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Agroinfiltration of Nicotiana Benthamiana


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#1 cahornsey

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Posted 21 December 2009 - 05:15 AM

Hi,
I have been using agroinnoculation to introduce my test construct into plants. I am using agrobacterium strain LBA4404 plus my plasmid at an OD600 of 0.1 in infiltration media (50 mM MES, pH 5.6, 0.5% glucose, 2 mM NaPO4, 100 μM acetosyringone). I then leave this 72 hours before measuring luciferase activity from my constructs. However, sometimes after infiltration the leaves shrivel up particularly round the edges and part of the leaf seems to die? I know the infiltration can cause temporary wilting but this is 3 days post innoculation and the leaves do not recover. It doesn't always happen and it does not affect leaves that haven't been infiltrated. It also seems to happen with just infiltration media without any bacteria? The infiltration media is filtered through a 0.2uM filter just before use but the acetosyringone is added afterwards.
Does anyone have any ideas why this might be happening or what I can do to stop it?
Thanks for your help
Crystal
PhD student

#2 smu

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Posted 21 December 2009 - 06:57 AM

Hi,
I have been using agroinnoculation to introduce my test construct into plants. I am using agrobacterium strain LBA4404 plus my plasmid at an OD600 of 0.1 in infiltration media (50 mM MES, pH 5.6, 0.5% glucose, 2 mM NaPO4, 100 μM acetosyringone). I then leave this 72 hours before measuring luciferase activity from my constructs. However, sometimes after infiltration the leaves shrivel up particularly round the edges and part of the leaf seems to die? I know the infiltration can cause temporary wilting but this is 3 days post innoculation and the leaves do not recover. It doesn't always happen and it does not affect leaves that haven't been infiltrated. It also seems to happen with just infiltration media without any bacteria? The infiltration media is filtered through a 0.2uM filter just before use but the acetosyringone is added afterwards.
Does anyone have any ideas why this might be happening or what I can do to stop it?
Thanks for your help
Crystal
PhD student


Hi. I don't know if this will help AT ALL. I've only started doing infiltrations myself, but your buffer is a little different from mine. I use 10mM MES, 10 mM Mgcl2 and 150 uM acetosyringone with the bacteria at an OD of 0.4. After resuspending the agro, I culture it for two more hours in this buffer. I haven't noticed parts of the leaf dying except sometimes around the innoculation site. I have no idea if this will help you or not, but I just thought that you might want to try a different buffer. Good luck with it.

smu

#3 cahornsey

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Posted 21 December 2009 - 08:06 AM

Thank you for your suggestion Smu. This buffer is from the paper An in vivo, luciferase-based, Agrobacterium -infiltration assay system: implications for post-transcriptional gene silencing by Christopher Ian Cazzonelli and Jeff Velten and is also the same as other people at my university use with Nicotiana Tabacum. I have considered changing the buffer but the fact that it does not always happen makes me think that it is perhaps something else? Also I do not understand what the problem would be with the buffer? thank you for your help.
Crystal




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