Do you mean callus clumps? Can you give us a photo?

Dear Natalia, yes it's a callus clump. I attached 2 photos.

Dear Diako,

You mentioned that not using any PGRs, but in some crops high levels of endogenous hormones will show their effect in in vitro.  To reduce the effect of cytokinins you may supplement your media with auxins like NAA. However, the concentrations need to be standardised

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Dear Diako,

Thank you for photos. I agree with Ravindra Kumar, try the addition of auxins, but in this case I would recommend IAA (since it's natural), try 2 mg/l added before autoclaving of medium.

By the way, are your Stevia shoots really inhibited by clumps? They look quite nice, although they do not branch...

Yes, I agree with Natalia, so far your plants look ok. I saw some roots out there (a few in the air and a few are touching the medium). Once the roots touch and penetrate the medium, the plants will grow better. However, I am not sure where these roots derived from-- the cut shoots or the calli?

I have seen a few of my tissue-culturing shoots growing callus like that. I just cut partial of the clump away using a sterilized scalpel if I feel I need to. Be very careful not to break the roots. I am not sure the texture of Stevia's calli. Mine is soft and very easy to be removed by cutting. Your plants in the jars are much bigger than mine (mine were in deep petri-dishes), extra care need to be taken when you do the cutting to avoid possible contamination.

Dear Ravindra,

Thank you for your advice. I 'll try it.

Dear Natalia,

Thank you for your suggestion.

I think some of my shoot cultures was prevented by clumps because the clumps grow more than stems and after a while, their growth stopped.

Dear Yuan-Yeu Yau,

Thank you. Your comments were very valuable. First of all, these roots derived from the calli but some of them didn't have any root.

Moreover, you're right I think cutting the clumps is a good idea but there is a risk of contamination. Despite that, should I cut all of the clumps or parts of it?

From the review and research, to induce micro shoot in stevia from nodes through shoot proliferation method, it has to add cytokinins such as BA and Kinetin at a low rate (less than 1.5 mg/l) to prevent callus and root formation, then adding auxin to promote root formation. But in this case, you can carry out subculture on medium contains low cytokinin after removing callus.

Braz. arch. biol. technol. vol.57 no.1 Curitiba Jan./Feb. 2014

http://dx.doi.org/10.1590/S1516-89132014000100004

on which medium they are growing now?

You said without added hormones. Did you use 0.5 MS?

Why this fear of contamination? One can take out the plant (from a jar?) put it into a sterile petridish and cut the clumps off in there and return to the Jar.Anyway I only work with test tubes. If plantlets in one tube are contaminated there are still more in other tubes.

I would simply cut the callus away with a sharp scalpel, using aseptic technique, and cut into nodal sections if you are wanting to increase numbers of plants. They look good to me but  depending on the medium they are growing on  and your expected outcome (i.e. are you growing for proliferation or rooting?)you might want to reduce the auxin concentration to help prevent large callus production. 

During our experiment with Stevia we observed similar callus lump at the base of the stems. But then we got rid of that by reducing the BAP concentration in the medium.

Dear Daiko

As you know stevia is easy to multiply. so development of multiple shoots is common. But if u want to get healthy plants, then u need to remove excessively growing side shoots. Try to keep only 2-3 shoots/explant. Then your plants grow fast and u will get healty plants.

As you mentioned you are not using any PGRs, then reduce the strength of MS medium upto half to control excessive multiplication.

Finally, The most important point, Pl watch your culture carefully. The shoots are developing from axillary buds or from adventitios buds.

if shoots are comming from adventitious buds or from base callus of main shoot, then don't use it for multiplication. Because in these type of shoots genetic variation level is more and not good for commercial tissue culture.

Thanking you

Regards

Pati R.

Dear Rajesh,

Thank you for your recomendation. 

Dear Diako,

U can cut the callus under aseptic condition (but be careful as if very less callus is left at base then again it will proliferate and hinder shot growth), and use culture tubes instead of jar so at the time of callus removal, another explant will not get affected and in turn chances of contamination is reduced. if you are afraid of getting contamination then u can:

if you are afraid of getting contamination during above process, then u can:

-Reduce the strength of MS to half or quarter which may help in reducing the growth of callus.

-Also add different PGRs like cytokinins (BA, Kn) and auxins (IAA, NAA) individually and in combination, this will decrease the growth of callus as well as increase the number of shoots.

-Gibberellic acid and adenine sulfate can be also used for elongation and multiplication, this may also nullify the effect of callus. 

-u can also cut the in vitro grown shoots and use the in vitro nodes for further multiplication, as may be the use of juvenile explant form less/no callus at base.

Hope this will help u.

Dear Ashutosh,

Thank you for your recommendations. Your suggestions were great. 

Which hormones diid you use? For leaves from outdoor plants I guess.

It is not clear from your questionif the clumps are of shoots or callus.

If the clumps are of shoots, in that case you may regularly devide and subculture in small groups (2-3 shoots) or you may like to incorporate some GA in to the medium.

Dear Anil,

The clumps are from shoots. By the way, I couldn't  regularly separate the clumps because they were too hard.