Dear Buhara,

Your question is really interesting. Would you please clarify what do you mean with protein sequence alignment?...do you want to compare just cDNAs, then it is transcriptomics?or are you doing proteomics comparing callus with embryogenic potential to leaves?. If you want to validate that during the production of plants by cell tissue culture, you do not induce any changes in the genome, I would say that yes, the genome does not change much, but there are changes involving epigenetic regulation of gene expression, so, it is nice you want to know the transcriptome but I do not quite understand how you want to do it. You can use whole transcriptome sequencing using 454 sequencing or Illumina sequencing. I worked with somatic embryogenesis but focused only on the induction of the process and yes, indeed the leaf has to change all its genetic expression program to become "callus" and also, the "callus" have to change its gene expression program to become "somatic embryos"...but if you compare original leaf and cell tissue culture obtained new plants, the changes are less dramatic but present.

I hope it helps.

Best regards.

As per my knowledge the comparesion between in vivo and in vitro leaf -leaf, root- root can be made to get the authentic results but callus is a un-differentiated mass of cells how it can be compared with in vivo leaf.

Dear Buhara,

Your question is really interesting. Would you please clarify what do you mean with protein sequence alignment?...do you want to compare just cDNAs, then it is transcriptomics?or are you doing proteomics comparing callus with embryogenic potential to leaves?. If you want to validate that during the production of plants by cell tissue culture, you do not induce any changes in the genome, I would say that yes, the genome does not change much, but there are changes involving epigenetic regulation of gene expression, so, it is nice you want to know the transcriptome but I do not quite understand how you want to do it. You can use whole transcriptome sequencing using 454 sequencing or Illumina sequencing. I worked with somatic embryogenesis but focused only on the induction of the process and yes, indeed the leaf has to change all its genetic expression program to become "callus" and also, the "callus" have to change its gene expression program to become "somatic embryos"...but if you compare original leaf and cell tissue culture obtained new plants, the changes are less dramatic but present.

I hope it helps.

Best regards.

It depends what protein or protein family you are referring to. E. g. isoenzymes of different tissues might differ in their protein sequence. Is it something like this you were interested in?

I just wanted to correct your terminology. I think you can call "callus" a tissue, but leaves are organs and contain many tissue types. These are epidermis, mesophyll, xylem and phloem.

Sorry me...You all are right, question is not clear... I revised the question in order to avoid any misunderstanding... Hope it is ok...

Very precisely explained by Maria Julissa Ek Ramos as per question statement.

Hi,

The study of somaclonal variation (SV) is difficult and not very straight forward.

Even looking for somaclonal variation using molecular markers is still problematic since only a fraction of the DNA is screened.

So my opinion is that you cannot base on one protein to say that the callus shows no SV. The final proof is still the regenation of plants from the callus and see wether the plant are still true to type.

Cheers

Bart

Dear Buhara,

First of all, I would like to know if you have isolated the enzyme and did protein sequencing or studied the activity of the enzyme (progesteron 5beta reductase) or isolated the cDNA for the enzyme. If you have done any of these, it means that, your callus sample is producing the enzyme. There are reports which say, callus tissue of certain plants are capable of producing specialized metabolites even if they are not in the organogenetic stage. Either ways, from your question, I think you are not concerned about the SV araising from your culture, you want to know if you can compare two different stages of development which surely makes sense, but the marker you choose should also be carefully done.

Hope my reply helps you

cheers

Kalai

Dear Buhara and all you guys,

I agree with Bart. You can not say that there is not somaclonal variation based on one gene even it has been used as a marker in other systems. If it is part of a paper you want to submit, I think you should write your discussion taking in count that. I believe that the merit of your work is being able to have a protocol to produce plants in vitro in your system. Please correct me if I am wrong, but this is the first time I know about a protocol involving callus, to propagate foxglove. I would focus on tell everybody how cool the protocol is and how it can be used. If you want to talk about somaclonal variation, I would be clear about why it was necessary to use a marker "to explore" the existence of somaclonal variation in the system. However I would not make strong conclusions based on that one gene and even I would propose global analysis of the transcriptome as I mentioned before. A great and realistic discussion would be appreciated by the reviewers.

But if you want to apply this plant production protocol massively and you are telling that there is not somaclonal variation based on one gene, I do not think it would be accepted. You would have to make deeper analysis of the transcriptome and follow up on the performance of the in vitro generated plants to have some idea about how different they are.

I think they the same type of cell due to using explant from leaf,so protein inside that cells are the same. Do your plant has any chimera? If it has ,it will has many variation. It depend on what cell that differentiated or what cells that you used to culture and has some section or cells from chimera.

Anchalee Jala

In fact ( a reply to Maria), thnx for your clear explanations. I was writing a paper on in vitro propagation of an endemic foxglove. Callus cultures on a different shoot induction media was highl effective for shooting. However, I was also working on that of gene (but this part is different from my tissue culture study) and saw that no genetic variability compared to natural population. I just wonder and shared with you all "does it make sense for a discussion part touching slightly on Somaclonal variation"...

On the otherhand, tissue culture techniques if you are using callus cultures might be critized due to SV. I wanted to avoid (upto some extent) the critics from reviewers.

Thank you all... Now, I can figure out something related to this issue...

this enzyme (progesteron 5beta reductase) takes a role in cardenolide metabolism. It is not homologues to the animals but very impotant for foxgloves, and some limited plant groups. Because it makes 5beta forms of cardenolides (cardiac glycosides) and makes them toxic plants to the aves and mamals. This enzyme active in callus, but no cardenolide accumulation was detected in callus tissue. After differention into shoot (or regenerant), cardenolide accumulation was detected. I try to undesrtand why callus do not produce cardenolides, although the enzyme is active. Earlier works say that cardenolide accumulaton was correlated to cardenolide accumulation in foxgloves...

There are certain unique features of plant, which are yet not resolved. For example- Tryptophan is in animal without Auxin. ABA in pig brain . Well, lack of cardenolides in callus , please look for whole pathway of its synthesis. Comparing the squence ( transcriptome) will certainly give the genomics of that plant, which has temporal & spacial expression. hence exhibition accordingly