LABORATORY PROCEDURES FOR PLANT CELL VIRUSES
REFERENCE NO.: PVC/1998/3.00
TITLE: PRESERVATION OF VIRUS INFECTED PLANT MATERIALS, MAINTENANCE AND STOCK CONTROL
Plant Viruses require special preservation methods to ensure long term viability/infectivity of the sample and to maintain the original characteristics. Preservation methods used in a CABRI Plant Virus Collection are: Freezing and low temperature storage in or above liquid nitrogen (PVC/1998/3.01), freeze drying and low temperature storage (PVC/1998/3.02), and virus maintenance in situ (PVC/1998/3.03). The latter is needed when virus can otherwise not be preserved, however, authenticity of virus isolates kept in situ can not be guaranteed. Detailed protocols for the different types of preservation methods are available at the Plant Virus Collection.
Samples of all virus isolates in the collection, independent of their mechanical transmissibility are preserved in liquid nitrogen. As a plant virus sample usually symptomatic leave tissue of virus infected plants is taken and kept as ORIGINAL SAMPLE for future comparison purposes. After a successful authentication process (PVC/1998/2.04), the original sample is discarded and replaced by the virus material for which an ordinary database entry has been assigned. The samples stored in liquid nitrogen serve as nuclear virus seed stocks in cases where infectivity is lost after prolonged storage, or when stock to be replenished fails authentication due to contamination with other viruses. Virus materials for distribution are stored in aliquots. Stock is controlled through the database administration managing replenishment of samples and procedures indicated. Virus material for replenishment has to be re-authenticated, as virus cultures may be subjected to changes, since preservation and subculture conditions present a selective pressure on the virus and certain features. For example, vector transmissibility can be lost during prolonged subculturing by mechanical transmission.
Virus materials for distribution are stored in aliquots of freeze dried tissues in labelled vials for distribution. An inventory entry in the database indicates batch date and number of vials in the particular preparation. Batch size depends on demand, but usually 30 vials containing virus infected tissues are prepared in a batch. When less than 5 vials remain, virus from the distribution stock is propagated on suitable propagation hosts and the material is authenticated according toPVC/1998/2.04
After successful authentication, batches are prepared and stored as described inPVC/1998/3.02. Stock management and inventory control is accomplished through database administration stating batch dates and vial numbers. For viruses that are not in high demand, a batch list is prepared stating virus samples kept in preservation for longer than 2 years. According to the list and virus specified, a vial from the distribution stock is taken for an infectivity test on suitable host plants (PVC/1998/2.02, Appendix 3) combined with EM (PVC/1998/2.02, Appendix 5).
Guidelines prepared for CABRI by DSMZ, 3 Feb. 1998
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