i want to confirm whether my cotton callus is embryogenic or not simply through light microscope observation. pls suggest me a good protocol (i.e.staining) for that. is it enough to confirm the embryogenic nature?
Hi, Premkumar, the best way of confirmation is to get somatic embryos from callus! Under light microscope you can observed small cell size with small vacuole, in many case embryogenic cell contain protein storage vacuole which you can detect after staining with tioludine blue. .
As pointed out by Taras, the surest way is to actually induce regeneration from the callus and look out for any of the known stages of somatic embryos such as globular, heart-shaped, torpedo or cotyledonary stages. However, you may need to do histological examination in which case the embryogenic cells will appear as small cells with densely-staining cytoplasm and roughly isodiametric shape, and more closely packed and often epidermal or sub-epidermal, as opposed to ordinary callus cells which are large, loosely arranged with large air spaces, poorly staining. Safranin stain followed by fast green counter-stain should do well.
Hello
Premkumar to get somatic embryos from callus! Under light microscope you can observed vacuole,n and Several types of callus were distinguishable based on the physical appearance.
Embryogenic callus are visually different from normal callus. Soon as you see them you will learn the difference. However, there is a strong genotyipic effect - recalcitrance also occurs... Treat every seed as a different individual.
Hi Premkumar,
It will be best if you can see different stages (globular, heart, torpedo or cotyledonary stage) by naked eye and best way if you can get bipolar structure (radicle and plumule from same cell). You can also confirm with microscopic studies as per suggestions.
All the best,
Aloka
The very simple method to identify embryogenic callus or not, is to see embryos forming in the callus on the medium.
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