The floral-dip method of Arabidopsis works very well and is widely used. Please see Clough and Bent, 1998, Plant J 16:753

For the Arabidopsis floral dip method it is important that you have your plants at just the right stage of flower bud formation in order for the transformation to occur efficiently. Other things like using Silwet L-77 are important. It is almost ten years since I have used this method, but there are a number of important references on this method. A good reference is Zhang et al. (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocols 1(2) 1-6. The screening of the seeds can be done on large agar plates after seed sterilisation or more simply in seedling raising trays with spraying of the seedlings with antibiotic to select for transformants.

Tobacco is probably the easiest plant to transform, with Agrobacterium being the easiest method for this species. I would check by PCR that both your plasmids are in fact present in your agrobacterium cells. It would probably be easier to try a co-transformation experiment whereby two agrobacterium cultures containing one binary vector each are mixed and inoculated onto your tobacco leaf explants. If would be worth getting a control plasmid from another lab that has say a gus or gfp gene as well as nptII or hyg gene to run as a control on your experiments. You need to ensure you have good quality tobacco shoot cultures as your starting material. I use leaves from sterile shoot cultures about three weeks after they have been subcultured. Ensure you run shoot regeneration controls each experiment to make sure your explants regenerate shoots to indicate that the material and tissue culture medium is fine. I think you kanaymcin and hygromycin concentrations are incorrect. To select for transformants of tobacco, I use 200 mg/L kanamycin or 30 mg/L hygryomycin.

It is difficult to provide a good answer as I don't know which genes you are working with, but here are some general guidelines regarding transgenic plants:

1) Both Arabidopsis floral dip and tobacco leaf disc transformations are routinely done when you follow the protocols, but if you have not done it before, I suggest you include a positive control (an Agro strain from someone else that successfully mediates plant transformation with the same selectable markers) to see if there are any technical issues. This is true for any new technique you try, always use positive and negative controls along side your unknown test objects.

2) More specifically for your case, depending on what your "genes" are, the resulting transformants might be unhappy and don't regenerate. In my lab every new recombinant gene is first tested by transient expression (either electroporation of a plasmid-borne gene into protoplasts, or Agrobacterium-mediated infiltration of leaf epidermis cells) and by detection of the gene-product (Western blot), We don't engage in long term experiments unless we know that we can detect the protein encoded by the introduced gene. It is popular for people to check "expression" by RT-PCR, but only the detection of the gene product itself reports on how much gene product you get. When we don't have an assay for the gene product established, we first do that (like make an antibody, or test biological activity) and then we carry on. So when we don't manage to detect the gene product, we ask the question if the resulting gene product is toxic to the cells. This can be tested by co-transfecting a reporter gene with the test object, if expression of the reporter gene is down-regulated, there could be toxicity. Making transgenics may then be impossible, unless you use tightly regulated promoters

3) If you want to transform multiple genes, consider cloning them into the same T-DNA, that is much better than mixing strains and hoping for dual transformation events. Alternatively, transform genes individually and then cross the resulting transgenic plants.

Good luck :)

Gregory stated the most important point for Arabidopsis: stage of the flower.

For tobacco: Try Pathi, Tula and Tuteja (2013) High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco.

And Kutty, Parveez & Huyop (2011) Kuttyetal_Agrobacterium tumefaciens-infection Strategies for Greater Transgenic Recovery in Nicotiana tabacum

Dear Homa, I also co- transformed tobacco leaf-discs by co-cultivation with A. tumefaciens strains carrying each one of the two binary vectors built [Irdani et al., 1998 PMB]. Co-transformants were selected on carbenicillin [500 microgr/ml + either 3 ng/ml MTX and 50 microgr/ml KAN]. After shoots appearing (4-5 weeks later) were transferred to rooting medium containing 3 ng/ml MTX and 100 microgr/ml KAN. I can suggest you more details in co-trasformation technique: leave 50-60 leaf-explants in 1:10 A. tumefaciens culture, in slight agitation at 25°C. Put them gently on sterile filter paper under flow-hood and then transfer them on regeneration medium (NAA 0.1 mg/l + BAP 1 mg/l). Let me know your progress ! Ciao!

Dear Homa,

You problem appears to be Agrobacterium or your T-DNA transformation vector. Arabidopsis flower dip transformation should have worked. It is a well tested technology. In case you wan to try here is another simple protocol for transformation "An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol". Published in Plant methods

http://www.plantmethods.com/content/2/1/16

T-DNA transformation vectors and use of appropriate strains are very critical. I can imagine that you are using Agrobacterium strains perhaps not carrying the so called helper plasmid which has all the genes necessary for T-DNA transfer except for T-DNA vector. This one you must supply. A very critical thing is that you must make sure that the right and left T-DNA borders are still intact and that there is no deletion or mutation in your selectable marker genes for Agrobacterium/plant selections.

Make sure you sequence your T-DNA vector and your insert. Use a plasmid from someone else which works and test if you also get transgenic plants. If you give the names, sequences or maps of the plasmid and Agro strains you used we could help you better to figure out the problem.

Best of luck

I forgot to mention also that selection of Agrobacterium transformants with Hygromycin is tricky as there is only little difference between a resistant plant and sussceptibe plant. I highly recommend to look at the literaure on this and find the best way of differentiating transformants with hygromycin selection.

Thanks everyone. I am trying to apply your advice to solve problem.

Tiziana I am very interested in your method. Can you explain more to me how you prepare your Agrobacterium solution. Do you mix two types of Agro (each containing one of binary vectors) together? what solution do you use for transformation? I used NT1. Do you use the same media?

Hi Homa,

thanks for your kind interest. As reported above, I used a hundred leaf-explants (in co-transformation) joining 1:10 A. tumefaciens (o/n) cultures (at least 108 cell/ml), in LS media (16 ml+2 ml each cultures) and slight agitation at 25°C for 25'. Then, put them gently on sterile filter paper under flow-hood before to transfer them on regeneration medium (NAA 0.1 mg/l + BAP 1 mg/l) and kept in the dark, in a growth chamber.

After 48h you can move the explants on new fresh plates LS+1gr/lt Cb+hormones. Later on, after other 48h, transfer them on new "selective" plates (plus antibiotics). These details should help you to get success. Cross the fingers.

Thanks Tiziana. That was a good one.

Novel AHP antibodies available at: http://www.protean.cz/en/recombinant-protein/51/anti-ahp-panel