Biology Reference
In-Depth Information
Lines that do not rescue mutant phenotypes and/or show abnormal phenotypes
should not be used in experiments. For microscopy experiments, fluorescent protein
fusions need to accumulate at detectable levels. Ultimately, it is preferable to choose
multiple transgenic lines that express the least amount of visible fluorescent protein
and compliment the null mutant phenotype.
The location of T-DNA insertion into the genome is uncontrolled and therefore
the T-DNA can act as a mutagen if inserted into a coding or regulatory sequence, or if
the addition of the fluorescent tag changes protein function. When creating fluores-
cent protein fusions, a typical goal is to observe the protein in its native environment
and so T-DNA-induced mutation is undesirable. Therefore, it is important to screen
multiple transgenic lines to find those that do not display unexpected phenotypes.
Mutant phenotypes can be observed in the T1 and T2 generation. We have observed
organ twisting, swelling, dwarfing, and reduced fertility when GFP-tagged versions
of tubulin and some MAPs are expressed. Observation of other nonwild-type pheno-
types may indicate that the transgene has caused a genetic mutation.
The expression of the transgenewill vary from line to line due to the number of trans-
genes inserted and the position of the transgene relative to transcriptional promoters,
enhancers, and suppressors. This phenomenon is referred to as the “positional” effect.
In some lines, no transgene expression will be observed. Screening for fluorescence can
be accomplished with an epifluorescence or confocal microscope. When screening for
mCherry transgenes, it is useful to remove the red chlorophyll autofluorescent signal
with appropriate filters. Finally, some MAPs are very difficult to detect because they
are naturally expressed at very low levels and/or have a high cytoplasmic localization.
15.1.2
Sample preparation
Many cellular and developmental processes that involve microtubules occur in the
A. thaliana
seedling thereby making the seedling an excellent system for experimen-
tation. Upon germination, growth of the seedling stem (hypocotyl) is driven almost
exclusively by cell expansion (
Derbyshire, Findlay, McCann, & Roberts, 2007;
Refregier, Pelletier, Jaillard, & Hofte, 2004
). Cell divisions primarily occur at the api-
cal meristems at the shoot and root tip. The root meristem is an accessible tissue and the
patterns of cell division are predictable (
Sedbrook & Kaloriti, 2008
). The shoot apical
meristem is shielded from view by young leaves that make live-cell imaging challeng-
ing, but possible
(Hamant et al., 2008; Heisler et al., 2010
). Stereotyped asymmetric
divisions occur during the formation of stomata in the developing leaf epidermis
(Lucas et al., 2006). Concurrent with stomatal formation, small box-like epidermal
cells develop into large puzzle-piece-shaped cells (
Fu, Gu, Zheng, Wasteneys, &
Yang, 2005
). All of these processes are available to researchers because the entire
A. thaliana
seedling fits between a standard slide and square cover glass. Next, we will
discuss general sample preparation techniques for the
A. thaliana
seedling.
Plant shoots typically grow upward toward the light, and therefore it can be a
fairly traumatic life event for a seedling to be sandwiched horizontally between a
slide and a coverslip. The impact of this situation should be minimized to avoid
