If you got embryogenic callus ,it is easy to get somatic embryos.You can try the same medium with increased level of phytagel (any gelling agent) ,in order to induce osmotic stress.Alternatively you can add some stress inducing compounds such as PEG or mannitol.You can also try different concentrations of ABA ranging from 0.1-3mg/l)
expose the callus to green luminescence and hope you will get your target.
I assume you used 2,4-D to induce embryogenic callus. For the callus to convert to embryo masses, you should remove the auxin from media and use higher sucrose (6%) concentration and incorporate active carbon also. Then there are many ingredients you can incorporate to get the larger food reserves once you have conversion.
Try to transfer the callus on a medium (MS / 2 or VA / 2) containing BAP (0.1 and 0.5 mg / l), AIB (0, 0.5 and 0.05 mg / l) and sucrose (30 g / l)
Hi Siva,
If you say 'embryogenic callus', that means these calli can generate a number of 'somatic embryo (or embryoids)' later on. If you don't get any somatic embryos for a long time, those are not 'embryogenic' calli. The medium may need your attention. There are some good papers out there with COTTON-specific protocols for embryo induction from callus. If you follow the protocols, you should have no problem getting somatic embryos. I have done so from cotton callus freshly induced from the seedling explants, and regenerated into hundred transgenic lines.
Hi Siva,
Just found.....this is a good cotton paper with detailed protocols you can follow. I followed the protocols described in the paper. It gave good results. They also used Gossypium hirsutum L.
Hi Siva
To obtaining somatic embryo you could subculture callus clusters onto PGR-free medium for realization of somatic embryo. Also you can utilize different stress such as mannitol, PEG, glutamic acid, ABA and ... To selection of the best medium, You can follow many tested protocol by other researchers.
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