I used bamboo axillary nodes as explants, after inoculation of 10 days bacterial contamination was identified, shall I reuse the plant by giving chemical treatment or should it be discarded?
If you're working in vitro, you can simply transfer the explants to fresh nutrition media containing some broad spectrum antibiotic that doesn't harm plants (in my lab we're using cefotaxime 500 mg/L). It works with most plant species, I hope it doesn't harm bamboo.
If you've been doing transformation with Agrobacterium and that's the reason why you have bacterial infection now, next time just make sure that the explants are inoculated with agrobacteria for no longer than 3 days, after which you should rinse them in sterile water with cefotaxime and transfer them to a cefotaxime-containing medium to kill the remaining bacteria.
If you're working in vitro, you can simply transfer the explants to fresh nutrition media containing some broad spectrum antibiotic that doesn't harm plants (in my lab we're using cefotaxime 500 mg/L). It works with most plant species, I hope it doesn't harm bamboo.
If you've been doing transformation with Agrobacterium and that's the reason why you have bacterial infection now, next time just make sure that the explants are inoculated with agrobacteria for no longer than 3 days, after which you should rinse them in sterile water with cefotaxime and transfer them to a cefotaxime-containing medium to kill the remaining bacteria.
If you can get fresh explants then it is better to do away with the infected ones - really found it very irritating to recover infected explants in vitro especally when you are looking at something like micropropagation - secondary metabolite production - where additions all to the media have to be accounted for and will influence your outcome.
Another aspect is the getting to the reason behind geting bacteria after 10 days - check you explant source and give proper sterlisation.
From my own experience, I would agree with Dipjyoti-- start over another batch of transformation experiment if the contamination is serious. However, if the contamination is limited to a very small area, you can try to rescue the explants by cutting away the 'contaminant part' of the explants (very carefully) and remove the 'clean part' to a fresh plate. I had rescued some explants this way.
It depends. If the plant is too damaged, discard it. I have experience with bacteria and fungi contaminated bulbous and grassland plants, cultivated in vitro. Sometimes just a rinse in 70% ethanol for 1-2 minutes, and transfer on a new media was enough. Sometimes, the transfer on medium with activated charcoal worked a good deal - the contamination appeared again, but with faint growth, and totally disappeared on the second transfer on charcoal medium. Sometimes, I cut a great part of the damaged plant, and it stimulated it to develop faster. I didn't have the experiment set with this objective, but I observed elicitation of the secondary metabolites in some of the contaminated cultivation lines.
Phillip and Lyubov have it right. It really depends on your purpose for growing these. Bacterial contamination is very likely going to lead to physiological and transcriptional changes. (Things like xylem clogging or induction of an SAR response (systemic aquired resistance)). That would argue for discarding them and starting over if they are for research purposes. But if you are just micropropagating, then 1) discard and start over if easy or 2) give a quick series of dips in 10% bleach, sterile water, 70% ethanol, sterile water and back to clean media. The length of each dip would depend on how deep the infection goes into your explant. Lyubov's and Yuan-Yeu's trimming suggestions would also help if practical in your situation. And following Dipjyoti's suggestion to identify the source of your contamination would certainly help with future efforts. Good luck- bamboos are great plants!
@ Leelavathy,
Bamboo plants are monocot. If you are using Agrobacteria for bamboo transformation, are the 'bamboo axillary nodes' the preferred explants for Agrobacterium-medium transformation? Just curious, because Agrobacterium prefers dicots although some monocot species (such as rice,...etc) have now been routinely transformed with Agrobacterium usingimmature embryos.
Martin provided a wonderful commentary on this subject over a wide range.
I know its painful but it's always good idea not to proceed with contaminated one. I suggest you to start new culture with proper sterilization. Here you did not mentioned your sterilization protocol but for grass seeds I used 70% ethanol for 30 sec followed by 50% bleach with 2 drop of tween20 and washed five times with SDW. Hopefully it will work for you as well.
All the best.
You can disinfest the epidermis of the plants without too much damage to plant tissue using a 1:50 Oxidate (H2O2) solution. Soak plants for about 10 minutes to eliminate all microorganisms on the plant surface. You may experience some leaf loss, but this is a generally safe and nontoxic method.
Any antibiotic does not kill bacteria, but supress their growth. In theory you can select a piece of young tissue without bactera after certain time of growth on medium with antibacterial compounds, but it is worth to do only with valuable genotypes of plants.
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