitive to both vitrification solu-tions and growth recovery of cryopreserved samples was generallylow (5–20%). No significant influence of PVS treatment (both natureof solution and treatment duration) on regrowth of cryopreservedshoot tips was observed.

When applying encapsulation–dehydration to P. dulcis (rootstockM51and cv. ‘Ferragnes’), about 60% survivalwas obtained after cryopres-ervation following desiccation to 20% MC for the cultivar and 19% forthe rootstock (Shatnawi et al., 2000). Vitrification and encapsulation–dehydration produced similar regrowth (76–78%) with P. aviumshoot tips (Shatnawi et al., 2007). De Boucaud et al. (2002) usedencapsulation–dehydration with shoot tips of three plum cultivars(‘Ferley’, ‘Ferlenain’ and ‘GF8I’), showing regrowth between 40 and55%. In the case of bitter almond, about 60% survival was achievedfollowing 5 weeks cold-acclimation of mother-plants, pretreatmentwith 0.75 M sucrose for 1 day and dehydration of beads for 6 h(bead MC 20%) before LN exposure (Al-Ababneh et al., 2003).

3.6. Pyrus

3.6.1. Shoot tipsIn vitro shoot apices of four Pyrus species were successfully cryo-

preserved for the first time in 1990 using slow freezing (Reed, 1990)and encapsulation–dehydration (Dereuddre et al., 1990). A compari-son of slow freezing and vitrification performed on 28 Pyrus genotypesshowed that slow freezing (0.1 °C/min) regrowth produced highersurvival (61%), compared to vitrification (43%) (Luo et al., 1995;Reed et al., 1998).

In the last 12 years, five reports were published on cryopreservationof shoot tips of various Pyrus species using different methods (Table 1).Several of these reports showed that cold acclimation or treatmentwith abscisic acid (ABA) ofmother-plantswas necessary for cryopreser-vation of many pear genotypes. Alternating temperatures (22 °C light/−1 °C dark with various photo- and thermoperiods) for 2 to 5 weekssignificantly increased regrowth of cryopreserved shoot tips in compar-ison with those sampled on mother-plants kept at constant tempera-ture (Chang and Reed, 2000). A 3-week pretreatment on medium

with 50 μMABA or high sucrose (5–7%) followed by a 2-week exposureto low temperature was optimal to obtain high regrowth (75%) aftercryopreservation of shoot tips of P. cordata (Chang and Reed, 2001).Using vitrification, survival of cryopreserved Pyrus spp. shoot tipswas 71% after dehydration with 60% PVS2 for 20 min followed by100% PVS2 for 2 h (Wang et al., 2008). Shoot tips of P. pyraster werecryopreserved by encapsulation–dehydration (Condello et al., 2009).After preculture for 2 days in liquidmediumwith 0.75 M sucrose, dehy-dration of the beads with silica gel to 20% MC and immersion in LN,shoot tip survival was 60%. In this study, no differences betweencryopreserved plants and mother-plants were detected using RAPDand SSR markers. In another study, the stability of the transgenic GUSconstruct in cv. ‘Okusankichi’ (P. pyrifolia) shoots cryopreserved usingencapsulation–dehydration was assessed (Hao et al., 2005). Survivalwas very high (82%); single-strand conformation polymorphism(SSCP) analysis did not detect any sequence variation in GUS fragmentand transient gene expression was present normally in recoveredshoots.

3.6.2. Dormant budsOnly one report has been published on cryopreservation of Pyrus

dormant buds (Guyader et al., 2012) (Table 2). The protocolemployed was adapted from that developed for apple dormant budsat NCGRP, USA (Towill and Ellis, 2008; Towill et al., 2004). Nodal sec-tions, 35 mm in length, were sampled from trees of 15 different cul-tivars in January 2010, stored at −5 °C until they reached 23% MC,slowly cooled at 1 °C/h to −30 °C, kept at this temperature for 24 hbefore immersion in LN. After slow rewarming and rehydration,buds were grafted on rootstocks using the chip budding technique.Regrowth of cryopreserved buds ranged between 0% and 92%, withan average recovery percentage after grafting of 30% for the 15 culti-vars tested.

3.7. Vitis

Apices from in vitro shoots of Vitis were cryopreserved more than20 years ago using encapsulation–dehydration (Plessis et al., 1991).More recently, Wang et al. (2000) obtained 60% and 40% survivalwith shoot tips of ‘LN33’ hybrid and cv. ‘Superior’, respectively, usingthe following protocol: preculture of encapsulated shoot tips in liquidmedium with daily increasing sucrose concentrations (0.25, 0.5, 0.75,1 M), dehydration to 15.6–17.6% MC and direct immersion in LN. Op-timal recovery of ‘LN33’ hybrid shoot tips cryopreserved by encapsula-tion–dehydration was achieved with the addition of 3–4 μM BA to theregrowth medium (Wang et al., 2003a). In another study, encapsulat-ed axillary buds of three cultivars (‘Cabernet franc’, ‘Chardonnay’,‘Fengh 51’) and of one rootstock (LN33) were precultured at 5 °C onmedium with daily increasing sucrose concentrations (0.1, 0.3, 0.7,1.0 M), desiccated to 21%MC and cryopreserved using a two-step pro-tocol (prefreezing down to −40 °C followed immersion in LN) (Zhaoet al., 2001). The highest regrowth after cryopreservation was 41%.The multiplication rate of in vitro plantlets regrown from cryo-preserved samples was lower than that of non-cryopreserved controlsafter the first subculture. However, it became equivalent to that ofnon-cryopreserved shoot tips at the end of the sixth subculture. Pre-liminary experiments using encapsulation–vitrification with shoottips of the rootstock ‘Kober 5BB’ showed that survival and regrowthafter rewarming was sporadic (Benelli et al., 2003).

Vitis spp. shoot tips were also cryopreserved using the vitrificationtechnique, involving either one-step dehydration with PVS2 for80 min at 0 °C before immersion in LN (about 60% shoot tip survival)or two-step dehydration (50% PVS2 for 30 min, followed by PVS2 for50 min at 0 °C), which increased survival to 80% (Matsumoto andSakai, 2000, 2003).

Genetic stability of in vitro plants regenerated from shoot-tipscryopreserved by encapsulation–dehydration (regrowth 36%), was


assessed using RAPD markers; the profiles observed were similar be-tween control and cryopreserved explants (Zhai et al., 2003).

4. Concluding remarks and future prospects

In this paper, we have shown that cryopreservation of economical-ly important fruit trees has made dramatic progress over recent years,using both dormant buds and in vitro shoot tips. Routine application ofthis technique for long-term germplasm storage in genebanks is cur-rently limited to apple and pear, for which large cryopreserved collec-tions have been established at NCGRP, Fort Collins (USA), usingdormant buds and in vitro shoot tips, respectively. However, thereare a growing number of examples of pilot scale testing experimentsunder way for different species in various countries. Progress in thefurther development and application of cryopreservation techniqueswill be made through a better understanding of the mechanisms in-volved in the induction of tolerance to dehydration and cryopreserva-tion in frozen explants. Research focused on different aspects of thesemechanisms is ongoing in an increasing number of laboratoriesworldwide.

Recently, it has been shown that cryopreservation can be em-ployed for other uses than germplasm conservation. Cryopreservationhas been used for eradicating viruses (cryotherapy), as a substitute orin complement to classical virus eradication techniques such as mer-istem culture and thermotherapy (Wang et al., 2008; Wang et al.,2009). Cryotherapy of shoot tips was used for the first time on a Pru-nus rootstock for elimination of the Plum Pox Virus (Brison et al.,1997). More recently, it was found efficient for elimination of BananaStreak Virus (BSV) and Cucumber mosaic virus (CMV) in banana(Helliot et al., 2002), Grape virus A (GVA) in grape (Wang et al.,2003b), and Potato virus Y (PVY) from potato. The Sweet potato littleleaf (SPLL) phytoplasma in sweet potato and Citrus Huanglongbing(HLB), caused by a Gram-negative bacterium, were both successfullyeliminated applying cryotherapy of shoot tips (Ding et al., 2008;Wang and Valkonen, 2009). Wang et al. (2003b) showed that cryo-preservation led to 97% elimination of grape virus A, against only12% by meristem culture, thereby illustrating the high potential ofcryotherapy. The hypothesis is that cryotherapy is based on selectivecell destruction by cryopreservation: the more differentiated cellswhich contain viruses have a high water content and are killed byformation crystals ice during cooling and rewarming, whereasmore meristematic cells, which have a lower water content andgenerally do not contain viruses withstand cryopreservation (Wanget al., 2009). In genebanks, cryotherapy could be adopted inpathogen-eradication schemes for species and genotypes that aregoing to be cryopreserved, thus offering the simultaneous opportuni-ty to conserve fruit trees displaying interesting features and to re-cover virus-free plants.

In conclusion, it is now well recognized that an appropriate con-servation strategy for a particular plant genepool requires a holisticapproach, combining the different ex situ and in situ conservationtechniques available in a complementary manner (Engelmann,2012). In situ and ex situ methods are options available for the differ-ent genepool elements. Selection of the appropriate methods shouldbe based on a range of criteria, including the biological nature of thespecies in question, practicality and feasibility of the particularmethods chosen (which depend on the availability of the necessaryinfrastructures) as well as the cost-effectiveness and security affordedby their application. Considerations of complementarity with respectto efficiency and cost-effectiveness of the various conservationmethods chosen are also important. In this regard, it is important tomention that a detailed economic study of the costs of establishingandmaintaining a coffee germplasm collection, either as whole plantsin the field or as seeds under cryopreservation clearly showed the ben-efits of using cryopreservation (Dulloo et al., 2009). Inmany instances,the development of appropriate complementary conservation

strategieswill still require further research to define the criteria, refinethemethods and test their application for a range of genepools and sit-uations. In this context, it is important to stress that the increasinglyefficient cryopreservation techniques developed over recent yearsare not seen as a replacements for conventional ex situ approaches.They offer curators of fruit tree species (and other species) genebanksadditional tools to allow them optimizing the conservation of germ-plasm collections placed under their responsibility.

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