Biology Reference
In-Depth Information
Inducible Expression of DUO1
T2 seed from transgenic Col-O plants were grown in MSO plates containing 20
µg/ml hygromycin in standard conditions for 12 days. 25 seedlings were trans-
ferred to either control plants containing 0.002% v/v DMSO or induction plates
contain 2 µM 17
β
-estradiol dissolved in DMSO. Plants were returned to the
growth room for a further 24 h, before being snap frozen in liquid nitrogen.
RT-PCR Analysis
Pollen from ecotype Landsberg erecta at different stages of development was iso-
lated and RNA extracted as described [31]. For RT-PCR on seedling ectopically
expressing mDUO1 and for DUO1 promoter analysis, RNA was extracted from
frozen samples using the Qiagen RNeasy Kit. Samples of 750 ng or 1 µg of total
RNA for pollen stages and seedlings, respectively, were reverse transcribed in a 20
µl reaction using Superscript II RNase H reverse transcriptase (Invitrogen) and
an oligodT primer as per the manufactures instructions. For PCR amplification
1 µl of a 10× (pollen stages) or 5× (seedling) diluted cDNA was used in a 25 µl
reaction using Biotaq DNA polymerase (Bioline) and 12.5 pmol of each primer.
PCR conditions were: 96°C for 1 min, 30 to 40 cycles at 96°C for 30 s, 55°C
for 30 s, 72°C for 40 s followed by 5 min at 72°C. Histone H3 (At4g40040) was
used as a control.
Analysis and Imaging of Pollen
Mature pollen was stained with DAPI (4
′
-6-Diamidino-2-phenylindole) as de-
scribed previously [35]. Staining for GUS activity was performed as described
[36] with inflorescences incubated in GUS buffer (100 mM sodium-phosphate,
pH 7; 5 mM EDTA, 0.1% Triton X-100) with 1 mM X-gluc (5-bromo-4-chlo-
ro-3-indolyl b-D-glucuronide) and 0.5 mM K
3
Fe[CN]
6
, at 37°C for 1-3 days.
Stained inflorescences were then cleared with 70% ethanol. Pollen was dissected
out and stained with 0.8 µg/ml DAPI in GUS buffer. Phenotypic analysis of pol-
len was conducted on a Nikon TE2000-E inverted microscope (Nikon, Japan).
Bright field and DIC images were captured with a Nikon-D100 camera (Model
MH-18, Japan) and fluorescence images were captured with HAMAMATSU -
ORCA-ER digital camera (Model C4742-95, Japan) using Openlab software ver-
sion 5.0.2. (Improvision).
For confocal laser scanning microscopy (CLSM) pollen from buds at different
stages of development was teased out of the anther with a needle and mounted
in 0.3 M mannitol and mature pollen was released directly into 0.3 M mannitol.
