5. Plant

So each plant transformation system is unique and I don't have time to delve into them all. So instead, I want to share some general considerations for your agrobacterium-based plant transformation!



With the construct you will express in your plant...

What kind of promoter are you going to use?

  • Do you intend to use a monocot/dicot promoter?
  • Is it inducible?
  • Is it cell-type specific
  • Be sure to match your desired level of expression to your promoter. You don't want something that is slightly toxic to be highly expressed

For your signal peptides:

  • Do you need to perform genome editing and would therefore need a nuclear localization signal?
  • Is it a gene that needs to be expressed in a particular organelle?

For your coding sequence:

  • Is this something that may need to be codon optimized for your plant?
  • If you run into accidental expression in bacteria, try adding an intron
  • Don't forget your start and stop codons!

For your terminator:

  • This is more of a slow down than a guaranteed stop, so at least be aware of the risk of transcriptional leakage 
  • So chances are, you don't speak 9 languages, but you can see that the signs below indicate stop. It might take you a second to realize it though. You should think of your terminators like this, a sequence that is understandable even if not in your native tongue, but you may not understand them all!
https://www.frontsigns.com/blog/the-difference-of-world-traffic-signs/





In terms of markers...







Antibiotics
  • Make sure your antibiotic will work in your monocot/dicot
  • Some antibiotics need light, does yours? And will that impact any dark-culturing your plant will need?

Visual
  • Does your plant have autofluorescence that could be mistaken for this marker?
  • Do you have the flashlight system in place for it? (and how much is it if not)


"Oh my God, there's so many antibiotics, I don't understand"


Don't get discouraged with all the antibiotics, we've all plated something on the wrong medium at some point.
Here's are some examples.
Here's a general outline for the antibiotic usage, though it is not a one-size fits all system.



Plasmid A has a bacterial promoter driving spectinomycin resistance, a plant promoter driving kanamycin resistance and is in Agro strain GV3101

Plasmid B has a bacterial promoter driving kanamycin resistance, a plant promoter driving hygromycin resistance and is in Agro strain LBA4404

When growing up a plasmid in E. coli....

            Make sure your plate has any antibiotics that your plasmid has resistance correlating to under the control of a BACTERIAL promoter.
           
        Plasmid A should have an LB plate with spec
        Plasmid B should have an LB plate with kan

When growing your Agro colonies....

            Be sure to use any antibiotics your Agro has resistance to as well as the bacterial resistance you may have.

        Plasmid A should have an LB/YM plate with spec as well as gentamycin and rifamycin (both are resistances GV3101 has innately)
        Plasmid B should have an LB plate with kan as well rifamycin (an innate resistance for LBA4404)           

When co-cultivating your Agro with your plant....

         It is transformation protocol specific, but this is usually antibiotic free.


When washing/cleaning your Agro off of your plant....

        Be sure to use antibiotics to kill your agrobacterium after transformation. 

        Plasmid A could have meropenem used to clean the plant cultures, don't forget your plant selection
        Plasmid B could have meropenem used to clean the plant cultures, don't forget your plant selection






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