Scientia Horticulturae 120 (2009) 84–88

Contents lists available at ScienceDirect

Scientia Horticulturae

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

Cryopreservation of shoot apices of hawthorn in vitro cultures originating fromEast Asia

D. Kami a,*, L. Shi a,1, T. Sato b,2, T. Suzuki a,1, K. Oosawa a,1

a Department of Horticultural Science and Landscape Architecture, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japanb Hokkaido Forestry Research Center, Koshunai-Cho, Bibai, Hokkaido 079-0198, Japan

A R T I C L E I N F O

Article history:

Received 21 September 2007

Received in revised form 20 September 2008

Accepted 22 September 2008

Keywords:

Cryopreservation

Encapsulation–dehydration

Encapsulation–vitrification

Glycerol

Hawthorn

Vitrification

A B S T R A C T

The objective of this study was to establish a cryopreservation protocol for hawthorn shoot apices

(Crataegus pinnatifida Bge.). Cryopreservation was carried out via encapsulation–dehydration, vitrifica-

tion, and encapsulation–vitrification on shoot apices excised from in vitro cultures. We began by showing

that cold-acclimation enhanced the regrowth of cryopreserved apices from 10.0 to 65.5% in

encapsulation–dehydration. We then decided that the encapsulation–dehydration method was an

optimal cryopreservation method for hawthorn shoot apices in terms of its high recovery after

cryopreservation as well as its ease of use compared with vitrification and encapsulation–vitrification. In

encapsulation–dehydration, the protocol leading to optimal regrowth was as follows: after cold-

acclimation at 5 8C in the dark for 2 weeks, excised shoot tips were pretreated for 24 h at 25 8C on

hormone-free Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth

and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497] (MS) basal medium with 0.4 mol/L

sucrose, then encapsulated and precultured in liquid MS medium with 0.8 mol/L sucrose for 16 h at 25 8C.

Precultured beads were dehydrated for 6 h at 25 8C in the dessicator containing 50 g silica gel to a

moisture content of 15.3% (fresh-weight basis) before cryostorage for 1 h. In addition, we examined the

effect of adding glycerol to both the alginate beads and loading solution to enhance regrowth after

cryopreservation in encapsulation–dehydration. In the present study, it was shown that adding 0.5 mol/L

glycerol resulted in high regrowth percentages (82.5–90.0%) in four Crataegus species.

� 2008 Elsevier B.V. All rights reserved.

1. Introduction

Hawthorn (Crataegus spp.) is a kind of small fruit treeoriginating in North America and has importance for landscapingand other ornamental purposes in Europe and Asia (Mi et al.,1992). In China, Crataegus pinnatifida is a commercially importanttree whose fruit is used for making jam, juice, and confectioneries(Mi et al., 1992). In addition, hawthorn has generated keeninterest as a functional material around the world because itsleaves or flowers are rich in flavonoids (Nikolov et al., 1982;Rigelsky and Sweet, 2002). Moreover, Shi et al. (2003) have alsoreported that hawthorn fruit that grew wild in Japan containedhigh levels of minerals, b-carotene and anthocyanin. Therefore,the production of interspecific hybrids of hawthorn has beenexamined using an embryoculture (Shi et al., 2004a, b). However,

* Corresponding author. Tel.: +81 11 706 2450; fax: +81 11 706 2450.

E-mail address: cryopreservation@hotmail.co.jp (D. Kami).1 Tel.: +81 11 706 2450; fax: +81 11 706 2450.2 Tel.: +81 126 63 4164; fax: +81 126 63 4166.

0304-4238/$ – see front matter � 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2008.09.019

since the subculture process of in vitro cultures is so time-, space-and labor-consuming, there is impetus to establish a practicalprocedure for preserving in vitro hawthorn apices over a longperiod. Cryopreservation has become important as a means ofensuring the long-term preservation of plant germplasms (Karthaand Engelmann, 1994; Reed and Hummer, 1995). AlthoughDamiano et al. (2006) reported a survival rate of 25% fromcryopreservation of shoot apices of Crataegus azarolus, an evenmore efficient cryopreservation protocol (regrowth percentageabove 80%) of hawthorn apices has yet to be found. Since thesecond half of the 1980s, various cryopreservation methods, i.e.,vitrification (Uragami et al., 1989; Sakai et al., 1990), encapsula-tion–dehydration (Fabre and Dereuddre, 1990; Gonzalez-Arnaoet al., 2003; Kami et al., 2005) and encapsulation–vitrification(Matsumoto et al., 1995; Hirai et al., 1998; Tanaka et al., 2004)have been developed for plant germplasm. Furthermore, in orderto promote the regrowth of cryopreserved apices, cold-acclima-tion of in vitro plants (Seibert and Wetherbee, 1977; Chang andReed, 2000) or adding glycerol to beads or loading solution(Matsumoto and Sakai, 1995; Sakai et al., 2000) have beeninvestigated.

D. Kami et al. / Scientia Horticulturae 120 (2009) 84–88 85

To select the most useful cryopreservation method of hawthornshoot apices, we investigated the effects of cold-acclimation, themoisture content of shoot apices, the exposure to the vitrificationsolution (duration of loading treatment), the addition of glycerol tobeads and loading solution and their interaction with shootregrowth from cryopreserved shoot tips of four Crataegus species(Crataegus pinnatifida Bge., Crataegus maximowiczii Schneid., Cra-

taegus dahurica Koehne. and Crataegus chlorosarca Maxim.) follow-ing cryopreservation using the above-mentioned three procedures.

2. Material and methods

2.1. Plant materials and preculture

Hawthorn (C. pinnatifida Bge.) subcultured for 4 years wereprimarily used in the present study. In the latest experiment todetermine the applicability of the established protocol, we usedthree other Crataegus species (C. maximowiczii Schneid., C. dahurica

Koehne. and C. chlorosarca Maxim.) originating from East Asia thatwere maintained for 4 years at Hokkaido University (Sapporo,Japan). As plant material, we used shoot apices (approximately1 mm in length) excised from axillary buds of 1-month-old culturesthat had been cold-acclimated for the preceding 2 weeks at 5 8C indarkness for cryopreservation experiments according to Kami et al.(2005). In addition, in encapsulation–dehydration, to show clearlywhether cold-hardiness increases regrowth after cryopreservation,apices without cold-acclimation were also used as plant materials.

The tissue cultures were carried out using Murashige and Skoog(1962) [MS] basal medium supplemented with 10�4 g/L 6-benzy-laminopurine (BAP), 30.0 g/L sucrose and 7.0 g/L agar (pH adjustedto 5.7) at 25 8C under 16-h illumination (60 mmol m�2 s�1 fromfluorescent tubes) according to Shi et al. (2003). Before freezing,apices were precultured for 24 h on hormone-free MS medium with0.4 mol/L sucrose under the same subculture conditions.

2.2. Encapsulation, loading, dehydration and cryopreservation

2.2.1. Encapsulation–dehydration

Our procedures were the same as those described by Kami et al.(2005). Apices were suspended in 50 mL of calcium-free MS liquidmedium with 30 g/L sodium alginate. The mixture was added dropby drop to the liquid MS medium containing 0.1 mol/L calciumchloride, forming beads about 5 mm in diameter. The above-mentioned MS liquid mediums (30.0 g/L sodium alginate and0.1 mol/L calcium chloride) contained 0.4 mol/L sucrose (pH 5.7),but without plant growth regulators. Alginate-coated apices wereimmersed for 16 h at 25 8C in the liquid MS medium (pH 5.7)containing 0.8 mol/L sucrose (the loading solution; it was plantgrowth regulator-free). Beads were air-dried for 60, 120, 180, 240,300, 360 and 420 min in a glass laboratory dish (12 cm in diameterand 3 cm in height) with 50 g silica gel (30 beads/dish) at 25 8C. Tocounteract the effects of adding different glycerol concentrationsto both the loading solution and beads, the components of both theformer and the latter were changed to contain 0.5, 1.0 and 2.0 mol/L glycerol. In this case, alginate-coated apices were dried for360 min in a glass dish with 50 g silica gel (30 beads/desiccator) at25 8C. The dried beads packed in a 5-mL plastic tube were plungeddirectly into liquid nitrogen (LN, �196 8C) and kept there for 1 h.Apices were then warmed immediately in 38 8C water for 2 min.

2.2.2. Vitrification

Procedures were identical to those described by Sakai et al.(1990). Ten apices were placed in a 5-mL plastic tube for 20 min at25 8C in 2 mL of liquid MS medium containing 2.0 mol/L glyceroland 0.4 mol/L sucrose (the loading solution; it was adjusted to pH

5.7 and plant growth regulators-free) and then dehydrated at 0 8Cfor 20, 40, 60, 80 and 100 min with 2 mL of plant vitrificationsolution 2 (PVS2; Sakai et al., 1990, pH adjusted to 5.7). Whendehydrating apices, PVS2 in a plastic tube was exchanged for freshPVS2 at intervals of 20 min to prevent its deterioration (anoccurrence previously reported by Kami et al., 2005). Ten apices ina tube filled with PVS2 were plunged directly into LN and keptthere for 1 h. Apices were then warmed in 38 8C water for 2 minand washed in the hormone-free liquid MS medium with 1.2 mol/Lsucrose for 20 min.

2.2.3. Encapsulation–vitrification

Procedures were the same as those described by Matsumoto et al.(1995). Apices were encapsulated and loaded under the sameconditions as those for encapsulation–dehydration. In this method,30.0 g/L alginate medium, 0.1 mol/L calcium chloride liquidmedium, and a loading solution were supplemented with1.0 mol/L glycerol. Ten loaded apices were placed in a 20-mL glasstest tube and then dehydrated at 0 8C for 60, 120, 180, 240, 300 and360 min with 10 mL of PVS2. When dehydrating apices, the PVS2 in atest tube was exchanged for fresh PVS2 at intervals of 30 min. Tocounteract the effects of adding glycerol concentrations to both theloading solution and beads, apices were encapsulated and loadedunder the same conditions (0.5, 1.0 and 2.0 mol/L glycerol) as thosefor encapsulation–dehydration. In this case, alginate-coated apiceswere dehydrated for 240 min in PVS2 at 0 8C. Ten encapsulatedapices packed in a 5-mL plastic tube filled with PVS2 were plungeddirectly into LN and kept there for 1 h. They were then warmed in38 8C water for 2 min and washed in the hormone-free liquid MSmedium with 1.2 mol/L sucrose for 20 min.

2.3. Evaluation of survival and regrowth

Apices with or without encapsulation were cultured on thestandard MS medium under the same culture conditions. Twoweeks after culture, apices without green tissue were counted asdead. In encapsulation–dehydration and encapsulation–vitrifica-tion, apices were stripped from the alginate beads 2 weeks afterculture and recultured in fresh culture medium. Apices withoutcryopreservation were used as a control. Regrowth of the apiceswas evaluated 2 months after the culture, and those withelongated shoots (above 5 mm) were counted as regrowing.

2.4. Determination of moisture content in alginate beads

Percentages of the moisture content in beads were determinedby calculating the weights of the beads. Complete drying wasachieved by heating fresh beads in a drying oven at 70 8C for 2 days.Moisture content in beads was calculated on a fresh-weight basis.

2.5. Statistical analyses of data

Regrowth percentages were independently determined fourtimes using 10 apices each. Moisture content in the beads wasdetermined by weighing four sets of 10 beads. Data wererepresented as average � S.E. Statistical differences in the data wereanalyzed by ANOVA and Tukey’s HSD at p < 0.05.

3. Results

3.1. Effects of cold-acclimation and cryopreservation methods on

regrowth after rewarming

In encapsulation–dehydration, regrowth changed with respectto both the dessication time and moisture content of beads. In cold-

D. Kami et al. / Scientia Horticulturae 120 (2009) 84–8886

acclimated apices, the regrowth percentage of control apiceshardly changed during the silica gel desiccation. On the other hand,in non-cold-acclimated samples, the regrowth have decreasedgradually during the first 240 min of dessication. Since the 240-min desiccation, the regrowth percentage remained 40%, and fellsignificantly (p < 0.05) compared with that of the 0-min desicca-tion.

After cryopreservation, in both cold-acclimated and non-acclimated shoot apices, following the first 180 min in dessication,no survival was observed. In cold-acclimated specimens, theregrowth percentage climbed quickly after 240-min desiccation,reaching more than 50%. The maximum regrowth percentage was65.5 � 9.6% at 360-min desiccation, though that showed nosignificant difference (p < 0.05) compared with that of 300-minand 420-min desiccation. On the other hand, in non-cold-acclimatedapices, apex regrowth was achieved after the 240-min silica geldesiccation, and regrowth increased to 10.0 � 6.5% after 360 min. Theoptimal regrowth conditions in both samples were acquired when themoisture content of apices was 15.3% at 360-min silica gel desiccation(Fig. 1).

The effects of the loading time of PVS2 (vitrification andencapsulation–vitrification) on the regrowth of control andcryopreserved apices were shown in Fig. 2. In the case ofvitrification, the regrowth rate of control apices tended to decreasealong with the PVS2 loading time. The regrowth of apices after

Fig. 1. Effects of silica gel desiccation at 25 8C on the bead moisture content and

regrowth of shoot apices immersed in LN using encapsulation–dehydration. Apices

were dehydrated on silica gel for various lengths of time prior to cooling

(Cryopreserved) or without cooling to �196 8C (Control). Apices were excised from

in vitro plants after cold-acclimation (b) or without it (a) at 5 8C in the dark for 2

weeks. Excised apices were precultured with MS medium containing 0.4 mol/L

sucrose for 24 h at 25 8C, then encapsulated with alginate beads containing 0.4 mol/

L sucrose, and loaded with the liquid MS medium (loading solution) with 0.8 mol/L

sucrose for 16 h at 25 8C before dehydration on silica gel; n = 40. Values represent

mean � S.E. of four determinations. Differences in mean values of control and

cryopreseved apices with different letters are statistically significant (Tukey’s HSD at

p < 0.05) in each figure.

Fig. 2. Effect of exposure time to PVS2 at 0 8C on the regrowth of shoot apices

immersed in LN using vitrification (a) and encapsulation–vitrification (b). Apices

were dehydrated with PVS2 solution at 0 8C for various lengths of time prior to

cooling (Cryopreserved) or without cooling to �196 8C (Control); n = 40. Values

represent mean � S.E. of four determinations. Differences in mean values of control

and cryopreseved apices with different letters are statistically significant (Tukey’s HSD

at p < 0.05) in each cryopreservation procedure.

cryopreservation increased with PVS2 loading time, with themaximum value (12.5 � 9.6%) obtained at a 60-min loading, thoughnot significantly different (p < 0.05) compared with other loadingtimes.

With encapsulation–vitrification, the regrowth percentages ofcontrol apices did not change significantly during PVS2 loadingtime. In cryopreserved apices, the regrowth percentage increasedquickly after the 120 min-PVS2 loading, reaching over 50%. Theoptimal regrowth percentage was 62.5% at a 240-min loading.

In three cryopreservation methods, the survival of apices withor without cryopreservation could be confirmed after 2 weeks ofculture. In encapsulation–dehydration and encapsulation–vitrifi-cation, apices were unable to break beads at 2 weeks. Apices werethen excised from alginate beads, and transplanted to a basalmedium. Surviving specimens always elongated their shoots by 2months of culture regardless of the cryopreservation methods used(Fig. 3).

3.2. Improvement of encapsulation–dehydration and encapsulation–

vitrification methods to increase regrowth after cryopreservation

To enhance regrowth after encapsulation–dehydration andencapsulation–vitrification, the effects of suitable glycerol con-centrations added to beads and loading solution on the regrowth ofcryopreserved apices were investigated. In both methods, thepercentage of regrowing apices increased with the glycerolconcentration added to beads and loading solution. Withencapsulation–dehydration, the addition of 0.5 mol/L glycerolincreased the regrowth percentage to 82.5 � 2.5%. However, adding2.0 mol/L glycerol caused a more significant decrease (p < 0.05) than

Fig. 3. Plantlets developed from shoot apices cooled to �196 8C using the encapsulation–dehydration procedure. Photos were taken 2 weeks (a) and 2 months (b) after

reculture. Bars indicate 5 mm in (a) and (b). Material: Crataegus pinnatifida.

D. Kami et al. / Scientia Horticulturae 120 (2009) 84–88 87

other concentrations. In the case of encapsulation–vitrification, theaddition of 1.0 mol/L glycerol resulted in a higher regrowth(60.0 � 5.8%) than that of 0 and 0.5 mol/L glycerol additions, thoughits value was significantly lower than that of 0.5 mol/L glycerol inencapsulation–dehydration (Fig. 4).

Using the optimal conditions of encapsulation–dehydrationestablished in the present study, the regrowth of shoot apices offour Crataegus species were compared after cryopreservation, andall four were found to be above 80%, ranging from 82.5% for C.

pinnatifida and C. maximowiczii to 90.0% for C. dahurica, with nosignificant difference at the 5% level (data not shown).

4. Discussion

The aim of this study is to establish a practical procedure forcryopreservation of in vitro-cultured tissues of hawthorn. We firstshowed that cold-acclimation was an effective treatment in thecryopreservation of hawthorn apices by demonstrating that theregrowth of cold-acclimated apices after desiccation and cryopre-servation was higher than non-acclimated apices in encapsulation–dehydration. This result coincided with that of the previous studies(Seibert and Wetherbee, 1977; Chang and Reed, 2000). Next, thethree cryopreservation methods were examined in cold-acclimatedspecimens, and high regrowth (above 50%) was achieved in bothencapsulation–dehydration and encapsulation–vitirification. The

Fig. 4. Effects on regrowth of cryopreserved hawthorn shoot apices by adding

different glycerol concentrations to both beads and loading solution using

encapsulation–dehydration or encapsulation–vitrification. Encapsulated samples

were desiccated on silica gel for 360 min in encapsulation–dehydration or with

PVS2 for 240 min in encapsulation–vitrification. Dehydrated samples were

immersed in LN for 1 h; n = 40. Values represent mean � S.E. of four

determinations. Differences in mean values of glycerol concentrations labeled with

different letters are statistically significant (Tukey’s HSD at p < 0.05) in two

cryopreservation procedures.

regrowth of apices immersed in LN was somewhat lower than thecontrol due to some type of cryopreservation injury following thesetwo techniques.

However, in encapsulation–dehydration, the regrowth ofencapsulated apices desiccated for more than 300 min did notdiffer significantly compared with control, regardless of the LNimmersion. Obviously, the regrowth of encapsulated apicesdepends on residual moisture in the beads, and the optimalregrowth condition after cryopreservation was obtained at amoisture content range of 14.3–17.2%, which is close to those inprevious reports on the cryopreservation of seed (Chandel et al.,1995; Kim et al., 2002). It seems possible that the vitrified state ofthe cytoplasm of hawthorn cells occurred at a bead moisture of lessthan 17.2% because of the reduction of free water. In this study, itwas determined that the optimal desiccation time was 360 minbecause its regrowth percentage was stable compared with that of300 or 420 min. As this stage, it appeared to us that encapsulation–dehydration was superior to encapsulation–vitrification as amethod of cryopreservation since the procedure of the formerwas easier than that of the latter, although maximum regrowth ofthe two methods were almost same (encapsulation–dehydration:65.0 � 6.5% and encapsulation–vitrification: 62.5 � 7.5%). However,treatments that raise regrowth percentage after cryopreservation tomore than 80% are needed. Matsumoto and Sakai (1995) reported thatthe addition of glycerol to beads and loading solution increased theregrowth of cryopreserved wasabi apices after rewarming.

Next, we examined the effect of added glycerol in beads andloading solution on the regrowth of hawthorn apices in encapsula-tion–dehydration and encapsulation–vitrification. As a result, theaddition of 0.5 mol/L glycerol to beads and loading solutionresulted in high recovery (82.5 � 2.5%) of shoot apices in encapsula-tion–dehydration, a value that proved to be higher than themaximum regrowth of encapsulation–vitrification (60.0 � 5.8%). Thisresult coincided with that of Kami et al. (2005), since they reported,using encapsulation–dehydration, 77% of blue honeysuckle apicesregrew after cryopreservation when 0.5 mol/L glycerol was added tothe loading solution only. Thus, it seemed that the effect of glycerol onshoot regrowth after cryopreservation depends more on loadingsolution than on alginate beads. With respect to the mechanism ofglycerol on shoot regrowth, Morris et al. (2006) estimated thatglycerol suppressed the modification of the cell after dehydrationand/or freezing by restricting the osmotic loss of water from cellssince they discovered that the viscosity of a glycerol aqueous solution(100 g/L) had exceeded 1000 cP at �45 8C. In this study, glycerolloaded-beads, unlike control beads, retained their soft form duringdesiccation. It is conceivable that glycerol acts to reduce beadshrinkage and related tissue damage during silica gel desiccation. Onthe other hand, glycerol might induce dehydration resistance and/or

D. Kami et al. / Scientia Horticulturae 120 (2009) 84–8888

freezing resistance of the tissues, since Matsumoto et al. (1998)discovered that an incorporation of glycerol enhanced the prolinecontent of tissues in the apical meristems of wasabi. Therefore, it isalso necessary to monitor the cells of encapsulated apices whendehydrating or cryopreserving to clarify any relation betweenglycerol and the physical properties (texture, etc.) of alginate beads.

It has been reported that regrowth after cryopreservation aredifferent between cultivars and related species (Niwata, 1995;Kuranhuki and Yoshida, 1996; Hirai et al., 1998; Tanaka et al.,2004). However, encapsulation–dehydration using glycerolseemed to be a suitable cryopreservation protocol for hawthornshoot apices, as evidenced by the four Crataegus species thatshowed 82.5–90.0% regrowth after rewarming.

In summary, a practical cryopreservation method using theencapsulation–dehydration technique was established for theshoot apices of hawthorns that grow wild in Japan. In particular,the addition of 0.5 mol/L glycerol to the beads and loading solutionwas very effective in increasing the regrowth rate of apices. It isexpected that this modified encapsulation–dehydration procedurewill be used widely for more successful cryopreservation inadditional plant germplasm.

References

Chandel, K.P.S., Chaudhury, R., Radhamani, J., Malik, S.K., 1995. Dessication andfreezing sensitivity in recalcitrant seeds of tea, cocoa and jackfruit. Ann. Bot. 76,443–450.

Chang, Y., Reed, B.M., 2000. Extended alternating-temperature cold acclimation andculture duration improve pear shoot cryopreservation. Cryobiology 40, 311–322.

Damiano, C., Dolores, M., Padro, A., Frattarelli, A., 2006. Experiences in cryopre-servation of temperate small fruit plants. Abstracts: Proceedings of the 27thInternational Horticultural Congress & Exhibition (IHC 2006), p. 5.

Fabre, J., Dereuddre, J., 1990. Encapsulation dehydration–a new approach to cryo-preservation of Solanum shoot-tips. Cryoletters 11, 413–426.

Gonzalez-Arnao, M.T., Juarez, J., Ortega, C., Navarro, L., Duran-Vila, N., 2003.Cryopreservation of ovules and somatic embryos of citrus using the encapsula-tion–dehydration technique. Cryoletters 24, 85–94.

Hirai, D., Shirai, K., Shirai, S., Sakai, A., 1998. Cryopreservation of in vitro grownmeristems of strawberry (Fragaria � ananassa Duch.) by encapsulation–vitri-fication. Euphytica 101, 109–115.

Kami, D., Suzuki, T., Oosawa, K., 2005. Cryopreservation of blue honeysuckle in vitro-cultured tissue using encapsulation–dehydration and vitrification. Cryobiol.Cryotechnol. 51, 63–68.

Kartha, K.K., Engelmann, F., 1994. In: Vasil, I.K., Thorpe, T.A. (Eds.), Plant Cell andTissue Culture. Kluwer Academic Publishers, Dordrecht, pp. 195–230.

Kim, H.H., Cha, Y.S., Baek, H.J., Cho, E.G., Chae, Y.A., Engelmann, F., 2002. Cryopre-servation of tea (Camellia sinensis L.) seeds and embryonic axes. Cryoletters 23,209–216.

Kuranhuki, Y., Yoshida, Y., 1996. Different responses of embryonic axis and coty-ledons from tea seeds to dessication and cryoexposure. Breeding Sci. 46, 149–154.

Matsumoto, T., Sakai, A., Takahashi, C., Yamada, K., 1995. Cryopreservation of invitro-grown apical meristems of wasabi (Wasabia japonica) by encapsulation–vitrification method. Cryoletters 16, 189–196.

Matsumoto, T., Sakai, A., 1995. An approach to enhance dehydration tolerance ofalginate-coated dried meristems cooled to �196 8C. Cryoletters 16, 299–306.

Matsumoto, T., Sakai, A., Nako, Y., 1998. A novel preculturing for enhancing thesurvival of in vitro-grown apical meristems of wasabi (Wasabia japonica) cooledto �196 8C by vitrification. Cryoletters 19, 27–36.

Mi, W.G., Zhang, Y.Z., Sanada, T., 1992. Genetic resources of hawthorn and its use inChina. Agric. Hortic. 67, 991–995 (in Japanese).

Morris, G.J., Goodrich, M., Acton, E., Fonseca, F., 2006. The high viscosity encoun-tered during freezing in glycerol solutions: effects on cryopreservation. Cryo-biology 52, 323–334.

Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassayswith tobacco tissue culture. Physiol. Plant. 15, 473–497.

Nikolov, N., Seligmann, O., Wagner, H., Horowitz, R., Gentili, B., 1982. Neue flavo-noid-glykoside aus Crataegus monogyna und Crataegus pentagyna. Planta Med.44, 50–53.

Niwata, E., 1995. Cryopreservation of apical meri-stems of garlic (Allium sativum L.)and high sub-sequent plant re-generation. Cryoletters 16, 102–107.

Reed, B.M., Hummer, K., 1995. In: Bajaj, Y.P.S. (Ed.), Cryopreservation of PlantGermplasm I. Biotechnology Agriculture and Forestry, vol. 32. Springer Verlag,Berlin, pp. 354–370.

Rigelsky, J.M., Sweet, B.W., 2002. Hawthorn: pharmacology and therapeutic uses.Am. J. Health Syst. Pharm. 59, 417–422.

Sakai, A., Kobayashi, K., Oiyama, I., 1990. Cryopreservatioin of nucellar cells of navelorange (Citrus sinensis Osb. var Brasiliensis Tanaka) by vitrification. Plant CellRep. 9, 30–33.

Sakai, A., Matsumoto, T., Hirai, D., Niino, T., 2000. Newly developed encapsulation–dehydration protocol for plant cryopreservation. Cryoletters 21, 53–62.

Seibert, M., Wetherbee, P.J., 1977. Increased survival and differentiation of frozenherbaceous plant organ cultures through cold treatment. Plant Physiol. 59,1043–1046.

Shi, L., Suzuki, T., Satoh, T., Oosawa, K., 2003. Vegetable propagation of hawthorn(Crataegus sp.) by stem tip culture. J. Jpn. Soc. Hortic. Sci. 72 (Suppl. 1), 391 (inJapanese).

Shi, L., Tanaka, A., Tanaka, T., Satoh, T., Suzuki, T., 2004a. Characteristics of chemicalcomponents of fruits of three Crataegus species originating in East Asia. Hortic.Res. (Jpn.) 3, 333–338 (in Japanese with English summary).

Shi, L., Murata, N., Satoh, T., Suzuki, T., Oosawa, K., 2004b. Interspecific hybrids ofhawthorn (Crataegus spp.) obtained by embryo culture. J. Jpn. Soc. Hortic. Sci. 73(Suppl. 1), 391–1391 (in Japanese).

Tanaka, D., Niino, T., Isuzugawa, K., Hikage, T., Uemura, M., 2004. Cryopreservationof shoot apices of in vitro-grown Gentiana plants: a comparison of vitrificationand encapsulation–vitrification protocols. Cryoletters 25, 167–176.

Uragami, A., Sakai, A., Nagai, M., Takahashi, T., 1989. Survival of cultured cells andsomatic embryos of Asparagus officinalis cryopreserved by vitrification. PlantCell Rep. 8, 418–421.