Need to add other information. I did transformation in tobacco using 25 mg/l hygromycin and 2 mg/l PPT and it worked like charm. The plasmid constructs and Agrobacterium strain are the same as in tomato. I use LBA4404.

We tried Basta in tomato transformation and failed. I am not sure about hygromycin but kanamycin is easy.

Hi Cahya!!!

Tomato is a gentle plant and hygromycin and phosphinothricin are too hard. You need to utilized Kanamaycin better,

All the best,

Martin

It seems like the system is discouraging, I wonder people have gone any success with this and the aberrant morphology is still puzzling to me as well.

Hi,

PPT (phosphinothricin, ammonium glufosinate, BASTA) is an analog of glutamic acid and then a competitive inhibitor of the enzyme glutamine synthetase. In the presence of PPT plants cannot produce glutamine, which is an aminoacid commonly found in protocols related to morphogenesis.

Also, the enzyme glutamine synthetase is the only one, in plants, that permits ammonia assimilation. It catalises the following reaction:

Glutamate + ATP + NH3 → Glutamine + ADP + phosphate

Therefore when you add a competitive inhibitor you produce a buildup of ammonia, which changes the equilibrium with ammonium (NH4) in the plant and is released to the medium,raising the pH. Also, all nitrogen that gets in the plant has to come through nitrate assimilation. However when it reaches plastids or chloroplasts it is reduced to ammonia that as shown above cannot be incorporated into aminoacids. In other words, in the presence of PPT a plant cannot incorporate any nitrogen and has to deal with an excess of ammonia.

To my knowledge it's not very clear, but it seems that the toxicity of PPT is more due to lack of production of glutamine than to ammonia-induced toxicity when you are working in vitro. NH3 uncouples photosynthesis (by sequestering H+ when producing NH4. In heterotrophic conditions this is not terrible, but you still see chlorosis in the leaves, and the rise in medium pH will affect morphogenesis for sure, apart from nutrient assimilation, unless you have it buffered (e.g. by adding MES, morpholine-ethanesulfonic acid). The main problem then is that there is no incorporation of nitrogen.

Bear in mind that when you transform only a few cells will receive the transgene. Therefore when you select a whole lot of them will be dying, and that can be extremely stressful for transgenic cells.

Also, when you get aberrant explants they can be physiologically in a very bad condition, and this can leave epigenetic scars, i.e. you cannot do anything with them after.

As an example of morphogenical effect of PPT, years ago I used it in selection with somatic embryos. Without it, the embryos would develop normally till a cotyledonary stage. With it they all stuck to embryogenic callus or globular stage.

The message to take, really, is: if you are really interested in the morphogenical effect and want to develop your research into it, go ahead and design experiments to get your answers. If what you want is to obtain transgenic tomato to carry on with your research program, forget about hyg and PPT and use kanamycin, because it's a well-established system and as other people here have already said it works just fine.

Cheers,

Ruben

Hi,

You did not say anything about your positive regeneration control in your question. I assume the control plants regenerated normally in the absence of selection, so you are sure that the aberrancies of your transgenics are due to the selection?

I'm just asking because I took for granted you are using the best regeneration protocol and that it works. Additionally some growth regulators are delicate (see http://www.sigmaaldrich.com/life-science/molecular-biology/plant-biotechnology/tissue-culture-protocols/growth-regulators.html). For instance IAA is sensitive to light, and both IAA and Zeatin are best filtered than autoclaved.

Cheers,

Ruben

Hi Ruben,

Thanks for your advice. I included non-transformed, non-selected controls and they developed into normal plants. However, I did observe "blind shoots" (aberrant shoots) in the controls as well, but the number was less than on the selection ones. I noticed certain levels of vitrification though. One or two papers reported this "blind shoot" phenomenon in the cultivar that I'm using now. I don't know whether abnormality was hormonal or genotype-specific. I used zeatin riboside 1 mg/l and IAA 0.1 mg/l; both were filter-sterilised and added post-autoclave. zeatin was dissolved in NaOH + water while IAA in ethanol + water. In my next experiment I will try another hormonal combination i.e. BAP + NAA. I know some people use DMSO to dissolve these hormones instead of NaOH and alcohol, the reason being DMSO is easily absorbed into plant tissue. I used BAP + NAA in DMSO for my tobacco transformation. 

I've been thinking, in response to how PPT works as you explained, to transfer the calli more often (shorter period) than usual, i.e. transfer them every week instead of fortnightly or every three weeks, to avoid extra toxicity caused by ammonia build up. Also to isolate green microshoots or calli from dying tissue as soon as possible.

If I used kanamycin, the vectors already contain KanR for bacterial selection, is it going to be a problem? The whole purpose is to get transgenic plants, so I'm thinking to modify the vectors with KanR.

Cheers,

Cahya

Hi,

I was in doubt about the hormones, but it seems that there are a lot of different protocols out there. Some have only cytokinins (zeatin seems to be crucial?), and some add also auxins like IAA. If there is not much published for your genotype you might have to do an experiment to test the best regeneration conditions, before going into transformation and selection.

I think your idea to keep explants as healthy as possible is very good, and frequent transfer for the first month could help. Fortunately although hyperhydricity is there it does not seem to be a major problem, although it would be worth it to try to prevent it.

As to the kan selection, it depends on the promoter. Your kanR for bacterial should have a bacterial promoter, and you need a plant one to express the gene in plants. Maybe you could raise another thread and ask for a vector with kanR in plants, instead of modifying the vector yourself (unless it's straightforward)?

Cheers,

Ruben