Dear Mohammad,

What species are exactly you referring?

Hi Ricardo Julian Licea-Moreno

Grapevine, coniferous (pine, norway spruce)...

I'm not sure that the recalcitrance depends only whether plants are perennial-lignified, or not. As you say, many factors might influence the in vitro response. I don't have experience with conifers neither grape vine; however, it has been reported that the germination of zygotic embryos is close related with their ontegenic states. As far as I know, the zygotic embryo germination is used like a model for the same process with somatic ones.

I have been working with all of that: apple trees, strawberries, spruces, larches, etc.. You go from grapevine to conifers! Seriously? A good start would be to try to understand the differences between Angiosperms and Gymnoperms! Really!

Do not ever think that YOU decide. In In vitro culture, plants are the bosses, surely not you, happily for human surviving. A good start would be that you try to understand PLANTS. You obviously haven’t started yet. You seem to ask for a cake recipe. Sorry, Tissue Culture is a specialty of Plant Physiology. The worst in the worst. Bad news.

I mostly agree with Francine, except that TC is a compendium of disciplines including genetic, botanic, cellular biology, biochemestry, plant nutrition... etc (and of course, plant physiology). I remember that some time the Prof. Chuck Maynard said that TC is an art. In the same way that different people play different melodies from the same music, the results of two different tissue culturist could be completely different using the same protocol.

I agree with you Francine M. Tremblay .

You should go step by step to understand plants; especially in vitro culture as Ricardo Julian Licea-Moreno said: " the results of two different tissue culturist could be completely different using the same protocol ".

According to the bibliography and the testimonies of confirmed researchers, the germination of in vitro cultured seeds of perennial woody plants (wether they belong to gymnosperm or angiosperm groups), especially coniferous, is subject to failure. WHY??? maybe because high genetic variability of the seeds (low fertility of natural hybrid seeds), hard seed coat, seed contamination with fungal colonnies...?

So, there is an evident focus on clonal propagation that is done using shoot, bud, as well as somatic embryos as starting material.

What do you think Dr. Tremblay?

Well dear Mohamad Issaoui , of course the need is to clone any of these plant species. However, in the starting question, there is no information as to WHY germinate seeds under in vitro conditions. To do what? They don’t germinate at all under in vivo conditions? Species rescue?

However, if ever it is to clone them, the worst thing that one can do is to germinate them. And that’s where the difference between Angiosperms and Gymnosperms come in the discussion. Any Angiosperm sp can do something after germination, what is not the case for Gymnosperms. And ontogeny has nothing to do in the story. It is a question of physiological age. Yes, physiology is the expression of genes through biochemistry, cytology, morphology, etc. Nothing new! Except for geneticists!

What is the point?

Dear Francine M. Tremblay , let's say that we want to study the responses of Douglas-fir (Pseudotsuga menziesii) to abiotic stress such as heavy metals. Someone could sow seeds of Douglas-fir on MS medium containing defined amount of cadmium while somebody else could use somatic embryos as starting material on cadmium-rich medium. Then the effects of cadmium toxicity on germination (of seeds or embryos) will be assessed. Here, the seeds of douglas-fir will not primarily germinate and that is not related to the abiotic stress! two questions are raising here; 1-why do the seeds of such coniferous species not germinate on petri dishes? 2-could somatic embryos be considered as good starting material to study the physiological responses to abiotic stress?

Dear Mohamad Issaoui , you should first verify if Pseudotsuga has seed dormancy. If so, you have to relieve it before germination but thereafter, sterilization becomes extremely difficult. Second, MS medium is not appropriate for germination. Too high in salts which inhibit germination. Somatic embryos are excellent for this type of work. However, if you have never work with somatic embryogenesis of coniferous species, it is not that easy, particularly that all you want is any material to study cadmium.

Dear Francine M. Tremblay ,

Thank you for this valuable discussion.

Yes seeds of Pseudotsuga have dormancy and we should subject them to cold stratification for two month before sowing them.

MS medium is more appropriate for angiosperms; I sowed seeds of arabidopsis and tomato and they germinated successfully with high percentage.

Seeds of conifeous germinate efficiently on soil substrate. I think that we are far away from finding a TC recipe to induce coniferous seed germination.

Best regards,

Mohammad

And how do you sterilize seeds after a 2-months cold stratification?

The purchased seeds are already sterilized. Then we proceed as following:

Seeds were mixed with wetted peat (peat and water were already autoclaved) then wrapped with sterile gauze pads then stored in plastic bags containing wetted peat and placed in the dark at 4 ° C for two month. Afterthat, stratified seeds were sown in pots containing soil in greenhouse.

So perfect! But your seeds still do not germinate under in vitro conditions. What is the percentage of germination of THESE super seeds under in vivo conditions? I am waiting for a %.

By by the way, I germinate conifer seeds without problems but I do not use MS medium.

Francine M. Tremblay

On soil substrates, under greenhouse, our seeds germinate with high rate: nearly 80%.

In fact, I did not find any study or reports about coniferous seed germination in in vitro culture. Can you give us more informations or suggestions on your successful seed germination?? That may be very useful for the scientific community.

I published it! Bad news, you will have to read.

Ricardo Julian Licea-Moreno On june 25, you explained to ME how I am wrong in my understanding of plant physiology and TC!

Plant physiology ALREADY includes cytology,, morphogenesis, biochemistry, genetics, etc. And you know what? Maynard, in my point of view (of course I accept that you are superior to me in everything including physiology and TC), was the most stupid person on earth. With this extremely stupid sentence, he brought TC to an art, meaning that TC is not anymore a science! And, all of a suddent, TC which is a subdivision of plant physiology, stopped to be a science.

By the way, I made an experiment on human influence in TC. Publishable? No. Why? Who knows?

I respect your opinion. Is not my problem who was stupid or not. Of course TC is a compendium of sciences, thus it's also science. However, the results of TC are highly influenced by humans and it's a fact. I'm not more wise than anyone, but none can know more than me about my own experiences. And yes, I consider that exist art in every aspect of our life, included the sciences. It's my opinion.

You do not respect me. You are a young researcher and you try to look superior to me. However, you have 12 citations while I have about 1500.

If you take time to read my response, I said that I MADE AN EXPERIMENT on human influence in TC. I compared 22 persons, being the treatments, on their effect on in vitro culture. The stats were clear: human is the main factor influencing the results. That was about 20 years ago. How awfull, I did not published it? No because it was unbublishable! And YOU give ME a lesson on human influence in In Vitro Culture? I insist, you are very young!

By the way, I come from a family of artists: painting, music, etc. And I am excellent in both. I always considered that I am excellent in cell, tissue and organ culture because of me artist point of view.

I maintain that Maynard (and I hope that you do not think anymore that you made me discovered Maynard) made a mistake. I am sure that he was well intentioned. However, his sentence was used by ... let me think .. geneticists?

Thanks for visit my profile. Congratulations for your results!!!!!

Ricardo Julian Licea-Moreno I am not sure that we follow the same conversation, which is not about you. Yes, I saw that you have 12 citations. That’s all. You do not interest me.

You congratulate me for my results? Thank you!

You are the second one to congratule me. The first one was intelligent and honnest (he saw the data) and saw the scientific point of view, of course, of my experiment . These data should have been published because they were the first on human influence in tissue culture. They had been obtained with a perfect experimental design. But, it was not in fashion!

You should, at least, be polite.

Dear Dr. Francine M. Tremblay , we should respect the opinion of Ricardo Julian Licea-Moreno even if you do not agree with him. Sorry, but you are a little bit agressive! "young researcher" , "12 citations" , "very young" , "you do not interest me" , "try to look superior" !!!!!!!!!!

In addition, you can answer my question by giving your valuable informations, even they were published! or at least you can show your corresponding articles (DOI, links). This is one of the reasons for such discussion on researchgate.

Dear @Mohammad Issawi, I'm agree with you. It's an space to discuss about science as well as to share our knowledge and to help to those that who need. Thanks.