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Cryopreservation of shoot apices of hawthorn in vitro cultures originating from East Asia
Scientia Horticulturae 120 (2009) 84–88
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Cryopreservation of shoot apices of hawthorn in vitro cultures originating from East Asia D. Kami a,*, L. Shi a,1, T. Sato b,2, T. Suzuki a,1, K. Oosawa a,1 a b
Department of Horticultural Science and Landscape Architecture, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan Hokkaido Forestry Research Center, Koshunai-Cho, Bibai, Hokkaido 079-0198, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 September 2007 Received in revised form 20 September 2008 Accepted 22 September 2008
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, vitrification, 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 coldacclimation 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. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Cryopreservation Encapsulation–dehydration Encapsulation–vitrification Glycerol Hawthorn Vitrification
1. Introduction Hawthorn (Crataegus spp.) is a kind of small fruit tree originating in North America and has importance for landscaping and other ornamental purposes in Europe and Asia (Mi et al., 1992). In China, Crataegus pinnatifida is a commercially important tree whose fruit is used for making jam, juice, and confectioneries (Mi et al., 1992). In addition, hawthorn has generated keen interest as a functional material around the world because its leaves or flowers are rich in flavonoids (Nikolov et al., 1982; Rigelsky and Sweet, 2002). Moreover, Shi et al. (2003) have also reported that hawthorn fruit that grew wild in Japan contained high levels of minerals, b-carotene and anthocyanin. Therefore, the production of interspecific hybrids of hawthorn has been examined using an embryoculture (Shi et al., 2004a, b). However,
* Corresponding author. Tel.: +81 11 706 2450; fax: +81 11 706 2450. E-mail address: [email protected] (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 ß 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-, spaceand labor-consuming, there is impetus to establish a practical procedure for preserving in vitro hawthorn apices over a long period. Cryopreservation has become important as a means of ensuring the long-term preservation of plant germplasms (Kartha and Engelmann, 1994; Reed and Hummer, 1995). Although Damiano et al. (2006) reported a survival rate of 25% from cryopreservation of shoot apices of Crataegus azarolus, an even more efficient cryopreservation protocol (regrowth percentage above 80%) of hawthorn apices has yet to be found. Since the second half of the 1980s, various cryopreservation methods, i.e., vitrification (Uragami et al., 1989; Sakai et al., 1990), encapsulation–dehydration (Fabre and Dereuddre, 1990; Gonzalez-Arnao et 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 order to promote the regrowth of cryopreserved apices, cold-acclimation of in vitro plants (Seibert and Wetherbee, 1977; Chang and Reed, 2000) or adding glycerol to beads or loading solution (Matsumoto and Sakai, 1995; Sakai et al., 2000) have been investigated.
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To select the most useful cryopreservation method of hawthorn shoot apices, we investigated the effects of cold-acclimation, the moisture content of shoot apices, the exposure to the vitrification solution (duration of loading treatment), the addition of glycerol to beads and loading solution and their interaction with shoot regrowth from cryopreserved shoot tips of four Crataegus species (Crataegus pinnatifida Bge., Crataegus maximowiczii Schneid., Crataegus dahurica Koehne. and Crataegus chlorosarca Maxim.) following cryopreservation using the above-mentioned three procedures.
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5.7 and plant growth regulators-free) and then dehydrated at 0 8C for 20, 40, 60, 80 and 100 min with 2 mL of plant vitrification solution 2 (PVS2; Sakai et al., 1990, pH adjusted to 5.7). When dehydrating apices, PVS2 in a plastic tube was exchanged for fresh PVS2 at intervals of 20 min to prevent its deterioration (an occurrence previously reported by Kami et al., 2005). Ten apices in a tube filled with PVS2 were plunged directly into LN and kept there for 1 h. Apices were then warmed in 38 8C water for 2 min and washed in the hormone-free liquid MS medium with 1.2 mol/L sucrose for 20 min.
2. Material and methods 2.1. Plant materials and preculture Hawthorn (C. pinnatifida Bge.) subcultured for 4 years were primarily used in the present study. In the latest experiment to determine the applicability of the established protocol, we used three other Crataegus species (C. maximowiczii Schneid., C. dahurica Koehne. and C. chlorosarca Maxim.) originating from East Asia that were maintained for 4 years at Hokkaido University (Sapporo, Japan). As plant material, we used shoot apices (approximately 1 mm in length) excised from axillary buds of 1-month-old cultures that had been cold-acclimated for the preceding 2 weeks at 5 8C in darkness for cryopreservation experiments according to Kami et al. (2005). In addition, in encapsulation–dehydration, to show clearly whether 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-benzylaminopurine (BAP), 30.0 g/L sucrose and 7.0 g/L agar (pH adjusted to 5.7) at 25 8C under 16-h illumination (60 mmol m 2 s 1 from fluorescent tubes) according to Shi et al. (2003). Before freezing, apices were precultured for 24 h on hormone-free MS medium with 0.4 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 liquid medium with 30 g/L sodium alginate. The mixture was added drop by drop to the liquid MS medium containing 0.1 mol/L calcium chloride, forming beads about 5 mm in diameter. The abovementioned MS liquid mediums (30.0 g/L sodium alginate and 0.1 mol/L calcium chloride) contained 0.4 mol/L sucrose (pH 5.7), but without plant growth regulators. Alginate-coated apices were immersed 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 plant growth regulator-free). Beads were air-dried for 60, 120, 180, 240, 300, 360 and 420 min in a glass laboratory dish (12 cm in diameter and 3 cm in height) with 50 g silica gel (30 beads/dish) at 25 8C. To counteract the effects of adding different glycerol concentrations to both the loading solution and beads, the components of both the former 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 for 360 min in a glass dish with 50 g silica gel (30 beads/desiccator) at 25 8C. The dried beads packed in a 5-mL plastic tube were plunged directly 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 at 25 8C in 2 mL of liquid MS medium containing 2.0 mol/L glycerol and 0.4 mol/L sucrose (the loading solution; it was adjusted to pH
2.2.3. Encapsulation–vitrification Procedures were the same as those described by Matsumoto et al. (1995). Apices were encapsulated and loaded under the same conditions as those for encapsulation–dehydration. In this method, 30.0 g/L alginate medium, 0.1 mol/L calcium chloride liquid medium, and a loading solution were supplemented with 1.0 mol/L glycerol. Ten loaded apices were placed in a 20-mL glass test tube and then dehydrated at 0 8C for 60, 120, 180, 240, 300 and 360 min with 10 mL of PVS2. When dehydrating apices, the PVS2 in a test tube was exchanged for fresh PVS2 at intervals of 30 min. To counteract the effects of adding glycerol concentrations to both the loading solution and beads, apices were encapsulated and loaded under the same conditions (0.5, 1.0 and 2.0 mol/L glycerol) as those for encapsulation–dehydration. In this case, alginate-coated apices were dehydrated for 240 min in PVS2 at 0 8C. Ten encapsulated apices packed in a 5-mL plastic tube filled with PVS2 were plunged directly into LN and kept there for 1 h. They were then warmed in 38 8C water for 2 min and washed in the hormone-free liquid MS medium with 1.2 mol/L sucrose for 20 min. 2.3. Evaluation of survival and regrowth Apices with or without encapsulation were cultured on the standard MS medium under the same culture conditions. Two weeks after culture, apices without green tissue were counted as dead. In encapsulation–dehydration and encapsulation–vitrification, apices were stripped from the alginate beads 2 weeks after culture and recultured in fresh culture medium. Apices without cryopreservation were used as a control. Regrowth of the apices was evaluated 2 months after the culture, and those with elongated 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 determined by calculating the weights of the beads. Complete drying was achieved 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 four times using 10 apices each. Moisture content in the beads was determined by weighing four sets of 10 beads. Data were represented as average S.E. Statistical differences in the data were analyzed 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 respect to both the dessication time and moisture content of beads. In cold-
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acclimated apices, the regrowth percentage of control apices hardly changed during the silica gel desiccation. On the other hand, in non-cold-acclimated samples, the regrowth have decreased gradually during the first 240 min of dessication. Since the 240min desiccation, the regrowth percentage remained 40%, and fell significantly (p < 0.05) compared with that of the 0-min desiccation. After cryopreservation, in both cold-acclimated and nonacclimated shoot apices, following the first 180 min in dessication, no survival was observed. In cold-acclimated specimens, the regrowth percentage climbed quickly after 240-min desiccation, reaching more than 50%. The maximum regrowth percentage was 65.5 9.6% at 360-min desiccation, though that showed no significant difference (p < 0.05) compared with that of 300-min and 420-min desiccation. On the other hand, in non-cold-acclimated apices, apex regrowth was achieved after the 240-min silica gel desiccation, and regrowth increased to 10.0 6.5% after 360 min. The optimal regrowth conditions in both samples were acquired when the moisture content of apices was 15.3% at 360-min silica gel desiccation (Fig. 1). The effects of the loading time of PVS2 (vitrification and encapsulation–vitrification) on the regrowth of control and cryopreserved apices were shown in Fig. 2. In the case of vitrification, the regrowth rate of control apices tended to decrease along with the PVS2 loading time. The regrowth of apices after
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 the maximum value (12.5 9.6%) obtained at a 60-min loading, though not significantly different (p < 0.05) compared with other loading times. With encapsulation–vitrification, the regrowth percentages of control apices did not change significantly during PVS2 loading time. In cryopreserved apices, the regrowth percentage increased quickly after the 120 min-PVS2 loading, reaching over 50%. The optimal regrowth percentage was 62.5% at a 240-min loading. In three cryopreservation methods, the survival of apices with or without cryopreservation could be confirmed after 2 weeks of culture. In encapsulation–dehydration and encapsulation–vitrification, apices were unable to break beads at 2 weeks. Apices were then excised from alginate beads, and transplanted to a basal medium. Surviving specimens always elongated their shoots by 2 months 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 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.
To enhance regrowth after encapsulation–dehydration and encapsulation–vitrification, the effects of suitable glycerol concentrations added to beads and loading solution on the regrowth of cryopreserved apices were investigated. In both methods, the percentage of regrowing apices increased with the glycerol concentration added to beads and loading solution. With encapsulation–dehydration, the addition of 0.5 mol/L glycerol increased the regrowth percentage to 82.5 2.5%. However, adding 2.0 mol/L glycerol caused a more significant decrease (p < 0.05) than
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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.
other concentrations. In the case of encapsulation–vitrification, the addition 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, though its value was significantly lower than that of 0.5 mol/L glycerol in encapsulation–dehydration (Fig. 4). Using the optimal conditions of encapsulation–dehydration established in the present study, the regrowth of shoot apices of four Crataegus species were compared after cryopreservation, and all four were found to be above 80%, ranging from 82.5% for C. pinnatifida and C. maximowiczii to 90.0% for C. dahurica, with no significant difference at the 5% level (data not shown). 4. Discussion The aim of this study is to establish a practical procedure for cryopreservation of in vitro-cultured tissues of hawthorn. We first showed that cold-acclimation was an effective treatment in the cryopreservation of hawthorn apices by demonstrating that the regrowth of cold-acclimated apices after desiccation and cryopreservation 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, the three cryopreservation methods were examined in cold-acclimated specimens, and high regrowth (above 50%) was achieved in both encapsulation–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 the control due to some type of cryopreservation injury following these two techniques. However, in encapsulation–dehydration, the regrowth of encapsulated apices desiccated for more than 300 min did not differ significantly compared with control, regardless of the LN immersion. Obviously, the regrowth of encapsulated apices depends on residual moisture in the beads, and the optimal regrowth condition after cryopreservation was obtained at a moisture content range of 14.3–17.2%, which is close to those in previous reports on the cryopreservation of seed (Chandel et al., 1995; Kim et al., 2002). It seems possible that the vitrified state of the cytoplasm of hawthorn cells occurred at a bead moisture of less than 17.2% because of the reduction of free water. In this study, it was determined that the optimal desiccation time was 360 min because its regrowth percentage was stable compared with that of 300 or 420 min. As this stage, it appeared to us that encapsulation– dehydration was superior to encapsulation–vitrification as a method of cryopreservation since the procedure of the former was easier than that of the latter, although maximum regrowth of the 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 to more than 80% are needed. Matsumoto and Sakai (1995) reported that the addition of glycerol to beads and loading solution increased the regrowth of cryopreserved wasabi apices after rewarming. Next, we examined the effect of added glycerol in beads and loading solution on the regrowth of hawthorn apices in encapsulation–dehydration and encapsulation–vitrification. As a result, the addition of 0.5 mol/L glycerol to beads and loading solution resulted in high recovery (82.5 2.5%) of shoot apices in encapsulation–dehydration, a value that proved to be higher than the maximum regrowth of encapsulation–vitrification (60.0 5.8%). This result coincided with that of Kami et al. (2005), since they reported, using encapsulation–dehydration, 77% of blue honeysuckle apices regrew after cryopreservation when 0.5 mol/L glycerol was added to the loading solution only. Thus, it seemed that the effect of glycerol on shoot regrowth after cryopreservation depends more on loading solution than on alginate beads. With respect to the mechanism of glycerol on shoot regrowth, Morris et al. (2006) estimated that glycerol suppressed the modification of the cell after dehydration and/or freezing by restricting the osmotic loss of water from cells since they discovered that the viscosity of a glycerol aqueous solution (100 g/L) had exceeded 1000 cP at 45 8C. In this study, glycerol loaded-beads, unlike control beads, retained their soft form during desiccation. It is conceivable that glycerol acts to reduce bead shrinkage and related tissue damage during silica gel desiccation. On the other hand, glycerol might induce dehydration resistance and/or
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freezing resistance of the tissues, since Matsumoto et al. (1998) discovered that an incorporation of glycerol enhanced the proline content of tissues in the apical meristems of wasabi. Therefore, it is also necessary to monitor the cells of encapsulated apices when dehydrating or cryopreserving to clarify any relation between glycerol and the physical properties (texture, etc.) of alginate beads. It has been reported that regrowth after cryopreservation are different between cultivars and related species (Niwata, 1995; Kuranhuki and Yoshida, 1996; Hirai et al., 1998; Tanaka et al., 2004). However, encapsulation–dehydration using glycerol seemed to be a suitable cryopreservation protocol for hawthorn shoot apices, as evidenced by the four Crataegus species that showed 82.5–90.0% regrowth after rewarming. In summary, a practical cryopreservation method using the encapsulation–dehydration technique was established for the shoot apices of hawthorns that grow wild in Japan. In particular, the addition of 0.5 mol/L glycerol to the beads and loading solution was very effective in increasing the regrowth rate of apices. It is expected that this modified encapsulation–dehydration procedure will be used widely for more successful cryopreservation in additional plant germplasm. References Chandel, K.P.S., Chaudhury, R., Radhamani, J., Malik, S.K., 1995. Dessication and freezing 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 and culture duration improve pear shoot cryopreservation. Cryobiology 40, 311– 322. Damiano, C., Dolores, M., Padro, A., Frattarelli, A., 2006. Experiences in cryopreservation of temperate small fruit plants. Abstracts: Proceedings of the 27th International Horticultural Congress & Exhibition (IHC 2006), p. 5. Fabre, J., Dereuddre, J., 1990. Encapsulation dehydration–a new approach to cryopreservation 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 encapsulation–dehydration technique. Cryoletters 24, 85–94. Hirai, D., Shirai, K., Shirai, S., Sakai, A., 1998. Cryopreservation of in vitro grown meristems of strawberry (Fragaria ananassa Duch.) by encapsulation–vitrification. Euphytica 101, 109–115. Kami, D., Suzuki, T., Oosawa, K., 2005. Cryopreservation of blue honeysuckle in vitrocultured 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 and Tissue 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. Cryopreservation of tea (Camellia sinensis L.) seeds and embryonic axes. Cryoletters 23, 209–216. Kuranhuki, Y., Yoshida, Y., 1996. Different responses of embryonic axis and cotyledons from tea seeds to dessication and cryoexposure. Breeding Sci. 46, 149– 154. Matsumoto, T., Sakai, A., Takahashi, C., Yamada, K., 1995. Cryopreservation of in vitro-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 of alginate-coated dried meristems cooled to 196 8C. Cryoletters 16, 299–306. Matsumoto, T., Sakai, A., Nako, Y., 1998. A novel preculturing for enhancing the survival of in vitro-grown apical meristems of wasabi (Wasabia japonica) cooled to 196 8C by vitrification. Cryoletters 19, 27–36. Mi, W.G., Zhang, Y.Z., Sanada, T., 1992. Genetic resources of hawthorn and its use in China. Agric. Hortic. 67, 991–995 (in Japanese). Morris, G.J., Goodrich, M., Acton, E., Fonseca, F., 2006. The high viscosity encountered during freezing in glycerol solutions: effects on cryopreservation. Cryobiology 52, 323–334. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497. Nikolov, N., Seligmann, O., Wagner, H., Horowitz, R., Gentili, B., 1982. Neue flavonoid-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 Plant Germplasm 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 navel orange (Citrus sinensis Osb. var Brasiliensis Tanaka) by vitrification. Plant Cell Rep. 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 frozen herbaceous 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 (in Japanese). Shi, L., Tanaka, A., Tanaka, T., Satoh, T., Suzuki, T., 2004a. Characteristics of chemical components 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 of hawthorn (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. Cryopreservation of shoot apices of in vitro-grown Gentiana plants: a comparison of vitrification and encapsulation–vitrification protocols. Cryoletters 25, 167–176. Uragami, A., Sakai, A., Nagai, M., Takahashi, T., 1989. Survival of cultured cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification. Plant Cell Rep. 8, 418–421.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Cryopreservation of shoot apices of hawthorn in vitro cultures originating from East Asia D. Kami a,*, L. Shi a,1, T. Sato b,2, T. Suzuki a,1, K. Oosawa a,1 a b
Department of Horticultural Science and Landscape Architecture, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan Hokkaido Forestry Research Center, Koshunai-Cho, Bibai, Hokkaido 079-0198, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 September 2007 Received in revised form 20 September 2008 Accepted 22 September 2008
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, vitrification, 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 coldacclimation 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. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Cryopreservation Encapsulation–dehydration Encapsulation–vitrification Glycerol Hawthorn Vitrification
1. Introduction Hawthorn (Crataegus spp.) is a kind of small fruit tree originating in North America and has importance for landscaping and other ornamental purposes in Europe and Asia (Mi et al., 1992). In China, Crataegus pinnatifida is a commercially important tree whose fruit is used for making jam, juice, and confectioneries (Mi et al., 1992). In addition, hawthorn has generated keen interest as a functional material around the world because its leaves or flowers are rich in flavonoids (Nikolov et al., 1982; Rigelsky and Sweet, 2002). Moreover, Shi et al. (2003) have also reported that hawthorn fruit that grew wild in Japan contained high levels of minerals, b-carotene and anthocyanin. Therefore, the production of interspecific hybrids of hawthorn has been examined using an embryoculture (Shi et al., 2004a, b). However,
* Corresponding author. Tel.: +81 11 706 2450; fax: +81 11 706 2450. E-mail address: [email protected] (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 ß 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-, spaceand labor-consuming, there is impetus to establish a practical procedure for preserving in vitro hawthorn apices over a long period. Cryopreservation has become important as a means of ensuring the long-term preservation of plant germplasms (Kartha and Engelmann, 1994; Reed and Hummer, 1995). Although Damiano et al. (2006) reported a survival rate of 25% from cryopreservation of shoot apices of Crataegus azarolus, an even more efficient cryopreservation protocol (regrowth percentage above 80%) of hawthorn apices has yet to be found. Since the second half of the 1980s, various cryopreservation methods, i.e., vitrification (Uragami et al., 1989; Sakai et al., 1990), encapsulation–dehydration (Fabre and Dereuddre, 1990; Gonzalez-Arnao et 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 order to promote the regrowth of cryopreserved apices, cold-acclimation of in vitro plants (Seibert and Wetherbee, 1977; Chang and Reed, 2000) or adding glycerol to beads or loading solution (Matsumoto and Sakai, 1995; Sakai et al., 2000) have been investigated.
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To select the most useful cryopreservation method of hawthorn shoot apices, we investigated the effects of cold-acclimation, the moisture content of shoot apices, the exposure to the vitrification solution (duration of loading treatment), the addition of glycerol to beads and loading solution and their interaction with shoot regrowth from cryopreserved shoot tips of four Crataegus species (Crataegus pinnatifida Bge., Crataegus maximowiczii Schneid., Crataegus dahurica Koehne. and Crataegus chlorosarca Maxim.) following cryopreservation using the above-mentioned three procedures.
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5.7 and plant growth regulators-free) and then dehydrated at 0 8C for 20, 40, 60, 80 and 100 min with 2 mL of plant vitrification solution 2 (PVS2; Sakai et al., 1990, pH adjusted to 5.7). When dehydrating apices, PVS2 in a plastic tube was exchanged for fresh PVS2 at intervals of 20 min to prevent its deterioration (an occurrence previously reported by Kami et al., 2005). Ten apices in a tube filled with PVS2 were plunged directly into LN and kept there for 1 h. Apices were then warmed in 38 8C water for 2 min and washed in the hormone-free liquid MS medium with 1.2 mol/L sucrose for 20 min.
2. Material and methods 2.1. Plant materials and preculture Hawthorn (C. pinnatifida Bge.) subcultured for 4 years were primarily used in the present study. In the latest experiment to determine the applicability of the established protocol, we used three other Crataegus species (C. maximowiczii Schneid., C. dahurica Koehne. and C. chlorosarca Maxim.) originating from East Asia that were maintained for 4 years at Hokkaido University (Sapporo, Japan). As plant material, we used shoot apices (approximately 1 mm in length) excised from axillary buds of 1-month-old cultures that had been cold-acclimated for the preceding 2 weeks at 5 8C in darkness for cryopreservation experiments according to Kami et al. (2005). In addition, in encapsulation–dehydration, to show clearly whether 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-benzylaminopurine (BAP), 30.0 g/L sucrose and 7.0 g/L agar (pH adjusted to 5.7) at 25 8C under 16-h illumination (60 mmol m 2 s 1 from fluorescent tubes) according to Shi et al. (2003). Before freezing, apices were precultured for 24 h on hormone-free MS medium with 0.4 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 liquid medium with 30 g/L sodium alginate. The mixture was added drop by drop to the liquid MS medium containing 0.1 mol/L calcium chloride, forming beads about 5 mm in diameter. The abovementioned MS liquid mediums (30.0 g/L sodium alginate and 0.1 mol/L calcium chloride) contained 0.4 mol/L sucrose (pH 5.7), but without plant growth regulators. Alginate-coated apices were immersed 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 plant growth regulator-free). Beads were air-dried for 60, 120, 180, 240, 300, 360 and 420 min in a glass laboratory dish (12 cm in diameter and 3 cm in height) with 50 g silica gel (30 beads/dish) at 25 8C. To counteract the effects of adding different glycerol concentrations to both the loading solution and beads, the components of both the former 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 for 360 min in a glass dish with 50 g silica gel (30 beads/desiccator) at 25 8C. The dried beads packed in a 5-mL plastic tube were plunged directly 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 at 25 8C in 2 mL of liquid MS medium containing 2.0 mol/L glycerol and 0.4 mol/L sucrose (the loading solution; it was adjusted to pH
2.2.3. Encapsulation–vitrification Procedures were the same as those described by Matsumoto et al. (1995). Apices were encapsulated and loaded under the same conditions as those for encapsulation–dehydration. In this method, 30.0 g/L alginate medium, 0.1 mol/L calcium chloride liquid medium, and a loading solution were supplemented with 1.0 mol/L glycerol. Ten loaded apices were placed in a 20-mL glass test tube and then dehydrated at 0 8C for 60, 120, 180, 240, 300 and 360 min with 10 mL of PVS2. When dehydrating apices, the PVS2 in a test tube was exchanged for fresh PVS2 at intervals of 30 min. To counteract the effects of adding glycerol concentrations to both the loading solution and beads, apices were encapsulated and loaded under the same conditions (0.5, 1.0 and 2.0 mol/L glycerol) as those for encapsulation–dehydration. In this case, alginate-coated apices were dehydrated for 240 min in PVS2 at 0 8C. Ten encapsulated apices packed in a 5-mL plastic tube filled with PVS2 were plunged directly into LN and kept there for 1 h. They were then warmed in 38 8C water for 2 min and washed in the hormone-free liquid MS medium with 1.2 mol/L sucrose for 20 min. 2.3. Evaluation of survival and regrowth Apices with or without encapsulation were cultured on the standard MS medium under the same culture conditions. Two weeks after culture, apices without green tissue were counted as dead. In encapsulation–dehydration and encapsulation–vitrification, apices were stripped from the alginate beads 2 weeks after culture and recultured in fresh culture medium. Apices without cryopreservation were used as a control. Regrowth of the apices was evaluated 2 months after the culture, and those with elongated 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 determined by calculating the weights of the beads. Complete drying was achieved 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 four times using 10 apices each. Moisture content in the beads was determined by weighing four sets of 10 beads. Data were represented as average S.E. Statistical differences in the data were analyzed 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 respect to both the dessication time and moisture content of beads. In cold-
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acclimated apices, the regrowth percentage of control apices hardly changed during the silica gel desiccation. On the other hand, in non-cold-acclimated samples, the regrowth have decreased gradually during the first 240 min of dessication. Since the 240min desiccation, the regrowth percentage remained 40%, and fell significantly (p < 0.05) compared with that of the 0-min desiccation. After cryopreservation, in both cold-acclimated and nonacclimated shoot apices, following the first 180 min in dessication, no survival was observed. In cold-acclimated specimens, the regrowth percentage climbed quickly after 240-min desiccation, reaching more than 50%. The maximum regrowth percentage was 65.5 9.6% at 360-min desiccation, though that showed no significant difference (p < 0.05) compared with that of 300-min and 420-min desiccation. On the other hand, in non-cold-acclimated apices, apex regrowth was achieved after the 240-min silica gel desiccation, and regrowth increased to 10.0 6.5% after 360 min. The optimal regrowth conditions in both samples were acquired when the moisture content of apices was 15.3% at 360-min silica gel desiccation (Fig. 1). The effects of the loading time of PVS2 (vitrification and encapsulation–vitrification) on the regrowth of control and cryopreserved apices were shown in Fig. 2. In the case of vitrification, the regrowth rate of control apices tended to decrease along with the PVS2 loading time. The regrowth of apices after
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 the maximum value (12.5 9.6%) obtained at a 60-min loading, though not significantly different (p < 0.05) compared with other loading times. With encapsulation–vitrification, the regrowth percentages of control apices did not change significantly during PVS2 loading time. In cryopreserved apices, the regrowth percentage increased quickly after the 120 min-PVS2 loading, reaching over 50%. The optimal regrowth percentage was 62.5% at a 240-min loading. In three cryopreservation methods, the survival of apices with or without cryopreservation could be confirmed after 2 weeks of culture. In encapsulation–dehydration and encapsulation–vitrification, apices were unable to break beads at 2 weeks. Apices were then excised from alginate beads, and transplanted to a basal medium. Surviving specimens always elongated their shoots by 2 months 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 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.
To enhance regrowth after encapsulation–dehydration and encapsulation–vitrification, the effects of suitable glycerol concentrations added to beads and loading solution on the regrowth of cryopreserved apices were investigated. In both methods, the percentage of regrowing apices increased with the glycerol concentration added to beads and loading solution. With encapsulation–dehydration, the addition of 0.5 mol/L glycerol increased the regrowth percentage to 82.5 2.5%. However, adding 2.0 mol/L glycerol caused a more significant decrease (p < 0.05) than
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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.
other concentrations. In the case of encapsulation–vitrification, the addition 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, though its value was significantly lower than that of 0.5 mol/L glycerol in encapsulation–dehydration (Fig. 4). Using the optimal conditions of encapsulation–dehydration established in the present study, the regrowth of shoot apices of four Crataegus species were compared after cryopreservation, and all four were found to be above 80%, ranging from 82.5% for C. pinnatifida and C. maximowiczii to 90.0% for C. dahurica, with no significant difference at the 5% level (data not shown). 4. Discussion The aim of this study is to establish a practical procedure for cryopreservation of in vitro-cultured tissues of hawthorn. We first showed that cold-acclimation was an effective treatment in the cryopreservation of hawthorn apices by demonstrating that the regrowth of cold-acclimated apices after desiccation and cryopreservation 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, the three cryopreservation methods were examined in cold-acclimated specimens, and high regrowth (above 50%) was achieved in both encapsulation–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 the control due to some type of cryopreservation injury following these two techniques. However, in encapsulation–dehydration, the regrowth of encapsulated apices desiccated for more than 300 min did not differ significantly compared with control, regardless of the LN immersion. Obviously, the regrowth of encapsulated apices depends on residual moisture in the beads, and the optimal regrowth condition after cryopreservation was obtained at a moisture content range of 14.3–17.2%, which is close to those in previous reports on the cryopreservation of seed (Chandel et al., 1995; Kim et al., 2002). It seems possible that the vitrified state of the cytoplasm of hawthorn cells occurred at a bead moisture of less than 17.2% because of the reduction of free water. In this study, it was determined that the optimal desiccation time was 360 min because its regrowth percentage was stable compared with that of 300 or 420 min. As this stage, it appeared to us that encapsulation– dehydration was superior to encapsulation–vitrification as a method of cryopreservation since the procedure of the former was easier than that of the latter, although maximum regrowth of the 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 to more than 80% are needed. Matsumoto and Sakai (1995) reported that the addition of glycerol to beads and loading solution increased the regrowth of cryopreserved wasabi apices after rewarming. Next, we examined the effect of added glycerol in beads and loading solution on the regrowth of hawthorn apices in encapsulation–dehydration and encapsulation–vitrification. As a result, the addition of 0.5 mol/L glycerol to beads and loading solution resulted in high recovery (82.5 2.5%) of shoot apices in encapsulation–dehydration, a value that proved to be higher than the maximum regrowth of encapsulation–vitrification (60.0 5.8%). This result coincided with that of Kami et al. (2005), since they reported, using encapsulation–dehydration, 77% of blue honeysuckle apices regrew after cryopreservation when 0.5 mol/L glycerol was added to the loading solution only. Thus, it seemed that the effect of glycerol on shoot regrowth after cryopreservation depends more on loading solution than on alginate beads. With respect to the mechanism of glycerol on shoot regrowth, Morris et al. (2006) estimated that glycerol suppressed the modification of the cell after dehydration and/or freezing by restricting the osmotic loss of water from cells since they discovered that the viscosity of a glycerol aqueous solution (100 g/L) had exceeded 1000 cP at 45 8C. In this study, glycerol loaded-beads, unlike control beads, retained their soft form during desiccation. It is conceivable that glycerol acts to reduce bead shrinkage and related tissue damage during silica gel desiccation. On the other hand, glycerol might induce dehydration resistance and/or
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freezing resistance of the tissues, since Matsumoto et al. (1998) discovered that an incorporation of glycerol enhanced the proline content of tissues in the apical meristems of wasabi. Therefore, it is also necessary to monitor the cells of encapsulated apices when dehydrating or cryopreserving to clarify any relation between glycerol and the physical properties (texture, etc.) of alginate beads. It has been reported that regrowth after cryopreservation are different between cultivars and related species (Niwata, 1995; Kuranhuki and Yoshida, 1996; Hirai et al., 1998; Tanaka et al., 2004). However, encapsulation–dehydration using glycerol seemed to be a suitable cryopreservation protocol for hawthorn shoot apices, as evidenced by the four Crataegus species that showed 82.5–90.0% regrowth after rewarming. In summary, a practical cryopreservation method using the encapsulation–dehydration technique was established for the shoot apices of hawthorns that grow wild in Japan. In particular, the addition of 0.5 mol/L glycerol to the beads and loading solution was very effective in increasing the regrowth rate of apices. It is expected that this modified encapsulation–dehydration procedure will be used widely for more successful cryopreservation in additional plant germplasm. References Chandel, K.P.S., Chaudhury, R., Radhamani, J., Malik, S.K., 1995. Dessication and freezing 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 and culture duration improve pear shoot cryopreservation. Cryobiology 40, 311– 322. Damiano, C., Dolores, M., Padro, A., Frattarelli, A., 2006. Experiences in cryopreservation of temperate small fruit plants. Abstracts: Proceedings of the 27th International Horticultural Congress & Exhibition (IHC 2006), p. 5. Fabre, J., Dereuddre, J., 1990. Encapsulation dehydration–a new approach to cryopreservation 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 encapsulation–dehydration technique. Cryoletters 24, 85–94. Hirai, D., Shirai, K., Shirai, S., Sakai, A., 1998. Cryopreservation of in vitro grown meristems of strawberry (Fragaria ananassa Duch.) by encapsulation–vitrification. Euphytica 101, 109–115. Kami, D., Suzuki, T., Oosawa, K., 2005. Cryopreservation of blue honeysuckle in vitrocultured 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 and Tissue Culture. Kluwer Academic Publishers, Dordrecht, pp. 195–230.
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