Perhaps Because you have not planted the artificial seed !!!!

Just as seeds should be planted in the soil and medium, the artificial seed culture medium may have too and MS medium was culture medium for them!!

or Of MS medium, nutrients the artificial seeds are absorbing.

If your explenation is correct, the "seeds" in th eoutermost plates are dehydrating since they do not have medium (empty plates). They will continue doing so until you provide nutrientes and water (medium)

Water agar plate controls

Hi!

Your question itself is telling the answer,"Only difference was the central ones placed on MS germination medium, while others were not, and they were stored in growth chamber in empty plates".

Did you get the point!!, the central ones are supplied with germination medium the other ones are not. so growth rate is very clearly noticed, where all nutrients are there, they are growing fast, where no nutrients growth is arrested.

Hope your question is answered or I did not follow your question!!

Your tissue that is without medium is likely to be stressed. It is hard to know the exact reason, but moisture, gas exchange, or nutrient availability could all contribute to what you are seeing.

Maybe, if into the cover of the empty petri dish a wet filter paper would be placed to achieve chamber (H2O) saturation, then you could tell if low water (moisture) was one of the stress factors, as reasonably suggested by George.

Physiological state or age of the embryos should also be considered, besides as explained above, the difference is due to germination medium or no medium. When SE are asynchronous, germination behavior is different.

thanx for the answers. I've forgotten to remind you that the tissues used in this experiment were axillary meristem tissues, mainly lateral buds.

All the answers above are correct. The seeds planted onto media with MS the growth is rapid because it is enrich media, micro and macro nutrients are present, unlike the seeds planted on water- agar alone will have slow growth and high mortality rate because it is deficient with nutrients which needs to encourage growth. (unless proven otherwise)

Might be the empty plates did absorb some of the fluid from the beads. Thus the seed inside does have less moisture and does not or much later germinate. To prove this idea one could put beaded seeds onto just plain agar in the petri dish. I do not think that the beads absorb nutrients from the MS medium beneath. Like normal seeds they go along for some time with too much new nutrients.

İn my opininon, MS medium in petri dishes has more water than alginate beads, so nutrients (minerals, carbon source, vitamins … which are needed for plant germination and growth) are solved easily in water, and alginate beads in petri dishes contain MS can absorbe easily than alginate beads in empty dishes....

most of the answers were given above. i have little doubt have you noticed any changes on the encapsulated tissues in both MS medium supplemented and empty plated ones?

I am happy to note that even a simple question is answered by so many scientists.

Thanks to Research Gate, and the ever helping scientists.

For me the problem is not so clear, I think that it’s more complex. I’ll try to explain me. How strength is your alginate? Is it enough soft to allow the budding? Did you facilitate a uniform polymerization of alginate? What will happen in the plates without culture medium if capsules are as hard as it’s an impediment to growth?.... On the other hand, when you put the capsules on the culture medium suddenly is established a gradient of water potential so if water potential in the capsule is more negative then the water will move from culture medium to the capsule allowing its softening and facilitating the growing of buds. I don’t know which experiments are you performed but I advice you to conduct a trial combining alginate concentration and time in polymerizing agent. I also I recommend you to improve the technique to obtain a uniform and smooth capsule. If you like I can prepare for you some pictures describing the way in which I do it. I hope it help you. Good luck.

it seems going deeper... all beads contained 3% Na-alginate+MS+3% sucrose and were dropped into 1.5% CaCl2 and mixed very slightly about 20 min. Medium composition in Petris MS+3%sucrose. Dear Ricardo, thanks your points but all are under same conditions. Considering the answers as above, May I ask the question as how MS germiantion medium acts on the tissues?

See similar photo after 3wk of culture inition as above... Left one (at margin) was on MS germination, others were not.

I like preceding answers, they all seem possible.

Let me give another thought in the discussion.

Maybe explants grow similarly at the beginning. Since they attain certain size, they break thorugh beads, and:

- if they meet empty Petri dish - they stop growing, due to the lack of nutrients

- if they find a rich medium around themselves - they continue to grow.

Encapsulated seeds grow better when they imbibe. Therefore, external medium is generally required for its better germination and growth. Absence of external medium acts as stress and it is recommended for increasing the storage period of such seeds.

i supported Buhara Yucesan in used axillary merstimes ; also Conrado Mamaril ,i like youer answer with adding, Ricardo Licea answer which consider to compatible Ergun Kaya andi like they answer.

The correct answer was given by Nishi Kumari, Banaras Hindu University without making any complication.

I will try this experiment with Rodney's recommendation in which water and agar compostion used only. any suggestion?

Let us know the results ;-)

If plants still won't grow better on agar and water medium compared to empty Petris, then the "nutritional" hypothesis may become more possible than "physical" one.

Good luck anyway!

hyy I think the problem with the empty plates is that the tissues can't get water from the ouside to enlarge as the other ones that take the water from the MS beds

Hallo. I do encapsulation studies also. If the beads are put in empty petri dishes at 10-25C for storage, my beads will shrink and eventually die. How can I improve the storage method?

Hassan: In a wet environment, your capsules are shrank as a consequence of water lost.

Dear Hassan, have you ever stored them in a fridge at 4 - 6 C?

Thank you. I dare not store them in 4-6 C because even at 10C, they are all shrunk. At 25C that is our culture room temperature, I got some survivals but only for 1 day storage. Storage at lower than 25C & longer than 1 day did not give any survivals.

to: Nor Asmah Hassan: How can I improve the storage method?

see my post 6 days ago

Dear Dr. Thomas Baumann, I would appreciate if you could share the post you mentioned again. I might have overlooked & accidentally deleted them. Thank you.

here it is!

Thomas Baumann · 23.74 · 60.61 · Universität Zürich

Maybe, if into the cover of the empty petri dish a wet filter paper would be placed to achieve chamber (H2O) saturation, then you could tell if low water (moisture) was one of the stress factors, as reasonably suggested by George.

Hassam sorry but wich explant ans specie are you using? how is the survival without encapsulation? because if you only recover so low at 25C may encapsulation is not the best way.

For increasing storage period you please try various substrate for growing encapsulated seeds, devoid of sugar in encapsulation mixture, devoid of medium in encapsulation mixture, storage at low temperature, dessication of encapsulated seeds, etc.

I believe that only the MS medium of the capsule is not suficient for the germination process. In my opinion that is the reason.

MS media can be served as the best medium for the encapsulation study in my opinion the plates containing the seed may be maintained at low temperature for better response.

We work on encapsulation and synthetic seed. We used Petri dishes only during the cold storage of many species and at least we determined some crucial matter:

1) the alginate matrix tends to lose water during the storage;

2) the vegetative regrowth can depend on the environmental conditions (relative umidity, nutritive conditions, light);

3) an equilibrate composition of the artificial endosperm is essential to stimulate a good vegetative activity of the encapsulated propagules;

4) we found a positive effect of an high content of sucrose (5%) added to artificial endosperm;

5) the composition of sowing medium not always is decisive for regrowth: we often use hormone free media.

Thx Micheli. I tried experiment considering some suggestions as above. In that, sowing medium was water-agar only. I obtained very weak or no regrowth for encapsulated meristem. But over 45 days storage in fridge, bulblets are still germinating on MS medium, but not water-agar.

Sowing medium is water agar. Cultivation period is 6 wk.

Sowing medium is MS. Cultivation perid is 5 wk

The starting point is that the use of water-agar dilute the nutritive compounds of the artificial endosperm. So, the sowing supports have to be enriched by nutritive formulation similar to those added to alginate matrix.

If you need to maintain the capsules/synthetic seeds for a long time storage in dishes, you have to dip them in a few amount of storage-solution to avoid dehidration. But the solution has to be enriched with same nutritive compounds of the storage-solution. We think so.

Hi, Buhara!

Thank you for loading your pictures and results! I think that your previous and present results sugest, that the lack of nutrients is a limitting factor for your growing "seedlings". Summerizing: your artificial seeds don't "germinate" neither when they were sown onto empty Petris nor when sown onto agar-water medium. So, the water loss is not the reason, neither is the loss of nutrients from the bead as a result of nutrient dilution (it wouldn't limit the growth of plants in empty Petris).

My idea for explaining this is that:

Tiny plantlets, as they apear after the "germination", are still heterotrophic one. It is similar to natural seeds' germination - the seedling, at the beginning, is dependent on the supplies of nutrients from endosperm or cotyledons. While growing it consumes reserves and becomes gradually photoautotrophic.

In typical tissue culture conditions, plants are either mixotrophic or heterotrophic (small explants, like the one you used in your experiment). Light conditions do not allow for eficient photosynthesis, so after consuming all nutrients from the bead, plantlet has no source of carbon available if cultured onto empty Petris as well as on simple agar-water medium.

I wonder if it could grow better under photoatutrophic conditions (higher photon flux density and CO2 concetration).

How do you think?

And thank you for the interesting case;-)

Best regards

Natalia Miler wrote: "Light conditions do not allow for eficient photosynthesis, so after consuming all nutrients from the bead, plantlet has no source of carbon available..."

So, she agree that the nutritive function of the bead has to be optimal for supllying the nutrients to plantlet during the initial development of plantlet.

I think all the answers have been given, but they are mixed. First, something that happens in all fields of science is that we focus in the subject we want to study or we are specialist in, but forget that reality is not subjected to that constraint. If a problem is complex, there is no guarantee that we will succeed in simplifying it to study one particular aspect. In this case, I think the reasons are simple but not single, and have already been offered. What do plant tissues need to grow? First of all, they need water as tissue growth is mainly maintained with water and, in fact, tissue is mainly formed by water, so to double size you need at least double water, and from your picture of empty dishes it is clear that beads did not contain enough water yo sustain growth. A lateral problem of these dihes is why do day shrink, and the answer is again water: when a tissue shrinks the reason is always water leaving the tissue (tissue shrinkage is synonimous to loss of turgor, and turgor is maintained by water pushing the cell walls from inside and elastically stretching them like a spring). The particular reason in this case is probably related, as referred above, to water evaporation to the dish, or even out of the dish if the closure is not hermetic. You might think than not enough water can escape to the dish atmosphere to produce such shrinkage, but the differerence in the amount of water needed to loss turgor in a plant tissue is very small as turgor grows (or decreases) exponentially with water (is the cell wall stretching, like a spring...). You can test that, as adviced, trying to maintain the air inside the dish saturated with a wet paper disk, but this will work only in the empty dishes and will just delay shrinkage, not allow growth as for that you need water.

Now, coming to the water-agar dishes, you have water there (provided water potential is high enough [not low as another answer wrote], to allow water extraction by the tissue, but this should not be an issue with the ordinary formulations). Here nutrients come: tissue is formed mainly by water, but not only, and those claiming for nutrients are right: in a nutrientless medium like agar-water, growth will begin with water, but soon nutrients (first of all nitrogen and phosphate) will became too diluted to sustain growth in form of the building of new membranes, protein/enzime etc, so growth will stop.

So, as Ockham wrote, if two explanations are equaly good at explaining results, take the simplest one... But only if they are equally good! So, in summary, if water only or nutrients only cannont explain everything, think in a more complex explanation in which each of them has a role, although not necessarily of equal importance.

Xavier

@ Maurizio Micheli, you're right, but can you produce so big beads (or so ... concentrated and rich) to provide nutrients for the whole plant development, without any external source of nutrients? They (beads) will be at last not sufficient, I suppose.

@ Xavier, I like your profound understanding of the problem. But I wonder - did I miss something? Was there anything about plantlets' shrinkage before?

And you wrote: "So, as Ockham wrote, if two explanations are equaly good at explaining results, take the simplest one... But only if they are equally good!" And I think of what in fact means "good explaining" in that case.

Best regards

The bead covers the whole propagule. Natalia Miler demands about its effectiveness as source of nutrients. I think we have to analyze the type of propagule. In fact, in relation with our experiences, we separate two product of encapsulation: it is possible to encapsulate unipolar propagules (nodes-buds) or bipolar structures, as microbulblets or somatic embryos. The regrowth of u.p. allow to develop the vegetative organs (shoots and leaves), consuming a specific amount of water and nutrients. But, when the bipolar propagule starts to grow, it has to complete the development in plantlet, producing vegetative organs and roots, with different nutrients-water consumption. But we have to consider always the genotype and its requirements.

@Natalia

About shrinkage, I am sorry, I mixed Buhara's question with Nor Asmah Hassan's answer. But this just simplifies my answer: if your only source of water is that contained in the beads, as for the empty dishes, those plants will never grow, as you need water for the mechanical process of cell growth. When those plantets are place in the water-agar medium, were water should be available, then the reason must be different (nutrients...). Of course, if the bead has not enough nutrients in the water-agar medium, it does not have either in the empty dishes. So the MS medium, were plantlets grow well, is solving not one but two problems (water and nutrients). This is what I mean by citing Ockham: you need to explain all the issues; in this case, reducing the answer to only nutrients or only water would not be enough. This could be checked, as I understand you suggest, with bigger or richer beads that, in the agar-water medium should at least allow to initiate growth, altough if another nutrient source is not provided, it will not last for long.

I agree with Dr Natalia's views by increasing the photon flux density and CO2 concentration to promote growt and raise up the maximum CO2 assimilation rate (Amax). Can try to place the culture upside down to avoid dehydration of cultures. Shrinkage of tissues might be due to change in water contents in the culture as time passed by.