1 INTERNATIONAL SYMPOSIUM CRYOPRESERVATION IN HORTICULTURAL SPECIES

st 1 INTERNATIONAL SYMPOSIUM CRYOPRESERVATION IN HORTICULTURAL SPECIES Leuven, Belgium 5-8 April 2009 Programme and Abstracts 1st International S...
Author: Clement Lester
7 downloads 4 Views 2MB Size
st

1 INTERNATIONAL SYMPOSIUM CRYOPRESERVATION IN HORTICULTURAL SPECIES

Leuven, Belgium 5-8 April 2009

Programme and Abstracts

1st International Symposium on Cryopreservation in Horticultural Species

Contents

Contents Page Welcome

1

Organisers

3

Sponsors

4

Scientific committee

5

Organising committee

6

Programme

7

Abstracts oral presentations

13

Poster abstracts

61

List participants

143

1st International Symposium on Cryopreservation in Horticultural Species

Welcome

WELCOME The organizing committee is welcoming you to Leuven and the First International Symposium on Cryopreservation in Horticultural Species. This unique event is being co-organized by the Catholic University of Leuven, the International Society for Horticultural Science (ISHS) and COST Action 871, CRYOPLANET of the European Science Foundation. We are extremely happy to welcome 149 registered participants from 43 countries from all parts of the world (see world map), so we can truly called it an “international” meeting. Since this meeting is also linked to the EU COST Action 871 ‘Cryopreservation of Crop species in Europe’, most European countries are also well represented. The response to the announcement of the meeting was in fact so overwhelming that we even had to change the meeting location.

One of the main aims for organizing this meeting is “bringing together scientists that are dealing with more fundamental science as well as scientists that are developing cryopreservation protocols and plant germplasm curators”. The fact that participants from renown research centers and universities as well as institutes holding the world largest collections of plants (Bioversity International, CIP ,CIAT, IITA, SPC, the Vavilov Institute, IPK, USDA-ARS and many others) are present at this symposium proves that we succeeded already partially. Whether at the end of the meeting, we will be able to call it a “successful” meeting, will depend on you. Since we have 41 oral and 75 poster presentations, there will be surely no scarcity of discussion topics. Last but not least, I would like to acknowledge everyone; colleagues, staff (with special thanks to Marleen and Suzy) and students from the Laboratory for Tropical Crop Improvement for their time, energy and expertise that made that the organization of this meeting took place in an efficient and pleasant atmosphere. Enjoy the meeting! Bart

 

1

1st International Symposium on Cryopreservation in Horticultural Species

Organisers/Sponsors

Organisers

COST is an intergovernmental European framework for international cooperation between nationally funded research activities. COST creates scientific networks and enables scientists to collaborate in a wide spectrum of activities in research and technology.

COST Action 871: Cryopreservation of Crop Species in Europe

EFS provides the COST offices through an EC contract

Catholic University of Leuven www.kuleuven.be

3

1st International Symposium on Cryopreservation in Horticultural Species

Organisers/Sponsors

Sponsors

Biochemicals Plant cell and tissue culture - Plant Molecular biochemicals – Phytopathology Seed Health Testing - Antibiotics www.duchefa.com

Your power for health. Greiner Bio-One is a leading designer, manufacturer and supplier of high-quality consumables to biotechnology, diagnostic and pharmaceutical partners world wide. www.gbo.com/bioscience

“Your partner in Life Science” Supplier for duchefa www.labconsult.be

For family holiday or business trip www.novotel.com

Supplier Partnerships for Customer Solutions www.vwr.com

4

1st International Symposium on Cryopreservation in Horticultural Species

Committees

Scientific Committee Luis Mroginski, Instituto de Botánica del Nordeste (IBONE), Corrientes, Argentina Sarah Ashmore, Griffith University, Nathan Queensland, Australia Danny Geelen, Ghent University, Ghent, Belgium Sebastien Carpentier, Katholieke Universiteit Leuven, Leuven, Belgium Bart Panis, Katholieke Universiteit Leuven, Leuven, Belgium Rony Swennen, Katholieke Universiteit Leuven, Leuven, Belgium Qiaochun Wang, Northwest Agricultural and Forest University, Yangling, Shaanxi, P. R. China Milos Faltus, Institute of Crop Production, Prague, Czech Republic Hely Haggman, University of Oulu, Oulu, Finland Florent Engelmann, IRD (Institut de Recherche pour le Développement), Montpellier, France Joachim Keller, Genebank Department Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany Maurizio Lambardi, CNR, IVALSA/Istituto per la Valorizzazione del Legno e delle Specie Arboree, Florence, Italy Anuradha Agrawal, NBPGR (National Bureau for Plant Genetic resources), New Delhi, India Takao Niino, National Institute of Agrobiological Science, Japan Normah Noor, Universiti Kebangsaan Malaysia, Malaysia Dominique Dumet, IITA (International Institute for Tropical Agriculture), Ibadan, Nigeria David Tay, CIP (International Potato Centre), Lima, Peru M. Angeles Revilla Bahillo, University of Oviedo, Oviedo, Spain Paul Lynch, University of Derby, Derby, UK Barbara Reed, National Clonal Germplasm Repository, United States Department of Agriculture, Corvallis, USA Dave Ellis, National Center for Genetic Resources Preservation, Fort Collins, USA

5

1st International Symposium on Cryopreservation in Horticultural Species

Organising Committee Edwige Andre,- Catholic University of Leuven - Belgium Sebastien Carpentier, Catholic University of Leuven - Belgium Johan De Rue, Catholic University of Leuven - Belgium Danny Geelen, University of Gent- Belgium Bart Panis, Catholic University of Leuven - Belgium Bart Piette, Catholic University of Leuven - Belgium Marleen Stockmans, Catholic University of Leuven - Belgium Rony Swennen, Catholic University of Leuven - Belgium Ines Van den houwe, Catholic University of Leuven - Belgium Annelies Vertommen- Catholic University of Leuven - Belgium Suzy Voets, Catholic University of Leuven - Belgium Paul Lynch, University of Derby - UK

6

Committees

1st International Symposium on Cryopreservation in Horticultural Species

Programme

1st International Symposium on Cryopreservation in Horticultural Species Programme

Symposium venue

• Auditorium Oude Molen, Kasteelpark Arenberg 50, Heverlee • Coffee breaks and poster sessions: Arenberg Castle, Kasteelpark Arenberg 1, Heverlee

Sunday 5 April

17.00 – 19.00h: Registrations and welcome drink at Novotel Leuven

7

1st International Symposium on Cryopreservation in Horticultural Species

Programme

Monday 6 April- Day 1 ISHS symposium 08.00h.

Registrations (+ mounting of posters)

09.00h.

Welcome B. Goddeeris, Chairman Department Biosystems

09.10h.

Opening lecture E. Frison, Director General Bioversity International

Session 1: Fundamental aspect of cryopreservation and cryoprotection 09.40h.

Effect of low temperatures and cryoprotectants on plant plasma membranes M. Uemura (invited speaker)

10.15h.

Improved potato cryopreservation based on the identification of biochemical cell compounds linked to response to abiotic stress A. Panta

10.35h.

Ultrastructural changes in suspension culture cells of banana (Musa spp. AAA) during cryopreservation by vitrification Xu Chunxiang

10.55h.

Coffee break

11.25h.

Will proteomics contribute to a better understanding of cryopreservation survival? S. Carpentier

11.45h.

Cryopreservation and proteomic analysis of vanilla (V. planifolia A.) apices treated with osmoprotectants M.T. González-Arnao

12.05h.

The changes of total soluble proteins and Ca2+ in cryopreservation pollen of Prunus mume Y. Liu

12.25h.

Glass transition determination in Allium shoot tips after dehydration J. Zamecnik

12.45h.

Lunch (Alma III) + poster session

Session 2: Genetic stability and traceability 14.30h.

Epigenetic changes and somaclonal variation in tissue culture-propagated plants R. Smulders (invited speaker)

15.05h.

Cryobanking of banana (Musa sp.) germplasm in India: Evaluation of agronomic and molecular traits of cryopreserved plants A. Agrawal

15.25h.

Gene expression in response to cryoprotectant and liquid nitrogen exposure in Arabidopsis shoot tips G.M. Volk

8

1st International Symposium on Cryopreservation in Horticultural Species

Programme

15.45h.

Coffee break

16.15h.

Survival of cryogenically-stored dormant apple buds: A 20 year assessment C. Walters

16.35h.

Effect of cryoprotectant exposure on post-thaw recovery, growth and genetic stability of Pinus nigra Arn. embryogenic tissues I. Matusikova

16.55h.

Assessment of Gentiana cruciata and G. tibetica regenerants derived from cryopreserved cell suspensions A. Mikula

17.15h.

DNA sequences of variable bands found after cryopreservation of chrysanthemum apices E. González Benito

17.35h.

END

19.30h.

Guided visit in Leuven

Tuesday 7 April - Day 2 ISHS symposium

Session 3: Technology aspects of cryopreservation 09.00h.

Choosing and Applying Cryopreservation Protocols to New Species or Tissues B. Reed (invited speaker)

09.35h.

Cryopreservation of ancient apple cultivars of Veneto: a comparison between PVS2-vitrification and dormant-bud techniques M. Lambardi

09.55h.

Cryopreservation by vitrification of embryogenic callus of wild crocus (Crocus hyemalis and Crocus moabiticus) R. Shibli

10.15h.

Droplet vitrification of shoot tips of fraser photinia (Photinia x fraseri Dress.) Y. Ozden Tokatli

10.35h.

Coffee break

9

1st International Symposium on Cryopreservation in Horticultural Species

Programme

11.05h.

Cryo-conservation of genetic diversity of recalcitrant-seeded species via zygotic embryonic axes: successes and problems N. Pammenter

11.25h.

Cryopreservation in the Vitaceae: seed cryopreservation is effective for wild species accessions Ph. Chatelet

11.45h.

In vitro propagation and cryopreservation of Stevia rebaudian a medicinal plants using vitrification method M. Shatnawi

12.05h.

Lunch (Alma III)

Session 4: Impact and applications of cryopreservation in plants 13.30h.

Cryotherapy of shoot tips: an efficient method for eradication of plant pathogens Q. Wang (invited speaker)

14.05h.

Cryobanking cassava germplasm at IITA D. Dumet

14.25h.

The Potato Cryo-Bank at the International Potato Center D. Tay

14.45h.

Cryopreservation of garlic germplasm collections using the droplet-vitrification technique H-H. Kim

15.05h.

Coffee break + poster session

16.00h.

Assessing variability between accessions, cryovials and over time of cryopreserved shoot tips M. Jenderek

16.20h.

Cassava cryopreservation activities at the International Center for Tropical Agriculture R. Escobar

16.40h.

Cryopreservation of native Australian fruits and crop wild relatives S. Ashmore

17.00h.

Business meeting ISHS

17.15h.

End

19.00h.

Conference dinner at Novotel

10

1st International Symposium on Cryopreservation in Horticultural Species

Programme

Wednesday 8 April - Day 3 ISHS symposium combined with COST Action 871 (CRYOPLANET) WG2 meeting

Session 5: Technology aspects of cryopreservation (CRYOPLANET) 09.00h.

Alliaceae in cryopreservation, achievements and constraints J. Keller (invited speaker)

09.35h.

Cryopreservation of Fraxinus spp. embryogenic cultures by slow cooling and vitrification techniques A. Ozudogru

09.55h.

Development of cryopreservation procedures for dwarf irises (Iris spp.) S. Jevremovic

10.15h.

Cryopreservation of Quercus robur plumules M. Michalak

10.35h.

Coffee break

11.05h.

Cryopreservation of dormant buds from temperate fruit crops - optimising working collection resources T. Toldam-Andersen

11.25h.

Cryopreservation of hairy root cultures of Maesa lanceolata and Medicago truncatula E. Lambert

11.45h.

Cryopreservation of date palm highly regenerable tissues L. Fki (invited speaker)

12.05h.

Encapsulation-dehydration: past, present and future F. Engelmann

12.25h.

Lunch (Alma III) + poster session

11

1st International Symposium on Cryopreservation in Horticultural Species

Programme

Session 6: Impact and applications of cryopreservation in plants (CRYOPLANET) 14.30h.

Current development and application of plant cryopreservation in Latin America and Caribbean M.T. González-Arnao (invited speaker)

15.05h.

Conservation strategy of genetic resources in strawberry in Germany M. Höfer

15.25h.

Immulocalisation of 2 viruses (PFBV and PLPV) in Pelargonium apices and study of their potential eradication by cryopreservation A. Grapin

15.45h.

Coffee break

16.15h.

Efficacy of cryotherapy for control of Cocoa Swollen Shoot Virus in cocoa somatic embryos A. Wetten

16.35h.

Droplet vitrification: the first generic cryopreservation protocol for organized plant tissues? B. Panis

16.55h.

Presentation of ISHS by the Executive Director J. Van Assche

17.05h.

Closing address B. Panis

17.15h.

End

12

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Abstracts

Oral Presentations

The abstracts are arranged according to the programme

13

1st International Symposium on Cryopreservation in Horticultural Species

14

Oral abstracts

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Effect of low temperatures and cryoprotectants on plant plasma membranes M. Uemura, A. Minami and Y. Kawamura Cryobiofrontier Research Center, Iwate University, Morioka 020-8550, Japan

The plasma membrane has been considered to be one of the most important determinants for survival at low temperatures. Failure of the maintenance of the plasma membrane leads to the imbalance of cytoplasmic components, the intrusion of extracellular ice crystals, and many serious injuries in plant cells. Thus, plants must increase the cryostability of the plasma membrane to withstand various stresses imposed by freezing and accelerate the recovery process after thawing. Under natural (as well as laboratory) conditions, many temperate plants increase their freezing tolerance when subjected to low but non-freezing temperatures, which is referred to as cold acclimation. Our group has been trying to understand how the plasma membrane dynamically responds to cold acclimation so that plants can survive under severe freezing conditions. We have reported that both lipid and protein compositions of the plasma membrane dynamically alter during cold acclimation, which ultimately results in an increase in the cryostability of the plasma membrane. High survival of plant cells after cryopreservation likely requires maintaining the intactness of the plasma membrane, too. During the process of cryopreservation, the plasma membrane is subjected to severe, multiple stresses due to various treatments which are prerequisite for survival. To help plant cells alive, cryoprotectants are often included in the system. There are many studies demonstrating that some of the cryoprotectants increase stability or intactness of the plasma membrane through their direct interactions or alterations of water distribution inside/outside cells. In either case, plant cells must keep their plasma membrane active and functional for survival. Based on recent studies on the role of the plasma membrane in cell survival at low temperatures obtained in our laboratory, we will discuss how function of the plasma membrane is associated with survival of plant cells under low temperature conditions. Full name of presenting author: Matsuo Uemura E-mail: [email protected]

15

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Improved potato cryopreservation based on the identification of biochemical cell compounds linked to response to abiotic stress A. Panta, B. Panis1, D. Sanchez, P. Canepa, C. Ynouye, J. Geuns2, R. Swennen1, W. Roca, and D. Tay International Potato Center, Genetic Resources Conservation and Characterization Division, Apartado 1558, Lima 12, Peru 1 Laboratory of Tropical Crop improvement, KULeuven, Kasteelpark Arenberg 13, 3001 Leuven, Belgium 2 Department of Biology, KULeuven, Kasteelpark Arenberg 31, 3001 Leuven, Belgium

The world largest potato collection is maintained in vitro at the International Potato Center (CIP). It mainly comprises Andean potatoes (4,601 acc) which are very diverse and hold valuable nutritive characteristics and adaptability to severe environment conditions. CIP’s current strategy is improving and establishing cryoprocedures for assuring the long term conservation of these genetic resources. Within this research, we have linked the response of a potato cultivar to abiotic stresses with its cryo-ability since plant tissues processed for cryopreservation are submitted to stress conditions similar to drought, salinity and frost. Six genotypes belonging to cultivated species (Solanum tuberosum spp., S. tuberosum subsp andigena, S. x juzepczukii and S. x ajanhuiri), and two genotypes from a wild frost tolerant species (S. commersonii) were cryopreserved using the droplet PVS2 vitrification method following temperature (6°C) and sugar (0.3M) hardening treatments. From each treatment, apical tips were sampled prior to cryopreservation, and analyzed for phospholipid, glycolipid, aromatic amine and polyamine composition and content. Later on, treatments were tested that involve the addition of specific compounds found to be linked with cryopreservation ability to the precultured medium. Results revealed that frost resistant genotypes have significant higher regeneration rates after cryopreservation and that the linoleic acid content is positively correlated with tolerance towards cryopreservation. Also it was demonstrated that the addition of putrescine to the pre-culture medium can improve the response of cryopreservation of potato accessions that originally show very low recovery rate. Full name of presenting author: Ana Panta E-mail: [email protected]

16

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Ultrastructural Changes in Suspension Culture Cells of Banana (Musa spp. AAA) during cryopreservation by vitrification C. Xu1, Y. Li1, B. Panis2, X. Deng1 and H. Chen1 1

Laboratory of Tropical and Subtropical Fruit Research, South China Agricultural University, Guangzhou 510642, China; 2 Laboratory of Tropical Crop improvement, KULeuven, Kasteelpark Arenberg 13, 3001 Leuven, Belgium

Using transmission electron microscopy, ultrastructural changes taking place during the application of a vitrification protocol of embryogenic cells of ‘Beida Aijiao’ were studied. The results showed that the control cells contained a lot of organelles, a regular nucleolus envelope and intact plasma membranes. After treatment with 25% of PVS2, some changes could be observed, such as smaller vacuoles, more phenolic compounds, swollen organelles and appearance of many lipid bodies. Also multivesicular membranous structures with vesicles appeared between the plasma membrane and the cell wall. Moreover, the phenomenon of plasmolysis remained limited. Only after dehydration with 100% PVS2 plasmolysis became more severe. Nucleus envelopes in these cells were malformed. Though the cytoplasm and the nucleus became more electron-dense and a lot of heterchromatin appeared, the plasma membrane was still intact. The ultrastructure of cells after freezing, thawing and unloading was similar to that of those cells after dehydration. After 1-2 weeks’ of post-thaw recovery, the ultrastructure of surviving cells was similar to that of the control cells. Full name of presenting author: Chunxiang Xu E-mail: [email protected]

17

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

WILL PROTEOMICS CONTRIBUTE TO A BETTER UNDERSTANDING OF CRYOPRESERVATION SURVIVAL? S.C. Carpentier, A. Vertommen, R. Swennen and B. Panis Faculty of Bioscience Engineering, Division of Crop Biotechnics, KULeuven, Kasteelpark Arenberg 13, 3001 Leuven, Belgium

We (K.U.Leuven) host the International Musa Bioversity collection under in vitro as well as cryopreserved conditions. We have shown that an osmotic acclimation phase on 0.4M sucrose medium is essential for the regeneration of cryopreserved meristems (Panis et al. 1996). Using a two-dimensional gel electrophoresis protocol developed for small amounts of tissue and mass spectrometric based cross species polypeptide identification (Carpentier et al. 2005), we have investigated the influence of this sucrose mediated osmotic stress acclimation at the protein level. For this purpose multiple genotypes, ranging from a dehydration tolerant variety to a dehydration sensitive variety, were compared. Our results suggested that the maintenance of an osmoprotective intracellular sucrose concentration, the enhanced expression of particular genes of the energy-conserving glycolysis and the conservation of the cell wall integrity may be essential to maintain homeostasis and to survive dehydration. We observed a genotype specific expression of certain proteins (isoforms) involved in energy metabolism and abscisic-acid- and salt-stressresponsive proteins (Carpentier et al. 2007). In order to distinguish the different aspects of the sucrose mediated acclimation, we included two sugar alcohols and PEG in the experimental design at the same osmotic pressure. Our results suggest that the penetration of the osmotic molecule to act as compatible solute and the metabolizing of the osmotic molecule are both crucial. To unravel the dynamics of acclimation process, we focused on the most tolerant variety (i.e. Cachaco) and have investigated the proteome over time using the 2D-DIGE proteomic approach. This enabled us to study for the first time the different aspects of osmotic stress acclimation of meristems by characterizing hundreds of proteins over time in a kinetic proteome investigation. Twenty eight proteins were correlated to general osmotic stress and fifty nine proteins were exclusively correlated to the sucrose treatment. Will proteomics contribute to a better understanding of cryopreservation survival? Yes, we do believe that proteomics –in combination with other complementary approaches- will contribute towards a better understanding and will lead to more efficient cryopreservation protocols. Full name of presenting author: Sebastien Carpentier E-mail: [email protected]

18

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cryopreservation and proteomic analysis of vanilla (V. planifolia A.) apices treated with osmoprotectants M.T. González-Arnao1, S.E. Valdés-Rodríguez2, B. Durán-Sánchez1, B. JiménezFrancisco1, A. Guerrero2 and C.E. Lázaro-Vallejo1 1

Laboratory of Biotechnology and Cryobiology, Universidad Veracruzana, Prolongación Oriente 6, No. 1009, CP 94340, Orizaba, Veracruz, México. 2 Laboratory of Molecular Biochemistry of Proteins, CINVESTAV. Campus Guanajuato. Km 9.6, Libramiento Norte, CP 36821, Irapuato, Gto, México.

Apices isolated from in vitro plantlets of Vanilla planifolia Andrews were subjected to cryopreservation using a droplet-vitrification approach. Apices were precultured on solid MS medium supplemented with 0.3M sucrose for 1 day, then loaded in different solutions, including 2M glycerol with 0.4M sucrose, 0.4M trehalose or both sugars at 0.2M. After loading, samples were exposed to the vitrification solution PVS3 for 0, 30 or 60 min at room temperature, and frozen directly in liquid nitrogen in 0.15 µl droplets of PVS3 placed on aluminium foil strips. For rewarming, the foil strips were immersed in liquid culture medium supplemented with 1.2M sucrose at room temperature. Survival (30%) and regeneration (10%) of new multiple shoots were achieved with 1 day pregrowth of apices on solid medium with 0.3M sucrose, loading with 0.4M sucrose + 2M glycerol solution for 20-30 min and exposure to PVS3 for 30 min at room temperature. This is the first successful report of cryopreservation of vanilla apices. For proteomic analysis, it was firstly required to optimize the protein extraction method from vanilla apices in order to reproducibly capture the most possible of proteins from samples either subjected to 1 day pregrowth on solid medium with 0.3M sucrose (CRTL) or from samples precultured for 1 day with 0.3 M sucrose, loaded and exposed to PVS3 up to 30 min (PVS). Using the phenol extraction method/ammonium acetate precipitation, proteins were separated by twodimensional gel electrophoresis, stained gels scanned and then analysed with Image-Master 2D platinum. Image analysis consistently reproduced 206 spots of proteins, from which 15 showed quantitative differential changes (13 increased the abundance of their expressions using the % volume criterion, and 2 decreased) in PVS samples. Qualitative changes were also detected in 2 proteins (1 induced and 1 repressed) of the same PVS samples. Full name of presenting author: María Teresa González Arnao E-mail: [email protected] or [email protected]

19

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

The Changes of Total Soluble Proteins and Ca2+ in Cryopreservation Pollen of Prunus mume Y.L. Zhang1,2, 3, B.L. Li 1,2, H. Wang1 and Y. Liu 1,2 1

College of Landscape Architecture Beijing Forestry University,Beijing, 100083, China National Flower engineering technology center 3 Institute of Plant Physiology & Ecology, Shanghai Institute for Biological Science, Chinese Academy of Science, Shanghai , 200231, China. 2

The Mei (Prunus mume Sieb. et Zucc.) is one of the most widely used landscape plants and important germplasm resources in China. From 2003 to 2007, the pollen from 51 Mei cultivars were cryopreserved and a pollen bank was constructed. To further study the pollen germination changes of cryopreserved pollen, the total soluble proteins and free Ca2+ were detected. Results indicated:1.Two-dimensional gel electrophoresis map of the total soluble proteins from cryopreserved pollen and fresh pollen are different in the 3 cultivars that represented higher, lower or unchanged germination rate after cryopreservation, respectively. There are a few same changing protein expression dots from cryopreserved pollen and also some are different among the cultivars. Result indicated that the different pollen germination rates after cryopreservation perhaps have some relationship with the protein change. 2.The relative fluorescence intensity of Ca2+ in cryopreserved pollen detected by Flow Cytometry was higher than in fresh pollen. After different periods of cryopreservation, the changing trends of Ca2+ in pollen were different with different cultivars 3. The free Ca2+ in the culture media during different periods of pollen germination were detected by ICP-AES. The content of free Ca2+ in the culture media increased, indicating that the free Ca2+ in pollen moved into the culture media and promoted pollen germination. Meanwhile the dynamic changes of Ca2+ may affect the level of pollen germination. Full name of presenting author: Yan Liu E-mail: [email protected]

20

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Glass transition determination in Allium shoot tips after dehydration J. Zamecnik 1, M. Faltus1 and R. Kotkova2 1

Crop Research Institute, Drnovska 507, CZ 16106 Prague, The Czech Republic Czech University of Life Sciences Prague, Kamycka 129, CZ 16521 Prague, The Czech Republic

2

The “Glassy state” is the goal status of cryopreservation methods named vitrification. A low water content leads to a lower ice content potentially dangerous for plant cells and to increasing temperatures of glass transition, supposing that changes of water status is not limiting for plants regeneration. Plant Vitrification Solutions (PVS) that are numbered according to the specific mixture of basic cryoprotectants and concentration are usually used for osmotic dehydration. A second method for desiccation is using a flow box with a defined air flow rate, temperature and humidity or alternatively desiccation over various saturated salt solutions. The experimental determination of the glassy state in plants is complicated by the overlap of endothermic reaction with glass transition. Ice crystallization as a first order reaction has a discontinuous change in heat capacity in contradiction to glass transition which is characterized only by heat capacity change. The main problem is to distinguish the endothermic reaction from the glass transition during the measurement of thermal events. Three methods for glass transition determination and ice melting in plant tissue were compared; the standard DSC method, the Temperature Modulated Differential Scanning Calorimeter (TMDSC) and the Quasi-isothermal Temperature Modulated Differential Scanning Calorimeter (QITMDSC). The results can be summarized as follows (i) both the TMDSC and the QITMDSC methods based on temperature modulation gave clearer and more unambiguous results and (ii) from these methods the QITMDSC gave a better results for distinguishing between heat capacity for endothermic ice melting and glass transition because the heat capacity is measured at quasi-isothermal temperature while thermal response of plant sample is under temperature modulation. Knowledge of glass transition temperatures is useful not only for improving methods involving a glass phase but also is essential for synchronous information for the long term storage of shoot tips. Full name of presenting author: Jiri Zamecnik E-mail: [email protected]

21

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Epigenetic changes and somaclonal variation in tissue culturepropagated plants M.J.M. Smulders and G.J. de Klerk Plant Research International, Wageningen UR Plant Breeding, P.O. Box 16, NL-6700 AA Wageningen, The Netherlands

In tissue culture abnormal plants may be produced. Aberrations occur especially in plants that have been generated after an intermediate callus phase. Multiplication from pre-existing meristems appears to be relatively ‘safe’. The frequency of aberrations depends on the genotype, the tissue from which the adventitious plantlets have been regenerated, and tissue culture conditions like the number of subcultures, the type of intermediate callus etc. Two classes of off-types are usually distinguished: genetic and epigenetic ones. Genetic changes, also referred to as somaclonal variation, include polyploidy, aneuploidy, (point) mutations, and new insertions of (retro)transposons. Genetic changes behave as Mendelian traits in crosses. Epigenetic changes are brought about by alterations in DNA methylation, by changes in histone modifications, or by a combination of these epigenetic mechanisms that both may influence transcription. They are in theory temporary (plants may ‘revert’ to normal phenotype), but changes are nevertheless sometimes transferred to the progeny. Typical for epigenetic changes is also that in a population of regenerated plants the same aberration often occurs at a high frequency (whereas genetic changes occur, in principle, at random). The distinction between genetic and epigenetic changes is, however, an oversimplification. We will discuss recent studies that have applied molecular genetic and genomic tools. These studies have uncovered complex epigenetic modifications in model systems, including cell suspension cultures, protoplasts, callus, and regenerated plants, with transcriptional silencing of genes and many classes of transposable elements. However, some become activated. Full name of presenting author: René Smulders E-mail: [email protected]

22

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cryobanking of banana (Musa sp.) germplasm in India: Evaluation of agronomic and molecular traits of cryopreserved plants A. Agrawal1, S. Uma2, R.K. Tyagi1, M.S. Saraswathi2, R. Goswami1 and P. Durai2 1

National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi-110 012, India 2 National Research Centre for Banana (NRCB), Thayanur Post, Thogamalai Road, Tiruchirapalli-620102, Tamil Nadu, India

Bananas and plantains (Musa spp.) are crops of immense horticultural importance grown in more than 130 countries around the tropical and sub-tropical parts of the world. India is the largest producer (19 MT) of the fruit and some species are believed to have Indian origin. Immense diversity exists in the Indian subcontinent. Conservation of banana diversity is imperative as anthropogenic activities coupled with threats of climate change are causing fast depletion. Cryopreservation, using in vitro derived shoot meristems, is a safe long-term conservation method for Musa germplasm. The aim of the present study was to compare the performance of cryopreserved banana germplasm for agronomic and molecular traits (using simple sequence repeats; SSR) to that of its parental type. Banana cv. Sommrani Monthan (AAB, Monthan subgroup, IC250538) was collected from the state of Bihar during 1994 and raised in field genebank at NRCB and in vitro shoot tip cultures were established at NBPGR in 2003 and conserved till date. Experiments were initiated for its cryopreservation in 2004-05 using vitrification technique on proliferating meristems (Panis et al., 2002, Cryoletters, 23, 375–384.; Agrawal et al., 2008 Curr. Sci. 94, 1125-1128.). The cryopreserved meristems were stored in liquid nitrogen (LN) for 1 month and subsequently thawed and regenerated into whole plants. Genetic stability of original mother plants maintained in the field genebank (control 1), in vitro-conserved plants (control 2), PVS2-treated plants (control 3) and cryopreserved plants was assessed using 12 agronomic traits and 9 SSR primers. A total of 20 plants per treatment were used. Shoot tip cultures were conserved on MS + BAP (10 µM) + IAA (1 µM) + 3% sucrose + 0.8% agar medium for 12 months with 1.2 shoots/culture. In the in vitro-derived shoot meristem cultures processed for cryopreservation, the mean shoot regeneration in PVS2-treated (without LN exposure) was 56.5%. In the LN-treated explants, 41% shoot regeneration was obtained in cultures treated with PVS2 for 120 min, which could be recovered successfully into complete plantlets. Results indicated that no significant differences existed for the major growth and yield parameters analyzed in all except one plant. In one of the plants raised from cryopreserved meristems, the fruit colour was found to be green instead of ash green, as in the control plants. Among the nine primer pairs tested, seven primers pairs namely AGMI-33,34, AGMI-35,36, AGMI-67,68, AGMI-93,94, AGMI-95,96, AGMI-129,130 and MbSSR 1-149 amplified products resulting in discrete repeatable amplicons, wherein a total of 13 alleles were identified with a mean of 1.86 alleles per primer. All the alleles were found to be monomorphic. On the basis of the tested SSR primer pairs, the morphological green-fruited variant also did not show any variation as compared with control 1, 2 and 3 plants. The study indicates that 100% of the in vitroconserved and 95% of the cryopreserved Musa germplasm was genetically stable. Further studies are in progress to determine the cause of green mutant observed under this study and markers associated with fruit ashyness. Full name of presenting author: Anuradha Agrawal E-mail: [email protected] or [email protected]

23

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Gene expression in response to cryoprotectant and liquid nitrogen exposure in Arabidopsis shoot tips G.M. Volk1, A. Henk1and C. Basu2 1

USDA-ARS-National Center for Genetic Resources Preservation, 1111 S. Mason St., Ft. Collins, CO 80521 U.S.A. 2 School of Biological Sciences, University of Northern Colorado, Greeley, CO 80639 U.S.A.

Arabidopsis thaliana serves as an ideal model system to study cryopreservation at the molecular level. We have developed reliable cryopreservation methods for Arabidopsis shoot tips using Plant Vitrification Solution 2, Plant Vitrification Solution 3 and polyethylene glycol-glucose-dimethylsulfoxide cryoprotectants. We have made use of the fully sequenced Arabidopsis genome and readily available microarray slides with 26,000 genes to compare the gene expression patterns among dissected control shoot tips, cryoprotectant-treated shoot tips, liquid nitrogen-treated shoot tips, and post-exposure recovering shoot tips. We have identified key gene families that show changes in gene expression in fully replicated experiments and have confirmed gene expression patterns using real-time PCR. Key gene families encode proteins involved in lipid transport, dehydration responses, seed maturation, and detoxification. Some genes are specifically upregulated in response to cryoprotectant treatment while others have a more general response expressed during shoot tip recovery. Our results reveal that cryoprotectant treatments induce gene expression and critical pathways may include those involved in lipid transport and osmoregulation. Full name of presenting author: Gayle M. Volk Email: [email protected]

24

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Survival of cryogenically-stored dormant apple buds: A 20 year assessment C. Walters1, G.M. Volk1, L.E. Towill1 and Ph.L. Forsline2 1

USDA-ARS National Center for Genetic Resources Preservation, Fort Collins, CO USDA- ARS, Plant Genetic Resources Unit Geneva, NY, USA

2

Cryobiologists assume that the extreme low temperatures of liquid nitrogen stop chemical and physical reactions that lead to sample aging and loss of viability. This assumption, based on extrapolations of temperature – reaction kinetic relationships, is not completely supported by accumulating evidence that dried seeds can deteriorate during cryogenic storage. After 30 years of cryogenic storage, seeds of some species exhibited quantitatively lower viability and vigor. In this paper, we ask whether loss of viability also occurs in cryogenically stored dormant buds of apple. The same thermodynamic models used for seeds apply to apple buds, suggesting the possibility that decline may occur within decades rather than centuries. Evaluating the empirical evidence of long term survival of apple buds is difficult because of the numerous sources of variation that confound viability assessments. Survival of buds dried to 25-30% water (fresh weight basis), slowly cooled to -30oC and then plunged into liquid nitrogen varies with genetic line, month of harvest and year of harvest. Viability assessments use budding techniques, which rely on consistency and skill of the grafter. Many of these variables were controlled in a 15 year long experiment in which the same eight genotypes were harvested yearly, cryogenically stored, and periodically evaluated for survival. For the most part, apple buds survived cryostorage. However, there are some indications of change in survival that may arise from uncontrolled experimental variation or deterioration during storage. Cryogenic storage of dormant buds is a highly efficient way to back up orchard collections, but assumptions about the power of cryogenics to prevent change in biological material must still be viewed with some caution. Full name of presenting author: Christina Walters E-mail: [email protected]

25

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Effect of cryoprotectant exposure on post-thaw recovery, growth and genetic stability of Pinus nigra Arn. embryogenic tissues. T. Salaj1, I. Matušíková1 and B. Piršelová2 1

Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, 950 07 Nitra, Slovak Republic 2 Faculty of Natural Sciences, Department of Botany and Genetics, Costantine the Philosopher University, Nitra, Slovak Republic

Embryogenic tissues of a conifer Pinus nigra Arn. were cryopreserved by slowfreezing method. The effect of cryoprotective compounds (maltose, sucrose, sorbitol in 0.5M combined with 7.5% DMSO) was tested. The tissues were pretreated with maltose, sucrose and sorbitol for 24 hours, and after gradually in 3 steps 15% DMSO was added to reach the final concentration 7.5%. The suspension in cryovials was plunged into liquid nitrogen for 1 hour. After thawing the following characteristics of tissues were evaluated: tissue recovery (6 weeks after thawing), post-thaw growth and genetic stability (2-3 months after thawing). The preliminary results show tissue regeneration after cryopreservation was cell line dependent. For cell line E223 each treatment resulted in high survival (100%). For some tissues (cell lines E227, E247) the maltose treatment was excellent giving 100% survival over the sucrose or sobitol treatments. The cell line E231 showed no survival. In general, out of 7 cell lines tested, 6 of them regenerated after cryopreservation, although in dependence on cell line and treatments. Two or three months after thawing, the fresh and dry weights of regenerated tissues were evaluated. No statistical differences in given growth parameters were found between the cryopreserved and control tissues (non-pretreated and non-frozen). Thirteen different RAPD primers were used to study the genetic stability of cultures following cryopreservation. We observed genetic variation in DMSO-treated but nonfrozen samples of E224 and E247. In addition, sorbitol treatment appeared to cause changes in genetic fidelity of E224 embryogenic tissues. Full name of presenting author: Ildikó Matušíková E-mail: [email protected]

26

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Assessment of Gentiana cruciata and G. tibetica regenerants derived from cryopreserved cell suspensions A. Mikuła and J.J. Rybczyński Botanical Garden – Center for Biological Diversity Conservation, Polish Academy of Sciences, ul. Prawdziwka 2, 02-973 Warsaw, Poland

The aim of this presentation is to show the effect of the tissue culture strategy and cryopreservation procedure to achieve “true – to - type” regenerants of Gentiana cruciata and G. tibetica. Initially, encapsulation appeared to be a reliable procedure for preserving viability and embryogenic competence of both cell suspensions. However, all steps of the cryopreservation resulted in lack of effect of ultralow temperature treatment on characteristics of the cell suspensions. Their re-growth after thawing and number of somatic embryos produced and quality of regenerants confirmed uniformity with control on morphogenetic level. Flow cytometry showed similarity of genome size of the PEM and regenerants to the control. 2C ranged from 8.69pg and 9.0pg for G. cruciata and G. tibetica, respectively. Molecular analysis of DNA with the help of restriction enzymes showed that the methylation changes were more narrow in post freezing regenerants than those derived from non-treated cell suspension in case of both studied species. Above results were obtained using the following materials, liquid or agar media: MM (maintain medium - MS (1962) medium supplemented with 1.0 mg/l Dicamba + 0.1 mg/l NAA + 2.0 mg/l BAP + 80 mg/l AS), RM (regeneration medium - MS supplemented with 0.5 mg/l GA3 + 1.0 mg/l Kin + 80.0 mg/l AS), CM (conversion medium - hormone-free medium 0.5MS medium). The level of cell suspension water content during experiments was controlled and assessed. For cell suspension encapsulation in 3% alginate was employed. Full name of presenting author: Anna Mikula E-mail: [email protected]

27

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

DNA sequences of variable bands found after cryopreservation of chrysanthemum apices C. Martín and M.E. González-Benito Departamento de Biología Vegetal, Universidad Politécnica de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain

Chrysanthemum (Dendranthema grandiflora Tzvelev) is one of the most important crops in the world for cut flowers and pot plants. Due to its economical interest, many new technologies and breeding programmes have been applied to this crop (i.e.: Teixeira da Silva JA (2003) Biotechnology Advances 21: 715-766). Although cryopreservation was supposed to be a guarantee for the genetic stability in comparison with other in vitro long term storage procedures, genetic variation in cryopreserved material has been found (Harding K (2004) CryoLetters 25: 3-22.). Genetic instability of cryopreserved chrysanthemum apices using two different methods has been detected (Martín C & González-Benito ME (2005) Cryobiology 51: 281-289). In order to evaluate the step of the cryopreservation protocol associated to the variation, we have studied the stability along the encapsulation-dehydration cryopreservation process (pre-culture, encapsulation, dehydration, freeze-treatment and recovery) using RAPDs markers. RAPDs markers were obtained from the amplification of the total genomic DNA isolated from samples (shoots) regenerated after the different steps. Differences in the band pattern were detected not only in cryopreserved samples, but also in the different previous steps. The sequences of the variable bands were analysed through MegAlign (DNAStar) and BLAST/FASTA programmes. Full name of presenting author: M.Elena González-Benito E-mail: [email protected] or [email protected]

28

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Choosing and Applying Cryopreservation Protocols to New Species or Tissues B.M. Reed USDA-ARS National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97333-2521 USA

Some have proposed that it is necessary to develop new cryopreservation protocols for each new plant or tissue. We have found that standard protocols can be applied to many plants with few if any changes. Screening of groups of plants in a genus show that many protocols are easily applied to large groups of plants. The protocol to use can be chosen from those developed for similar plants or several standard protocols can be tested. We often compare controlled rate cooling, PVS2 vitrification, and encapsulation-dehydration techniques. Comparison of these techniques on diverse germplasm of pear, grass, blueberry and mint provided clear choices of the best protocol to use for storing large collections. Once a protocol is chosen, some critical points can be adjusted to improve the plant response as needed. Each of these methods has some basic steps that can be modified to make them effective for many types of plants. Finding a protocol for use with a new plant species may be as simple as testing available protocols for similar plants. Adjustments at critical points allow relatively quick adaptation of standard protocols to new groups of plants. Preliminary knowledge of the plant species can also provide tools for choosing a technique and certain parameters may help predict success or failure for a particular group of plants. These parameters may also provide clues for future protocol improvements. Full name of presenting author: Barbara M. Reed E-mail: [email protected]

29

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cryopreservation of ancient apple cultivars of Veneto: a comparison between PVS2-vitrification and dormant-bud techniques M. Lambardi1, A. Previati2, C. Benelli1, F. Da Re2, A. De Carlo1 and D. Ellis3 1

IVALSA/Istituto per la Valorizzazione del Legno e delle Specie Arboree, National Research Council (CNR), 50019 Sesto Fiorentino (Florence), Italy. 2 Centro Sperimentale Ortofloricolo “Po di Tramontana”, Veneto Agricoltura, 45010 Rosolina (Rovigo), Italy. 3 National Center for Genetic Resources Preservation USDA-ARS, Fort Collins, CO 80521, USA.

Although the cryopreservation of vegetatively-propagated fruit species has mostly used PVS2-based vitrification procedures of shoot tips, the conservation in liquid nitrogen of dormant buds, followed by chip budding, has also been very successful for specific applications. This study has focused on the assessment of PVS2vitrification and dormant-bud techniques on ancient apple cultivars, recovered in Veneto (North-East of Italy) and preserved in field by the Veneto Agricoltura regional agency. For PVS2-vitrification, after cold-hardening of stock cultures at 4°C for 3 weeks, shoot tips were excised and cold-hardened at 4°C for additional 2 days. The shoot tips were then exposed to a cryoprotective solution (2 M glycerol and 0.4 M sucrose) for 30 min at 25°C, treated with PVS2 for 60 or 90 min at 0°C and then directly immersed in liquid nitrogen. After thawing, the shoot tips were plated on semi-solid media, containing various combinations of growth regulators and activated charcoal. The best results of three ancient cultivars (‘Campanin’, ‘Rosetta Mantovana’ and ‘Pom dell’Oio’) ranged from 57% to 100% of shoot-tip survival after cryopreservation. The dormant-bud protocol was based on winter collection of scions from which uni-nodal sections were cut, desiccated, slow cooled (-1°C/h) up to -30°C, stored in liquid nitrogen for a minimum of 48 hours, thawed, rehydrated and chip budded onto clonal rootstocks in a greenhouse. Of the three cultivars tested (‘San Piero’, ‘Canada Ruden’ and ‘Rosa Gentile’), best result was achieved with the cv San Piero, for which 79% of the buds survived ultra-freezing and 100% of the grafted plants regrew successfully when 26%-desiccated and cryopreserved buds were used for chip budding. A comparison between the two techniques was made based on resources required for the introduction and recover from liquid nitrogen of a single apple accession. Full name of presenting author: Maurizio Lambardi E-mail: [email protected]

30

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cryopreservation by vitrification of embryogenic callus of wild crocus (Crocus hyemalis and Crocus moabiticus) S. Baghdadi1, R. Shibli2, M. Syouf3, M. Shatnawai4, A. Arabiat3 and I.Makhadmeh1 1

Department of Plant Production, Faculty of Agriculture, Jordan University of Science and Technology; P.O. Box 3030, Irbid- 22110, Jordan. 2 Department of Horticulture and Crop Science, College of Agriculture, University of Jordan, Amman, Jordan. 3 National Center for Agricultural Research and Extension (NCARE), Al-Baqa’a, Jordan. 4 College of Agriculture, Al-Balqa’a Applied University, As-Salt, Jordan.

Cryopreservation of wild crocus species (Crocus hyemalis and Crocus moabiticus) was studied via vitrification. The effect of PVS2 concentration on the viability of crocus calli before and after cryopreservation was examined. Higher viability of calli was obtained using step-wise vitrification than direct exposure to a 100% PVS2. Complete survival and 91.2-94.3% regrowth rates of C. hyemalis and C. moabiticus calli, respectively, were achieved with four-step dehydration by PVS2 for 20 min prior to freezing. The effect of different cryoprotectants on survival of crocus calli was also tested. The use of 2.0 M glycerol plus 0.4 M sucrose resulted in 92.2% survival of C. hyemalis calli and 97.4% survival of C. moabiticus prior to freezing. Furthermore, the effect of combinations of different cryoprotectants and vitrification solutions on viability of calli was tested. Cryoprotection by 2.0 M glycerol plus 0.4 M sucrose followed by dehydration with 15% DMSO plus 1.0 M sucrose vitrification solution gave complete survival and maximum regrowth (21.1-25.0%) rates after freezing for C. hyemalis and C. moabiticus calli, respectively. The influence of duration of exposure of embryogenic calli to 2.0 M glycerol plus 0.4 M sucrose before dehydration with a highly concentrated PVS2 solution on viability of calli was also examined. Cryoprotection of calli for 20 min by using 2.0 M glycerol plus 0.4 M sucrose produced complete survival without or with freezing. However, increased duration of exposure to the cryoprotectant solution was observed to decrease the viability of calli. Full name of presenting author: Rida A. Shibli E-mail: [email protected]

31

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Droplet vitrification of shoot tips of fraser photinia (Photinia × fraseri Dress.) Y. Ozden Tokatli and H. Akdemir Gebze Institute of Technology, Faculty of Science, Department of Biology, 41400, Gebze (Kocaeli), Turkey.

Photinia genus consists of many well-known shrubs that are grown not only for ornamental purposes, but also for their fruit and foliage. As it is evergreen and attractive all year long, numerous hybrids and cultivars are available. Among them fraser photinia (Photinia × fraseri Dress.) with a 3-5 m height, is commonly used as ornamental plant of green areas. Although there are several reports about in vitro micropropagation of some hybrid species of this genus including Photinia fraseri, there are few reports about its medium- and long-term conservation. Therefore, it is important to develop efficient cryopreservation techniques that enable to preserve plant germplasm for a theoretically unlimited period of time at ultra low temperatures (-196 °C). Hence in this study, droplet-vitrification protocol was used for cryopreservation of shoot-tips of in vitro-grown Photinia × fraseri Dress. The optimised protocol consists of excision of cold-hardened (8 weeks at 4ºC) shoot tips, preculture in Murashige and Skoog (MS) semi-solid medium containing 0.5 M sucrose (3 days at 25ºC), dehydration with concentrated ice-cooled PVS2 vitrification solution for various period of time (0, 30, 60, 90, 120, 150, 180, 210 min at 0°C) followed by rapid freezing in individual droplets of the vitrification solution placed on aluminium foils, which were immersed rapidly in liquid nitrogen for at least 1 hour. Then frozen samples were rapidly thawed by direct dipping of the strips into unloading solution containing 1.2 M sucrose and the shoot-tips were transferred directly to QP culture medium containing 1 mg l-1 BA. The optimised droplet vitrification protocol improved the mean post-thraw Photinia regeneration rates to more than 30% from 12.5% obtained with previously reported encapsulationdehydration protocol. Full name of presenting author: Yelda Ozden Tokatli E-mail: [email protected]

32

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cryo-conservation of genetic diversity of recalcitrant-seeded species via zygotic embryonic axes: successes and problems P. Berjak, N.W. Pammenter, M. Goveia and Sershen Plant Genetic Resources Conservation, School of Biological & Conservation Sciences, University of KwaZulu-Natal, Durban, 4001 South Africa

While ex situ seed storage offers the most convenient means of genetic resources conservation, this is impossible – except in the short-term – for species producing desiccation-sensitive (recalcitrant) seeds. In these cases, the most obvious candidate explant for long-term conservation is the excised embryonic axis, with optimum storage conditions afforded by cryostorage. This has been achieved with some success for a spectrum of monocotyledenous amaryllid species, for which a delicate balance among use of cryoprotectants or not, water content preceding immersion in LN, and cooling rate needed to be achieved, with parameters differing across species. In contrast, axes from dicotyledonous seeds having fleshy cotyledons, present a significant problem when excised for cryopreservation, where explants are pared down to minimum size by excision of the cotyledons flush with the axis surface. We have found for a variety of tropical/sub-tropical woody species, that shoot development fails to occur from such explants, with the apical meristem becoming necrotic. This is the consequence of excision injury, thus generally seeming to preclude the use of such explants. While the problem of excision injury can be overcome by leaving blocks of cotyledon tissue attached to the axis, this will increase explant thermal mass unfavourably. Other means, which will be discussed, are presently being sought to ameliorate the problem of shoot tip necrosis, which appears to be associated with a burst of ROS in response to excision. The factor apparently obviating similar excision damage to amaryllid axes, is that the single, non-fleshy cotyledon is able to be retained, without compromising the responses of the explant to dehydration and cooling. Full name of presenting author: Patricia Berjak E-mail: [email protected]

33

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cryopreservation in the Vitaceae : Seed cryopreservation is effective for wild species accessions P. Chatelet, N. Chabrillange, J.P. Peros, P. Ortigosa, A. Peyriere, T. Lacombe, P. This and S. Dussert UMR DiAPC, INRA/IRD, Montpellier, France

Curators of large field-maintained plant collections have to consider how to address best the problem of safety duplicates. Seed cryopreservation could provide a secure, cost-effective response towards safeguarding wild Vitaceae accessions in which gene pools only are to be preserved. However, little relevant data, if any, is available for this group of lignose species forming dormant oily seeds. This prompted the initiation of various experiments to set up an effective cryopreservation protocol for seeds of Vitaceae species. Studying germination percentage after seed equilibration under various relative humidities, together with Differential Scanning Calorimetry analyses and longevity predictive models proposed elsewhere, allowed selecting 75% RH for optimal seed tolerance to LN exposure. Total lipid content was assessed in seeds of various contrasting genotypes. The value of seed unfrozen water content calculated from heating thermogram data corresponded to that predicted on the basis of seed lipid content. Further qualitative analysis of lipids (fatty acid composition) was of great help to interpret seed calorimetric profiles. Additional trials were performed to determine whether a dormancy breaking treatment remained necessary in cryopreserved seeds and if it should be best performed before of after cryopreservation. Germination of LN-treated seed lots decreased in less than half of the genotypes tested, while it was either not impaired or even improved in others, provided that the dormancy breaking treatment was performed immediately after seed thawing. The protocol retained is currently being implemented on material from the Vassal Vitaceae collection and has already been successfully applied to over one hundred accessions. Further studies are underway to extend cryopreservation techniques to different organs (pollen, dormant buds) in order to address the differing strategies for gene pool or true-to-type conservation. Full name of presenting author: Philippe J. Chatelet E-mail: [email protected]

34

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

In vitro propagation and cryopreservation of Stevia rebaudian a medicinal plants using vitrification method M. Shatnawi1, R. Shibli2, H. Al-Dmoor1, M. Ateyat1 and S. Abubaker 1 1

Al-Balqa Applied University, Biotechnology Department. Faculty of Agriculture Technology. Al-Salt 19117, Jordan 2 Department of Horticulture and Agronomy, Faculty of Agriculture. University of Jordan Amman, Jordan

The present study aims to develop a reliable method for in vitro micropropagation and long-term storage of shoot tips in Stevia rebaudian using a vitrification method. Successful multiplication was achieved on Murashige and Skoog (MS) medium supplemented with benzylamino purine (BAP). The different cytokinin concentration resulted in differences in number of new shoot / explant, shoot height and number of new leaf per explants. For cryopreservation of shoot tips in liquid nitrogen, several pretreatments prior cryopreservation were conducted to enhance shoot tips recovery after storage in liquid nitrogen; culturing plantlets in vitro for four weeks in MS medium containing 30 g/L sucrose, excision of shoot tips; preculture of shoot tips on 0.4 M sorbitol for 2 d, followed by loaded shoot tips with 80% PVS2 for 20 min; then dehydrated with a highly concentrated vitrifiction solution (PVS2) for 60 min at 0 ˚C prior to a plunge into liquid nitrogen. The highest post thaw re-growth obtained was 68.8% using a water bath at 45 ºC for 2 min. In a follow-up study of growth 6 months after cryopreservation, photosynthetic pigments were used to assess the physiological stress of the in vitro plantlets. All of the photosynthetic pigment parameters were lower in the plantlets, which had been cryopreserved, although none of the growth parameters had been significantly affected by the cryopreservation process. The procedure developed in this study is easy to handle and produced a high levels of shoot formation. Thus, the protocol is promising for long-term storage of S. rebaudian germplasm in liquid nitrogen. Full name of presenting author: Mohamad Shatnawi E-mail: [email protected] or [email protected]

35

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cryotherapy of shoot tips: an efficient method for eradication of plant pathogens Q. Wang1, F. Engelmann2,3, M. Lambardi4, Z. Yin1,, B. Wang1 and B. Panis5 1

Key Laboratory of Plant Genetic Improvement of Northwest China, College of Horticulture, Northwest Agricultural & Forest University, Yangling 712100, Shaanxi, P.R. China 2 Institut de Recherche pour le dćveloppement (IRD), UMR DIA-PC, 911 avenue Agropolis, BP 64501, Montpellier, Cedex 534394, France 3 Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy 4 IVALSA/Istituto per la Valorizzazione del Legno e delle Specie Arboree, Consiglio Nazionale delle Ricerche, Via Madonna del Piano n° 10, 50019 Sesto Fiorentino (Florence), Italy 5 Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, Katholieke University Leuven, Kasteelpark Arenberg 13, B-3001, Leuven, Belgium

Cryopreservation is considered an ideal means for the long-term preservation of plant germplasm and is now successfully applied to a great number of plant species originating from tropical to temperate regions. Routine application of cryopreservation of plants such as potato and banana has already started in several countries like Germany and Belgium. Recently, cryotherapy of shoot tips has proven to be an efficient method to eradicate plant pathogens such as viruses, phytoplasmas and bacteria. To date, nine different plant viruses as well as sweet potato little leaf phytoplasma and Huanglongbing bacterium have been successfully eradicated by this technique from important staple and horticultural crops. As such, cryotherapy of shoot tips can be used for the simultaneous production and long-term storage of pathogen-free plant genetic resources. Histological and ultrastructural studies on cryotherapy-treated shoot tips as well as pathogen localization in shoot tips provided some answers as to why cryotherapy of shoot tips can efficiently eliminate plant pathogens. Full name of presenting author: Qiaochun Wang E-mail: [email protected]

36

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cryobanking cassava germplasm at IITA D. Dumet, S. Korie and A. Adeyemi International Institute of Tropical Agriculture, PMB 5320, Ibadan, Nigeria

IITA maintains over 4,000 accessions of cassava (Manihot esculanta Crantz) mainly collected in Africa. During the past few years, over two-thirds of the collection was transferred to in vitro storage. To further rationalize in vitro conservation, cassava cryobanking is presently studied. A droplet vitrification procedure based on the work of Panis et al. (2005) was applied to 48 accessions of cassava randomly chosen from the in vitro collection. To mimic cryobanking procedures, the freeze/thaw cycle included, or not, transfer into a cryotank. Survival after cryopreservation was observed for most accessions (95%) when meristems were thawed shortly after freezing (= no transfer to cryostorage conditions). Within this group, the percentage of meristems that produced a shoot varied from 6% to 86%. In contrast, only 75% of the accessions survived liquid nitrogen exposure when meristems were retrieved from a cryotank. In the latter case, the percentage of meristems that produced a shoot after freezing ranged from 10% to 82%. The droplet vitrification procedure looks promising for cryobanking cassava. Some disparity in meristem survival observed from one thawing batch to another suggests that thawing needs further standardization. Full name of presenting author: Dumet Dominique E-mail: [email protected]

37

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

The Potato Cryo-Bank at the International Potato Center D.Tay, A. Panta, B. Zea, C. Ynouye and W.Roca International Potato Center, Genetic Resources Conservation and Characterization Division, Apartado 1558, Lima 12, Peru

The International Potato Center (CIP) genebank was established in 1973 following the formation of CIP. It has the CGIAR mandate to collect, conserve, characterize, evaluate, document and distribute the world FAO-International Treaty designated potato and sweet potato collections that include both cultivated and wild species. In addition, nine minor Andean root and tuber crops are also conserved. Currently, more than 15,000 accessions of about 200 species are maintained mainly clonally as field and tuber collection, in vitro collection, cryo- collection and DNA collection. In January 2008, CIP became the first genebank in the world to obtain an ISO 17025 accreditation for its in vitro germplasm conservation and distribution system. The cryo-bank is an integrated part of the total genebank safety backup management system. Research on cryo-preservation of potato was initiated in the mid-1970s in collaboration with the University of Birmingham, England and the success in fast freezing technique of potato shoot-tips was reported. From 1994 to 1999, the vitrification protocol developed by Peter Steponkus at Cornell University was applied to 400 potato genotypes at CIP and 121 accessions were successfully cryopreserved. The overall viability decreased as more genotypes were tested. During 2004-2006, with the aim of establishing a cryo-bank for the long-term conservation of potato landraces, research for improving cryo-procedures was conducted and as the result a revised PVS2 vitrification method was identified. Currently, the droplet PVS2 vitrification method is the standard procedure applied to put the 4,640-accession cultivated potato collection into the cryo-bank which tallies 670 accessions to date. This protocol gave 91 % recovery rate of the genotypes tested so far and of these 74% with shoot-tip recovery rate of 20-100% of two replicates of 50 shoot-tips each. The strategy is thus to put priority in applying this protocol to the whole cultivated potato collection while modified protocols are being developed later for the next cycle of application. With this management strategy we anticipate that at least 1,550 accessions will be in the cryo-bank by 2020. Full name of presenting author: David Tay E-mail: [email protected]

38

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

CRYOPRESERVATION OF GARLIC GERMPLASM COLLECTIONS USING THE DROPLET-VITRIFICATION TECHNIQUE H.-H. Kim1, D.-J. Shin1, N-Y. No1, M.-K. Yoon2, H.-S. Choi2, J.-S. Lee3 and F. Engelmann3, 4 1

National Academy of Agricultural Science, RDA, Suwon 441-707, Korea. National Institute of Horticultural & Herbal Science, RDA, Suwon 441-707, Korea. 3 Danyang Garlic Research Institute, Danyang 395-840, Korea. 4 Institut de recherche pour le développement (IRD), UMR DIA-PC, BP 64501, 34394 Montpellier cedex 5, France. 5 Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy. 2

The droplet-vitrification protocol was applied to unripe inflorescences of plants of four Korean garlic field collections at Danyang, Suwon, Mokpo and Namhae, to establish a cryopreserved germplasm collection. The protocol applied was consisted of preculture for 3-5 days at 10°C on solid MS medium with 0.3 M sucrose, loading for 40 min in liquid medium with 35 % PVS3, dehydration with PVS3 vitrification solution for 150 min, cooling in 5 µl droplets of PVS3 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated (40°C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 40 min. Among the various materials tested, unripe inflorescences which hold bulbil primordia display several advantages i.e. lower risks of contamination with bacteria and soil borne viruses, rapid and high plantlet formation during post-culture. Alternative materials, i.e. clove shoot apices and fully matured bulbils were used, when varieties do not produce appropriate inflorescences. A total of over 800 accessions of five clonal Allium species, including garlic, were stored in liquid nitrogen for long-term conservation using unripe inflorescences, cloves or bulbils. Full name of presenting author: Haeng-Hoon Kim E-mail: [email protected]

39

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Assessing Variability Between Accessions, Cryovials and Over Time of Cryopreserved Shoot Tips D. Ellis, B. Ambruzs, G. Holman, E. Staats and M. Jenderek USDA-ARS, National Center for Genetic Resources Preservation, 1111 S. Mason Street, Fort Collins, CO, USA 80521

The development of a long-term cryopreservation program for plant genetic resources relies heavily on the ability to predictably reproduce plants from cryopreserved explants. Since explants are cryostored as single entities, but rather as multiple explants in vials or tubes, the predictability depends on estimation of the number of units (vials or tubes) needed to obtain at least one live explant after thawing and regeneration. Further, the program needs to know if survival changes over time and the rate this change occurs to adjust where needed the number of cryopreserved units pulled to ensure a live explant. To address how many cryopreserved units are needed to obtain a single living explant, variability between individual cryopreserved vials was accessed in cryopreserved shoot tips of Fragaria, Ribes, Rubus and Pyrus. For each species three accessions were used with 100 shoot tips evenly distributed into 10 cryovials and replicated three times (300 shoot tips per accession). All vials yielded viable shoot tips, with Pyrus and Rubus showing the largest variability in viable shoots per vial (100-50% and 100-20% respectively). These results suggest that for these species one vial can be safely pulled to yield at least one viable shoot tip. To access changes in the viability of cryopreserved shoot tips over time, a set of 40 vials (10 shoot tips per vial) were set up for each of the three accessions of the four species used in the study. Based on the relatively low variability between cryovials, it was determined that accessing viability in 5 cryovials at each time point would suffice to determine if viability decreased over time. Five vials/accession will be accessed after 1 year and then at each 10-20 year interval. If pulled every 10 years, the final cryovial will be accessed in 2068 (60 years from the start of the experiment). Full name of presenting author: Dave Ellis E-mail: [email protected]

40

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cassava cryopreservation activities at the International Center for Tropical Agriculture R.H. Escobar, N. Manrique, L. Muñoz, A. Rios, D. Debouck and J. Tohme. Biotechnology and Agrobiodiversity Project. International Center for Tropical Agriculture (CIAT). Km 17 Recta Cali - Palmira. A.A. 6713 Cali, Colombia, South America.

The International Center for Tropical Agriculture (CIAT in Spanish) maintains the most important cassava collection from Latin American, Caribbean and Asian countries. The in-vitro collection, with more than 6,000 clones, is conserved using slow-grow conditions, under which the materials are kept for 14-24 months without regeneration. Since 1988, CIAT with IBPGR (now Bioversity International) support launched an initiative of Cryopreservation research as a part of cassava genetic resources management strategy. In 1997 we reported on a classical methodology, but for some of clones it was not possible to obtain a successful recovery after freezing steps. Then, new methodologies (encapsulation/ dehydration; encapsulation/ vitrification) were implemented on a wider number of clones. The most promising technology was the encapsulation/ dehydration methodology, with which we obtained better results after freezing in most of the clones tested. Currently CIAT has tested the new technique on the cassava core collection (640 accessions), finding that more than 75% of the clones tested showed at least 30% plant recovery after liquid nitrogen step. A new initiative is being implemented to get better ideas about the reproducibility and liability among laboratories and researchers. Full name of presenting author: Roosevelt Humberto Escobar Pérez E-mail: [email protected]

41

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

CRYOPRESERVATION OF NATIVE AUSTRALIAN FRUITS AND CROP WILD RELATIVES S.E. Ashmore, A. Parisi2 and K. Hamilton3 1

School of Biomolecular and Physical Sciences, Griffith University, Nathan, QLD 4111, Australia 2 Department of Primary Industries and Fisheries, University of Queensland, St Lucia, QLD 4067, Australia. 3 Botanic Gardens Trust Sydney, Mount Annan Botanic Garden, Mount Annan, NSW 2567, Australia.

Target 8 of the Global Strategy on Plant Conservation (GSPC) for 2010 is to achieve '60 per cent of threatened plant species in accessible ex situ collections' and ‘technology development and transfer, especially for species with recalcitrant seeds’. It is estimated that 25% (2000 spp.) of the flowering plants of Queensland may have recalcitrant or seeds. These indigenous species include wild diversity of major horticultural crops (Macadamia spp.), minor commercial crops (Davidsonia spp., Citrus spp., Diploglottis spp., Syzygium spp.) and crop wild relatives (CWRs) of major horticultural crops (Garcinia spp.). Many of these are currently under severe threat in situ and/or restricted to forest fragments and thus vulnerable under a changing climate. This highlights the urgent need for technology development to facilitate longterm ex situ conservation of the flora of this region. This paper will highlight some recent advances in conservation biotechnologies, including cryopreservation, for selected Australian native fruits and CRWs. This includes improved scientific understandings of seed desiccation and storage responses and associated seed chemistry and cryobiology studies. Full name of presenting author: Sarah E. Ashmore E-mail: [email protected]

42

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Alliaceae in cryopreservation, achievements and constraints E. R. J. Keller, A. Senula and C. Zanke Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany

The family Alliaceae contains several crop species. Some of them do not form seeds (garlic, A. hookeri, top onions, other primary hybrids) or are, for other reasons, multiplied as clones (shallot, ornamental alliums, Tulbaghia). Furthermore, the outbreeding character causes complications in management of large collections. As field maintenance is labour-intensive and risky and in vitro storage suffers from accumulation of endophytes, cryopreservation is the only safe method for long-term storage of Alliaceae germplasm. Several methods help to improve the cryopreservation success. Thus, preculture at alternating temperatures (25/-1 °C) increased the regeneration percentages in garlic clones from 40-50 up to 60-100 %. Using such preculture now regularly, a number of 31 accessions have been introduced into cryopreservation considering a statistical model (Dussert S, Engelmann F & Noirot M (2003) CryoLett. 24: 149-160) for safety storage. Several clones of wild species, A. obliquum and A. hookeri, as well as hybrids were cryopreserved. Two methods were used: vitrification and droplet vitrification using PVS3. The latter method resulted in 64 % survival and 15 % regeneration in A. obliquum. Preculture of donor plantlets of the hybrid A. cepa x saxatile on higher sucrose (8% vs. 3%) resulted in better regeneration (12 vs. 5%). Hidden endophytes are a problem in Allium cryopreservation. They lead sometimes to unexpected bacteria outbreaks. Although the safety conditions require larger samples of clonal material, storing of populations consisting of smaller clones might be sensible in case of emergency rescue actions. Full name of presenting author: E. R. Joachim Keller E-mail: [email protected]

43

1st International Symposium on Cryopreservation in Horticultural Species

Cryopreservation of Fraxinus spp. cooling and vitrification techniques

Oral abstracts

embryogenic cultures by slow

M. Capuana1, M. Lambardi2, E. A. Ozudogru3 and B. Panis4 1

IGV / Istituto di Genetica Vegetale, CNR, Via Madonna del Piano 10, 50019, Sesto Fiorentino, Florence, Italy 2 IVALSA / Istituto per la Valorizzazione del Legno e delle Specie Arboree, CNR, Via Madonna del Piano 10, 50019, Sesto Fiorentino, Florence, Italy 3 GYTE / Gebze Yuksek Teknoloji Enstitusu, Istanbul Caddesi, No 101, 41400, Gebze, Kocaeli, Turkey 4 Laboratory of Tropical Crop Improvement, K.U. Leuven, Kasteelpark Arenberg 13, 3001, Leuven, Belgium

Narrowleaf ash (Fraxinus angustifolia L.) and common ash (Fraxinus excelsior L.) are timber species, used widely for furniture and tool manufacturing. They are commonly propagated by seed. However, as the quality of trees developed from seeds is variable, the species can benefit from the production of embryogenic callus cultures which can be used both for in vitro propagation and genetic transformation. Repeated proliferation of such embryogenic callus cultures can lead to a gradual reduction of embryogenic potential, as well as to loss of the cultures due to contamination. Hence, cryopreservation, i.e., the storage of cells, tissues and organs at the extremely low temperatures (generally the temperature of liquid nitrogen), is a sound method to enable the safe storage of embryogenic cells for theoretically unlimited periods of time. The present study was conducted to develop an effective cryopreservation protocol for embryogenic callus cultures of F. angustifolia and F. excelsior. When encapsulated (in 3% alginate), callus clusters of F. angustifolia were treated with PVS2 (90 min at 0°C), directly immersed in liquid nitrogen, thawed and plated on a regrowth medium, 27% of the samples recovered. In addition, isolated somatic embryos could be successfully cryopreserved by a PVS2 vitrification protocol (40 min at 0°C). The vitrification technique, however, was totally ineffective when applied to a callus culture of F. excelsior. Here, slow-cooling of cell suspensions down to -40°C (1°C/min) before immersion in liquid nitrogen showed to be effective in terms of cell survival and recovering proliferation. The procedure, originally developed for banana embryogenic cell suspensions, involves the incubation at 0°C of cell cultures in liquid medium, supplemented with different sucrose concentrations (120-210 g/l) and 7.5% DMSO. Slow cooling of the samples is achieved by placing the cryovials (containing 1 ml of solution) in the cells of Mr. Frosty® (Sigma) and keeping the devise in -70°C freezer until the temperature drops down to -40°C, indicated by a temperature probe inserted in a representative cryotube. The cryovials are then rapidly plunged into liquid nitrogen for storage. The thawing is done at 40°C water bath. Cell survival is monitored soon after thawing by TTC test and, in time, the potential to recover proliferation activity is evaluated. Full name of presenting author: Elif Aylin Ozudogru E-mail: [email protected]

44

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Development of cryopreservation procedures for dwarf irises (Iris spp.) S. Jevremovic1, C. Benelli2, A. De Carlo2, A. Subotic1 and M. Lambardi2 1

Institute for Biological Research “Sinisa Stankovic”, Bulevar despota Stefana 142, 11060 Belgrade, Serbia. 2 IVALSA/Istituto per la Valorizzazione del Legno e delle Specie Arboree, National Research Council (CNR), 50019 Sesto Fiorentino (Florence), Italy.

Aiming to optimize cryopreservation procedures for shoot tips and embryogenic calli of two dwarf iris species (I. pumila and I. reichenbachii), several experiments were carried out using the vitrification, the encapsulation-dehydration and the dropletvitrification methods. Best results were obtained with the vitrification method for shoot tips and the encapsulation-dehydration method for embryogenic calli. Shoot tips, excised from in vitro shoot cultures of irises, were successfully cryopreserved after the development of a vitrification procedure based on the treatment with the PVS2 solution. Following thawing and plating, survived shoot tips resumed growth within 3 weeks without any production of callus, and afterwards developed in well-formed shoots within 5 weeks. Maximum shoot survivals (75% for I. pumila and 93% for I. reichenbachii) were achieved when the shoot tips were treated with PVS2 for 15-20 min at 25°C before the direct immersion in liquid nitrogen. However, in terms of shoot development and further rapid shoot multiplication, best results (55%) were achieved for I. pumila when shoot tips are treated with PVS2 for 20 min at 0°C. When the encapsulation-dehydration method was applied, best survival (63%) was achieved with I. reichenbachii shoot tips. Experiments are still in progress to develop also a droplet-vitrification method for shoot-tip cryopreservation, with promising preliminary results. For cryopreservation of embryogenic calli, the possibility to desiccate samples of non-encapsulated and encapsulated calli in 3% alginate beads was first evaluated. Non-encapsulated and encapsulated callus samples of both the species showed to tolerate well the exposure to the sterile air of a laminar flow hood for up to 4 hours. When longer desiccation times were applied, proliferation activity was resumed only by I. reichenbachii calli. Among the different iris callus typologies, a yellow-white type showed the highest morphogenetic potential after dehydration and regrowth on 2,4-D containing medium. Experiments on conservation in liquid nitrogen of callus samples, following the application of the developed dehydration procedure, are in progress. Full name of presenting author: Sladjana Jevremović E-mail: [email protected]

45

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cryopreservation of Quercus robur plumules P. Chmielarz1, M. Michalak1, M. Pałucka2, C. Kozioł2 1 2

Polish Academy of Sciences, Institute of Dendrology, Parkowa 5, 62-035 Kórnik, Poland The Kostrzyca Forest Gene Bank, Miłków nr 300, 58-535 Miłków

Whole seeds of Quercus robur L. (pedunculate oak) are classified as recalcitrant, i.e. sensitive to dehydration and low temperatures, so they cannot be stored longer than for 1–2 years without loss of germinability. Thus plumules (embryonic shoots) were isolated from acorns and then cryopreserved. Two methods of plumule drying and two methods of freezing in liquid nitrogen (LN) were compared. Survival rate after thawing was assessed by in vitro culture. Plumules were dried in a stream of gaseous nitrogen or in petri dishes over silica gel. Next, the plumules were frozen by shooting into sub-cooled nitrogen (~ -210ºC) or directly in vials filled with LN (-196ºC). After freezing, they were stored in LN for 24 h. In all variants, the same method of plumule cryoprotection was applied, i.e. successively in solutions of sucrose and glycerine. The survival rate of plumules after thawing ranged from 21 to 67%. Their survival was significantly affected by the drying method. Also the interaction between freezing method and drying method was significant. The highest survival rate of plumules frozen in LN was reached only if they were dried over silica gel, and next were frozen directly in vials filled with LN. The survival rate of plumules dried in a stream of gaseous nitrogen was significantly lower: 21% if frozen directly in vials and 37% if frozen by shooting. The latter freezing method ensured plumule survival at the level of 37–49% (differences not significant), irrespective of the drying method. Shoot regeneration was observed in 14% of all the plumules used in the experiment. This study showed that genetic resources of Q. robur can be preserved by means of cryopreservation. Full name of presenting author: Marcin Michalak E-mail: [email protected]

46

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cryopreservation of dormant buds from temperate fruit crops – optimising working collection resources B.W.W. Grout1, J. Green2, C. Vogiatzi1 and T.B. Toldam-Andersen1 1

Agriculture and Ecology, University of Copenhagen, Højbakkegård Alle 21, 2630 Taastrup, Denmark 2 CIC bioGUNE , Parque Tecnológico de Vizcaya Ed. 801 A Unidad de Biología Estructural Edificio 800, 48160 DERIO Vizcaya, Spain

Any extensive use of in vitro maintained explants for cryoconservation commits large repositories of fruit crops (e.g University of Copenhagen >1700 accessions) to significant human and material resource costs, and limits the speed at which accessions can be taken into frozen storage. Dormant bud cryopreservation (35mm of scion wood with a single bud), with grafting as the recovery technique, reduces resource requirements as field staff can complete the entire preservation process with little additional equipment, and at a time (winter) when workload is most manageable. The major disadvantage is that the disease status of the material has to be established after recovery. The challenge is to ensure survival of both the bud and the cambial tissue, to provide the graft union, in a single protocol. Our data for Malus, Pyrus and Ribes indicates that success of existing protocols is variable between accessions, with post-thaw electrolyte leakage evidence from Ribes indicating cambial tissue damage as a significant factor in graft failure. However, immediate post-thaw, in vitro bud culture can, in some instances, retrieve viable plantlets where graft success is compromised. Successful, protective dehydration and subsequent rehydration of the thawed explant is essential for bud and graft viability. Symptoms of xylem embolism in thawed scions of Ribes, where the graft has failed, may indicate rehydration problems relating to water movement in the scion tissues. Patterns of water redistribution and the sites of loss from explants will be described for both surviving and lethally injured explants in an attempt to use this in a predictive way with regard to probable explant survival. Full name of presenting author: Brian Grout E-mail: [email protected]

47

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cryopreservation of hairy root cultures of Maesa lanceolata and Medicago truncatula E. Lambert, A. Goossens, B. Panis, M.-C. Van Labeke and D. Geelen University of Ghent, faculty of bioscience engineering, department of plant production, Coupure links 653, 9000 Ghent, Belgium

Maesa lanceolata is a medicinal shrub growing in the tropics of Africa and Asia. It produces a number of different saponins which have biological activities that are potentially interesting for the pharmaceutical industry. Medicago truncatula presents an interesting model species for study and modulation of saponin biosynthesis. To our knowledge, this species does not produce pharmaceutically important saponins. Instead, the Medicago saponins are mainly insecticidal and antinutritional components. To modulate the production of secondary metabolites of Maesa lanceolata and Medicago truncatula, transgenic root cultures of both species were established. Maintenance of large numbers of hairy roots is labour intensive and costly. In addition there’s also a risk on contamination and genetic changes. Therefore we developed a cryopreservation protocol to store different transgenic lines over time. Using encapsulation-dehydration, we obtained high survival rates for both Maesa and Medicago hairy roots. Root tips (2-3 mm) were isolated and encapsulated in calciumalginate beads, containing 0.1M sucrose. These encapsulated root tips were precultured for 3 days using basal medium, in which the sucrose concentration was increased every day (from 0.5M sucrose on the first day to 1M on the last day). Preculture of Medicago root tips during 3 days lead to unwanted growth out of the beads, which could be tempered by addition of plant growth inhibitors. After preculturing, the beads were dehydrated for 2-5 hours in a laminar flow until 35-40% of the initial bead weight was reached. Dehydrated beads were plunged into liquid nitrogen and were thawed in a water bath at 40°C after 2-3 days. The survival rates were 90% for Maesa lanceolata and 53% for Medicago truncatula, which are sufficient to allow implementation of the technique for storage of large numbers of hairy roots. Full name of presenting author: Ellen Lambert E-mail: [email protected]

48

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Cryopreservation of date palm highly regenerable tissues L. Fki*, N. Sahnoun*, N. Bouaziz*, O. Bo uattour*, M. Guarbaya*, B. Panis** and N. Drira* *Laboratoire des Biotechnologies Végétales Appliquées à l’Amélioration des Cultures. Faculté des Sciences de Sfax- Route Sokra BP : 1171, 3000 Sfax, Tunisie. **Laboratory of Tropical Crop Improvement. Katholieke Universiteit Leuven (K.U.Leuven), Kasteelpark Arenberg 13, Bus 2455, 3001 Leuven, Belgium.

Date palm genetic resources are today subject to severe erosion due to the spread of lethal diseases like “bayoud” and the extension of the monoclonal cultivations. In view of this critical situation, and also due to the high cost and risks of in situ conservation, the preservation of date palm genetic resources has become an important topic. Safe storage of this germplasm can be achieved through the application of a wide variety of tissue culture techniques, among them crypreservation. In the first part of this work, we investigated date palm somatic embryogenesis and caulogenesis. Such study is important to ensure the availability of highly regenerable tissues which can resist the cryogenic treatments. In date palm, buds and proembryogenic masses (PEMs) can be induced using several explants and low concentrations of 2,4-D. The maintenance of the in vitro-cultured multiple bud clusters and the proembryos by successive subcultures could, however, increase the risk of genetic and physiological variations. The cryoconservation of this kind of material soon after their initiation is therefore necessary and will be a practical and effective way to produce certified in vitro plants with a genetic conformity. Next, we studied the cryopreservation of these highly proliferating meristem cultures and proembryos using the ultra-rapid droplet freezing and the encapsulationvitrification methods. These techniques, based on a high-speed cooling of the dehydrated meristems and proembryos, proved to be efficient. Survival rates of about 70 % could be obtained for Barhee cultivar PEMs that are treated during 30 min with the PVS2 solution. Moreover, we observed that the cryogenic treatment does not affect the morphogenetic capacity of the tissues and several in vitro plants could be regenerated. The morphological and molecular analyses confirm the genetic stability of the cryopreserved tissues. Full name of presenting author: Lotfi Fki E-mail: [email protected] or [email protected]

49

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Encapsulation-dehydration: past, present and future F. Engelmann IRD, BP 64501, 34394 Montpellier cedex 5, France & Bioversity International, Via dei Tre Denari 472a, 00057 Maccarese, Rome, Italy

The encapsulation-dehydration technique has been established in France through the pioneering work of the team of the late Jean Dereuddre (Dereuddre J, Blandin S & Hassen N (1991) CryoLetters, 12,125-13; Dereuddre J, Scottez C, Arnaud Y & Duron M (1990) Comptes Rendus de l’Académie des Sciences Paris, t. 310, Série III, 317-323 ; Fabre J & Dereuddre J (1990) CryoLetters, 11, 413-426). This method is based on the technology developed for producing synthetic seeds, i.e. the encapsulation of explants in calcium alginate beads. Encapsulated explants are then precultured in liquid medium with a high sucrose concentration and partially desiccated before freezing. Encapsulating the explants allows submitting them to very drastic treatments including preculture with high sucrose concentrations and desiccation to low moisture contents, which would be highly damaging or lethal to non-encapsulated samples. The encapsulation-dehydration technique was developed firstly with species from temperate climates, including notably pear, potato, grape, eucalyptus and carrot. Later, it was experimented with plants from tropical origin, notably cassava, sugarcane and coffee. Today, it has been successfully extended to over 40 plant species using cell suspensions, shoo tips, zygotic, somatic and microspore embryos, ovules and seeds, as well as to microalgae. An example of its routine, large scale application is found in CIAT (Colombia) with the work performed on cryopreservation of cassava germplasm. Mode of action, costs, advantages, disadvantages, examples as well as future prospects of the encapsulation-dehydration technique will be discussed. Full name of presenting author: Florent Engelmann E-mail: [email protected]

50

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Current development and application of plant cryopreservation in Latin America and Caribbean. M.-T. González-Arnao1, F. Engelmann2,3, A. Abdelnour4, M. E. Aguilar5, V. Arahana6, E. Aranzales7, A. Arias6, T. Avila8; A. Clausen9, A. Diamante10, A. Digilio9, N. Dolce11, R. Escobar7, E. Flachsland11, J. F. Cedeño12, Z. Fundora13, R. Gámez1, L. García14, P. J. González6, J. Izquierdo15, S. Korneva12; C. Luna11, N. Manrique7, R. Maribona12 K., R.H. Maribona12, Y. Martínez1, M. Martinez16, J. Mendoza12; G.l Mercado17; J.E. Montoya7, L. Mroginski11, A. N. Salomão18, A. Panta19, E. Peralta12, F. Piña12, M. Ramirez20, H. Rey11, W. Roca19, O. Ruiz12, J. Salcedo20, M. Sanchez21, L. G. Santos7, A. Scocchi11, M. de los Ángeles Torres13, M. de Lourdes Torres6, R. Valbuena22, S. Vila10, V. Villalobos23 and B. Zea19 1: Universidad Veracruzana, Mexico; 2: IRD, France; 3: Bioversity International, Italy; 4: Instituto Tecnológico de Costa Rica; 5: CATIE, Costa Rica; 6: Universidad San Francisco de Quito, Ecuador; 7: CIAT, Colombia; 8: Centro de Investigaciones Fitoecogenéticas de Pairumani, Bolivia; 9: INTA, Argentina; 10: REDBIO, International Foundation, Chile; 11: IBONE, Argentina; 12: CIBE, Ecuador; 13: INIFAT, Cuba; 14: Instituto de Biotecnología de las Plantas, Cuba; 15: FAO REDBIO, Chile; 16: Centro de Bioplantas, Cuba; 17: Fundación PROINPA, Bolivia; 18: CENARGEN, Brazil; 19: CIP, Peru; 20: Bioversity International, Colombia; 21: Univ. Nacional de Colombia; 22: CORPOICA, Colombia; 23: SAGARPA, Mexico.

The large majority of species for which cryopreservation protocols need to be developed are located in tropical countries. It is therefore necessary to develop cryopreservation research activities in these countries, and to promote the dissemination of relevant scientific and technical information as a prerequisite to the initiation of such research activities. Central America, South America and the Caribbean host several world biodiversity hotspots. Many of the plant species found in such areas have recalcitrant seeds or are propagated vegetatively. They are thus relevant candidates for the development and application of cryopreservation protocols. At the moment, cryopreservation research activities are limited in Latin American and Caribbean (LAC) countries. Their development is hampered notably by the absence of specialized literature in Spanish. The availability of such literature would be instrumental in informing scientists and decision makers in the region of the current state of the art of plant cryopreservation, thereby stimulating the development of research activities in this area. It is in this context that the Universidad Veracruzana (UV, Orizaba, Mexico) has initiated a project in collaboration with the Institut de Recherche pour le Développement (IRD, Montpellier, France) aiming at producing a scientific publication in Spanish describing the current development and application of cryopreservation in LAC countries. Cryopreservation specialists from Mexico, Argentina, Brazil, Bolivia, Colombia, Costa Rica, Cuba, Ecuador, Peru, as well as representatives of international institutions (FAO, Bioversity International) are contributing to this publication. It is hoped that, in addition to the publication planned, the implementation of this project will lead to the establishment of a regional network of cryopreservation researchers, which will stimulate research in this area in LAC countries and lead to the development of regional collaborative projects. Full name of presenting author: María Teresa González-Arnao. E-mail: [email protected] or [email protected]

51

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Conservation strategy of genetic resources in strawberry in Germany M. Höfer Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, Institute for Breeding Research on Horticultural and Fruit Crops, Pillnitzer Platz 3a, D-01326 Dresden, Germany

Strawberry Fragaria x ananassa is the most favoured berry crop in Germany. Consumption per capita is continuously increasing. Fruit production is about 60% domestic and dominated by only one cultivar ‘Elsanta’. Little is known about the diversity available in the species which consists of more than 1,000 cultivars. The ‘National Program for Genetic Resources of Agricultural and Horticultural Plants’ is designed to provide long-term preservation, utilization, research and development for these species. The German Fruit Genebank was established as a network of collections held at several locations. Based on an assessment performed in different institutions, 369 strawberry cultivars were selected to be included into the National German Strawberry Genebank. Only 43 cultivars were duplicated and maintained in field collections at two locations at the Julius-Kuehn Institute Dresden-Pillnitz and at the Bundessortenamt Wurzen. High operating expenses and budget limitations for field collections do not allow further duplication of theses collections. The conservation strategy requires the application of several different methods for safety duplication. First, in vitro cold storage was elaborated and adapted. The average storage duration at 4°C for a range of strawberry cultivars was 22 months using five chamber bags as a storage container and a combined medium without plant growth regulators or vitamins. A calculation required to establish and maintain a safety duplication of the collection demonstrated that the application of the in vitro cold storage is too labor intensive to be maintained. It was determined that the development of an effective method for cryopreservation is required for cost effective long-term storage. Both results on in vitro cold storage and on cryopreservation will be presented. Full name of presenting author: Monika Höfer E-mail: [email protected]

52

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Immulocalisation of 2 viruses (PFBV and PLPV) in Pelargonium apices and study of their potential eradication by cryopreservation A. Gallard1, R.Mallet2, R. Filmon2, M. Chevallier3, N. Dorion1 and A.Grapin1 1

UMR GenHort, AGROCAMPUS-OUEST, centre d’Angers, France Service Commun d’Imagerie et d’Animation Médicale, Faculté de médecine de l’université d’Angers, France 3 UMR GenHort, Centre INRA Angers-Nantes, France 2

The effect of a possible eradication of the Pelargonium Flower Break Virus (PFBV) and Pelargonium Line Pattern Virus (PLPV) by apex “droplet-vitrification” cryopreservation was investigated using 5 different cultivars. The virus detection was based on DAS-ELISA tests and immunolocalisations were carried out for virus localisation. Immunolocalisations realised on control explants excised from DAS-ELISA positive plants showed that PFBV and PLPV were present in the apices, even in the meristematic dome. However, viral particles were more numerous in the cells of the basal zone than in the more meristematic ones. A simple apex culture did not permit to eliminate the viruses. Immediately after cryopreservation, virus eradication was neither observed. Plants regenerated from cryopreserved apices were tested by DAS-ELISA after a 3 months growing period. Viruses were not detected in 25 % and 50 % of the tested plants for PFBV and PLPV respectively. But, immunolocalisations realised on apices from the DAS-ELISA negative cryoregenerated plants showed the viruses being still present. Our results firstly demonstrated that PFBV and PLPV are even present inside meristematic cells and secondly that cryopreservation could partially eradicate 2 viruses in Pelargonium plants but without eliminate them totally. In the future, our investigations would more specifically study the cryopreservation impact on virus replication. More knowledge on virus behaviour during cryopreservation processes could permit to optimize the management of genetic resources using this conservation method. Full name of presenting author: Agnès Grapin E-mail: [email protected] and [email protected]

53

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Efficacy of cryotherapy for control of Cocoa Swollen Shoot Virus in cocoa somatic embryos A. Wetten1, R. Adu-Gyamfi1, R. Edward1, G. Ameyaw1 and J. Allainguillaume2 1

University of Reading, School of Biological Sciences, Whiteknights, PO Box 221, RG6 6AS, UK. 2 University of Wales Aberystwyth, Institute of Biological Sciences, Edward Llwyd Building, Penglais, Ceredigion SY23 3DA, UK.

Cocoa Swollen Shoot Virus (CSSV) is one of the most debilitating plant pathogens affecting cocoa production in West Africa, the most virulent isolates Agou 1 (Togo) and New Juaben (Ghana) of which cause defoliation, yield reduction and ultimately death of affected trees. The pathogen is a badnavirus, spread by a number of mealy bug species and is now severely impacting cocoa production in Togo, Ghana and Ivory Coast (the world’s largest cocoa exporter). CSSV-free propagules would benefit re-planting programmes and are essential for the establishment of germplasm collections and floral-derived somatic embryos go some way to addressing this need as, when initiated from virus infected trees, the majority give rise to virus-free plants. In an effort to improve the efficiency of CSSV elimination a PVS2-based vitrification system was applied for the cryopreservation of virus infected cocoa somatic embryos. Enhanced virus elimination has been observed but in order to test the efficacy of this cryotherapy protocol it has proved necessary to develop an artificial inoculation system involving the infection of embryogenic cotyledonary explants so as to generate sufficient numbers of CSSV-infected secondary somatic embryos for the cryopreservation test population. It was also crucial to sample tissues from somatic embryos prior to cryopreservation in such a way that their virus status could be established without reducing their ability to subsequently regenerate. PCR-based detection was used for virus screening and a degenerate approach to primer design is described which, using a single test, has allowed for the recognition of 47 out of 56 of the CSSV isolates so far screened. Full name of presenting author: Andy Wetten E-mail: [email protected]

54

1st International Symposium on Cryopreservation in Horticultural Species

Oral abstracts

Droplet vitrification: the first generic cryopreservation protocol for organized plant tissues? B. Panis, B. Piette, E. André, I. Van den houwe and R. Swennen Laboratory of Tropical Crop improvement, KULeuven, Kasteelpark Arenberg 13, 3001 Leuven, Belgium

Cryopreservation procedures are now available for about 150-200 different plant species, but until now for each species and tissue type, cryopreservation protocols needed to be empirically adapted in function of the natural frost/dehydration resistance of the species under investigation, explant size, explant type and water content. Most of the work on cryopreservation of plants has been performed in the framework of academic studies and involves only one or a few genotypes. Only few plant germplasm collections stored in liquid nitrogen currently exist but the number is increasing. The unavailability of an efficient and robust cryopreservation protocol applicable to many plant species and diverse germplasm types is one of the major bottlenecks for the wide application of cryopreservation to plant germplasm. Droplet vitrification is now one of the methods that receives much attention and is moreover on a large scale applied to germplasm collections of for example banana, potato and garlic. The method is also now under investigation to be applied in collections from other plant species such as pelargonium, taro, ulluco and sweet potato. Mode of action, costs, advantages, disadvantages, examples as well as future prospects of the droplet vitrification method will be discussed. Full name of presenting author: Bart Panis E-mail: [email protected]

55

1st International Symposium on Cryopreservation in Horticultural Species

Notes:

56

Oral abstracts

1st International Symposium on Cryopreservation in Horticultural Species

Notes:

57

Oral abstracts

1st International Symposium on Cryopreservation in Horticultural Species

Notes:

58

Oral abstracts

1st International Symposium on Cryopreservation in Horticultural Species

Notes:

59

Oral abstracts

1st International Symposium on Cryopreservation in Horticultural Species

Notes:

60

Oral abstracts

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

Poster abstracts

The abstracts are arranged alphabetically according to presenting (underlined) author

61

1st International Symposium on Cryopreservation in Horticultural Species

62

Poster abstracts

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

1. The current status of conservation of agricultural and horticultural genetic resources and its cryopreservation future in Syria S. Abdullateef, I. Pinker and M. Böhme Department of Horticultural Plant Systems, Humboldt-University of Berlin Lentzeallee 75, D-14195 Berlin, Germany

Syria located at the eastern side of the Mediterranean Sea has a unique positing and richness in genetic diversity. There are over 3150 plant species among them of world-wide important plants e.g. wild olive, pear, apple, date palm, pistachio, potato and pomegranate. Because of numerous man-made and natural pressures losses in biological diversity especially for wild olive and apples are recorded. Therefore, in collaboration with IPGRI and FAO a national program for plant genetic resources conservation is established since 10 years including in situ, ex situ and in vitro conservation approaches. In the frame of this program characterization of the genetic diversity of local Syrian cultivars is started. The in situ conservation of wild plant species is carried out through over 30 protected areas and two national parks of 315.221 ha in total. Ex situ conservation includes Seed gene banks to storage seeds at the long-term (-18, - 22°C) and medium-term (0, – 4°C), Field gene banks to storage wild olive, apple, pistachio etc., and Botanical gardens. In vitro conservation is used mainly to conserve local cultivars of potato, date palm and sweet potato. For vegetatively propagated plants the cryopreservation would be a more promising technique for the long-term conservation. However, cryopreservation protocols are available in Syria there is no use of these techniques so far due to lack of coordinated research, unavailability of efficient and robust cryopreservation protocols and technology. Using the example of potato we start to develop cryopreservation for Syrian cultivars to adopt this technique for Syrian conditions. Full name of presenting author: Shaher Abdullattef E-mail: [email protected]

63

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

2. Preservation of genetic resources in Switzerland – clarification of the

options J. Angstl Institute of Natural Resource Sciences, Zurich University of Applied Sciences, Schloss, P.O. Box, Waedenswil 8820, Zurich, Switzerland

Nowadays in Switzerland fruit trees are exclusively obtained in so called on farm collections. More than 2500 apple varieties have been collected and are conserved within the framework of a governmental program. To assure these collections, duplicates are planted in various regions of Switzerland. Nevertheless there is a high risk that this backup is insufficient. Because of the immense occurrence of fire blight (Erwinia amylovora) in 2007 these variety collections could be infected and must be cleared. There are 3 different options to preserve Swiss apple varieties being clarified: • Cryopreservation • In vitro (tissue culture) • Nuclear stock in greenhouses Full name of presenting author: Julia Angstl E-mail: [email protected]

64

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

3. Slow growth conditions of Ranunculus in vitro shoots M. Beruto*, M. Fibiani**, A. Smeraldi* and R. Lo Scalzo** * Istituto Regionale per la Floricoltura (IRF), Via Carducci 12, 18038 Sanremo (IM), Italy ** Agricultural Research Council - Food Technology Research unit (CRA – IAA), Via G. Venezian 26, 20133 Milano, Italy

Ranunculus is an important cut flower production. Its importance is testified by the fact that the commercial turn-over has greatly increased during the last 10 years in the Sanremo area where 90-100 ha and 430 growers are addressed to this cultivation. The Regional Institute for Floriculture (IRF) studied the in vitro protocol to micropropagate Ranunculus which reached an industrial application. Nowadays, the in vitro cloned material, with a production of about 2 millions of plants, represents 15% of the whole Ranunculus production in the Sanremo area. Since the cultivation unit, in the Mediterranean area, is represented by the tuberous roots gathered from the micropropagated plantlets at the end of the in vivo cultural cycle, it is very important to perform the acclimatization phase for the ex vitro plantlets in a short period (2-3 months) in order to allow the formation of tuberous roots of quality. This represents an important drawback in the productive flow of the commercial laboratory which is forced to work a large amount of plant material in a very short period. In vitro slow growth conditions could be a valuable alternative to the cryopreservation. Aim of the present work was to evaluate the growth of in vitro Ranunculus plantlets (cv. Alfa Success®) at 2°C, 70% RH, PAR 27 μmol.m-2.s-1, 16 h photoperiod under normal atmosphere or reduced oxygen conditions (2% O2). The usual conditions adopted during the micropropagation cycle (19°C, PAR 50-60 μmol.m-2.s-1, 16 h photoperiod) were considered as control. In vitro plantlets (fresh weight 152 ± 13 mg, 2,3± 0.2 cm height and 2-3 leaves), at the end of the multiplication phase, were incubated in a solution of IBA (3 mg L-1) overnight. After the rooting induction, plantlets were transferred to gelled media with no hormones and supplemented with different MS salts and different sucrose concentrations (30 or 60 g/l). Cultures were maintained over different periods (1, 2 and 3 months) under the above described slow growth conditions and, at the end of the conservation, fresh and dry weight, plant height and number of leaves and roots were scored; chlorophyll fluorescence parameters were measured before and after cold storing too. Plantlet growth was followed over the acclimatization phase and further on also during the cultivation in the field. Promising results (survival percentage 80-100%) were obtained when the slow growth was carried out over a two months period; indeed plantlets showed a satisfactory rooting efficiency (about 100%) and number of roots per shoots (7-9 roots/plant versus 11 roots/ plant in the control). Maximum quantum yield (Fv/Fm) showed a reduction with respect to the initial value, but a satisfactory stability, with no differences between normal and low oxygen atmosphere, was observed over two months of storage. In the field their performance was even better than the control with a little increase in the flowers number per plant (7 versus 5) and an increased weight of the tuberous roots gathered at the end of the cultural cycle. Full name of presenting author: Margherita BERUTO E-mail: [email protected]

65

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

4. Cryopreservation of

fruit tree species through the encapsulationdehydration of in vitro shoot tips at the CRA – Fruit Research Centre of Rome C. Damiano, E.Caboni, A. Frattarelli, E. Condello, MA Palombi, F. Engelman and M. Arias CRA-Fruit Tree Research Centre, via di Fioranello, 52 00134 Rome – Italy

The CRA-Fruit Tree Research Centre of Rome is the repository of the in vivo national collection of the Fruit tree germoplasm with about 8.000 genotypes planted by now. Since cryopreservartion and slow-growth storage of in vitro material, reducing costs and risks of loss of genotypes respect to the in vivo collections, could be valuable complementary tools for germoplasm conservation, programs for the application of this strategies were also developed in the CRA-FRU and here are presented the results so far obtained, at experimental level, with the application of the cryopreservation by the encapsulation dehydration method. In strawberry the best results (60%of re-growth) were obtained using alginate encapsulated apices, excised from 2-week cold acclimatised shoots, treated with 0.75 M sucrose added liquid medium for 24 h, desiccated 8-h in silica-gel to ~20% moisture content, then plunged in liquid nitrogen (LN). In vitro apices of mulberry (Morus alba L.), blackberry (Rubus fruticosus L.) cv “Kotata” and raspberry (Rubus idaeus L.), cv “M. Exploit” were also successfully cryo-preserved. The highest survival was obtained using sucrose concentrations of 0.75M or 1M for 1-3 days, according with the species, and silica treatment to reduce water content (fresh weight basis) of beads between 23% and 17% before immersion in LN. The survival rates were 62 % for mulberry, 57 % for blackberry and 43 % for raspberry. Apices of in vitro plantlets of almond (Rootstock M51 and cv Ferragnes), apple (Cv MacIntosh, Gala e Annurca), peach (Sel P16 and cv Babygold) and pear (wild pear, rootstock Farold 40, William) were also cryopreserved through the encapsulationdehydration technique. In both genotypes of almond the highest survival rates (50-62%) were achieved with conditions including pre-growth of encapsulated apices for 1 to 3 d in liquid medium with 0.75M sucrose, desiccation of beads to 19-20% moisture content followed by direct immersion in LN. In peach and apple the best results were obtained with dehydration of Na-alginate beads for 1 day in 0.75M sucrose (plus 0.75M glycerol in peach) and desiccation to 20% moisture content. Dehydration of Na-alginate beads for 2 days in 0.75M sucrose and desiccation to 20% moisture content (fresh weight basis), gave 60% recovery after exposure to LN in wild pear, while the highest recovery so far obtained with commercial cultivars was of 26% in William with a dehydration in sucrose 1M for 3 days and a silica desiccation of 10 hours. Fifteen of the cryopreserved lines of wild pear were used for genetic analyses by RAPDs and SSRs, in comparison with the mother plant. Both RAPDs and SSRs did not reveal any polymorphism between cryopreserved shoots and the origin genotype, suggesting that cryopreservation, using encapsulation-dehydration, does not affect genetic stability. Now attempt of application of this method to hazelnut shoot tips are also in progress. Full name of presenting author: Emilia CABONI E-mail: [email protected]

66

1st International Symposium on Cryopreservation in Horticultural Species

Cryopreservation embryonic axes

5.

of

Anadenanthera

colubrina

Poster abstracts

(Vell.)

Brenan

F. C. Nery, R. Paiva, A. C. A. L. Campos, G. F. Nogueira, V. C. Stein and A. A. Alvarenga Federal University of Lavras, Department of Biology, Sector of Plant Physiology, 37.200-000 - Lavras - MG, Brazil.

The ex situ preservation and rational use of Anadenanthera colubrina demands conclusive information related to seed storage. The objective of this study was to determine a protocol to cryopreserve embryonic axes of A. colubrina seeds. Embryonic axes treated with three concentrations (5%, 10% and 15%) of DMSO and glycerol were maintained in liquid nitrogen (-196° C), ultra freezer (-80° C) and freezer (-20° C) during 15, 30 and 150 days. The embryonic axes moisture content was determined prior and after each storage period. After the storage period, the embryonic axes were thawed and inoculated in WPM supplemented with 30.0 g L-1 sucrose, 4.0 g L-1 agar and pH adjusted to 5.8. Embryonic axes immersed in chemical cryoprotectors and maintained at -196, -80 and -20 ºC, absorbed the solutions and presented moisture content higher than 65%. On these conditions, no germination was observed. Embryonic axes that were stored in the absence of cryoprotectors presented viability around 100%, independent of the cryostorage period. Supported by FAPEMIG, CNPq and CAPES, Brazil Full name of presenting author: Ana Carolina Atala Lombelo Campos E-mail: [email protected]

67

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

6. Viability of castor bean pollen using different cytological dyes 1

D. P. Vargas, 1R. Paiva, 1A. C. A. L. Campos, 1G. F. Nogueira, 2V. L. Bobrowski and 1 L. V. Paiva 1

Federal University of Lavras, Department of Biology, Sector of Plant Physiology, 37.200-00 Lavras - MG, Brazil. 2 Federal University of Pelotas, Department of Zoology and Genetics, 96.010-900 - Pelotas RS, Brazil.

The growth of castor bean (Ricinus communis L.) in Brazil has been substantially increased in recent years based on its medicinal properties and the use for the production of biodiesel. The great genetic diversity observed in the castor bean germoplasm provides the development of breeding programs, despite the need for an adequate preservation of this diversity. For the preservation of biological materials some methods such cryopreservation are more efficient. This technique enables the storage of biological materials for an undetermined period based on the maintenance in liquid nitrogen (-196 º C). The objective of this study was to determine the effect of the cryoprotector glycerol 10% (v/v) on the viability of castor bean pollen extracted from male flowers and maintained in liquid nitrogen at -196º C using Acetic-Carmim and Reactive of Alexander as cytological dyes. The pollen grain analysis with these two dyes presented no significant difference regarding viable, non-viable and burst pollen. Supported by FAPEMIG, CNPq and CAPES, Brazil Full name of presenting author: Ana Carolina Atala Lombelo Campos E-mail: [email protected]

68

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

7. The effect of antioxidants on the cryopreservation recovery of two potato cultivars P. Canepa, A. Panta and D. Tay International Potato Center, Genetic Resource Conservation and Characterization Division Apartado 1558, Lima 12, Peru

The International Potato Center (CIP) maintains the largest worldwide in vitro potato collection comprising 4,571 accessions and is continuously looking for more efficient genetic resources conservation methods. Currently, CIP is using PVS2 vitrification droplet cryopreservation technique for long-term potato conservation. It was found by using this method, some post-thawing shoot tip samples survived but turned brown followed by necrosis during recovery stage, suggesting that oxidation processes are involved in the viability decline. With this hypothesis we investigated the effect of two antioxidants, ascorbic acid and glutathione, on tissue survival and plantlets recovery. Apical shoot tips from 3 weeks old in vitro plantlets of S. tuberosum subsp. andigenum cvs. ‘Ccompis’ and S. tuberosum subsp. tuberosum x andigenum cvs. ‘Tacna’ were subjected to cryo-process with the droplet PVS2 vitrification method. One experiment was conducted using three doses of ascorbic acid (50, 100 and 150 mg/l) and another with glutathione (5, 10, 15 mg/l) added to the post-thawing medium. The ascorbic acid showed a significant negative effect on survival and recovery of shoot tips of the two cultivars, without any genotype differences. With glutathione, ‘Ccompis’ had better survival and recovery of the plantlets than in ‘Tacna’ but the differences were not statistically significant. Full name of presenting author: Patricia Maria Canepa M. E-mail: [email protected]

69

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

Applications of molecular markers and flow cytometry prior cryopreservation actions 8.

E. Cires1, E.L. Peredo2, C. Cuesta2, J. A. Fernández Prieto1 and M. Á. Revilla2 1

Área de Botánica. Universidad de Oviedo, Departamento de Biología de Organismos y Sistemas, Catedrático Rodrigo Uría s/n, 33071 Oviedo, Spain. 2 Área de Fisiología Vegetal. Universidad de Oviedo, Departamento de Biología de Organismos y Sistemas, Catedrático Rodrigo Uría s/n, 33071 Oviedo, Spain.

It is a well-known fact that the cryopreservation is a useful tool for long-term maintenance of selected plants. The growing loss of plant diversity in wild habitats has promoted great efforts to investigate protocols for conservation in many botanical centres. However, sometimes we are not able to answer the question: what species/populations should be cryopreserved? Indeed, and before conservationists embark on a project, they must be clear about why the taxon warrants conservation. The reason for the selection of taxa/populations to conserve should be based on a series of measurable criteria, such as genetic and morphological distinctness or the current conservation status. One example, if the objective is to conserve threatened species, is Ranunculus parnassifolius s.l. an orophilous plant distributed throughout Central and South Western Europe (Alps, the Pyrenees and the Cantabrian Mountains) where its evolutionary history and taxonomy are often complicated, having been little studied. At least five races have been recognized, treated either at the species or subspecies level mainly based in morphological and ecological traits, with different levels of ploidy (diploid and tetraploid cytotypes). Nonetheless, difficulties with taxonomic classification may stem from undetected polyploidy; therefore in this case, the ploidy level is one of the main criteria used for discriminating among closely related species. Techniques such as flow cytometry have become a frequently used method in the estimation of DNA ploidy, offering a rapid and precise method for identifying taxa of different ploidy. In addition, a study of the genotypic variability applying different molecular markers will allow the establishment of proper conservation programs (exsitu and/or in-situ conservation), inferring about the most appropriate systematics. This example shows how the conservationist can use distinct tools to select complementary target taxa to conserve the maximum range of biodiversity. Full name of presenting author: Eduardo Cires E-mail: [email protected]

70

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

9. Progresses in the cryopreservation of apple cultivars through the

droplet vitrification method E. Condello1, E. Andrè2, B. Piette2, P. Druart3, E.Caboni1, C. Damiano1 and B. Panis2 1

CRA - Fruit Tree Research Institute. Via di Fioranello, 52 - Rome - Italy K.U. Leuven - Laboratory of Tropical Crop Improvement, Kasteelpark Arenberg ,13- LeuvenBelgium 3 CRA-W Department Biotechnology, Chaussée de Charleroi, 234 - Gembloux - Belgium 2

The Fruit Research Center of Rome hosts an on field fruit plant germoplasm collection. The Propagation Group has been gradually establishing an in vitro collection to be conserved through “slow growth” method. Cryopreservation protocols for various fruit species have been also established and recently a National Project was financed to support the establishment of protocols for old pear and apple varieties. In the framework of a STSM of COST 871 we developed a cryopreservation method that utilizes axillary buds in a droplet vitrification protocol. We tested the recovery after liquid nitrogen immersion of two in vitro grown apple cultivars (Pinova and Jonagold). The establishment of the protocol was performed evaluating the effect of various factors: length of application of the PVS2 solution, application of high sugar pre-treatments and hormonal composition of the medium. In cultivar Pinova we obtained 46.7% of surviving buds after liquid nitrogen immersion, treating axillary buds, without cold or high sugar pretreatment, for 30 min in PVS2 solution; 64% of which developed plants. In cultivar Jonagold, we obtained 70% of surviving buds with 45 min of PVS2 treatment, 30% of which developed plants. With this method no cold acclimation period and pre-culture medium were required and the most recent results showed that the method can be applied with success to different apple cultivars. Moreover, the use of axillary buds increase the availability of material to be stored as compared to the use of only the apical tips. Full name of presenting author: Emiliano Condello E-mail: [email protected]

71

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

Nuclear DNA integrity of cryopreserved embryonic axes of Anadenanthera colubrina (Vell.) Brenan 10. 1

F. C. Nery, 1R. Paiva, 1D. P. C. da Silva, 1A. C. A. L. Campos, 2J. M. S. de Campos and 1A. Chalfun-Júnior

1

Federal University of Lavras, Department of Biology, Sector of Plant Physiology, 37.200-000, Lavras – MG, Brazil. 2 Empresa Brasileira de Pesquisa Agropecuária/Gado de Leite, 36038-330 - Juiz de Fora, MG, Brazil

Storage in liquid nitrogen is a potential method for the preservation of plant species for unlimited periods of time. The objective of this work was to evaluate the nuclear DNA quantification of embryonic axes of Anadenanthera colubrina stored in liquid nitrogen. Embryonic axes were stored in the absence and presence of different concentrations of DMSO and glycerol. Nuclear DNA content and integrity were evaluated. Axes stored in cold chamber and in liquid nitrogen presented differences. The percentage of sub-G1 was relatively high, showing that the material was not perfectly cryopreserved since this fraction indicates of nucleus fragments or nucleus smaller than the standard. Intact nucleus was observed in all treatments (%G1). Axes stored in liquid nitrogen in the absence of cryoprotectors preserved the integrity of nuclear DNA. The use of chemical cryoprotectors resulted on a lower percentage of intact nucleus in embryonic axes. Higher concentrations of the cryoprotector may benefit the conservation of embryonic axes nucleus integrity of A. colubrina. Higher percentage of nucleus in G1 phase was observed using glycerol. Supported by FAPEMIG, CNPq and CAPES, Brazil Full name of presenting author: Diogo Pedrosa Corrêa da Silva E-mail: [email protected]

72

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

11. Evaluation of some physiological markers in Balkan endemic

Hypericum rumeliacum Boiss. regenerated after cryopreservation K. Danova1, M. Urbanová2, M. Skyba2, E. Čellárová2 and V. Kapchina1 1

Department of Plant Physiology, Faculty of Biology, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria 2 Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik University in Košice, Slovakia

Oxidative stress generated during the cryogenic treatment is considered as the major stress affecting vital cell functions of the plants. Reactive oxygen species (ROS) attack the lipid fraction of membranes, resulting in the formation of lipid peroxides which, being unstable, break down to form toxic secondary oxidation products. The aldehydic products of lipid peroxidation are especially cytotoxic and one of the most damaging, malondialdehdye (MDA), is known to impair cell function as it cross-links to macromolecules such as DNA and proteins. There is a considerable evidence to suggest that free radical mediated oxidative stress occurs during cryopreservation and in seed germplasm exposed to low-temperature storage [Hendry, G.A.F. 1993. Oxygen, free radical processes and seed longevity. Seed Sci Res. 3:141–153]. It can also occur in the other steps of cryopreservation protocols that are associated with desiccation and tissue culture manipulations [2. Robertson, L., W.J. Magill, E.E. Benson, D.H. Bremner and T.E.J. Buultjens. 1995. Oxidative stress in the plant tissue culture environment. Biochem Soc Trans. 23:263S]. In this communication we report on in vitro cultivated Balkan endemic Hypericum rumeliacum subjected to cryopreservation by means of vitrification. Shoot tip meristems were pretreated with 0.076µM ABA solution for 3, 7 and 10 days, cryoprotected with plant vitrification solution (PVS3) and immersed directly in LN. We are presenting the results related to the influence of cryopreservation on MDA and H2O2 levels in Hypericum rumeliacum, as markers of physiological status. These parameters are measured 10 months after their regeneration, in order to estimate the long-term recovery capacity of the plants subjected to freeze-injury stress. Estimation is presented on the basis of effectiveness of the pretreatment approaches. Full name of presenting author: Katalina Danova E-mail: [email protected]

73

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

12. Effect of DMSO on castor bean pollen grain viability 1

D. P. Vargas, 1R. Paiva, 1D. P. C. da Silva, 1A. C. A. L. Campos, 1G. F. Nogueira and 2M. L. M. de Carvalho 1

Federal University of Lavras, Department of Biology, Sector of Plant Physiology, 37.200-000 - Lavras - MG, Brazil. 2 Federal University of Lavras, Department of Agriculture, Sector of Seeds, 37.200-000 Lavras - MG, Brazil.

The castor bean (Ricinus communis L.) is an oleaginous plant of high economic value in Brazil due to its use in the industrial (biodiesel production) and medicinal areas. In this context, techniques of cryopreservation have been used in the preservation of the species considered as an important Brazilian genetic resource. The objective of this study was to evaluate the effect of the use of the cryoprotector DMSO in the maintenance of castor bean pollen grain integrity under different storage temperatures. Pollen grains were extracted from anthers of male flowers. The pollen grain viability was determined using the cytological dye acetic-carmim 1% and maintained in liquid nitrogen (-196º C), ultra freezer (-80º C) and freezer (-20 ºC) during 30 days in a cryoprotector solution of DMSO 10% (v/v). Viability of 85.5 and 86.5% was obtained in pollen cryopreserved at -80º C and -20º C, respectively. The maintenance in liquid nitrogen promoted high number of burst pollen. Pollen viability in all treatments was higher than 70%. Supported by FAPEMIG, CNPq and CAPES, Brazil Full name of presenting author:: Diogo Pedrosa Corrêa da Silva E-mail: [email protected]

74

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

13. Cryopreservation and survival ability of different Elite embryogenic

lines of Norway spruce(Picea abies (L.) Karst.) B. Dedicova*, U. Egertsdotter ** and O. Nilsson* * Department of Forest Genetics and Physiology, Swedish University of Agricultural Sciences SE- 901 83 Umea, Sweden. **School of Biology, Georgia Institute of Technology 500 Tenth Street, NW, Atlanta, GA.

Calli from six different Elite embryogenic cell lines of Norway spruce originated from the controlled crossing conducted by Skogforsk (Forest Research Institute of Sweden) have been cryopreserved in liquid nitrogen using the slow freezing method and sorbitol (0.2 and 0.4 M ) and DMSO (5%) as cryoprotectants. Freezing of samples was performed in a programmable freezer Cryo Med 7452 . The cryo vials were inserted at +4 ˚C and frozen at a rate 0.3 C˚ /min to -16 ˚C . A hold for 15 min at - 16 ˚C was inserted to avoid rapid cooling and crystal formation in the cells followed by further cooling to -35˚C at a rate of 0.3 ˚C/min . At the end of freezing program the vials were transferred and stored in a CBS V1500 liquid nitrogen storage unit at -196 ˚C. The results indicate that the cell survival after the storage in liquid nitrogen reached values between 40- 80%. After a short lag phase, the recovered tissues continued to grow on proliferation medium followed by transfer to pre- maturation and maturation medium. Somatic embryo maturation and plantlet regeneration occurred in all selected lines with different frequencies not related to survival rates after cryogenic storage. Full name of presenting author: Beata Dedicova E-mail: [email protected]

75

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

14. Cryopreservation of Byrsonima intermedia A. Juss. embryos using

different moisture contents 1

G. F. Nogueira, 1R. Paiva, 1M. A. de F. Carvalho, 1D. P. C. da Silva, 1L. C. Silva and R. C. Nogueira

2 1

Federal University of Lavras, Department of Biology, Sector of Plant Physiology, Lavras – MG, Brazil. 2 Federal University of Pará, 68.372-040 - Altamira, PA, Brazil.

Byrsonima intermedia is a native medicinal species of Brazilian Cerrado that presents difficulties of sexual propagation due to a reduced germination rate and seedling emergence. The cryopreservation technique of embryonic axes is a viable alternative for the conservation genetic diversity. In this context, the objective of this study was to evaluate the effect of freezing in liquid nitrogen, using the cryoprotector PVS2, of zygotic embryos of B. intermedia presenting 15% and 25% moisture contents. After isolation, embryos were disinfested and inoculated directly on the culture medium, prior frozen for 20 minutes in PVS2 solution or cryostored directly in liquid nitrogen. After submitted to these treatments, embryos were inoculated in WPM 50% and transferred to a growth room. No significant differences between the treatments and moisture contents were observed. Embryos inoculated directly in the culture medium showed 70% regeneration. Embryos directly immersed in liquid nitrogen presented low germination rate. The use of PVS2 promoted the in vitro regeneration embryos that were cryostored in liquid nitrogen. Supported by FAPEMIG, CNPq and CAPES, Brazil Full name of presenting author: Milene Alves de Figueiredo Carvalho E-mail: [email protected]

76

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

15. Cryopreservation of friable callus of Byrsonima intermedia A. Juss. 1

G. F. Nogueira, 1R. Paiva, 1M. A. de F. Carvalho, 1D. P. C. da Silva, 1D. P. Vargas and 2G. A. da Silva 1

Federal University of Lavras, Department of Biology, Sector of Plant Physiology, 37.200-000 - Lavras - MG, Brazil. 2 Federal University of Alfenas, Departarment of Pharmacy, 37.130-000 - Alfenas, MG, Brazil.

The B. intermedia is a Cerrado shrub which belongs to the Malpighiaceae Family. It is a medicinal plant rich in tannins, which has difficulties to be propagated sexually. One of the ways of multiplication of this species is through indirect somatic embryogenesis, though, due to successive cultivation the callus usually loose their embryogenic capacity. The objective of this work was to evaluate the effect of different cryoprotector solutions [A (MS + 2.0M glycerol + 0.4M sucrose), B (MS + 8% DMSO + 0.4M sucrose) and PVS2] on the process of cryopreservation of B. intermedia friable callus induced from leaf explants. After thawing, the callus were cultivated in MS medium supplemented with 1.0 mg L-1 2.4D, 30.0 g L-1 sucrose, 6.0 g L-1 agar and the pH adjusted to 5.8. Callus cryopreserved with cryoprotectors presented no differences resulting on an average increase of 16.2% in fresh matter. However, callus cryopreserved directly in liquid nitrogen, lead to a decrease in fresh matter of 20%. These results suggest the possibility of cryopreserve callus of B. intermedia at extremely low temperatures. Supported by FAPEMIG, CNPq and CAPES, Brazil. Full name of presenting author: Milene Alves de Figueiredo Carvalho E-mail: [email protected]

77

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

16. Cryopreservation of Juglans cinerea (butternut) dormant buds D. Ellis1, M. Ostry2, M. Moore2, B. Ambruzs1 and M. Jenderek1 1

USDA-ARS, National Center for Genetic Resources Preservation, 1111 S. Mason Street, Fort Collins, CO, USA 80521 2 USDA-FS, Northern Research Station, 1561 Lindig Street, St. Paul, MN USA 55108

Juglans cinerea (butternut) is a deciduous tree native to the United States and Canada with oblong-lemon shaped nuts with oily texture and pleasant flavor. In North America butternut is threatened by a canker disease caused by the introduced fungus (Sirococcus clavigignenti-juglandacearum) which has killed 90% of the native butternut in some areas. The USDA-Agricultural Research Service (ARS) National Plant Germplasm System maintains a germplasm collection of 26 selected accessions while the USDA-Forest Service (FS) maintains numerous field sites of selected accessions varying in canker resistance. The development of a cryopreservation protocol for dormant buds of butternut serves as a good model for the development of protocols for dormant bud cryopreservation of other woody plants. In 2006, a feasibility study was done on the cryopreservation of dormant butternut buds with five accessions and despite the use of subpar rootstock left over from other studies and limited replications for each treatment, four of the accessions had at least one out of ten buds survive cryopreservation. The first year further defined that survival after slow cooling to -35oC was greater when buds were dehydrated to 25-30%MC vs. 30-35%MC. In 2007 the focus was to increase sample sizes and survival rates. Utilizing the same five accessions, survival of buds after cryopreservation increased to 53% in one accession and ranged from 13% to 53%. Results from a third year (2008), were comparable to those obtained in 2007 with survival ranging from 20% to 53% in 2008). Chip grafting in butternut is difficult and could be a limitation in cryopreservation of butternut. However, as evidenced from these studies, the species is amenable to cryopreservation of dormant buds. Future work will focus on cryopreservation of both the ARS and the FS butternut collections. Full name of presenting author: Dave Ellis E-mail: [email protected]

78

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

17. Cryopreservation of French plant genetic resources collections

(CRYOVEG) F. Engelmann1,2, E. Balsemin3, T. Barreneche3, Ph. Chatelet4, J.-E. Chauvin5, E. Couturon6, F. Curk7, M.-A. Dantec5, J.-P. Dantec5, S. Dussert1, L. Feugey8, Y. Froelicher9, L. Fouilhaux3, F. Gamiette10, A. Grapin11, M. Grisoni12, Ph. Guérif8, A. Guyader8, A. Label5, F. Luro7, B. Moulin5, M. Muller5, A. Peyrière4, Y. Prigent5, M. Renard5, M. Roux-Cuvelier12, D. Roques13, S. Rubens10, J. Sapotille13, C. Souchet5 and D. Teyssedre12 1 : IRD Montpellier ; 2 : Bioversity International ; 3 : INRA Bordeaux ; 4 : INRA Montpellier ; 5 : INRA Ploudaniel ; 6 : IRD la Réunion ; 7 : INRA Corse ; 8 : INRA Angers ; 9 : CIRAD Corse ; 10 : INRA Guadeloupe ; 11 : INHP Angers ; 12 : CIRAD La Réunion ; 13 : CIRAD Guadeloupe

In France, cryopreservation is currently used for a limited number of forest tree species, but has been little applied yet to horticultural or other agricultural species. The CRYOVEG project aims at 1) developing or optimizing cryopreservation techniques in a range of selected species; 2) establishing a national scientific and technical network of plant biological resource centers (CRBs) using cryopreservation. The project has a network organization, with IRD/INRA Montpellier as the cryopreservation expertise centre and partners in continental France and overseas departments in charge of genetic resources conservation for various species: INRA Petit Bourg, Guadeloupe (yam); INRA San Giuliano, Corsica (Citrus); INRA Bordeaux (Prunus); INRA Angers (apple and pear); INRA Montpellier (grapevine); INRA Ploudaniel (potato, Brassica); IRD La Réunion (coffee); CIRAD Roujol, Guadeloupe (sugarcane); and CIRAD La Réunion (vanilla, garlic). Deliverables will vary depending on the state-of-the-art of cryopreservation for each species: for potato, yam, sugarcane, garlic, Citrus, Brassica and coffee, optimized protocols will be tested on a range of representative accessions. For grapevine, Prunus, apple, pear and vanilla, operational protocols will be developed. The group will work as a thinktank on the integration of cryopreservation in the national plant genetic resources conservation strategy and an action plan for its implementation will be collectively designed. The project will work towards establishing a French research network on plant genetic resource cryopreservation, open to all partners interested in cryopreservation. The group will prepare research and development projects whose results will be disseminated through European plant genetic resource conservation networks e.g. ECP/GR). Finally, network members will train French and foreign (from North and South countries) researchers and technicians in cryopreservation techniques. This project is supported by a French competitive grant (Call for Projects 2008 "Biological Resource Centers") of the Consultative Committee for Biological Resources / Infrastructures in the Biology, Health and Agronomy sectors (CCRB/IBiSA). Full name of presenting author: Florent Engelmann E-mail: [email protected]

79

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

18. Morpho-histological study of banana (Musa spp. cv. Grande Naine

[AAA]) cell suspension during cryopreservation during cryopreservation and regeneration F. Georget1, F. Engelmann2, 3, R. Domergue1 and F. Côte1 1: CIRAD, Boulevard de la Lironde - TA B-26 / PS4 - 34398 Montpellier Cedex 5 France. 2: IRD, 911 avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France. 3: Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.

A morpho-histological study of banana (Musa spp. cv. Grande Naine [AAA]) embryogenic cell suspensions was performed during cryopreservation and regeneration. It was demonstrated that the regeneration process of somatic embryos originating from cryopreserved cell suspensions was different from that of control cell suspensions. Somatic embryos originating from cryopreserved cell suspensions had a unicellular origin, whereas those originating from control embryos had a pluricellular origin. The regeneration process was modified not only by freezing in liquid nitrogen but also by the plasmolyzing effect of the 0.5 M sucrose solution employed during pretreatment. This result explained the high number of embryonic structures formed on medium M3, compared with control. Proembryos blocked at the globular stage could pursue their development when they were plated on new culture medium at a lower density after 30 days of culture on medium M3. The unicellular origin of somatic embryos produced from cryopreserved cell suspensions allows envisaging the use of cryopreservation to select non-chimeral transformed plants. Moreover, freezing selection of the more embryogenic cells would allow disposing of homogeneous, high quality material for transformation experiments. Full name of presenting author: Florent Engelmann E-mail: [email protected]

80

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

19. Progress in the Czech hop germplasm cryoconservation M. Faltus1, P. Svoboda2, J. Patzak2, J. Zamecnik1 and V. Nesvadba2 1

Dep.of Mol. Biol., CRI Prague, Drnovska 507, CZ16106 Prague, Czech Republic HRI Zatec, Kadanska 2525, CZ43846 Zatec, Czech Republic

2

Hop is traditional and important crop in the Czech Republic. To keep the Czech hop germplasm without risk of an accession lost the cryocollection of hops was established in the Czech Crop Cryobank (CCC) at the Crop Research Institute (CRI) in Prague. The process of hop cryoconservation includes collection of material in the field conditions, evaluation of its healthy state, plant material introduction into in vitro conditions, pathogens (viruses) elimination, explants multiplication, cryoprotocol application, explant cryoconservation and genetic stability verification. The hop collection in the Czech Republic is kept in the field conditions at the Hop Research Institute (HRI) in Zatec. Plant material is introduced into “in vitro” conditions to produce and to keep virus free plants. The material is tested on a presence of the most important pathogens according to EPPO scheme (ApMV, PNRSV, ArMV, HpMV, SLRV, CMV, PAMV, TNV and CLRV) of hop plants. Infected plants are involved into the process of pathogen eradication by a meristem culture. After virus elimination, the explants are multiplied and sent to the specialized cryo-laboratory at the CRI in Prague for their cryopreservation. The cryopreservation method is based on explants hardening with a low temperature, an osmotic adjustment with sucrose solution and following air-dehydration. The phytohormone study was done to improve plant recovery after cryopreservation. Hop accessions are stored in the CCC at the CRI. Plants recovered after cryopreservation are sent back to the HRI to verify the genotype identity by molecular methods (STS or SSR markers). Currently, the Czech hop collection at the HRI includes 327 accessions and 53 of them has been cryopreserved and stored in the CCC in Prague. This study was supported by research projects of the Ministry of Agriculture of the Czech Republic QF3039 and 0002700602. Full name of presenting author: Milos Faltus E-mail: [email protected]

81

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

20. Conservation of potato germplasm M. Faltus1, J. Domkarova2, L. Kreuz2, V. Horackova2 and J. Zamecnik1 1

Dep.of Mol. Biol., CRI Prague, Drnovska 507, CZ16106 Prague, Czech Republic PRI Havlickuv Brod, Dobrovskeho 2366, CZ58001 Havlickuv Brod, Czech Republic

2

Potato belongs to the vegetatively propagated crops without possibility of germplasm keeping in seed form. The potato germplasm collection stored in the field conditions is engaged by abiotic and biotic factors. To eliminate risk of genotype lost the Czech potato collection was introduced into in vitro-collection. Currently, whole potato germplasm collection is stored in in vitro-bank at the Potato Research Institute (PRI) in Havlickuv Brod. The potato collection is in the process of pathogen elimination by meristem cultures, thermotherapy and chemotherapy. Original sub-collection of old potato varieties of the Czech origin was recovered and the process of pathogen elimination was finished in this collection. ELISA method is used for detection of important viral pathogens (PVM, PVS, PVA, PVX, PVY and PLRV). To improve stability and safety of the potato collection, the cryo-collection of potato germplasm was established. The sub-collection of old potato varieties of the Czech origin was selected as the most important part of potato germplasm kept in Czech in vitro-bank of potato. Selected genotypes are multiplied and transferred to the Crop Research Institute (CRI) in Prague for cryoconservation. The cryoprotocol for potato cryopreservation is based on osmotic adjustment of explants with sucrose and following air-dehydration. Cryopreserved samples are kept in the Czech Crop Cryobank (CCC) at the CRI in Prague. Recovered potato explants are sent back to the PRI in Havlickuv Brod for genotype identity evaluation by morphological, biochemical and genetic markers. Currently, there are 2225 accessions of Solanum genus in in vitro-bank at the PRI in Havlickuv Brod and 50 potato vatieties of the Czech origin in cryo-collection at the CRI. This study was supported by research projects of the Ministry of Agriculture of the Czech Republic QF3039 and 0002700602. Full name of presenting author: Milos Faltus E-mail: [email protected]

82

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

21. Cryopreservation of mulberry winter buds in Japan K. Fukui, K. Shirata, I. M. Kashif and T. Niino Genebank, NIAS(National Institute of Agribiological SCienses), Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan

NIAS Genebank has 1,470 accessions of Morus species (mulberry plants) in field genebank, including M. bombycis K., M. latifolia, P., M. alba, L., M. australis P. and tropical/sub-tropical zone mulberries. Among these, 1,236 accessions were originated in template areas and were able to be applied for the technique of dormant buds preservation. Then, these accessions had been practically cryostoraged in LN tanks (vapor phase, -160 °C) from 2003 to 2008. The 160 mulberry winter buds per each accession, packed in 8 cryotubes, were cryostoraged following a protocol developed for mulberry winter buds. For practical storage, it is very important to confirm that cryopreserved shoot tips can maintain viability during long-term storage. We examined the survival rate of mulberry winter buds in LN tank stored for 11 years and obtained more than 70 % survival rate. Also, we checked the survival rates of 24 accessions, which were randomly selected 4 accessions each year from 1,236 accessions cryostoraged from 2003 to 2008. The survival rates of 24 accessions ranged from 70 to 100 %. These results show the technique of dormant bud preservation developed for mulberry is appropriate and mulberry plants can be preserved for long term in LN tank in safety. Full name of presenting author: Kuniaki Fukui E-mail: [email protected]

83

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

Application of anti-ice nucleation substances from deep supercooling xylem cells in trees in order to regulate freezing condition for cryopreservation 22.

D. Kami1, J. Kasuga2, T. Suzuki3 and S. Fujikawa2 1

National Agricultural Research Center for Hokkaido Region, Sapporo 062-8555, Japan Laboratory of Woody Plant Biology, Research Faculty and Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan 3 Laboratory of Horticultural Science, Research Faculty and Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan 2

Deep supercooling xylem parenchyma cells of trees contain diverse kinds of supercooling-facilitating (anti-ice nucleation) substances (Kasuga et al.: Plant Cell Env., 2008). Among them, several flavonol-glycosides, especially kaempferol-7-Oglucoside (KF7G), had the highest supercooling-facilitating activity. The KF7G was applied to improve survival of plant shoot apices that were cryopreserved by vitrification procedures. One of injurious factors that reduce survival of cryopreserved materials by vitrification procedures is chemical toxicity due to high concentration of vitrification solution (VS). Shoot apices of cranberry and hawthorn were seriously injured during treatment by 100% concentration of VS (PVS2 or PVS3) before freezing, and almost no survival was obtained after cryopreservation at practical cooling and warming rates. By reducing concentration of VS to 75%, injury by chemical toxicity was significantly reduced, but after cryopreservation the survival was poor. However, when KF7G was supplementary added to 75% VS, high survival was obtained after cryopreservation. It is suggested that dilution of VS reduced chemical toxicity of VS, but such diluted VS produced crystallization during cryopreservation. However, addition of supercooling-facilitating KF7G to diluted VS provided vitrification duringcryopreservation, resulting in high survival. It is expected that KF7G and the related compounds will be used widely for more successful cryopreservation by vitrification procedures in various biological systems. Full name of presenting author: Seizo Fujikawa E-mail: [email protected]

84

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

23. Testing of microplants from VIR in vitro collection for presence of endogenous bacterial contamination Y. Osledkin, S. Levchuk, V.F., S. Dunaeva, Y. Lupysheva, V. Rogovaya and T. Gavrilenko N.I.Vavilov Institute of Plant Industry (VIR), 42-44, B.Morskaya Street, St.-Petersburg, 190000, Russia

In vitro collection of VIR consists of 680 accessions of vegetatively propagated crops (potato, raspberries, blackberries, black currant, blue honeysuckle, cherries, garlic, onion). The microplants - source of meristems and shoot tips for cryopreservation have to be free from endogenous bacterial contamination because it can reduce the surviving and regeneration rates after cryopreservation. In connection with that screening for bacterial contaminants was done for microplants of small berry and fruit crops. Testing of microplants on the presence of bacterial contamination was performed based on protocol B.Reed, 1999. Two groups of microplants were used in this study: 1) visually non-infected, 2) contaminated plants (with ‘galo’-effect into the root area). We tested 171 accessions that were presented by 822 clones or by 1369 microplants of raspberries, blackberries and cherries. The solidified Viss medium and liquid LB medium were used for the testing procedures. The results has shown 87,6% of healthy microplants. 184 microplants had bacterial infections. 24 bacterial species were identified in the contaminated microplants using traditional bacteriological methods, most of them were belong to Arthrobacter, Bacillus and Pseudomonas species. Full name of presenting author: Tatjana Gavrilenko E-mail: [email protected]

85

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

24. SUCCESSFUL CRYOPRESERVATION OF MUSA AAA CV. GRANDE NAINE CELL SUSPENSIONS AND REGENERATION OF PLANTLETS F. Georget1, R. Domergue1, H. Etienne2, F. Côte1 and F. Engelmann3, 4 1: CIRAD, Boulevard de la Lironde - TA B-26 / PS4 - 34398 Montpellier Cedex 5 France. 2: CIRAD, c/o IRD, 911 avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France. 3: IRD, 911 avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France. 4: Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.

A cryopreservation protocol has been established for embryogenic cell suspensions of the triploid banana cv. Grande Naine, adapted from that developed by Panis et al. (1994). The main modifications concerned the following points: cell suspensions were precultured for 1 h in medium containing 180 g/l sucrose. Freezing took place in a Nalgene® box filled with isopropanol, which ensured an average cooling rate of 1°C/min between 0°C and -40°C, when the box was placed in a -80°C freezer. This simple freezing procedure avoided using a programmable freezer. After rapid warming in a 40°C water-bath, the content of the cryotubes was poured on a filter paper placed on recovery medium. After 20 days, the recovering suspensions were plated on regeneration medium at a 3% density (v/v), compared with 10% density for non-frozen control suspensions, and placed in a culture room at 28±1°C under a light intensity of 40 µmole/m2/s with a 12 h light/12 h dark photoperiod. The output of this cryopreservation protocol in terms of production of fully developed plantlets was close to 1. This simple and efficient method allows envisaging the establishment of pre-commercial cryopreserved stocks and constitutes a first step towards the control and management of the somatic embryogenesis process for this banana cultivar. Full name of presenting author: Fréderic Georget E-mail: [email protected]

86

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

25. Thermal events in calcium alginate beads during encapsulationdehydration and encapsulation-vitrification protocols R. Gámez-Pastrana1, Engelmann2

M.T.

González-Arnao1,

Y.

Martínez-Ocampo

and

F.

1. Universidad Veracruzana, Prolongación Oriente 6, No. 1009, CP 94340, Orizaba, Veracruz, México. 2. IRD, 911 avenue Agropolis, BP 64501, 34394, Montpellier Cedex 5, France.

The effect of dehydration conditions employed in encapsulation-dehydration (ED) and encapsulation-vitrification (EV) protocols on thermal events occurring during freezing was studied on empty calcium alginate beads using Differential Scanning Calorimetry (DSC). With ED, beads were submitted to 1 day pregrowth in liquid medium supplemented with 0.75M sucrose and desiccated for 1, 2, 4, 6, 8 or 24h with silicagel. With EV, beads were subjected to a loading treatment in 0.4M sucrose + 1M glycerol solution for 20 min, and then exposed to vitrification solutions PVS2 or PVS3 for 0, 5, 10, 15 or 30 min at 0°C or room temperature. Thermal analyses were performed using a DSC Q2000 V23.4 apparatus (TA Instruments), with a ramp of 5 °C.min-1 from 0°C to -70 °C and 10 °C.min-1 from -70°C to +70 °C. The thermal profiles obtained revealed that penetration of sucrose in beads during preculture with 0.75 M sucrose resulted in a decrease of the melting temperature from -0.65 °C to 4.14 °C, compared with that of untreated beads. Exposure of samples to PVS at 0°C produced a slower removal of osmotically active water from beads compared with water removal at room temperature. However, after 30 min exposure, all freezable water had been extracted from the beads, irrespective of the PVS employed and of temperature. It was observed that more freezable water was removed using PVS3, compared with PVS2 under the same treatment conditions. Beads dehydrated with PVSs displayed a glass transition (Tg) temperature below -65°C, while Tg of beads dehydrated with silicagel was above -50°C. Full name of presenting author: María Teresa González-Arnao. E-mail: [email protected] or [email protected]

87

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

26. Cryopreservation of in vitro-grown shoot tip of Morus spp. and Pyrus

cossonii Rehder by encapsulation dehydration S. Gupta Tissue culture and Cryopreservation Unit, National Bureau of Plant genetic Resources, New Delhi-110012 India

Encapsulation-dehydration technique was applied for standardizing a routine longterm conservation protocol of two diverse economical important crops namely, Morus spp. and Pyrus cossonii Rehder. Morus (mulberry) is important for the sericulture industry, being the main source of feed for silk worm (Bombyx mori); Pyrus (pear) is one of the most important temperate fruits. In Morus spp. (M. alba, M. indica and M. sinensis), the shoot tips explants were excised from 2 weeks cold acclimated plantlets, encapsulated in sodium alginate beads and pretreated in 0.1M, 0.3M and 0.75 M sucrose for 20 h, desiccated for 6 h under laminar flow so as to have ~20% moisture content, then plunged in liquid nitrogen and rapidly warmed. The osmotic dehydration in 0.75M sucrose solution was found the best because the encapsulated shoot tips showed 50-80% regrowth after air dehydration of beads. The shoot tips of M. indica and M. alba gave 35% regrowth while M. sinensis gave 30% regrowth after liquid nitrogen treatment. In P. cossonii Rehder, the shoot tips explants were excised from 1-week cold acclimated plantlets, encapsulated in sodium alginate beads and pretreated in 0.75M sucrose for 20 h. The beads were desiccated for 6 h under laminar flow then plunged in liquid nitrogen and rapidly warmed. Osmotic dehydration produced 60% regrowth. The 6 h air-dehydration of beads reduced the moisture content of beads to 17%. No regrowth of encapsulated explants was obtained either after air-dehydration or airdehydration followed by liquid nitrogen treatment. Full name of presenting author: Sandhya Gupta E-mail: [email protected]

88

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

27. Application of Cryopreservation Technique for Green house Grown

Biota orientalis L. Seeds 1

2

3

2

F. Hatami , M. A. Naderi Shahab , M. Tabari and A. Ghamarizare

1.Post-Graduate Student, Faculty of Natural Resources, Tarbiat Modares University, Noor, Iran 2.Research Institute of Forests and Ranges, Tehran, Iran 3.Faculty of Natural Resources, Tarbiat Modares University, Noor, Iran

Biota orientalis L. is an ever green forest species. Small and limited growth habitate, regeneration difficulties and degredation of growth sites, cause to pay more attention on conservation of this endangered species. Cryopreservation technique is an important approach to preserve plant seed, organ and cells, for long-term period at 196 ºC . In order to evaluate long-term preservation of Biota orientalis seeds, effects of cryoprotectants, such as Glycerol 30%, PVS2 and Desiccation, as pre-treatments was evaluated. The treated seeds transferred in to liquid nitrogen for periods of 7 days. Subsequently cryopreserved seeds taken out of liquid nitrogen, imposed to heat shock, transferred to green hous and caltivated to pot. The results including cryopreservation periods, effects of cryoprotectants and other treatments showed that, cryopreserved seeds germinate at highest rate and developed to shoots and roots normally. Overall results showed that, cryopreservation technology is an important approach to preserve the seeds of plant species and diversity as well as conservation of endangered species. This approach is most important regarding conservation of ecosystems and natural resources via preservation of plant species and biodiversity. Full name of presenting author: Firouzeh Hatami E-mail: [email protected]

89

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

28. Primary metabolism of abiotic stress underlying cryopreservation in

potato B. Criel, B. Panis*, M. Oufir, R. Swennen*, J. Renaut and J.F. Hausman Centre de Recherche Public - Gabriel Lippmann, Department 'Environment and Agrobiotechnologies' (EVA), Belvaux, GD Luxembourg *Faculty of Bioscience Engineering, Division of Crop biotechnics, KULeuven, Belgium

Cryopreservation complements classical conservation methods, which are carried out in the field or in vitro. It involves the storage of biological material in liquid nitrogen (-196°C). The aim of our work is to understand the effects of pretreatments, by increasing the osmotic pressure of the medium or by chilling exposure of the plantlets, on the cryopreservation ability of potato shoot tips. This works aims as well at trying to evaluate the effects of such pretreatments on the primary metabolism of potato. Indeed, drought acclimation is known to improve recovery after cryopreservation in potato and other species. Less is known about the effects of chilling exposure on this process. In vitro Désirée potato shoots were precultured for 21 days on regular MS and MS complemented with 0.055M, 0.11M, and 0.22M sorbitol for drought pretreatment or shoots were exposed for 21 days at 4°C on regular MS. Directly after pre-treatment, leaf and shoot tip samples were taken and stored at –80°C for carbohydrate analyses. In addition, cryopreservation was carried on the precultured shoot-tips. For the cryopreservation, potato shoot tips were cut from pre-treated plants and treated according to the droplet-vitrification technique according to the method developed by anis and coworkers (Panis B., Piette B. & Swennen R. (2005) Plant Sci. 168, 45-55). Thawing was done in a highly osmotic Recovery Solution at room temperature, to prevent osmotic shock. After cryopreservation, shoot tips were transferred into the dark for 1 week. During the initial days of post-culture, shoot tips were maintained on MS media containing 0.3M sucrose. Afterwards, regular MS media were used. After 30 days, recovery was calculated as the percentage of shoot-tips forming new shoot. The following carbohydrates and polyos were measured: sucrose, glucose, fructose, galactose, stachyose, arabinose, melibiose, maltose, trehalose, inositol, mannitol, galactinol and sorbitol. Carbohydrates were analyzed on a high performance anion exchanger chromatography couple with a pulsed amperometric detection. Results indicated changes affecting the primary metabolism. Further experiments including also chilling pretreatments will lead to a better understanding of the physiological status of the tissue and will hopefully explain the reason of the better results of cryopreservation. Full name of presenting author: Jean-Francois HAUSMAN E-mail: [email protected]

90

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

29. Desiccation and freezing studies of dormant buds from selected

horticultural woody plant species M.M. Jenderek1, J. Postman2, E. Stover3 and D. Ellis1 1

USDA-ARS, National Center for Genetic Resources Preservation, 1111 S. Mason Street, Fort Collins, CO, USA 80521 2 National Clonal Germplasm Repository, USDA-ARS, 33447 Peoria Rd., Corvallis OR 97333 3 National Germplasm Repository, USDA-ARS, Straloch Rd., Davis, CA 95616

Several reports have demonstrated the advantages of backing up field maintained collections in liquid nitrogen; however the application of cryopreservation protocols to a wide range of genetic diversity found in germplasm collections has only been reported in a few instances. Long-term preservation of dormant buds is less expensive than cryopreservation of shoot or meristem cultures; hence it presents an option for a back up of woody plant species. Dormant bud cryopreservation procedures can be species dependent but in general they require an ability of the buds to survive desiccation to 80% of sweet cherry, >21% of walnut and >12% of blueberry dormant buds survived desiccation and slow-cooling. In contrast, pomegranate dormant buds did not survive desiccation suggesting other preservation methods may be more applicable for this species. Dormant buds present a difficult experimental system due to many sources of variation which can confound results including the level of dormancy of the buds and the grafting step where success can depend on the personnel doing the grafting as well as the source and variety of the rootstock. Development of dependable cryopreservation protocols would increase the number of woody plant accessions being backed up at the National Center in a considerable shorter time than preserving the accessions via tissue culture material. Full name of presenting author: Maria Jenderek E-mail: [email protected]

91

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

30. Cryopreservation of Vanda coerulea protocorm-like bodies by

droplet-vitrification N. Jitsopakul1, K. Thammasiri2, 3 and K. Ishikawa4 1

Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. 2Department of Plant Science, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. 3Institute of Science and Technology for Research and Development, Mahidol University, Nakhonpathom 73170, Thailand. 4 Department of Research and Development, Japan Horticultural Production and Research Institute, Chiba 270-2221, Japan.

Vanda coerulea is the most popular Vanda species of Thailand that has become endangered because of wild orchid trade, deforestation and environmental changes. To preserve this orchid, droplet-vitrification was studied for cryopreservation of protocorm-like bodies. Protocorm-like bodies were developed from one protocrom in ND liquid medium supplemented with 1 mg/l BA in combination with 0.5 mg/l NAA and 30 g/l maltose. Protocorm-like bodies (3.0 mm in diameter) were precultured in ND liquid medium supplemented with 0.5 M sucrose for 1 day on a shaker, then dehydrated with loading solution for 15 min and exposed to PVS2 solution for 30 min at 25°C. The plant materials were then soaked in liquid nitrogen, rewarmed rapidly and cultured on solidified ND medium for recovery. After cryopreservation, the survival rate of cryopreserved protocorm-like bodies was 5%. The assessment of the genetic stability of cryopreserved protocorm-like bodies was developed using RAPD markers, size and sequence of the trnL (UAA) non-coding region of cpDNA. Result showed the same RAPD patterns of non-cryopreserved and cryopreserved protocorm-like bodies. The size and sequences of the trnL (UAA) non-coding region of cpDNA for non-cryopreserved and cryopreserved protocorm-like bodies were not different. There was no difference in morphology and similar patterns of ploidy levels of plantlets developed from non-cryopreserved and cryopreserved protocorm-like bodies. Full name of presenting author: Nipawan Jitsopakul E-mail: [email protected]

92

1st International Symposium on Cryopreservation in Horticultural Species

Thermal Analyses by Differential Cryopreservation of Potato Shoot Tips

31.

Scanning

Poster abstracts

Calorimetry

for

A. Kaczmarczyk, C. Zanke, A. Senula, M. Grübe and E.R.J. Keller Leibniz Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, 06466 Gatersleben, Germany

Differential scanning calorimetry (DSC) belongs to thermal analysis methods that can be used for measurement and determination of glass transitions and crystallization in tissues used for cryopreservation. The aim of this study was to analyze the physical state of water in potato shoot tips during cooling and rewarming applying the DMSO droplet method. Results should give insight into the function of the method. Thermal characterization was done with freshly isolated and cryoprotected shoot tips as well as for the applied solutions proper. Two accessions were compared. These were the frost-resistant, wild potato Solanum demissum and the frost-sensitive, cultivated potato S. tuberosum ‘Désirée’. Influence of cooling speed on thermal characteristics of free water was compared for slowly (10 °C/min) and ultra-rapid (ca. 12.000 °C/min) directly in liquid nitrogen cooled samples. Results showed glass transitions for the 10 % DMSO solution and cryoprotected samples of S. tuberosum ‘Désirée’. In contrast, S. demissum did not show glass transition. Freshly isolated and cryoprotected shoot tips as well as the cryoprotectant solution proper showed melting of ice during rewarming. Of the three investigated materials, the amount of freezable water was the smallest in the cryoprotected samples. It can be concluded that DMSO lowers the freezing point and the amount of freezable water in the tissue. The comparison of cooling speeds in S. tuberosum ‘Désirée’ showed two glass transitions during rewarming of samples cooled directly in liquid nitrogen in comparison to one transition in samples cooled within the DSC device. Therefore, it can be concluded that the DMSO droplet method is based on ultra-rapid cooling with partial ice formation in the tissue. Full name of presenting author: Anja Kaczmarczyk E-mail: [email protected]

93

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

32. Tomato pollen storage at freeze and cryogenic temperature. Effects

on viability and fecundity Ch. Karipidis and D. Douma Technological Educational Institute of Epirus, Department of Crop Production, Arta Greece

Storage at cryogenic temperatures is considered a successful method of retaining pollen viability in many plant species. Storage at -20° C has been also reported as a convenient method of pollen preservation with satisfactory results. In this study, the effects of two storage methods on tomato pollen viability and fecundity have been compared. Tomato pollen was stored at -20° C using CaCl2 as desiccant and at -196° C after modification of its moisture content at 6.5 -7% (dry/fresh basis). After 6, 8, 10, 12 and 18 months, in vitro pollen germination and pollen tube growth were tested following rehydration for 3 h in 100% R.H. at 15° C, and incubation for 6 h on semisolid substrate (1% agar, 12% sucrose and 50 ppm HBO3). Respiration activity was measured after storage for 6 and 12 months in liquid substrate (12% sucrose and 50 ppm HBO3). Fecundity of stored pollen was assessed by artificial pollinations conducted at the same time. Pollen stored at -20° C retained high in vitro germination for 12 months. However, after 6 months, pollen tube length and respiration activity declined with a negative impact on pollen fecundity, as indicated by the reduced number of seeds per fruit in fruits obtained by artificial pollination. Pollen germination ability significantly declined after 12 months. On the other hand, pollen stored at -196° C for 18 months exhibited similar in vitro germination, pollen tube growth and respiration activity with those recorded for fresh pollen. Furthermore, artificial pollination with cryogenically stored pollen resulted in similar fruit set and number of seeds per fruit to those observed after artificial pollination with fresh pollen. Full name of presenting author: Charalambos Karipidis E-mail: [email protected]

94

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

33. Cryopreservation of Embryonic Axes of Melia azedarach L. and

Camellia sinensis L. by Encapsulation-Dehydration B. Kaviani Department of Horticulture, Faculty of Agriculture, Islamic Azad University, Rasht Branch, Rasht, Iran

It is estimated that up to 100,000 plants are currently threatened or face extinction in the wild. Melia azedarach L. and Camellia sinensis L. was evaluated for long-term storage in LN at -196°C. Cryopreservation of germplasm in LN is a perfect method for the long-term conservation of plant genetic resources. Encapsulation within alginate beads was shown to be beneficial to the technique. Embryonic axes of M. azedarach and C. sinensis were encapsulated in MS medium supplemented with 3% Na-alginate, 100 mM CaCl2 and 0.75 M sucrose and desiccation for 1 h under the laminar air flow. Embryonic axes were plunged into LN and held for at least 1 h. Following cryopreservation, embryonic axes were cultured on solid MS medium. In M. azedarach survival after freezing was nil for non-pretreated, non-encapsulated embryonic axes, also nil for pretreated, non-encapsulated embryonic axes and 70% for pretreated, encapsulated embryonic axes. In C. sinensis, non-encapsulated embryonic axes even those pretreated with sucrose and dehydration did not survive. But pretreated, encapsulated embryonic axes may offer a better resistance after exposure to LN. To conclude, encapsulation may be suitable to reinforce the tolerance of tissues. Also the encapsulation-dehydration technique has been continuously improved and applied to an increasing number of species. Full name of presenting author: Behzad Kaviani E-mail: [email protected]

95

1st International Symposium on Cryopreservation in Horticultural Species

Cryopreservation of Lily [Lilium ledebourii Germplasm by Encapsulation-Dehydration

34.

Poster abstracts

(Baker)

Bioss.]

B. Kaviani Department of Horticulture, Faculty of Agriculture, Islamic Azad University, Rasht Branch, Rasht, Iran

In vitro conservation of germplasm is one of the best methods for long-term storage of valuable genetic resources of many crop and forest species. Cryopreservation at ultra-low temperature (-196ºC) is a perfect method for the long-term conservation of plant genetic resources. Cryopreservation of tissues can be successful only if intracellular ice crystal formation is avoided. Also encapsulation within alginate beads was shown to be beneficial to the technique. Lilium ledebourii (Baker) Bioss., is distributed in the Damash of Ammarloo and Kalchooleh of Dorfak areas (ca. 2100) of Guilan province in the north of Iran. Seeds of lily were isolated from the ripe capsules and disinfected. For encapsulation, seeds were suspended in MS medium supplemented with 3% (w/v) sodium alginate and 0.6 M sucrose. After 30 min seeds were picked individually and dropped into MS medium containing 100 mM CaCl2 (w/v) and 0.6 M sucrose for 30 min. Encapsulated and non- encapsulated seeds were desiccated in the air current of a laminar flow chamber for 1 h. For cryopreservation after dehydration samples were transferred into sterile polypropylene tubes which were directly immersed in LN for 1 h. Then frozen samples were thawed rapidly by placing the tubes into a water bath at 37ºC for 2 min and transferred directly to MS culture medium. Control seeds do not survive after LN treatment. The rate of viability in non- encapsulated seeds, pretreated with sucrose and dehydration was 25% . But if seeds is encapsulated in complement to sucrose and dehydration the survey is increased until 50%. To conclude, encapsulation may be suitable to reinforce the tolerance of tissues. Also the encapsulation-dehydration technique has been continuously improved and applied to an increasing number of species. Full name of presenting author: Behzad Kaviani E-mail: [email protected]

96

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

35. Development of alternative plant vitrification solutions in droplet-

vitrification procedures H.-H. Kim1, Y.-G. Lee1, D.-J. Shin1, H.-C. Ko1, J.-G. Gwag1, E.-G. Cho1 and Engelmann2,3

F.

1

2 National Academy of Agricultural Science, RDA, Suwon 441-707, Korea. Institut de recherche pour le développement (IRD), UMR DIAPC, BP 64501, 34394 Montpellier cedex 5, 3 Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), France. Rome, Italy.

This study aimed at developing alternative vitrification solutions, modified either from the original PVS2 vitrification solution by increasing glycerol and sucrose and/or decreasing dimethylsulfoxide and ethylene glycol concentration, or from the original PVS3 vitrification solution by decreasing glycerol and sucrose concentration. The application of these vitrification solutions to two model species, i.e. garlic and chrysanthemum in a droplet-vitrification procedure, revealed that PVS3 and variants were superior to PVS2 and variants and that most PVS2 variants were comparable to the original PVS2. Both species were sensitive to chemical toxicity of permeating cryoprotectants and chrysanthemum was also sensitive to osmotic stress. The lower recovery of cryopreserved garlic shoot apices dehydrated with PVS2 and variants compared with those dehydrated with PVS3 and variants seemed attributed to cytotoxicity of the vitrification solutions tested as well as to insufficient protection against freezing injury. The present study revealed that some of the PVS2 variants tested which have increased glycerol and sucrose and/or decreased dimethylsulfoxide and ethylene glycol concentration can be applied when explants are of medium size, tolerant to chemical toxicity and moderately sensitive to osmotic stress. PVS3 and variants can be used widely when samples are heterogeneous, of large size and/or very sensitive to chemical toxicity and tolerant to osmotic stress. Acknowledgements: This work was supported by a grant (20080401034060) from BioGreen 21 Program, Rural Development Administration, Korea. Full name of presenting author: Haeng-Hoon Kim E-mail : [email protected]

97

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

36. Development of alternative loading solutions in droplet-vitrification

procedures H.-H. Kim1, Y.-G. Lee1, S.-U. Park2, H.-J. Baek1, S.-J. Seok1, E.-G. Cho1 and F. Engelmann3,4 1

2 National Academy of Agricultural Science, RDA, Suwon 441-707, Korea. Chungnam National University, Daejon, 305-764, Korea. 3 Institut de recherche pour le développement (IRD), UMR DIA-PC, BP 64501, 34394 4 Bioversity International, Via dei Tre Denari 472/a, 00057 Montpellier cedex 5, France. Maccarese (Fiumicino), Rome, Italy.

In normal plant vitrification protocols, loading treatment (introduction of moderate concentrated cryoprotectants into tissues and cells) is preceded dehydration of explants with vitrification solution to reduce the toxicity which can be induced by direct exposure of samples to a highly concentrated vitrification solution. The study aims to develop alternative loading solutions, which are composed of glycerol and sucrose, applicable for droplet-vitrification procedure. Differential scanning calorimetry running of loading solutions and of explants loaded and dehydrated were applied to assay thermal events. Application of the loading solutions to two model species, i.e. garlic and chrysanthemum in droplet-vitrification procedure noted that loading treatment is beneficial for both garlic and chrysanthemum to increase the recovery growth of cryopreserved explants. But, responses to diverse loading solutions tested are different: the former has no preference, the latter is significantly influenced by composition of the loading solutions tested and a 35 %-diluted PVS3 solution was the best. The beneficial effect of loading treatment may osmotic neutralizer and/or physiological adaptation of tissues/cells, including membranes, before both dehydration and freezing. Acknowledgements: This work was supported by a grant (20080401034060) from BioGreen 21 Program, Rural Development Administration, Korea. Full name of presenting author: Haeng-Hoon Kim E-mail: [email protected]

98

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

37. Cryopreservation of different biological material obtained from plants

of genre Musa spp . S.Korneva, R. Maribona, J. Mendoza, F. Piña, O.Ruiz. and R.H.Maribona Escuela Superior Politecnica de Litoral (ESPOL) Biotechnology Research Center of Ecuador(CIBE),Guayaquil, Ecuador

Banana and plantains, tropical fruits of great acceptance in the international market, are an important source of food, being among the main economic lines in more than 100 countries, including Ecuador. The food shortage due to severe climatic changes, the spread of diseases and the demographic explosion prompt scientists to create different programs to obtain new varieties of Musa spp, with high crop yield and resistance. A gene bank is indispensable for that purposes. The cryopreservation of a biological material in liquid nitrogen is a simple and effective method for medium and long term storage (Panis, 2002). Cryopreservation not only allow the preserve the biologic material for genetic improve; it can be used as starting material for the rapid multiplication of plants in vitro for commercial purposes. The objective of this work was the cryopreservation in liquid nitrogen of apical meristems, scalps and suspension culture of Musa spp and the regeneration of plants from cryopreserved material. The biological material, -80 accessions of Musa spp received from the INIBAP Transit Center (Belgium)-, were multiplied in vitro in the MS medium supplemented with 2,25 mg/l of BAP. The apical meristems of 15 different accessions of 0,8-1mm, were explanted from rooted plants previously cultured during 6 weeks in the presence of sucrose at 6%. The scalps of 54 accessions were obtained through various subcultures of vitroplantlets microcorms in the MS medium supplemented with 2,2 mg/l TDZ and then were pretreated in the PCM medium(0,4M sucrose). The banana cell suspensions of William (national) and Calcutta - 4 varieties were established through disaggregation of embryogenic calli in the liquid medium. For inducing the tolerance in the cells for dehydration during the frost process, the cell suspensions were pretreated with different cryoprotective solutions, rapidly cooled and frozen in liquid nitrogen. After 30 minutes of cryopreservation at -196°C, the biological material was quickly thawed at 40°C and subcultured in the MS medium at the presence of sucrose at 0,3M for neoformation of embryoids and regeneration into plants. Obtained results showed variety dependence in the neoformation of plants from the meristems and scalps and have from 5 to 84,6% of plant regeneration rate and between 0 -72,2 % in the case of scalps. The regeneration of plants from embryoids formed in the cryopreserved cells of banana William and Calcutta-4 was more than 80%y 48% respectively. Full name of presenting author: Sofia Korneva E-mail: [email protected]

99

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

38. Long-term cryopreservation of Abies alba Mill. embryogenic cell lines J. Krajňáková1,2, H. Häggman 3, D. Gömöry4 and A. Vianello2 1

present address University of Udine, Dept. Biology and Plant Protection, Unit of Plant Biology, Via Cotonificio 108, Udine, 33100, Italy 2 permanent address National Forest Center, Forest Research Institute, T. G. Masaryka 22, Zvolen, SK-960 92 Slovakia 3 Department of Biology, University of Oulu, P.O. Box 3000, FIN-90014 Oulu, Finland 4 Technical University Zvolen, Faculty of Forestry, T. G. Masaryka 24, Zvolen, SK- 960 53 Slovakia

Conifer somatic embryogenesis, with the potential for capturing genetic diversity and providing for selection of elite genotypes, is emerging as a key component of advanced forestry programmes and cryo-preservation is an active part of them. Abies alba Mill., in past the most productive indigenous forest tree species in Central Europe, has exhibited a serious decline as a result of its sensitivity to drought and other environmental factors. It became very quickly the subject of tree breeding programs and biotechnological methods being the active component of them. Twelve embryogenic cell lines of silver fir were cold-hardened on MS-based proliferation medium, pre-treated on proliferation medium with 0.2 M sucrose for 24 h and with 24 h for additional 24 h. During the cryo-preservation procedure, samples were incubated in series of polyethylene glycol (PEG), glucose, and DMSO solutions of increasing concentrations and were frozen using a programmable controlled temperature cooling chamber, as described by Aronen et al. (1999). The samples were thawed after 6 years of storage in liquid nitrogen. The recovery of samples was assessed by means of proliferation rate after 3, 5 and 7 week after thawing and microscopical analysis of embryogenic tissue. Four out of twelve cell lines reached the stable proliferation rate 3 months after thawing. The recovered embryogenic cell lines were subjected to series of maturation experiments. Regeneration of plantlets was obtained with one cell line. Two cell lines have not responded positively to maturation treatments and continued with proliferation. These are the first data presenting the long-term cryo-preservation of embryogenic cell lines of A. alba. Full name of presenting author: Jana Krajňáková E-mail: [email protected]

100

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

39. Preparative culture medium and procedures affect the success of

cryopresevation of Dioscorea opposita Thunb. cv. Tiegun plantlet nodes using encapsulation vitrification M. J. Li, X.T. Zhao, X.L. Zhang, H.B. Li, P.P. Song, C.Y. Zhou, X. Li and M.M. Li College of Life Sciences, Henan Normal University, Xinxiang 453007, China.

The effect of preparative culture medium and procedures on the success of cryopreservation with encapsulation vitrification of Dioscorea opposita Thunb. cv. Tiegun plantlet nodes, which is a famous Chinese medicinal plant, had been probed into in this paper. All the preculture experiments were carried out at 4 ℃. The results showed that: preparative culture medium and procedures had significant effect on relative viability and survival rate of nodes after cryopreservation by encapsulation vitrification; Preculturing the nodes for 3 d on the Murashige-Skoog(MS) medium containing 0.2 mol·L-1 sucrose (Suc) , glycerol(Gly), inose, glucose, mannitol, fructose respectively and 0.2 mol·L-1 Suc 50 g·L-1 DMSO, nodes on the preparative culture medium containing 0.2 mol·L-1 Suc had the best highest relative viability (82%) and survival rate(41%) after cryopreservation. As for preparative procedures, the four kinds procedures were designed: (1) the nodes were precultured for 1 d in turn on the MS containing 0.3 mol·L-1, 0.5 mol·L-1and 0.75 mol·L-1 Suc respectively, (2) the nodes were precultured for 1 d in turn on the MS containing 0.1 mol·L-1 0.2 mol·L-1 and 0.3 mol·L-1 Suc respectively, (3) the nodes were pre-cultured for 3 d on the MS containing 0.2 mol·L-1 Suc, (4) the nodes were precultured for 3 d on the MS containing 0.1 mol·L-1 Suc + 0.1 mol·L-1Gly. Comparative analysis, it was found that the nodes being precultured for 3 d on the MS containing 0.1 mol·L-1 Suc + Gly 0.1 mol·L-1 could got the best highest relative viability (89%) and survival rate(52%) after cryopreservation. So, in order to obtain the success of cryopreservation, plantlet nodes should be precultured on the MS containing 0.1 mol·L-1 Suc and 0.1 mol·L-1Gly for 3 d at 4 ℃. Full name of presenting author: Mingjun Li E-mail: [email protected]

101

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

40. Pollen Cryopreservation of Camellia B. Li1,2, H. Wang1,2 and Y. Liu1,2 1College of Landscape Architecture Beijing Forestry University –Beijing – 100083 - China 2 National Engineering Research Center For Floriculture

Camellia is one of the traditional famous flowers in China with numerous species and cultivars. Pollen is importance for camellia breeding and germplasm preservation. Pollen from three species (C. chekiangoleosa Hu, C. plexicaulis Chang and C. euryoides Lindl.) and four cultivars (C. japonica ‘DaHongJinXin’, ‘DaHuaJinXin’, ‘LiangYeJinXin’ and ‘ZiHuDie’ ) was cryopreserved in liquid nitrogen for evaluating the reliability of germplasm long-term preservation in camellia. In vitro germination of fresh pollen and pollen stored in LN2 for one or five years was compared by hanging drop method , The liquid medium contains 15% sucrose and 0.3% H3BO3. The germination lever of cryopreserved pollen in LN2 for one year was not diminished than that of the fresh one. But after five years’ storage in liquid nitrogen, pollen germination level of most species and cultivars decreased significantly except ‘LiangYeJinXin’, however, the germination lever of all the pollen still exceeded 16%. Field pollination with one-year cryopreserved pollen showed no significantly difference from fresh pollen in fruit and seed set. Full name of presenting author: Liu Y E-mail: [email protected]; [email protected]

102

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

41. Cryopreservation as a tool for the management of coconut

germplasm B. Malaurie1,*, O. N’Nan3, HD. D. Bandupriya4, M. Borges5, J-L Verdeil2 and J. Tregear1 1

IRD, Palm Development Group, UMR DIAPC, F-34000 Montpellier, France,. 2CIRAD, UMR DAP, F-34000 Montpellier, France. 3Université d’Abidjan Cocody, laboratoire de génétique, UFR Biosciences 22 BP 582 Abidjan 22 Côte d’Ivoire. 4Coconut Research Institute, Tissue Culture Division, LUNUWILA, 61150, Sri Lanka. 5 Universidad de Granma, Facultad de Ciencias Agrícolas, Centro de Estudios de Biotecnología Vegetal, Bayamo 85 100, Granma, Cuba.

The feasibility of coconut zygotic embryo cryopreservation has been confirmed using 10 different ecotypes. Because of the presence of phloem vascular bundles in the haustorial part of the zygotic embryo, this material is suspected to be capable of transmitting phytoplasma, the lethal yellowing disease agent. Histological observations of plumule (shoot apical meristem and leaf primordia) tissues have shown that they are characterized by the presence of an undifferentiated vascular system consisting of procambium. This characteristic should limit the possibility of phytoplasma transmission and offer a best guarantee of plant health. Cryopreservation of plumules by the encapsulation-dehydration technique allows successful (20%-40%) regrowth level into leafy shoots, and the potential to regenerate a whole plant (approximately 90% success rate) free of diseases. The approach described here should therefore increase the scope for obtaining material free of pathogenic agents, an essential prerequisite for the conservation and exchange of germplasm. Full name presenting author: Bernard Malaurie E-mail: [email protected]

103

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

42. Flow cytometry and DNA molecular analysis confirmed true – to -

type regenerants of frozen Gentiana kurroo cell suspension. A. Mikuła*, K. Tomiczak*, P. Bednarek** and J.J. Rybczyński* *Botanical Garden – Center for Biological Diversity Conservation PAS, Prawdziwka 2 str., 02973 Warsaw, Poland ** Institute for Plant Breeding and Acclimatization, 05-870 Błonie, Radzików, Poland

The aim of presentation is to show the uniformity of regenerants obtained in post thawing culture of frozen G. kurroo cell suspension. Derived from hypocotyl callus. The culture was maintained in MS(1962) basal liquid medium supplemented with 1.0 mg/l Dicamba+ 0.1 mg/l NAA + 2.0 mg/l BAP + 80 mg/l AS (MM medium). Two different cell suspension were analysed. First one being frozen with the help of LN for 18 months long period and second one only 48 hrs treated. Fractioned cell suspension aggregates closed in alginate beads were performed as experimental material for both control and NL2 treated. Post – thawing and control cultures were initiated by 14 day long agar culture of beads what resulted in the initial cell proliferation. To stimulate process of early somatic embryogenesis beads were transferred to the liquid culture consists on MM medium. After next four weeks newly formed structure were subcultured on agar regeneration medium (RM medium) composed of MS supplemented with 0.5 mg/l GA3 + 1.0 mg/l Kin + 80.0 mg/l AS. Later, completely formed somatic embryos were transferred into jars with hormonefree 0.5MS medium for plantlet achieving. Regenerants presented no differences in total DNA content measured in nucleuses of leaf blade mesophyll cells. All analysed plants contained 5.89 ± 0.1 pg DNA. Preliminary results of metAFLP analysis indicated that site methylation changes induced higher variation than DNA sequence mutations . The regenerants derived from cryopreserved tissue differed from control in 3% based on Acc65I/MseI data. At the DNA sequence level. regenerants that cell suspension underwent cryopreservation differed from those which suspension was not kept in LN in about 0.04% as depicted by KpnI/MseI based data. Full name presenting author: Anna Mikula E-mail: [email protected]

104

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

43. Cryopreservation of the Ilex dumosa (Aquifoliaceae) germplasm N. Dolce, H.Y.Rey and L.A. Mroginski Instituto de Botánica del Nordeste (IBONE) , Facultad de Ciencias Agrarias ( UNNE), Sargento Cabral 2131, Corrientes (3400), Argentina.

Most of the subtropical Ilex species have recalcitrant seeds. and therefore not suitable for long term preservation using conventional seed storage methods. Thus, the germplasm of Ilex spp. is maintained in the field as ex–situ genebanks. This work describes experiments demonstrating the feasibility of long-term conservation of I. dumosa var. dumosa R. through cryopreservation of both whole seeds or zygotic rudimentary embryos. Lately, this specie has received the most attention from plant breeders because, besides of the quality of its leaves for making the stimulatory beverage named “mate” with less caffeine than the ones from I.paraguariensis, the plants are resistant to some pests. For cryopreservation of zygotic embryos and apical shoot tips: The explants were aseptically removed, encapsulated in 3% calcium alginate beads and pretreated at 24 h intervals in liquid medium supplemented with progressively increasing sucrose concentrations (0.5, 0.75 and 1 M). Beads were dehydrated for with silicagel to 25% water content and then placed plunged rapidly in liquid nitrogen. The beads were rewarmed by immersion in a water bath at 30ºC. Finally, beads were transferred onto culture medium (1/4MS, 3% sucrose, 0.1 mg/l Zeatin, ( solidified with 0.8% agar) and incubated in a growth room at 27 ºC under a 14 h light (116 mol. m-2.s-1)/ 10 h dark. By culturing cryopreserved embryos, as much as 42% of them produced plants. However, no plants were recovery when apical shoot tips were cryopreserved. In addition, a procedure for cryopreservation of mature seeds of I.dumosa by desiccation with silicagel y rapid cooling was developed. As much as 40% of the cryopreserved seed. Full name of presenting author: Luis Mroginski E-mail: [email protected]

105

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

44. Applications of differential Scanning Calorimetry in developing

cryopreservation strategies for Parkia speciosa, a tropical tree species J. Nadarajan1,2,a, M. Mansor2, B. Krishnapillay2, H. Staines1 , E. Benson3 and K. Harding3 1

University of Abertay Dundee, Bell Street. Dundee DD1 1HG, Scotland, UK Forest Research Institute of Malaysia (FRIM), Kepong, 52109 Kuala Lumpur, Malaysia 3 Research Scientists, Conservation, Environmental Science and Biotechnology, Damar, Drum Road, Cupar Muir, Fife, KY15 5RJ, Scotland, UK a (Current contact address) Royal Botanic Gardens Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK 2

Shoot-tips of Parkia speciosa, a recalcitrant seed producing tropical leguminous tree withstood cryopreservation using encapsulation-vitrification in combination with trehalose preculture. Differential Scanning Calorimetry (DSC) revealed that trehalose moderated the thermal characteristics of the shoot-tips. A 30 min PVS2 treatment had the lowest glass transition temperature (Tg) (-50.21 ± 1.07ºC) when applied in combination with 5% (w/v) trehalose. The Tg increased to -40.22 ± 0.95ºC as the sugar’s concentration was decreased to 2.5% (w/v). Tg heat capacity for shoot-tips treated with 2.5% and 5% (w/v) trehalose and exposed to PVS2 for 30 min increased from 0.17 ± 0.05 to 0.23 ± 0.01 J.g-1 respectively. Enthalpies of the melt-endotherm varied in proportion to trehalose concentration, for the 30 min PVS2 treatment, whereas the melt enthalpy for control shoots was >150 J.g-1 and decreased to ca. 60 J.g-1 with 2.5% (w/v) trehalose. For 5% and 10% (w/v) trehalose treatments, enthalpy declined to ca. 24 and 12 J.g-1 respectively and freezing points were depressed to – 75ºC and –85ºC with 2.5% and 5% trehalose (w/v), respectively. DSC elucidated the critical points at which vitrification occurred in germplasm exposed to trehalose and PVS2. A 60 min PVS2 treatment supporting ca. 70% survival was found optimal for stable glass formation during cooling and on rewarming. Full name of presenting author: Jayanthi Nadarajan E-mail: [email protected]

106

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

45. Cryopreservation of In vitro Grown Black Chokeberry Shoot Tips by

Droplet Vitrification D. Tanaka1, T. Matsumoto2, A. Nishiuchi1 and T. Niino1 1 2

Gene Bank, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan Shimane Agricultural Technology Center, Izumo Shimane 693-0035, Japan

The shoot tips from in-vitro grown black chokeberry (Aronia melanocarpa Elliot) using droplet vitrification procedure were successfully cryopreserved. The 30 day coldhardened shoots were excised and precultured on a solidified MS medium containing 0.3 M sucrose for 1 day at 5ºC. Then the shoot tips were treated with LS for 30 min at 25ºC, and dehydrated by PVS2 for 15 - 55 min at 25ºC before immersed into nitrogen slush (-210ºC). The survival rate of vitrified shoot tips were 70-80% after 30 days regrowth. Even only 15 min dehydration by PVS2 treatment followed droplet vitrification was enough to obtain high survival rate, in spite of no survival by vitrification protocol. The spectrum of optimal exposure time in droplet vitrification protocol was widened in comparison with one in vitrification protocol. This droplet vitrification protocol appears to be an applicable technique for cryopreservation of plant genetic resources. Full name of presenting author: Takao Niino E-mail: [email protected]

107

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

46. Cryopreservation of embryonic axes of castor bean cv. AL-Guarany

2002 G. F. Nogueira, R. Paiva, A. C. A. L. Campos, F. C. Nery, D. P. Vargas and P. D. de O. Paiva Federal University of Lavras, Department of Biology, Sector of Plant Physiology, 37.200-000 Lavras - MG, Brazil.

The castor bean (Ricinus communis L.) is an oleaginous plant with outstanding social and economical importance. Castor bean seeds tolerate storage on seed banks for a lower period compared with orthodox seeds, probably due to its high lipid content. The objective of this study was to elaborate a cryopreservation protocol of castor bean embryonic axes. After seed disinfection, embryonic axes of the cultivar ALGuarany 2002 were isolated, dehydrated and inoculated directly in the culture MS medium (control) or cryostored in liquid nitrogen at -196º C, during one hour, in the presence or absence of cryoprotector solutions (PVS2 or PVS2 modified) prior inoculation in MS medium supplemented with 30.0 g L-1 sucrose, 6.0 g L-1 agar, and pH adjusted to 5.8. The results showed 100% in vitro regeneration for all treatments. After acclimatization however embryonic axes cryostored only in liquid nitrogen presented a surviving rate of 100%. Axes stored in the presence of PVS2 presented 35% survival rate. Supported by FAPEMIG, CNPq and CAPES, Brazil. Full name of presenting author: Gabriela Ferreira Nogueira E-mail: [email protected]

108

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

47. Ultra structural aspects of Anadenanthera colubrina (Vell.) Brenan

embryonic axes cryopreservation 1

F. C. Nery, 1R. Paiva, 1G. F. Nogueira, 1D. P. C. Silva, 1D. P. Vargas and 2E. Alves

1

Federal University of Lavras, Department of Biology, 37.200-000 - Lavras - MG, Brazil. Federal University of Lavras, Department of Phitopathology, Lavras - MG, Brazil.

2

Cryopreservation techniques have been used to conserve seeds of several species for long periods in germoplasm banks. The use of this technique is important mainly to species that present difficulties to be propagated sexually or are considered endangered. It this study, ultra structural aspects of Anadenanthera colubrina embryonic axes cells were analyzed, prior (control) and after cryostorage in liquid nitrogen (-196ºC), -80ºC and -20ºC and also embryonic axes cryopreserved in liquid nitrogen using the cryoprotectors DMSO 15% and glycerol 15%. Observations were performed in axes maintained for 30 days in these conditions, fixed in modified Karnovisky with sections examined in a Zeiss (model EM 902 at 80Kv) electronic microscope of transmission. The results show no apparent damage after cryopreservation at the different temperatures, being possible to observe the presence of bodies of similar size. However, the pattern of distribution of these bodies was modified occurring more randomly in the cells possibly due to the freezing effect on the cytoskeleton, causing disturbs in the distribution of cell components. The embryonic axes stored at temperatures below zero using cryoprotectors presented no regeneration, although there were no evident visual signals of necrosis, confirmed by ultra structural analyses. Cell alterations during cryopreservation of embryonic axes may occur due to the disorganization of cell content and cells damages such folds in the cell wall and cytoplasm fragmentation. These results suggest that the cytotoxic effect of cryoprotectors in contact with the cell membrane may result on irreversible damages to the plasmatic membrane. Supported by FAPEMIG, CNPq and CAPES, Brazil. Full name of presenting author: Gabriela Ferreira Nogueira E-mail: [email protected]

109

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

48. Two-step preconditioning - a feasible method for cryopreservation of

Fortunella polyandra shoot tips using vitrification technique D.C. Chandrabalan1, M.N. Normah1,2 and M.M. Clyde1 1

Faculty of Science and Technology,2 Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600UKM, Bangi, Selangor, Malaysia

A study was conducted to develop an optimized methodology for cryopreservation of in vitro derived Fortunella polyandra shoot tips using vitrification-based techniques. Cryopreservation of F. polyandra shoot tips was successfully performed with the emphasis placed upon the aspects of preconditioning involving the pregrowth and preculture of explants. Preculture alone on Murashige and Skoog (MS) medium with 0.3M sucrose for one day was not sufficient in giving survival after cryopreservation. A preliminary experiment involving pregrowth on MS medium with 0.72M sucrose for three days produced a survival of 23.4% after cryopreservation. The usage of a combination of cryoprotectants in the preculture medium (MS with 0.3M sucrose and 0.5M ethylene glycol) proved to be beneficial in enhancing survival percentage of cryopreserved explants up to 40% when precultured for one day. The highest percentage of shoot tip survival after cryopreservation (56.6%) was achieved when two-step preconditioning method (pregrowth on MS medium with 0.72M sucrose for three days followed by preculture on MS medium with 0.3M sucrose for one day) was employed. Full name of presenting author: Normah M. Noor E-mail: [email protected]

110

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

49. A preliminary study of droplet vitrification method on lilac

cryopreservation Nukari, J. Laamanen and M. Uosukainen MTT Plant Production Research, Antinniementie 1, FI-41330 Vihtavuori, Finland

MTT Plant Production Research had the need to transfer some lilac (Syringa sp.) accessions from active vegetative nursery propagation to long-term preservation. Droplet vitrification method was tested for cryopreservation of lilac. This method has already been used at MTT for cryopreservation of raspberry and strawberry. At present, virus free mother plants of lilac are maintained at MTT in an isolated greenhouse. In vitro cultures, maintained at normal growth conditions, are being used for propagation. Cryopreservation is an alternative for long-term preservation of the lilac genetic resources. Apical buds of a virus tested Hungarian lilac type (Syringa sp.) were excised from micropropagated plantlets. Buds were pretreated on high sucrose Murashige and Skoog gelrite-gelled medium for three days, either with stable or stepwise increasing sucrose concentrations (0,25 M, 0,50 M and 0,75 M sucrose). Buds were vitrified using loading solution treatment for 30 minutes and PVS2 solution treatment for 45 minutes and frozen on aluminium foil strips. Pretreatment on stable 0,50 M sucrose MS plates induced the best regrowth rates. No benefit was achieved by using preculture on stepwise increasing sucrose concentration. As a conclusion, droplet vitrification combined with sucrose pretreatment was a suitable method for long-term cryopreservation of lilac. Further research is needed to obtain an optimal regeneration medium for lilac. Full name of presenting author: Anna Nukari E-mail: [email protected]

111

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

50. Effect of glycerol on castor bean pollen grain viability under different

periods of storage 1

D. P. Vargas, 1R. Paiva, 1G. F. Nogueira, 1F. C. Nery, 1J. M. P. Porto and G. A. da Silva

2 1

Federal University of Lavras, Department of Biology, Sector of Plant Physiology, 37.200-000 - Lavras – MG, Brazil. 2 Federal University of Alfenas, Departarment of Pharmacy, 37.130-000 - Alfenas, MG, Brazil.

The cultivation of castor bean (Ricinus communis L.) in Brazil has been considered strategic due to an enormous increase of its use in the production of biodiesel. The techniques of cryoconservation show a great potential to preserve cultivars or varieties of this species. Considering the importance of using pollen grains as conservation elements of genetic resources, the objective of this study was to develop a protocol to cryopreserve male gamete structure of Ricinus communis L. Cytological viability of pollen grains using acetic-carmim 1% as dye was evaluated. Pollen grains with 5-7% relative moisture content cryopreserved on a 10%glycerol solution were stored in cryotubes (2.0 mL) during 1hour, 15 days, 30 days and 180 days at -196 ºC in liquid nitrogen. Viable, non-viable and burst pollen grain number were analyzed. Pollen grains stored for one hour in liquid nitrogen showed higher viability and lower number of burst pollen. Viability higher than 70% was observed on pollen cryopreserved up to 180 days. Supported by FAPEMIG, CNPq and CAPES, Brazil Full name of presenting author: Renato Paiva E-mail: [email protected]

112

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

51. Cryopreservation of in vitro shoot bases of Allium tuberosum (L.)

Rottl. ex Spreng. by vitrification technique R. Pandey, A. Pandey*, N. Sharma and K.S. Negi** Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi-110012, India *Germplasm Exploration Division, NBPGR, New Delhi-110012, India **NBPGR Regional Station, Bhowali (Uttarakhand), India

The preservation of valuable germplasm of crops and their wild relatives is important for the maintenance of a broad genetic base in order to meet the future demand. Chinese chives (Allium tuberosum Rottl. ex Spreng.), a non-bulbous species, is cultivated for food and medicine in India and Southeast Asia. Chinese chives germplasm has been maintained in field and in vitro gene bank at NBPGR headquarter at New Delhi and at its regional station in Bhowali, respectively. However, there are many difficulties with in vitro storage, including risks of loss of material due to contamination, high maintenance cost and space requirement and the possible occurrence of somaclonal variation. There are no reports in the literature regarding cryopreservation of this medicinally important Allium species. Shoot bases obtained from 3 or 4 week-old in vitro plantlets of Chinese chives (Allium tuberosum Rottl. ex Spreng.) were successfully cryopreserved by vitrification. Shoot bases (about 2 mm in length ) were precultured on MS medium containing high sucrose, for 2 days at 250C. They were then dehydrated with the PVS2 solution for 10-40 mins at 250C. The dehydrated shoot bases were directly immersed into liquid nitrogen. After rapid warming in water at 420C, the shoot bases were quickly washed with 1.2M sucrose solution and then plated on a solidified culture medium. Successfully vitrified shoot bases resumed growth and developed shoots without intermediary callus formation. This vitrification procedure is very simple and appears promising for the long term conservation of A. tuberosum germplasm. Present paper highlights the factors affecting in vitro cryopreservation of A. tuberosum, at NBPGR in India. Full name of presenting author: Ruchira Pandey E-mail: [email protected]

113

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

52. Cryopreservation by encapsulation of in vitro grown shoot tips of

rosa ‘New Dawn’ B. Pawłowska and A. Bach Department of Ornamental Plants, University of Agriculture, Al. 29 Listopada 54, 31-425 Krakow, Poland

In the present studies, the protocol of preservation in liquid nitrogen by encapsulation- dehydratation method of shoot meristems park Rosa ‘New Dawn’ has been developed. Shoot apical and axillary meristems about 4 mm in diameter were collected from plants propagated in vitro in the laboratory of the Department of Ornamental Plants on the mineral medium according to Quoirin et al. (1977), supplemented with 5 µM BA, 0.3 µM GA3, 0.5 µM NAA and sucrose at concentration 0.06 M. Moreover, some explants for cryopreservation were pre-cultured on the medium containing higher sucrose level (0.25 M) for 8 weeks. After encapsulation plant material was dehydrated by quick method (capsules were placed in liquid medium containing 0.75 M sucrose for 18 h) or by gradual method (capsules were transferred to liquid solutions of media with increasing sucrose concentrations from 0.3 M to 1 M for consecutive 7 days). The obtained results indicate that survival rate of plant tissue after freezing in liquid nitrogen depended on plant material: apical meristems of roses regenerated callus and shoots but axillary meristems did not survive the freezing. Cutting capsules after freezing did not influence regeneration of the cryopreserved plant explants. It was shown that slow dehydration method, used for preparation of plant material for cryopreservation, allowed to obtain a viable tissue of Rosa ‘New Dawn’. Full name of presenting author: Bozena Pawlowska E-mail: [email protected]

114

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

53. Cryopreservation of Fragaria x ananassa cv. ‘Senga Sengana’ by

droplet-vitrification – Effects of sucrose preculture I. Pinker1, A. Halmagyi2, K. Olbricht3,4 and F. Ehrig4 1

Humboldt University, Institute for Horticultural Sciences, Albrecht-Thaer-Weg 1, 14195 Berlin, Germany 2 Institute of Biological Research, 400015 Cluj-Napoca, Republicii str. 48, Romania 3 Hansabred GmbH & Co. KG, Radeburger Landstrasse 12, 01108 Dresden, Germany 4 JKI – Federal Research Centre for Cultivated Plants, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany

Shoot tips (2 - 3 mm in length) of micropropagated strawberry (Fragaria × ananassa DUCH. cv. ‘Senga Sengana’) plants were precultured in sucrose enriched media for 24 and 48 h. Subsequently, shoot tips were transferred into 6 µl droplets of PVS2 vitrification solution for 20 min and plunged afterwards into liquid nitrogen. The effect of sucrose concentration (0.1, 0.25, 0.5, 0.75, 1.0 M) used in preculture of the shoot tips regarding their regrowth, histological changes and the morpholocical stability of the regenerants in the field was analysed. After freezing, the highest recovering rate (60%) was achieved after a preculture with 0.25 M sucrose for 24 h. The number of off-type plants after acclimatisation was clearly affected by the sucrose concentration. After preculture in 0.1 M sucrose no off-types were observed, while after preculture in the highest sucrose concentration the number of off-types reached 20%. This fact could be of common interest for application of this method in gene banks regarding the selection of the appropriate sucrose concentration for the preculture. The observations made by light and electron microscope should help to understand the reasons for long term effect of the sucrose treatment. During preculture in 0.01 M, 0.25 M sucrose, the sugar is taken up by plant cells and starch is accumulated in parenchymatic and meristematic cells while treatment with 0.75 M sucrose resulted immediately in severe plasmolysis even in meristematic cells. The ultrastructure of cells, however, seems to be affected also by the treatment with 0.25 M sucrose. These effects on serveral cell membranes could explain the off-type plants observed in the field. Full name of presenting author: Ina Pinker E-mail: [email protected]

115

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

54. Cryopreservation of Coriandrum sativum somatic embryos and plant

regeneration E.V. Popova, Z.-W. Lu, E.-J. Hahn and K.-Y. Paek Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Korea

Coriander (Coriandrum sativum L.) is a world-wide popular spice and medicinal herb, the source of unusual (unsaturation at C6) petroselinic acid, essential oils and oleoresin for aroma industry. Despite of high importance, long-term germplasm collections of coriander are still rare and do not comprise cryopreservation. In this report, we present a simple and easy-to-apply desiccation method for cryopreservation of coriander somatic embryos with subsequent regeneration of plants. Clumps comprised of 3 to 5 embryos, mostly in globular or heart-shape stages, were precultured on MS medium with 1 mg l-1 2,4-D and 30 g l-1 sucrose (BM) or 100 g l-1 sucrose without growth regulators for 3 days. Precultured clumps were air-desiccated to water content below 25% (FW base) followed by direct cryopreservation in liquid nitrogen. Preculture on sucrose-enriched medium prior to cryopreservation led to 95% survival and the highest number of newly formed embryos (14 embryos per clump). Sucrose preculture also increased the ration of embryo/callus formation from cryopreserved clumps. After maturation of embryos, GA3 was essential for regeneration of healthy plants. Among six concentrations tested, 1 mg l-1 GA3 was found optimal and resulted in formation of normal plants that exhibited the same profiles of relative DNA contents as plants regenerated from noncryopreserved embryos. Full name of presenting author: Elena Popova E-mail: [email protected]

116

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

55. Characterization of cryoprotective activity of protein in Acer, Fagus

and Quercus embryonic axes P.M. Pukacki, M. Jarząbek and S. Pukacka Physiology of Abiotic Stress Laboratory, Institute of Dendrology, Polish Academy of Sciences, PL-62-035 Kórnik, Poland The possible role of Acer platanoides, A. pseudoplatanus, Fagus sylvatica and Quercus robur embryonic axes protein in cryoprotection was investigated. It was found that cold-labile enzyme lactate dehydrogenase (LDH) activity was higher after freezing in the presence of embryonic axes proteins. After several freeze/thaw cycles at -196ºC/20ºC of LDH, in the presence of embryonic axes protein, it was shown their cryoprotective activity, which was comparable to that of the antifreeze proteins of spruces needles (Jarzabek et al. 2009). Especially, high cryoprotective effect indicated protein fraction below 30 kDa (cpf30). It was about 3-18 times more effective than glucose, sucrose and other proteins including bovine serum albumin. Our further research on the protein of fraction cpf30 should broaden our knowledge of the range and nature of the cryoprotective function of this fraction. Literature: Jarzabek M., P.M. Pukacki, K. Nuc. (2009). Cold-regulated proteins with potent antifreeze and cryoprotective activities in spruces (Picea spp.), Cryobiology. (doi:10.1016/j.cryobiol.2009.01.007). Full name of presenting author: Pawel M Pukacki E-mail: [email protected]

117

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

56. A case study on Ribes cryopreservation methods S. Rantala, J. Laamanen, M. Uosukainen and A. Nukari MTT Plant Production Research, Antinniementie 1, FI-41330 Vihtavuori, Finland

At MTT Plant Production Research the transfer of Ribes accessions from active vegetative nursery propagation to long-term preservation is a current issue. The cryopreservation of a group of accessions representing such a taxonomically diverse group as the Ribes species, it will not be possible to apply only one method. The accession can be grouped by their cryoability. The methods need to be chosen separately for each species group and even for each accession. The phytosanitary status of the accessions needs to be checked prior to long-term preservation. At present, virus free mother plants of Ribes species are maintained at MTT in an isolated greenhouse. Cuttings or in vitro cultures, maintained at normal growth conditions, are being used for propagation. Either dormant buds or in vitro propagated shoot tips are available and can be chosen as the target of cryopreservation. If possible, the accessions are being cryopreserved using the prevailing method, which at MTT is droplet vitrification method. It has been tested for cryopreservation of in vitro propagated Ribes. Apical buds of virus tested black currants (Ribes nigrum) and red currants (Ribes rubrum group) were excised from micropropagated plantlets. In case the prevailing method doesn’t give satisfactory results, an alternative method, cryopreservation of dormant buds can be taken into use. The testing of this method has been started for black and red currants. Full name of presenting author: Saija Rantala E-mail: [email protected]

118

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

57. Is the in vitro establishment a critical point in the (epi)genetic

stability of the cryopreserved plant material? M.A. Revilla1, R. Arroyo-García2 and E.L. Peredo1 1

Universidad de Oviedo, Departamento de Biología de Organismos y Sistemas, C/ Catedrático Rodrigo s/n 33071 Oviedo, Spain 2 Instituto Nacional de Investigación Agraria y Alimentaria, Departamento de Biotecnología, Carretera de A Coruña km 7.5, 28040 Madrid, Spain

Micropropagation is a basic technique used as a previous step in many of the plant cold storage and cryopreservation procedures. It has been reported that tissue culture induces genetic changes referred as somaclonal variation and previous studies in hop detected epigenetic variation of the genome during in vitro adventitious regeneration, cold storage and cryopreservation. Obviously this variation could be a severe treat as the maintenance of the genetic fidelity of the recovery plants is the main goal in the conservation process. In the present work the changes produced along the in vitro establishment of the hop plant material are analyzed. Three molecular markers, RAPD, MSAP and REMAP (Random Amplified DNA Polymorphism, Methylation Sensitive Amplification Polymorphism and Retrotransposon Microsatellite Amplified Polymorphism) were used to asses in first place the differences between field and in vitro plants and secondly, the variation produced along twelve sequential subcultures of the microplants performed along two years. No genetic variation among control and treated plants was found, even after 12 cycles of micropropagation. However, epigenetic variation was detected, firstly, when field and in vitro samples were compared. Nearly a 30% of the detected fragments presented the same alteration in all the vitroplants. Secondly, lower levels of epigenetic variation were detected among plants from the different subcultures and part of this detected variation seemed to be accumulated along the twelve sequential subcultures tested. Future analysis are needed to confirm whether the variation present in the in vitro plants reverts in field conditions as well as to evaluate the influence of the detected epigenetic variation in the phenotypes of the plants. Full name of presenting author: Angeles Revilla E-mail: [email protected]

119

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

58. Cryopreservation tolerance of seeds of the endangered species Tuberaria major S. Gonçalves1, L. Fernandes1, F. Pérez-García2, M.E. González-Benito2 and A. Romano1 a

IBB/CGB, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal Departamento de Biología Vegetal, Escuela Universitaria de Ingeniería Técnica Agrícola, Universidad Politécnica de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain

b

In this work we studied the cryopreservation tolerance of Tuberaria major (Cistaceae) seeds, an endangered plant species endemic from the Algarve region (South of Portugal). Seeds were transferred to 2 ml polypropylene cryovials and directly plunged in liquid nitrogen (LN) for 1 month. Seeds were removed from storage and allowed to warm at room temperature for few hours and then surface sterilized, heated at 100ºC during 60 min and sown; a group of seeds were sown without heat treatment. Low germination percentages were attained from cryopreserved seeds that were not submitted to any pre-treatment before sowing. This percentage increased to values similar to those of non-cryopreserved seeds when the heat treatment was applied. These results demonstrate that cryopreservation did not affect the germination capacity but, a heat shock is also essential after cryopreservation to enhance the final germination percentage. In conclusion, it was observed that heat stimulated germination of T. major seeds and cryopreservation could provide a method for long-term storage of germplasm from this endangered species, since no decrease on germination percentage was observed after cryopreservation and the embryos grew into normal seedlings without morphological abnormalities. Full name of presenting author: Anabela Romano E-mail: [email protected]

120

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

59. Cryopreservation of ulluco (Ullucus tuberosus Cal.) and oca (Oxalis

tuberosa Mol.) by PVS2 droplet-vitrification D. Sánchez, A. Panta, W. Roca and D. Tay International Potato Center Genetic Resources Conservation and Characterization Division Apartado 1558, Lima 12, Peru

Ulluco and oca are tuber crops from the Andean region of high importance for the nutrition and subsistence of Andean farmers. These crops present a wide genetic diversity which is affected by social, biotic and abiotic factors risking their conservation. The world largest germplasm collections, comprising 495 and 591 accessions of ulluco and oca, respectively, are maintained in vitro at the International Potato Center (CIP). For long-term conservation of these genetic resources, CIP is currently developing cryopreservation methods. The effect of cryoprotectant using Plant Vitrification Solution (PVS2) and preculture of the mother plants in different temperature regimes on shoot survival and recovery after cryopreservation was investigated. Four accessions each of ulluco and oca, and 3 replications of 10 2-mm long apical shoot tips (extracted from 3-week-old in vitro plantlets) each were cryopreserved by droplet-vitrification protocol. Apical shoot tips were incubated in loading solution (LS) for 20 min, dehydrated with PVS2 for 0, 15, 30, 45, 60 and 75 min at 0ºC, and then plunged on foils into liquid nitrogen. Thawing was carried out in liquid MS with 1.2M sucrose and shoot tips were rehydrated in the same medium for 20 min and then plated on postculture medium for regrowth. Surviving shoot tips developed into plantlets 30 days later with normal growth. The best percentages of recovery after cryopreservation were 13–65 % for ulluco and 6- 8 % for oca obtained with 60 min exposition to PVS2. In vitro plantlets cultured at constant temperature at 6ºC or alternating temperature of 18ºCin week 1 and 6ºC in week 2 did not increase the survival and recovery of shoot tips. This cryopreservation protocols will be tried on a broad range of genotypes. Full name of presenting author: Dino Favio Sánchez Verano E-mail: [email protected]

121

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

60. Cryopreservation of avocado embryogenic cultures E. Guzmán-García1, F. Bradaï2, B. Panis3 and C. Sánchez-Romero2 1

IFAPA Centro de Churriana, Cortijo de la Cruz s/n, 29140 Churriana-Málaga, SPAIN Dpto. Biología Vegetal, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, 29071 Málaga, SPAIN. 3 Laboratory of Tropical Crop Improvement, KU Leuven, Kasteelpark Arenberg 13, 3001 Leuven, BELGIUM 2

Avocado is a commercially important tropical crop very valued by the high price of its fruits in the market. Biotechnological techniques, such as transformation, somaclonal variation or somatic hybridization, have been applied in this species, all of them depending on the use of embryogenic cultures. However, genetic instability and loss of embryogenic competence are often observed in cultures that are maintained in proliferation medium for a prolonged time. Therefore, the establishment of a cryopreservation protocol for valuable avocado embryogenic cell lines is considered of outermost importance. In this study, three different cryopreservation protocols have been tested using two avocado embryogenic cell lines: a slow freezing method (1oC/min) and two vitrification-based methods, the classic protocol and an ultra fast freezing protocol using droplet vitrification on aluminium foil strips. The results obtained were different depending on the embryogenic cell line. While in one of them high survival and recovery percentages were achieved independently of the cryopreservation method used, in the other one, satisfying results were obtained only using the two vitrification-based protocols. The influence of the developmental stage of the starting material was also tested using the droplet vitrification method. Somatic embryos, tested at two different stages, gave rise to inferior results compared to embryogenic callus or the mixture of callus and somatic embryos at early developmental stages that usually is used for maintenance of cultures. Full name of presenting author: Carolina Sánchez-Romero E-mail: [email protected]

122

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

61. Evaluation of strategies for the cryopreservation of shoot tips of

cassava (Manihot esculenta KRANTZ) I. R. I. Santos and R. Caldas Mundim Embrapa Genetic Resources and Biotechnology (Cenargen), Brasília-DF, Brasil.

Cassava (Manihot esculenta Krantz) is a tropical perennial shrub, propagated vegetatively, which produces tubers with high starch content. It provides food for millions of people in many countries. The objective of this work was to compare the effect of three cryopreservation strategies on survival of cassava shoot tips, i. e. a) vitrification with PVS2 (15% DMSO, 15% ethleneglycol, 30% glycerol and 0,4 M sucrose), followed by freezing in liquid nitrogen; b) encapsulation/dehydration, in which encapsulated shoot tips were subcultured in liquid MS-62 containing high concentrations of sucrose, desiccated and submerged in liquid nitrogen; and c) encapsulation/vitrification, in which encapsulated shoot tips were precultured in PVS2 before freezing in liquid nitrogen. For all experiments shoot tips (1,0 mm) were isolated asseptically, at room temperature (21±2 ºC), from plants (two accessions from the in vitro collection of cassava maintained at Cenargen, Brasília-DF) cultivated on solid basic MS-62 (Murashige & Skoog, 1962). Thawing following each procedure was carried out by plunging cryovials containing shoot tips on a water bath at 40 ºC. After warming shoot tips were transferred into Petri dishes containing basic MS-62 for regeneration. Shoot tips of accession BGM 603 showed 72% survival after the vitrification alone and 33% survival after the encapsulation/vitrification. Survival of shoot tips of accession CG 1489 - 23 was 80% following vitrification alone and 73% after encapsulation/vitrification. These results suggest that cryopreservation is a viable approach for the preservation of cassava shoot tips. However, further studies must be carried out to improve survival of cassava shoot tips after cryopreservation and to test these strategies with other cassava accessions. Full name of presenting author: Izulmé Rita Imaculada Santos E-mail: [email protected]

123

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

62. Cryopreservation of mint – routine application in a Genebank,

experience and problems A. Senula and E.R.J. Keller Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany

The genebank of the IPK contains 207 mint accessions, which are maintained in the field or in vitro. In the year 2005, the routine cryopreservation of seed-sterile mint was started, using a simple droplet-vitrification method with PVS 2 (Senula, A., E.R.J. Keller, T. Sanduijav & T. Yohannes (2007). CryoLett. 28: 1-12). Axillary shoot tips, growing from cold-acclimated nodal segments were used. At present, 35 accessions of different species are cryopreserved by this method. The average regeneration rate is 65 %. Strong variation was found between 15 to 98 %, both between the accessions and the two repetitions of one accession (150 shoot tips each). This is caused by the genotype and endophytes in the in vitro donor plants that were previously found to be free of visible infections. After rewarming bacterial infections often broke out of the explants and reduced or inhibited regeneration of plantlets. A screening on endophytes using single donor plants prior to multiplication and cryopreservation was done. Since bacteria are not homogenously distributed in the plants, the results of the screening test reflected only the given situation in the tested material proper, but not that in the whole plant. Therefore, the use of these pre-tests alone would not be conclusive enough to rely only on them. Furthermore, some endophytes are not cultivable on culture media alone, which makes detection difficult. The preventive use of antibiotics in the regeneration medium after cryopreservation resulted in higher plant regeneration, when the donor plants were infected. When the cryopreserved shoot tips had been clean before, the antibiotics so far tested (Cefotaxim, Ticarcillin, Vancomycin) delayed the regeneration process, but did not reduce the rates. Full name of presenting author: Angelika Senula E-mail: [email protected]

124

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

63. Cryopreservation of Shoot Tips of Bacopa monnieri (L.) Wettst. – A

Commercially Important Medicinal Plant, by vitrification technique N. Sharma, R. Satsangi and R. Pandey Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi-110012, India

An attempt was made in this investigation to develop a protocol for cryopreservation of in vitro shoot tips of Bacopa monnieri (L.) Wettst., a medicinal plant of high commercial potential as a memory vitalizer. There are no reports in the literature regarding conservation of this precious herb in the natural habitat. Insufficient seed availability, problems associated with seed propagation and short seed viability severely limit the application of seed conservation methods. A simple one-step method for in vitro clonal multiplication and conservation of Bacopa has been developed. To our knowledge, successful cryopreservation of this medicinal plant has not yet been reported. Shoot tips (about 1 mm in length) excised from 4-24-week-old proliferating shoot cultures were pretreated on MS medium supplemented with various osmotica before dehydrating with PVS2 solution at 0ºC. The dehydrated shoot tips were directly immersed in LN2. Following cryopreservation, and after rapid rewarming at 45ºC, shoot tips were quickly washed with 1.2 M sucrose solution and then plated on solidified shoot culture medium. Shoot tips were successfully cryopreserved by vitrification, when they were precultured on medium supplemented with 5% DMSO at 4ºC for two days before dehydrating in PVS2 for 10-40 minutes at 0ºC. Pretreatment of shoot cultures with sucrose at 25ºC significantly improved the survival and shoot regeneration of cryopreserved shoot tips. Plantlets regenerated from cryopreseved shoot tips were successfully transferred to pots. Impact of various parameters such as age of explants, type and/or duration of preculture treatment and dehydration on cryopreservation will be the focus of discussion. Full name of presenting author: Dr. Neelam Sharma E-mail: [email protected]

125

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

64. Ultrastructural evidence for the effects of rapid cooling on plant

shoot apices by the vitrification based protocol using cryo-scanning electron microscopy D. Tanaka1, T. Niino2, T. Matsumoto3 and S. Fujikawa4 1

Laboratory of Gene Research, Faculty of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan, 2 Gene Bank, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan, 3 Shimane Agricultural Technology Center, Izumo Shimane 693-0035, Japan, 4 Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Although there are several long-term preservation methods for plant germplasm, cryopreservation of shoot tips has been recognized as the most reliable, cost- and space-efficient method. However, it is not yet widely employed as a reliable long-term preservation protocol due to the lack of basic knowledge on the cellular and water behavior in tissues when immersed in LN. In the present study, cryo-scanning electron microscopy combined with freeze-substitution was employed to examine ultrastructure of cells of plant shoot apices that were cooled to the temperature of LN after exposure to various steps of the vitrification based protocol. When the shoot apices were immersed in LN without vitrification solution (PVS2) treatment, largesized ice crystal formation was observed throughout the cells, indicating severe injury occurring during rapid cooling. In contrast, when the shoot apices were immersed in LN after optimal PVS2 treatment under conditions that resulted in a high survival rate, there was no visible ice crystals formed in cells. In addition, cells of the PVS2treated shoot apices exhibited considerable plasmolysis and small vesicles in cytoplasm. The results clearly demonstrated that the PVS2 treatment is essential for preventing damage caused by ice formation and for successful cryopreservation of plant shoot apices. Full name of presenting author: Daisuke Tanaka E-mail: [email protected]

126

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

65. A comparison of shoot meristem cryopreservation in banana derived

from in vitro cultures and suckers from field R. Sanayaima, A. Agrawal and R.K. Tyagi* National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi-110 012, India

Amongst the methods of cryopreservation in banana (Musa spp.), the droplet vitrification method applied to individual meristems derived from in vitro shoot cultures is reported to be the most successful (Panis et al 2005, Plant Sci. 168, 4555). Depending on the genotype, cryopreservation of meristems, starting from excising explants from field (suckers) to plunge into liquid nitrogen (LN), would require a minimum of 1 to 2 year(s). The objectives of present study were (i) whether total cryopreservation duration could be reduced and (ii) to establish the protocol for cryopreservation using sucker-derived meristems for those laboratories where abundantly suckers are available in field but have inadequate in vitro facilities. Dual purpose banana cv. Karpura Chakkarakeli (AAB; Mysore subgroup; IC 250497) was selected. Shoot recovery between the crypreserved meristems from in vitroshoots (IVM) and suckers (SM) were compared. For SM, standard disinfection procedures were adopted and suckers containing shoot tips trimmed to 1x1 cm size and cultured on MS + BAP (10µM) + IAA (1 µM) + Ascorbic acid (10 mg/l) + 3% sucrose for 4-6 weeks. Subsequently, these were transferred to the same medium, but with 6% sucrose for 4 weeks. Cryopreservation of IVM and SM was done (Panis et al 2005). Post-thaw regeneration was compared between (IVM) and (SM). No significant difference was recorded in mean shoot regeneration in PVS2-treated IVM (83.3+7.5%) and SM (60+11.5%). Similarly in LN-treated explants, regenerated shoots from IVM (54.4+5.8%) were significantly not different from SM (60.0+11.55 %). Callus formation was significantly higher in the SM as compared to IVM. Importantly, the total duration for cryopreserving plants from SM was about 2 months; whereas it took 6-8 months for IVM. The study indicates that meristems from suckers can be directly used for cryopreservation in banana, without undergoing the elaborate in vitro multiplication phase and facilities. Full name of presenting author: Rishi Kumar Tyagi E-mail: [email protected], [email protected]

127

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

66. Endogenous polyamines in Norway spruce embryogenic cultures

during cryopreservation M. Cvikrová, Z. Vondráková, K. Eliášová, O. Martincová and M. Vágner Institute of Experimental Botany, AS CR, Rozvojová 263, CZ 16502 Prague 6, Czech Republic

Norway spruce embryogenic cultures of various cryotolerances were subjected to cryopreservation in liquid nitrogen. The procedure comprises preculture in a liquid medium with a rising concentration of sorbitol, DMSO treatment and driven temperature reduction before storing in liquid nitrogen. After thawing, we monitored the cultures’ re-growth. Morphology and viability of embryogenic cultures were studied using simple trypane blue staining and green-fluorescent fluoroscein diacetate/red-fluorescent propidium iodide double staining. Endogenous free polyamines (putrescine, spermine and spermidine) and their bonded and conjugated forms were analyzed during the process. Putrescine and subsequently spermidine levels declined during the precultivation in sorbitol. Conversely, spermine level (considered a stress marker) and level of abscisic acid increased. The small compact cells of embryo meristems were the only ones to survive cryopreservation. Developing regeneration of early embryos became apparent only 4 d after thawing. At this stage, although vivid cells comprise but a small fraction of analyzed material, the levels of polyamines still changed dramatically. The level of spermine increased only after 1 d of re-growth. Bond polyamine levels rose first followed by conjugated polyamine levels (6th d). The culture characterized by the growth of new embryos showed declining levels of conjugated polyamines and escalating levels of free polyamines. Full name of presenting author: Martin Vagner E-mail: [email protected]

128

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

67. Cryopreservation of an azalea germplasm collection J. Van Huylenbroeck and E. Calsyn Institute for Agricultural and Fisheries Research (ILVO), Plant Unit, Applied Genetics and Breeding, Caritasstraat 21, 9090 Melle, Belgium

Belgium is a renowned centre for pot azalea (Rhododendron simsii) propagation and production with around 150 nurseries supplying 40 million plants annually. The Institute for Agricultural and Fisheries Research hosts the largest in vivo germplasm collection for pot azaleas in the world. Besides 350 accessions of Rhododendron simsii, also around 250 related Rhododendron species and varieties are present. This collection is not only important for research and plant breeders, but also represents a substantial part of Flanders’ horticultural history and cultural heritage. Recently it was decided to cryopreserve part of the collection in order to reduce conservation costs as well as to safeguard the most (historically) important and valuable genetic material. Prior research has shown that cryopreservation of Rhododendron might be an efficient technique. Acceptable and reliable survival percentages were obtained (25 to 70%) in experiments with six different cultivars. In vivo characteristics as drought and cold tolerance had an important influence on survival percentages. True-to-type plant material was obtained and no sport formation was observed after regeneration of cryopreserved mersitems. Full name of presenting author: Johan Van Huylenbroeck E-mail: [email protected]

129

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

68. CRYOPRESERVATION ON CITRUS GERMPLASM F. Vidigal Duarte Souza1, M. Jenderek2 and D. Ellis2 1

Embrapa Mandioca e Fruticultura, EMBRAPA – BRAZIL, rua Embrapa s/n - Bairoo de Chapadinha, Cruz das Almas, 44380-000 Bahia, Brazil 2 USDA-ARS, National Center for Genetic Resources Preservation, 1111 S. Mason Street, Fort Collins, CO, USA 80521

Citrus is one of the most important fruit crops in Brazil with research efforts looking at a wide a diversity species, each with its own challenges in cultivation. Embrapa Cassava and Tropical Fruits has developed a breeding program aimed at generating new hybrids with superior traits. To support this breeding work, a germplasm collection has been made and maintained as a field planting since the early 70’s. Losses of important germplasm have occurred in this collection due mainly to biotic factors such as pests and diseases. These losses have intensified research efforts in developing alternative strategies for the conservation of this valuable germplasm. Cryopreservation is an important tool for the long-term storage of vegetativelypropagated plant genetic resources, as it requires a minimum of space and maintenance. Preliminary studies on the cryopreservation of embryogenic cell clusters and somatic embryos from Citrus sunki Hort and Citrus reshni Hort have been initiated. These studies include the use of encapsulation–dehydration, encapsulation–vitrification with PVS2 and droplet vitrification with PVS2 Although no survival in liquid nitrogen was obtained, both embryos and embryogenic cell clusters survived desiccation and somatic embryos were more sensitive to PVS2 than embryogenic cell clusters. Encapsulation allowed survival with longer exposure to PVS2, however LN2 survival was no different. Full name of presenting author: Fernanda Vidigal Duarte Souza E-mail: [email protected]

130

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

69. Viability and multiplication of blackberry and raspberry shoots upon encapsulation Đ. Ružić and T. Vujović Fruit Research Institute, Kralja Petra I, No 9, 32000 Čačak, Serbia

The paper deals with the viability of cultures and multiplication parameters upon encapsulation. Shoot tips of blackberry cv Čačanska Bestrna and raspberry cv Meeker were initially encapsulated in calcium alginate beads at 5oC (in darkness). As regards cvs Čačanska Bestrna and Meeker, 18.8% and 6.3% of plants respectively survived one-month encapsulation and gave regrowth, the latter resulting in additional explants after 2 months. Multiplication index in blackberry cv Čačanska Bestrna decreased gradually from the first to the sixth subculture, the latter being the point of increase, nonetheless did not reach the value of the first subculture. Identical rising trend towards the sixth subculture was observed in the length of axial shoot. Fresh and dry shoot weights of encapsulated shoots were almost equal as compared to unencapsulated ones, whereas the rooting parameters were markedly superior in encapsulated shoots. In raspberry cv Meeker, multiplication index did not result in any major increase, except in the eigth subculture (two-month encapsulation) when it was even higher than in unencapsulated shoots. Fresh and dry shoot weights in encapsulated shoots showed no great difference in comparison to the unencapsulated ones. However, as for the rooting parameters of encapsulated shoots, these had lower values in comparison with unencapsulated ones, though displaying very high acclimatization rate (100%). In both genotypes, shoots maintained their earlier morphology. The obtained results suggest that keeping shoot tips in alginate pearls neither reduces multiplication rate nor influences morphology and viability of cultures and even contributes to the rooting parameters. Full name of presenting author: Tatjana Vujović E-mail: [email protected]

131

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

70. Cryostorage of spores and gametophytes of royal fern (Osmunda

regalis L.) A. Wójcik, A. Mikuła, D. Makowski and J.J. Rybczyński Botanical Garden – Center for Biological Diversity Conservation, Polish Academy of Sciences, Prawdziwka 2, 02-973 Warsaw, Poland

Osmunda regalis L. (royal fern) is a protected species of Polish flora. Its chlorophyllous spores quickly germinate (within 40h) but also their viability fast decline during storage. Our investigation showed that the spore viability drop by 70% after three months of storage at 22oC. Five-month refrigeration (+4oC) reduced their germination ability only by 10%, however, germination initiation required at least 96hrs or even more. Direct immersion in the liquid nitrogen of the non-disinfected and non-dried spores protected their viability. Alternative system of biodiversity conservation developed in this studies based on in vitro culture of spore and gametophyte. For spore sterilization 2% H2O2 for 20 min and followed by three washes in sterile distilled water were used. Frozen and nonfrozen spores germinated and developed into gametophytes on 0.5MS medium supplemented with 2 % (w/v) sucrose. Non differences in development of those gametophytes were recognized. On the other hand, in vitro derived gametophytes could serve as experimental material for the cryostorage purpose. Physical and chemical conditions of the gametophyte culture were examined as followed: time of preculture, consistency of medium (liquid or agar) and ABA. All these factors influenced on the O. regalis gametophyte survival. Independently of preculture treatment the viability after cryopreservation reached 80% and the majority of cells of particular gametophyte explants was alive. We conclude that besides of spores gametophytes could be similarly exploited for O. regalis biodiversity conservation. Full name of presenting author: Anna Wójcik E-mail: [email protected]

132

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

71. Preparation of shoot tips by sucrose and dehydration pretreatment of

Ullucus tuberosus for cryopreservation J. Zamecnikova1, J.Zamecnik2,M.Faltus2, E.Fernandez1 and I.Viehmannová1 1

Institute of Tropics and Subtropics, Czech University of Life Sciences Prague, Kamycka 129, CZ16521 Prague, The Czech Republic 2 Crop Research Institute, Drnovska 507, CZ 16106 Prague, The Czech Republic

Ullucus tuberosus is a vegetatively propagated tuber crop of Andean region with high values of proteins, calcium and carotene. Plants are mostly vegetatively propagated and that is why to store them for long time is difficult. Cryopreservation can be an appropriate method for long term storage of U. tuberosus genetic resources. The excess of water was removed by simple dehydration of shoot tips but plants were not able to recover from water stress caused by 4 hours desiccation. How sucrose increases tolerance to desiccation and how it acts as cryoprotectant was tested in experiments with various pre-treatments. Plants were grown on various sucrose concentrations during pre-culture. Subsequently, shoot tips were isolated and treated in sucrose solution. The pre-treatment consists of two steps: (i) in vitro growing plants were overlaid by 0.7 M or 2 M sucrose; after that (ii) shoot tips were treated in 0.7 M solution for 12 hours before desiccation. Thereafter the shoot tips were dehydrated over silica gel. The shoot tips lost excess water during first hour of desiccation by a rate of 2 gH2O/gDW hour to the level of 22 % of initial water content. After two hours of desiccation the regeneration of thawed shoot tips from liquid nitrogen increased up to 10%. Ice nucleation of shoot tips decreased from -30 °C in non-desiccated to minimum -36 °C in shoot tips after 2.5 hours of desiccation. From these results it is obvious that sucrose pre-treatment increased the tolerance of plants to desiccation. The combination of sucrose pre-treatment and desiccation over silica gel after 2 hour desiccation increased regeneration of shoot tips after thawing from liquid nitrogen up to 10%. Nevertheless this regeneration rate is unsatisfactory and further research is needed. Full name of presenting author : Jana Zamecnikova Email: [email protected]

133

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

72. Cryopreservation of vegetative garlic for the establishment of a

European Core collection C. D. Zanke1, E. R. J. Keller1, T. Kotlińska2, M. Olas2 and J. Zámečník3 1

Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany; 2Research Institute of Vegetable Crops (RIVC), Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland; 3Crop Research Institute (CRI), Drnovska 507, 161 06 Prague, Czech Republic

In April 2007, a European project named EURALLIVEG started under Council Regulation EC 870/2004. Its main task is the development of a European integrated core collection of garlic provided by national programmes of the partners. For its central part, a number of 200 most important accessions will be cryopreserved by three project participants. The final aim is to establish a Tripartite Cryopreservation Genebank. The garlic collection at IPK is the third in Europe after the collections of Spain and Czech Republic according to the number of entries in the European Allium Database. The source organs for garlic cryopreservation depend on the type of the material (bolting or non-bolting garlic). In bolting forms, the bulbils can be used. For nonbolting accessions in vitro cultures need to be established. After different durations of cold preculture (25/-1 °C day/night temperatures) explants from in vitro cultures can be cryopreserved using the vitrification method with the cryoprotectant solution PVS3. In most cases when bulbils are used, regeneration after cryopreservation is lower than by using in vitro cultures after cold preculture. Therefore, IPK used in vitro preculture not only for non-bolting but also for bolting accessions. So far, this amounted in total to 36 accessions. The weighted average survival rate of 24 accessions was 72 % and the regrowth rate 54 %. Differences in regrowth were not only found between but also within the accessions. Sometimes the regrowth rates of different repetitions of one accession ranged from 45 to 83 %. These results will be discussed in relation to data previously recorded in the IPK genebank (in total 49 clones cryopreserved at IPK). Full name of presenting author: Christine Zanke E-mail: [email protected]

134

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

73. Evaluation of genetic stability in cryopreserved Solanum tuberosum R. Zarghami1 and Sh. Sepahpour 2 1

Agricultural Biotechnology Research Institute of Iran, Karaj, Iran Department of Plant Breeding, College of Agriculture, Tarbiat Modares University

2

Cryopreservation is a technology of high importance in the storage of plant germplasm for long periods; however, the practical application of this technology for the preservation of plant materials is useful only if it does not lead to the genetic changes in the plant of interest. In the present investigation, the genetic stability of potato (Solanum tuberosum L.) plantlets of the cultivars Agria and Marphona stored under cryopreservation and noncryopreservation conditions was studied using amplified fragment length polymorphism (AFLP) technique. Also, flow cytometric studies were performed to detect if there were probably any changes in the level of polyploidy. Seven primer combinations were used in AFLP studies. Agria plantlets kept under noncryopreserved conditions were approximately of average 97% genetic similarity to those of the same cultivar but stored under cryopreservation conditions. With the cultivar of Marphona, full (100%) homology was found between plantlets stored under cryopreservation and non-cryopreservation conditions. Comparative studies on the polyploidy levels of the plantlets of both cultivars conserved under two above-mentioned storage conditions indicated that cryopreservation technique did not cause any changes in polyploidy levels. Full name of presenting author: Reza Zarghami E-mail: [email protected]

135

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

Evaluation of morphological cryopreserved Solanum tuberosum

changes

74.

and

physiological

in

R. Zarghami1 and Sh. Sepahpour 2 1

Agricultural Biotechnology Research Institute of Iran, Karaj, Iran Department of Plant Breeding, College of Agriculture, Tarbiat Modares University

2

Cryopreservation is one of great important techniques in long-term preservation of plant germplasms. This technique is useful for plant preservation only if morphological and physiological changes don't occur. Morphological and physiological changes in plantlets of two potato varieties, Agrya and Marfona, preserved in freezing and normal conditions were studied in this research. Twentythree morphological characteristics were assessed using a descriptor with a scale of 0 to 9 for each trait. Free proline, soluble carbohydrates, soluble proteins and a and b chlorophyll content were measured for physiological studies. The results indicated a general homology for morphological traits of Agrya and Marfona plantlets preserved in freezing condition compared to non-freezing condition. The plants studied under various preservation conditions revealed no significant difference for assessed physiological characteristics excepting free proline. Content of free proline in shoots preserved in freezing condition increased significantly in comparison with non-freezing condition which this increment may be due to cold and oxidative stresses induced in plants. Full name of presenting author: Reza Zarghami E-mail: [email protected]

136

1st International Symposium on Cryopreservation in Horticultural Species

Poster abstracts

75. Cryopreservation of sweetpotato: comparison of PVS2 droplet

vitrification with encapsulation-vitrification method B. Zea, A. Panta and D. Tay International Potato Center Genetic Resources Conservation and Characterization Division Apartado 1558, Lima 12, Peru

The International Potato Center (CIP) maintains the largest in vitro sweetpotato (Ipomoea batatas) collection in the world comprising 6,855 accessions. CIP’s experience on potato cryopreservation has paved the way for developing efficient cryo-methods for the long-term sweetpotato genetic resources conservation. The droplet vitrification protocol which uses the Loading (LS) and Plant Vitrification (PVS2) cryoprotective solutions was tested. In the first assay, response of meristematic tips of cultivars, Jewel and Trujillano, to LS and PVS2 was evaluated. Apical shoot tips of 0.8-1.5 mm from 4 weeks old in vitro plantlets were incubated in LS for 45, 75, 90 min and over night, and then exposed to PVS2 for 10, 20, 30, 35, 40, 45, and 50 min at 0°C before rapidly frozen in liquid nitrogen. In a second assay, the droplet method using the best LS and PVS2 treatments was compared with the encapsulation-vitrification method. Three cultivars, Jewel, Trujillano and Cinitavo, were used. Both assay followed a complete randomized design with 3 replications. Each treatment used 48 meristematic tips. The highest tissue survival (58%) and shoot regeneration (49%) rates were observed using the droplet method with 45 min LS in combination with 75 min PVS2 treatment compared to 4% tissue survival and 0% shoot regeneration using the encapsulation-vitrification method. In addition, testing some pre-culture media supplements had indicated that casamin acid increases the tissue survival and shoot recovery. Full name of presenting author: Brenda C. Zea Obando E-mail: [email protected]

137

1st International Symposium on Cryopreservation in Horticultural Species

Notes:

138

Poster abstracts

1st International Symposium on Cryopreservation in Horticultural Species

Notes:

139

Poster abstracts

1st International Symposium on Cryopreservation in Horticultural Species

Notes:

140

Poster abstracts

1st International Symposium on Cryopreservation in Horticultural Species

Notes:

141

Poster abstracts

1st International Symposium on Cryopreservation in Horticultural Species

Notes:

142

Poster abstracts

1st International Symposium on Cryopreservation in Horticultural Species

List of participants

List of participants

143

1st International Symposium on Cryopreservation in Horticultural Species

144

List of participants

1st International Symposium on Cryopreservation in Horticultural Species

List of participants

Shaher Abdullateef

Humboldt University of Berlin GERMANY E-mail: [email protected]

Anuradha Agrawal

Tissue Culture and Cryopreservation Unit National Bureau of Plant Genetic Resources, INDIA E-mail: [email protected]

Hulya Akdemir

Gebze Institute of Technology, TURKEY E-mail: [email protected]

Edwige André

K.U.Leuven, Laboratory of Tropical Crop Improvement BELGIUM E-mail: [email protected]

Julia Angstl

Institute of Natural Resource Sciences Zurich University of Applied Sciences SWITZERLAND E-mail: [email protected]

Ericson Aranzales Rondon

International Center for Tropical Agriculture (CIAT) COLOMBIA E-mail: [email protected]

Sarah Ashmore

School of Biomolecular & Physical Sciences Griffith University AUSTRALIA E-mail: [email protected]

Ana Carolina Atala Lobelo Campos Plant Science Group Wageningen University THE NETHERLANDS E-mail: [email protected] Anna Bach

Department of Ornamental Plants University of Agriculture POLAND E-mail: [email protected]

Carla Benelli

IVALSA/Istituto per la Valorizzazione del Legno e delle Specie Arboree CNR (National Research Council) ITALY E-mail: [email protected]

Patricia Berjak

School of Biological and Conservation Sciences University of KwaZulu-Natal SOUTH AFRICA E-mail: [email protected]

Margherita Beruto

Istituto Regionale per la Floricoltura ITALY E-mail: [email protected]

Emilia Caboni

Fruit Tree Research Institute CRA ITALY E-mail: [email protected]

Pablo Caceres

K.U.Leuven, Laboratory of Tropical Crop Improvement BELGIUM E-mail: [email protected]

145

1st International Symposium on Cryopreservation in Horticultural Species

List of participants

Evelien Calseyn

ILVO-PLANT Genetica en Veredeling BELGIUM E-mail: [email protected]

Manuel Cantos Barragán

CSIC Instituto de Recursos Naturales y Agrobiologia de Sevilla SPAIN E-mail: [email protected]

Maurizio Capuana

Plant Genetics Institute C.N.R. ITALY E-mail: [email protected]

Sebastien Carpentier

K.U.Leuven, Laboratory of Tropical Crop Improvement BELGIUM E-mail: [email protected]

Milene Carvalho

K.U.Leuven, Laboratory of Tropical Crop Improvement BELGIUM E-mail: [email protected]

Karipidis Charalambos

Epirus Institute of Technology GREECE E-mail: [email protected]

Philipppe Chatelet

INRA FRANCE E-mail: [email protected], [email protected]

Pawel Chmielarz

Institute of Dendrology POLAND E-mail: [email protected]

Eduardo Cires

Biology of Organisms and Systems Oviedo University SPAIN E-mail: [email protected]

Emiliano Condello

Fruit Tree Research Institute CRA ITALY E-mail: [email protected]

Charlotte Crahay

Université Catholique de Louvain-la-Neuve BELGIUM E-mail: [email protected]

Carmine Damiano

Fruit Tree Research Institute CRA ITALY E-mail: [email protected]

Katalina Danova

Department of Plant Physiology Faculty of Biology Sofia University BULGARIA E-mail: [email protected]

Edmond De Langhe

Em. K.U.Leuven BELGIUM E-mail: [email protected]

Beata Dedicova

Department of Forest Genetics and Physiology Swedish University of Agricultural Sciences (SLU) SWEDEN E-mail: [email protected]

146

1st International Symposium on Cryopreservation in Horticultural Species

List of participants

Elohor Diebiru

International Institute of Tropical Agriculture (IITA) NIGERIA E-mail: [email protected]

Dimitra Douma

Epirus Institute of Technology GREECE E-mail: [email protected]

Dominique Dumet

International Institute of Tropical Agriculture (IITA) NIGERIA E-mail: [email protected]

Florent Engelmann

Institut de Recherche pour le Développment (IRD) FRANCE E-mail: [email protected]

Roosevelt Humberto Escobar Pérez International Center for Tropical Agriculture (CIAT) COLOMBIA E-mail: [email protected] Milos Faltus

Department of Molecular Biology Crop Research Insitute CZECH REPUBLIC E-mail: [email protected]

Lotfi Fki

Faculté des Sciences de Sfax-Tunisia Dép. de Biologie, Lab. des Biotechnologies Végétales, TUNISIA E-mail: [email protected] or [email protected]

Emile Frison

Bioversity International ITALY E-mail: [email protected]

Seizo Fujikawa

Research Faculty and Graduate School of Agriculture Hokkaido University JAPAN E-mail: [email protected]

Kuniaki Fukui

National Institute of Agrobiological Sciences JAPAN E-mail: [email protected]

José Luis Garcia Fernández

CSIC Instituto de Recursos Naturales y Agrobiologia de Sevilla SPAIN E-mail: [email protected]

Tatiana Gavrilenko

N.I. Vavilov Institute of Plant Industry (VIR) RUSSIA E-mail: [email protected]

Danny Geelen

University of Ghent Faculty of Bioscience Engineering - Dept. of Plant Production BELGIUM E-mail: [email protected]

Bruno Goddeeris

K.U.Leuven BELGIUM E-mail: [email protected]

147

1st International Symposium on Cryopreservation in Horticultural Species

List of participants

Maria Teresa Gonzalez Arnao

Facultad de Ciencias Quimicas, Laboratorio de biotecnologia y Cryiobiologia Vegetal Universidad Veracruzana MEXICO E-mail: [email protected] or [email protected]

M. Elena Gonzalez-Benito

Dpto. de Biologia Vegetal Universidad Politecnica de Madrid SPAIN E-mail: [email protected]

Agnès Grapin

UMR GenHort AGROCAMPUS-OUEST - centre d'Angers - INHP, FRANCE E-mail: [email protected]

Badara Gueye

International Institute of Tropical Agriculture (IITA) NIGERIA E-mail: [email protected]

Sandhya Gupta

National Bureau of Plant Genetic Resources Tissue Culture & Cryopreservation Unit INDIA E-mail: [email protected]

Hely Häggman

Department of biology University of Oulu FINLAND E-mail: [email protected]

Jean-Francois Hausman

CRP - Gabriel Lippmann LUXEMBOURG E-mail: [email protected]

Karin Hoedemaekers

Nunhems Netherlands BV THE NETHERLANDS E-mail: [email protected] or [email protected]

Monika Höfer

Julius Kuehn-Institute Institute for Breeding Research on Horticultural and Fruit Crops GERMANY E-mail: [email protected]

Femke Hoogervorst

Anthura Research bv THE NETHERLANDS E-mail: [email protected]

Maria Jenderek

USDA-ARS National Center for Genetic Resources Preservation USA E-mail: [email protected]

Sladjana Jevremovic

Department for Plant Physiology Insitute for Biological Research "Sinisa Stankovic SERBIA E-mail: [email protected]

Nipawan Jitsopakul

Department of Science and Mathematics Faculty of Agriculture and Technology, Rajamangkala University of Technology Isan, Surin Campus THAILAND E-mail: [email protected]

148

1st International Symposium on Cryopreservation in Horticultural Species

List of participants

Anja Kaczmarczyk

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) GERMANY E-mail: [email protected]

Behzad Kaviani Livani

Department of Horticulture Islamic Azad University, Faculty of Agriculture IRAN E-mail: [email protected]

Joachim Keller

Genebank Department Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) GERMANY E-mail: [email protected]

Haeng-Hoon Kim

National Agrobiodiversity Center National Academy of Agricultural Sciences REPUBLIC OF KOREA E-mail: [email protected]; [email protected]

Sofia Korneva

Centro de Investigaciones Cientificas de Ecuador (CIBE) Escuela Superior Politecnica de Litoral (ESPOL) ECUADOR E-mail: [email protected]

Frank T.M. Kors

Duchefa Biochemicals THE NETHERLANDS E-mail: [email protected]

Jana Krajnaková

Forest Research Institute National Forestry Center SLOVAKIA E-mail: [email protected] or [email protected]

Ismahen Lalaymia

Université Catholique de Louvain-la-Neuve BELGIUM E-mail: [email protected]

Maurizio Lambardi

IVALSA/Istituto per la Valorizzazione del Legno e delle Specie Arboree, CNR (National Research Council) ITALY E-mail: [email protected]

Yves Lambeens

K.U.Leuven, Laboratory of Tropical Crop Improvement BELGIUM E-mail: [email protected]

Ellen Lambert

University of Ghent Faculty of Bioscience Engineering - Dept. of Plant Production BELGIUM E-mail: [email protected]

Mira Langegger Hohle

ZHAW SWITZERLAND E-mail: [email protected]

Yan Liu

College of Landscape Architecture Beijing Forestry University CHINA E-mail: [email protected]

Bingling Liu

College of Landscape Architecture Beijing Forestry University CHINA E-mail: [email protected]

149

1st International Symposium on Cryopreservation in Horticultural Species

List of participants

Charlotte Lusty

Global Crop Diversity Trust ITALY E-mail: [email protected]

Ulamila Lutu

Centre for pacific Crops and Trees (CePaCT) Secretariat of the Pacific Community (SPC), Suva FIJI ISLANDS E-mail: [email protected]

Paul Lynch

Biological Sciences Research Group University of Derby UK E-mail: [email protected]

Bernard Malaurie

Palm Development Group, UMR DIAPC Institut de Recherche pour le Développment (IRD) FRANCE E-mail: [email protected]

Ildikó Matusiková

Department of Molecular Biology and Biotechnology Institution of Plant Genetics and Biotechnology, Slovak Academy of Sciences SLOVAKIA E-mail: [email protected]

Marcin Michalak

Institute of Dendrology POLAND E-mail: [email protected]

Anna Mikula

Botanical Garden, Center for Biological Diversity Conservation Polish Academy of Sciences POLAND E-mail: [email protected]

Tim Minkhorst

Seminis/ Monsanto Holland THE NETHERLANDS E-mail: [email protected]

Normah Mohd. Noor

Institute of systems Biology Universiti Kebangsaan Malysia MALAYSIA E-mail: [email protected]

Luis Mroginski

IBONE ARGENTINA E-mail: [email protected]

Jayanthi Nadarajan

Seed Conservation Department Royal Botanic Gardens Kew UK E-mail: [email protected]

Takao Niino

National Institute of Agrobiological Sciences JAPAN E-mail: [email protected]

Gabriela Nogueira

Department of Biology, Sector of Plant Physiology Federal University of Lavras BRAZIL E-mail: [email protected]

Anna Nukari

Plant Production Research, Nursery Group MTT FINLAND E-mail: [email protected]

150

1st International Symposium on Cryopreservation in Horticultural Species

List of participants

Marta Olas

Plant Genetic Resources Lab. Research Institute of Vegetable Crops POLAND E-mail: [email protected]

Yelda Ozden Tokatli

Gebze Institute of Technology TURKEY E-mail: [email protected]

Elif Aylin Ozudogru

Gebze Institute of Technology Faculty of Science Biology Department TURKEY E-mail: [email protected] or [email protected]

Norman Pammenter

School of Biological and Conservation Sciences University of KwaZulu-Natal SOUTH AFRICA E-mail: [email protected]

Ruchira Pandey

National Bureau of Plant Genetic Resources Indian Council of Agricultural Research, Government of India INDIA E-mail: [email protected] or [email protected]

Bart Panis

K.U.Leuven, Laboratory of Tropical Crop Improvement BELGIUM E-mail: [email protected]

Ana Panta

International Potato Center (CIP) PERU E-mail: [email protected]

Bozena Pawlowska

Department of Ornamental Plants University of Agriculture POLAND E-mail: [email protected]

Diogo Pedrosa Corrêa da Silva

Department of Biology, Sector of Plant Physiology Federal University of Lavras BRAZIL E-mail: [email protected]

Bart Piette

K.U.Leuven, Laboratory of Tropical Crop Improvement BELGIUM E-mail: [email protected]

Ina Pinker

Institute of Horticultural Sciences Humboldt University of Berlin GERMANY E-mail: [email protected]

Elena Popova

Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University REPUBLIC OF KOREA E-mail: [email protected]

Alberto Previati

Centro Sperimentale Ortofloricolo "Po di Tramontana" Veneto Agricoltura ITALY E-mail: [email protected]

151

1st International Symposium on Cryopreservation in Horticultural Species

List of participants

Pawel Pukacki

Physiology of Abiotic Stress Laboratory Institute of Dendrology Polish Academy of Sciences POLAND E-mail: [email protected]

Saija Rantala

Plant Production Research, Nursery Group MTT FINLAND E-mail: [email protected]

Barbara Reed

National Clonal Germplasm Repository USDA - ARS USA E-mail: [email protected]

M. Angeles Revilla

Biology of Organisms and Systems, Oviedo University SPAIN E-mail: [email protected]

Anabela Romano - Lopez

Faculty of Sciences and Technology, University of Algarve PORTUGAL E-mail: [email protected]

Nicolas Roux

Bioversity International, Commodities for Livelihoods Programme FRANCE E-mail: [email protected]

Djurdjina Ružić

Fruit Physiology Department, Fruit Research Institute SERBIA E-mail: [email protected]

Jan Rybczyński

Botanical Garden, Center for Biological Diversity Conservation Polish Academy of Sciences POLAND E-mail: [email protected]

Carolina Sanchez

Dept. Biologia Vegetal University of Malaga SPAIN E-mail: [email protected]

Angelika Senula

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) GERMANY E-mail: [email protected]

Neelam Sharma

National Bureau of Plant Genetic Resources Indian Council of Agricultural Research, Government of India INDIA E-mail: [email protected] or [email protected]

Mohamad Shatnawi

Biotechnology Department, Faculty of Agricult. Technology Al-balqa applied University JORDAN E-mail: [email protected] or [email protected]

Rida Shibli

University of Jordan College of Agriculture JORDAN E-mail: [email protected]

Marinus J.M. (René) Smulders

Plant Research International Wageningen UR Plant Breeding THE NETHERLANDS E-mail: [email protected]

152

1st International Symposium on Cryopreservation in Horticultural Species

List of participants

Petr Svoboda

Hop Research Institute, Co., Ltd CZECH REPUBLIC E-mail: [email protected]

Rony Swennen

K.U.Leuven, Laboratory of Tropical Crop Improvement BELGIUM E-mail: [email protected]

Daisuke Tanaka

Faculty of Bioresource Sciences, Akita prefectural university JAPAN E-mail: [email protected]

David Tay

International Potato Center (CIP) PERU E-mail: [email protected]

Torben Toldam-Andersen

Department of Agriculture and Ecology, Copenhagen University DENMARK E-mail: [email protected]

Matsuo Uemura

Cryobiofrontier Research Center, Iwate University JAPAN E-mail: [email protected]

Marjatta Uosukainen

Plant Production Research, Nursery Group MTT FINLAND E-mail: [email protected]

Martin Vagner

Lab. of Biologically Active Compounds Insitute of Experimental botany AS CR CZECH REPUBLIC E-mail: [email protected]

Jo Van Assche

ISHS (International Society for Horticultural Science) BELGIUM E-mail: [email protected]

Anna Van de Vyver

National Botanic Garden of Belgium BELGIUM E-mail: [email protected]

Ines Van den houwe

K.U.Leuven, Laboratory of Tropical Crop Improvement BELGIUM E-mail: [email protected]

Peter van der Ende

Sagaplant AS NORWAY E-mail: [email protected]

Toon van doormalen

Nunhems Netherlands BV THE NETHERLANDS E-mail: [email protected] or [email protected]

Johan Van Huylenbroeck

ILVO-PLANT Genetica en Veredeling BELGIUM E-mail: [email protected]

Maurice van Veen

Anthura Research bv THE NETHERLANDS E-mail: [email protected]

153

1st International Symposium on Cryopreservation in Horticultural Species

List of participants

Annelies Vertommen

K.U.Leuven, Laboratory of Tropical Crop Improvement BELGIUM E-mail: [email protected]

Fernanda Vidigal Souza

Embrapa Mandioca e Fruticultura EMBRAPA Brazil E-mail: [email protected]

Maarten Vliex

Nunhems Netherlands BV THE NETHERLANDS E-mail: [email protected] en [email protected]

Christina Vogiatzi

Department of Agriculture and Ecology LIFE University of Copenhagen DENMARK E-mail: [email protected]

Gayle Volk

USDA-ARS National Center for Genetic Resources Preservation USA E-mail: [email protected]

Tatjana Vujović

Fruit Physiology Department, Fruit Research Institute SERBIA E-mail: [email protected]

Christina Walters

USDA-ARS National Center for Genetic Resources Preservation USA E-mail: [email protected]

Qiaochun Wang

College of Horticulture Northwest Agricultural and Forest University CHINA E-mail: [email protected]

Andy Wetten

School of Biological Sciences, Reading University UK E-mail: [email protected]

Anna Wójcik

Botanical Garden, Center for Biological Diversity Conservation Polish Academy of Sciences POLAND E-mail: [email protected]

Chunxiang Xu

College of Horticulture/IMB Institute of Celluar and Molecular Botany GERMANY E-mail: [email protected]

Jiri Zamecnik

Department of Molecular Biology Crop Research Insitute CZECH REPUBLIC E-mail: [email protected]

Jana Zamecnikova

Institute of Tropics and Subtropics Czech University of Life Sciences Prague CZECH REPUBLIC E-mail: [email protected]

Christine Zanke

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) GERMANY E-mail: [email protected]

154

1st International Symposium on Cryopreservation in Horticultural Species

List of participants

Reza Zarghami

Agriculture Biotechnology Research Institute IRAN E-mail: [email protected]

Brenda Zea

International Potato Center (CIP) PERU E-mail: [email protected]

Ya-li Zhang

Institute of Plant Physiology & Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences CHINA E-mail: [email protected]

155