My Arabidopsis plants growing on MS media got contaminated by both fungus and bacteria after the transfer to fresh media. Please suggest some method to remove theses contamination.
Thank you
Hi Nikhil,
1. I assume that you sterile the seeds before you place those seeds in the MS medium.
2. From my own experience, most contamination of subculturing the plantlets to fresh medium are caused by the hood condition (hood should be certified/checked by a contractor once in a while) or the tools used for transferring those seedlings (such as incompletely sterilized). The forceps we used are usually autoclaved. They are then dipped into 70% ethanol and briefly sterilized under the flame in the hood while doing the transferring/subculturing.
3. We have encountered some contaminated transgenic tobacco plantlets while tissue-culture them in MS medium; we tried to rescue them by just transferring directly into soil. For some reasons, the soil can 'inhibit' the worsen of the contamination and provide the seedlings to further grow. We have rescued important transgenic lines by this way. Of course, if you need sterile plants as start materials for experiments, such as Arabidopsis protoplast genetic transformation; probably the point #2 above is more helpful.
Good luck on your research,
Hi Nikhil
for sterilising the seedlings before subculturing, I agree with Yuan Yeu-Yau.
1. First of all, the position of laminar flow is very important. it should not be directly facing to the doors whether windows or main doors.
2. there should be proper cleanliness maintained in the lab. no entry should be allowed to outer shoes or slippers.
3. proper disinfection of hands should be taken care of.
4. seedlings should be washed with antifungal and antibacterial solutions for at least 1-5 min after that final washings should be with autoclaved distilled water.
5. the forceps and surgical blades should be again sterilized in flame after a dip in 70% ethanol.
6. the media plate on which the seedligs are to be subcultured, should not be open for more than 10sec in single transfer.
Thank you ,
But I am having a different problem all my seed sterilization is good and there is no contamination in primary culture after transferring the grown seedlings to a new medium only I got huge amount of contamination. I agree with you all that my forceps was not sterile .
Now I want to same my rare mutants ,but currently I cannot transfer them to soil. So please suggest me some sterilization protocol to sterilize plants and transfer them to fresh MS media.
Does the plants survive? Does it affect the plant normal physiological functions?
Hi Nikhil,
This is tough..., also you need to make sure that the bag of plates you use are sterile, not broken or with defect.
Maybe you can try one of these two ways to rescue the plants:
1. Sub-culturing them to fresh MS medium everyday, until they reach the size ready for transferring to soil. I have tried this before.
2. First, "seedlings should be washed with antifungal and antibacterial solutions for at least 1-5 min after that final washings should be with autoclaved distilled water (described by Anila above)". Then, quickly blot away the excess solution on an autoclaved paper towel, and place the seedlings back into fresh MS medium (don't let the plantlets dry out). Do this everyday or every other day, depending on the improvement of the contamination). Unfortunately, I have not done the antifungal and antibacterial wash before, so you may need Anila's help.
To be honest, we cannot predict if the plants will survive, but it is worth a try.
** Note, after you flame the forceps, let it cool down before you touch the seedlings (they are weak now, don't burn them).
Best,
Also,
From your first paragraph description:
(1) Your seed sterilization is good, (2) There was no contamination in primary culture and (3) only got contamination after the seedlings were sub-cultured to fresh medium. In this case, you also need to keep an eye on the autoclave machine. Make sure it does not breakdown, and the condition for steam autoclave (121 C, 15 PSI) is reached for the whole time of sterilization.
You can transfer your Arabidopsis plants from tissue cultures to potting mix at 7-10 days after germination. You do not need to subculture them. When transferring plants to potting mix, please make sure that the potting mix are wet. Keep Arabidopsis plants in a tray with about 1 cm of diluted fertilizer water and cover them for at least 3 days to keep humidity. And if your plants is contaminated, just transfer them to potting mix because potting mix is not sterile.
Try to use a plant extract Vegelys which in my lab succesfully stablished against pathogens.
www.phytoauxilium.com
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