Agrobacterium-based transformations of Arabidopsis and other plants?

[DavidCampen]

I was researching the use of organosilicone wetting agents as pesticide adjuvants and came across this:
Vac-In-Stuff (Silwet L-77) for vacuum infiltration

I take it that this is using the wetting agent to facilitate the bacterium entering the plant? Is this to deliver genetic material? Anyone know more about this?

[Leafmite]

I thought PCR or something but when I read the lab steps, I see that it is not.

[camille1585]

We do this sort of stuff in the lab. I know how it works roughly, but not in detail since I'm not one of the molecular scientists. I use mutated Arabidopsis lines that are already available in our 'library'.

Agrobacterium is a very nice bacteria to use for genetic engineering because it is extremely good at transfering DNA to plants. First you transform the Agrobacterium plasmid (circular) DNA by inserting the gene of interest that you ultimately want in the plant. The reason it's so good at inserting genes into plant DNA is because certain strains of the bacteria normally causes tumors/galls by inserting a piece of its own DNA into the plant. So science has taken advantage of this.

Silwet L-77 is a surfactant which greatly increases the chance of a successful transformation. Tween-20 (a detergent) is another one.

Plants or inflorescences (to get transformed seed) are dipped or sprayed with the surfactant+bacterium solution and then placed under a vacuum pump, and the quick release of the vacuum will draw the solution into the plant tissue, where hopefully it will get integrated into cells. I think the success rate of transformation is still somewhere around 1-2%, maybe lower even.

To make GMO crops they frequently use what's called a 'gene gun', which doesn't involve the surfactant. Very small gold pellets are coated with the transforimed plasmid DNA, then using a meachine, shot them at cell calluses. With any luck some of the DNA will end up in a plant cell without destroying it in the process, and the bacterium will 'give' the gene to the plant genome.

Afterwards there are various techniques used to select the successful transformations. That's also where PCR ( that Leafmite brought up) can come into play to see if new plant tissues contain the inserted gene or not.

I hope I explained this in sufficiently lay terms and didn't say anything silly, since my knowledge is only theoretical.

[DavidCampen]

Thank you Camille, an excellent explanation.

[Leafmite]

I am the fool! I saw the plant mentioned and I knew it was well-known for being one of the first completely sequenced and guessed this was genetics-related based on the polymers involved but, well, I never really studied the logistics of this method of genetic engineering in much detail. With such a low success rate, it would seem more logical to use another method unless, in specific cases, only this one works.

[camille1585]

I double checked, with the surfactant the success rate is around 3%. And I think that this is only one of many ways for making mutant lines. But I don't know what is used and when...

[camille1585]

And 3% is actually quite good. I dug a bit further, here is the abstract of a paper named "Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method":

"Collective efforts of several laboratories in the past two decades have resulted in the development of various methods for Agrobacterium tumefaciens–mediated transformation of Arabidopsis thaliana. Among these, the floral dip method is the most facile protocol and widely used for producing transgenic Arabidopsis plants. In this method, transformation of female gametes is accomplished by simply dipping developing Arabidopsis inflorescences for a few seconds into a 5% sucrose solution containing 0.01–0.05% (vol/vol) Silwet L-77 and resuspended Agrobacterium cells carrying the genes to be transferred. Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants. A transformation frequency of at least 1% can be routinely obtained and a minimum of several hundred independent transgenic lines generated from just two pots of infiltrated plants (20–30 plants per pot) within 2–3 months. Here, we describe the protocol routinely used in our laboratory for the floral dip method for Arabidopsis transformation. Transgenic Arabidopsis plants can be obtained in approximately 3 months."