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To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.
Cite this Article
Pasquali, M., Kistler, C. Gibberella zeae Ascospore Production and Collection for Microarray Experiments.. J. Vis. Exp. (1), e115, doi:10.3791/115 (2006).
- Carrot agar (Klittich and Leslie, 1988) was prepared in 9-cm diameter Petri dishes.
- A 2 mm cube of agar containing fresh mycelium grown on potato dextrose agar was applied to the centre of each Petri dish.
- Cultures were grown at 25°C and a 12-h photoperiod for 96 hrs.
- One ml of aqueous 2.5% Tween 60 was applied to the surface of the culture (Trail and Common, 2000) and spread across the culture with a plastic rod, by gently pressing.
- Perithecia shooting ascospores developed 7-10 days after this induction treatment when kept in a high humidity environment at 25°C with the same photoperiod.
- Spore collection is performed by changing the Petri plate lid, leaving perithecia to shoot for a limited time (for example 12 hrs) in the dark.
- Sterile water is added to the lid, suspending ascospores by gently moving water on the surface.
- Ascospores are collected in sterile Falcon tubes, washed and used for further analysis.
The protocol presented here is based on previous procedures used for perithecial production for Fusarium spp. (Klittich and Leslie, 1988; Trail and Common, 2000). Standardization of the procedure by which large quantities of ascospores (sufficient for microarray analysis) were generated was essential for the reproducibility of the experiment. It has been reported that environmental factors, age and substrate differences can change the biological character of ascospores (Beyer and Verreet, 2005). Therefore the use of video may highlight small details in the way spores are produced and collected that should facilitate reproducibility. In particular the video of the procedure should improve the level of standardization among laboratories and facilitate the comparison of whole genome transcription studies which require ascospore production. The amount of RNA necessary for experiment procedure is relatively high so a large number of Petri dish should be processed synchronously. Video is a particularly suitable tool when it is necessary to implement whole genome transcriptional studies on new biological material, setting a standard for future experiments.
The authors thank Karen Hilburn for excellent technical support. This project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service (Award #2004-35604-14327). Matias Pasquali is supported by Branco Weiss Fellowship. The US Wheat and Barley Scab Initiative is also acknowledged for the continuing support of research.
|Petri dish 9 cm diameter||Tool|
|Tween 60 (20%)||Reagent|
|Carrot agar (20% w/v organic carrots and 1.5% w/v agar)||Reagent|
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