Nowadays, with Agrobacterium transformation, people transform tobacco using 'leaf discs' infection (to obtain transgenic shoots), and using 'floral dipping' (to obtain transgenic seeds) for Arabidopsis transformation.

Yes, I know this but still, I was just wondering that what is the reason behind recalcitrant nature of A.t. for this method.

If you want to transform it from leaf tissues. See this attached article, reference (6). I do not have the full text. I think that you can type in the authors' names and Arabidopsis transformation as key words to search online.

Dear Yuan-Yeu, thanks for pointing out my publication.

Dear Panchsheela, I don't have the pdf for the book chapter, but the leaf transformation protocol is also described in the attached paper. Transforming Arabidopsis leaves is certainly more difficult than Tobacco. If possible use the floral dip method, I would not waste time anymore on tissue culture methods for Arabidopsis transformation.

You are welcome, Eric.

Sir Yuan-Yeu Yau and Eric van der Graaff, Thank you so much. 

Actually, I have tried both the plants but could not understand that  what was the reason that Arabidopsis was not efficient, is it because of the leaf type which was very fragile and thin compared to nicotiana or it was just an experimental error that I could not regenerate ago infected (agroinfection was performed for 20-30 minutes) Arabidopsis leave explants. 

I am not able to find out any paper which shows the reason behind this. 

Hi Panchsheela,

That is what I think too. The leaf type of Arabidopsis are very fragile and thin compared to Nicotiana's. That could be one of the reasons. However, with extra care you should be able to transform them as some papers have published.

Sir Yuan Yeu Yau,

That means there is no scientific reason behind this, its just that nicotiana is easy to work with. 

Apart from being fragile, I only obtained cali from vascular tissue, ie the midvein. With tobacco, basically every wounded site in the leaf pieces or discs produced cali. When I transformed tobacco I wounded the leaf pieces additionally with a forceps, which gave more cali/transformants. Further, the antibiotics used for stopping the Agrobacterium after co-cultivation have a strong impact on the success of the Arabidopsis transformation.

Sir Eric and Yuan,

The use of cefotaxime and longer incubation time (30 minutes) with agrobacterium could be the main reason in my study for not getting any successful regeneration of transformants. I could not find any recent papers in which people have used A.t. for coculture method, if you can suggest any papers, it would be a great help. 

@ Panchsheela,

The method in the paper Eric attached is using tissue co-culturing.

You are right, probably it is very hard to find a 'recent' paper which use tissue co-culturing method for A.t transformation. Because, majority of researchers have switched to use floral dipping. It is a simple way for transformation.

Sir Yuan,

I have read sir Eric's paper, it is the only reference I have. 

I think one must be careful to say there is no scientific basis for the unusual receptivity of members of the Nicotiana genus to Agrobacterium mediated transformation, which might be a consequence of having been naturally transformed during evolutionary history. See Horizontal gene transfer and mutation: Ngrol genes in the genome of Nicotiana glauca PNAS v 96 no 23 p13229-13234. See also from Professor Gelvin "Improving plant genetic engineering by manipulating the host" Trends in Biotechnology" v21 no 3 p. 95-98 2003.

Sir Ray,

Thank you for the valuable suggestions, yes there should be always a scientific reason. I will go through the papers suggested by you.

@ Ray,

Interesting paper you listed: Ngrol genes in the genome of Nicotiana glauca PNAS v 96 no 23 p13229-13234.

The situation (T-DNA components present in plant genomes without transformation) is similar to that published in this 2015 PNAS paper:

"The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop."

See attached paper.

I believe that there should be more plants out there which are 'natural transformed' by Agrobacterium. The question is how many species and how frequently this occur. And is this good or bad for plants in the sense of evolution?

@ Ray,

1. In terms of evolution. I think Agrobacterium must have co-evolved with plants. For example, in general, Agrobacterium tumefaciens usually transforms a low numbers of T-DNA (or transgenes) into plant genomes. Why not 100 copies or even more? Nowadays, we know that multiple copies of gene transfer can lead to 'gene silencing'. The original purpose of Agrobacterium to transfer its own wild-type T-DNA into plants is to make foods for themselves. If gene silencing occurs, Agrobacteria would have no foods to maintain their survive. So, they 'learned' to transfer only a few T-DNA into plants, to guarantee expression of genes in the T-DNA and make proteins for them. This just my personal opinion.

I have talked to Dr. Professor Gelvin (who is an expert of Agrobacterium in the field) about this recently, but he said that there are still a lot of things we don't know about Agrobacterium.

2. Is Agrobacterium rhizogenes useful for 'monocot' transformation? We know Agrobacterium tumefaciens have been used for some important monocot plant species for genetic transformation, such as rice and wheat, using immature embryos.

@Yuan-Yeu Yau, short answer yes, disarmed Agrobacterium rhizogenes 18r12v (which  is a disarmed version of Agrobacterium rhizogenes strain NCPPB 2659 which I generated in late 2004 at the Donald Danforth Plant Science Center in St. Louis, Missouri) works for Brachypodium and Switchgrass (we published this at USDA this year, see Front. Plant Sci. 7:716. doi: 10.3389/fpls.2016.00716).

I was aware of the sweet potato publication, thanks for sharing though. That sort of finding immediately grabs my attention, because I too feel that there must be more of this naturally occurring transgenesis occurring in nature, we just haven't intensively looked for it. As more and more plant genomes are completed I imagine we will see more evidence of naturally transformed taxa.

As far as the impact on plant fitness goes it is a complicated question, and I think it is dependent on the plant species. I have personally observed the impact of rol gene expression in a variety of plants (mostly composite plants, but in I have seen both Arabidopsis and sweet potato that were stable transgenics carrying the NCPPB 2659 root inducing T-DNA) and there is a wide range of phenotypes.

Your logic seems sound regarding the number of T-DNA copies. I guess gene silencing should be a very ancient process, and I'm not knowledgeable about when the first known instance of crown gall or hairy root occurred (or if there are fossils of galls or hairy roots), but if the diseases predicated on transferred DNA forcing plants into an unproductive metabolic shunt occurred in the context of gene silencing machinery that was already established (ostensibly as a defense mechanism against viruses, which I assume would have developed first), then yes, the triggering of silencing would have acted as a selective pressure limiting the quantity of T-DNA molecules that could be productively integrated. There is an interesting parallel discussion to be had about the strength of the promoters driving the oncogenes; none of the Agrobacterium promoters are especially strong, which might also have been a result of selection to subvert host silencing mechanisms.