Automated Microscopy in Forward Genetic Screening of Arabidopsis - Plant Cell Morphogenesis: Methods and Protocols - page 56

Fig. 1 The .slide automated microscopy system consisting of slide loader for 50
slides ( left ), BX51 microscope ( middle ), and the control unit with computer ( right )

3 Methods

Unless otherwise specifi ed, all procedures are performed at labora-
tory temperature.

3.1 Plant Cultivation

1. Temper freshly autoclaved MS media to 50-70 °C in a pre-
warmed water bath, then pour approximately 25 ml of the
media into cylindrical Petri dishes. Work in a fl ow box under
sterile conditions.
2. Place approximately 50 seeds from each M2 family separately
into a 1.5 ml tube. Add 1 ml of 70 % ethanol per tube. Shake
the tube with seeds several times for maximally 5 min ( see
Note 10 ). Transfer the seeds by pipetting (1,000 ml pipette)
onto sterile fi lter papers placed in the fl ow box and let them
dry. Transfer dry seeds by “dusting” (use sterile tweezers and
a glass stick) onto prepared Petri dishes with MS media and
seal the lid using air-permeable tape. For each biological rep-
lica and/or separate experiment, use the non-mutagenized
background line (WT) as a control.
3. Store dishes with seeds at 4 °C in darkness for 2 days
( see Note 11 ).
4. Place dishes into a growth chamber and cultivate for 7 days in
vertical orientation ( see Note 12 ).

3.2 GUS Staining

1. Always prepare fresh GUS staining buffer immediately before
use. Add 1 ml of the GUS staining buffer to each well of the
staining plate ( see Notes 3 , 5 , and 6 ). This amount is suffi cient
for staining about 50 Arabidopsis seedlings 7 days old.