Hi Damian,

It is highly unlikely that Agrobac will survive in open air for so long.

However it is not uncommon for meristematic tissues to become infected during the floral dip procedure producing transformed flowers until degeneration. it all depends of how dip did your Agro gets to go. But more commonly the transformation is not permanent and only a few flowers will be successfully transformed.

some authors will suggest to repeat the floral dip every few days to maximize the transformation efficiency.

for optimization of protocol look for this two articles.

"Floral dip: a simplified method for Agrobacterium-mediated

transformation of Arabidopsis thaliana", Steven J. Clough and Andrew F. Bent 1998

http://www.scirp.org/journal/PaperInformation.aspx?paperID=4334#.VMdyOWisV1Y

"Minor modifications in obtainable Arabidopsis floral dip

method enhances transformation efficiency and production of

homozygous transgenic lines harboring a single copy of

transgene", Das et all 2011

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-313x.1998.00343.x/pdf

Good luck

Hi Damian,

It is highly unlikely that Agrobac will survive in open air for so long.

However it is not uncommon for meristematic tissues to become infected during the floral dip procedure producing transformed flowers until degeneration. it all depends of how dip did your Agro gets to go. But more commonly the transformation is not permanent and only a few flowers will be successfully transformed.

some authors will suggest to repeat the floral dip every few days to maximize the transformation efficiency.

for optimization of protocol look for this two articles.

"Floral dip: a simplified method for Agrobacterium-mediated

transformation of Arabidopsis thaliana", Steven J. Clough and Andrew F. Bent 1998

http://www.scirp.org/journal/PaperInformation.aspx?paperID=4334#.VMdyOWisV1Y

"Minor modifications in obtainable Arabidopsis floral dip

method enhances transformation efficiency and production of

homozygous transgenic lines harboring a single copy of

transgene", Das et all 2011

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-313x.1998.00343.x/pdf

Good luck

Transformation capacity of Agrobacterium decreases with time without additional stimulation. If you analyze dynamic of transient expression after plant tissue infiltration with activated Agrobacteria, you can see the maximum of product accumulation at 3-5 dpi, then it decreases. So I would estimate the maximum of transformation capacity at 1-3 dpi.     

Hi, I usually conduct floral dip twice on the same flower, 3 days interval from the first floral dip to increase the transformation rate. 

http://www.nature.com/nprot/journal/v1/n2/full/nprot.2006.97.html

Floral dip experiments for Agro infection have varied success rates. One can try the dip procedures twice at an interval of 24 h.

Try to avoid infecting the same plants repeatedly, This does not improve transformation efficiency very much. It would be much better to grow plants with many coline flower branches and then do the infection one tine after remove open flowers or young siliques. Unusually it takes about 10-15 days for a silique to get mature for releasing seeds after full flow or opening.

As you have seen through responses, transformation frequency changes with the lab and personal experience. All have shared their experience with the model plant- Arabidopsis. Parameters will change once you try another species.

It will be crucial to monitor the stage of flower buds when you hit them with Agro and of course a time course study for co-cultivation.

Dear Damian D Guerra!

My name is Victoria Tsygankova. I am ScD in Plant Biotechnology and I am Head of group of screening of synthetic compounds of Department for Chemistry of Bioactive Nitrogen-Containing Heterocyclic Compounds, Institute of Bio-organic Chemistry and Petrochemistry, NAS, Ukraine, 2014. I study actual problems of modern agriculture - increase of plant development and productivity and increase of plant resistance to various pathogenic organisms and insect pests with use of new effective growth regulators of synthetic and natural origin as well as genetic engineering methods.Concerning your question I would like to offer to you my research article in which you can see in details of dip floral method of genetic transformation. You can see this article on-line:

https://www.academia.edu/4344993/Increasing_of_plant_resistance_to_nematodes_using_RNAi-technology

I send you also my original article in attached file. I hope that my article will help you to understand much better this problem.

Sincerely yours, ScD Victoria Tsygankova

In case you are interested on the practical method, I have successfully transformed Arabidopsis by dipping the all flowers every five days over a 2-weeks period, covering the whole plants after dipping and keeping them overnight at 30 C.

I agree with Costa, Wei and Rodriguez. You can follow their suggestion and can get good results.

Just a note to add (we did this a long time ago), but we had been able to isolate and regrow what we suspected to be A. tumefaciens, from seeds collected of vacuum infiltrated A. thaliana plants, on agar plates containing kanamycin and rifampicin, the two antibiotics we used to grow the A. tumefaciens used for vacuum infiltration. We could not get these from non-infiltrated plants, but we did not confirm that the isolated bacteria indeed were A. tumefaciens, nor if they still contained the correct binary vectors. It did prompt us to always sterilize seeds after floral dip transformation.

I agree with Wei, try not to repeatly infect the sample flowers again and again. Do so, it may increase the risk of producing higher percentage of transgenic lines with multiple-transgene insertion, which is a source of causing gene silencing. Ideally, we want to generate more single-copy transgenic lines for analysis.

@ Yuan-Yeu Yau: repeated transformation is unlikely to lead to multiple-transgene insertions and even less likely to result in silencing. What I understood of what is published on this, transformation by dipping in general appears to transform the ovules, so the female germline, resulting in transgenic seed which is hemizygous for the T-DNA. Silencing (RNAi) will need a source of double strand RNA to act on. This can happen by chance for single T-DNA insertions, depending on the insertion site of the T-DNA, but it will happen more frequently with multiple insertions at the same site, especially if they insert in inverted orientation. This seems to increase the chance of read-through of highly transcribed sequences, eg. driven by the CaMV 35S promoter, and generation of double strand RNAs.

Since only ovules at the right stage seem to be susceptible to A. tum. transformation, I think the chance of finding newly transformed flowers days after dipping will be low. This is also what one sees when transforming with a T-DNA expressing a seed specific fluorescent marker gene: most transgenic seeds are found in only few siliques per inflorescence. Since the transformation frequency seems to be higher when plants are healthier, and since Arabidopsis does not particularly like dipping, it seems more efficient to dip more plants than to dip repeatedly, as suggested by Wei.