2Botanic Gardens and Parks Authority (Kings Park and Botanic Garden), Perth, WA 6005, Australia.
3Faculty of Natural and Agricultural Sciences (Plant Science), University of Western Australia, Perth, WA 6907, Australia.
Ex situ conservation of endangered plants is an important aim in order to preserve biodiversity of flora in threatened ecosystems. Among the biotechnological techniques which can be used, cryopreservation is emerging as a preferred option in many instances. This study describes a cryopreservation technique developed for shoot tips of the endangered species Centaurea ultreiae (Compositae) using a vitrification procedure. Basal medium (BM) for preculture and loading phases consisted of 1/2 MS basal salts with modified vitamins (3 μ thiamine). For preculturing shoot tips, BM with five osmotic treatments were investigated: 0.3 M sucrose � 20 μ ABA, 0.6 M glycerol � 20 μ ABA and 0.25 M sucrose + 0.25 M glycerol + 10 μ ABA. A loading solution treatment (BM with 2 M glycerol and 0.4 M sucrose) was applied prior to exposure of shoot tips to PVS2 and found to be indispensable to obtaining successful post-LN recovery. Highest (95.5%) regrowth of LN immersed shoot tips was obtained following incubation on BM + 0.3 M sucrose + 20 μ ABA or 0.25 M sucrose + 0.25 M glycerol + 10 μ ABA, with loading treatment and PVS2 exposure for 20 minutes at 0�C.
An iterative method has been proposed to determine the relationship between the temperature depression of intracellular ice formation (IIF) and the equilibrium melting point depression for initial cryoprotective agent (CPA) concentrations larger than 1.5M. Using the iterative method coupling with a water transport model for freezing induced cell dehydration and intracellular ice growth, the temperature of IIF has been determined. The new model of temperature of IIF has been applied to predict nucleation parameters at various temperature and initial CPA concentrations according to Karlsson's approach. A geometrical model of soft impingement proposed by Bruna has been incorporated into Karlsson's diffusion limited crystal growth model to include the effect of soft impingement. The new crystal growth model has been verified by a comparison between the predicted critical cooling rates for vitrification with the reported values in literature. With the new crystal growth model, it has been found that the limiting value of the crystallized volume fraction increases as cooling progresses and decreases as the initial CPA concentration increases. A comparison of simulated crystallized volume fractions when soft impingement, hard impingement and no corrections are used has also been made and the result shows that soft impingement could not be omitted in the prediction of intracellular ice formation and growth, especially when the final crystallized volume fraction is larger than 0.1.
2Present address: Divisi�n Acad�mica de Ciencias Biol�gicas, UJAT, C.P. 86150 Villahermosa, Tabasco, M�xico.
146 � 12s) and hatching rates (29 � 04%) were higher than those by other rates. Progressive motility (83�5%) did not differ significantly from fresh samples.
An optimum cholesterol content in cells could provide the benefit of lowering or eliminating the lipid phase transition temperature, while maintaining membrane fluidity and strength; thus, making cells less sensitive to chilling injury and more amenable to cryopreservation. Such effects were shown in some gametes and embryos of certain mammalian species, however, some other cell types, benefited from cholesterol removal. The experiments developed in this study aimed to determine the effect of incubating Pacific oyster (Crassostrea gigas) oocytes in cholesterol-addition or removal solutions prior to cryopreservation on their post-thaw fertilization ability. The results showed a positive association of cholesterol with the oocytes when assessed by fluorescent microscopy. However, this uptake was not reflected by an increase in cholesterol as determined by colorimetric analysis or in the post-thaw fertilization rate of treated oocytes. It is presumed either that oyster oocytes already contain a substantial amount of cholesterol or other lipids in their plasma membranes and do not benefit from any additional cholesterol or there is no lipid phase transition temperature in oyster oocytes.
2701 S. Zarzamora, Texas Diabetes Institute, San Antonio, Texas 78207-5209, USA (current address).
3 Centre for Biotechnology, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India.
The aim of this study was to develop a cryopreservation protocol for Dioscorea rotundata with maintenance of genetic stability of regenerated plants after cryopreservation. In vitro shoot tips were cryopreserved using vitrification and encapsulation-dehydration to compare the efficacy of the two methods. Both methods produced high levels of plant regeneration from cryopreserved shoot tips. The regeneration level obtained using vitrification (71%) was not significantly different from that obtained using encapsulation-dehydration (67%). Genetic stability of plants derived from cryopreserved shoot tips was evaluated using RAPD markers. Analysis of 50 cryopreserved-derived and 20 in vitro- maintained (control) plantlets showed that 10 primers produced 77 clear, reproducible bands, with the amplification products being monomorphic for all the plantlets tested. A total of 5,390 bands obtained from this study exhibited no aberration in RAPD banding. Thus, the present study showed that both vitrification and encapsulation-dehydration methods are equally applicable to D. rotundata for cryopreservation. The in vitro plantlets derived from cryopreservation were genetically stable at the molecular level tested.
Key words: Dioscorea rotundata, cryopreservation, vitrification, encapsulation dehydration, RAPD, genetic stability.
A reliable technique for cryopreservation by encapsulation was developed for two suspension cultures of gentian species (Gentiana tibetica and G. cruciata) of different ages and embryogenic potential. The effect of water content, aggregate size and the subculture time on viability was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) test. Regrowth of a proembryogenic mass (PEM) on agar, liquid or agar/liquid media was assayed by measuring the increase in biomass. A water content of 24-30% (fresh weight basis) after 5-6 h dehydration of encapsulated cells of gentians yielded the highest survival (68% for G. tibetica and 83% for G. cruciata) after cryopreservation. Regardless of species, aggregate size and subculture time, the lowest PEM survival was 44%. These parameters did not influence the survival of G. tibetica PEM, but the survival of G. cruciata was higher when the smaller aggregates were cryopreserved on the 5th day of culture. Agar/liquid culture caused the greatest biomass increase. Cryopreservation did not affect the characteristics of suspension cultures and their regrowth after thawing, nor the number and dynamics of somatic embryos formed. Flow cytometry showed that cryopreservation did not change the genome size of the PEMs or regenerants.
2KT & G Central Research Institute, Suwon 441-480, Korea.
Korean ginseng germplasm is maintained as clonal germplasm since there is no practical method for long-term seed conservation. The aim of this study was to establish a cryopreservation protocol for Korean ginseng seeds. Desiccation of undehisced ginseng seeds to a moisture content (MC) of 7.1 % did not decrease their dehiscence and germination. After cryopreservation, the dehiscence percentage of desiccated seeds decreased for MC above 12.5%; it was 26% for 22.6% seed MC and nil for 41.9% seed MC. Germination percentage did not decrease significantly between 12.5-22.6% seed MC, while germination percentage of dehisced seeds decreased below 7.2% MC, reaching 25.8% at 3.8% MC. After cryopreservation, the germination percentage decreased from 90.5-92.9% at 8.3-10.6% MC to 84.8% at 12.5% MC. At MCs below 8.3%, germination rapidly decreased from 85.0% at 7.2% MC to 34.9% at 5.3% MC. Therefore, the hydration window for cryopreservation of ginseng seeds is around 8-11% MC. Undehisced Korean ginseng seeds were characterized by their high lipid and protein content (lipids, 42.6% FW; proteins, 41.0% FW). When using thermal analysis, during the cooling phase, exothermic ice crystallization peaks were observed with dehisced ginseng seeds above 13.5% MCs (3.3 J g-1 FW). A second crystallization peak was detected following ice crystallization peaks.
Keywords: after-ripening, desiccation, germination, Panax ginseng C.A. Meyer.
1. Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.
2. Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan.
We established a simple cryopreservation protocol for Arabidopsis T87 cells using an encapsulation-dehydration method. T87 cells were encapsulated into alginate beads containing 2 M glycerol and 0.4 M sucrose. Alginate beads containing T87 cells were dehydrated with silica gel for 2 h (to c. 0.7 g H2O g DW-1) followed by immersed in LN. After rewarming at 35�C for 3 min and 1-d incubation under continuous illumination at 22�C, cryopreserved T87 cells exhibited considerable regrowth. Exponentially-grown 7-d-old T87 cells regrew more vigorously (86% of control) than 14-d-old cells after cryopreservation without preculture in medium containing 0.3 M sucrose. Genetic stability of cryopreserved T87 cells was demonstrated by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and principal component analysis (PCA). Transformed T87 cells were cryopreserved using established protocols, and GUS expression was maintained within a 2-fold variance. These results indicate that cryopreservation of T87 cells is useful for comprehensive metabolomics research and for the large scale collection of transformed cultured cell lines for functional genomics research.
Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka 564-8680 Japan.
Some organisms like bacteria and plants have a cryoprotective protein to cryopreserve the freeze-labile enzyme under stable conditions having high activity. By screening of the cryoprotective activities of various food-industrial yeasts, we found that the cell membrane component that was a glucanase extractable component in Pichia anomala NBRC 141 had a high level of cryoprotective activity against freeze-labile lactate dehydrogenase (LDH). The absorption of the active compound in the crude extract by ConA-Sepharose chromatography suggested that this active compound might be a glycoprotein (COGP). Strain NBRC had the COGP constantly without the treatment of cold acclimation. The active compound, that is, a COGP, could be homogeneously purified using DEAE-TOYOPEARL and Sephacryl S-400 chromatography. The purified COGP had a cryoprotective activity of 50.9% at a sugar concentration of 17.9 μ/ml. The molecular weight of purified COGP was 83,000, which was composed of one protein with a molecular weight of 22,000 and polysaccharide. Furthermore, the constituent sugar of COGP was only D-mannose based on HPLC analysis of its acid hydrolysate. Also, we confirmed that the cryoprotective activity of COGP was higher than those of the commercial cell membrane components. The CP50 of COGP was 2.8 X 10 2 nM, which was half to the CP50 of BSA. This is the first report, to our knowledge, that the cell membrane component of Pichia anomala had a high level of cryoprotective activity against a freeze-labile enzyme.