|
|
|
|
| In vitro Storage and Cryopreservation
|
|
|
|
|
 |
The genebank collection contains plants which have to be propagated and maintained solely vegetatively. At Gatersleben, these are species of the genus Allium (garlic, shallots, top onions etc.) as well as several other vegetables and herbs. Accumulation of diseases has been found in accessions which have been maintained vegetatively in field plots. However, plants, which do not form storable seeds or which have to be maintained as clones for breeding or research can be maintained apart from sources of infection by in vitro culture and cryopreservation. The practical application of both methods is in different states of development. Therefore, a whole range of applied as well as fundamental investigations are still necessary for the optimisation of the processes. This is especially important for cryopreservation.
|
|
The in vitro maintenance is based on culture cycles consisting of micropropagation, isolation of single shoots, plant growth, and cold storage. Such cycles have been established for various vegetatively propagated Allium forms. Further development is in progress. In Allium, multiple shoot bunches, single plantlets or in vitro induced storage organs (bulblets) can be maintained under cold storage conditions. Recently, vegetatively propagated accessions were introduced of Mentha, Brassica, Dioscorea, Artemisia, Antirrhinum and Orthosiphon for which cycles of in vitro maintenance have been developed.
In vitro culture is also applied in exploiting the large genetic diversity within the Gatersleben collections. Novel hybrids have been produced with the aid of embryo rescue after crossing the onion with distant Allium species. These plants are in permanent maintenance in the genebank. They will be analysed and are ready for further utilisation.
The potentially safest method to maintain vegetative germplasm and recalcitrant seeds for long time periods without changes is the storage in or above liquid nitrogen (cryopreservation). A cryo-collection of potato has been established, comprising shoot tips of material from the IPK branch at Groß Lüsewitz and of accessions introduced in the former BAZ/FAL genebank at Braunschweig. Other cryo-collections have been established for garlic and mint. The cryo-genebank increased already up to 1140 accessions of potato, 67 of Allium and 40 of mint. For further crops, however, research input is necessary to make cryopreservation methods applicable for practical use. Organised structures may be stored for germplasm preservation such as meristems, embryos, pollen etc. Further Allium species, yams and other vegetatively propagated materials are research targets in the group.
|
|
Special research was conducted in potato. It was focussed on physiological, biochemical and ultrastructural analyses after different preculture temperatures and after exposure to liquid nitrogen. Cold-tolerant wild species are compared with cold-sensitive cultivated varieties. Light- and electron microscopic observations (Collaboration Research Group Structural Cell Biology) were used to investigate the behaviour of the cell structure in the different steps of the cryopreservation protocol and to localise the regeneration processes after rewarming. Patterns of protein expression (Collaboration Research Group Applied Biochemistry), soluble sugars, starch and amino acids were used as indicators of differences in the reactions in cryopreservation after variation of the preculture. The increase of regeneration after rewarming in material cultivated at alternating temperatures prior to cryopreservation is obviously not caused by cold hardening proper but by a complex reaction consisting of accumulation of soluble sugars and a general vigour increase in the tissue. The regeneration takes place after rewarming mainly in meristematic regions of the youngest leaf primordia.
Biophysical parameters within the tissues of the material to be preserved are investigated by differential scanning calorimetry (DSC). Measured heat flow changes are used for exploration of dehydration and glass transition processes, which are necessary for preservation of the tissues in liquid nitrogen. The glassy state is a condition of solutions within the cells in which they are solidified in an amorphous structure, which is much less damaging for the tissue than ice crystallisation taking place without cryoprotection.
|
|
| In Allium, the research group is also participating in the general germplasm characterisation and it has contributed to the development of plant descriptors. An image database of the garlic core collection of IPK gives an overview about 124 garlic accessions regarding important characters, ontogensis of plants in selected accessions and the classification into infra-specific groups.
|
|
| Reconstruction of the inflorescence reduction in course of the domestication of garlic demonstrated with morphological pictures of various accessions of the garlic core collection. Above the pictures are the respective infra-specific groups.
An international project is co-ordinated by the research group within the EU regulation 870/2004 concerning the management of genetic resources. The project EURALLIVEG comprises molecular-genetic characterisation for duplicate elimination, cryopreservation and virus elimination of garlic as well as molecular-genetic characterisation of shallot. This project is dedicated to rationalise the collections and to increase maintenance safety of both crop species.
|
|
|
DUPLICATE SCREENING
Leaf pieces for DNA isolation
|
CRYOPRESERVATION
Transfer of the samples in
into liquid nitrogen
|
VIRUS ELIMINATION
ELISA test on virus-free status
|
Pictures of Cryopreservation
|
Potato droplet-freezing: 1) Droplets on aluminium foil containing explants prior to freezing; 2) Regenerants on solid medium 2 weeks after rewarming; 3) Regenerant plantlet 5 weeks after rewarming. |
Garlic vitrification: 4) explant for vitrification; 5) first regeneration stage 2 weeks after rewarming; 6) Petri dish containing regenerating explants 3 weeks after rewarming; 7) Comparative experiment, plantlets taken out of the culture vessels 4 months after rewarming (-LN control without liquid nitrogen, +LN after cryopreservation). |
Mint droplet vitrification: 8) and 9) two regenerants 5 days after rewarming; 10) In vitro shoots 8 weeks after rewarming; 11) Plants in the greenhouse 4 months after rewarming. |
|
| [ ^ ]
|
|
|
|
| 2009 |
KACZMARCZYK, A., M. GRÜBE & E.R.J. KELLER
| Present state of potato cryopreservation – new aspects reviewed. CryoLetters: 30: 81-82. |
|
|
| 2009 |
KACZMARCZYK, A., A. SENULA, M. GRÜBE & E.R.J. KELLER
| DSC - Anwendung in der Kryokonservierung vegetativer pflanzengenetischer Ressourcen. In: KUNZE, W. (Ed.): Anwendungen der Thermischen Analyse, Mikrokalorimetrie & Rheologie im Bereich der Pharmazie, Biotechnologie, Lebensmitteltechnologie & Kosmetik. TA Instruments, Waters GmbH, Eschborn, Deutschland. pp. 15-29. |
|
|
| 2008 |
KACZMARCZYK, A., T. RUTTEN, M. MELZER & E.R.J. KELLER
| Ultrastructural changes associated with cryopreservation of potato (Solanum tuberosum L.) shoot tips. CryoLetters 29: 145-156. |
|
|
| 2008 |
KACZMARCZYK, A., N. SHVACHKO, Y. LUPYSHEVA, M.-R. HAJIREZAEI & E. R. J. KELLER
| Influence of alternating temperature preculture on cryopreservation results for potato shoot tips. Plant Cell Rep. 27: 1551-1558. . |
|
|
| 2008 |
KELLER, E. R. J., A. SENULA & A. KACZMARCZYK
| Cryopreservation of herbaceous dicots. In: REED, B. M. (ed) Plant Cryopreservation: A Practical Guide. Springer, New York. pp. 281-332. |
|
|
| 2008 |
KELLER, E.R.J., A. KACZMARCZYK & A. SENULA
| Cryopreservation for plant genebanks - A matter between high expectations and cautious reservation. CryoLetters 29: 53-62. |
|
|
| 2007 |
KACZMARCZYK, A., M. GRÜBE & E.R.J. KELLER
| Cryopreservation of potato: New Results from the IPK Gatersleben, Germany. Mem. Int. Congr. Plant Biotechnol. & Agricult. (BioVeg2007). Centro de Bioplantas, Ciego de Avila, Cuba, May 7-11, CD ROM, 8 pp. |
|
|
| 2007 |
KELLER, E. R. J.
| Cryopreservation for maintenance of plant germplasm in Germany. Adv. Hort. Sci. 21: 228-231. |
|
|
| 2007 |
SENULA, A., E.R.J. KELLER, T. SANDUIJAV & T. YOHANNES
| Cryopreservation of cold-acclimated mint (Mentha spp.) shoot tips using a simple vitrification protocol. CryoLetters 28: 1-12. |
|
|
| 2006 |
ASTLEY, D., N. BAS, J. KELLER & E. ROSA
| AEGIS discussions for Allium and Brassica subgroups in Prague. IPGRI Newsl. Europe 32: 10-11. |
|
|
| 2006 |
KELLER, E.R.J.
| Die Erhöhung von Effektivität und Sicherheit bei der Erhaltung permanent vegetativer Genbank-Akzessionen durch In-vitro-Kultur und Kryokonservierung. Vortr. Pflanzenzüchtg. 70: 16-26. |
|
|
| 2006 |
KELLER, J. &. D. ASTLEY
| Rich genepool of vegetatively propagated Allium L. in Europe. IPGRI Newsl. Europe 32: 7. |
|
|
| 2006 |
KELLER, E.R.J. & R. M. FRITSCH
| Current status of the Allium collections at IPK Gatersleben, Germany. In: Thomas, G., D. Astley, I. Boukema, M. C. Daunay, A. del Greco, M. J. Díez, W. van Dooijewert, J. Keller, T. Kotlinska, A. Lebeda, E. Lipman, L. Maggioni & E. Rosa (eds) Report of a vegetables network. Joint meeting with an ad hoc group of leafy vegetables, 22-24 May 2003, Skierniewice, Poland. IPGRI, Rome: 25-28 pp. |
|
|
| 2006 |
KELLER, E. R. J., A. SENULA, S. LEUNUFNA & M. GRÜBE
| Slow growth storage and cryopreservation - tools to facilitate germplasm maintenance of vegetatively propagated crops in living plant collections. Int. J. Refrigeration 29: 411-417. |
|
|
| 2006 |
KRYSZCZUK, A., J. KELLER, M. GRÜBE & E. ZIMNOCH-GUZOWSKA
| Cryopreservation of potato (Solanum tuberosum L.) shoot tips using vitrification and droplet method. Food, Agriculture Environm. 4:196-200. |
|
|
| 2006 |
THOMAS, G., D. ASTLEY, I. BOUKEMA, M. C. DAUNAY, A. DEL GRECO, M. J. DÍEZ, W. VAN DOOIJEWERT, J. KELLER, T. KOTLINSKA, A. LEBEDA, E. LIPMAN, L. MAGGIONI & E. ROSA (EDS)
| Report of a vegetables network. Joint meeting with an ad hoc group of leafy vegetables, 22-24 May 2003, Skierniewice, Poland. IPGRI, Rome: 146 pp. |
|
|
| 2006 |
ZIEGERT, K., W. SCHÜTZE, H. SCHULZ, M. KEUSGEN, E. GUN & E.R.J. KELLER
| Efficient determination of cysteine sulphoxides in Allium plants applying new biosensor and HPLC-MS2 methods. J. Appl. Bot. Food Quality 80: 31-35. |
|
|
| 2005 |
ISLAM, M. T., D. P. DEMBELE & E. R. J. KELLER
| Influence of explant, temperature and different culture vessels on in vitro culture for germplasm maintenance of four mint accessions. Plant Cell, Tissue, Organ Cult. 81: 123-130. |
|
|
| 2005 |
KELLER, E. R. J.
| Improvement of cryopreservation results in garlic using low temperature preculture and high-quality in vitro plantlets CryoLetters 26: 357-366. |
|
|
| 2005 |
KELLER, E. R. J., M. GRÜBE & A. SENULA
| Cryopreservation in the Gatersleben genebank - state of the art in potato, garlic and mint. Mem. Congr. Internat. Biotecnología y Agricultura (Bioveg 2005). Centro de Bioplantas, Ciego de Avila, Cuba, pp. 97-105. |
|
|
| 2005 |
KELLER, E. R. J., A. SENULA & M. DREILING
| Genebanking of vegetatively propagated medicinal plants - two cases: Allium and Mentha. Acta Hortic. 676: 103-109. |
|
|
| 2005 |
LEUNUFNA, S. & E. R. J. KELLER
| Cryopreservation of yams using vitrification modified by including droplet method: effects of cold acclimation and sucrose. CryoLetters 26: 93-102. |
|
|
| 2004 |
KELLER, E.R.J. & A. SENULA
| In vitro techniques to improve the germplasm preservation - case studies for three temperate crops and some general remarks. In: ISLAM, A.S. (ed.) In Vitro Culture, Transformation and Molecular Markers for Crop Improvement. Sci. Publ. Enfield, USA, Plymouth, UK, 107-117. |
|
|
| 2004 |
KIM, H.-H., E.-G. CHO, H.-J. BAEK, C.-Y. KIM, E.R.J. KELLER & F. ENGELMANN
| Cryopreservation of garlic shoot tips by vitrification: effects of dehydration, rewarming, unloading and regrowth conditions. Cryo-Letters 25 59-70. |
|
|
| 2004 |
STORSBERG, J., H. SCHULZ, M. KEUSGEN, F. TANNOUS, K.J. DEHMER & E.R.J. KELLER
| Chemical Characterization of Interspecific Hybrids between Allium cepa L. and Allium kermesinum Rchb. J. Agric. Food Chem. 52: 5499-5505. |
|
|
| [ ^ ]
|
|
|
|
| [ ^ ]
|
|
|
|
| in vitro genebank, in vitro maintenance, cryopreservation, slow growth culture, virus elimination, potato (Solanum tuberosum), garlic (Allium sativum), Allium species, Mentha species, Dioscorea species
|
|
|
|
