Automated Microscopy in Forward Genetic Screening of Arabidopsis - Plant Cell Morphogenesis: Methods and Protocols - page 53

We adapted a “classical” EMS mutagenesis approach in combi-
nation with automated microscopy screening for isolation of muta-
tions affecting spatiotemporal expression of genes of interest
(GOI). The method is based on the identifi cation of mutants in
the expression profi le of a transgenic line carrying a reporter under
control of a GOI promoter. Alternatively, the translational fusion
of a reporter with GOI coding sequence under control of the GOI
promoter could be used.
As an example, we report a mutant screen directed to identify-
ing factors regulating signaling via multistep phosphorelay (MSP)
in Arabidopsis (reviewed in ref. [ 8 ]). To identify factors modulat-
ing expression of CKI1 encoding one of the sensor histidine kinases
initiating MSP signaling, we performed a forward genetic screen
using the ProCKI1:GUS reporter line in a combination with EMS
mutagenesis. This approach would be diffi cult without employing
automated microscopy and innovative high-throughput imaging
technology, thus enabling rapid acquisition and analysis of large
datasets.
Here, we present a use of the “dot slide” (.slide) system for
digital virtual microscopy (Olympus, http://www.olympus-
europa.com ) that was developed primarily for automated imaging
of tissue sections in biomedical applications. We adapted this tech-
nology for effective scanning of specimens with very low contrast,
i.e., Arabidopsis seedlings after GUS staining and tissue clearing
[ 9 ]. This experimental setup has substantially accelerated screening
the M2 population of an EMS-mutagenized ProCKI1:GUS trans-
genic line. In our hands, the entire process of screening approxi-
mately 2,000 M2 families by a single researcher can be completed
within 6 months. Together with the following rescreen of interest-
ing mutant lines, it is possible to begin mapping the mutation of
interest after 1 year of work. The use of automated microscopy not
only facilitates the data acquisition but also makes the subsequent
data analysis less time-consuming. In any case, of course, the speci-
men preparation remains the most time- and labor-intensive part
of the work.
This technology is suitable for use also with other types of
reporters, including fl uorescent proteins that can be visualized
using additional, optional hardware. Finally, in addition to
Arabidopsis seedlings, the automated microscopy can be used for
detailed analysis of diverse tissue types and identifi cation of rather
moderate anatomical changes at microscopic level while screening
for morphology mutants even in reporter-less lines.
The automated microscopy could be performed with other
recently commercially available systems. However, we fi nd the
.slide system as the most open and therefore as the most suitable
for diverse research applications. We are open to share the .slide
system in our lab on either collaborative or commercial basis.