I'm currently performing Agrobacterium transformations of immature wheat embryos for a CRISPR-Cas9 project. I do not have any colleagues with experience of working with wheat transformations and I have several questions to ask. Does anyone here have experience with the protocol and is willing to answer several questions about it?
The protocol is:
Ishida Y., Tsunashima M., Hiei Y., Komari T. (2015) Wheat (Triticum aestivum L.) Transformation Using Immature Embryos. In: Wang K. (eds) Agrobacterium Protocols. Methods in Molecular Biology, vol 1223. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1695-5_15
Questions include:
Thank you.
Hello Tua,
I am using the same protocol for my wheat embryo transformations. Although I haven't succeeded so far, I can answer your questions 3 and 4.
3. Centrifugation step is performed to induce minor injuries to allow efficient Agrobacterium-mediated delivery of Ti plasmid. In your centrifuge, set a speed of 20000g and check how much it translates to RPM and use that speed for your experiments. For the centrifugation step, you can refer to Hayta et al 2019 Plant Methods article. They have successfully used 13000 rpm in their experiments.
4. To remove excess of Agrobacterium growth, I wash explants after infection with 3-4 rounds of autoclaved water with gentle shaking/inverting and then wash the explants with WLS-medium supplemented with Cefotaxime at 200mg/l conc for 20 minutes with gentle shaking. Dry the embryos on filter paper and transfer to WLS-Resting medium. You can also try the infection step on filter paper to avoid excess Agro growth.
For the 4 questions…
1. Not sure but maybe it was done to reduce the stress encountered for easier regeneration.
2. If you mean the two temperatures as 23 and 25 C, I guess the low temperature is due to efficient T-DNA transfer by Agrobacterium. (https://pubmed.ncbi.nlm.nih.gov/8626274/#:~:text=Early%20studies%20on%20Agrobacterium%20tumefaciens,temperatures%20above%2029%20degrees%20C.) and the higher temperature is the optimized temperature for resting. I remember that we used a temperature of 22-24 C during the 2-day co-cultivation period for the cotton plant.
3. I think you meant to say that you need 20000 g while your machine can go up to 17000 g at most. If I'm right, I think you can continue with 17000 g in THIS experiment.
4. In our experiment (cotton plant) we washed the explants with antibiotics and also added the antibiotic to the regeneration medium.
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