Re: [DIYbio] A question about generating plant calluses

People have used DIY gene guns, which doesn't require agrobacterium which is considered a plant pest. And it's really just pressurized gass shooting DNA-coated metal-nanoparticles into plant cells. Although it's unlikely that that disarmed lab strain remains viable in the wild (they lose their advantage of forcing plant cells to feed them) it may be smart to circumvent any regulatory issues. 

Depending on the strain and plasmids you work with, some antibiotics might work. Kanamycin or streptomycin. Tetracyclin is not recommended as they can develop natural resistance very easily, for what I heard. 

In practiuce you have to subculture the regenerated plantlets several times with antibiotics to make reasonably sure all the agrobacterium will be gone.

On Sunday, May 28, 2017 at 2:36:51 AM UTC+2, Jeff Backstrom wrote:

On Fri, May 26, 2017 at 4:33 PM, Patricia P <> wrote:
 1.  Do calluses need light to grow?

Most do. Not all.
Some protocols grow them in the dark, so I am confused.  If so, what lights/cycle do you recommend?

By the time you generate calli, you will have enough to run experiments. Some in the light, some in the dark. Try 12/12 for those in the light.
My purpose for for growing calluses (calli?) would be two-fold: somatic embryogenesis, and transformation with Agrobacter in Crassula. 
 2.  Can I use parafilm to seal the callus-growing petri dishes? What about gas exchange? 

To seal the dishes around the edges? Yes. Parafilm is not air-tight. Carbon dioxide and ethylene will diffuse in and out; ditto with water and oxygen. Parafilm tends to embrittle and get useless after a period of time. You may want to try other plastic wrap like Saran Wrap or other plastic films, or medical paper tape.

 3. Would the PPM in the callus media kill Agrobacter?

Never count on PPM being -cidal. You'll have to run tests on media without selective agents to demonstrate the agro is either gone, or hiding so well that you can't easily find it.

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