Inoculation of sugar beet protoplasts with beet necrotic yellow vein virus particles by mild sonication.
A novel procedure employing mild sonication for in vitro inoculation of plant protoplasts with virus particles has been established. Sugar beet (Beta vulgaris L.) protoplasts were briefly exposed to 20 kHz ultrasound in the presence of beet necrotic yellow vein virus (BNYVV) particles. The accumulation of BNYVV coat protein was analyzed by an enzyme-linked immunosorbent assay. The infection was detectable after 16 h and reached maximum 3-3 1/2 days after inoculation. Maximum levels of BNYVV coat protein in inoculated protoplasts were obtained by sonication for 500-600 ms at 45-60 W. This reduced the viability to 15-30%. The efficiency of the inoculation increased with the concentration of BNYVV particles up to 28 micrograms/ml. Infection was found to be optimal when the BNYVV particles were added just before sonication but low levels of infection could be obtained by addition of virus particles up to 60 min after sonication.
DANISCO A/S, Biotechnology Research Division, Copenhagen, Denmark.
M
M
Joersbo
J
J
Brunstedt
D002213:Capsid / Q000302:isolation & purification; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D010942:Plant Viruses / Q000254:growth & development*; D010944:Plants; D011523:Protoplasts; D013010:Sonication; D014776:Virus Cultivation / Q000379:methods*