Graduate Thesis Or Dissertation
 

Evaluation of hop (Humulus lupulus L.) protoplast inoculation with Prunus necrotic ringspot virus

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  • Hop tissue (Humulus lupulus L.) was grown in vitro in callus, suspension, and protoplast cultures. Medium components were evaluated for optimal callus growth. Solid Murashige and Skoog (MS) medium with picloram at 1.5 uM, 6-benzylaminopurine (BAP) at 10 uM, 1 g/1 casein hydrolysate, and 100 mg/1 citric acid induced white, relatively friable callus from petioles of the cultivar Cascade. Suspension cultures were initiated and sustained using liquid Gamborg's B5 medium with 1 g/1 casein hydrolysate, 100 mg/1 citric acid, picloram at 1.5 uM, BAP at 10 uM, 250 mg/1 L-glutamine, and 250 mg/1 NH₄NO₃. Protoplasts were isolated from suspension cultures to test procedures and conditions for inoculation with Prunus necrotic ringspot virus (NRSV). Successful infection was not verified; however, 0.0 to over 1,000 ng of virus were adsorbed per 50,000 protoplasts, the amount adsorbed depending on the inoculation treatment. Poly-L-ornithine and protamine sulfate, included with inoculum as infection facilitators, both adversely affected protoplast viability, particularly with protamine sulfate concentrations above 10 ug/ml. This effect was moderated by adding 147 mg/1 CaCl₂ to inoculum buffer. Enzyme linked immunosorbent assay (ELISA), an immunofluorescent slide assay, and nucleic acid hybridization were used to detect NRSV in or on protoplasts. ELISA was the most reliable and sensitive method. Results from hybridization using a cDNA probe, reverse transcribed from NRSV RNA, were difficult to interpret because of non-specific hybridization with host RNA. Extraction with 2M LiC1 was found to be an efficient way to extract total RNA from small plant samples. Possible reasons for negative results from protoplast inoculation are discussed.
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