supplement Gibberrallic acid,try various concentrations

As Dr Bahadur told you, you should use Gibberellic acid because it is responsible of the growing and elongation of the shoots of novo. And if you are looking for a morphogenetic response you should use a combination of auxins and cytokinins.

Hello Pandarinath,

Without seeing the protocol you are following I can only speculate and suggest you questions to ask yourself:

1. How is the positive control (tissue in the same regenerative conditions, but not inoculated with agrobacterium) behaving? If it is regenerating shoots, the problem is not the culturing conditions but the transformation. If the shoots are not elongating either, you should optimise shoot regeneration first.

2. Have you tested a dilution series of the selective agent? You should aim for a concentration of selective agent that slows or stops growth of non-transformed cells. If you completely kill them, since they are more abundant than the transformed ones in an experiment, that cell death can be deleterious or lethal for the transformed cells (e.g. in an hypersensitive response). On the contrary, if the concentration is too low this could lead to escapes (non-transformed cells or chimeras of transformed/non-transformed cells if the morphogenetic response in your system works this way, that regenerate and may carry on growing or die, depending on the long-term sensitivity to the selective agent).

3. How do you know the shoots you are obtained are transformed? Do they express a reporter gene? Have you done PCR to check for the insertion? It could just be that the tissue is regenerating and they are just non-transformed escapes that die after a while in the presence of the selective agent, in which case you should revise the transformation protocol.

Good luck,

Ruben

Dear Dr.Ruben Alvarez  Fernandez

Thank you ,

As for you are suggesting  

1.We  optimize shoot regeneration,positive  control also good

2.We optimize selective agent (kanamycin) initially

3. We conformed with PCR,

But here the major problem in capsicum regeneration after 2-3 subculture the shoot buds formed Rossetti then not elongated……

In comparing to regeneration shoot buds, the transformed shoot buds are not elongated

Hello,

When you say that you confirmed by PCR: agrobacterium could be contaminating the shoot sample, so you could be amplifying the agrobacterium T-DNA and the shoots could be non-transformed. Have you added a PCR control that amplifies something specific to agrobacterium (e.g. a vir gene, or a chromosomal gene) to rule this out?

Have you set up a transformation control using agrobacterium with an empty plasmid on selective plates? This will rule out the possibility that you gene of interest is detrimental for regeneration. This same control on non-selective plates will tell you whether agrobacterium is affecting the regeneration procedure.

In every experiment I would set up a non-transformed control on kanamycin plates, to rule out the chance that the kanamycin stock is bad. This will tell you the percentage of escapes, because sensitivity to the antibiotic can be tissue-dependent. I would also set up a control on non-selective plates with the antibiotics you use to kill agrobacterium (cefotaxime, ampicillin, vancomycin... whichever you are using), because this will tell you if the regeneration process is impaired by them (e.g. cefotaxime has a cytokinin-like activity, and sometimes it promotes regeneration. But in other cases you could get a different result).

Which gene are you transforming with? Could it be affecting regeneration?

If the regeneration control is working fine I don't see why you should add gibberelings for elongation to the transformed control though. Also, if the 'transformed' shoots not only not elongate but eventually turn yellow/white, that means that they were not transformed and the kanamycin is killing them.

Cheers,

Ruben