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112. The cryogenic collection of fruit biodiversity in Kazakhstan
Abstracts / Cryobiology 63 (2011) 306–342 equipped with probes to control chamber and sample temperature. To define the experimental cryoprotectant efficacy we performed vitality and death assays after 0h and 24h from thawing by trypan blue exclusion and FACS counting (GuavaÒ EasyCyte; Via Count test), using CryoStoreÒ CS10 (Biolife Solution) as reference freezing medium. Good cell recovery was obtained in all cell lines showing the following survival rates: 47% (t = 0 h) and 41% (t = 24 h) in NS46C; 50% (t = 0 h) and 40% (t = 24 h) in CB660sp; 41% (t = 0 h) and 30% (t = 24 h) in G179; and 58% (t = 0 h) and 91% (t = 24 h) in AF22. Conflict of interest: The authors declare no conflict of interest. Acknowledgments: Research activities are supported by NeuroStemcell (European Community’s Seventh Framework Programme grant agreement no. 222943) to I.B. doi:10.1016/j.cryobiol.2011.09.114
112. The cryogenic collection of fruit biodiversity in Kazakhstan. I. Kovalchuk * a, B. Reed * b, T. Turdiyev a, S. Frolov a, G. Madiyeva a, a Institute of Plant Biology and Biotechnology at National Center of Biotechnology, Timiryazev Str. 45, 050040, Almaty, Kazakhstan, b United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97333, United States Conservation of the biodiversity of fruit crops is important to the future of horticulture in Kazakhstan. A field collection of fruit germplasm with more than 4000 cultivars and wild selections is grown in the Pomological Garden of the Institute of Horticulture and Viticulture near Almaty, to preserve the native species, as well as cultivated fruit. These plants are at risk from environmental as well as biotic threats. Cryopreservation in liquid nitrogen ( 196 °C) is used for reliable long-term preservation of plant genetic resources. A cryopreserved collection allows preservation for an unlimited term, retention of viability, regeneration potential and genetic stability and provides a backup of the collection that is secure from anthropogenic and abiotic factors, or attack by insects and diseases. A cryo genebank is now being established based on experiments that defined optimal cryopreservation approaches for liquid nitrogen ( 196 °C) storage of these cultures. A PVS2 vitrification protocol was used with in vitro cultured shoot tips. Shoot tips ( 1 mm) were precultured for 2 days in cold acclimation conditions on MS medium with 0.3 M sucrose. Shoot tips were then placed in loading solution of 2 M glycerol in 0.4 M sucrose for 20 min, then placed in 1.2 ml cryovials on ice with 1 ml PVS2 for 80 min and submerged in liquid nitrogen. Rewarming of samples was carried out in a 45 °C water bath for 1 min, then in 22 °C water for 1 min. Dormant buds were cryopreserved with the following protocol: Scion wood was collected after the temperature 10 °C and below. The acclimated scions were dehydrated to 30% moisture at 3 °C to 5 °C, then cooled at 1 °C/h to 25 °C and held at this temperature for 24 h before placing in vapor phase over LN at 150 °C. Scions were rewarmed for 24 h in cold room at +4 °C. Buds from the scions were grafted onto rootstocks or regenerated in vitro. The cryogenic collection includes the following plants stored as dormant buds: 124 apple genotypes [Malus x domestica Borkh. and Malus sieversii (Ledeb.) M. Roem)] (cultivars, hybrids, wild forms) with recovery from 92% to 100%; 60 sweet cherry genotypes (Prunus avium L.) with recovery from 62% to 75%; 17 pear genotypes (Pyrus communis L.) with recovery from 90% to 100%; 25 cherry genotypes (Prunus cerasus L.) with recovery from 75% to 82%. Collections stored as shoot tips by PVS2 vitrification of in vitro shoot tips include: 5 pear genotypes (P. communis L.) with 83.3% to 84.6 % regrowth; 6 cherry genotypes (P. cerasus L.), with 40% to 80% regrowth; 15 black currant genotypes (Ribes nigrum L.) with 46.7% to 100% regrowth; 25 raspberry genotypes (Rubus idaeus L., Rubus slesvicensis Lange) with 50% to 80% regrowth. Encapsulation-dehydration was used to cryopreserve 28 strawberry genotypes (Fragaria x ananassa Duch.) with 40% to 75% regrowth. This collection is the beginning of a national germplasm storage system for Kazakhstan. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.115
113. Desiccation at ambient temperature and storage of mouse sperm in 3-O-methyl-Dglucose medium without freezing. Gloria Lee, Jie Liu *, Joel Lawitts, Mehmet Toner, John Biggers, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital and Shriners Hospitals for Children, Boston, MA, USA, Beth Israel Deaconess Medical Center, Boston, MA, USA, Harvard Medical School, Boston, MA, USA Previously we reported that mouse sperm can be dried at ambient temperature and stored in medium containing trehalose for up to 3 months above 0 °C. However, it was necessary to permeate the sperm plasma membrane with the enzyme, alpha-hemolysin, to allow trehalose entry. We now report that 3-O-methyl-D-glucose (3-OMG), a metabolically inactive sugar transported into the sperm, can confer
337
protection against desiccation. Recovered sperm heads after storage above 0 °C can fertilize ova capable of development into newborn pups. Mouse sperm in 100 mM 3-O-methyl-D-glucose medium were convectively dried with ultra-pure dry nitrogen gas for 6 min at 10 l per minute, stored at 4 °C and 22 °C for 1, 2, and 3 months in different relative humidity levels, and tested for functionality by intracytoplasmic sperm injection (ICSI) into mouse oocytes. Sorption jars of stable relative humidity (RH) levels were achieved in glass jars with varying salt to water ratios of NaBr (RH 61%), MgCl2 (RH 35%), and LiCl (RH 12%). The percentage of blastocysts that developed from sperm dried in 3-OMG medium and stored in 12% RH at 22 °C for 1, 2, and 3 months were 28.8%, 26.6% and 12.2%, respectively, while those at 4 °C for 1, 2, and 3 months were 62.6%, 53.4% and 39.6%, respectively. The rates that blastocysts developed using dried sperm samples stored in the higher humidity conditions of NaBr and MgCl2 sorption jars at either 4 °C or 22 °C were lower, and directly related to increasing humidity and storage duration as compared with those stored in LiCl sorption jars. There was very little change in moisture contents of the dried sperm samples stored in 12% relative humidity LiCl sorption jars for 3 months, with a gain of 0.02 gH2O/g anhydrous weight at 4 °C, and a loss of 0.05 gH2O/g anhydrous weight at 22 °C. Transfers of two-four cell embryos derived by ICSI from convectively dried mouse sperm in 3-OMG medium and stored for 3 months at 22 °C produced two live pups from five recipient mothers while those stored at 4 °C produced nine live pups from two recipients. The results showed that 3-O-methy-D-glucose offered significant protection of desiccated mouse spermatozoa in storage above 0 °C without the need for poration of cell membrane. Source of funding: This research was supported by NIH/NCRR 5 R24 RR018934. Conflicts of interest: None declared. doi:10.1016/j.cryobiol.2011.09.116
114. Genetic stability assessment of long-term cryopreservation using wasabi plant by morphological, biochemical and molecular analysis for three years. Toshikazu Matsumoto * a, Takashi Akihiro a, Shinya Maki b, Kouhei Mochida c, Masaru Kitagawa c, Daisuke Tanaka d, Takao Niino d, a Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-2059, Japan, b Niihama National College of Technology, Niihama, Ehime 792-8580, Japan, c Shimane Agricultural Technology Center, Izumo, Shimane 693-0035, Japan, d National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8518, Japan Wasabi, Japanese horseradish (fam. Cruciferae), is an important crop in Japan. The plant contains a pungent ingredient. Myrosinase is responsible for the development of the flavor and pungency of wasabi root. Since the seed of wasabi is sensitive to desiccation, the cultivars and strains are maintained as living material in the field of some repositories in Japan. Shoot tips of wasabi were cryopreserved using the following vitrification method. Excised shoot tips from in vitro grown plants were precultured on a modified MS medium (half strength KNO3 and NH4NO3; termed 1/2 MS) containing 0.3 M sucrose and 0.2% gellan gum for 16 h at 25 °C, and osmo-protected with 1/2 MS liquid medium supplemented with 2 M glycerol plus 0.4 M sucrose for 20 min at 25 °C. Then the shoot tips were dehydrated with PVS2 for 50 min at 0 °C, and immersed into liquid nitrogen for 2 h (LN2h) or for 1 h before storage in a 150 °C deep freezer for 10 years (LN10yr). The survival rates of cryopreserved shoot tips for 10 years (LN10yr), for 2 h (LN2hr), treated control plants (TC) and control plants (Cont) were checked, and genetic stability of plants regenerated above shoot tips was assessed for 3 years using morphological, biochemical and molecular analysis. The survival rates ranged between 93% and 100%, irrespective of the treatment. Regenerated plants were acclimated for 2 months, then grown in a greenhouse. Morphological analysis of plants revealed no significant difference among the plants tested. Biochemical components such as sinigrin (pungent component), sucrose, glucose, fructose and some amino acids were measured using high performance liquid chromatography. No significant difference in sucrose content was observed between Cont. For sinigrin, glucose, fructose, aspartic acid and glutamic acid contents statistical differences (by Tukey multiple range test) were observed between LN10yr or LN2hr and Cont. However, it was not clear whether these differences were caused by genetic variation or not. In this study, we confirmed that cryopreserved wasabi shoot tips maintained high survival rate after 10 years of storage in a 150 °C deep freezer. To determine the genetic relationship among each regenerated plant, we carried out random amplified polymorphic DNA (RAPD) PCR using 0-mer oligonucleotide primers set. Total DNA was extracted from each leaf of plant by PVP method with some modifications. The results show that genetic variations were not detected by RAPD. These results indicated that wasabi plants derived from shoot tips cryopreserved for 10 years might be genetically stable. Source of funding: This study was partly supported by a research grant from Genebank, National Institute of Agrobiological Sciences, Japan. Conflicts of interest: None declared. doi:10.1016/j.cryobiol.2011.09.117
112. The cryogenic collection of fruit biodiversity in Kazakhstan. I. Kovalchuk * a, B. Reed * b, T. Turdiyev a, S. Frolov a, G. Madiyeva a, a Institute of Plant Biology and Biotechnology at National Center of Biotechnology, Timiryazev Str. 45, 050040, Almaty, Kazakhstan, b United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97333, United States Conservation of the biodiversity of fruit crops is important to the future of horticulture in Kazakhstan. A field collection of fruit germplasm with more than 4000 cultivars and wild selections is grown in the Pomological Garden of the Institute of Horticulture and Viticulture near Almaty, to preserve the native species, as well as cultivated fruit. These plants are at risk from environmental as well as biotic threats. Cryopreservation in liquid nitrogen ( 196 °C) is used for reliable long-term preservation of plant genetic resources. A cryopreserved collection allows preservation for an unlimited term, retention of viability, regeneration potential and genetic stability and provides a backup of the collection that is secure from anthropogenic and abiotic factors, or attack by insects and diseases. A cryo genebank is now being established based on experiments that defined optimal cryopreservation approaches for liquid nitrogen ( 196 °C) storage of these cultures. A PVS2 vitrification protocol was used with in vitro cultured shoot tips. Shoot tips ( 1 mm) were precultured for 2 days in cold acclimation conditions on MS medium with 0.3 M sucrose. Shoot tips were then placed in loading solution of 2 M glycerol in 0.4 M sucrose for 20 min, then placed in 1.2 ml cryovials on ice with 1 ml PVS2 for 80 min and submerged in liquid nitrogen. Rewarming of samples was carried out in a 45 °C water bath for 1 min, then in 22 °C water for 1 min. Dormant buds were cryopreserved with the following protocol: Scion wood was collected after the temperature 10 °C and below. The acclimated scions were dehydrated to 30% moisture at 3 °C to 5 °C, then cooled at 1 °C/h to 25 °C and held at this temperature for 24 h before placing in vapor phase over LN at 150 °C. Scions were rewarmed for 24 h in cold room at +4 °C. Buds from the scions were grafted onto rootstocks or regenerated in vitro. The cryogenic collection includes the following plants stored as dormant buds: 124 apple genotypes [Malus x domestica Borkh. and Malus sieversii (Ledeb.) M. Roem)] (cultivars, hybrids, wild forms) with recovery from 92% to 100%; 60 sweet cherry genotypes (Prunus avium L.) with recovery from 62% to 75%; 17 pear genotypes (Pyrus communis L.) with recovery from 90% to 100%; 25 cherry genotypes (Prunus cerasus L.) with recovery from 75% to 82%. Collections stored as shoot tips by PVS2 vitrification of in vitro shoot tips include: 5 pear genotypes (P. communis L.) with 83.3% to 84.6 % regrowth; 6 cherry genotypes (P. cerasus L.), with 40% to 80% regrowth; 15 black currant genotypes (Ribes nigrum L.) with 46.7% to 100% regrowth; 25 raspberry genotypes (Rubus idaeus L., Rubus slesvicensis Lange) with 50% to 80% regrowth. Encapsulation-dehydration was used to cryopreserve 28 strawberry genotypes (Fragaria x ananassa Duch.) with 40% to 75% regrowth. This collection is the beginning of a national germplasm storage system for Kazakhstan. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.115
113. Desiccation at ambient temperature and storage of mouse sperm in 3-O-methyl-Dglucose medium without freezing. Gloria Lee, Jie Liu *, Joel Lawitts, Mehmet Toner, John Biggers, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital and Shriners Hospitals for Children, Boston, MA, USA, Beth Israel Deaconess Medical Center, Boston, MA, USA, Harvard Medical School, Boston, MA, USA Previously we reported that mouse sperm can be dried at ambient temperature and stored in medium containing trehalose for up to 3 months above 0 °C. However, it was necessary to permeate the sperm plasma membrane with the enzyme, alpha-hemolysin, to allow trehalose entry. We now report that 3-O-methyl-D-glucose (3-OMG), a metabolically inactive sugar transported into the sperm, can confer
337
protection against desiccation. Recovered sperm heads after storage above 0 °C can fertilize ova capable of development into newborn pups. Mouse sperm in 100 mM 3-O-methyl-D-glucose medium were convectively dried with ultra-pure dry nitrogen gas for 6 min at 10 l per minute, stored at 4 °C and 22 °C for 1, 2, and 3 months in different relative humidity levels, and tested for functionality by intracytoplasmic sperm injection (ICSI) into mouse oocytes. Sorption jars of stable relative humidity (RH) levels were achieved in glass jars with varying salt to water ratios of NaBr (RH 61%), MgCl2 (RH 35%), and LiCl (RH 12%). The percentage of blastocysts that developed from sperm dried in 3-OMG medium and stored in 12% RH at 22 °C for 1, 2, and 3 months were 28.8%, 26.6% and 12.2%, respectively, while those at 4 °C for 1, 2, and 3 months were 62.6%, 53.4% and 39.6%, respectively. The rates that blastocysts developed using dried sperm samples stored in the higher humidity conditions of NaBr and MgCl2 sorption jars at either 4 °C or 22 °C were lower, and directly related to increasing humidity and storage duration as compared with those stored in LiCl sorption jars. There was very little change in moisture contents of the dried sperm samples stored in 12% relative humidity LiCl sorption jars for 3 months, with a gain of 0.02 gH2O/g anhydrous weight at 4 °C, and a loss of 0.05 gH2O/g anhydrous weight at 22 °C. Transfers of two-four cell embryos derived by ICSI from convectively dried mouse sperm in 3-OMG medium and stored for 3 months at 22 °C produced two live pups from five recipient mothers while those stored at 4 °C produced nine live pups from two recipients. The results showed that 3-O-methy-D-glucose offered significant protection of desiccated mouse spermatozoa in storage above 0 °C without the need for poration of cell membrane. Source of funding: This research was supported by NIH/NCRR 5 R24 RR018934. Conflicts of interest: None declared. doi:10.1016/j.cryobiol.2011.09.116
114. Genetic stability assessment of long-term cryopreservation using wasabi plant by morphological, biochemical and molecular analysis for three years. Toshikazu Matsumoto * a, Takashi Akihiro a, Shinya Maki b, Kouhei Mochida c, Masaru Kitagawa c, Daisuke Tanaka d, Takao Niino d, a Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-2059, Japan, b Niihama National College of Technology, Niihama, Ehime 792-8580, Japan, c Shimane Agricultural Technology Center, Izumo, Shimane 693-0035, Japan, d National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8518, Japan Wasabi, Japanese horseradish (fam. Cruciferae), is an important crop in Japan. The plant contains a pungent ingredient. Myrosinase is responsible for the development of the flavor and pungency of wasabi root. Since the seed of wasabi is sensitive to desiccation, the cultivars and strains are maintained as living material in the field of some repositories in Japan. Shoot tips of wasabi were cryopreserved using the following vitrification method. Excised shoot tips from in vitro grown plants were precultured on a modified MS medium (half strength KNO3 and NH4NO3; termed 1/2 MS) containing 0.3 M sucrose and 0.2% gellan gum for 16 h at 25 °C, and osmo-protected with 1/2 MS liquid medium supplemented with 2 M glycerol plus 0.4 M sucrose for 20 min at 25 °C. Then the shoot tips were dehydrated with PVS2 for 50 min at 0 °C, and immersed into liquid nitrogen for 2 h (LN2h) or for 1 h before storage in a 150 °C deep freezer for 10 years (LN10yr). The survival rates of cryopreserved shoot tips for 10 years (LN10yr), for 2 h (LN2hr), treated control plants (TC) and control plants (Cont) were checked, and genetic stability of plants regenerated above shoot tips was assessed for 3 years using morphological, biochemical and molecular analysis. The survival rates ranged between 93% and 100%, irrespective of the treatment. Regenerated plants were acclimated for 2 months, then grown in a greenhouse. Morphological analysis of plants revealed no significant difference among the plants tested. Biochemical components such as sinigrin (pungent component), sucrose, glucose, fructose and some amino acids were measured using high performance liquid chromatography. No significant difference in sucrose content was observed between Cont. For sinigrin, glucose, fructose, aspartic acid and glutamic acid contents statistical differences (by Tukey multiple range test) were observed between LN10yr or LN2hr and Cont. However, it was not clear whether these differences were caused by genetic variation or not. In this study, we confirmed that cryopreserved wasabi shoot tips maintained high survival rate after 10 years of storage in a 150 °C deep freezer. To determine the genetic relationship among each regenerated plant, we carried out random amplified polymorphic DNA (RAPD) PCR using 0-mer oligonucleotide primers set. Total DNA was extracted from each leaf of plant by PVP method with some modifications. The results show that genetic variations were not detected by RAPD. These results indicated that wasabi plants derived from shoot tips cryopreserved for 10 years might be genetically stable. Source of funding: This study was partly supported by a research grant from Genebank, National Institute of Agrobiological Sciences, Japan. Conflicts of interest: None declared. doi:10.1016/j.cryobiol.2011.09.117