Dear Researchers,
I am doing transformation in cotton plant. Now I want to check GFP expression in transformed tissue. Can anyone suggest a standard protocol for preparing tissue sample to visualize GFP under fluorescence microscope? Thanks in advance.
Dear Prem Kumar G
Pls. find the attached files
http://onlinelibrary.wiley.com/doi/10.1111/j.1467-7652.2005.00145.x/full
http://www.biotechniques.com/multimedia/archive/00170/BTN_A_000113778_O_170833a.pdf
http://link.springer.com/article/10.1186/1746-4811-9-40
http://www.biotechniques.com/BiotechniquesJournal/2012/January/A-cryohistological-protocol-for-preparation-of-large-plant-tissue-sections-for----screening-intracellular-fluorescent-protein-expression/biotechniques-324974.html
http://link.springer.com/article/10.1023/A:1011906701925
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC92827/
http://jhc.sagepub.com/content/51/1/5.full
https://www.jove.com/video/50521/efficient-agroinfiltration-plants-for-high-level-transient-expression
http://www.jove.com/visualize/abstract/23143487/estimating-copy-number-transgenes-transformed-cotton-real-time
https://www.ncbi.nlm.nih.gov/pubmed/22229725
Hoping this will be helpful
Regards
Prof. Houda Kawas
I did GFP transformation of cotton before. I did not use a specific protocol. I just took the embryoids (or germinated embryoids) to check under fluorescence microscope. The transformed ones showed very good green fluorescence, while the wild-type (non-transformed) had no fluorescence.
If you use green tissues, the auto-fluorescence can be generated and cover the GFP-produced green fluorescence. So, be aware auto-fluorescence.
You can directly visualize your plant tissue expressing GFP if your fluorescence microscope has a filter for GFP which will detect fluorescence that is above the background (auto-fluorescence from green tissue). You may refer:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854347/
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