PRESERVATION OF VIRUS INFECTED PLANT MATERIAL BY FREEZE-DRYING AND LOW TEMPERATURE STORAGE

LABORATORY PROCEDURES FOR PLANT CELL VIRUSES

REFERENCE NO.: PVC/1998/3.02

TITLE: PRESERVATION OF VIRUS INFECTED PLANT MATERIAL BY FREEZE-DRYING AND LOW TEMPERATURE STORAGE

INTRODUCTION

Virus infected material for distribution purposes is preserved by freeze-drying of virus infected leaves for virus inocula or, as homogenates of virus infected plants, for virus reference and standardisation purposes.

PROCEDURE

Symptomatic leaves of plants authenticated for infection with a specific virus strain or virus isolate are excised from the plants, and further processed under semi-sterile conditions eliminating problems of contaminations with other viruses. Leave midribs are removed and the material is sliced into small 1-2 cm long stripes. Using a sterile forceps approx. 300 µg of leave material is filled in 5ml glass vials which are topped with a rubber stopper to be subjected to the freeze drying process that is ended by closing the vials under vacuo. A batch of 30 vials are processed per sample and only one virus specimen is subjected to the process at a time to prevent any spill and contamination at any phase of the sample preparation. After freeze-drying the labelled vials are stored at –60 °C. An inventory remark is made indicating batch date, number and size to assist in stock management. An infectivity test with this material is made only when batch date indicates for storage longer than 2 years or, after customer complaints.

For preparation of freeze-dried virus infected plant homogenates, symptomatic leaves are excised and a virus suspension is prepared by homogenising the material 1:10 (w/v) in 0.1M sodium phosphate buffer pH 7.0 with a sterile pestle and mortar. After filtering of the homogenate through 3 layers of muslin to remove plant debris, 0.5ml aliquots are pipetted into 5 ml vials and processed as above stated by freeze-drying. The labelled virus reference vials are stored at –20 °C.

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