Dear researchers,
I used Agrobacterium tumefaciens to transform Arabidopsis thaliana, and the vectors were constructed on pCAMBIA1301 and pCAMBIA1302 with a hygromycin resistance gene on them.
I have harvested T0 seeds, and planted them on a selective medium (MS agar + 50mg/L hygromycin B), but after more than a week, all the seedlings looked the same as the negative control (col-0). (Attach pictures)
Even though I repeated the transformation experiment and harvested the seeds (To), used freshly prepared MS medium, and used freshly prepared hygromycin (bought a new one),
So even the second time the results were the same as the first time, all the seedlings looked the same as the negative control (col-0).
Has anyone encountered this kind of problem?
What should I do when I choose existing transformed seedlings?
Thank you very much.
The following may help. Transformants and non-transformants look different:
Article A rapid and robust method of identifying transformed Arabido...
https://www.researchgate.net/post/How_to_select_transformed_arabidopsis_with_Hygromycin_B
Lidia Samarina Added the antibiotic after cooling the media,
Shin Murakami Thanks for sharing, i have been following these,
Dr- Hafiz Muhammad Rizwan
Looks like no one died? Is the hygromycin agent still effective? As Lidia Samarina mentioned, try adding hygromycin solution into medium while the medium is cool down a bit. And, swirl the medium nicely, so the hygromycin solution can be evenly distributed in the medium. I would also try not to put too many seeds one a same plate (?).
Yuan-Yeu Yau Sir Thanks for your answer,
Yes I added the Hygromycin when the medium was cool down, next time will try not to put too many seeds,
It is the same problem in the repeated experiment, so don't know where is the problem,
Dr- Hafiz Muhammad Rizwan
Did you include a plate of control (non-transformants)? What did controls look like? They should die. Is this T1 seeds after floral dip?
Yuan-Yeu Yau i did not include the control (non-transformants),
Yes, these areT0 seeds after floral dip
You should make sure those seeds you used for transformation are not already transgenic (hygr). Do a test. How old is your hygromycin? Where do you store it?
Yuan-Yeu Yau
How old is your hygromycin? Where do you store it?
About 1 month old, after prepared the stock stored at -20
Dr- Hafiz Muhammad Rizwan
what are you going to do next, about this problem?
Yuan-Yeu Yau prepared fresh MS + Hyg (80mg/L, trying with high concentration) Petri plates, and transferring the seedlings, lets see if will be successful by this or not,
Sometimes freezing hygromycin can make it become less effective. I keep mine at 4 degrees.
Try a tiny subset before testing the larger batch
You can also try putting your plates with seeds at 4 degrees in the dark for a day or two, stresses the plants a bit, seems to make the selection more effective
Here's a protocol for a faster selection so you don't have to grow your plants on plates for as long.
Article A rapid and robust method of identifying transformed Arabido...
Might want to double-check your math to make sure the concentration is correct. It's easy to be off by a factor of 10.
Good luck!
Amy Klocko The company mentioned on the Hyg bottle that after prepared stock store at -20,
Amy Klocko Thanks for your answer,
But the company mentioned on the Hyg bottle that after prepared stock store at -20,
I usually put the seeds at 4 C for 24-48h prior to poring on media ,
so after poring on MS should again do for 2 days dark in 4 C?
Dr- Hafiz Muhammad Rizwan
We bought liquid Hyg (sterile), and directly added it to medium. We stored it at 4C.
It's the method cited by Katie A Burnette
Stratify (cold and dark) 2 days (on the plates, no need to pre-stratify)
light for 6 hours
cold and dark 2 days
then grow in the light and select
This method makes the sensitive seedlings a pale green, while the resistant ones are darker green
It sounds like you are putting your sterilized seeds in liquid (sterile water or low % agar) prior to plating
keep in mind that whatever volume of liquid is being added with the seeds will dilute away the herbicide in that area and reduce the overall concentration
an alternate approach would be to ethanol sterilize the seeds and sprinkle the dry seeds directly onto the plate
Sorry for being vague, but I would say it is Hygromycin, whoever, I cannot provide a paper to support this answer. In a second isntance I would like to say it is the plasmid. Maybe the combination of both (a cassette?). Is there a way for you to be sure the gene is inserted? this could make it totally as the negative control.
Dr- Hafiz Muhammad Rizwan The company is telling you to store the ORIGINAL bottle at -20, not your liquid stock solution.
I recommend a met rapid and robusthod of the article of Harrison et al: " A rapid and robust method of identifying transformed at seedlings following floral dip transformation ". Successes.......
Amy Klocko
I put the seeds in 2-4 ml of sterilized MS + Hygromycin (50mg/L) and then spread on plates, that way seems to be convenient for seed spreading,
so if you would like to recommened some other way that will be appreciated,
Only 75% ethanol for 3 minutes and then washing 5 times will be enough for seed sterilization and then directly to spread onto plates?
Mª Angeles Zorrilla Lopez-Perea
First, I used sequencing to ensure that my gene was successfully inserted, and also through the restriction enzyme cuts with the control,
After that, I started the transformation,
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