I want to transform BY-2 cells with the agrobacterium that carries Gentamycin resistance gene and the construct which is kanamycin resistant. In almost all protocols I am looking, two antibiotic called carbenicillin and timentin are used as plant selectable antibiotic. It looks they are used after transformation process and when plating the transformed cells for inhibiting the agrobacterium. I was wondering that if I am in a right way? Is it right to use the antibiotic Kanamycin based on my construct and then carbenicillin and timentin after transformation?
best,
Kannamycin and Gentamycin should be good for transformation. You need to ensure you can select the construct and avoid any other bacterial growth, hence the selections.
1. Kanamycin-resistant gene (nptII) [or selectable marker] and your gene-of-interest should be transformed through T-DNA cassette (on your construct) and integrated into plant genome.
2. Carbenicillin or timentin is used to suppress Agrobacterium after transformation
3. So, after co-cultivation with Agrobacterium, your explants should be cultured in medium with kanamycin (for selecting transformants) and Carbenicillin (to eliminate Agrobacterium).
Just to add to Yuan-Yeu's anwer:
1. Kanamycin and Gentamycin are both aminoglycoside antibiotics. They inhibit translation at ribosome level, and are active in both bacteria and plants.
2. Carbenicillin and ticarcillin are B-lactam antibiotics that inhibit bacterial cell wall formation and do not affect plants negatively (however, it has been shown that in some plants they can have e.g. cytokinin activity and then they may interfere with regeneration, so it is important to try several antibiotics before settling for one, such as vancomycin and cefotaxime, which have different action mechanisms). Timentin is a mix of ticarcillin and clavulanate, the latter being an inhibitor or B-lactamases.
Therefore the mechanisms of action of the selectable antibiotics and the ones you use to kill agro are different and compatible. As long as the plant system is also compatible with that selection, and the bacteria does not have resistance to b-lactamic antibiotics (e.g. agrobacterium AGL1 has a chromosomal gene called 'bla', which confers resistance to carbenicillin), you are OK.
Cheers,
Ruben
And to lump onto Ruben:
Not all Agrobacterium strains are the same. There are some strange ones out there with curious resistance and susceptibility profiles, so you would be wise to do an empirical test with the strain with which you are working to confirm that it is indeed Carb sensitive. Timentin seems to be universally effective for the Agro strains with which I have worked, but sometimes the Agro is able to finally overcome this single method of chemical control (maybe the effective concentration of the Timentin is simply too low when the plates reach a certain age, every compound has a half life). Agro, in spite of these chemical controls, is remarkably resilient and can actually persist in low numbers even after the tissue has been cultured on media formulated to prevent this. It seems like tissue culture is really more about managing the infection than absolutely stopping it.
You are right. Carbenicillin and timentin was used to suppress and eventually kill bacterium if possible. Keep it with these antibiotics as long as you can. You may still detect Agro using PCR after 1-2 mo.
You use another antibiotic to select you plant cells based on what selective marker in the T-DNA of your binary vector.
Agreed with Ruben and Ray. Additionally while using antibiotics (e.g. Timetin, widely used) to suppress agro growth, be careful with its concentration to be used in your transformed cultures. Sometines, high concentration of your antibiotic affects plant regeneration capacity. Good Luck
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