I'm working with walnut rootstock. I noticed a lot of plants have white streaks/fuzzy white clouds around the callus. They made the plants grow slowly and made the callus turn dark brown/black. Sometimes, no callus form at all. Could you guys suggest some ways to control it? Would PPM or streptomycin help? Thanks for any thoughts.
yeh streptomycin will work, Yoy should add the antibiotic after sterilization of media. Use 100 ppm final concentration and standardize the conc. Better to use syringe filter for antibiotic sterlization
Try 8 HQC. Surface sterilize the explants with 200 ppm of 8- hydro Quinnoline citrate for 1 hour
We use often a combination of Penicillin and Streptomycin. Final concentration of 100ppm Penicillin and 200ppm Streptomycin.We normally make stocks solutions that we can add to the medium after sterilization. (We make 2ml stocks containing 50mg Penicillin and 100mg Streptomycin in sterile water. We add this to 500ml medium. Further sterilization is in our case never necessary. We store the stocks in the -20°C freezer)
There is a small possibility that your plants will not like the antibiotics, in that case you can try a lower concentration. This might also work because you use a mix of two antibiotics. Succes with the experiments.
I suggest that you isolate your contaminant(s) and perform a simple inhibtion test with the antibiotics you have available, like the ones indicated by other collaborators. Then, you can add to the growth medium the most efficient ones at the lowest inhibitory concentrations. Treating the source of the explants with these antibiotics also helps considerably in getting less contaminated explants.
I am also agree with Carlos, but the another way you can try that is you first choose few surface sterilizing agent according to your explant from the literature and screened it on the basis of time and concentrations. I think it will solve your problem.
Go for multi-step surface sterilization protocol. Before using any antibiotic, you can try to improve the mother plant maintenance, which is the source of most of the contaminants. If the load of contaminants is high, then you can drench the antibiotics in the mother plant base itself. You may isolate the bacteria from infected cultures and test different antibiotics at varied concentrations to determine the most effective limits. Apart from killing the contaminants, antibiotics are known to cause phyto-toxicity in some plants. So the preliminary work is required before using those antibiotics. Plantlets cultured on antibiotic containing medium need to be incubated for one or two cycles in antibiotic free media prior to hardening.
In a nutshell, incorporating antibiotics in media could be your last option.
I agree with previous answer, for surface sterilization I recommend mercuric chloride (0.1 - 0.2 % for about 10 min), If you need to eliminate endogenous contamination, the test of its sensitivity is good idea, may be this is not bacteria only, It could be a mixture with fungi or yeasts. You can also try to change temperature, for elimination termotherapy in about 45°C; and for slowing down the growth of microorganisms - lower temperature and lower sugar content. Then you could separate the youngest, good growing parts of shoots to new medium and so decrease the level of endogenous microorganisms to minimum. Good luck!
You can apply combination of antibiotics cefotaxime and canamicyne with dosage 50-100 ppm adding to culture medium. Use filter sterilized for those antibiotics. Before sub-culturing the callus please remove brown or dark part of callus and then placing to sterilized tissue paper. Good luck with your experiment
I use chloramphenicol to control bacterial contamination but use it during surface decontamination. if incorporated in the culture medium it can slow the growth of cultures
Hi,
endogenous contamination in your culture is not easy to overcome.
I do not recommend: transfer of visible contaminated shoots - try to sellect clear ones and try to change condition to slow down endogenous microorganisms growth:
1/ decrease temperature
2/ decrease sugar (amino acids) content in medium
3/ put active charcoal (1-3 g.L-1) into medium
If it does not help, use antibiotic and/or antimycotic, but at first I recommend screening - prepare medium in Petri dishes and transfer pieces of contaminated plants to find optimal treatment to stop growth of colonies, because it could be yeast, bacterie or fungi and also their mixture...I have good experience with combination of timentin (ticarciline) 0,5-1,5g.L-1 and nystatin 40 - 100 mg.L-1 but I hope you will be succesfull in finding the optimal mixture for your culture.
Good luck!
Shoot tip cultures helps out to reduce contamination. Somebody with good eyesight should be on the look out and screen out containers with contamination. Cefotaxime and other antibiotics should be a last resort because most of these are bacteriostatic.
Having worked with trees for years, the only way I found with contaminations is to detect them as soon as possible to get rid of contaminated cultures. Since most microorganisms do not like high-salt media, they grow very slowly and it takes time and sometimes several subcultures before you see them. Not only you waste time but also the danger is that you can spread them in clean cultures. For early detection of contaminations, I always add 125 mg/L of yeast extract in my initiation medium. It does not affect the plant materials but contaminations (bacteria, yeast, etc.) just love it! Thrust me, you will see them fast!
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