Cryopreservation as a tool used in long-term storage ...

Scientia Horticulturae 168 (2014) 88–107

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Review

Cryopreservation as a tool used in long-term storage of ornamental species – A review Dariusz Kulus ∗ , Małgorzata Zalewska University of Technology and Life Sciences in Bydgoszcz, Department of Ornamental Plants and Vegetable Crops – Laboratory of Biotechnology, Bernardy´ nska St. 6, PL-85-029 Bydgoszcz, Poland

a r t i c l e

i n f o

Article history: Received 18 March 2013 Received in revised form 9 January 2014 Accepted 11 January 2014 Keywords: Cryopreservation Genetic stability Ornamental plants Storage

a b s t r a c t The market for ornamental plants is growing every year, becoming an important part of the economy. Every year hundreds of new cultivars, replacing the current assortment, are produced. However, since consumer preferences are changing rapidly, the cultivars considered old-fashioned today may become popular once again. They are also a valuable breeding material source. Bearing that in mind, there is a great need to develop a strategy for their long-term conservation. Storage in gene banks under in vitro cultures, although offering many advantages, is expensive and threatened with somaclonal variation and contamination loss. Cryopreservation is believed to be a more promising method. It has been successfully used with many agricultural species. Unlike micropropagation, cryopreservation has not yet found wider employment with ornamental plants. The upcoming years and progress in cryobiology may, however, change this situation and broaden the potential of cryoconservation. Over years several freezing methods have been introduced. The first one developed, based on slow cooling, had limited usefulness in temperate species. Today the encapsulation-dehydration technique is most often used with ornamental plants. In the future, however, combined techniques will probably be the most popular. So far insufficient attention has been paid to the problem of the genetic stability of cryopreserved ornamental species, especially chimeras. The aim of this paper is to present different cryopreservation techniques and their use for the storage, protection and breeding of ornamental plants. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2.

3.

4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Material selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Cryopreservation of shoot and root tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cryopreservation of germplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cryopreservation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Traditional methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Vitrification-based methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Desiccation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Preculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. Vitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Droplet-vitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.5. Encapsulation-dehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.6. Combined techniques, encapsulation-vitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thawing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Survival analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolic activity after cryostorage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +48 0523749522. E-mail address: [email protected] (D. Kulus). http://dx.doi.org/10.1016/j.scienta.2014.01.014 0304-4238/© 2014 Elsevier B.V. All rights reserved.

89 89 89 91 91 92 92 92 98 99 99 100 101 101 101 102 102

D. Kulus, M. Zalewska / Scientia Horticulturae 168 (2014) 88–107

8. 9.

Genetic stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Ornamental plants are produced mainly for their aesthetic value. They have been an important element of human culture and economy since ancient times (Ozudogru et al., 2010). Thanks to their unique beauty, they accompany us at every stage of our lives. The commercial production of ornamental species is constantly growing. Still the market for ornamental plants is subjected to periodical trend-driven changes. Indeed, every year, hundreds of new cultivars, replacing the current assortment, are produced. However, due to changes in consumer preferences, cultivars unfashionable today may in the future once again be attractive for potential buyers. Furthermore, very often they constitute a great breeding material source. For this reason, the protection and storage of those valuable genetic resources is of great importance in order always to be able to meet market demands. Nevertheless it is difficult for breeders and horticulturalists to provide enough space and funds for traditional cultivation of such numerous cultivars, which is laborious and threatened with biotic and abiotic stresses (Sekizawa et al., 2011). Traditional genetic conservation in the field or greenhouse requires intensive care of pot cultures or carefully separated field plots (Reed, 2006). Haploids (important in breeding) and transgenic cultivars, which are gaining popularity among ornamental plants (Rosa L., Dianthus L., Gladiolus L.), require isolation to protect them from cross-breeding (Joung et al., 2006; Rajasekharan et al., 1994). Additionally many ornamental species (Orchidaceae, Cactaceae, Gentianaceae) are on the brink of extinction. Fast and easy access to high quality gene banks of large material variety is the key for ornamental plant producers and so an efficient method for long-time conservation of the plant material may be extremely valuable for breeding and horticultural production (Halmagyi et al., 2004). Today, cryopreservation, developing rapidly over the last 25 years, is believed to be the most promising and valuable long-term storage method. Cryopreservation techniques are based on tissue storage (usually with high cell division rates and low water content, e.g. meristems, seeds, zygotic and somatic embryos or pollen but also callus cultures and cell suspensions) at ultra-low temperature of liquid nitrogen (LN, −196 ◦ C/−321 ◦ F) or, seldom, its vapor phase (−150 ◦ C/−238 ◦ F). At this temperature the biochemical metabolic and cell division activities are arrested, allowing for long-term storage. The main advantage of this method is the reduction of in vitro culture costs, required space, contamination and somaclonal variation risk. The long-term cryoconservation of embryogenic cell lines could be a valuable tool in genetic transformation. Storage in LN would also help in preserving genetic diversity by storing wild species (e.g. for the purpose of breeding), some of which are endangered already (Winkelmann et al., 2004). Moreover, cryotherapy may be used to reduce the number of pathogens, as proven with Pelargonium L’Hér (Gallard et al., 2011; Grapin et al., 2011). Consequently the method is attracting more and more interest among researchers. Cryopreservation has been successfully used for many agricultural species (Forsline et al., 1998; Xue et al., 2007; Zhao et al., 2005). The first information on cryopreservation of ornamental species was reported by Fukai (1989) and regarded Dianthus hybrida. Whit its short history, the significance of cryopreservation for ornamental plants is less, although growing every year. The aim of this review is to present the previous achievements and difficulties with the cryopreservation of ornamental plants, as

89

103 103 104

well as the popularization of this method for the purpose of breeding, production and protection of these species. 2. Material selection It is possible to freeze biological material (e.g. buds of Chrysanhtemum × grandiflorum/Ramat./Kitam., spores of Cyathea australis (R. BR.) Domin, or seeds and bulblets of Lilium ledebouri [Baker] Boiss) excized directly from ex vivo conditions (Fukai, 1990; Kaviani et al., 2009, 2010; Mikuła et al., 2009a). Such material, due to its greater size, is easier to isolate and very often has a better survival rate (Mikuła et al., 2009a). It is, however, more exposed to loss due to contaminations for disinfection is performed after thawing (Fukai, 1990). Furthermore, because nowadays the commercial production of ornamental plants is based mainly on micropropagation (about 160 genera [Rout et al., 2006]), the use of in vitro derived explants seems well-founded. The selected material should be young, demonstrating meristematic potential (or regeneration potential, in the case of non-meristematic explants, such as callus cultures and cell suspensions, which should be in the linear growth phase 7–10 days after subculture) since only cells with dense cytoplasm and small vacuoles can survive freezing. Moreover, the use of a meristematic explant provides a greater chance of avoiding any variation. 2.1. Cryopreservation of shoot and root tips Different explants are used for cryostorage e.g. apical or axillary shoot tips, seeds, spores, gametophytes, rhizomes or even protoplasts (Yamazaki et al., 2009; Table 1). Among them, shoot tips are used most often. As for vegetatively propagated, sterile plants (e.g. Crocus L., Chrysanthemum L. – crops with a high economic value) shoot tips constitute the best initial material (Fukai, 1990; Zadeh et al., 2009). As a result it is necessary to prepare the shoot tips first. As for plants of elongated growth (e.g. Dianthus, Chrysanthemum), they are very easy to obtain by inoculating single-node explants on the Murashige and Skoog (MS; 1962) medium for 14 days. The in vitro phase cannot be a source of any variation at that point. The optimal age of the cryopreserved buds should be between 2 (for shoot apices) and 7 weeks (for axillary buds) (Popova et al., 2010). Shoot tips of older plants are more difficult to isolate, due to the development of covering leaves, although Takagi et al. (1997) reported better results when using 2–3-month-old mother plants of Colocasia esculenta L. Schott. Many authors emphasize the significance of the frozen explant size for protocol success. This phenomenon, however, seems to be genotype-dependent in the case of crocus (Crocus sativus L.) and chrysanthemum (Chrysanthemum × grandiflorum); satisfying results were reported for both smaller (0.5–1.0 mm) and bigger (3–4 mm) shoot tips (Fukai, 1990; Zadeh et al., 2009) using different techniques. With rose (Rosa × hybrida L.) smaller explants (1–2 mm) were less efficient than bigger ones (3–4 mm) with 2–4 leaf primordia after applying the droplet-vitrification technique (Halmagyi and Pinker, 2006). Similar results were observed with encapsulated protocorms of orchids (Yin et al., 2011). Still, the latter are easier to isolate and less susceptible to injury (Shuhaida et al., 2009). However, as for vitrified Colocasia esculenta, smaller (0.8 mm) shoot tips with one leaf primordium provided a 5× higher survival than the bigger ones (2.0 mm) with two leaves primordia (Takagi et al., 1997). It could be assumed that when

90

D. Kulus, M. Zalewska / Scientia Horticulturae 168 (2014) 88–107

Table 1 Examples of explant types of ornamental plants used for different cryopreservation techniques. Plant species

Material

Technique

Survival [%]

Reference

Asplenium cuneifolium

Gametophytes Zygotic embryogenic axesa Somatic embryosb Pollenc

Camellia sinensis Chrysanthemum × grandiflorum

Suspension culture Shoot tips excized ex vivo Shoot tips excized ex vitro Nodal segment with axillary buds excized ex vitro Shoot tips excized ex vitro Shoot tips excized ex vitro Axillary buds excized ex vitro Shoot tips excized ex vitro Shoot tips Axillary buds Axial meristems Non-disinfected immature spores Non-disinfected mature spores Disinfected matures pores Young gametophytes obtained from in vitro spore germination Mature gametophytes obtained from in vitro spore germination Hairy roots Proembryogenic masses (PEMs) derived from hypocotyl callusa PEMs derived from cotyledon callusb Axillary buds excized from in vitro-grown plants Shoot tips

100 80 58–69 31 0 0 16–61 76 87 (48) 80 (10) 24 (0)

Makowski et al. (2011)

Camellia japonica

Encapsulation-dehydration Encapsulation-vitrification Desiccationa Direct immersion in LNa Vitrificationb Encapsulation-dehydrationc Encapsulationc Vitrification Slow cooling

Vitrification Droplet-vitrification

(0–70) (82) (85) (30–45) 25 4 86 (41)a (80)a (0)a 73–80b 50b

Halmagyi et al. (2004) Lee et al. (2011)

Encapsulation-vitrification Slow cooling Preculture-slow cooling Vitrification Encapsulation-dehydration

0 0a,b 2.5a –2.7b 86a –91b 65b

Xue et al. (2007) Mikuła et al. (2005)

Preculture-desiccation

5–90

Suzuki et al. (2006)

Vitrification Encapsulation-dehydration Encapsulation-dehydration Vitrification

75–93 63 69 (54) 60–90 90 0a /10c /50d /75e 0a/b /10c 0a/c 0a/c 58–90 (53–88)f

Jevremovic´ et al. (2009)

C. esculenta Crocus sativus C. australis

Gentiana macrophylla G. cruciata

Gentiana scabra Iris sp. I. nigricans Lilium sp.

Somatic embryos Shoot tips Root tips Seedsa,c,d,e Embryogenic axesa,b,c Lateral budsa,c Bulbleta,c Shoot tips from adventitious budsf Apical meristems from adventitious buds

L. ledebouri

Lilium × siberia Lilium × lancifolium Lilium × longiflorum Orchidaceae

Encapsulation-dehydration Vitrification Vitrification Direct immersion in LNa Preculture-encapsulationb

Direct immersion in LNa / Vitrificationb /encapsulationvitrificationc /encapsulationdehydrationdd / Preculture-desiccatione Droplet-vitrificationf Vitrification/ droplet-vitrification

Janeiro et al. (1996) Li et al. (2011a)

YaJun et al. (2009) Fukai (1990)

Halmagyi et al. (2004) Takagi et al. (1997) Zadeh et al. (2009) Mikuła et al. (2009a)

Shibli (2000) Bouman et al. (2003) Kaviani et al. (2008, 2009, 2010) Yi et al. (2013)

35 (35)/65 (62) 45 (36)/84 (68) 42 (33)/43 (35) 8 81–92 (64)

Chen et al. (2011)

Jitsopakul et al. (2008a)

Vitrification encapsulation-dehydration Encapsulation-vitrification

93 91 84 33 27 15

Vitrification

19

Vitrification

67

Mature seeds

Direct plunge in LN Vitrification

(10) (14)

Jitsopakul et al. (2012)

P. lactiflora

Zygotic embryos Somatic embryos Dormant buds

Encapsulation-dehydration Air desiccation Air desiccation

85 (>66) (66–74)

Kim et al. (2004) Kim et al. (2006) Seo et al. (2007)

Rosa × hybrida

Shoot tips excized ex vitroa Axillary buds excized ex vitrob

Droplet-vitrification Encapsulation-dehydration

(58–64)a (12)a (0)b

Halmagyi and Pinker (2006), Pawłowska and Bach (2011)

Bletilla striata

Immature seeds

C. hatschbachii

Immature seeds

Dendrobium cruentum Dendrobium cariniferum Rhynchostylis gigantean Seidenfadenia mitrata V. tricolor

Mature seeds Germinating seeds Protocorms Protocorms

Direct plunge in LN vitrification Encapsulation-dehydration Droplet vitrification

Lower-case letters refer to the applied cryopreservation technique, values in parenthesis refer to regrowth.

Hirano et al. (2005) Surenciski et al. (2007)

Thammasiri (2008)

D. Kulus, M. Zalewska / Scientia Horticulturae 168 (2014) 88–107

utilizing vitrification solutions, in order to ensure the deepest penetration of cryoprotectants, a smaller explant is preferred. With encapsulation-based techniques, bigger explants are also suitable. Interestingly, even though dormant buds of cold-hardy species might also be useful for cryopreservation, as proven with several woody plants (Forsline et al., 1998; Volk et al., 2009), there is only one study performed by Seo et al. (2007) with Paeonia lactiflora Pall., that addressed the use of cryopreservation to preserve dormant buds of ornamental species. Therefore more work should done in that direction. High precision and technical skills are necessary to isolate shoot tips or meristems. Any injury to the tissue at that point may be fatal. Moreover, isolation must be performed rapidly, in order to protect the explants from excessive desiccation. Therefore it is recommended to find a source of easier-to-obtain explants. An interesting alternative was proposed by Bouman et al. (2003), who developed an efficient vitrification method for root tips of some Lilium species and cultivars. Root tips require only a slight modification of the protocol applicable for shoot tips. Besides easier isolation, cryopreservation of root tips is a bigger genetic stability guarantee in the case of chimeric plants, for root meristems are characterized by a simpler structure with only one histogen layer. Therefore, more attention should be paid to this possibility in the future. 2.2. Cryopreservation of germplasm With endangered species (e.g. Lilium ledebourii and members of the Orchidaceae or Cactaceae family), it is important to derive explants without destroying the mother plant. Seeds seem to be a well-founded choice. The seeds are very often stored at subzero temperatures (Liu et al., 2001). Popov et al. (2004), Tarre et al. (2007), Veiga-Barbosa et al. (2010) and Surenciski et al. (2012) confirmed a high tolerance of Orchidacae, Bromeliaceae and Cactaceae family species to drying and freezing (germination higher than or similar to control). Application of cryopreservation in germplasm was successful in Dendrobium chrysotoxum Lindl. (99% survival, vitrification), D. cruentum Rchb. (32%, vitrification), D. draconis Rchb. (95%, vitrification), D. hercoglossum Rchb. (80%, encapsulation-vitrification), Doritis pulcherrima Rchb. (62%, vitrification), Rhynchostylis coelestis Rchb. (85%, vitrification), Vanda coerulea Griff. ex Lindl. (67%, vitrification) (Thammasiri, 2008). One should keep in mind though, that unlike shoot tips, more developed mature seeds (3–4 months after pollination) are preferable, since they show a decrease in water content with increasing time after pollination (Hirano et al., 2005). Still, their age should not exceed 6 months, since older seeds show lower germination capacity, as observed with Vanda tricolor Lindl. (Jitsopakul et al., 2012). Similarly somatic embryos in cotyledonary stage can be used for cryoconservation provided that efficient embryogenesis induction and embryos synchronization systems are developed, and that the embryos are converted into plants. As for species which have recalcitrant seeds and their tissue culture and micropropagation system were not established, the pollen cryopreservation of just-opening flowers (after enclosing approximately 0.5 g of sample in gelatin capsules) is a feasible alternative. Long-term pollen storage is important for germplasm preservation, pollen research, germplasm exchange, and improved efficiency in plant breeding, by helping to overcome the problems of geographic isolation and flowering asynchronism (Geng et al., 2011). Viability and fertility profiles of Rosa, Lilium L. and Gladiolus pollen have shown that it is possible to use cryogenic methods for conservation and management of the haploid gene pool in these species (Geng et al., 2013; Marchant et al., 1993; Rajasekharan et al., 1994). Ajeeshkumar and Decruse (2013) used cryopreserved pollinia (10 min PVS2) of Luisia macrantha Blatt. for hybridization with Vanda tessellate (Roxb.) Hook., which gave 87% fruit set and

91

21% viable seeds. The viable seeds germinated and developed into healthy seedlings. Thus cryopreservation has been proved useful for utilization in breeding.

3. Cryopreservation techniques In nature many plant species develop special protective mechanisms associated with the activation of specific genes, which allows them to survive at sub-zero temperatures. Tropical plants, however, do not have such abilities. This was a major problem for cryobiologists for many years. Only the breakthrough discoveries made in the late twentieth century facilitated extending the applicability of cryopreservation techniques. Over the years it was proven that not only cold tolerance but also desiccation resistance are the main factors affecting the usefulness of material for cryopreservation. Even though water removal to a certain level (