modifications in populations of cells can be screened by flow cytometry for desired rare events, and these cells sorted, if CRISPR
action is coupled to a fluorescent phenotype, such as FP expression/repression [124]. Prolonged (<24 h) culture of protoplasts is
unfortunately accompanied by resynthesis of the cell wall, the onset
of cell division, and the formation of cell clusters [125, 126], which
confounds cytometric analysis and sorting of single cells. Inclusion
of 2,4-dichlorobenzonitrile in the culture medium, which inhibits
wall formation without deleterious effects on other cellular metabolic processes [127], may be useful in this situation.
1.5 Dealing with
Large Cells
Protoplasts from plant tissues are typically larger than the animal
cells around which flow cytometric instrumentation was originally
designed. Protoplast diameters frequently exceed 20–30 μm, and
standard flow tips (50–70 μm diameter) cannot be used, being
replaced by large flow tips (diameters in the range of
100–200 μm). The use of large flow tips limits the rate of sorting,
since laws of physics constrain the upper limit of the actuation
frequency that drives droplet production [128, 129]. For maintenance of protoplast viability during sorting, employing compatible
sheath fluids is essential. Most core facilities operate instruments
using some variant of standard phosphate-buffered saline (PBS) as
the sheath fluid. This is certainly non-physiological for plants, and
inclusion of phosphate conflicts with a common requirement for
millimolar levels of Ca
2+ in protoplast preparation and culture
media. The general question of the effects of protoplasting on
“cellular state” remains a subject of debate: on the one hand,
removal of the cell wall, imposition of water stress in the form of
an osmoticum, and employing complex mixtures of lytic enzymes
to dissolve the walls are most likely to affect protoplast physiology.
On the other hand, imposition of these biotic and abiotic stresses
onto intact plants is not known to change developmental cellular
fate, at least in the short term, and some experiments demonstrate
this point directly [25]. Circumspect pragmatism appears to be in
order, in terms of experimental design and interpretation of the
results. However, in all cases, and as a general rule, employing
“clean” populations of obviously viable protoplasts as input samples
is strongly recommended, and to this end the experimenter should
become intimately familiar with recognizing cell biological and
anatomical indicators of cellular health through the use of light
and fluorescence microscopy.
1.6 Dealing with
Superlarge Objects,
such as Seed
Specialized instrumentation has been designed for flow analysis and
sorting of biological objects that are much larger than cells, such as
Arabidopsis seeds. This niche is occupied by Union Biometrica,
with its COPAS, BioSorter, and COPAS VISION families of instruments, which achieve high-throughput analysis and sorting of
objects up to 1500 μm in diameter. Sorting involves pneumatic
266
David W. Galbraith and Guiling Sun
action is coupled to a fluorescent phenotype, such as FP expression/repression [124]. Prolonged (<24 h) culture of protoplasts is
unfortunately accompanied by resynthesis of the cell wall, the onset
of cell division, and the formation of cell clusters [125, 126], which
confounds cytometric analysis and sorting of single cells. Inclusion
of 2,4-dichlorobenzonitrile in the culture medium, which inhibits
wall formation without deleterious effects on other cellular metabolic processes [127], may be useful in this situation.
1.5 Dealing with
Large Cells
Protoplasts from plant tissues are typically larger than the animal
cells around which flow cytometric instrumentation was originally
designed. Protoplast diameters frequently exceed 20–30 μm, and
standard flow tips (50–70 μm diameter) cannot be used, being
replaced by large flow tips (diameters in the range of
100–200 μm). The use of large flow tips limits the rate of sorting,
since laws of physics constrain the upper limit of the actuation
frequency that drives droplet production [128, 129]. For maintenance of protoplast viability during sorting, employing compatible
sheath fluids is essential. Most core facilities operate instruments
using some variant of standard phosphate-buffered saline (PBS) as
the sheath fluid. This is certainly non-physiological for plants, and
inclusion of phosphate conflicts with a common requirement for
millimolar levels of Ca
2+ in protoplast preparation and culture
media. The general question of the effects of protoplasting on
“cellular state” remains a subject of debate: on the one hand,
removal of the cell wall, imposition of water stress in the form of
an osmoticum, and employing complex mixtures of lytic enzymes
to dissolve the walls are most likely to affect protoplast physiology.
On the other hand, imposition of these biotic and abiotic stresses
onto intact plants is not known to change developmental cellular
fate, at least in the short term, and some experiments demonstrate
this point directly [25]. Circumspect pragmatism appears to be in
order, in terms of experimental design and interpretation of the
results. However, in all cases, and as a general rule, employing
“clean” populations of obviously viable protoplasts as input samples
is strongly recommended, and to this end the experimenter should
become intimately familiar with recognizing cell biological and
anatomical indicators of cellular health through the use of light
and fluorescence microscopy.
1.6 Dealing with
Superlarge Objects,
such as Seed
Specialized instrumentation has been designed for flow analysis and
sorting of biological objects that are much larger than cells, such as
Arabidopsis seeds. This niche is occupied by Union Biometrica,
with its COPAS, BioSorter, and COPAS VISION families of instruments, which achieve high-throughput analysis and sorting of
objects up to 1500 μm in diameter. Sorting involves pneumatic
266
David W. Galbraith and Guiling Sun