Sorry，this quesition is not my major. So I don't know how to do?

Healthy leaves of E. scaber does'nt has internal contamination.If it is, then leaves are deceased one which apparently can be identified morphologically and it should not be used as explant 

• what kind of internal contamination you are talking about??

For plant tissue culture (PTC), it is better you can obtain the leaf explant from seedlings germinated from properly sterilized seeds. Sterilize the seeds with disinfectants such as ethanol and bleach (with proper concentrations).

you can use antibiotics in the PTC medium which help the growth of explant and on the other hand adversely affects the contaminants.

Meristem culture is the only way to obtain infection free cultures from field grown plants, especially viruses.

Inducement of faster growth of the shoots will ensure an active meristem with proportionally high % of clean tissue. Treatment of the mother plant to maintain infection under control (thermotheraphy and/or chemotherapy) is to be complemented.

When one attempts meristem culture, the medium has to be appropriate and the techniques to explant adopted with great care.

Have a look at this paper. http://sweetpotatoknowledge.org/seedsystem/research-methods-tools/utility%20of%20thermotheraby.pdf

Personal understanding of each plant gives insight to handle it better.

In our experience, if the infection in bacterial, first sterilize explant with low concentration of bleach solution (approx. 5%) for longer period of time, maybe 30 min.Then put it on the media with wide spectrum antibiotics, like cefotaxime

But, I suppose that your infection is fungal, which is usualy the case with field explants. The only thing that worked for us is actually treating the plants in the field with fungicide 48h prior taking the samples for PTC. When you take samples, you can also stir them for 30 min - 1 h in the fungicide water solution. 

Then proceed with bleach and antibiotics in medium ...

Sorry but you have to figure out which concentrations and washing times work for you - different explants and plant species have different requirements.

Good luck!

Thanks to all

Eradication of internal contamination is a major challenging task. Normal surface sterilization procedure may not proof fruitful. following approaches can be undertaken overcome the problem.

1) Use of antifungal and antibacterial of broad spectrum.

2) Apical meristematic culture.

3) Nano Silver mediated sterilization.

4) Treating explants in bavistin solution.

5) Indirect organogenesis through callus culture.

Sir which crop you are working with? To eliminate endogenous contamination antibiotics can be added  to medium but not when leaf is an explant. You can try with the axillary bud , shoot tip or mature explant. if it is not a woody plant like datepalm or sandalwood try different explants which can be easily surface sterilized

Dear Sopan,

You can able to avoid internal contamination if, you will use seed properly sterilized (depends on plant seed) and use that seedlings fresh new seedling for tissue culture.

Thanks with regards,

Aloka

Dear Madam,

Thank you...

Sir,

Go for gamma irradiation.

Dear Sopan,

you can try broad spectrum antibiotic during surface sterilization or use media fortified with antibiotic against endophytic bacteria/ fungi.