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H E A L T H Y P L A N T S A N D M IC R O P R O P A G A T IO N

MOLECULARDIAGNOSTICS OFPOTATO SPINDLE TUBERVIROID

(PSTV) INFECTION IN VIRUS-FREE STOCK-PLANTS COLLECTION

S.Musin, V.Bojko, A.Babosha

All-Russia Potato Research Institute, KorenevoMoscow Region

A new DNA-probe, labeled non-radioactively by Pt(dien), was applied

to detect PSTV. A sample containing total plant nucleic acids was applied

onto a membrane filter and fixed there. The filter was put into a hybridization

solution with a DNA-probe labeled by Pt(dien). DNA probe hybridysed only

to PSTV-containing samples from infected plants. To found specific hybrid

duplex by IEA the filters with DNA probes hybridised were put into blocking

solution and after that to another one, containing prime monospecific

antibodies to Pt(dien). Further after washing and blocking samples were

incubated with conjugates o f secondary antibodies w ith alcali phospatase,

which interacted with prime antibodies. The visualization system included a

buffer, phosphatase substrat and paint. An enzime reaction resulted in

formation of a non-soluble product, painting samples, containing secondary

antibodies conjugates, thus being

such a test permitted

to visually determ in PSTV infection, the intensivity o f colouration being in

accordance w ith degree o f infection. Sensitivity o f indication o f the

recombinant pGEM-3Z (PSTV) plasmid by the method was 0.1 picograms.

The method described was applied to test 166 samples from a collection

o f virus-free plants. It was found that unfortunately 56% o f tube virus-free

plants initial for seed production in Russian federation show positive PSTV

reaction. Historically the technology fo r obtaining virus-free potato plants,

developed and applied earlier, was direscted opposit viruses concidered to

be the most harmful at that time, and absolutely didn't take into concideration

a possibility of PSTV and other possible infection. All this and also non-

suffisient attention put to tarditional clones selection methods which must

be applied in complex with virus eradiation led to a situation when PSTV

became a main reason o f unqualitative seed potato in Russian Federation.

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