Identifying Subcellular Protein Localization with Fluorescent Protein Fusions After Transient Expression in Onion Epidermal Cells - Plant Cell Morphogenesis: Methods and Protocols - page 77

localization and protein-protein interaction studies. In fact, a num-
ber of collections of organelle marker constructs are available from
stock centers (e.g., refs. [ 5 , 6 ]) that make identifi cation of subcel-
lular localization studies relatively straightforward.
Stable transformation of plants with genes encoding fl uores-
cently tagged proteins, preferably under control of their native
promoters, is clearly desirable since this approach will allow for
observation of long-term effects and, ideally, complementation of
mutant phenotypes. This approach, however, requires a substantial
investment of time. Transient expression approaches, on the other
hand, can already yield important insights into protein localization
and function and can be achieved without much technical effort by
Agrobacterium infi ltration [ 7 - 9 ]. In this chapter, however, we
describe the use of transient expression by means of particle bom-
bardment. As long as the necessary equipment is available, particle
bombardment is usually faster than Agrobacterium-mediated
approaches since it does not require integration of the gene con-
struct into a binary plasmid and its mobilization into an appropri-
ate Agrobacterium strain. The second part of the protocol describes
basic epifl uorescence techniques to visualize fl uorescent proteins in
living plant tissues which apply to all transformation techniques.
The methods presented here can also be modifi ed to accommodate
more complex microscopy techniques such as laser scanning or
spinning disk confocal microscopy.

2 Materials

This protocol is based on the PDS1000/He system (BioRad):

2.1 Particle
Bombardment

1. For onion tissues, M17 tungsten particles (1.0 μ m, BioRad)

work best.
2. A supply of macrocarriers, stopping screens, and rupture disks.
3. Agar plates with standard growth medium (for example, 1/2×
Murashige-Skoog medium, 1 % sucrose, pH 6.0); one per
sample.
4. 2.5 M MgCl 2 .
5. 200 mM spermidine (store in −20 °C freezer).
6. 70 % and 100 % ethanol.
7. Purifi ed plasmids encoding the expression constructs (approx.
100 ng/ l). Miniprep DNA is usually suffi cient.

8. Fresh onion.
9. Fine forceps.
10. Razor blades or scalpel.

μ