I think, the Ti plasmid can precipitate very fast during PEG mediation. But if you can prevent it, it will work. Direct Ti plasmid injection works, and can produce transgenic cells.

Well, direct transformation has been shown to work, though generally with a lower efficiency. If PEG does not work (as Akos mentioned) you could try electroporation - there are some (rather old) protocols around, including my own youthfull follies :-)

Since direct transformation efficiency is rather low (in comparison to Agrobacterium co-transfection protocols) one usually needs a highly efficient protoplast regeneration protocol to make it work. What species are you working with? Is there a reason you can't use Agrobacteria protocols? They are usually more effective.

Good luck,

Klaus

We have been doing protoplast transformation using PEG for years in our lab. Our transformation efficiency is not too bad, most of the time, but it depens on the insert :).

We are using "normal" Ti-plasmids (the ones you can usually also use with Agrobacteria) if I am not totally mistaken.

Acetosyringone on the other hand works with agrobacteria and helps them to connect to the cells, as far as I know. So that would not help in getting your Plasmid inside. :)

I would rather suggest something in the line of DMSO or, like Klaus Eimert suggested, electroporation (which we also use) to help with the uptake.

What we foud was crucial though is, to be super careful, when handling the cells, as they are quite fragile, once the cell-wall is gone :).

I agree with what has been said. The Ti-plasmid can integrate into the protoplast genome. The efficiency could vary with your method of handling and age of the protoplasts, size of the insert and etc. Acetosyringone is useful only when you use Agrobacterium for gene delivery.

Hyunbae Lee

1. PEG can be used to transform both Ti plasmid and non-Ti plasmid. Are you transforming *Ti-plasmid* or *binary vector with T-DNA*? These are 2 different things.

2. Acetosyringone has nothing to do with PEG-mediated protoplast transformation. It is frequently used for Agrobacterium-mediate genetic transformation with plant explants.

Plasmids can be transformed (or "transfect") into plant protoplasts, through PEG method or electroporation method. However, electroporation can damage/kill more protoplasts than PEG method.

You are totally right Yuan-Yeu Yau . Thanks for pointing it out: I also think, that we are not using Ti-Plasmid, as this are the originals and nowadays there are the smaller and easier to use binary vectors.

I read this nice review Article T-DNA binary vectors and systems

Teresa Wulf

That is correct. But, I think Hyunbae Lee has updated his question (or I missed it earlier) that " I'm trying to directly transfect plant protoplasts with purified Ti-plasmid isolated from Agrobacterium. " So, I think he is working on the original Ti-plasmid.

Hyunbae Lee

Now, your question is clear. So, to your questions:

1. < Can the Ti-plasmid be integrated to the chromosomes.>

Yes. We used to use salmon DNA fragments and a vector (with our gene-of-interest (GOI)) to co-transform tobacco protoplasts. We found our vector was integrated into the plant genome. And, in rare case, the short fish DNA fragments were detected (through sequencing). The salmon DNA was used to minimize the chance our GOI vectors get degraded by cellular enzymes.

2. < Can the presence of acetosyringone affect the efficiency of transfection? >

In my opinion, no. Because Acetosyringone (AS) is useful for Agrobacterium (see the paragraph from [1] below), no effect on PEG (a chemical). You are using PEG for transfect.

"The processing and transfer of T-DNA (Note: inside Agrobacterium) are controlled by the activity of the virulence (vir) genes. The virA gene encodes a membrane-bound kinase that perceives chemical signals, such as the phenolic compound, acetosyringone (AS), from wounded plant cells. [1]" A figure attached.

Article Acetosyringone treatment duration affects large T-DNA molecu...

Hi Yuan-Yeu,

I was wondering - if you use the whole Ti-Plasmid (is it a dis-armed version?), is the integration of it (or parts of it) random? Since the processing, border-recognition etc. probably won't work.

Cheers,

Klaus

Yuan-Yeu Yau Oh... I was totally mistaken. The one I was going to use is pCambia1302 which is a binary vector. I guess introduction of this plasmid to protoplasts leads to transient expression of GFP if it works. What I'm looking for is a single vector that can introduce a foreign gene and be stably integrated to the chromosome. Many articles that utilized PEG or electroporation for protoplast transfection seems to use a single vector. I want these kinds of vectors because I am also going to use Chlamydomonas, an alga whose transformation efficiencies are calculated after spreading on solid medium which I think would require stable expression of the introduced gene.

Then... if I purify Vir helper plasmid from Agrobacterium and co-introduce it with pCambia1302, would it work?

Hyunbae Lee

No, 'bi'nary vector does not refer to '2' vectors here. Binary vector such as pCambian vector is a single vector. You DON'T need Vir helper plasmid to co-transfect. You are not using Agrobacteria here. Vir helper plasmid is something to do with Agrobacteria transformation. Just transfect the pCambia1302 alone into protoplasts with PEG. We have done that.

Yuan-Yeu Yau Then pCambia1302 would stably transfect the protoplast? That's a great news if the GOI can be integrated to the protoplast chromosome!

Many thanks to you!

Hyunbae Lee Yes. After transfection, observe them under a GFP microscope after 1, or 2 or 3 days (or longer if you wish). Transient expression is easy. If you want stably transgenic lines, you need to go through selection, and callus induction from the transformed protoplasts. It is time-consuming. Besides, you need to isolate protoplasts from tissue-culturing plants (from sterile-environment). I wonder why don't you just use regular Agrobacterium transformation method, but instead going through protoplast method?

Yuan-Yeu Yau I'm already doing transient expression with other GFP vector (pBI221-GFP). I want a stable transfection vector because I wouldn't have enough time to grow and examine regenerated plants.

Besides, I am going to introduce the same gene to plant protoplasts and Chlamydomas. The expression of the gene would be checked from the protoplast itself for plants and from the culture of selected colonies for Chlamydomonas. For the plant protoplasts, I can still use transient expression vector but I need stable one for Chlamydomonas. The expression levels of GOI would be quite different in the two experiment group so I wish to have a vector that is more stubborn in plant protoplasts.

Hi Hyunbae,

that makes it "easier" :-) As Yuan-Yeu said, transfection with binary vectors should work and beside transient transformation you should see some chromosomal integration. In our hands the integration events were rather few and we saw a lot of incomplete integration events. Thus, your approach with the observation of GFP for successful integration seems reasonable. It probably depends on the plant species, though. We were not very successful observing GFP flourescence (from the CAMBIA vectors) - too low intensity or too insensitive equipment and pretty high auto-fluorescence in our cells. eGFP worked better for us. You even might think about other colors - there is now a whole range of different colors available (though, I am not sure about the plant adaptation of those).

Good luck,

Klaus

Hyunbae Lee What did you mean by " I want a stable transfection vector "?

Klaus Eimert < Thus, your approach with the observation of GFP for successful integration seems reasonable. >

In conventional transformation, induced callus with GFP can also be viewed and selected. The GFP+ protoplasts can form cell wall, then develop into callus. But the bad thing is that the calli are also very easy to crumble (I don't know Lee's plant species' callus will do the same. Mine was tobacco callus) into several pieces. That means it might be hard for you to obtain as many as independent-event individuals for further growing. Independent-event individuals are few, most are some same-clones from the crumbled callus clumps.