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131. Cold acclimation influence on recovery of cryopreserved apple shoot tips and meristem cells ultrastructure
Abstracts / Cryobiology 53 (2006) 367–446 130. Controlled rate freezing of dedifferentiated plant cell lines—A mini-test system for quick evaluation of parameters. Elke HeineDobbernack, Stephanie Seufert, Heinz M. Schumacher, Plant Cell Cultures, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, D-38124 Braunschweig, Germany Controlled rate freezing is still the most widely applied method used to freeze dedifferentiated plant cell lines. The method offers a lot of practical advantages for this type of cell line, especially in terms of handling, such as the ease of producing a large number of frozen samples under standardized conditions in one experiment. However, controlled rate freezing comprises three different steps each with several parameters which in combination have to be optimized for each specific cell line. This is time consuming and requires a lot of cell material. Therefore, a minitest system was developed using the wells of a 24-multiwell-titerplate as culture vessels. The mini-test system is a reproducible procedure which allows testing several cryopreservation parameters at the same time under identical cultivation conditions, using only small amounts of cell material. The wells have a total volume of 3 mL. 1.5 mL of cell suspension can be reproducibly cultivated for up to 7 days and exposed to pre-culture procedures as well as different cryoprotectants. The amount of cell suspension in one well is used to fill one 2 mL cryovial. Reproducible inoculation with cells is carried out by placing the multiwell dishes on a balance and transferring a specific fresh weight of cells with a little spoon into each well. For several cell lines tested, an inoculum of approximately 0.4 mg cells adding 1.0 mL medium yielding 1.5 mL total volume of the suspension (30% PCV) has been used. If the PCV value exceeds 50% a reproducible cultivation becomes difficult. Exchange of the culture medium without removing the cells from the wells was done by soaking up the medium using sterilized tissue plugs obtained from cigarette filters. To test, for example, the effect of five different concentrations of a pre-culture osmotic in relation to controls needs only 13.2 g of cells. This calculation includes already four replicas for each concentration as well as the controls. During the method-evaluation phase a quick TTC assay is carried out directly in the multiwells. It could be demonstrated that the parameters which lead to successful cryopreservation in the mini-test system turned out to be successful also after a scale up of the procedure to normal Erlenmeyer flask level. This is important since a large number of cryovials is normally needed for the final cryostorage. Therefore, the mini-test system allows speeding up the development of a controlled rate freezing procedure adequate to a specific cell line. Nevertheless it may happen that in cases where the use of different conditions does not show a distinct optimum in the mini-test the conditions for the optimum results may be shifted. Using this mini-test method controlled rate freezing procedures for dedifferentiated cell lines from Catharanthus roseus, Lycopersion esculentum, Atropa belladonna, Cytisus canariense, and Nicotiana benthamiana were all worked out. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.131
131. Cold acclimation influence on recovery of cryopreserved apple shoot tips and meristem cells ultrastructure. Svetlana V. Kushnarenko a, Irina Y. Kovalchuk a, Natalia V. Romadanova a, Elena V. Rakhimova a, Barbara M. Reed b, a Genetics and Bioengineering, Laboratory of Germplasm Cryopreservation,
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Institute of Plant Physiology, 050040 Almaty, Kazakhstan; USDA-ARS National Clonal Germplasm Repository, 34447 Corvallis, OR, USA The effect of cold acclimation on cryopreservation of apple (Malus · domestica Borkh.) shoot tips and cell ultrastructure was studied. Two apple cultivars with different winter resistance were compared using two cryopreservation protocols: PVS2 vitrification and encapsulation-dehydration. In vitrogrown shoots were cold acclimated for up to 6 weeks at alternating temperatures (22 C with 8 h light and 1 C with 16 h darkness). Compared with nonacclimated controls, cold acclimation significantly improved post-cryopreservation regrowth of both cultivars with both protocols. The cultivars were distinguished by cold acclimation duration and percentages of cryopreserved shoot recovery. The best mean shoot regrowth of Grushovka Vernenskaya, which has weak winter resistance, was 54.1% for encapsulation–dehydration and 58.3% for PVS2 vitrification. The optimal cold acclimation duration for recovery of this genotype cryopreserved shoot tips was 1–3 weeks; longer acclimation resulted in decline of shoot tip regrowth. High winter resistant Voskhod was characterized by significantly better post-cryopreservation regrowth: 77.6% for encapsulation-dehydration and 74.2% for PVS2 vitrification. Recovery remained high for up to 5 weeks acclimation. Electron microscopic examination of cell ultrastructure of the apple cultivars was carried out for detailed elucidation of intracellular changes induced by cold acclimation. An intense starch accumulation inside plastids was noted after three weeks cold acclimation of Grushovka Vernenskaya. In the plastids of Voskhod, characterized with high cold hardiness, small starch grains and two types of plastoglobules were observed. The mean linear sizes of Grushovka Vernenskaya meristem cells increased slightly from 7.8 · 5.0 lm in nonacclimated controls to 10.9 · 6.9 lm in 3 weeks acclimated samples. On the contrary, the cell sizes of Voskhod acclimated meristems decreased from 18.2 · 9.7 lm in the controls to 12.7 · 7.8 lm. Two types of small vacuole in acclimated meristem cells of Grushovka Vernenskaya cultivar were noted: the first was electron-transparent with separate vesicles and sediments, and the second was composed of vacuoles with electron-opaque contents. There were parts and interweaving of membranes and occasional dark flecks in the vacuoles of Voskhod acclimated cells. Sometimes large globules with density and structure similar to lipids were observed. (Conflict of interest: None declared. Source of funding: ISTC Project.) b
doi:10.1016/j.cryobiol.2006.10.132
132. Cryopreservation of fruit and small fruit cultures in Kazakhstan. Irina Y. Kovalchuk a, Svetlana V. Kushnarenko a, Timur Turdiev a, Gulnar Karycheva a, Barbara M. Reed b, a Genetics and Bioengineering, Science and Education, Institute of Plant Physiology, 050040 Almaty, Kazakhstan; b USDA-ARS National Clonal Germplasm Repository, Agriculture, 34447 Corvallis, OR, USA The submountain area of Kazakhstan is a center of origin and diversity for apples, apricots and other fruit and small fruit cultures. For preservation of these important genetic resources, acknowledged as a source of resistance genes for breeding, we are developing cryopreservation techniques. Cryopreservation
131. Cold acclimation influence on recovery of cryopreserved apple shoot tips and meristem cells ultrastructure. Svetlana V. Kushnarenko a, Irina Y. Kovalchuk a, Natalia V. Romadanova a, Elena V. Rakhimova a, Barbara M. Reed b, a Genetics and Bioengineering, Laboratory of Germplasm Cryopreservation,
423
Institute of Plant Physiology, 050040 Almaty, Kazakhstan; USDA-ARS National Clonal Germplasm Repository, 34447 Corvallis, OR, USA The effect of cold acclimation on cryopreservation of apple (Malus · domestica Borkh.) shoot tips and cell ultrastructure was studied. Two apple cultivars with different winter resistance were compared using two cryopreservation protocols: PVS2 vitrification and encapsulation-dehydration. In vitrogrown shoots were cold acclimated for up to 6 weeks at alternating temperatures (22 C with 8 h light and 1 C with 16 h darkness). Compared with nonacclimated controls, cold acclimation significantly improved post-cryopreservation regrowth of both cultivars with both protocols. The cultivars were distinguished by cold acclimation duration and percentages of cryopreserved shoot recovery. The best mean shoot regrowth of Grushovka Vernenskaya, which has weak winter resistance, was 54.1% for encapsulation–dehydration and 58.3% for PVS2 vitrification. The optimal cold acclimation duration for recovery of this genotype cryopreserved shoot tips was 1–3 weeks; longer acclimation resulted in decline of shoot tip regrowth. High winter resistant Voskhod was characterized by significantly better post-cryopreservation regrowth: 77.6% for encapsulation-dehydration and 74.2% for PVS2 vitrification. Recovery remained high for up to 5 weeks acclimation. Electron microscopic examination of cell ultrastructure of the apple cultivars was carried out for detailed elucidation of intracellular changes induced by cold acclimation. An intense starch accumulation inside plastids was noted after three weeks cold acclimation of Grushovka Vernenskaya. In the plastids of Voskhod, characterized with high cold hardiness, small starch grains and two types of plastoglobules were observed. The mean linear sizes of Grushovka Vernenskaya meristem cells increased slightly from 7.8 · 5.0 lm in nonacclimated controls to 10.9 · 6.9 lm in 3 weeks acclimated samples. On the contrary, the cell sizes of Voskhod acclimated meristems decreased from 18.2 · 9.7 lm in the controls to 12.7 · 7.8 lm. Two types of small vacuole in acclimated meristem cells of Grushovka Vernenskaya cultivar were noted: the first was electron-transparent with separate vesicles and sediments, and the second was composed of vacuoles with electron-opaque contents. There were parts and interweaving of membranes and occasional dark flecks in the vacuoles of Voskhod acclimated cells. Sometimes large globules with density and structure similar to lipids were observed. (Conflict of interest: None declared. Source of funding: ISTC Project.) b
doi:10.1016/j.cryobiol.2006.10.132
132. Cryopreservation of fruit and small fruit cultures in Kazakhstan. Irina Y. Kovalchuk a, Svetlana V. Kushnarenko a, Timur Turdiev a, Gulnar Karycheva a, Barbara M. Reed b, a Genetics and Bioengineering, Science and Education, Institute of Plant Physiology, 050040 Almaty, Kazakhstan; b USDA-ARS National Clonal Germplasm Repository, Agriculture, 34447 Corvallis, OR, USA The submountain area of Kazakhstan is a center of origin and diversity for apples, apricots and other fruit and small fruit cultures. For preservation of these important genetic resources, acknowledged as a source of resistance genes for breeding, we are developing cryopreservation techniques. Cryopreservation