I am using agrobacterium tumefaciens to transform some arabidopsis, the vector is constructed on pCAMBIA1305.1 so there is a hygromycin-resistant gene on it.
I have harvested the seeds, does anyone know how to select the seeds with hygromycin B?
I have planted some on a hygromycin B, but seems all seedlings look like my negative controls (col-0).
Has anyone done this before?
Thanks a lot.
Hi, Shi, have you plated sterile seeds om agar with Hygromicin? Which concentration have you used? Perhaps, if you did not get a "positive" plants, the main problem is not a selection, but transformation. How much seeds did you put on selection? To be sure that you got several independent lines, you have to put quite a lot seeds (500 at least).
Good luck!
Hi, Shi, in this paper they described all points, just have a look. It is Open Access! http://www.plantmethods.com/content/2/1/19 After 1 week you should see differences, but I suggest you to check details of your transformation methods as the first step. If transformation was not enough effective, you will need a lot of seeds to select transformatnts! Good luck!
Hygromycin is somewhat light sensitive. Use low fluence of light for seed germination. The best indicator is root growth. The roots of hygromycin sensitive control plants should hardly grow beyond a stub and some rudimentary root hair, while roots of the resistant plants should grow longer, may branch more, exhibit more root hair, and penetrate the agar plates deeply. It might take a few more days. Ultimately the control plants cotyledons should bleach and primary leaves should not form, which resistant plants should make green cotyledons and further leaves.
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