Disruption of leaves and initial extraction of wildtype CPMV virus as a basis for producing vaccines from plants

Wildtype cowpea mosaic virus (CPMV) was extracted from fresh and frozen plant material by methods suitable for large-scale application. Deep freezing, crushing, and thawing in water or buffers gave 0.6+/-0.2 mg g(-1) of virus after up to 24 h. Release from sliced fresh leaves was lower at 0.14+/-0.03 mg g(-1). Homogenisation of frozen leaves for 1 min increased yield to a maximum, on average of 3.5 mg g(-1) but varying between batches from 2.2 to 4.8 mg g(-1) virus Long term storage at -80 degrees C increased subsequent yield by 2 mg g(-1) per year on average; the maximum was 10.4+/-1.9 mg g(-1) (665 days storage). Within a batch, similar yields were obtained between individual fresh plants, and from frozen versus fresh leaves. After homogenisation for 1 min, 90% of debris particles were smaller than 12 microm, half under 5 microm and 10% less than 1 microm. Homogenate (4% dry weight) was rheologically complex, exhibiting shear thinning with hysteresis at low shear rates which bears on subsequent processing. At shear rates above 200 s(-1), its apparent viscosity was 0.02 N s m(-2).