Steps Towards the Validation of Allium and Strawberry Cryopreservation

To enable the routine utilisation and wider application of plant germplasm cryoconservation techniques by other laboratories, there is a need to identify critical factors that effect germplasm survival and implement best practice procedures for their validation. This is of fundamental importance for a protocol to be effective and reliable in the context of genebanking and transfer of germplasm, however the validation of cryopreservation protocols between different laboratories is not yet commonplace. Cryopreservation protocols for clonally propagated crop species have been developed, at the University of Derby for the use of Allium sativum (garlic) stem disc explants and in vitro shoot tips of Fragaria x ananassa Duch. (strawberry) and demonstrated to be applicable to a number of genotypes. However, to indicate their potential for more widespread use these protocols require to be validated to facilitate their application to germplasm conservation. To assist the validation process, the encapsulation/dehydration technique was optimised using cultivars supplied by IPK Genebank to cryopreserve garlic stem disc explants during a technology exchange programme between IPK Genebank and the University of Derby. A detailed description of the protocol was sent to the IPK Genebank and using stored garlic cloves that were physiologically standardised validation experiments took place simultaneously between both laboratories. Following the validation process, several parameters were identified as critical factors during the technological exchange, including: (i) differing technical experience in relation to garlic explant tissue excision; (ii) encapsulation of the stem-discs and (iii) differences in facilities, particularly relating to in vitro culture rooms. Nonetheless, this exercise proved to be of paramount importance to validate this encapsulation/dehydration protocol for the cryopreservation of garlic stem-disc explants between the IPK Genebank and University of Derby laboratories. Moreover, a further cryopreservation validation exercise is currently being conducted using a Plant Vitrification Solution 2 (PVS2) based protocol for the vitrification of strawberry in vitro shoot tips cultures, in a technology exchange programme between the University of Derby and the Institute of Fruit Breeding, Dresden-Pillnitz. This cryopreservation exchange programme has been initiated through a Short-Term Scientific Mission, as part of COST Action 871 by Dr.Monika Höfer with the University of Derby. This Mission s outcome, so far has enabled Dr. Höfer to gain specific and essential hands on experience and technical advice regarding the application of the PVS2 protocol. Perhaps, more significantly, this opportunity to visit colleagues at the University of Derby has enabled both collaborators to compare and contrast differing technical experiences, particularly in terms of explant characteristics crucial to the survival of strawberry in vitro shoot tips cultures and successful application of the PVS2 protocol. Additionally, the COST Mission provided the occasion to identify potentially critical factors including variation in size of explant and in the use of micropropagation media assisting in the transfer of this protocol to Dr. Höfer institution. An assessment of the significance of these factors will be undertaken as the basis for future validation experiments which will be conducted between both Institutions. The value of this exchange programme will be instrumental in the future design of experiments to use newly initiated in vitro cultures from the Institute of Fruit Breeding, Dresden-Pillnitz. Studies will incorporate strawberry varieties which have been shown at the University of Derby to be responsive to the PVS2 protocol and ones which are significant types representative of the strawberry germplasm collection from the Institute of Fruit Breeding, Dresden-Pillnitz. Based on these two case studies illustrating the cryopreservation of clonally propagated crop species for A. sativum stem disc explants and in vitro shoot tips of Fragaria x ananassa the issues associated with effective technology transfer and the validation of cryopreservation protocols will be discussed.

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