Good day all
I am having difficulty with my agrobacterium transformations with regard to agro overgrowth after co-cultivation. I am currently using 100 mg/L timentin along with 400 mg/L cefotaxime as my antibiotics. I have also tried using 400 mg/L cef and 400 mg/L carbenicillin. These all have failed to get rid of the agro. My process for agro transformation takes place from newly streaked glycerol stocks, and my infection culture is maintained at an OD of 0.6. I then infect the leaf cuttings and allow them to incubate on co-cultivation media for 2 days before I blot the leaves on sterile filter paper and transfer them to regeneration media with antibiotics. Here I monitor the leaves and transfer ones with a few spots of agro to new plates of regen media although overgrowth usually occurs from these. If agro is overgrown, I wash the cuttings with sterile water 4 to 5 times and then incubate them for 1 hour in liquid media with 400 mg/L cef. I do this 2 times and then dry the leaves on sterile filter paper then transfer them to regeneration media.
I have conducted 6 to 7 agro transformations in the last 3 months and the results remain the same. Can anyone possibly help me?
Buren Logan Moodley
Cut the leaf discs smaller, so the leaf disc can completely touch the medium with antibiotics. large leaf discs tend to become curvy, and most parts of the leaf discs won't touch the medium. When I did it, I pushed the 4 corners of leaf discs into medium (w/antibiotics) to make the leaf disc contact the medium well. It works fine with me (w/o overgrowth). Besides, after co-cultivation, I subcultured them more frequently, every 2 weeks on regeneration medium (especially for the first 2 subcultures).
By the way, I used both cef and carbenicillin at the same time, 500 mg/L. That means, I added 250 mg of cef and 250 mg of carbenicillin into 1 L of medium simultaneously. LBA4404 should be less overgrowing than others like GV3101. GV3101 caused me some troubles, but not LBA4405. To my impression, once overgrowth appears on a leaf disc, it seems very hard to get rid of.
You said, "I then infect the leaf cuttings and allow them to incubate on co-cultivation media for 2 days."
After I infected leaf discs in Agrobacterium solution, I moved these leaf discs onto a sterile filter paper membrane to blot them dry **before** I put them on co-cultivation media for 2 days. It seems to me that you blotted it dry after 2-days' co-cultivation (I think this might be the root of the problem.)
Buren Logan Moodley
In a same co-cultivation by LBA4404 on tomato, I used 20 mg/l "Meropenem" antibiotic for controlling Agrobacterium in all regeneration steps and we never experienced contamination or overgrowth by Agrobacterium.
You just may need to play with Meropenem concentration for your experiment to optimize the best one.
Good luck
Danial Shokouhi
Thanks for the info. I have not tried Meropenem for any of my transformations (carrots, tobacco, cotton; with LBA4404 and GV3101). It is good to know that it works on Agrobacterium too.
🔶Meropenem: https://www.sciencedirect.com/science/article/abs/pii/S1671292706601338#:~:text=A%20case%20of%20Meropenem%20as%20a%20novel%20antibacterial,bodies%20%28PLBs%29%20has%20been%20reported%20in%20this%20article.
Buren Logan Moodley,
I have done tobacco transformation using AGL1 strain. I used leaf disc of about 1 cm dimeter and I infect with agrobacterium culture (O.D = 0.6-0.7) for 5 min and then blot dry the excess agrobacterium on sterile filter paper and after that co-cultivate. After co-cultivation I can see little agro growth. Then I washed them with cef (250 mg/L) and blot dry before keeping on selection media. I did not get any agrobacterium overgrowth.
Good day all
I would firstly like to thank you for all the help you provided. I have managed to identify the limiting factor in my work. It turns out that the growth on my tobacco plates was not agrobacterium but yeast. This explains the extreme resistance to most antibiotics. I have recreated glycerol stocks and redone the transformation using 400 mg/L cef. This has proved successful.
Thank you again for your replies!
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