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Cryobank of plant genetic resources in Russian Academy of Sciences
International Journal of Refrigeration 29 (2006) 403–410 www.elsevier.com/locate/ijrefrig
Cryobank of plant genetic resources in Russian Academy of Sciences A.S. Popov*, E.V. Popova, T.V. Nikishina, O.N. Vysotskaya Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul, 35, 127276 Moscow, Russian Federation Received 12 December 2004; received in revised form 18 March 2005; accepted 9 July 2005 Available online 20 February 2006
Abstract Cryopreservation is the most reliable method for long-term storage of plant genetic resources. A review of cell injury by ice crystals and dehydration during a freeze–thaw cycle is given. For successful regeneration of plants and cultures after cryopreservation of their cells, the development of reliable cryopreservation procedure is required including preliminary cultivation, treatment by cryoprotectors, freezing by different methods, thawing and recultivation. Up to now 27 cell lines successfully resumed their growth after storage in liquid nitrogen and preserved their specific features and biosynthetic potential. Besides, shoot tips of 40 cultivars of potato, rose, strawberry and raspberry regenerated plants both in vitro and in vivo after cryopreservation. The longest storage duration was 25 years. Now in liquid nitrogen we continuously store 24 cell strains of rare medicinal plants, shoot tips of seven cultivars of strawberry and raspberry and seeds of 250 endangered plant species collected over all Russian territory. q 2005 Elsevier Ltd and IIR. All rights reserved. Keywords: Cryogenics; Cryopreservation; Data bank; Liquid nitrogen; Chilling injury; Cell; Dehydration; Storage life
Cryobanque pour la cryoconservation des ressources ge´ne´tiques ve´ge´tales a` I’Acade´mie russe des sciences Mots cle´s: Cryoge´nie; Cryoconservation; Banque de donne´es; Azote liquide; Maladie due au froid; Cellule; De´shydratation; Dure´e de conservation
1. Introduction Long ago the mankind has faced the problem of rare and endangered plant species. During last decades, longterm preservation of plant genetic resources became one
* Corresponding author. E-mail addresses: [email protected] (A.S. Popov), [email protected] (A.S. Popov).
0140-7007/$35.00 q 2005 Elsevier Ltd and IIR. All rights reserved. doi:10.1016/j.ijrefrig.2005.07.011
of the most important aims and even common responsibility because of the surprisingly rapid changes in the environment. More than 100,000 of about 300,000 higher plant species can disappear by the middle of this century as it was claimed at the XVI International Botanical Congress (USA, 1999). In vitro cultures of rare crops, medicinal and other plants can ensure short- and middle-term preservation of their genomes, but long-term culturing runs risk of contamination, somaclonal variation and, ultimately, loss of
404
A.S. Popov et al. / International Journal of Refrigeration 29 (2006) 403–410
Fig. 1. Influence of high osmolarity and freezing on the number of cells with damaged plasmalemma. Cell suspensions of Panax ginseng (upper) and Dioscorea deltoidea (lower) were cultivated on rotary-shaker at 25 8C in the dark and were taken for experiments in the exponential growth phase. (A) CaCl2 was added step by step to cells at 2 8C without freezing with the rate approximately equal to freezing rate (0.5 8C/min) in slowfreezing experiments. (B) Panax cells were cold-hardened first at 10 8C and then at 4–5 8C in the dark. Sucrose concentration in the medium was simultaneously increased to 20% during 3 weeks. Slow freezing was performed at the rate of 0.5 8C/min to K40 8C, then at 9 8C/min to K70 8C, and ampules were immersed into liquid nitrogen. Before all experiments fluoresceine diacetate (FDA) was added to cells. Ampules were taken from the freezer each 10 min during freezing and then at K70 and K196 8C. Cells were filtered and concentration of fluoresceine in supernatant was determined with a fluorimeter. This concentration showed the percent of cells with damaged plasma membranes. For Panax ginseng: 1— strain IFR Zh-1, resistant; 2—strain IFR Zh-2, cold hardened culture; 3—strain IFR Zh-2, non-hardened culture. For Dioscorea deltoidea: 1— strain IFR D-1, very sensitive; 2—strain IFR DM-0.5, sensitive; 3—strain IFR DM-8, resistant.
A.S. Popov et al. / International Journal of Refrigeration 29 (2006) 403–410
405
Fig. 2. (A) Root development in Dioscorea balcanica calluses regenerated after slow freezing and storage in liquid nitrogen. (B), (C) Plants of Dioscorea caucasica (B) and D. balcanica (C) in the greenhouse after regeneration from cryopreserved cells.
regenerative and biosynthetic potential [1]. Cryopreservation of plant cells, shoot tips, embryos, pollen and seeds in liquid nitrogen (K196 8C) is the most reliable method for a long-term germplasm storage. The minimization of cell injury during freezing-thaw cycle and simplification of the whole procedure are the main points that should be taken into account by cryobank developers. 2. Methodical bases Freezing is the most important step of cryopreservation. This step is not complicated only for orthodox seeds, which are sufficiently dry or can be adequately dehydrated. But this
is a severe stress for cells and tissues, which have high water content. The main difficulty—the large amount of freezable water—is predetermined by the specific features of plant cells because even young plant cells have volume at least 1000 fold larger than the volume of animal cells [2]. Most volume of plant cell is occupied by central vacuole that contains freezable water. Cell damages during freezing and subsequent thawing can be caused, on the one hand, by the formation of intracellular ice crystals with acute facets disintegrating cell membranes [3,4], and, on the other hand, by dehydration. Therefore the most dangerous process, which may occur during freezing is intracellular ice formation. It can be avoided by the preliminary dehydration and by freezing at very slow cooling rates [5] that inevitably
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Fig. 4. Berries of strawberry plants (cv. Kokinskaya pozdnyaya) regenerated from the meristems after several month storage in liquid nitrogen.
result in significant water loss and protoplast shrinkage (plasmolysis) [6]. The phenomenon is a result of ‘solution effects’, the term introduced by Mazur [5] for more general phenomena. Among them protoplast shrinkage is most dangerous for cell surviving because of plasma membrane injures [7–10]. Preliminary dehydration is often achieved by a vitrification method in the course of deep freezing, with or without encapsulating the biological material into alginate grains. This method does not need expensive electronic freezers, but the treatments with loading and vitrification solutions must be time-limited (from a few minutes for cell suspensions to dozen minutes for apexes, etc.) because of the extremely high concentrations of cryoprotectors. These solutions dehydrate the cells and convert to a glass state during ultrafast deeping in liquid nitrogen. Ice crystals are either absent or almost absent, being not dangerous. However, the intensive dehydration results in a very strong plasmolysis, phase transitions of membrane lipids and plasmalemma rupture leading to cell death [7–11].
Fig. 3. In vitro regeneration of potato (cv. Tawa) from young cell suspension after slow freezing with cryoprotector solution (DMSOCsucroseCtrehalose) and storage in our cryobank for several months. The following investigators took part: L.A. Volkova (cell suspension and callus cultures), A.S. Popov (slow freezing), A.B. Burgutin (plant regeneration). Fig. 5. Renewal of carrot cells growth on agar medium after 25 years of storage in liquid nitrogen.
A.S. Popov et al. / International Journal of Refrigeration 29 (2006) 403–410
407
Table 1 Plant materials in the cryobank of Timiryazev Institute of Plant Physiology of Russian Academy of Sciences, Moscow
Table 1 (continued) Species
Year of freezing
Species
Strain
Year of freezing
2004
Daucus carota Dioscorea deltoidea
M-34 IFR D-1 IFR DM-0.5 IFR DM-1 IFR DM-8 IFR Zh-1 IFR Zh-2 IFR Zh-3 IFR Zh-10 NrEs-1 L-1 KT-4
1977 1984–1989 1984–1989 1984–1989 1984–1991 1983–1992 1985–1991 1984–1988 1985–1987 1984 1985–1989 1991
Dactylorhiza fuchsii Dactylorhiza maculata Gymnodenia conopsea
Rhs-1
1992
Rhs-2 Rhs-8 IFR T-1 TsU-194 Callus Callus B-233 PZ-4 R-1 1C BFT 01-95
1999 1997–1999 1994 1994 1995 1995 1998–1999 1999 1998 1999 1998
Panax ginseng
Panax quinquefolius Nicotiana sylvestris Medicago sativa Solanum tuberosum, cv. Tawa Rhaponticum carthamoides
Triticum timofeevi Triticum aestivum Dioscorea caucasica Dioscorea balcanica Thalictrum minus Panax japonicus Panax ginseng Panax ginseng Polyscias filicifolia Species Orchid seeds Tropical Angraecum magdalenae Bratonia, F1 Calanthe spp. Encyclia cochleata Cattleya spp. Dendrobium stratictes Dendrobium ochreatum Liparis nigra Mischobulbum cordifolium Phaius flavus Pholidota guibertae Polystachya sp. Trichopilia tortilis Vanda coerulea Russian Cypripedium macronthon Platanthera bifolia Epipactis helleborine Dactylorhiza longifolia Dactylorhiza incarnata
Year of freezing
2001 2001 2000–2001 2001 2002 2003 2003 2004 2004 2004 2004 2004 2004 2004 2004 2004 2002 2002 2004 2004
2004 2004
Besides we continuously store in liquid nitrogen shoot tips of strawberry (6 cultivars), raspberry (2 cultivars) and rose (3 cultivars) and seeds of 230 endangered plant species collected over the whole Russian territory and studied by Prof Valentina L. Tikhonova (the Main Botanical Garden, Moscow).
The slow freezing methods has some advantages such as: lower concentration of cryoprotector solutions, slow dehydration, which are particularly important for some objects. According to the above stated, young cell strains with proembryo cell clusters are more eligible for deep freezing because they contain small and almost nonvacuolated cells. Other cell lines need special preliminary cultivation procedures such as short subculturing times (for cell suspensions), cold acclimation, specific nutrient media supplemented with sucrose, mannitol, sorbitol, amino acids and other substances [12–15]. Often it is important to concentrate cells in suspension before freezing [16]. 3. Results and discussion To demonstrate the integrity of plasma membrane during treatment, a new fluorometric method was developed based on selective plasmalemma permeability for fluoresceine and its diacetate [17]. This method is not a tool for distinguising live and death cells in suspension culture, but is specially aimed at estimating the number of cells with plasmalemma destructions [18]. In original Widholm’s method, one counts the fluorescent cells under a microscope because fluoresceine is still localized into the cells. In our method, the fluorescence is measured with a fluorimeter in ambient solution, using in parallel two control specimens. The temperature and time specifications must be rigidly met in this method [18]. During cell freezing and thawing, the fluoresceine released from cells, thus indicating the damage to plasmalemma that increased linearly when temperature decreased to K30 and K40 8C. Under dehydration stress in CaCl2 or sorbitol solutions (their concentrations were increased step by step) at 2 8C, the active release of fluoresceine (and thus plasmalemma injury) occurred at 16.12 osm (equal to K30 8C) and 21.5 osm (K40 8C). By using this method, the correlation between cryo- and osmoresistance was shown for five cell lines of Dioscorea deltoidea and Panax ginseng (Fig. 1) [18–20]. Thus, it became clear that the osmotic stress caused by active cell
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without cryopreservation after cryopreservation m 10–6
2000 1800 1600 1400 1200 1000 800 600 400 200 0 0
40
80
120
160
days after seed sowing Fig. 6. Growth of Dactylorhiza longifolia protocorms on agar medium after seed storage in liquid nitrogen. Y axis shows the diameter of protocorms, mm.
dehydration plays the main role in the injury of cell plasma membrane and cell death during freezing. In Cryopreservation Group of Department of Cell Biology and Biotechnology in the Institute of Plant Physiology, cryoresistance of 34 cell strains of 21 plant species was studied. Cryopreservation technique, developed for these lines, included special conditions of preliminary cultivation and recultivation of meristem and cell cultures, different cryoprotectors and artificial initiation of crystallization during slow freezing. For the latter, a special automatic device was constructed. For example, the cells of D. deltoidea original strain (IFR D-1) were preliminary cultivated with asparagine, proline or alanine [12]; the cells of P. ginseng mutant strains IFR Zh-2 and Zh-3, of Panax quinquefolius IFR Zh-10, the donor in vitro plants and excised meristems of Fragaria and Rubus were preliminarily cold hardened with elevated (step by step) sucrose concentration in nutrient medium [12]. The cells of Rhaponticum carthamoides and Thalictrum minus were cultivated with mannitol. Other cell strains did not demand any special preliminary cultivation. Cryoprotector solutions involved a DMSO (dimethylsulfoxide), glycerol, sucrose, trehalose, and glucose at various concentrations and combinations. Slow freezing was performed at a rate of 0.3–0.5 8C/min by the freezers constructed in Kharkov (Institute of Cryobiology and Cryomedecine Problems,
Fig. 7. Flowering of Encyclia cochleata obtained from cryopreserved seed. After in vitro regeneration by T.V. Nikishina, G.L. Kolomeytseva grew the plants in a greenhouse of Main Botanical Garden of Russian Academy of Sciences.
Ukrainian National Academy of Sciences). Now we use the vitrification method of ultrafast freezing for the cell culture of Stephania glabra and obtained the first success, but these results are still not published. The seeds of rare plants, involving orchids, were freezed without cryoprotectors by direct immersion into liquid nitrogen, being sufficiently dry. As a result, 27 cell lines of 16 plant species successfully resumed their growth after cryogenic storage [12,21–23] and preserved their specific features and biosynthetic potential. Some of these strains are used in biotechnological industry as the producers of pharmaceutical preparations. Fig. 2 shows the plants of rare species Dioscorea caucasica and Dioscorea balcanica regenerated after cryopreservation of their embryogenic calluses [24,25]. Besides, shoot tips of about 40 cultivars of potato (Fig. 3), rose, strawberry (Fig. 4) [26] and raspberry [27] regenerated plants in vitro and in vivo after storage in liquid nitrogen. Potato plants regenerated after cryopreservation of their meristems showed the same protein profiles of tubers and leaves as the control ones [28]. The cell strains of
A.S. Popov et al. / International Journal of Refrigeration 29 (2006) 403–410
carrot, D. deltoidea and P. ginseng successfully resumed their growth and biosynthetic capacities after 6, 9, 12 and 14 years of storage in liquid nitrogen. The longest storage time exceeded 25 years (Fig. 5). 4. Conclusion Now we continuously store in liquid nitrogen 24 cell strains from 15 species of rare medicinal plants and crops, shoot tips of 7 cultivars of strawberry and raspberry and seeds of 230 endangered plant species collected over the whole Russian territory—from the South Kuril Islands to Baltic Sea (Table 1). The seeds were collected and their germination and growth after storage in liquid nitrogen were studied by Prof. Valentina L. Tikhonova and her coworkers in the Main Botanical Garden of Russian Academy of Sciences (Moscow). Recently, we have developed the methods for cryopreservation and in vitro cultivation of the microscopic seeds of tropical and Russian orchids [29–31]. Protocorms and juvenile plants obtained from cryopreserved seeds did not significantly differ from those obtained from control (unfrozen) seeds in growth rate (Fig. 6). Fig. 7 shows flowering of Encyclia cochleata, a tropical orchid, grown from cryopreserved seeds first in vitro and then in a greenhouse. Now we continuously are storing the seeds of 22 rare tropical and Russian orchid species in liquid nitrogen (Table 1).
References [1] D.K. Dougall, J.M. Johnson, G.H. Whitten, A clonal analysis of anthocyanin accumulation by cell cultures of wild carrot, Planta 149 (1980) 292–297. [2] V.A. Sidorov, Yu.Yu. Gleba, L.D. Krivokhatskaya, K.M. Sytnik, Ultrastructural analysis of the fusion of human cells with protoplasts of higher plants, Dokl Akad Nauk 240 (1978) 211–213 (Proceedings of the USSR Academy of Sciences). [3] T. Nei, Growth of ice crystals in frozen specimens, J Microscopie 99 (1973) 227–233. [4] K. Shimada, E. Asahina, Visualization of intracellular ice crystals formed in very rapidly frozen cells at K27 8C, Cryobiology 12 (1975) 209–218. [5] P. Mazur, Cryobiology: the freezing of biological systems, Science 168 (1970) 939–949. [6] P. Mazur, The role of intracellular freezing in the death of cells cooled at supraoptimal rates, Cryobiology 14 (1977) 251–272. [7] S. Wiest, P. Steponkus, Freeze–thaw injury to isolated spinach protoplasts and its simulation at above freezing temperatures, Plant Physiol 62 (1978) 699–705. [8] J. Wolfe, P. Steponkus, Mechanical properties of the plasma membrane of isolated plant protoplasts. Mechanism of hyperosmotic and extracellular freezing injury, Plant Physol 71 (1983) 276–285. [9] P. Steponkus, Role of the plasma membrane in freezing injury and cold acclimation, Ann Rev Plant Physiol 35 (1984) 543–584. [10] P. Steponkus, D. Lynch, Freeze–thaw-induced destabilization of the plasma membrane and the effects of cold acclimation, J Bioenerg Biomembr 21 (1989) 21–41.
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[11] A.S. Popov, Mechanisms of in vitro cryoinjury in plant cell cryopreservation, Russ J Plant Physiol 40 (1993) 421– 430. [12] R.G. Butenko, A.S. Popov, L.A. Volkova, N.D. Chernyak, A.M. Nosov, Recovery of cell cultures and their biosynthetic capacity after storage of Dioscorea deltoidea and Panax ginseng cells in liquid nitrogen, Plant Sci Lett 33 (1984) 285–292. [13] L. Withers, P. King, Proline: a novel cryoprotectant for the freeze preservation of cultured cells of Zea mays L, Plant Physiol 64 (1979) 675–678. [14] L. Withers, Low temperature storage of plant tissue cultures in: A. Fiechter (Ed.), Advances in Biochemical Engineering Plant Cell Cultures II, vol. 18, Springer, Heidelberg-Berlin, 1980, p. 101–150. [15] A. Sakai, Development of cryopreservation techniques in: F. Engelmann, H. Takagi (Eds.), Cryopreservation of Tropical Plant Germplasm. Current Research Progress and Application, JIRCAS, IPGRI, Tsukuba, Japan, 2000, p. 1–8. [16] A.S. Popov, R.G. Butenko, I.N. Glukhova, Effects of preparing conditions and deep freezing on the renewal of cell suspension cultures of wild carrot, Russ J Plant Physiol 25 (1978) 972–979. [17] J. Widholm, The use of fluoresceine diacetate and phenosafranine for determining viability of cultured plant cells, Stain Technol 47 (1972) 189–195. [18] A.S. Popov, D.N. Fedorovskii, Injuries to the plasmalemma of Dioscorea cells cultured in vitro incurred in the process of their cryopreservation, Russ J Plant Physiol 39 (1992) 211– 216. [19] D.N. Fedorovskii, A.S. Popov, Plasmalemma injuries in different strains of Dioscorea deltoidea during cryopreservation, Russ J Plant Physiol 39 (1992) 381–385. [20] D.N. Fedorovskii, N.D. Chernyak, A.S. Popov, The disturbing effects of cryopreservation on the plasma membranes of ginseng cells, Russ J Plant Physiol 40 (1993) 94–98. [21] L.A. Volkova, N.V. Gorskaya, A.S. Popov, V.N. Paukov, V.V. Urmantseva, Preservation of the main characteristics of Dioscorea mutant cell strains after storage at extremely low temperatures, Russ J Plant Physiol 33 (1986) 598– 604. [22] A.S. Popov, L.A. Volkova, R.G. Butenko, Cryopreservation of germplasm of Dioscorea deltoidea in: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry Cryopreservation of Plant Germplasm I, vol. 32, Springer, Berlin, 1995, p. 487–499. [23] O.Yu. Baskakova, N.M. Voinkova, T.V. Nikishina, E.A. Osipova, A.S. Popov, E.A. Zhivukhina, Freezing resistance and cryopreservation of cell strains of Rhaponticum carthamocdes and Thalictrum minus, Russ J Plant Physiol 50 (2003) 666–671. [24] L. Cˇulafic´, D. Grubishich, R. Vuiichich, L.A. Volkova, A.S. Popov, Somatic embryo production in vitro in Dioscorea caucasica Lipsky and Dioscorea balcanica Kosanin and cryopreservation of their organogenic callus tissues, Russ J Plant Physiol 41 (1994) 821–826. ˇ ulafic´, Cryopreservation of [25] A.S. Popov, L.A. Volkova, L. C in vitro plants and plant tissue genofond, Bull de l’Institut et du jardin botaniques de l’Universite de Beograd XXIX (1995– 1997) 1–9.
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[26] O.N. Vysotskaya, A.S. Popov, R.G. Butenko, The advantage of glucose over sucrose in cryopreservation of strawberry meristems, Russ J Plant Physiol 46 (1999) 255–257. [27] O.N. Vysotskaya, A.I. Mochammed, R.G. Butenko, Cryoconservation of red raspberry meristems (Rubus idaeus L.) isolated from in vitro plants, Biology Bulletin 26 (1999) 19–22. [28] N.V. Donets, S.M. Musin, A.S. Popov, Stability of composition of polymorphic proteins of potato plants produced from meristems after cryogenic storage, Sel’skokhosyastvennaya Biologiya 3 (1991) 76–83 (in Russian).
[29] T.V. Nikishina, A.S. Popov, G.L. Kolomeitseva, B.N. Golovkin, Effect of cryopresrvation on seed germination of rare tropical orchids, Russ J Plant Physiol 48 (2001) 810–815. [30] E.V. Popova, T.V. Nikishina, G.L. Kolomeytseva, A.S. Popov, The effect of seed cryopreservation on the development of protocorms by the hybrid orchid Bratonia, Russ J Plant Physiol 50 (2003) 672–677. [31] A.S. Popov, E.V. Popova, T.V. Nikishina, G.L. Kolomeytseva, The development of juvenile plants of the hybrid orchid Bratonia after seed cryopreservation, Cryo Lett 25 (2004) 205–212.
Cryobank of plant genetic resources in Russian Academy of Sciences A.S. Popov*, E.V. Popova, T.V. Nikishina, O.N. Vysotskaya Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul, 35, 127276 Moscow, Russian Federation Received 12 December 2004; received in revised form 18 March 2005; accepted 9 July 2005 Available online 20 February 2006
Abstract Cryopreservation is the most reliable method for long-term storage of plant genetic resources. A review of cell injury by ice crystals and dehydration during a freeze–thaw cycle is given. For successful regeneration of plants and cultures after cryopreservation of their cells, the development of reliable cryopreservation procedure is required including preliminary cultivation, treatment by cryoprotectors, freezing by different methods, thawing and recultivation. Up to now 27 cell lines successfully resumed their growth after storage in liquid nitrogen and preserved their specific features and biosynthetic potential. Besides, shoot tips of 40 cultivars of potato, rose, strawberry and raspberry regenerated plants both in vitro and in vivo after cryopreservation. The longest storage duration was 25 years. Now in liquid nitrogen we continuously store 24 cell strains of rare medicinal plants, shoot tips of seven cultivars of strawberry and raspberry and seeds of 250 endangered plant species collected over all Russian territory. q 2005 Elsevier Ltd and IIR. All rights reserved. Keywords: Cryogenics; Cryopreservation; Data bank; Liquid nitrogen; Chilling injury; Cell; Dehydration; Storage life
Cryobanque pour la cryoconservation des ressources ge´ne´tiques ve´ge´tales a` I’Acade´mie russe des sciences Mots cle´s: Cryoge´nie; Cryoconservation; Banque de donne´es; Azote liquide; Maladie due au froid; Cellule; De´shydratation; Dure´e de conservation
1. Introduction Long ago the mankind has faced the problem of rare and endangered plant species. During last decades, longterm preservation of plant genetic resources became one
* Corresponding author. E-mail addresses: [email protected] (A.S. Popov), [email protected] (A.S. Popov).
0140-7007/$35.00 q 2005 Elsevier Ltd and IIR. All rights reserved. doi:10.1016/j.ijrefrig.2005.07.011
of the most important aims and even common responsibility because of the surprisingly rapid changes in the environment. More than 100,000 of about 300,000 higher plant species can disappear by the middle of this century as it was claimed at the XVI International Botanical Congress (USA, 1999). In vitro cultures of rare crops, medicinal and other plants can ensure short- and middle-term preservation of their genomes, but long-term culturing runs risk of contamination, somaclonal variation and, ultimately, loss of
404
A.S. Popov et al. / International Journal of Refrigeration 29 (2006) 403–410
Fig. 1. Influence of high osmolarity and freezing on the number of cells with damaged plasmalemma. Cell suspensions of Panax ginseng (upper) and Dioscorea deltoidea (lower) were cultivated on rotary-shaker at 25 8C in the dark and were taken for experiments in the exponential growth phase. (A) CaCl2 was added step by step to cells at 2 8C without freezing with the rate approximately equal to freezing rate (0.5 8C/min) in slowfreezing experiments. (B) Panax cells were cold-hardened first at 10 8C and then at 4–5 8C in the dark. Sucrose concentration in the medium was simultaneously increased to 20% during 3 weeks. Slow freezing was performed at the rate of 0.5 8C/min to K40 8C, then at 9 8C/min to K70 8C, and ampules were immersed into liquid nitrogen. Before all experiments fluoresceine diacetate (FDA) was added to cells. Ampules were taken from the freezer each 10 min during freezing and then at K70 and K196 8C. Cells were filtered and concentration of fluoresceine in supernatant was determined with a fluorimeter. This concentration showed the percent of cells with damaged plasma membranes. For Panax ginseng: 1— strain IFR Zh-1, resistant; 2—strain IFR Zh-2, cold hardened culture; 3—strain IFR Zh-2, non-hardened culture. For Dioscorea deltoidea: 1— strain IFR D-1, very sensitive; 2—strain IFR DM-0.5, sensitive; 3—strain IFR DM-8, resistant.
A.S. Popov et al. / International Journal of Refrigeration 29 (2006) 403–410
405
Fig. 2. (A) Root development in Dioscorea balcanica calluses regenerated after slow freezing and storage in liquid nitrogen. (B), (C) Plants of Dioscorea caucasica (B) and D. balcanica (C) in the greenhouse after regeneration from cryopreserved cells.
regenerative and biosynthetic potential [1]. Cryopreservation of plant cells, shoot tips, embryos, pollen and seeds in liquid nitrogen (K196 8C) is the most reliable method for a long-term germplasm storage. The minimization of cell injury during freezing-thaw cycle and simplification of the whole procedure are the main points that should be taken into account by cryobank developers. 2. Methodical bases Freezing is the most important step of cryopreservation. This step is not complicated only for orthodox seeds, which are sufficiently dry or can be adequately dehydrated. But this
is a severe stress for cells and tissues, which have high water content. The main difficulty—the large amount of freezable water—is predetermined by the specific features of plant cells because even young plant cells have volume at least 1000 fold larger than the volume of animal cells [2]. Most volume of plant cell is occupied by central vacuole that contains freezable water. Cell damages during freezing and subsequent thawing can be caused, on the one hand, by the formation of intracellular ice crystals with acute facets disintegrating cell membranes [3,4], and, on the other hand, by dehydration. Therefore the most dangerous process, which may occur during freezing is intracellular ice formation. It can be avoided by the preliminary dehydration and by freezing at very slow cooling rates [5] that inevitably
406
A.S. Popov et al. / International Journal of Refrigeration 29 (2006) 403–410
Fig. 4. Berries of strawberry plants (cv. Kokinskaya pozdnyaya) regenerated from the meristems after several month storage in liquid nitrogen.
result in significant water loss and protoplast shrinkage (plasmolysis) [6]. The phenomenon is a result of ‘solution effects’, the term introduced by Mazur [5] for more general phenomena. Among them protoplast shrinkage is most dangerous for cell surviving because of plasma membrane injures [7–10]. Preliminary dehydration is often achieved by a vitrification method in the course of deep freezing, with or without encapsulating the biological material into alginate grains. This method does not need expensive electronic freezers, but the treatments with loading and vitrification solutions must be time-limited (from a few minutes for cell suspensions to dozen minutes for apexes, etc.) because of the extremely high concentrations of cryoprotectors. These solutions dehydrate the cells and convert to a glass state during ultrafast deeping in liquid nitrogen. Ice crystals are either absent or almost absent, being not dangerous. However, the intensive dehydration results in a very strong plasmolysis, phase transitions of membrane lipids and plasmalemma rupture leading to cell death [7–11].
Fig. 3. In vitro regeneration of potato (cv. Tawa) from young cell suspension after slow freezing with cryoprotector solution (DMSOCsucroseCtrehalose) and storage in our cryobank for several months. The following investigators took part: L.A. Volkova (cell suspension and callus cultures), A.S. Popov (slow freezing), A.B. Burgutin (plant regeneration). Fig. 5. Renewal of carrot cells growth on agar medium after 25 years of storage in liquid nitrogen.
A.S. Popov et al. / International Journal of Refrigeration 29 (2006) 403–410
407
Table 1 Plant materials in the cryobank of Timiryazev Institute of Plant Physiology of Russian Academy of Sciences, Moscow
Table 1 (continued) Species
Year of freezing
Species
Strain
Year of freezing
2004
Daucus carota Dioscorea deltoidea
M-34 IFR D-1 IFR DM-0.5 IFR DM-1 IFR DM-8 IFR Zh-1 IFR Zh-2 IFR Zh-3 IFR Zh-10 NrEs-1 L-1 KT-4
1977 1984–1989 1984–1989 1984–1989 1984–1991 1983–1992 1985–1991 1984–1988 1985–1987 1984 1985–1989 1991
Dactylorhiza fuchsii Dactylorhiza maculata Gymnodenia conopsea
Rhs-1
1992
Rhs-2 Rhs-8 IFR T-1 TsU-194 Callus Callus B-233 PZ-4 R-1 1C BFT 01-95
1999 1997–1999 1994 1994 1995 1995 1998–1999 1999 1998 1999 1998
Panax ginseng
Panax quinquefolius Nicotiana sylvestris Medicago sativa Solanum tuberosum, cv. Tawa Rhaponticum carthamoides
Triticum timofeevi Triticum aestivum Dioscorea caucasica Dioscorea balcanica Thalictrum minus Panax japonicus Panax ginseng Panax ginseng Polyscias filicifolia Species Orchid seeds Tropical Angraecum magdalenae Bratonia, F1 Calanthe spp. Encyclia cochleata Cattleya spp. Dendrobium stratictes Dendrobium ochreatum Liparis nigra Mischobulbum cordifolium Phaius flavus Pholidota guibertae Polystachya sp. Trichopilia tortilis Vanda coerulea Russian Cypripedium macronthon Platanthera bifolia Epipactis helleborine Dactylorhiza longifolia Dactylorhiza incarnata
Year of freezing
2001 2001 2000–2001 2001 2002 2003 2003 2004 2004 2004 2004 2004 2004 2004 2004 2004 2002 2002 2004 2004
2004 2004
Besides we continuously store in liquid nitrogen shoot tips of strawberry (6 cultivars), raspberry (2 cultivars) and rose (3 cultivars) and seeds of 230 endangered plant species collected over the whole Russian territory and studied by Prof Valentina L. Tikhonova (the Main Botanical Garden, Moscow).
The slow freezing methods has some advantages such as: lower concentration of cryoprotector solutions, slow dehydration, which are particularly important for some objects. According to the above stated, young cell strains with proembryo cell clusters are more eligible for deep freezing because they contain small and almost nonvacuolated cells. Other cell lines need special preliminary cultivation procedures such as short subculturing times (for cell suspensions), cold acclimation, specific nutrient media supplemented with sucrose, mannitol, sorbitol, amino acids and other substances [12–15]. Often it is important to concentrate cells in suspension before freezing [16]. 3. Results and discussion To demonstrate the integrity of plasma membrane during treatment, a new fluorometric method was developed based on selective plasmalemma permeability for fluoresceine and its diacetate [17]. This method is not a tool for distinguising live and death cells in suspension culture, but is specially aimed at estimating the number of cells with plasmalemma destructions [18]. In original Widholm’s method, one counts the fluorescent cells under a microscope because fluoresceine is still localized into the cells. In our method, the fluorescence is measured with a fluorimeter in ambient solution, using in parallel two control specimens. The temperature and time specifications must be rigidly met in this method [18]. During cell freezing and thawing, the fluoresceine released from cells, thus indicating the damage to plasmalemma that increased linearly when temperature decreased to K30 and K40 8C. Under dehydration stress in CaCl2 or sorbitol solutions (their concentrations were increased step by step) at 2 8C, the active release of fluoresceine (and thus plasmalemma injury) occurred at 16.12 osm (equal to K30 8C) and 21.5 osm (K40 8C). By using this method, the correlation between cryo- and osmoresistance was shown for five cell lines of Dioscorea deltoidea and Panax ginseng (Fig. 1) [18–20]. Thus, it became clear that the osmotic stress caused by active cell
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without cryopreservation after cryopreservation m 10–6
2000 1800 1600 1400 1200 1000 800 600 400 200 0 0
40
80
120
160
days after seed sowing Fig. 6. Growth of Dactylorhiza longifolia protocorms on agar medium after seed storage in liquid nitrogen. Y axis shows the diameter of protocorms, mm.
dehydration plays the main role in the injury of cell plasma membrane and cell death during freezing. In Cryopreservation Group of Department of Cell Biology and Biotechnology in the Institute of Plant Physiology, cryoresistance of 34 cell strains of 21 plant species was studied. Cryopreservation technique, developed for these lines, included special conditions of preliminary cultivation and recultivation of meristem and cell cultures, different cryoprotectors and artificial initiation of crystallization during slow freezing. For the latter, a special automatic device was constructed. For example, the cells of D. deltoidea original strain (IFR D-1) were preliminary cultivated with asparagine, proline or alanine [12]; the cells of P. ginseng mutant strains IFR Zh-2 and Zh-3, of Panax quinquefolius IFR Zh-10, the donor in vitro plants and excised meristems of Fragaria and Rubus were preliminarily cold hardened with elevated (step by step) sucrose concentration in nutrient medium [12]. The cells of Rhaponticum carthamoides and Thalictrum minus were cultivated with mannitol. Other cell strains did not demand any special preliminary cultivation. Cryoprotector solutions involved a DMSO (dimethylsulfoxide), glycerol, sucrose, trehalose, and glucose at various concentrations and combinations. Slow freezing was performed at a rate of 0.3–0.5 8C/min by the freezers constructed in Kharkov (Institute of Cryobiology and Cryomedecine Problems,
Fig. 7. Flowering of Encyclia cochleata obtained from cryopreserved seed. After in vitro regeneration by T.V. Nikishina, G.L. Kolomeytseva grew the plants in a greenhouse of Main Botanical Garden of Russian Academy of Sciences.
Ukrainian National Academy of Sciences). Now we use the vitrification method of ultrafast freezing for the cell culture of Stephania glabra and obtained the first success, but these results are still not published. The seeds of rare plants, involving orchids, were freezed without cryoprotectors by direct immersion into liquid nitrogen, being sufficiently dry. As a result, 27 cell lines of 16 plant species successfully resumed their growth after cryogenic storage [12,21–23] and preserved their specific features and biosynthetic potential. Some of these strains are used in biotechnological industry as the producers of pharmaceutical preparations. Fig. 2 shows the plants of rare species Dioscorea caucasica and Dioscorea balcanica regenerated after cryopreservation of their embryogenic calluses [24,25]. Besides, shoot tips of about 40 cultivars of potato (Fig. 3), rose, strawberry (Fig. 4) [26] and raspberry [27] regenerated plants in vitro and in vivo after storage in liquid nitrogen. Potato plants regenerated after cryopreservation of their meristems showed the same protein profiles of tubers and leaves as the control ones [28]. The cell strains of
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carrot, D. deltoidea and P. ginseng successfully resumed their growth and biosynthetic capacities after 6, 9, 12 and 14 years of storage in liquid nitrogen. The longest storage time exceeded 25 years (Fig. 5). 4. Conclusion Now we continuously store in liquid nitrogen 24 cell strains from 15 species of rare medicinal plants and crops, shoot tips of 7 cultivars of strawberry and raspberry and seeds of 230 endangered plant species collected over the whole Russian territory—from the South Kuril Islands to Baltic Sea (Table 1). The seeds were collected and their germination and growth after storage in liquid nitrogen were studied by Prof. Valentina L. Tikhonova and her coworkers in the Main Botanical Garden of Russian Academy of Sciences (Moscow). Recently, we have developed the methods for cryopreservation and in vitro cultivation of the microscopic seeds of tropical and Russian orchids [29–31]. Protocorms and juvenile plants obtained from cryopreserved seeds did not significantly differ from those obtained from control (unfrozen) seeds in growth rate (Fig. 6). Fig. 7 shows flowering of Encyclia cochleata, a tropical orchid, grown from cryopreserved seeds first in vitro and then in a greenhouse. Now we continuously are storing the seeds of 22 rare tropical and Russian orchid species in liquid nitrogen (Table 1).
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