MICROPROPAGATION

Shoot regeneration and cryopreservation of shoot tips of apple(Malus) by encapsulation–dehydration

Bai-Quan Li & Chao-Hong Feng & Ling-Yun Hu &

Min-Rui Wang & Long Chen & Qiao-Chun Wang

Received: 9 August 2013 /Accepted: 4 April 2014 / Editor: Barbara Reed# The Society for In Vitro Biology 2014

Abstract Here, we report an efficient and widely applicablemethod for cryopreservation of Malus shoot tips by encapsu-lation–dehydration using adventitious shoots. Shoots wereinduced from leaf segments cultured on a shoot inductionmedium containing 2–3 mg L−1 thidiazuron, depending ongenotype, and 0.5 mg L−1 indole-3-butyric acid. Shoot tips(3 mm in length) containing six leaf primordia excised from11-wk-old adventitious shoots were encapsulated andprecultured with 0.5 M sucrose for 5 d, followed by air-drying for 6 h prior to direct immersion in liquid nitrogen.With our protocol, we obtained a mean organogenesis rate of100%, a mean of 4.5 adventitious shoots per explant (leafsegment), and a mean shoot recovery of 57.0% from cryopre-served shoot tips in fourMalus species. Inter-simple sequencerepeat (ISSR) analysis did not reveal any polymorphic bandsin regenerants recovered from either leaf segments or cryo-preserved shoot tips of ‘Gala’. To the best of our knowledge,this is the first report on cryopreservation of Malus shoot tipsusing adventitious shoots derived from leaf segments and isthe most widely applicable protocol so far reported for cryo-preservation ofMalus. Establishment of this protocol providesan alternative means for cryopreservation of Malus.

Keywords Cryopreservation . Encapsulation–dehydration .

ISSRmarker .Malus . Shoot regeneration . Shoot tip

Introduction

Apple (Malus) is one of the most economically important fruitcrops in the world. The genus Malus contains about 27 spe-cies; among which, about 17 species are native to China (Li1999). Conservation and availability of genetic resourcesprovide basic support for breeding novel cultivars by bothtraditional and genetic engineering programs. Having severaldistinct advantages such as the capability for long-term stor-age, minimal requirement for storage space, and maintenanceof genetic integrity of stored materials, cryopreservation haslong been considered an ideal means for long-term conserva-tion of plant genetic resources (Benson 2008).

Since Kuo and Lineberger (1985) attempted for the firsttime to cryopreserve apple shoot tips, various cryopreserva-tion protocols have been developed such as two-step freezing(Wu et al. 1999), droplet (Zhao et al. 1999; Halmagyi et al.2010), vitrification (Niino et al. 1992; Kushnarenko et al.2009; Wu et al. 1999), encapsulation–dehydration (Niinoand Sakai 1992; Wu et al. 1999; Zhao et al. 1999; Paulet al. 2000; Kushnarenko et al. 2009; Feng et al. 2013),encapsulation–vitrification (Paul et al. 2000), and dropletvitrification (Condello et al. 2011; Halmagyi et al. 2010).Recently, cryo-banking of apple germplasm has beenestablished at the National Center for Genetic ResourcesPreservation, Fort Collins, CO, USA, in which 1,915 acces-sions, representing 30 species and 16 interspecific hybrids,have been cryopreserved using both dormant buds and in vitroshoot tips (Towill et al. 2004).

Several issues still need to be addressed with regard tocryopreservation of apple shoot tips. First, in most of theprevious studies, in vitro stock cultures had to be cold hard-ened at 5°C for 3–4 wk (Niino and Sakai 1992; Niino et al.1992; Wu et al. 1999; Zhao et al. 1999), at 4°C for 12 wk(Paul et al. 2000) or at alternating temperatures (22°C for 8 hin light and −1°C for 16 h in darkness) for 3 wk (Kushnarenko

Bai-Quan Li and Chao-Hong Feng contributed equally to the presentstudy

B.<Q. Li : C.<H. Feng : L.<Y. Hu :M.<R. Wang : L. Chen :Q.<C. Wang (*)State Key Laboratory of Crop Stress Biology for Arid Areas, KeyLaboratory of Genetic Improvement of Horticultural Crops ofNorthwest China of Ministry of Agriculture of China, College ofHorticulture, Northwest A&FUniversity, Yangling, 712100 Shaanxi,People’s Republic of Chinae-mail: qiaochunwang@nwsuaf.edu.cn

In Vitro Cell.Dev.Biol.—PlantDOI 10.1007/s11627-014-9616-2

et al. 2009) to ensure high shoot regrowth rates followingcryopreservation. Cold hardening requires a temperature-controlled growth chamber and is time-consuming. Recently,Halmagyi et al. (2010) and Condello et al. (2011) reportedsuccessful cryopreservation of apple shoot tips by a dropletvitrification method, in which cold-hardening of in vitro stockcultures was eliminated. However, the cultures had to bemaintained on the stock culture medium without subculturefor 2 mo (Halmagyi et al. 2010) or 4 mo (Condello et al.2011). More recently, Feng et al. (2013) described an encap-sulation–dehydration protocol for apple shoot tips using 4-wk-old stock cultures without a cold-hardening treatment,thus simplifying the cryopreservation procedure. Second, api-cal shoot tips were used in all previous studies, meaning thatonly one shoot tip could be obtained from each in vitro shoot.Efficiency of shoot tip production significantly affects the rateat which materials can be processed for cryopreservation.Adventitious buds derived from in vitro culture may providea more efficient means to produce shoot tips for cryopreser-vation (Matsumoto et al. 1995; Burritt 2008). In Malus, leafsegments are the most frequently used explant for efficientshoot regeneration (Dobránszki and Teixeira da Silva 2010;Magyar-Tábori et al. 2010). Apple shoot regeneration haspotential applications for micropropagation (Dobránszki andTeixeira da Silva 2010) and production of artificial seeds(Brischia et al. 2002), and has been widely used in applegenetic transformation (Aldwinckle andMalnoy 2009). Third,a widely applicable cryopreservation protocol is still lacking(Halmagyi et al. 2010; Condello et al. 2011; Feng et al. 2013),thus severely limiting the ability to cryo-bank applegermplasm.

The objective of the present study was therefore to developan efficient, widely applicable cryopreservation method forapple shoot tips using adventitious buds derived from leafsegments. The genetic integrity of regenerants from leaf seg-ments and cryopreserved shoot tips was assessed using inter-simple sequence repeat (ISSR) markers.

Materials and Methods

Plant material. Two apple scion cultivars, ‘Gala’ and ‘Fuji’(Malus × domestica), and two rootstocks, ‘M9’ and ‘M26’(Malus pumila paradisiaca), were used for optimizing thecombination of plant growth regulators (PGRs) for adventitiousshoot regeneration from leaf segments (Table 1). Five addition-al genotypes including three scion cultivars (M. × domestica)and two wild species (Malus robusta and Malus micromalus)were then used to test the new protocol (Table 2). M. robustaand M. micromalus are native to China, highly resistant todrought and mainly used for breeding drought-resistant root-stocks. ‘Gala’wasmainly used to establish the cryopreservation

procedure, which was then further tested for its potential appli-cation to the other nine genotypes.

In vitro shoot stock cultures were maintained on a basicmedium (BM) composed of Murashige and Skoog (MS)medium (Murashige and Skoog 1962) supplemented with30 g L−1 sucrose, 0.25 mg L−1 6-benzyladenine (BA),0.01 mg L−1 indole-3-butyric acid (IBA), and 8 g L−1 agar(Feng et al. 2013). The pH of the BMwas adjusted to 5.8 priorto autoclaving for 20 min at 121°C. Cultures were maintainedat 22±2°C under a 16-h photoperiod at 50 μmol m−2 s−1

provided by cool-white fluorescent tubes. Subculture wasperformed once every 4 wk.

Shoot regeneration. The first three fully opened leaves(Fig. 1a) were excised from 4-wk-old in vitro shoot stockcultures. Leaf segments were trimmed to 0.8×0.6 cm, andfour cuts at 1-mm intervals were made transversally to themidrib (Fig.1b). To select an optimal combination ofthidiazuron (TDZ) and IBA for shoot regeneration, leaf seg-ments were cultured with their adaxial surface in contact withshoot regeneration medium (SRM) containing 2, 3, or4 mg L−1 TDZ alone or in combination with 0, 1, 1.5, or2.0 mg L−1 IBA. The cultures were kept at 22±2°C in the darkfor 3 wk before transfer to the light conditions described forin vitro shoot stock cultures. Subculture was done once every4 wk. The organogenesis rate (defined as the percentage ofleaf segments regenerating shoots out of the total explantsused) and the number of shoots (≥3 mm in length with at leasttwo fully expanded leaves) per explant were recorded after8 wk of culture.

The shoot regeneration procedure developed above wasthen applied to other eight Malus genotypes (Table 2).Data in this experiment were recorded after 11 wk ofculture.

Cryopreservation and shoot recovery. The encapsulation–de-hydration procedure was performed according to Feng et al.(2013). Shoot tips 1, 2, or 3 mm (Fig. 1g) in length andcomprising two to three, four to five, or six leaf primordia(LPs) (Fig. 1g) were excised from adventitious shoots derivedfrom leaf segments that had been cultured for 8, 9, 10, 11, or12wk and placed onBM for 1 d. Shoot tips were suspended inMS medium supplemented with 2.5% (w/v) Na-alginate, 2 Mglycerol, and 0.4 M sucrose. The mixture, including shoottips, was dropped with a sterile pipette into MS inorganicmedium containing 0.1 M CaCl2, 2 M glycerol, and 0.4 Msucrose and left for 20 min to form beads, each being about4 mm in diameter and containing one shoot tip (Fig. 1h). Thebeads were precultured on MS medium containing 0.5 Msucrose for 1 to 7 d, followed by air-drying in a laminar flowcabinet (SW-CJ-2FD, Jiangshu, China) (Fig. 1i) for 6 h toreduce the water content of the beads to 20–22% (Feng et al.2013). The dehydrated beads were then transferred into

LI ETAL.

Table 1 Effect of different combinations of TDZ and IBA on adventitious shoot regeneration from leaf explants of four Malus genotypes

Growth regulators (mg/L) Genotype

Malus × domestica Malus pumila

‘Gala’ ‘Fuji’ ‘M9’ ‘M26’

TDZ IBA Organogenesis(%)

No. shoots/explant

Organogenesis(%)

No. shoots/explant

Organogenesis(%)

No. shoots/explant

Organogenesis(%)

No. shoots/explant

2 0 100a 1.5±0.2b 67±3c 0.1±0.1c –z – – –

0.5 100a 6.4±0.3a 100a 2.9±0.1a – – – –

1 90±10a 1.8±1.0b 96±3a 1.3±0.3b – – – –

1.5 100a 2.0±0.9b 100a 1.4±0.5b – – – –

3 0 100a 2.0±0.2b 77±3b 0.1±0.0c 93±5a 0.1±0.0c 100a 0.1±0.0d

0.5 100a 2.7±1.7b 90±10a 3.1±0.3a 100a 4.7±0.2a 97±3a 4.2±0.2a

1 100a 2.5±0.4b 97±3a 1.8±0.2b 100a 3.8±0.9a 100a 4.3±0.3a

1.5 100a 3.7±0.2b 60±6c 0.4±0.2c 97±3a 4.4±0.4a 100a 4.6±0.2a

2 – – – – 100a 1.5±0.3b 100a 1.6±0.3b

4 0 – – – – 80±12b 0.5±0.2c 70±8b 0.1±0.0d

0.5 – – – – 80±12b 2±0.2c 67±9b 0.2±0.1d

1 – – – – 90±6ab 1.2±0.6bc 77±15b 0.9±0.3c

1.5 – – – – 93±7ab 0.9±0.2bc 57±7b 0.3±0.2d

Leaf segments (∼0.8×0.6 cm) from the top three fully expanded leaves were cultured on SRM containing different concentrations of TDZ and IBA.Percent organogenesis and number of adventitious shoots (>3 mm in length) per leaf segment were recorded after 8 wk of culture and are presented asmean±SE. Data were analyzed using one-directional ANOVA and Student’s t test. Means in the same column followed by different letters indicatesignificant difference at P<0.05 by least significant difference (LSD) testz Not tested

Table 2 Adventitious shoot regeneration from leaf explants of nineMalus genotypes

Genotype TDZ/IBA (mg L−1)

2:0.5 3:0.5

Organogenesis (%) No. shoots/explant Organogenesis (%) No. shoots/explant

Malus × domestica

‘Gala’ 100 8.7±0.3 100 4.6±0.7

‘Fuji’ 100 5.6±0.7 100 5.3±0.3

‘Himekami’ 100 1.5±0.2 100 1.9±0.5

‘Greensleeves’ 100 4.9±0.4 100 4.7±0.3

‘Wangshanhong’ 100 1.2±0.1 100 2.0±0.5

Malus pumila paradisiaca

‘M9’ –z – 100 6.6±0.4

‘M26’ – – 100 6.3±0.5

Malus robusta 100 3.5±0.2 100 1.6±0.2

Malus micromalus 100 0.4±0.1 100 0.6±0.1

Mean from the best combination Organogenesis (%) No. of shoots/explant

100 4.5

Leaf segments (∼0.8×0.6 cm) from the top three fully expanded leaves were cultured on two types of SRM that gave good responses in the precedingstudy (Table 1). Percent organogenesis and number of adventitious shoots (>3 mm in length, with at least two fully expanded leaves) per leaf segmentwere recorded after 11 wk of induction and are presented as mean±SEzNot tested

CRYOPRESERVATION OFAPPLE SHOOT TIPS

1.8-mL cryogenic tubes (10 beads per tube) and directlyplunged into liquid nitrogen (LN) for 1 h of storage.

Cooled cryogenic tubes containing shoot tips were rapidlyrewarmed in a water bath at 38°C for 2 min. The shoot tipswere then post-cultured on BM for recovery. The cultureswere grown in the dark at 22±2°C for 3 d and then transferredto the light conditions as described for the in vitro stockcultures. During the 3 d of post-culture in the dark, the beadswere transferred twice (after 12 and 36 h) onto fresh BM to

prevent browning of the shoot tips. Shoot recovery was de-fined as the percentage of shoot tips that regenerated intonormal shoots (≥3 mm in length with at least two fully openedleaves) after 8 wk of post-culture. Subculture was done onceevery 4 wk. Recovered shoots were transferred onto a rootingmedium containing MS supplemented with 30 g L−1 sucrose,0.5 mg L−1 naphthalene acetic acid (NAA), and 8 g L−1 agar(pH 5.8). After 4 wk of rooting, shoots that had well-developed roots were transferred into soil and established

Figure 1 Shoot regenerationfrom leaf segments and shoot tipsof Malus domestica ‘Gala’cryopreserved by encapsulation–dehydration. (a)A 4-wk-old stockshoot culture. (b) A leaf segmentwith four transverse cuts acrossthe midvein on the abaxial side,used for shoot regeneration. (c)Small meristemoids (Me) formedfrom callus after 11 d of culture onSRM. (d)Magnified view of therectangular area in (c), showingmeristems. (e) An adventitiousbud with leaf primordia (LPs)after 16 d of culture on SRM. (f)Adventitious shoots regeneratedfrom leaf segments after 11 wk ofculture on SRM. (g) A typicalshoot tip (3 mm in length)containing six LPs excised froman 11-wk-old adventitious shootand used for cryopreservation. (h)Encapsulated shoot tips. (i) Air-drying of encapsulated shoot tipsin a laminar flow cabinet. (j) Asurviving shoot tip after 7 d ofpost-culture. (k) A recoveredshoot after 8 wk of post-culture.(l) A rooted shoot after 4 wk ofrooting on rooting medium.

LI ETAL.

under the greenhouse conditions according to Wang et al.(2005).

The encapsulation–dehydration procedure developedabove was further applied to other eight Malus genotypes(Table 3) to assess percent shoot recovery.

Assessment of genetic stability by inter-simple sequence re-peat analysis. Shoots from ‘Gala’ were either regeneratedfrom leaf segments that had been cultured for 11 wk orrecovered from cryopreserved shoot tips after 3 mo of post-culture. In vitro shoot stock cultures served as a control. Thirtysamples were randomly selected from each of 150 shootsregenerated from leaf segments, 100 from cryopreservedshoot tips, and 200 from in vitro stock cultures and subjectto ISSR analysis according to Yin et al. (2013).

Genomic DNA was extracted from 150 mg of fresh leaftissue using a Plant Genomic DNA Kit (Tiangen, Beijing,China), according to the manufacturer’s instructions. Purifiedtotal DNAwas quantified and its quality verified by ultravioletspectrophotometry. Each sample was diluted to 50 ng μL−1 inTris–EDTA buffer and stored at −20°C until use. Fifty ISSRprimers (Pathak and Dhawan, 2012) were screened to selectsuitable primers for assessment of genetic stability of theshoots. PCR was performed in a 25-μL reaction solutioncontaining 1 μL template DNA (50 ng μL−1), 1 μL primer(10 μM), 12.5 μL TaqMix (CoWin Biotech, Beijing, China),

and 10.5 μL H2O. DNA amplification was performed in aPCR instrument (Eppendorf, Hamburg, Germany) using thefollowing reaction conditions: initial denaturation step at 95°Cfor 5 min; followed by 45 cycles at 94°C for 45 s, 53°C for40 s, and 72°C for 70s; and followed by a final extension stepat 72°C for 7 min. The PCR products were separated byelectrophoresis in 1.5% agarose gel containing 0.1% ethidiumbromide and visualized under ultraviolet light. A molecularmass ladder (DNA marker DS2000; Dongsheng, Guangzhou,China) was used for estimating the size of the amplifiedproducts. ISSR analysis was manually scored for the presence(1) and absence (0) of each band. Bands of equal molecularweight and mobility generated by the same primer wereconsidered to represent the same locus. Both distinct mono-morphic bands and polymorphic bands were scored. Electro-phoretic DNA bands of low visual intensity that could not bereadily distinguished as present or absent were consideredambiguous markers and were not scored; only reliable andrepeatable bands were included in the data analysis.

Experimental design and statistical analysis. For shoot regen-eration and cryopreservation, each experiment included 10samples in each of three replicates. All experiments wereconducted twice. Data were presented as means with theirstandard errors and analyzed using one-directional ANOVAand Student’s t test. Least significant differences (LSD) werecalculated at P<0.05. The ISSR analysis was performed twiceto confirm the repeatability of the scored bands.

Results

Overall process of shoot regeneration from leaf segments. -When cultured on SRM, leaf segments of ‘Gala’ rolled upafter 2–3 d of culture and started to swell after about 4 d ofculture. Callus formation was observed at the cuts across themidvein and edge of the leaf segments after about 7 d ofculture. Small protuberances were visible from the callus afterabout 11 d of culture (Fig.1c, d). These protuberances contin-ued to grow and developed into adventitious buds (Fig. 1e)after about 16 d of culture. These adventitious buds continueddevelopment and growth and formed shoots (≥3 mm in lengthwith at least two to three fully opened leaves) after 8 wk ofculture. The number of adventitious buds increased and theirgrowth continued up to 11 wk of culture (Fig. 1f). The patternsof shoot regeneration of ‘Fuji’, ‘M9’, and ‘M26’ were similarto that of ‘Gala’. The shoot regeneration response was fastestin ‘Gala’, followed by ‘M9’, ‘M26’, and ‘Fuji’.

Effect of TDZ and IBA concentrations on shootregeneration. For ‘Gala’, although almost all leaf segmentsshowed an organogenic response when cultured on SRM

Table 3 Application of the encapsulation–dehydration procedure to nineMalus genotypes

Species and cultivars Shoot recovery (%)

+LN −LN

Malus pumila paradisiaca

‘M9’ 74.0±4.1 88.3±2.1

‘M26’ 52.5±4.8 70.0±4.1

Malus × domestica

‘Gala’ 79.3±6.2 81.3±5.5

‘Fuji’ 62.5±2.5 76.3±2.8

‘Himekami’ 27.5±2.5 48.3±4.4

‘Wangshanhong’ 43.8±2.4 60.0±4.1

‘Greensleeves’ 0 7.5±2.0

Malus robusta 50.0±0 57.5±2.5

Malus micromalus 66.3±5.5 90.0±4.1

Averagez 57.0 71.5

Shoot tips (3 mm in length) containing six leaf primordia were excisedfrom adventitious shoots regenerated from leaf segments that had beencultured on SRM for 11 wk. Encapsulated shoot tips were preculturedwith 0.5 M sucrose for 5 d and dehydrated by air-drying in a laminar flowcabinet for 6 h prior to direct immersion in LN for 1 h. Shoot regrowthfrequencies were recorded after 8 wk of post-culture and are presented asmean±SEzAverages were calculated without data from ‘Greensleeves’

CRYOPRESERVATION OFAPPLE SHOOT TIPS

containing 2 or 3 mg L−1 TDZ alone or in combination with0–1.5 mg L−1 IBA, the greatest number of shoots per explant(6.4) was obtained when a combination of 2.0 mg L−1 TDZand 0.5 mg L−1 IBA was used (Table 1). For ‘Fuji’, use ofTDZ alone at 2–3 mg L−1 markedly reduced the rate oforganogenesis (67–77%) (Table 1); significantly higher or-ganogenesis rates (96–100%) were obtained on SRM contain-ing either 2.0 mg L−1 TDZ in combination with 0.5–1.5 mg L−1 IBA or 3.0 mg L−1 TDZ in combination with0.5–1.0 mg L−1 IBA. The greatest number of adventitiousshoots per leaf segment (2.9–3.1) was obtained on SRMcontaining 2.0–3.0 mg L−1 TDZ in combination with0.5 mg L−1 IBA (Table 1). For the two rootstocks ‘M9’and ‘M26’, high organogenesis rates (93–100%) wereproduced in the leaf segments cultured on SRM contain-ing 3.0 mg L−1 TDZ alone or in combination with 0.5–2 mg L−1 IBA (Table 1). SRM containing 4.0 mg L−1

TDZ and 1.0–1.5 mg L−1 IBA also gave high organogen-esis rates (90–93%) in rootstock ‘M9’, but SRM contain-ing 4.0 mg L−1 TDZ resulted in markedly lower organo-genesis rates (≤77%) in rootstock ‘M26’, regardless ofIBA concentration (Table 1). The highest number ofshoots per leaf segment was obtained in rootstocks ‘M9’(3.8–4.7) and ‘M26’ (4.2–4.6) when a combination of3.0 mg L−1 TDZ and 0.5–1.5 mg L−1 IBA was used(Table 1).

Shoot regeneration from leaf segments of Malus. In total,nine Malus genotypes including five scion cultivars (M.× domestica), two rootstocks (M. pumila), and two wildspecies (M. robusta and M. micromalus) were tested inthis experiment (Table 2; Fig. 2). The two SRMs used inthis experiment (2 mg L−1 TDZ and 0.5 mg L−1 IBA;3 mg L−1 TDZ and 0.5 mg L−1 IBA) were selected fromthe first experiment. Both SRMs gave rates of 100%organogenesis in each of the Malus genotypes tested(Table 2; Fig. 2). SRM containing 2 mg L−1 TDZ and0.5 mg L−1 IBA resulted in the highest shoot number for‘Gala’ (8.7), ‘Fuji’ (5.6), ‘Greensleeves’ (4.9), andM. robusta (3.5), while that containing 3 mg L−1 TDZand 0.5 mg L−1 IBA gave the maximum shoot number in‘Himekami ’ (1 .9 ) , ‘Wangshanhong ’ (2 .0 ) andM. micromalus (0.6). The two rootstocks were tested onlyat 3 mg L−1 TDZ and 0.5 mg L−1 IBA; with this SRM,‘M9’, and ‘M26’ gave shoot numbers of 6.6 and 6.3,respectively. Thus, with the best combinations of TDZand IBA, a mean organogenesis rate of 100% and a meanof 4.5 shoots per explant could be obtained in the nineMalus genotypes tested.

Overall process of shoot recovery from cryopreserved shoottips. Cryopreserved shoot tips of ‘Gala’ started to brownduring the first 2 d of post-culture. To avoid browning, they

were transferred twice (after 12 and 36 h) onto fresh recoverymedium. Within 7 d of post-culture, cryopreserved shoot tipsbegan to show green color (Fig. 1j). Surviving shoot tipsresumed shoot growth, with shoot elongation and formationof new leaves, and finally regenerated into new shoots(≥3 mm long and with at least two fully opened leaves) after8 wk of post-culture (Fig. 1k). When transferred onto rootingmedium, more than 90% of the microshoots formed roots after4 wk (Fig. 1l). Rooted microshoots were successfullyestablished in soil under greenhouse conditions within 4 wk(data not shown).

Effect of adventitious shoot age on recovery. Shoot tips (3mmin length) containing six LPs (Fig. 1g) were excised fromadventitious shoots derived from ‘Gala’ leaf segments thathad been cultured on SRM for 8, 9, 10, 11, and 12 wk toinvestigate the effect of adventitious shoot age on shoot re-covery following cryopreservation. The morphologies of theadventitious shoots with ages ranging from 8 to 12 wk weresimilar, except that shoot size increased with increasing shootage. The 8-wk-old shoots were about 3 mm in length with twoto three fully opened leaves, while 12-wk-old shoots weremore than 5 mm in length with five to six leaves. Shoot agehad a marked influence on shoot recovery. For cryopreservedshoot tips (+LN), shoot recovery increased from 20.1 to79.3% as shoot age increased from 9 to 11 wk, and thenstarted to decrease as shoot age increased further (Fig. 3).The shoot recovery pattern of the treated control (−LN) wassimilar to that of cryopreserved shoot tips (+LN) (Fig. 3).

Effect of preculture duration on recovery. Shoot tips (3 mm inlength) containing six LPs that were excised from 11-wk-oldadventitious shoots were used in this experiment to test theeffects of preculture duration on recovery. For cryopreservedshoot tips (+LN), preculture with 0.5 M sucrose for 4–5 dresulted in the highest rate of shoot recovery (Fig. 4).Preculture for less than 4 d or more than 5 d significantlyreduced shoot recovery. A similar shoot recovery trend wasfound in the control shoot tips (−LN).

Effect of shoot tip size on recovery. In this experiment, 1-, 2-,and 3-mm-long shoot tips containing two to three, four to five,and six LPs, respectively, were excised from 11-wk-old ad-ventitious shoots and precultured on 0.5 M sucrose for 5 d.Shoot recovery from both cryopreserved (+LN) and control(−LN) shoot tips significantly increased with an increase inshoot tip size (Fig. 5). When 3-mm shoot tips containing sixLPs were used, shoot recovery rates of about 82 and 79%wereobtained in the control (−LN) and cryopreserved (+LN) shoottips, respectively.

Cryopreservation of nine Malus genotypes. Shoot recoveryfrom cryopreserved (+LN) and control (−LN) shoot tips

LI ETAL.

varied considerably among the nine genotypes tested(Table 3). With ‘Greensleeves’, the control (−LN) and cryo-preserved (+LN) shoot tips gave shoot recovery rates of 7.5and 0%, respectively. Among the other nineMalus genotypes,the highest and lowest shoot recovery rates in the control(−LN) shoot tips were obtained in M. micromalus (90.0%)and ‘Himekami’ (48.3%), respectively (Table 3). For cryopre-served shoot tips (+LN), ‘Gala’ and ‘Himekami’ produced thehighest (79.3%) and lowest (27.5%) shoot recovery, respec-tively (Table 3). Themean shoot recoverywas 57.0 and 71.5%for cryopreserved (+LN) and control (−LN) shoot tips of theeight Malus genotypes other than ‘Greensleeves’.

Assessment of genetic stability. Out of the 51 primers tested inthe ISSR analysis, eight produced strong, clear, reproduciblebands (Table 4). Assays with these eight primers generated 59scorable bands. The number of bands for each primer variedfrom 5 to 12, with an average of 7 bands produced per primer(Table 4, Fig. 6). A total of 1,770 bands were scored across allprimers and samples analyzed. No polymorphic bands wereobserved among the regenerants derived from leaf segments,shoots recovered from cryopreserved shoot tips or in vitrostock shoots (Fig. 6).

Figure 2 Shoots regeneratedfrom leaf segments of nineMalusgenotypes after 11 wk of cultureon SRM. (a) ‘Fuji’. (b) ‘M9’. (c)‘M26’. (d) ‘Wangshanhong’. (e)‘Himekami’. (f) M. robusta. (g)M. micromalus. (h)‘Greensleeves’. Bars represent1 mm. A comparable illustrationof ‘Gala’ is presented in Fig. 1f.

0

20

40

60

80

100

8 9 10 11 12Age of adventitious shoots (wk)

Sho

ot r

egro

wth

(%

)

+LN

-LN

a

z

y

y

a

b

a

z

cx

Figure 3 Effect of adventitious shoot age on shoot regrowth of cryopre-served shoot tips of ‘Gala’. Shoot tips (3 mm in length) containing sixleaves were excised from adventitious shoots regenerated from leafsegments that had been cultured on SRM for 8, 9, 10, 11, or 12 wk.Encapsulated shoot tips were precultured with 0.5 M sucrose for 5 d anddehydrated by air-drying in a laminar flow for 6 h, prior to directimmersion in LN for 1 h. Shoot regrowth was recorded after 8 wk ofpost-culture. Data are presented as mean±SE and analyzed using one-directional ANOVA and Student’s t test. Bars with different letters in thesame treatment indicate significant difference at P<0.05 by least signif-icant difference (LSD) test.

CRYOPRESERVATION OFAPPLE SHOOT TIPS

Discussion

The present study tested cryopreservation of shoot tips excisedfrom adventitious buds derived from leaf segments. On thebest medium identified for each genotype, the organogenesisrates reached 100% and the number of shoots per explantranged from 0.6 to 8.7, with an average number per explantof 4.5 obtained in the nine Malus genotypes, including fivecultivars (M. × domestica), two rootstocks (M. pumila), andtwo wild species (M. robusta andM. micromalus). To the bestof our knowledge, this is the first report on cryopreservation ofshoot tips using adventitious shoots derived from leaf seg-ments in Malus.

Two significant improvements were achieved in the presentstudy, compared with previous studies. First, cryopreservationof shoot tips using adventitious buds without cold hardening,as reported here, is highly efficient with regard to shoot tipproduction. Here, we take ‘Gala’, which is the most frequentlyused cultivar for cryopreservation (Hao et al. 2001; Liu et al.2004; Halmagyi et al. 2010; Condello et al. 2011; Feng et al.2013), as an example to compare the efficiency of shoot tipproduction between the present study and previous studies. Inseveral previous studies, the cultures had to remain on thestock culture medium without subculture for 4 wk (Feng et al.2013), 2 mo (Halmagyi et al. 2010), or 4 mo (Condello et al.2011) before excision of shoot tips for cryopreservation. Thus,only one shoot tip could be obtained from each stock shoot inthat length of time. In our study, 3 leaves from one stock shootwere used and produced at least 24 shoot tips (3 leaves×8shoots per leaf segment) within 11 wk. This production rate ofshoot tips is highly efficient when compared with these pre-vious studies (Halmagyi et al. 2010; Condello et al. 2011;Feng et al. 2013). Second, nine genotypes belonging to fourMalus species were successfully cryopreserved in the presentstudy. Other studies have obtained successful cryopreserva-tion of seven genotypes from four species and one hybrid(Feng et al. 2013), two genotypes from one species (Condelloet al. 2011), four genotypes from one species (Halmagyi et al.2010), five genotypes from two species (Kushnarenko et al.2009), seven genotypes from two species (Zhao et al. 1999),and five genotypes from three species (Niino et al. 1992);thus, the present method promises to be the most applicablecryopreservation procedure for the genus Malus.

In Malus, PGRs are critical for achieving efficient shootregeneration. A combination of a cytokinin such as TDZ orBAwith an auxin such as IBA or NAAwas frequently used toenhance shoot regeneration from leaf segments (Dobránszkiand Teixeira da Silva 2010; Magyar-Tábori et al. 2010). TDZwas more effective than BA for shoot regeneration (Fasoloet al. 1989; Korban et al. 1992; Sarwar and Skirvin 1997;Gamage and Nakanishi, 2000; Dobránszki et al. 2004, 2006;Mitić et al. 2012). The optimal TDZ concentration dependedon genotype (Fasolo et al. 1989; Korban et al. 1992; Sarwar

0

20

40

60

80

100

1 2 3

Size of shoot tips (mm in length)

Sho

ot r

egro

wth

(%

)

+LN

-LN

a

b

c

z

y

x

Figure 5 Effect of shoot tip size on shoot regrowth of cryopreservedshoot tips of ‘Gala’. Shoot tips of 1, 2, or 3 mm in length containing twoto three, four to five, or six leaf primordia, respectively, were excised from11-wk-old leaf segments. Encapsulated shoot tips were precultured with0.5 M sucrose for 5 d and dehydrated by air-drying in a laminar flowcabinet for 6 h, prior to direct immersion in LN for 1 h. Shoot regrowthwas recorded after 8 wk of post-culture. Data are presented as mean±SEand analyzed using one-directional ANOVA and Student’s t test. Barswith different letters within the same treatment indicate significant differ-ence at P<0.05 by least significant difference (LSD) test.

0

20

40

60

80

100

1 2 3 4 5 6 7

Preculture duration (d)

Sho

ot r

egro

wth

(%

)

+LN

-LN

z

x

xx

w

yy

a

bb

b

c

cd

d

Figure 4 Effect of preculture duration on shoot regrowth of cryopre-served shoot tips of ‘Gala’. Shoot tips (3 mm in length) containing six leafprimordia were excised from adventitious shoots regenerated from leafsegments that had been cultured on SRM for 11 wk. Encapsulated shoottips were precultured with 0.5 M sucrose for 1, 2, 3, 4, 5, 6, or 7 d anddehydrated by air-drying in a laminar flow for 5 h, prior to directimmersion in LN for 1 h. Shoot regrowth was recorded after 8 wk ofpost-culture. Data are presented as mean±SE and analyzed using one-directional ANOVA and Student’s t test. Bars with different letters withinthe same treatment indicate significant difference at P<0.05 by leastsignificant difference (LSD) test.

LI ETAL.

and Skirvin 1997; Dobránszki et al. 2004, 2006; Magyar-Tábori et al. 2010; Mitić et al. 2012). Fasolo et al. (1989)reported that 10 μM TDZ was beneficial for shoot regenera-tion in ‘McIntosh’, ‘Paladino SpurMcIntosh’, and ‘Triple RedDelicious’. Apple cultivars that responded best at higher TDZconcentrations (≥10.0 μM) for shoot regeneration include‘M7’, ‘Orine’, ‘Golden Delicious’, ‘Melrose’, ‘Macspur’,and ‘Strakrimson’ (Korban et al. 1992; Gamage andNakanishi, 2000; Magyar-Tábori et al. 2010; Mitić et al.2012), whereas most other cultivars responded best to lowerTDZ concentrations (≤5.0 μM) (Sriskandarajah et al. 1990;Theiler-Hedtrich and Theiler-Hedtrich 1990; Korban et al.1992; Sarwar and Skirvin 1997; Magyar-Tábori et al. 2010).A number of cultivars such as ‘Royal Gala’ and ‘Dayton’responded similarly to a wide range of TDZ levels (5–20 μM) (Korban et al. 1992; Magyar-Tábori et al. 2010).Our study showed that a suitable TDZ concentration was2 mg L−1 (9.08 μM) for ‘Gala’, 2–3 mg L−1 (9.08–13.6 μM)for ‘Fuji’, and 3 mg L−1 (13.6 μM) for ‘M9’ and ‘M26’. For‘Gala’, 3 μM TDZ was found suitable for shoot regenerationby Sriskandarajah et al. (1990), compared with 15 μM(Korban et al. 1992) and 2 mg L−1 (9.08 μM) in our study.These differences may be caused by variation in the length ofthe dark period used at the initial shoot regeneration stage inthe different studies: 4 wk in Sriskandarajah et al. (1990),3 wk in our study, and 1 wk in Korban et al. (1992) becauselight conditions at the initial stage of shoot regenerationproved to significantly affect shoot regeneration in Malus

(Liu et al. 1983; Welander 1988; Fasolo et al. 1989; Korbanet al. 1992; Famiani et al. 1994; Mitić et al. 2012).

For successful cryopreservation, shoot tips have to tolerateboth desiccation and very low (LN) temperature. Shoot tipsare frequently precultured with sugar to improve desiccationtolerance and cryo-tolerance (Jitsuyama et al. 2002; Wanget al. 2005; Pinker et al. 2009). Sucrose has been the sugarused most often to establish this tolerance, the most importantfactors being sucrose concentration and duration of preculture.High sucrose concentrations (0.3–1.0 M) and preculturedurations ranging from hr to 7–8 d were found suitable forMalus (Niino and Sakai 1992; Niino et al. 1992; Wu et al.1999; Paul et al. 2000; Kushnarenko et al. 2009; Halmagyiet al. 2010; Feng et al. 2013). In the present study, preculturewith 0.5 M sucrose for 4–5 d was found optimal for achievingthe highest shoot recovery of cryopreserved shoot tips of‘Gala’, whereas in our previous study, preculture with 0.5 Msucrose for 7–8 d was found optimal for the same purpose(Feng et al. 2013). The major difference between these twostudies was the type of shoot stock cultures: in vitro shootstocks in Feng et al. (2013) and adventitious shoots inducedfrom leaf segments in the present study. Shoot tips originatingfrom different types of source plants may require differentsucrose preculture conditions for optimal shoot regrowth fol-lowing cryopreservation.

The age of the stock cultures significantly affects shootrecovery from cryopreserved shoot tips (Wang and Perl 2006).Studies on Dianthus caryophyllus found that shoot regrowth

Table 4 ISSR primer names,primer sequences, and numbers ofamplified bands generated inregenerants derived from leafsegments and recovered fromcryopreserved shoot tips of ‘Gala’

Primer name Primer sequence (5′–3′) Number of amplified bands Number of polymorphic bands

UBC 811 (GA)8C 7 0

UBC 815 (CT)8G 12 0

UBC 834 (AG)8YT 10 0

UBC 835 (AG)8YC 5 0

UBC 836 (AG)8YA 7 0

UBC 843 (CT)8RA 5 0

UBC 845 (CT)8RG 7 0

UBC 873 (GACA)4 6 0

Total 59 0

Figure 6 ISSR banding patterns of the in vitro stock shoots, regenerantsderived from leaf segments and recovered from cryopreserved shoot tipsof ‘Gala’ using the primers UBC815 and UBC834. Mmarker: Lanes 1-3:

the in vitro stock shoots; Lanes 4-6: adventitious shoots derived from leafsegments; Lanes 7-9: shoots recovered from cryopreserved shoot tips.

CRYOPRESERVATION OFAPPLE SHOOT TIPS

of cryopreserved shoot tips increased from 30 to 94% as theage of the stock cultures increased from 0- to 14-d old(Dereuddre et al. 1988). In potato, Yoon et al. (2006) foundthat the effect of stock culture age on cryopreservation wascultivar specific: 5- and 7-wk-old stock shoots were suitablefor ‘STN13’ and ‘Dejima”, respectively. In the present study,the best shoot recovery from cryopreserved shoot tips wasobtained with 11-wk-old adventitious shoots. Stock cultureage is closely associated with the physiological and develop-mental status of the cultures, which has been shown to signif-icantly influence the success of cryopreservation (Engelmannet al. 1997; Wang and Perl 2006). The specific explanation forthis is not yet fully understood.

Shoot tip size was shown to considerably influence shootrecovery from cryopreserved shoot tips. In potato, Halmagyiet al. (2005) reported that the highest frequency of shootrecovery was found with 3–4-mm shoot tips; smaller (1–2 mm) and larger (5–6 mm) shoot tips reduced shoot recovery.Yoon et al. (2006) found that the optimal size of shoot tipsvaried among potato cultivars: 1.5–2.0 mm for ‘Dejima’ and1.0–1.5 mm for ‘STN13’. In sweet potato, Wang andValkonen (2008) reported that although shoot tips rangingfrom 0.5 to 1.5 mm produced similar survival rates (85–90%), shoot recovery was extremely low (15%) for 0.5-mmshoot tips and significantly higher (80–86%) for 1.0- to 1.5-mm shoot tips. In previous studies on apple, various sizes ofshoot tips were used, e.g., 0.8–1.0 mm (Kushnarenko et al.2009), 1 mm (Paul et al. 2000), 1.5–2.0 mm (Niino et al.1992), 2 mm (Zhao et al. 1999; Feng et al. 2013), and 2–3mm(Halmagyi et al. 2010). However, no comparative studies onthe effect of shoot size on shoot recovery had been conducted.In the present study, we found that the optimal shoot tip sizefor shoot recovery was 3.0 mm with six LPs. Therefore, theoptimum shoot tip size for obtaining the highest frequency ofshoot recovery in a given protocol needs to be determinedunder the specific conditions found in each laboratory.

The nine Malus genotypes responded significantly differ-ently to the encapsulation–dehydration procedure describedhere, with the highest (79.3%) and lowest (27.5%) rates ofshoot regrowth among responding genotypes obtained in ‘Ga-la’ and ‘Himekami’, respectively (Table 3). However, none ofthe cryopreserved shoot tips of ‘Greensleeves’ could regener-ate into shoots, thus suggesting that the cryopreservationprocedure must be further optimized for this genotype. Thesedata reconfirm that genotype-specific response is common incryopreservation studies (Benson 2008).

Genetic stability of the regenerants from cryopreservedtissues is an important concern. Cryopreservation steps suchas preculture with high sugar concentrations and dehydrationwith PVS2 cause stress to the samples and thus may result ingenetic alterations of regenerants following cryopreservation(Aronen et al. 1999; Harding 2004). In addition, our studyused the tips of adventitious shoots derived from leaf

segments for cryopreservation. Genetic variation may occurin in vitro-derived regenerants (Dobránszki and Teixeira daSilva 2010). Using ISSRmolecular markers, we did not detectany polymorphism in regenerants derived from leaf segmentsand recovered from cryopreserved shoot tips of ‘Gala’, indi-cating that the regenerants following shoot regeneration andcryopreservation were genetically stable. Pathak and Dhawan(2010, 2012) showed that the ISSR amplification profile washomogenous in plantlets regenerated from in vitro-derivedcultures and from mother shoots of apple rootstocks‘MM111’ and ‘Merton 793’. No genetic variation was foundby analysis of random amplified polymorphic DNA (RAPD)in regenerants derived from leaf segments of ‘Gala’(Montecelli et al. 2000) or from axillary buds of rootstock‘EMLA 111’ (Gupta et al. 2009). Using RAPD, Viršcek-Marnet al. (1998) detected somatic variation in regenerants fromleaf segments of ‘Golden Delicious Bovey’ and ‘Goldspur’.Off-type plants regenerated from axillary buds were alsodetected by RAPD in rootstock ‘MM106’ (Modgil et al.2005). Polymorphisms were detected byRAPD in regenerantsfrom leaf segments of ‘Golden Delicious Bovey’ and‘Goldspur’, but not in those from apical meristems of root-stock ‘Jork 9’ (Caboni et al. 2000). Three factors, i.e., types ofexplants, culture conditions, and genotype, were suggested tobe responsible for somaclonal variation in in vitro-derivedplants (Venkatachalam et al. 2007; Dobránszki and Teixeirada Silva 2010), but a number of other studies suggest thatvariation is governed more by the genotype than by cultureconditions. For example, Devarumath et al. (2002) observedgenetic stability in in vitro-derived plants of Camellia spp.genotypes U3 and U27, while variation was found in geno-typeU26when cultured under the same conditions. Genotypicdifferences in the frequency of somaclonal variation have alsobeen reported in Eucalyptus by Rani and Raina (1998) and inMusa by Ray et al. (2006). In the present study, no polymor-phism was detected by ISSR in plantlets regenerated fromleaf segments of ‘Gala’, indicating that this cultivar may bemore genetically stable, like rootstocks ‘MM111’ (Pathakand Dhawan 2010) and ‘Merton 793’ (Pathak and Dhawan2012), than other apple cultivars such as ‘Golden DeliciousBovey’ and ‘Goldspur’ (Viršcek-Marn et al. 1998) androotstock ‘MM106’ (Modgil et al. 2005). With Malus, anal-yses at the morphological, chromosomal, and molecularlevels have been conducted in regenerants recovered fromcryopreserved shoot tips, and all results demonstrated thatcryopreservation did not caused any variation (Hao et al.2001; Liu et al. 2004, 2008). The data reported here wereentirely consistent with those of Hao et al. (2001) and Liuet al. (2004, 2008), who showed that regenerants fromcryopreserved shoot tips were genetically stable by usingamplified fragment length polymorphism (AFLP: Hao et al.2001; Liu et al. 2004), RAPD (Liu et al. 2004), and ISSR(Liu et al. 2008) markers.

LI ETAL.

In conclusion, we have developed a simple and widelyapplicable protocol for cryopreservation of Malus shoot tipsby encapsulation–dehydration using adventitious buds with-out cold hardening. This protocol was successfully applied toeight Malus genotypes including four scion cultivars (M. ×domestica), two rootstocks (M. pumila), and two wild species(M. robusta and M. micromalus). This protocol markedlyincreased the efficiency of shoot tip production and largelysimplified the cryogenic procedure, therefore providing analternative method for Malus cryopreservation.

Acknowledgments The authors acknowledge financial support fromNorthwest A&F University (Z222020904) and from the Department ofFruit Industry of Shaanxi Province (K336021105).

References

Aldwinckle H, Malnoy M (2009) Plant regeneration and transformationin the Rosaceae. Transgenic Plant J 3:1–39

Aronen T, Krajnakova J, Häggman H, Ryynänen L (1999) Geneticfidelity of cryopreserved embryogenic cultures of open-pollinatedAbies cephalonica. Plant Sci 142:163–172

Benson EE (2008) Cryopreservation of phytodiversity: a critical appraisalof theory & practice. Crit Rev Plant Sci 27:141–219

Brischia R, Piccioni E, Standardi A (2002) Micropropagation and syn-thetic seed in M.26 apple rootstock (II): a new protocol for produc-tion of encapsulated differentiating propagules. Plant Cell TissueOrgan Cult 68:137–141

Burritt DJ (2008) Efficient cryopreservation of adventitious shoots ofBegonia × erythrophylla using encapsulation–dehydration requirespretreatment with both ABA and proline. Plant Cell Tissue OrganCult 95:209–215

Caboni E, Lauri P, Damiano C, D’Angeli S (2000) Somaclonal variationinduced by adventitious shoot regeneration in pear and apple. ActaHorticult 530:195–201

Condello E, Caboni E, Andre E, Piette B, Druart P, Swennen R, Panis B(2011) Cryopreservation of apple in vitro axillary buds usingdroplet-vitrification. CryoLetters 32:175–185

Dereuddre J, Fabre J, Bassaglia C (1988) Resistance to freezing in liquidnitrogen of carnation (Dianthus caryophyllus L. var. Eolo) apicaland axillary shoot tips excised from different aged in vitro plantlets.Plant Cell Rep 7:170–173

Devarumath RM, Nandy S, Rani V, Marimuthu S, Muraleedharan N,Raina SN (2002) RAPD, ISSR and RFLP fingerprints as usefulmarkers to evaluate genetic integrity of micropropagated plants ofthree diploid and triploid elite tea clones representing Camelliasinensis (China type) and C. assamica ssp. Assamica (Assam-India type). Plant Cell Rep 21:166–173

Dobránszki J, Hudák I, Magyar-Tábori K, Galli Z, Kiss E, Jámbor-Benczúr E (2006) How can different cytokinins influence the pro-cess of shoot regeneration from apple leaves in ‘Royal Gala’ and‘M.26’. Acta Horticult 725:191–196

Dobránszki J, Hudak I, Magyar-Tabori K, Jambor-Benczur E, Galli Z,Kiss E (2004) Effects of different cytokinins on the shoot regener-ation from apple leaves of ‘Royal Gala’ and ‘M.26’. Int J Hortic Sci101:69–75

Dobránszki J, Teixeira da Silva JA (2010) Micropropagation of apple—areview. Biotechnol Adv 28:462–488

Engelmann F, Callow JA, Ford-Lloyd BV, Newbury HJ (1997) In vitroconservation methods. In: Callow JA, Ford-Lloyd BV, Newbury HJ

(eds) Biotechnology and plant genetic resources: conservation anduse. Biotechnology in agriculture series, vol 19. CAB International,Oxon, pp 119–161

Famiani F, Ferradini N, Staffolani P, Standari A (1994) Effect of leafexcision time and age, BA concentration and dark treatments onin vitro shoot regeneration of M.26 apple rootstock. J Hortic Sci 69:679–685

Fasolo F, Zimmerman RH, Fordham I (1989) Adventitious shoot forma-tion on excised leaves of in vitro grown shoots of apple cultivars.Plant Cell Tissue Organ Cult 16:75–87

Feng CH, Cui ZH, Li BQ, Chen L, Ma YL, Zhao YH, Wang QC (2013)Duration of sucrose preculture is critical for shoot regrowth ofin vitro-grown apple shoot-tips cryopreserved by encapsulation-dehydration. Plant Cell Tissue Organ Cult 112:369–378

Gamage N, Nakanishi T (2000) In vitro shoot regeneration from leaftissue of apple (cultivar ‘Orine’): high shoot proliferation using carryover effect of TDZ. Acta Horticult 520:291–298

Gupta R, Modgil M, Chakrabarti SK (2009) Assessment of geneticfidelity of micropropagated apple rootstock plants, EMLA 111,using RAPD markers. Ind J Exp Bot 47:925–928

Halmagyi A, Deliu C, Coste A (2005) Plant regrowth from potato shoottips cryopreserved by a combined vitrification-droplet method.CryoLetters 26:313–322

Halmagyi A, Deliu C, Isac V (2010) Cryopreservation ofMalus cultivars:comparison of two droplet protocols. Sci Hortic 124:387–392

Hao YJ, Liu QL, Deng XX (2001) Effect of cryopreservation on applegenetic resources at morphological, chromosomal, and molecularlevels. Cryobiology 43:46–53

Harding K (2004) Genetic integrity of cryopreserved plant cells: a review.CryoLetters 25:3–22

Jitsuyama Y, Suzuki T, Harada T, Fujikawa S (2002) Sucrose incubationincreases freezing tolerance of asparagus (Asparagus officinalis L.)embryogenic cell suspensions. CryoLetters 23:103–112

Korban SS, O’Connor PA, Elobeidy A (1992) Effect ofthidiazuron, naphthalene acetic acid, dark incubation and ge-notype on shoot organogenesis of Malus leaves. J Hortic Sci67:341–349

Kuo CC, Lineberger RD (1985) Survival of in vitro cultured tissue of‘Jonathan’ apples exposed to −196°C. HortSci 20:764–767

Kushnarenko SV, Romadanova NV, Reed BM (2009) Cold acclimationimproves regrowth of cryopreserved apple shoot tips. CryoLetters30:47–54

Li YN (1999) Progress in research on the origin and evolution of thegenus Malus in the world. J Fruit Sci 16:8–19

Liu JR, Sink KC, Dennis FG (1983) Plant regeneration from appleseedling explants and callus cultures. Plant Cell Tissue Organ Cult2:293–304

Liu YG, Liu LX, Wang L, Gao AY (2008) Determination of geneticstability in surviving apple shoots following cryopreservation byvitrification. CryoLetters 29:7–14

Liu YG, Wang XY, Liu LX (2004) Analysis of genetic variation insurviving apple shoots following cryopreservation by vitrification.Plant Sci 166:677–685

Magyar-Tábori K, Dobránszki J, Teixeira da Silva JA, Bulley SM, HudákI (2010) The role of cytokinins in shoot organogenesis in apple.Plant Cell Tissue Organ Cult 101:251–267

Matsumoto T, Sakai A, Yamada K (1995) Cryopreservation of in vitro-grown apical meristems of lily by vitrification. Plant Cell TissueOrgan Cult 41:237–241

Mitić N, Stanišić M, Milojević J, Tubić L, Ćosić T, Nikolić R, NinkovićS, Miletić R (2012) Optimization of in vitro regeneration from leafexplants of apple cultivars ‘Golden Delicious’ and ‘Melrose’.HortSci 47:1117–1122

Modgil M, Mahajan K, Chakrabarti SK, Sharma DR, Sobti RC (2005)Molecular analysis of genetic stability in micropropagated applerootstock MM106. Sci Hortic 104:151–160

CRYOPRESERVATION OFAPPLE SHOOT TIPS

Montecelli S, Gentile A, Damino C (2000) In vitro shoot regeneration ofapple cultivar Gala. Acta Horticult 530:219–223

Murashige T, Skoog F (1962) A revised medium for rapid growthand bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

Niino T, Sakai A (1992) Cryopreservation of alginate-coated in vitro-grown shoot tips of apple, pear and mulberry. Plant Sci 87:199–206

Niino T, Sakai A, Yakuwa H, Nojiri K (1992) Cryopreservation ofin vitro-grown shoot tips of apple and pear by vitrification. PlantCell Tissue Organ Cult 28:261–266

Pathak H, Dhawan V (2010) Molecular analysis of micropropagatedapple rootstock MM111 using ISSR markers for ascertaining clonalfidelity. Acta Horticult 865:73–80

Pathak H, Dhawan V (2012) ISSR assay for ascertaining genetic fidelityof micropropagated plants of apple rootstock Merton 793. In VitroCell Dev Biol Plant 48:137–143

Paul H, Daigny G, Sangwan-Norreel BS (2000) Cryopreservation ofapple (Malus× domestica Borkh.) shoot tips followingencapsulation-dehydration or encapsulation-vitrification. Plant CellRep 19:768–774

Pinker I, Halmagyi A, Olbricht K (2009) Effects of sucrose preculture oncryopreservation by droplet-vitrification of strawberry cultivars andmorphological stability of cryopreserved plants. CryoLetters 30:202–211

Rani V, Raina SN (1998) Genetic analysis of enhanced axillary branchingderived Eucalyptus tereticornis and E. camaldulensis plants. PlantCell Rep 17:236–242

Ray T, Dutta I, Saha P, Das S, Roy SC (2006) Genetic stability ofthree economically important micropropagated banana (Musaspp.) cultivars of lower Indo-Gangetic plains, as assessed byRAPD and ISSR markers. Plant Cell Tissue Organ Cult 85:11–21

Sarwar M, Skirvin RM (1997) Effect of thidiazuron and 6-benzylaminopurine on adventitious shoot regeneration from leavesof three strains of ‘McIntosh’ apple (Malus×domestica Borkh.)in vitro. Sci Hortic 68:95–100

Sriskandarajah S, Skirvin RM, Abu-Qaoud H, Korban SS (1990)Factors involved in shoot elongation and growth of adventi-tious and axillary shoots of three apple scion cultivars in vitro.J Hortic Sci 65:113–121

Theiler-Hedtrich C, Theiler-Hedtrich R (1990) Influence of TDZ and BAon adventitious shoot regeneration from apple leaves. Acta Horticult280:196–199

Towill LE, Forshline PL, Walters C, Waddell JW, Laufmann J (2004)Cryopreservation ofMalus germplasm using a winter vegetative budmethod: results from 1915 accessions. CryoLetters 25:323–334

Venkatachalam L, Sreedhar RV, Bhagyalakshmi N (2007)Micropropagation in banana using high levels of cytokinins doesnot involve any genetic changes as revealed by RAPD and ISSRmarkers. Plant Growth Regul 51:193–205

Viršcek-Marn M, Javornik B, Štampar F, Bohanec B (1998) Assessmentof genetic variation among regenerants from in vitro apple leavesusing molecular markers. Acta Horticult 484:299–303

Wang QC, Laamanen J, Uosukainen M, Valkonen JP (2005)Cryopreservation of in vitro-grown shoot tips of raspberry (Rubusidaeus L.) by encapsulation–vitrification and encapsulation–dehy-dration. Plant Cell Rep 24:280–288

Wang QC, Perl A (2006) Cryopreservation in floricultural plants. In: daSilva T (ed) Floriculture, ornamental and plant biotechnology.Global Science Books, London, pp 523–539

Wang QC, Valkonen JPT (2008) Eradication of two synergisticallyinteracting viruses from sweetpotato using shoot tip culture andcryotherapy of shoot tips. J Virol Meth 154:135–145

Welander M (1988) Plant regeneration from leaf and stem segments ofshoots raised in vitro from mature apple trees. J Plant Physiol 132:738–744

Wu YJ, Engelmann F, Zhao YH, Zhou MD, Chen SY (1999)Cryopreservation of apple shoot tips: importance of cryopreservationtechnique and of conditioning of donor plants. CryoLetters 20:121–130

Yin ZF, Zhao B, Bi WL, Chen L, Wang QC (2013) Direct shoot regen-eration from basal leaf segments of Lilium and assessment of geneticstability in regenerants by ISSR and AFLP markers. In Vitro CellDev Biol Plant 49:333–342

Yoon JW, Kim HH, Ko HC, Hwang HS, Hong ES, Cho EG, EngelmannF (2006) Cryopreservation of cultivated and wild potato varieties bydroplet vitrification: effect of subculture of mother-plants and ofpreculture of shoot tips. CryoLetters 27:211–222

Zhao YH, Wu YJ, Engelmann F, Zhou MD, Chen SY (1999)Cryopreservation of apple in vitro shoot tips by the droplet freezingmethod. CryoLetters 20:109–112

LI ETAL.