Hi,
To screen different organelles in the cells, I am using CFP markers in agrobacterium based on the following protocol http://www.nature.com/nprot/journal/v1/n4/abs/nprot.2006.286.html. So, growing the bacteria overnight and then adding infiltration medium with several washing and centrifugation process and adjusting OD of the cells+ bacteria to 0.1, I allow the cells to grow within the medium supplemented with the resulting bacteria in infiltration medium. Then. I look at them under microscope. However, the organelles are not observed with this method. I thought since I am working with the cells not the plant tissue, there should be some changes in preparation process. Would be grateful any suggestion.
best,
Hi
In order to look at mitochondria you can use mitotracker, see my paper from 2012, plant journal or 2009 RNA journal, I did it in protoplasts, you can also fused to GFP or RFP the transit peptides of ssRubisco as a chloroplast signal and AOX for mitochondria, very known localization signals for these organelles
if you have more questions you can email me in person
good luck
Are you doing leaf infiltration? Maybe you are not waiting long enough after infiltration for full expression. Also what organelles are you trying to visualize?
Are you using the same species as the protocol (tobacco)? Other plant varieties may need alterations to the protocol, such as the time needed for expression.
Also, CFP can be a bit tricky to see. If you don't have the correct excitation lamp/laser and filter set then the signal may be really dim. Do have you have some control CFP positive tissues/cells that you can observe on your scope to check the settings?
If I understand you correctly - you are adding the agrobacterium to tissue culture cells? But the protocol that you cite you are following is for transformation into whole leaves. The article cited below is a protocol for transforming plant cells in tissue culture. Also, as stated above, CFP is a very weak fluorescent tag. You might want to try TFP or Cerulean if you are set on using a blue FP instead of RFP or YFP. I assume you have chosen a blue FP to either avoid overlap with autofluorescence or do some co-localization experiments. Hope the article below helps you out. Alternatively, you can transform protoplasts or into whole leaves.
Curr Protoc Cell Biol. 2003 Aug;Chapter 1:Unit 1.7. doi: 10.1002/0471143030.cb0107s19. BY-2 cells: culture and transformation for live cell imaging.
Thank you all of you for your suggestion.
Yes dear, Stacy you are right I am using a protocol which has been used for the whole plant while I am using the cells.
I was wondering if I could ask you to send me the paper you recommended, I can't get it!
best,
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