Biology Reference
In-Depth Information
.1 ProduCtIon oF VnPs In the greenhouSe
A broad range of plant VNPs has been exploited for nanotechnology and
biotechnology. The most extensively studied particles are the icosahedrons
Brome mosaic virus
(BMV),
Cowpea chlorotic mottle virus
(CCMV),
Cowpea
mosaic virus
(CPMV),
Red clover necrotic mottle virus
(RCNMV), and the rod-
shaped particle
(TMV), all of which can be produced in
large scales in their natural plant hosts. Plant viruses typically accumulate to
high titers in the plants and can be extracted in yields of 1-2 g/kg infected
leaf material. The production species for BMV is barley (
Tobacco mosaic virus
Hordeum vulgare
).
CCMV and CPMV are produced in black-eyed pea (
Vigna unguiculata
). RCNMV
is produced in the common pea (
Phaseolus vulgaris
), and TMV is produced in
tobacco plants (
). For further information on plant viruses
and their hosts, the reader is referred to the Description of Plant Viruses
database (DPV; http://www.dpvweb.net).
Infection of plants can be achieved by mechanical inoculation using
either homogenized plant tissue from infected leaves, or purified VNPs.
An infection can also be achieved using linearized plasmids containing
a complementary DNA (cDNA) copy of the viral genomes or
Nicotiana tabacum
RNA
transcripts of the genomes (the RNA transcript serves as a template for
protein expression during translation); these methods are typically used
when generating mutants or chimeras (Section 3.1.1). To infect about
50 plants, one begins with just a few infected leaves. The plant material
is homogenized using either mortar and pestle or a blender; the latter is
applicable for large-scale inoculations. The plant material is then suspended
in buffer and filtered through cheesecloth (the composition of the extraction
buffer depends on the VNP; for many viruses 0.01-0.1 M phosphate buffers
are used). The leaf homogenate is then rubbed onto the surface of leaves
in such a way to break cells on the surface but without causing too much
mechanical damage. Carborundum (silicon carbide, SiC) can be applied
onto the leaves to help create lesions in the cell walls on the leaf surface
and to facilitate viral cell entry.
After initial inoculation, viruses typically spread within the plant
via cell-to-cell and long-distance movement. Long-distance movement
is through the vascular tissue along with the photoassimilates and the
direction is source-to-sink (Mekuria
in vitro
et al
., 2008; Roberts
et al
., 1997; Silva
et al
., 2002). “Source” refers to leaves that are photosynthetically active
(these are developed leaves), and “sink” refers to the developing tissue
(young developing leaves). To facilitate systemic spread primary leaves of
young plants are infected, and the infection proceeds as the plant develops.
At 2-3 weeks post-inoculation, leaves can be harvested and stored frozen
indefinitely until further processing.
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