Agrobacterium-based transformations of Arabidopsis and other plants?

[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.