2. Selection of appropriate FPs, or combinations of FPs, for
transgenic expression in arabidopsis should be done thoughtfully [38]. It is particularly important to provide an accurate
provenance for the coding sequences that are ultimately
employed. The FPs listed in FPBase (https://www.fpbase.
org/) are an excellent starting point, with other factors being
the tissue or organ specificity of expression (including whether
or not protoplasts can be prepared from that tissue or organ),
the subcellular location for FP expression and accumulation,
and the optical configurations available in the flow instruments.
Although production of transgenic plants is quick and simple
for arabidopsis, considerable variation in expression may be
observed for the primary transformants. Thus, monitoring of
multiple founder lines while stabilizing expression within selfed
progeny will be necessary.
3. Methods of protoplast preparation may require empirical optimization. Issues associated with this include: (a) Source of
tissues. Plants can be grown in pots as exposed plants in the
greenhouse or growth chambers, or can be grown under axenic
conditions on media in tissue culture vessels. If the former, the
tissues will require surface sterilization prior to protoplast preparation. Tissue excision can induce systemic wound responses,
which may result in the source materials becoming recalcitrant
to protoplast production. The change from vegetative to reproductive growth can also make the leaves recalcitrant to digestion. (b) Composition and amounts of cell wall-degrading
enzymes and times and temperatures of incubation that are
employed for protoplast production. This is largely a matter of
trial-and-error, with the general aim being to employ the lowest possible levels of these enzymes, in part acknowledging
their potential to act as agents signaling pathogenic attack. In
general, lower levels of enzymes require longer periods of
incubation. Tissue digestion and protoplast release should be
done at room temperature. Vacuum infiltration can be
employed, and this has no obviously deleterious effects on the
tissues or protoplasts. (c) Protoplast purification. Isopycnic step
gradient flotation is strongly recommended to eliminate subcellular debris and broken and non-viable protoplasts, and
protoplast viability can be conveniently monitored during this
process using fluorochromatic dyes such as fluorescein
diacetate [19].
Acknowledgements
Part of the development of the methods described in this chapter
involved support to DG from the NSF Plant Genome Research
Flow Cytometry and Sorting in Arabidopsis
287
transgenic expression in arabidopsis should be done thoughtfully [38]. It is particularly important to provide an accurate
provenance for the coding sequences that are ultimately
employed. The FPs listed in FPBase (https://www.fpbase.
org/) are an excellent starting point, with other factors being
the tissue or organ specificity of expression (including whether
or not protoplasts can be prepared from that tissue or organ),
the subcellular location for FP expression and accumulation,
and the optical configurations available in the flow instruments.
Although production of transgenic plants is quick and simple
for arabidopsis, considerable variation in expression may be
observed for the primary transformants. Thus, monitoring of
multiple founder lines while stabilizing expression within selfed
progeny will be necessary.
3. Methods of protoplast preparation may require empirical optimization. Issues associated with this include: (a) Source of
tissues. Plants can be grown in pots as exposed plants in the
greenhouse or growth chambers, or can be grown under axenic
conditions on media in tissue culture vessels. If the former, the
tissues will require surface sterilization prior to protoplast preparation. Tissue excision can induce systemic wound responses,
which may result in the source materials becoming recalcitrant
to protoplast production. The change from vegetative to reproductive growth can also make the leaves recalcitrant to digestion. (b) Composition and amounts of cell wall-degrading
enzymes and times and temperatures of incubation that are
employed for protoplast production. This is largely a matter of
trial-and-error, with the general aim being to employ the lowest possible levels of these enzymes, in part acknowledging
their potential to act as agents signaling pathogenic attack. In
general, lower levels of enzymes require longer periods of
incubation. Tissue digestion and protoplast release should be
done at room temperature. Vacuum infiltration can be
employed, and this has no obviously deleterious effects on the
tissues or protoplasts. (c) Protoplast purification. Isopycnic step
gradient flotation is strongly recommended to eliminate subcellular debris and broken and non-viable protoplasts, and
protoplast viability can be conveniently monitored during this
process using fluorochromatic dyes such as fluorescein
diacetate [19].
Acknowledgements
Part of the development of the methods described in this chapter
involved support to DG from the NSF Plant Genome Research
Flow Cytometry and Sorting in Arabidopsis
287