Live Cell Imaging of Arabidopsis Root Hairs - Plant Cell Morphogenesis: Methods and Protocols - page 191

3.5 Plant Growth
and Microscopy

1. Petri dishes with Biofoil sandwiches are closed with Parafi lm
and Falcon tubes are closed by screwing on their lid before
transferring the material to an incubator. We incubate the
sandwiches at 25 °C under long-day conditions (16 h light,
8 h darkness) for 4-6 days ( see Note 2 ).
2. When seeds germinate, roots grow into the agar against the
coverslip ( see Note 3 ). As soon as the roots are at least 5 mm
in length, they can be used for imaging ( see Note 4 ). We image
the Biofoil sandwiches on an inverted microscope by clamping
them into a stage insert with adjustable sliders. Growing root
hairs can be imaged for prolonged time (at least 4 h).

Drugs and dyes can be applied to roots grown in Biofoil sand-
wiches by submerging the sandwich in liquid Hoagland's medium
containing the chemical of interest. It takes some time for chemi-
cals to diffuse into the sandwiches, but we have been able to suc-
cessfully depolymerize microtubules within 20 min after application
of 10

3.6 Drug Treatments
and Stainings

M oryzalin and we have observed FM4-64 internalization
within 15 min (Zhang and Ketelaar, unpublished results).

μ

4 Notes

1. If seeds on your Biofoil sandwich germinate, but do not form
roots, the seed sterilization may have damaged the root meri-
stem. Try reducing sterilization times or increase the number
of washes after sterilization.
2. If a white cloud appears after several days, seed sterilization has
not been suffi cient or contamination has occurred during the
procedure.
3. If roots do not grow into the medium, the Biofoil sandwich
may have dried too much. Try applying colder medium, cover
with Biofoil immediately after solidifi cation of the medium,
and do not leave the Biofoil sandwiches uncovered in the fl ow
hood for prolonged periods.
4. If roots grow nicely into the agar, but do not form root hairs,
the sandwich may be too thick or thin, or the number of seeds
is not high enough.

References

1. Schiefelbein J (2000) Specifi cation of root hair
cells. In: Ridge RW, Emons AMC (eds) Root
hairs. Cell and molecular biology. Springer,
Tokyo, Berlin, Heidelberg, New York, pp
197-210
2. Miller DD, De Ruijter NCA, Bisseling T et al
(1999) The role of actin in root hair morpho-
genesis: Studies with lipochitooligosaccharide

as a growth stimulator and cytochalasin as an
actin-perturbing drug. Plant J 17:141-154
3. Miller DD, Leferink-ten Klooster HB, Emons
AM (2000) Lipochito-oligosaccharide nodula-
tion factors stimulate cytoplasmic polarity
with longitudinal endoplasmic reticulum and
vesicles at the tip in vetch root hairs. Mol Plant
Microbe Interact 13:1385-1390