Joersbo M, Brunstedt J
DANISCO A/S, Biotechnology Research Division, Copenhagen, Denmark.
J Virol Methods. 1990 Jul;29(1):63-9. doi: 10.1016/0166-0934(90)90008-4.
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.
一种采用温和超声处理在体外将病毒颗粒接种到植物原生质体中的新方法已经建立。在甜菜坏死黄脉病毒（BNYVV）颗粒存在的情况下，将甜菜（Beta vulgaris L.）原生质体短暂暴露于20 kHz超声中。通过酶联免疫吸附测定法分析BNYVV外壳蛋白的积累情况。接种16小时后可检测到感染，接种后3 - 3.5天达到最大值。通过在45 - 60 W下超声处理500 - 600毫秒，可在接种的原生质体中获得BNYVV外壳蛋白的最大水平。这使活力降低至15 - 30%。接种效率随着BNYVV颗粒浓度增加至28微克/毫升而提高。发现当在超声处理前刚加入BNYVV颗粒时感染最为理想，但在超声处理后长达60分钟加入病毒颗粒也可获得低水平的感染。
