Hi,
I am selecting rice calli of Nipponbare on hygromycin (20mg/L) on selection as well as regeneration media. Some protocols suggest more than 30mg/L and even 50. What is the optimal concentration to select transgenic only and rule out any non-transformed cells? Very high concentration can be detrimental to the regeneration of the transgenic, I guess. Please advise, thanks.
I would suggest to set up experiment to optimize concentration of hygro for rice cultivar you are using (use various concentration) and even better to optimize at callus stage and later on at regereration step. Remember its an important step.
We normally use 2 steps...40mg/L in the calli selection and 50mg/L in the regeneration step. This would rule out the large majority of false positives. But if you are not getting any regenerant you can decrease the selection pressure to 25mg/L and you should be fine. You will always need to confirm through PCR, Southern blot, etc later. All the best
I would suggest to set up experiment to optimize concentration of hygro for rice cultivar you are using (use various concentration) and even better to optimize at callus stage and later on at regereration step. Remember its an important step.
I agree with Allan, you can set an experiment to determine the minimum Hyg concentration you need to kill the wild type material. Ideally, you can generate calli from transgenic rice and use it as a positive control. In our experience, different sources (brands or types) of Hyg can give different results, so it is important to test it.
I recommend 50 mg/l. If you go under 40 mg/l, the number of the untransformed survivor callus lines will increase dramatically.
I agree with Allan and Hugo, it will be better you can set up a series of Hyg concentrations to test your callus under your lab conditions, and the use of the particular Hyg brand you purchase. we also have the same experience that using different source of Hyg giving us different results (on tobacco). Since most people above suggested the suitable Hyg concentrations was ranged from 20 to 50 mg/L, it may be a good start to set up experiments containing concentrations of 20, 30, 40 and 50 mg/L for testing your rice calli.
Thanks to everyone for answering my question. I think apart from hygromycin brand, it might also depend upon the vector. But we are using pCAMBIA1301 on Nipponbare, which I think is universally used and has hpt gene driven by enhanced 35S promoter. Apart from that the other variables might not affect much and the selection procedure is pretty standard. please correct me if I am wrong. I think for regeneration also, we need to optimise the hygromycin concentration and people have used variable concentrations there too.
Calli derived from Japonica rice cultivars can be screened at 50 mg/l
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