Can anyone suggest me how to avoid fungal infection on regeneration media while doing agrobacterium mediated transformation of arabidopsis leaves by co culture method?
For agro removal i prefer to use cefotaxime and that works well but i immediately get fungal contamination hence no further callus initiation. This has happened several times with me in case of tobacco and arabidopsis both.
Thank You...I wanted to try both the methods...floral dip and coculture...
Did you filter-sterilized antibiotic cefotaxime before adding it into the medium?
Are these leaves used for transformation from tissue-cultured plants derived from sterilized seeds?
Try to use the leaves from tissue-cultured plants derived from sterilized seeds to minimize the contamination next time. Do you have the wild-type tissue-cultured plants you can use now? Or you can ask some from other labs, and do a transformation to see whether the contamination situation can be improved.
Thank You so much...but i think problem is something different because when i use these pot grown leaves for some other tissue culture work like callus induction etc then i didn't get any contamination but when i use these leaves for agro infection then they don't survive.
I suggest a combination of surface sterilization of your plants prior to co-cultivation, and after co-cultivation rinse your plants 5x with a solution of CuSO4 (1 gr/L) to avoid fungal contamination, and finally, put your plants in regeneration media with cefotaxime (check that your cefotaxime works properly with a some susceptible bacteria prior to its use).
Regards,
Hi Panchsheela,
1. That is odd. I know that you said it was fungal infection, but was it fungal infection or agrobacteria overgrowth to kill the explants?
2. Also, please see this related discussion on RG (see below). They are discussing the use of HgCl2 for seed sterilization but with 0.5%. I don't have experience of using HgCl2. But, there are other ways of sterilizing plant explants, such as the combination use of EtOH and Bleach. Have you tried that? Please read the discussion for further info. https://www.researchgate.net/post/Why_is_mercuric_chloride_used_as_a_seed_surface_sterilizer#view=54ec2d2cd2fd64031d8b45a0
Thank you Aaron, I will try cuso4 for fungal contamination.
Thank you Yuan for all your suggestions, I am not very sure but i thought it is fungal contamination, may be i can improve upon cefotaxime washing because i had done this for hardly 1-2 minutes, which seems to be insufficient. I will consider other aspects too.
1. If possible, can you post a 'contamination' photo for us to check-- to see it is 'fungal contamination' or 'agrobacterium overgrowth'
2. It also depends on which Agrobacterium you are using, for example, GV3101 or LBA4404. In my experience, GV3101 is more 'vigorous' and sometimes we experienced its overgrowth. Once it overgrows, it is very hard to get rid of them. LBA4404, on the other hand, is relatively 'mild'.
I am using LBA4404, although i had faced this issue with both the strains. At this moment i don't have any such contaminated tubes with me but i am going to repeat the experiment this week only so if i will have same problem then i will keep a pic of that. I have heard that GV is more efficient than LBA, so is it true???
@Panchsheela,
LBA4404 is less virulent. Although I tried both strains, I personally like to use LBA4404 for less overgrowth problem.
I quickly checked though online. There is one paper demonstrated that GV3101 has a better transformation efficiency than that from LBA4404. However, 3 papers showed that GUS gene has higher expression levels when LBA4404 was used for transformation than when GV3101 was used. Of course, there might have other different results from different species or genotypes out there.
@ Yuan
I had repeated the co culture experiment with arabidopsis and i am again facing the same issue of tissue death and fungal contamination, i am attaching herewith the respective photos. This time i had given cefotaxime wash for 30 minutes.
I have had this in my explants as well. It looks like a similar fungi. I have found that it is in the soil of my plants and the spores must be on my explants and hard to kill with surface sterilization. My next plan is to empty the soil, wash the roots and transplant into fresh soil after I sterilize my plants. I also use PPM 1ml/L in my media to help reduce fungal contamination. Fungizone can also be used in small concentrations, but I have not researched that concentration yet.
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