Simply, from callus appearance. Embryogenic callus white and granuular apperance while the other callus looking yellow and friable.

A couple of thoughts based on your observation that you suspect two types of calli -

1) Embryogenic cells are typically highly cytoplasmic (vs. large vacuoles). In citrus (what I work with), the cytoplasmic/vacuole-size difference between embryogenic and nonembryogenic cells is dramatic and easily recognized.

2) Try to induce embryogenesis in each of the 2 callus types. Somatic embryo induction in one but not the other would be the true test.

I suspect that if you can separate the two via cytoplasm density and/or somatic embryo induction that you will learn to visually distinguish between the two and easily identify the embryogenic calli.

Simply, from callus appearance. Embryogenic callus white and granuular apperance while the other callus looking yellow and friable.

Embryonic callus is fariable soft ,globular shape and write or yellowish colour

Non- Embryonic call us is sold , celluer shape and Brown color

@ Dung Tran,

By the way, how to distinguish embrogenic callus from non-embryogenic callus using scanning electron microscope? Is any report?

Dear Dung Tran,

All the best for your study and looking forward to hear the result of your study.

Regards:

Hom

Thank you so much for all suggestions. I still have some points that I can not clarify. Based on all your answers, I understood that embryogenic calli would be developed further into somatic embryo. But this process can occur in MS free medium. How about the non-embryogenic calli? It also can be developed further (for example they can be induced into shoot)?

Actually, during our experiment, we do not use plant growth regulators so that the regeneration rate is very low. My expectation is that when we can identify two kinds of callus, we will subculture in the medium supplementing with different plant growth regulators to increase the regeneration rate as well as to decrease the duration of culture.

Thank you again and I hope to receive more recommendations. They are really useful to me.

Dear Yuan Yeu Yau,

There are several articles of which the authors used scanning electron microscope to identify two kinds of callus. For example, this is one paper: https://link.springer.com/content/pdf/10.1007%2Fs11240-013-0330-2.pdf.

Under the microscope the sotic emryo cells are round in shape. Highly cytoplasmic densely stained more over the starch grains are accumulated in the cytoplasma. See the attached file please

Hi Dung Tran,

Thanks for pointing out the paper.

About your question in your later post: "But this process can occur in MS free medium. How about the non-embryogenic calli?"

These are my thoughts:

1. It should be 'pytohormone-free MS medium'.

2. 'non-embryogenic' calli means that these callus cells are not embryogenic and cannot develope into somatic embryos and therefore shoots

Non embryonic callus can not developed in any way to somtic embryos or even produce adventitious shoots because it has not the pre embryonic determind cells Which were responsible for this action . Clearly there is a physiological state and such Inner components are control in general the callus differentiation since the beginning.

What type of medium are you using for germinating Cucumis immature seeds ? Basal like half MS or Basal + hormone and/or sugar supplements ? Can you identify the origin of the calli ? Embryo or maternal tissues ? Frequently (but not always) embryogenic calli which haver been hormone induced developed into embryos after transfer on hormone free medium. Distinguishing between embryo germination or shoot emergence from a callus is not so easy, but generally you can see by lens that somatic embryos germinate (root then shoot emerged) when buds develop initially without roots.

Dear Mr. Jullien Marc,

Thank you so much for your answer. Actually, those questions you ask are also the problems that we are facing. We use MS without hormone for culturing immature seeds. I think among lots of them there were some seeds which germinated directly because I could see the cotyledon or hypocotyl of seeding. But almost of them developed through callus pathway. We are not sure the callus induced from embryo or maternal tissues. I think the callus is induced by wounding (we cut immature seeds into half).

Even from germinated one, it seems to be lack SAM (shoot apical meristem) to continue growth. From callus, it is very difficult for shoot emergence or embryogenesis. And even getting the bud cluster, they can not elongate into the shoot. It is easy to be vitrification. So that, the most serious issue of our experiment is that the plant regeneration rate is so low. That is the reason why I want to find the solution to improve. For the whole process, we do not use hormones except for the final step to induce root. Do you have any advice to me? For us, we have started the experiments involving to tissue culture just one year. We do not have much experience. Thank you in advance!

Dear all colleagues,

Thank you so much for the wonderful comments and suggestions. I hope Dung Tran, should have benefited a lot from these comments.

Regards:

Hom

Dear Dung Tran

Try to determine the way you want to regeneration your plant. If you decide to select direct way so try to add some cytokinines once seed start to germinate. Instead of this you can concentrate on in direct way and so you need to add some auxin or some combinations of auxin and cytokinines to promot callus tissues. Then free hormones meduim. And so on.The plant hormones you added will help cells to make the response you need.some plants can not servive in vitro without plant hotmones.

Good luck

Dear Dung Tran

The embryonic callus can be easily distinguished from non-embryonic callus, it is a white granule full of rotation and is grown on a nutrient medium containing auxin and cytokinine in balance to be total somatic embryos. While The non-embryonic can not developed.