Research Group In vitro Storage and Cryopreservation

Head: Dr. Joachim Keller

Research Interest

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 MenthaBrassicaDioscoreaArtemisiaAntirrhinum and Orthosiphon for which cycles of in vitro maintenance have been developed.

 

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). The cryo-genebank consists of 1403 accessions of potato, 112 of Allium and 97 of mint. Research input is necessary for further crops 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 and other vegetatively propagated materials are research targets in the group. Cryopreservation of Allium pollen was proved to be usable for genebanking. A number of 176 accessions have been stored already.

 

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. This was performed in collaboration with research groups Structural Cell Biology, Applied Biochemistry and Molecular Plant Physiology. Increase of regeneration after rewarming in material cultivated at alternating temperatures prior to cryopreservation is obviously not caused by cold hardening proper but by generally higher vigour of the tissue. The DMSO droplet method is successful despite presence of crystallizable water in the tissue.


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 395 garlic and shallot accessions regarding important characters, ontogenesis of plants in selected accessions and the classification into infra-specific groups. Accessions from the international EURALLIVEG project were also included.

 

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.

 

[link]An international project was co-ordinated and successfully concluded by the research group within the EU regulation 870/2004 concerning the management of genetic resources. The main result is the establishment of a cryopreserved garlic collection which is maintained in genebanks of three countries (Czech Republic, Germany, and Poland). It includes also safety duplicates of all accessions. This cryo-genebank comprises 220 accessions, 143 of which are flagged as European Accessions. A core collection of shallots from five genebanks was also circumscribed. Further work was done on virus elimination of garlic. The project was performed for rationalisation and increase of maintenance safety of both crops.

 

The research group is also participating in the European COST Action “Endophytes in Biotechnology and Agriculture”, where we provide research material. In the frame of the Leibniz Competition we are integrated in the Project “Cryostress - mechanisms of cellular adaptation to extremely low temperatures”. Working on potato and using the model organism Arabidopsis thaliana (L.) Heynh., interrelationships between physico-chemical parameters (cell water), biochemical alterations and genetics are investigated.

 

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.

12) Inflorescence of the model species Allium obliquum L.; 13) Anther with pollen grains which can be harvested; 14) Staining by fluorescein diacetate (FDA) gives an impression about pollen viability; 15) Even better is to count the pollen germination in a germination solution.