Biology Reference
In-Depth Information
3
Methods
Carry out all procedures at room temperature unless otherwise
specifi ed.
3.1 Pollen Tube
Cultivation and AODN
Treatment
For simple analysis of pollen tube growth parameters, optimal vol-
ume per sample is 200
μ
L of fi nal pollen tube culture.
1. Premix 1.5
μ
L of cytofectin aliquot (the fi nal concentration is
15
M fi nal ODN con-
centration in glass or polystyrene test tube and add 20
μ
g/ml) with ODNs to obtain 30-50
μ
l of
the cultivation medium. Incubate for 15 min. Do not use
polypropylene labware ( see Note 4 ).
2. Meanwhile, weigh 10 mg of fresh/frozen tobacco pollen per
1 ml of the fi nal pollen tube culture. If freeze-stored pollen is
used, keep it at room temperature for 10-15 min for recovery
before resuspension.
3. Mix the pollen with the cultivation medium and vortex vigor-
ously to obtain homogenous culture without any clumps of
pollen grains.
4. Add appropriate volume of the pollen suspension to ODN-
cytofectin premix to get the fi nal volume of 200
μ
l. Cultivate
for 1-4 h on horizontal shaker (~200 rpm) for effi cient aera-
tion ( see Note 5 ).
5. For subsequent analysis, living pollen tubes can be observed
immediately or the culture is fi xed by addition of formalde-
hyde solution (3.7 % fi nal concentration). Fixed pollen tube
culture can be stored at 4 °C.
μ
3.2 Data Collection
and Analysis
To monitor the effects of AODNs on pollen tube growth and mor-
phology, various approaches can be used. A fi rst insight is usually
obtained by the light microscope, whereby growth rate, morpho-
logical changes of width, or shape could be observed. For the
quantifi cation of these parameters, several programs for the image
analysis can be used (e.g., ImageJ, http://rsbweb.nih.gov/ij/ ;
NIS Elements, http://www.nis-elements.cz/en/front-page/ ).
Based on the questions studied, an appropriate biochemical analysis
(e.g., enzyme essays) of the pollen culture can be performed as well
( see Note 5 ). To get information about effi ciency of the AODN-
mediated gene knockdown, quantitative PCR could be utilized.
4
Notes
1. Second pollen mitosis that gives rise to sperm cells takes place
either before (in case of tricellular pollen) or after (bicellular
pollen) germination.
 
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