Hello Manjula,

Great to see you micropropagating trees. Why did you decide to use cotyledons and not the zygotic embryos? Do you want to maintain clones? I asked because it would be easier if you use zygotic embryos and induce multiple shoots and go from there - direct organogenesis pathway. For regeneration from cotyledons you have to follow indirect organogenesis, so the balance of auxin/cytokinin is important and it looks like yours is too much towards auxins (IBA), resulting in root formation. Without knowing the species it is difficult to explain any further.

Sorry, I had to describe fundamentals because the question was too broad.

Good luck

Ranjith P

I think your choice of IBA is not right. IBA can easily induce rooting. The Place where you cut the cotyledon may also have an effect. Don't forget that there will be endogenous auxin flow to the proximal end of the cotyledons. In some plants adding ethylene action inhibitors like silver thiosulphate may help.

try to use better cytokinin like TDZ or Zeatin to initiate shoots from cotyledons...IBA often used for callus induction and shoot initiation whereas BAP sometimes induce callus when you use leaf disc...better option is to use TDZ or zeatin...or supplementation of coconut water

Dear Ranjith Pathirana

Thank you so much. I am doing micropropagation of a dry forest tree species called "Mee". Its zygotic embryo is microscopic and turns to brown colour very soon due to polyphenolic substances. I am trying with other plant parts such as shoots and leaves. But for cotyledons, I observed rooting without IBA also.

Dear Bekele Abebie and Eyu Chan Hong. It would be a great help. Thank you so much.

I think addition of cytokinins like BA, Kn, TDZ, 2-ip (individual and in combination) will help in inducing shoots. You can add Adenine sulphate as an additive which facilitates organogenesis. And if you want to try cytokinin-auxin combination, use NAA, IAA instead of IBA.

If possible use nodal explants as it regenerate true to the plants.

It might be just a matter of shifting the ratio of cytokinin to auxin. That could be by reducing the auxin, or increasing the cytokinin. As others have suggested also, try different auxins and/or cytokinins.

I agree, cotyledons are a known source of auxin which is then transported to the petiole. That could explain your difficulty to regenerate shoots. You should take that in consideration and adjust your phytohormone ratio in favor of cytokinins.

Manjula Karunarathna if you have problems with phenolization after embryos extraction, I would suggest the in vitro germination of seeds; afterward you could use the rejuvenated in vitro-formed organs to continue with your experiments, with likely a lower phenolic releasing.