1.2 Flow Cytometry
and Sorting Applied
to Plant Protoplasts
Flow analysis and sorting of plant protoplasts and the successful
culture of sorted subpopulations was first reported 35 years ago
[8]. Although protoplasts are exceptionally fragile [9, 10], it was
found possible to configure a commercial flow sorter for protoplast
analysis and sorting, while maintaining their viabilities. This implies
the hydrodynamic, mechanical, and environmental forces experienced by the protoplasts during sorting are too low to cause serious
damage.
These basic cytometric methods were further used with protoplasts from maize leaves [11–13], arabidopsis roots [14, 15], aerial
tissues [16], and leaves [17]. The diameters of arabidopsis protoplasts vary according to source tissue and endoreduplication status:
10–20 μm for root protoplasts [18]; 30–50 μm for protoplasts
prepared from leaves [17], and 10–20 μm for protoplasts prepared
from aerial portions of plantlets grown on vertical agar plates
(Galbraith laboratory, unpublished observations). For tobacco,
the flow sorted protoplasts remained viable, as shown by culturing
them to form cell clusters and regenerating these into plants
[8]. Flow sorting was also used to directly isolate viable heterokaryons produced through induced protoplast fusion, from which
somatic hybrid plants were produced [19]. Thus, the physical
process of flow sorting seems innocuous even to protoplasts that
have been subjected to drastic membrane destabilization to induce
fusion.
1.3 Analyzing
Protoplasts Using Flow
Cytometry
Flow cytometers record the light scatter and fluorescence signals
produced by suspensions of cells, microscopic particles (such as
standard fluorospheres) or submicroscopic cellular and
non-cellular suspensions (including bacteria, large viruses, subcellular organelles, and extracellular vesicles), as they pass through one
or more points of illumination. Analysis of each particle is triggered
by the detection of the first pulse waveform produced as it transits
its points of illumination, and all pulse waveforms (scatter and
fluorescence) that coincide in time with this trigger waveform are
processed coordinately. Flow cytometers can be configured with
multiple laser illumination sources and optical detectors, and this
has greatly expanded the maximum numbers of fluorescent parameters that can be measured from a single object, assuming sufficient independent fluorescent dyes and delivery/specificity
methods (antibodies, etc.) are available. Analysis of around 30 different parameters is now routinely achieved, the vast majority of
these analyses involving mammalian (human, mouse) cell suspensions [20–22].
Fluorescence signals can be derived either from pre-existing
endogenous fluorochromes, chlorophyll being an obvious example
for plants, or from fluorochromes that are produced by transgenic
synthesis (c.f. the Fluorescent Proteins (FPs)), or simply added
exogenously, such as the standard fluorochrome labels (FITC,
Flow Cytometry and Sorting in Arabidopsis
257
and Sorting Applied
to Plant Protoplasts
Flow analysis and sorting of plant protoplasts and the successful
culture of sorted subpopulations was first reported 35 years ago
[8]. Although protoplasts are exceptionally fragile [9, 10], it was
found possible to configure a commercial flow sorter for protoplast
analysis and sorting, while maintaining their viabilities. This implies
the hydrodynamic, mechanical, and environmental forces experienced by the protoplasts during sorting are too low to cause serious
damage.
These basic cytometric methods were further used with protoplasts from maize leaves [11–13], arabidopsis roots [14, 15], aerial
tissues [16], and leaves [17]. The diameters of arabidopsis protoplasts vary according to source tissue and endoreduplication status:
10–20 μm for root protoplasts [18]; 30–50 μm for protoplasts
prepared from leaves [17], and 10–20 μm for protoplasts prepared
from aerial portions of plantlets grown on vertical agar plates
(Galbraith laboratory, unpublished observations). For tobacco,
the flow sorted protoplasts remained viable, as shown by culturing
them to form cell clusters and regenerating these into plants
[8]. Flow sorting was also used to directly isolate viable heterokaryons produced through induced protoplast fusion, from which
somatic hybrid plants were produced [19]. Thus, the physical
process of flow sorting seems innocuous even to protoplasts that
have been subjected to drastic membrane destabilization to induce
fusion.
1.3 Analyzing
Protoplasts Using Flow
Cytometry
Flow cytometers record the light scatter and fluorescence signals
produced by suspensions of cells, microscopic particles (such as
standard fluorospheres) or submicroscopic cellular and
non-cellular suspensions (including bacteria, large viruses, subcellular organelles, and extracellular vesicles), as they pass through one
or more points of illumination. Analysis of each particle is triggered
by the detection of the first pulse waveform produced as it transits
its points of illumination, and all pulse waveforms (scatter and
fluorescence) that coincide in time with this trigger waveform are
processed coordinately. Flow cytometers can be configured with
multiple laser illumination sources and optical detectors, and this
has greatly expanded the maximum numbers of fluorescent parameters that can be measured from a single object, assuming sufficient independent fluorescent dyes and delivery/specificity
methods (antibodies, etc.) are available. Analysis of around 30 different parameters is now routinely achieved, the vast majority of
these analyses involving mammalian (human, mouse) cell suspensions [20–22].
Fluorescence signals can be derived either from pre-existing
endogenous fluorochromes, chlorophyll being an obvious example
for plants, or from fluorochromes that are produced by transgenic
synthesis (c.f. the Fluorescent Proteins (FPs)), or simply added
exogenously, such as the standard fluorochrome labels (FITC,
Flow Cytometry and Sorting in Arabidopsis
257