What kind of Agrobacterium strain did you use? GV3101? Some of the high virulent strains, once they start 'overgrowth' on the explants, it is really hard to get rid of. Therefore, it is important to avoid them overgrowing at the first place.

I usually use both carbenicillin and cefotaxime in the medium to suppress Agrobacteria. For example, if calculation shows that I need 300 mg carbenicillin, I will opt to use 150 mg carbenicillin plus 150 mg cefotaxime. Although there is no systematic study on this, I find that when both are used together, it shows better results.

By the way, when you tell people that you used a 400 mg/mL 'stock' solution, it did not give much info to the readers. Because it depends on how much mL of the stock solution you add into the medium and what is the total volume of the medium you have. You should present the final concentration of the compound in your medium. What is the final concentration of Carbenicillin in your selection medium?

Dear Yuan,

Thanks so much for replying. I used Agrobacterium strain LB4404. Should i try using Cefoxime? because i have read that Cefoxime might cause the callus to not regenerate especially for indica rice. What ssp of your callus? For final concentration, i usually add 200microlit of 20 mg/ml Hygromycin in 200ml distilled water.

 I am just a novice in rice transformation field :)Please kindly advise. Tq

Hi Yunz,

I used to use LBA4404 a lot, it did not cause too much trouble of overgrowth. It is not considered as a highly virulent Agrobacterium strain. As long as a proper concentration of fresh carbenicillin and cefotaxime is applied, it should be no problem to suppress them. I work on carrot callus, cotton callus, tobacco, Arabidopsis..... but not rice. I don't know whether cefotaxime will affect the regeneration of Indica rice. What is the carbenicillin concentration did you use?

Dear Yunz, you can also use Augmentin at conc. of 300-500 mg/l to suppress Agro in ur cultures. Good Luck

Yunz,

Use Timentin, it is the best and does not cause any regeneration problem. You can use in the concentration of 300-400mg/L. You should wash the explants in antibiotic solution after transformation and co-cultivation. Washing with water first and rinsing a few times with antibiotic solution and drying on the filter paper  helps in reducing contamination. While washing you can use Cefotaxime and Carb combination.

I agree with Sachin, use Timentin. Already at 100mg/L this stops Agro growth where Cefotaxim or Vancomycin +Augmentin failed.

 For Agrobacterium strain LB4404.200-300 mg/l cefotaxime is sufficient to control overgrowth.

Each and every species has its own selection concentration for Timentin. For rice we use 400mg/L and tobacco and tomato we use 300mg/L. For Arabidopsis 100mg/L is sufficient. Which species you are working on?

Yuan,

The concentration carbenicillin that i used is 400mg/l. I just learn about tissue culture and the only plant that i have work on is only rice.

Eric, Srrinath and Allah Baqksh,

TQ so much :)

Sachin,

Thanks for responding :) Currently i am onlyworking on rice callus.

Yunz,

There are publications, I searched on it quite a bit, that timentin is better than cefotaxime as far as regeneration is concerned. Nipponbare and even other cultivars grow well on Timentin. Use 400mg/L at the selection stage and 250mg/L at the regeneration stage. However, you will also need to wash your callus after co-cultivation. I wash in 500mg/L carb + 500mg/L cefo, 5-6 times (until the solution is clear) and then dry for at least 2 hours in the hood on the paper towel. Then I put the calli on selection medium. It does wonders. If you don't wash well, no matter what antibiotic you use, Agro will keep coming back. try it, you will see the big difference. I started rice transformation some 6 months back and have been enormously successful in getting plenty of transformants. For rice, certainly carb is not  a good choice. If you have other questions I am happy to answer. 

Hi Yunz, I agree with the above - use timetin if possible.  However, if timentin is not available or within your budget, both carbenicillin and cefotaxime will work but one must keep in mind that they are not very stable for lengthy periods of time or at elevated temperatures (> 30 C).  If you have to use them, prepare them fresh every time you make your media.  Store the media, if possible, at 20-25 C, and use it up with 2 weeks.  Also, do not make freezer stocks of either antibiotic that is stored > 60 days. Even frozen those antibiotics will begin to degrade and exhibit reduced performance in controlling Agrobacterium overgrowth.  If you check the product information sheet for carbenicillin from Phytotechnologies Laboratories, it will tell you that stock carbenicllin solution should be stored for no longer than 72 hrs at 4 C.  So think about it... if you are making media and storing it at > 25 C for more than a week, then then antibiotic is beginning to degrade.  I witnessed this exact problem during my graduate work...many experiments were lost due to overgrowth. Once we eliminated "long term" storage of antibiotics and media, we had no further issues.  If you follow those stringent protocols for handling cefotaxime and carbenicillin, you shouldn't have any problems. Use the carbenicillin at 400 mg/L and the cefotaxime at 250 mg/L or greater.  If you can get your hands on timentin, by all means, use it.  Nonetheless, I would still follow a pretty strict schedule with the timentin.  If you do this, you will see that you'll never experience overgrowth problems EVER.

I totally agree with Joel Hague. I have also seen that when the cell density of Agrobacterial  suspension is not accurately adjusted , this happens. Also if the temperature exceeds 30 C we face similar problem. In our experience also Timentin/Augmentin is very effective.  We never prepare  the stock of these antibiotics. Whenever we prepare the media we weigh appropriate amount of antibiotic and add directly to autoclaved media. 

#Joel

Hi Joel, I thought that carbenicilline was supposed to be VERY stable? That's why it was proposed to use instead of ampicillin (for use with bacteria). For work with bacteria, we keep our selective plates in the fridge for months and they seem to work just fine. Did I miss something here?

Hi Klaus,

Yes, carbenicillin is MORE stable in relation to ampicillin; nonetheless, it's still problematic in relation to what we have discussed above.  It performs better than ampicillin in overnight plates using E. coli/blue-white screening, etc, but it's not great for eliminating Agrobacterium if you can't control the temperature of your media plates.

Notice you said "we keep our selective plates in the fridge."  Right, you are storing at 4 C or close to it, and that will likely work no problem.  However, if you stored those same plates at "room temperature" and that temperature gets higher than 28 C, particularly for several days in a row, you'll see the antibiotic drop out pretty quick.  Also, if you are using carb for screening transformed bacteria this is not such a big deal b/c the cells are in direct contact with the medium.  In tissue culture, there will be Agrobacteria "on top" of the explants, away from the medium, and the antibiotic reaches these cells by diffusion through the explant tissue.  Thus, there's actually gradient here (the same applies for all the tissue culture components, i.e., salts, sugar, growth regulators, etc), so if the concentration of the antibiotic dips via degradation below a level that is effective to control the bacterium on the explants , a small proportion of bacteria will survive.  Over time, and WHEN IT IS TOO LATE, suddenly the Agrobacterium "reappears' and has overrun your cultures.

Just try it out for yourself if you like - make some plates with carbenicillin and store one group of plates at 30 C and another at 20 C for 5 d.  Then plate out a somewhat dense culture on these plates and see what happens.  I'd be surprised if there wasn't  a difference.  I'm not trying to be argumentative, but controlling Agrobacterium growth on explants is a different animal then culturing bacteria transformed with a selective vector.  Lots of "nooks and crannies" for the bacteria to hide in on an explant, none when the bacteria are plated directly on the medium.

Tissue culture is somewhat like cooking (heck, most molecular biology is like this) - the best and freshest ingredients will get you the best results, every time.  FYI, the folks from Phytotechnolgies Laboratories in Missouri, USA were here in RI, USA for the ASPB meetings in 2013 and they had a number of posters on the degradation of various tissue culture components under differing storage conditions.  You might be surprised at what they found.  The tl;dr version is that many components start to lose peak activity faster than one might expect.  

Here is just one poster they presented (there were several others):http://phytotechlab.com/files_clients/data/technical_literature/SIVB2013CytokininStability.pdf.

Anyway, best regards to everyone on this thread.

Hi,

150 mg/Liter Ticarcillin disodium / clavulanate potassium (15:1) should solve your problem.

Best

Imani

Hi Joel,

what you said makes a lot of sense. I didn't doubt your words, I was just surprised to see that cabenicillin was also temperature sensitive. I just noticed that the recommendations of suppliers for storage temperatures are quite different. I just checked our stock - one supplier recommends storage of the dry form at 4°C while the other states RT. Stock solutions also are supposed to be stored at 4°C vs. "below 20°C". Well, I guess I will err on the safe side, then.

Concerning the Agrobacteria - I didn't work with rice. For other cultures I found that usually we can get rid of the agrobacteria with 250 mg/l carbenicillin + 250 mg/l cefotaxim (depending on the strain of course). In some species, 2 weeks are enough to kill the agrobacteria - in others we have to continously keep the plants on media with antibiotics to suppress the agrobacteria.  In some cultures, a primary wash in double concentration of antibiotics (5' shaking) helped in our hands - fewer plantlets show agrobacterial overgrowth. Once an overgrowth happens that doesn't help anymore, though. For some cultures though (apple, for instance), such "pre-wash" was deliterious to transformation efficiency. So, I guess you will have to find the right combination of antibiotics and duration of treatment depending on your own varieties and culture system.

Hi Klaus,

i agree with everything you said above.  Not only do the differing manufacturers differ on their storage and preparation guidelines, sometimes the same manufacturer will differ depending upon which bit of documentation you look at (e.g, catalog vs package insert).  It's a bit maddening when one is having contamination/overgrowth issues.  I agree with you that one has to do what works for your particular system.  

If growth of Agrobacterium is more on selection medium. Its simply mean that you have taken high inoculum of the bacteria. I suggest you that you reduced the inoculum and the time of co-cultivation. if co-culture time is 48 hours then it should be reduced to 24 hour or 12 hour. Further you should take fresh culture of Agrobacterium (10  to 12 hour old culture) rather than overnight grown culture.

Dear all,

Thank you so much for all your helps. Really appreciated it :) Since this is my first time doing rice transformation, i am very lacking at preparation and experiences. I am also having problems due to the limited resources. Will takes note of all your suggestions.

Btw, can you help me to view word file attached. The file contained the callus photo after the third selection. Hope you can give comments on this photo. I wondered whether this really the Agrobacterium culture growing on the callus or something else.

Have a nice day

Thank you.

This (the attached picture) does not look good; it looks like the calli are dipped in the Agrobacterium (severe overgrowth). I wonder whether the calli will survive. It will be a good idea to re-start another batch for backup.

These calli are full of Agro growth and will not regenerate. The only, but rare chance to save them now is to do washing as I suggested earlier. But washing close to regeneration stages is not good and severely affects regeneration, in my experience. My suggestion to you is to start all over and follow the suggestions I gave to you before. These calli are no good.

By the way, from the picture, it shows that not only the calli are full of Agrobacterium, most of the surface of your plates are covered with a film of Agrobacterium. Next time, dry the plates a little (before use for experiment), or try not to use plates with 'condensation' or condensation-induced liquid on the surface of the medium. It seems that after you placed your Agrobacterium-infected calli in the plate, when you tilt those plates, the liquid ran through those calli and carry the Agrobacterium all over the plates. This may cause detrimental effect on your experiments.

@Yunz and Sachin,

The overgrowth of the Agrobacterium on your calli looks pretty severe. I think  Mohammed raised a good point, the 'high inoculum of bacteria' can be a culprit. When we conduct tobacco leaf disc transformation, we (according to a protocol's suggestion) diluted the overnight grown Agrobacterium culture before using it. So, Yunz, did you dilute your Agrobacterium culture? Since I have not done rice transformation, Sachin, has you diluted your Agrobacterium culture before using it for rice transformation?

Yuan and Sachin,

Thank you. Yes,  i think i have to start all over again. After 3 rounds of antibiotic selection, i placed all callus on the pre-regeneration medium and the Agrobacterium was growing back again. So i just gave up on them i thought. Yuan, when you dilute your culture you have certain OD that you used for transformation? I only did 2 trial of transformation and did not do any dilution for the suspension. How about you Sachin? did you do the dilution? You have any reference for the OD reading?

Hello Yunz Yunus,

You should try the following thing to overcome from overgrowth problem of agrobacterium:

1). Check out that your bacterial culture should not be overgrown.

2). Use filter paper method for co-cultivation step, (first place sterilized filter paper in empty petri plate , then pour about 1ml solution containing 200 micromolar acetosyringone on filter paper), now place explants in this plate after removing excess inoculum. 

3). Reduce co-cultivation time

4). Use antibiotics combinations (Cefotaxime 250 mg/L + Timentin 160 mg/L) upto root induction step, otherwise it seems to be that bacteria will be revived again.

5). After co-cultivation wash explants with distilled water then with doubled concentrated antibiotic solution i.e.  Cefotaxime 500 mg/L + Timentin 320 mg/L, with  intermittent shaking.

All above solutions are tried by me for wheat tissue culture system. I think same will be applicable with rice (wheat is also monocot). And don't worry about the effect of antibiotics on regeneration. it will work...... 

Bakesh Basatia,

Thank you so much!Really appreciated your suggestion :)

HI Yunz,

Sorry for the late reply.

Yes, when we diluted, we diluted the overnight night(s) growing bacterium to 1/10x (in MS liquid medium), without measuring OD. We used it for tobacco leaf disc transformation. I also attached one article at the end of this post. They diluted Agrobacterium 1/20x for tomato transformation. We used to work at the same building. But, since the species are different from yours, if you want to try, you can try a few dilution factors (such as 1/5x, 1/10x, 1/20x..etc). Good luck.

0.2 OD agro 48h co-cultivation combined with washes suggested before will help a lot. In addition I add the antibiotic solution in liquid AAM medium 50 -100 uL on each callus pieces after it is plated on selection medium in addition to 300 mg/L timentin in the medium. We use 0.8% agarose instead of agar/gelrite/phytagel in all our media for all genotypes of rice. It works and greatly improve the efficiency most of the time even better than immature embryos!!

Hello everyone,

Thank all of you for your kind help!

I am working on the transformation in rice. If the callus survive through several selection of antibiotics (I use hygromycine 50mg/L, cefotaxime 400 mg/L and vancomycin 100mg/L) but when transferring to regeneration medium (without antibiotics) I still have problem with Agro ( it should have been discarded after 3 stages of antibiotic selection, what should I do?

Thank you in advance. I really need your experience.

Bests,

I find this surprising Ai, nonetheless it must be happening. Have you tried just streaking your Agrobacterium on control YEP (or similar) plates (one w/ cefotaxime, one without) just to test if your cefotaxime working solution is... actually working? Do not overlook the age of the stock, and trust no one - make it yourself fresh and see if it works. Also, vancomycin is effective against gram-positive bacteria, not gram-negative bacteria - and Agrobacterium is a gram-negative bacterium. Thus, vancomycin is only helpful to inhibit/prevent contamination of cultures with gram-positive bacteria, not kill off your Agrobacteria.

If possible, I would switch to timentin (150 mg/L) if you can. It works much better to eliminate Agrobacterium than cefotaxime (which I would avoid, it is claimed here that it reduces regeneration frequency in IR64, it is also known to inhibit somatice embryogenesis in maize Hi II:Article Effect of antibiotics on callus regeneration during transfor...

I hope that works for you!

Yes. Cefotaxime is known for affecting regeneration. Also, Yes, Timentin even at 200 mg/l do not affect regeneration, in my experience both with Kitaake and IR64 with our current ongoing experiments. I have no experience with other genotypes.

Kindly thanks to Joel Hague and Muthu Kumar for your advices.

I will test the working of antibiotics. Yesterday I try to save the callus just contaminated by Agro by washing them with the same media of regeneration (same concentration of hormone, without Agar and added higher concentration of cefotaxime). I will see if it works tomorrow or next week.

In our lab, they do not use timentin. I will ask if someone has it. Thanks.

Does Timentin induce fungal growth during transformation? Since when ever i try timentin it causes fungal growth in the medium after inoculation even with maximum sterility?

Hi Ai,

You're welcome and no problem. The rinsing idea can help. When I did rice transformation, we did this step, actually submerging the calli for 0.5 min in liquid medium + antibiotic after co-cultivation. We then blotted the calli dry on sterile Whatman filter paper and then placed them onto selection medium. You don't always have to do this (Yes, Agrobacterium density plays a role, too), but it works well. Also, I hope you are able to get Timentin as I have never had an overgrowth problem with it.

Jimmy John,

I don't know of any literature or reports of this. I would suspect something "the pipeline" of inoculation is the culprit.

Amoxycilin-potassium clavulanate or amoxyxillin clavulanate (400mg/Litre) with combination of low concentrations of cefotaxim (50-100mg/litre) has surprising results in control of growth of different agrobacterium strains

I'm having the same problem I will try all these advice and see if any will work

I seem not to stop these bad boys GV3101 from growing

Timentin antibiotic is a mixture of ticarcillin and clavulanic acid, and at concentrations of 200–500 mg/l were used to control the over growth of Agrobacterium strain on explant while transgenic work.

Can anyone here comment on the stability of Timentin in semisolid media when stored in RT versus 4C? I use about 200mg/L and store them in 4C currently and it controls contamination effectively, but I'd like to move to RT storage due to lack of space.

Actually Agrobacterium infect plants only when it is in very very small in number. It should not be seen through the eyes. If the infection increases the explant will never survive because it causes harm to the tissue cultured explants.

You can save your plants only when the Agrobacterium is in very very less concentration and explant appeared clear (no infection) by treating it with 500mg/l carbenicillin or cefotaxime for 1hour.