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77. Cryopreservation of pollen from 28 ornamental plants
Abstracts / Cryobiology 63 (2011) 306–342 A, and with PE: CD5, 7, 8, 10, 11c, 13, 19, 22, 33, and 34. The subcultures of colony cells were tried. The results: (1) Granulocyte–Macrophage CFU-C -and Erythroblast-CFU-C were observed as self-maintaining and differentiation capacity under favourable conditions in all cases, which had been preserved in liquid phase of liquid nitrogen for 29 years or more. (2) Overall recovery % of thawed marrow stem cells gave the regression equation: Y = 0.0002X + 0.0362, which suggests that freezing in liquid nitrogen hardly influences the CFU-C recovery. (3) The increased expression of surface markers: CD 11b, 13, and 33 clarified that the growth factors in the media effectively stimulated the proliferation and differentiation of those cells expressing those surface makers. But, the increased titers of those surface markers decreased again on Day 14 in some cases. (4) The cells, which had no stimulation by any colony stimulation factors, expressed few markers and appeared in vitro as the cell debris under the reversed microscopy and on the cytogram of flow cytometry. (5) Some colony cells were successfully sub-cultured to the second generation. (6) The mature dendritic cells were surrounded by many small nucleated cells that was reminiscent to antigen presentation and the another dendrites differentiated to spindle-shaped fibroblasts and made the colonies. (7) Mesenchymal stromal cells (BMSCs) or fibroblast were recruited to construct interesting frameworks on the bottom surface of Petri dish on Day 7 or later in culture. (8) Although 3 of 30 conservation bags were ruptured during thawing process in a +40 °C water bath, no obstacle including bacterial contamination was not caused in the culture preparation afterward. (9) The cryopreservation of stem cells and cord blood cells too at 60 to 80 °C in the mechanical deep freezer and in the vapour phase of liquid nitrogen did not constantly succeed the formation of colony, probably due to long-termed duration of unstable temperatures as already reported. Finally, the authors like to emphasize that the cryopreserved autologous bone marrow stem cells or peripheral progenitor cells will play an important and an indispensable role of therapeutic procedure not only for the patients of iatrogenic ablation of hematopoietic systems but also for the special workers of the accidental radiation injury encountered in the vicinity of the nuclear reactor by any chance. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.078
Plant biobanking
76. New data on the project – ‘‘Cryopreservation of plant seeds in the permafrost”. E.S. Khlebnyy *, G.V. Philippova, V.E. Repin, A.A. Shein, M.M. Shashurin, I.V. Voronov, Institute of Biological Problems of Cryolithozone, Siberian Branch of the Russian Academy of Science, Yakutsk, Russia The republic Sakha (Yakutia) and the Siberian branch of the Russian Academy of Sciences (SB RAS) represent to the government of Russia and the Russian Academy of Sciences the project of the organization federal seeds cryostorage on the basis of functioning underground laboratory of Institute of biological problems of permafrost zone SB RAS and Institute of permafrost SB RAS. In this connection the new data under the given project is of interest. In May, 2009 three-year experiment on storage of seeds of three species of quickly growing old cultural plants (onion, fennel, mustard) in the conditions of a long-term frozen ground (temperature 5.5 °C) is put at various structure of gas medium (Ar, CI2, N2) and in the presence of a sorbent – lichen thallus, and also under different temperature conditions of artificial cooling in a range from +2 to 17 °C. For conservation of viability of seeds, the tight containers excluding change of humidity of a seed material for a long time are used. Results of analyses have shown that 1.5-year storage of seeds of these species in the above-stated conditions has resulted following: (1) At storage at temperatures 6 to 20 °C germination of seeds even has increased on 7–20%, especially at storage in the environment of Ar, under the same storage conditions characteristics of functional activity and fastness of the genetic apparatus were the stablest. (2) At storage at temperature +2 °C germination of seeds practically hasnot changed in mediums Ar, CI2 and N2 and has started to decrease on 8–13% in the environment of atmospheric air. Similar tendencies obtained concerning characteristics of functional activity and fastness of the genetic apparatus. (3) At storage at room temperatures (+20 °C) germination of seeds decrease on 12–30%, besides, that characteristics of functional activity of the genetic apparatus (activity of replication systems, transcription, DNA reparation) and activity of antioxidant systems change so that fastness of the genetic apparatus, non linear image depending from them remained. It is necessary to note that only at storage in a temperature range 6 to 10 °C there was minimum a level of an abnormal mitosis 1.1–1.6%. Under other storage conditions (including 17 °C) and in control it has made 3.6–6.9%, and at storage in the Ar medium at 6 °C the level of an abnormal mitosis was 0–0.2%. At identical temperature, but in various gas medium percent of an abnormal mitosis varies in wide ranges (from 0% to 13.3% for fennel), therefore to find any communication between genetic disturbances and gas medium of containers yet it was not possible. As in a number of similar projects ‘‘stumbling-block” is conservation of microbiological cleanliness of a seed material at long storage, the microbiological analysis of seed material stored of 35 years,
327
and the premise cryostorage has been carried out. Our colleagues from laboratory of microbiology of Institute of chemical biology and fundamental medicine (head of department doctor Repin V. E) microbiological samples of the seeds stored in conditions of a long-term frozen ground within nearby 33 years, and smears from walls of underground laboratory are taken. Preliminary results the following: The blanket of grains after processing is disinfected, but in microcracks teleorgánic microorganisms which under conditions favorable for them start to breed remain; In cryostorage exists insignificant semination both bacteria, and mushrooms. Thus was not received bacteriemic, psychrophilic, psychrotolerant cultures. The specific structure of microorganisms in assays with seeds does not coincide with a variety of microorganisms in the storehouse. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.079
77. Cryopreservation of pollen from 28 ornamental plants. J. Xu, B.L. Li *, Y. Liu, College of Landscape Architecture, Beijing Forestry University, Beijing 100083, China, National Floriculture Engineering Research Center, Beijing, 100083, China Long-term storage of ornamental plant pollen is meaningful for overcoming the problems of flowering asynchronism and geographical isolation in cross breeding. This paper compares the pollen viability of 28 species ornamental plants, which are widely used in Beijing, China, at LN2 and room temperatures (20 ± 2 °C) by in vitro pollen germination. The results show that: (1) The germination levels of fresh pollen were different among 28 species. (2) The lifetime of pollen from 28 species was different at room temperature (20 ± 2 °C); 4 species had the shortest pollen lifetime, 8d, whereas Amygdalus persica var. persica f. atropurpurea had the longest one, 43d; the half of 28 species was between 16 30d. (3) After 3 day’s storage in 196 °C, all species pollen germinated normally. Compared to their fresh pollen, the germination levels of pollen from 8 species rose dramatically, 7 species were similar to the fresh pollen, and 13 species declined. (4) After 2 year’s cryopreservation, the pollen germination levels of 7 species significantly dropped to below 1%, 3 species increased remarkably, 3 species kept stable, and the other 15 species decreased in various degree. But those changes did not show the relation with that of 3 day’s stored in LN2. In our study, the pollen of 75% of 28 ornamental plants was available after 2 years storage in LN2, which indicates that cryopreservation is an effective method to prolong the lifetime of pollen from most ornamental plants. However the pollen germination level was very different among the species and storage periods in LN2. And the changes had no significant coherence with neither the fresh pollen lifetime at room temperature nor the storage period, which demonstrates that the speed of pollen vigour losing is different among the species. That also suggests that the evaluation of cryopreservation of pollen need an eligible time immerged in LN2. Conflict of interest: None declared. Source of funding: This work was supported by the National Natural Foundation (30972411). doi:10.1016/j.cryobiol.2011.09.080
78. Cryopreservation of Juglans sp. germplasm using winter dormant buds. M.M. Jenderek * 1, J. Postman 2, C. Leslie 3, M. Coggeshall 4, D. Ellis 1, 1 National Center for Genetic Resources Preservation, USDA-ARS, Fort Collins, CO, USA, 2 National Clonal Germplasm Repository, USDA-ARS, Corvallis, OR, USA, 3 Department of Plant Science, University of California, Davis, CA, USA, 4 Department of Forestry, Center of Agroforestry University of Missouri, Columbia, MO, USA The USDA-ARS, National Plant Germplasm System (NPGS), preserves 20 Juglans species with over 680 unique accessions that are maintained as field plantings. Cryopreservation of these genetic resources is an economic alternative to a costly back-up planting in another location. One of the major obstacles to using cryopreservation for Juglans germplasm is the lack of reliable cryo-processing protocols that produce sufficient post-cryo viability of the material. We used dormant winter buds of butternut (J. cinerea), black walnut (J. nigra) and Persian walnut (J. regia) to test the suitability of the cryopreservation method used for apple dormant buds to cryopreserve germplasm of these nut trees. That method uses dormant buds from twigs cut into one node segments, desiccated to 25–30% moisture content, slow cooled to 30 °C and exposed to liquid nitrogen vapors (LNV) for at least 24 h. The viability is tested by grafting the LNV exposed buds onto rootstocks. Modifications to the apple dormant bud cryopreservation method included twig cold-hardening under two light regimes, soaking in ABA or sucrose solutions (2 °C, 4 days) prior to desiccation and post-cryo viability testing by forcing shoot growth under mist instead of grafting. Post-cryo viability evaluated under misting conditions was 60–100% for black walnut and 10–60% for Persian walnut whereas the survival of controls (one-node segments stored at 5 °C) was 62.5–100% and 80–100%, respectively. Cryo-survival was also genotype dependent. These data suggest that cryopreservation of Juglans germplasm using dor-
Plant biobanking
76. New data on the project – ‘‘Cryopreservation of plant seeds in the permafrost”. E.S. Khlebnyy *, G.V. Philippova, V.E. Repin, A.A. Shein, M.M. Shashurin, I.V. Voronov, Institute of Biological Problems of Cryolithozone, Siberian Branch of the Russian Academy of Science, Yakutsk, Russia The republic Sakha (Yakutia) and the Siberian branch of the Russian Academy of Sciences (SB RAS) represent to the government of Russia and the Russian Academy of Sciences the project of the organization federal seeds cryostorage on the basis of functioning underground laboratory of Institute of biological problems of permafrost zone SB RAS and Institute of permafrost SB RAS. In this connection the new data under the given project is of interest. In May, 2009 three-year experiment on storage of seeds of three species of quickly growing old cultural plants (onion, fennel, mustard) in the conditions of a long-term frozen ground (temperature 5.5 °C) is put at various structure of gas medium (Ar, CI2, N2) and in the presence of a sorbent – lichen thallus, and also under different temperature conditions of artificial cooling in a range from +2 to 17 °C. For conservation of viability of seeds, the tight containers excluding change of humidity of a seed material for a long time are used. Results of analyses have shown that 1.5-year storage of seeds of these species in the above-stated conditions has resulted following: (1) At storage at temperatures 6 to 20 °C germination of seeds even has increased on 7–20%, especially at storage in the environment of Ar, under the same storage conditions characteristics of functional activity and fastness of the genetic apparatus were the stablest. (2) At storage at temperature +2 °C germination of seeds practically hasnot changed in mediums Ar, CI2 and N2 and has started to decrease on 8–13% in the environment of atmospheric air. Similar tendencies obtained concerning characteristics of functional activity and fastness of the genetic apparatus. (3) At storage at room temperatures (+20 °C) germination of seeds decrease on 12–30%, besides, that characteristics of functional activity of the genetic apparatus (activity of replication systems, transcription, DNA reparation) and activity of antioxidant systems change so that fastness of the genetic apparatus, non linear image depending from them remained. It is necessary to note that only at storage in a temperature range 6 to 10 °C there was minimum a level of an abnormal mitosis 1.1–1.6%. Under other storage conditions (including 17 °C) and in control it has made 3.6–6.9%, and at storage in the Ar medium at 6 °C the level of an abnormal mitosis was 0–0.2%. At identical temperature, but in various gas medium percent of an abnormal mitosis varies in wide ranges (from 0% to 13.3% for fennel), therefore to find any communication between genetic disturbances and gas medium of containers yet it was not possible. As in a number of similar projects ‘‘stumbling-block” is conservation of microbiological cleanliness of a seed material at long storage, the microbiological analysis of seed material stored of 35 years,
327
and the premise cryostorage has been carried out. Our colleagues from laboratory of microbiology of Institute of chemical biology and fundamental medicine (head of department doctor Repin V. E) microbiological samples of the seeds stored in conditions of a long-term frozen ground within nearby 33 years, and smears from walls of underground laboratory are taken. Preliminary results the following: The blanket of grains after processing is disinfected, but in microcracks teleorgánic microorganisms which under conditions favorable for them start to breed remain; In cryostorage exists insignificant semination both bacteria, and mushrooms. Thus was not received bacteriemic, psychrophilic, psychrotolerant cultures. The specific structure of microorganisms in assays with seeds does not coincide with a variety of microorganisms in the storehouse. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.079
77. Cryopreservation of pollen from 28 ornamental plants. J. Xu, B.L. Li *, Y. Liu, College of Landscape Architecture, Beijing Forestry University, Beijing 100083, China, National Floriculture Engineering Research Center, Beijing, 100083, China Long-term storage of ornamental plant pollen is meaningful for overcoming the problems of flowering asynchronism and geographical isolation in cross breeding. This paper compares the pollen viability of 28 species ornamental plants, which are widely used in Beijing, China, at LN2 and room temperatures (20 ± 2 °C) by in vitro pollen germination. The results show that: (1) The germination levels of fresh pollen were different among 28 species. (2) The lifetime of pollen from 28 species was different at room temperature (20 ± 2 °C); 4 species had the shortest pollen lifetime, 8d, whereas Amygdalus persica var. persica f. atropurpurea had the longest one, 43d; the half of 28 species was between 16 30d. (3) After 3 day’s storage in 196 °C, all species pollen germinated normally. Compared to their fresh pollen, the germination levels of pollen from 8 species rose dramatically, 7 species were similar to the fresh pollen, and 13 species declined. (4) After 2 year’s cryopreservation, the pollen germination levels of 7 species significantly dropped to below 1%, 3 species increased remarkably, 3 species kept stable, and the other 15 species decreased in various degree. But those changes did not show the relation with that of 3 day’s stored in LN2. In our study, the pollen of 75% of 28 ornamental plants was available after 2 years storage in LN2, which indicates that cryopreservation is an effective method to prolong the lifetime of pollen from most ornamental plants. However the pollen germination level was very different among the species and storage periods in LN2. And the changes had no significant coherence with neither the fresh pollen lifetime at room temperature nor the storage period, which demonstrates that the speed of pollen vigour losing is different among the species. That also suggests that the evaluation of cryopreservation of pollen need an eligible time immerged in LN2. Conflict of interest: None declared. Source of funding: This work was supported by the National Natural Foundation (30972411). doi:10.1016/j.cryobiol.2011.09.080
78. Cryopreservation of Juglans sp. germplasm using winter dormant buds. M.M. Jenderek * 1, J. Postman 2, C. Leslie 3, M. Coggeshall 4, D. Ellis 1, 1 National Center for Genetic Resources Preservation, USDA-ARS, Fort Collins, CO, USA, 2 National Clonal Germplasm Repository, USDA-ARS, Corvallis, OR, USA, 3 Department of Plant Science, University of California, Davis, CA, USA, 4 Department of Forestry, Center of Agroforestry University of Missouri, Columbia, MO, USA The USDA-ARS, National Plant Germplasm System (NPGS), preserves 20 Juglans species with over 680 unique accessions that are maintained as field plantings. Cryopreservation of these genetic resources is an economic alternative to a costly back-up planting in another location. One of the major obstacles to using cryopreservation for Juglans germplasm is the lack of reliable cryo-processing protocols that produce sufficient post-cryo viability of the material. We used dormant winter buds of butternut (J. cinerea), black walnut (J. nigra) and Persian walnut (J. regia) to test the suitability of the cryopreservation method used for apple dormant buds to cryopreserve germplasm of these nut trees. That method uses dormant buds from twigs cut into one node segments, desiccated to 25–30% moisture content, slow cooled to 30 °C and exposed to liquid nitrogen vapors (LNV) for at least 24 h. The viability is tested by grafting the LNV exposed buds onto rootstocks. Modifications to the apple dormant bud cryopreservation method included twig cold-hardening under two light regimes, soaking in ABA or sucrose solutions (2 °C, 4 days) prior to desiccation and post-cryo viability testing by forcing shoot growth under mist instead of grafting. Post-cryo viability evaluated under misting conditions was 60–100% for black walnut and 10–60% for Persian walnut whereas the survival of controls (one-node segments stored at 5 °C) was 62.5–100% and 80–100%, respectively. Cryo-survival was also genotype dependent. These data suggest that cryopreservation of Juglans germplasm using dor-