Picking the right strain of A. tumefasceins for 'gene jockeying' P. virgatum

[Nico B.]

Working on a hobby project to make a transgenic switchgrass with an expression of CYP2E1 and working with snap gene as a complete novice. I've found over a dozen varieties of A. tumefasceins species that seem to be dependent on the targeted plant whether that may be a poplar hybrid, petunia, etc., not sure what the considerations are for each of those, though.

How could I go about figuring out what strain is appropriate for my desired function per plant?

[scoc...@gmail.com]

I use GV3101. Other popular strain is EHA105. Swtchgrass, being monocot, is harder to regenerate and agro isn't hugry for it since it lacks large phenolic efflux during wounding which induces virulence in agro. Hardest part isn't strain but a reliable regeneration protocol. Leaves of grasses are tricky to induce somatic embryogenesis. Pardon grammar and brevity, on mobile but wanted to chime in.

Silver nitrate and alpha phytosulphokine works in maize for induction. Pricey medium and light sensitive but can be done. Take protocols with grain of salt since most unless well cited are hard to reproduce. Some cultivars of commonly used crops are distributed for free or low fee from the USDA to business and institutions. Try to replicate experiments using same tissue source or email author for materials.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

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From: Nico B.
Sent: ‎2/‎15/‎2015 6:17 PM
To: diy...@googlegroups.com
Subject: [DIYbio] Picking the right strain of A. tumefasceins for 'genejockeying' P. virgatum

Working on a hobby project to make a transgenic switchgrass with an expression of CYP2E1 and working with snap gene as a complete novice. I've found over a dozen varieties of A. tumefasceins species that seem to be dependent on the targeted plant whether that may be a poplar hybrid, petunia, etc., not sure what the considerations are for each of those, though.

How could I go about figuring out what strain is appropriate for my desired function per plant?

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[Mega [Andreas Stuermer]]

I'd say any agro strain you get from a lab will do the job. Add vanillin to the medium (it simulates a wound signal, so the agro activates its vir genes) and it should be able to transform monocots too. 

[Nico B.]

So if the strains are less of an issue than regeneration, what pushes you to use GV3101 over another strain? Or more generally speaking, what would be the factors to consider when pairing strains to species for this process?

Thanks for the tips on regeneration, got me going in a good direction. I also appreciate the density of your grammar.... helps me learn more, faster

[scoc...@gmail.com]

I say follow the paper trail. Whatever was published in switchgrass, try that strain. Stan Gelvin at Perdue "fathered" some of those strains and I learned with GV. EHA is slower and less aggressive thus lower efficiceny and GV is more aggressive but has a higher chance of causing pathogen defense. Personal experience and some snippets of papers I read long ago is what I'm basing the above statements/claims on. I do agro mediated tobacco mostly. Some floral dips.

My old adage is its all in the culture. Ralph Bock,a personal hero of mine working out of  Max Plank institute, has a nice paper on corn somatic embryo induction using silver nitrate and that awesome peptide  alpha psk (previous post) and was done from leaves. I always try to recommend getting a tissue culture system going before spending time on transformation since it will fail if you can't regenerate. Multiple strains, large sample size, and good experimental design can lead to a solid conclusion but you risk reinventing the wheel if you don't hit the books and see what's been done already.

Aim for articles cited multiple times or which are explicitly stated as the main method in a related paper. Citation is a rough indicator of practicality and function of the method as a reproducible protocol.

Get your hands dirty and report back! :)

Also there is a reason why crown gall is much less prevalent in monocots. Hard to find a natural agro tumor on grasses in the field. May be evidence for the lack of steamy romance between the soil bacterium and cereals in general.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

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From: Nico B.
Sent: ‎2/‎17/‎2015 1:57 PM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] Picking the right strain of A. tumefasceins for'genejockeying' P. virgatum

To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/cfdbf907-169a-4dce-986f-fbf31b2701d0%40googlegroups.com.