Ammonium vs Nitrate In Vitro

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eteson

Phragmad
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Dear friends, I would like to understand the Nitrate vs Ammonium absortion in invitro (sterile conditions) where the soil bacteria are absent. Do you have literature about this topic? Can the ammonia be used by the seedlings? Why the source of N is ammonium nitrate in most part of the comercial media?
Thanks a lot!
 
Dear friends, I would like to understand the Nitrate vs Ammonium absortion in invitro (sterile conditions) where the soil bacteria are absent. Do you have literature about this topic? Can the ammonia be used by the seedlings? Why the source of N is ammonium nitrate in most part of the comercial media?
Thanks a lot!

A I recall the ammonium is used to inhibit root growth by blocking Ca and Mg. Ammonium nitrate at the same time supplies enough NO3 to grow leaves. Without the NH4 the plants would grow roots too fast and destroy the invitro environment before the top part of plant has time to develop.
 
Thanks Lance. Does the pH changes while plant takes the Nitrogen from the NO3 and not from NH4?
Most precisely: If I use N from Ca/Mg nitrate to make my medium... the pH changes are going to be least important than if i use Ammonium nitrate?
 
Thanks Lance. Does the pH changes while plant takes the Nitrogen from the NO3 and not from NH4?
Most precisely: If I use N from Ca/Mg nitrate to make my medium... the pH changes are going to be least important than if i use Ammonium nitrate?

I assume pH change will depend on what phase the culture is in. If the pH has changed significantly in the media then wouldn't the media be also depleted of N and need to be changed anyway?

Why do you want to move away from NH4?
 
Eliseo, there are quite a few papers about this topic. But I'm afraid that you can't generalize it; different species respond differently to NH4:NO3 (this seems to be the general consensus). So you probably need to experiment.

I just looked at a couple which I found on my computer.

There are a lot more than pH. For example, the ratio could influence the hormonal balance. This is not in orchids, but in flasks of epiphytic bromeliads, more NO3 caused higher auxins/cytokinins (more auxins = more root) while more NH4 and urea caused the opposite.
Mercier, H., G. B. Kerbauy, B. Sotta, E. Miginiac. 1997. Effects of NO3, NH4 and urea nutrition on endogenous levels of IAA and four cytokinins in two epiphytic bromeliads. Plant, Cell and Environment 20:387-392.

In Bletilla, Ichihashi et al did experiments to see the optimum nutrient composition for in vitro. NO3:NH4 of about 4:1 (I think this is weight ratio, not the number of molecules) worked better.

In Phalaenopsis, pure NO3 seems to be better, and they observed the interference between NH4 vs other cations (Ca, Mg etc).
KUBOTA, S., YONEDA, K. and SUZUKI, Y., 2000. Effects of ammonium to nitrate ratio in culture medium on growth and nutrient absorption of Phalaenopsis seedlings in vitro. Environment Control in Biology, 38(4), pp.281-284.

But in Cymbidium, mixture of NH4 and NO3 was better (this was for cloning, but I'm sure it applies to in vitro seed germination):
da Silva, J.A.T., 2013. Ammonium to Nitrate Ratio Affects Protocorm Like Bodies PLB Formation In vitro of Hybrid Cymbidium. J Ornam Plants, 3(3), pp.155-160.

There are quite a few references in these papers. So if you are interested, let me know your email via PM.
 
Thanks Lance. Does the pH changes while plant takes the Nitrogen from the NO3 and not from NH4?
Most precisely: If I use N from Ca/Mg nitrate to make my medium... the pH changes are going to be least important than if i use Ammonium nitrate?

The simple answer is yes, ... but ignoring the issue as to whether the plants you are growing like a NO3 only medium, and if you take into account what Naoki has said then it's difficult to guess this.

There is the theory, and then there is what happens in practice, and they diverge a bit and I give you an example of this below. The theory is based on work with non-orchids, but the basic predictions do seem to hold up for orchids to some extent, if we accept that NH4 and NO3 uptake are not the only effectors of changes in medium pH, even if they are the more dominant ones.

The theory is that uptake of NH4+ involves export of H+ (so medium acidification). You can read in textbooks that with nitrate uptake an OH- is exported to the medium, but from my reading this is no longer supported by the evidence. It appears a consensus has emerged that anion uptake occurs by simultaneous uptake of H+ (called proton symport). This is sort of the same thing as leaving an OH- behind, so in a practical sense it is the same, so alkalinising. You might expect from this that a 50:50 :: NH4:NO3 medium should be pH balanced (w.r.t. N), but it's not that simple. NO3 uptake is less alkalinising than NH4 uptake is acidifying. I did read through an explanation of this, but it made my head spin and it wasn't worth the effort required to get a detailed grasp of it. But the basic issue is that there are different types of transporters that work at different nutrient concentrations and they are not all directly coupled to H+ transport in or out, it's more about how much membrane depolarisation is caused by ion uptake, as this is what the cell needs to correct by pumping H+ out of the cell to restore a negative charge on the inside. Anions have less effect on this.

Media like P668 have a NH4:NO3 of 1:2 (molar ratio) and this is supposed to be in the region of pH balanced with respect to N transport according to a "bunch of calculations" (although I've also seen a figure of 1:3), so the theoretical overall minimal contribution of N to pH changes. Now lets look at an example of pH changes from an orchid paper. This study looked at different NH4:NO3 ratios in Catasetum fimbriatum (Majerowitz,N, et al. 2000, Enviromental and Experimental Botany: 44, 195-206.). They show that this orchid acidifies the medium even when grown on NO3 alone pH 5.5 -> 4.62, but with NH4 alone the acidification is more extreme i.e. pH 5.5 -> 3.55. Note that they didn't add MES buffer. In this study the optimal ratio for both shoot and root growth was NH4:NO3 of 2:3 (final pH 3.98). Interestingly, they showed that glutamine was a better N source than the optimal ratio of inorganic nitrogen and with this the final pH was 4.6. So both the anionic N sources, NO3 and glutamine, do reduce the extent of acidification, but still the overall effect is acidification. Has anyone ever heard of anyone getting alkalinisation in orchid propagation media ? It seems the issue is always a case of acidification.

Getting back to the idea of a NO3 only medium. I did a bunch of reading on plant tissue culture when setting up to make my own medium for Neo propagation. All I could find was single paper in Korean, but with an English abstract. I also read some papers on Vanda propagation including a thesis on this topic. It was in the thesis that I read a comment about the requirement for a reduced N source for seed germination (so either NH4 or amino acid), because nitrate reductase only becomes active at some time during protocorm growth subsequent to germination. I didn't check the reference to this or whether it is known to be a general feature across orchid species ? If you are adding a peptone of some sort it's irrelevant anyway. There is even a medium designed for terrestrial orchids that has very low salt and no inorganic N at all (it's either BM-1 or BM-2, I can't remember which). It's sold by a German vendor of plant propagation stuff ( https://dephyte.com/Plant-Culture-Media ), and they recommend it for Paph's.
 
Myxodex, thanks a lot for your explanation it is very helpful. I have been using p668 for years and it works very fine for most part of Pharagmipedim species and hybrids. But a couple of years ago I started with Pk and Pk hybrids and also with Phrag anguloi seed sowing and something was wrong with the medium for this species. Plantlets grow fine for 3 weeks or so and then they stop growing and the tips of the leaves start to turn yellow... In the particular case of Pk I usually start with a pH of 6.4 and it drops to 5.5 in about two months! ... so I need to replate them very frequently. I have been testing several mediums without too good results so I started tailoring my own one... so far the best one has a NO3/NH4 ratio of about 12/1. This medium allows me less frequent replating and the pH changes are not significant. Your explanation made me understand why the changes in pH with P668, thanks a lot!
 
Suggestion: To prepare your media maybe you can use MSU fertilyser because its high content in nitrate. It contains all is necessary to prepare a media for In Vitro Culture.

Envoyé de mon Nexus 9 en utilisant Tapatalk
 
Suggestion: To prepare your media maybe you can use MSU fertilyser because its high content in nitrate. It contains all is necessary to prepare a media for In Vitro Culture.

Envoyé de mon Nexus 9 en utilisant Tapatalk

Thanks! Yes, I have used it in my formulations.
 
And ... Is it an improvement ?
Have you access to MES ? This a product which will buffer you media.

Brabantia, for some Phrag crosses I am using MSU adding MES, Peptone, vitamins and banana puree. This formulation allows me less frequent replating, but the growt rate is not as good as with P668 and more frequent replates.
Some years ago I used it also for delenatii with very good results.
 
Eliseo, it's nice to hear of someone else making up their own propagation media, it can be frustrating, but it's rewarding to solve a problem. It is also a good guess that commercial growers of Pk and it's hybrids have the answer to your problem.

Unlike you, I have no experience of phrag propagation, but I've been thinking about your problem and trying to figure out what I modifications I would try, and which I'd try first.

If I've understood your posts correctly, you have a medium based on P668, on which most of your phrags grow well, except Pk and it's hybrids. A reduced NH4 version of this supports Pk, but frequent replating is needed, in part due to rapid acidification. The rapid acidification suggests that the NH4 is taken up quite quickly and possibly depleted. You have a NO3 only medium based on MSU with banana puree that supports growth for longer but the growth on this is slower. One possibility is that phrags prefer a reduced form of N under in vitro conditions, just that some species are sensitive to the acidification caused by NH4 use. The only way to find out if this is the main issue, is to try a modification that gets around this problem.

There are two approaches that could work, the first is to replace NH4 with glutamine, either in a P668-like recipe or in your MSU formulation. It's a good guess that phrags will use glutamine. Glutamine is anionic so not acidifying, has two N atoms per molecule and feeds straight into a primary N assimilation pathway and it is the main source of N in the BM-1 medium that has been used for phrag propagation. The second approach that I favour much less, is to stick with the NH4 but add it as salts of organic acids (OA's) from the citric acid cycle (but I'll leave this alone for now as there are some complications with this). I think it could be worth your while to have a look at BM-1 just for ideas, rather than as a recommendation to use it as it is.

BM-1 is a totally different type of medium to P668. You can see the recipe here: https://dephyte.com/BM-1-Terrestrial-Orchid-Medium , just click the specification tab. This medium is recommended by this company for paphs and phrags. It has very low salt, no NO3 and no NH4. All the N comes from a combination of glutamine and casein hydrolysate. What is common between P668 and BM-1, well not a lot, they are almost totally different types of medium. They do both have a supply of reduced N, so NH4 in P668 and amino acids in BM-1. The issue I would have with this medium is no added Ca, so maybe this is a contaminant in the casein hydrolysate? It may seem strange to add glutamine when you are already adding peptone or protein hydrolysates. The answer is that protein hydrolysates can differ in their ability to support growth. Some amino acids, e.g. threonine, if too abundant in the mix can be slightly inhibitory to growth, so the profile or balance of different amino acids can be important. In BM-1 they are adding casein hydrolysate at 500 mg/l and glutamine at 100 mg/l , and hydrolysates also contain oligopeptides that are not all available, which means only a fraction of the 500 mg/l is as free amino acids by weight, and so glutamine will be by far the most abundant amino acid in this medium. On a practical level I note that people who use glutamine tend to filter sterilise it and add after autoclaving. I think this is because it can partially break down into glutamic acid and NH4 during autoclaving, however BM-1 is sold as complete medium in which case autoclaving it would be the only option, so I'm not sure how important this really is, but it's good to be aware of this issue.

Another issue is the frequency of replating needed on the media that give the best growth. It could be that you are working to a osmolality limit. The P668 medium might be near the osmolality optimum and your MSU medium above the optimum. If this is the case it might be possible to make adjustments in your recipes that get you to a better compromise between rapid growth and frequency of replating. I have developed a script that estimates the osmolality of media, it's only rough, but serves as a guide during formulation of media. For example the banana puree will contain some organic acids, sugars, salts, etc., and will add to the osmolality, so you might be able to reduce the sucrose a fraction if you're adding this supplement in order to offset the osmolality increase. So essentially if a compound added isn't used effectively as a nutrient, is present in excess or doesn't serve an essential function (like MES is needed as a buffer), then replacing it with something that is, would move your recipe towards a better balance between growth rate and replating frequency. Another example; P668 has a lot of chloride (3mM) ,and plants need only a tiny fraction of this, it's place can be taken by something that is more useful. If all the NO3 isn't being used efficiently then reducing it from 19.7 mM to 10 mM say, gives you another 9.7 mM to play with. We can discuss this more if you wish.
 
Myxodex thanks a lot for your comolete and detailed answer. You are opening my mind with very interesting topics... Just replated some flasks to MSU and KLite media this weekend, so will keep you updared. Will test also BM1.
Thanks a lot!
 

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