What are the genes-of-interest on the 2 failed binary vectors? The gene products might have produced in the Agrobacterium and toxic to them.

We have not inserted our GOI yet. They all currently have GUS (beta-glucoronidase gene).

Are these 3 binary vectors all pCambia binary vectors? Some confer kanamycin resistant (bacterial selection).

And what is Ubiquitin B.V. plate? Shouldn't they grow on LB medium (or other Agrobacterium medium), if they don't have your experimental gene (GOI) in there??

I checked pCambia 1301 map (see attachment). Looks like that the binary vector contains kanamycin-resistant (kanr) gene for the bacteria selection, and hygromycin selection for transformed plants (inside the T-DNA borders).

What antibiotic did you use for selection of the other two binary vectors??

Im using hygromyocin (50mg/L) to select for the binary vector and streptomyocin (100mg/L) to select for the LBA4404 agrobacterium I am electroporating (resistance to streptomyocin on the chromosome). The same antibiotics and concentrations are being used for the other two binary vectors.

Im unable to edit the annotation but Apha-3 and CDS on ubiquitin and LP2 respectively are kanamyacin resistance. all three binary vectors I am using have the exact same resistance.

I think you used the wrong antibiotic for bacterial selection. You should use 'kanamycin' (+ streptomycin) to select the Agrobacterium harboring the binary vectors. The hygromycin-resistant gene inside the T-DNA borders (RB and LB) will be transferred into plants, and will be used for transgenic plants selection. We used kan to select them.

Use kanamycin (+ streptomycin) to select the Agrobacterium with the two other binary vectors and find out.

**pCambia 1301, outside the T-DNA borders on the backbone,  there is a kan-resistance gene. This is the gene people use to select the binary vector.

See this attached picture of "General structure of pCambia vectors". You can see that the gene for bacterial selection (or what you said: "binary vector" selection) is outside the T-DNA borders (RB and LB).

Info from: http://www.cambia.org/daisy/cambia/585

The pCAMBIAs has kanamycin and hygromycin s-markers for bacterial and plant selection respectively. Whereas, Agrobacterium has the ability to grow on streptomycin plate (as it's screening plate). Hence, this is why your plate needs to have the two antibiotics (kan and sm).

Getting single colony on the selective plate didn't signify that your transformation is successful, until after selection analyses e.g. colony PCR and d-digestion !. Then how about the transformation efficiency ??

I suggest you should try a chemical method for the transformation as it also works well (base on my experience).

Yes, they should be selected on kan + streptomycin plate, not hygromycin + streptomycin plate. Update us your results @ Jackie Mettler if you re-try. Thanks.

So, you're saying the the agrobacterium are unable to express Hygromyocin resistance? How would the P. Cambia transformants be able to grow on my hygro plates if so? My only reason for doing so was that we don't have kanamyacin and my coworker said either hygro or kana would work.

@Jackie, Remember, pCAMBIAs has kanamycin and hygromycin selectable markers for bacteria and plant selection respectively. The Hyg is for plant selection after the plant transformation system, not for bacterial screening after it's (bacterium) transformation.

Though, it may grow on Hyg plate.

Wield, RG these days does not send out update emails. Sorry for the late response.

@ Jackie, kanamycin is pretty cheap (I think it is cheaper than hygromycin). Just buy one and grow the Agrobacterium with the other two constructs (binary vectors). You should be able to get what you want.