I am transforming a dicot with Agrobacterium tumefaciens. I am using two constructs one with hygromycin selection and other with PPT (BASTA) selection for transformation. Surviving shoots on selection media are not producing roots. Tried 1/2 Strength MS (resulting overgrowth of Agrobacterium), tried MS with 0.5 mg/l IBA with selection agent.
Depending on which dicot you are working with, you might try inducing root growth on soil instead of MS medium. Have you grown shoots from transgenic callus? If you have successfully grown transgenic shoots from this system you could stop the antibiotic selection, cut the shoots and transfer them to soil. This works for some species that can be propagated via cuttings. You can dip the stem in 1 % (w/v) IBA in talcum powder for root induction. Keep the shoots moist and protected from wind. I do not believe this works for Arabidopsis, though...
Hi
I recommend you to stop using antibiotic and hormone. I don't know your transgenic plants, but unfortunately in the case of some plants like canola, some transformants never can not producing roots.
God speed you
Increasing the IBA conentration might work, we obtained about 60% rooting in transgenic chilli shoots (Capsicum sp.) at 2.0 mg/L IBA. Omitting antibiotic might help, choose well elongated shoots, as they appear to form roots easily.
Reduce sugar content to 15 g/l, and also use auxin like hormone in your medium.
would like to know whether the shoot buds got rooted. Reducing the hygromycin / PPT concentration also may help. All the above suggestions are also very much useful. Whether the shoots are confirmed transgenic ?
As suggested exclude antibiotic from rooting media. Increase IBA concentration 1-2 mg/l and also try NAA in the same range.Use semisolid medium/liquid medium suitably half strength MS basal medium + 1-2 % sucrose. Hope this will work.
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