Switchgrass Lab Protocols: Difference between revisions
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==Diploid ''Panicum'' crossing technique== | ==Diploid ''Panicum'' crossing technique== | ||
Crossing the diploid ''Panicum'' species ''P. hallii'', ''P. filipes'', and ''P. capillare'' is difficult because the species are strongly inclined to self-fertilize. In order to make successful crosses, hand emasculation must be used. Because flowers are small I have always used a dissecting microscope. | Crossing the diploid ''Panicum'' species ''P. hallii'', ''P. filipes'', and ''P. capillare'' is difficult because the species are strongly inclined to self-fertilize. In order to make successful crosses, hand emasculation must be used. Because flowers are small I have always used a dissecting microscope. I confirm all crosses with microsat genotyping. Even when I am very careful, the about 5-15% of my putative hybrids end up being the selfed instead. | ||
The method I have found most successful is the following: | The method I have found most successful is the following: | ||
1) Place plants in the dark over night | 1) Place plants in the dark over night. This prevents flowers from opening too quickly the next morning. | ||
2) Remove plants from dark the next morning around 8-10am | 2) Remove plants from dark the next morning around 8-10am. | ||
3) Search for flowers that look as if they are about to open (i.e. lemma and palea starting to separate) | 3) Search for flowers that look as if they are about to open (i.e. lemma and palea starting to separate). | ||
4) Maneuver plant so that flower is under dissecting microscope | 4) Maneuver plant so that flower is under dissecting microscope. | ||
5) Using a fine forceps, carefully and gently separate lemma and palea. This will help the flower to fully open. The anthers and stigma should pop out of the flower and be accessible for emasculation and fertilization. If you damage the lemma or palea in the process you are unlikely to be successful, so only flowers that are almost open will work. | 5) Using a fine forceps, carefully and gently separate lemma and palea. This will help the flower to fully open. The anthers and stigma should pop out of the flower and be accessible for emasculation and fertilization. If you damage the lemma or palea in the process you are unlikely to be successful, so only flowers that are almost open will work. | ||
Revision as of 10:26, 18 March 2011
Diploid Panicum crossing technique
Crossing the diploid Panicum species P. hallii, P. filipes, and P. capillare is difficult because the species are strongly inclined to self-fertilize. In order to make successful crosses, hand emasculation must be used. Because flowers are small I have always used a dissecting microscope. I confirm all crosses with microsat genotyping. Even when I am very careful, the about 5-15% of my putative hybrids end up being the selfed instead.
The method I have found most successful is the following:
1) Place plants in the dark over night. This prevents flowers from opening too quickly the next morning.
2) Remove plants from dark the next morning around 8-10am.
3) Search for flowers that look as if they are about to open (i.e. lemma and palea starting to separate).
4) Maneuver plant so that flower is under dissecting microscope.
5) Using a fine forceps, carefully and gently separate lemma and palea. This will help the flower to fully open. The anthers and stigma should pop out of the flower and be accessible for emasculation and fertilization. If you damage the lemma or palea in the process you are unlikely to be successful, so only flowers that are almost open will work.
6) Use the fine forceps to remove all three anthers. Make sure that the anthers have not opened yet as this will lead to self-fertilization. Examine the stimgas carefully to make sure that no pollen is on them.
7) Clean forceps with ethanol to prevent any contamination
7) Once anthers have been removed, find nearly open flowers on the plant that you want to be the pollen donor (sire) of your cross. Remove the anthers from that fresh flower and wait until the anthers open. Usually, the anthers open only a minute or two after the flower opens. Note: Fresh pollen is absolutely necessary for a successful cross. I have tried crosses with older pollen and all of these failed. A researcher working on other Panicum species told me that pollen may only be viable for 15-30 minutes after the flower has opened, so move quickly.
8) Use the forceps to transfer pollen to the stigma. The stigmas look like little purple Christmas trees. I generally cover them with 50-100 grains of pollen such that the stigmas look like they are decorated with many ball ornaments.
9) Mark crosses with a small piece of lab tape
10) Clean forceps with ethanol and move onto the next cross.
Microsat Primers
Coming soon
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