Seeds were grown in sterile condition, but nevertheless bacteria emerged from their roots. Thus sure the leaves taken from the seedlings contain it too.
Tomatoes especially get me crazy.
Did someone experinece the same?
Dear Dr. Wenkart,
I guess by inborn you mean endogenous contaminants, those that are inside the explant and go into the médium given certain conditions.
If this is the case, we had the same trouble with in vitro fruit trees. Our problem was even worse becasue the type of bacteria we got was a plasmid acceptor and therefore very soonn all our marker genes were inside the bacteria and therefore all plants regardless of being transgenic or not became positive in PCR experiments.
Only solution is not to relay in PCR, used introns in your visual marker genes and treat plants with antibiotics before doing any analysis.
I hope this is useful for you.
Hello!
I used to do tomato transformation in former times. Protokol was easy going and I was able to obtain transgenic plants.
Like Lorenzo writing above, I never relied on PCR and analysed my plant on RNA level too. Usially 10-20 % of plants were true overexpressiors. So, it's not entirely excluded thar I had the similar problem (because of low yield of true transfromants) but was not aware of it...
You can maybe try to let the original seedlings grow on media containing antibiotics, e.g. ticarcillin? However you cannot expose your plants on the same media while transfroming with Agrobacteria, as latter will be killed to.
Best luck!
Thank you both. I came to the same conclusion, only now, and I certainly shall try, because it is logically.
Hello,
indeed endogenous bacteria (plant or seed coat derived) can affect transformation either by destroying the explant or outcompeting the agrobacteria. So people try to grow the donor material on antibiotics, logically this is not a good idea in the situation of an agrobacterium transformation and, it might not kill all of them. The only thing that helps is to produce a new batch of seeds of the material that is going to be used for transformation under clean conditions maybe even after an embryo rescue (grow in replica plate) and just propagate the clean ones.
Good luck
Thank you Iris, sounds really great and should be done when you have a lot of time. But the tomato should be ready and ... it is not in the slightest.
You may can try culture your tissue at higher temperature or lower temperature to see whether the endogenous bacteria still grow in the same level. Or just give a heat shock during your Argoinfection, like 42 degree for 2 minutes. The endogeneous definitely will afftets Agro-mediated transformation if it is shows over growth. Another way is you might can added a certain antibiotic, but not afect Agrobacterium and plant tissue growth.
Good luck
the certain antibiotic, - that's the critical question. Because of that idid not try to add antibiotics to the plant tissue culture in order that the antibiotics won't interfere with the antibiotics I need to use to get rid of the Agro. The good question is: how long do antibiotics stay in the tissue, that even refers to humans.....when they are ill.
I am not convinced that tomato seeds can contain endogenous bacteria, but bacterial spores on the seed coat can be difficult to eliminate or inactivate.
If your transformation procedure uses seeds, these can be submitted to harsh sterilization conditions. A short treatment with 70% ethanol, followed by bleach or hydrogen peroxide with a trace of detergent may help.
The use of seedlings as explants is always risky. If this is possible, you could try using a sterile shoot cultures as source of explants. These can most likely be cultured in large jars on half strength hormone free MS medium. It may be necessary to seal the jars with porous medical tape (UrgoporeR or something similar) to keep contaminants out, while allowing adequate gas exchange.
Thank you. I use cotyledons, 10 days old, They are in touch of course before coming out, with the seed coat.
I thought about using leaves instead of cotyledons. Hope overcoming the obstacles.
You might get rid of the inborn bacteria by add 0.5 g/l timentin into growth media. Transfer plants to fresh media + timentin every 2 weeks. Timentin is harmless for Arabidopsis and potato, but I do not know its effect on the other plants. After 1-2 months in media with Timentin, non-contaminated plants could be transfer to normal media without Timentin and are ready for transformation.
I think the ideas of Dr.s Dinh and JPH could solve your problem.
By the way, you might can use a strong "Chrolid Gas" based sterilize method. Only in a tight colsed drier make a chemical reaction by add 50 ml commercial Clorox and 3.5 ml concentrated hydrochloric acid, put your seed in it for about 6 hours will kill any bacteria but nearly no harm to seeds.
Hi,
I experienced a similar problem. I recommend you to change seed stocks. Some stocks of seeds are very old or have some special contaminants. Also, if you need the leaves, you may cut them carefully and use. You'd better to sterilize the cutted leaves with sodium hypochlorite 20% for 10 min. Some contamination in tissue culture media are yeast, not bacteria.
God speed you
some endophytes are having PGPR activities, they are helpful, if the contaminant is pathogenic organism, special care is required. You must be able to identify the organism as friend or foe
Good luck
Hi. I am also working on tomato and its a common problem especially with old seeds. i had tried various options for overcoming this problem, like using Hgcl2 instead of Naocl for seed sterilization, changing the variety of tomato plants etc, but the most effective one is using PPM in the media.
S. Wenkart Plant Preservative Mixture...sorry, I am a bit too late to see your query.
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