1) Organic acids can act as buffers in plant tissue culture media. The K or Na salts are used - e.g., succinate, malate, fumarate, and citrate. Of course, these can also act as chelates.

2) Nitrate and ammonium ions. The amount and ratio of these two ions can have a large effect on medium pH. NH4(+) uptake results in a lowering of pH (H+ ions are exchanged) and NO3(-) uptake results in an increase in pH (OH- are exchanged). I attached a paper on this effect (Plant Cell Tissue and Organ Culture).

3) Medium composition. This is more complex but can have an effect. Vacin and Went (Some pH changes in nutrient solutions. Botanical Gazette 110:605-613, 1949) looked at the effect of the various salts in their, now famous, formulation. The two salts having the largest effect on pH change (little buffering capacity) were FeSO4.7H2O and Ca(NO3)2.4H2O. They replaced these two salts with ferric tartrate and Ca3(PO4)2 and KNO3 to improve the buffering of the medium.

A side note comment - pH is a "tricky" variable when designing experiments because from an experimental design perspective it is inherently a dependent factor - i.e., pH can never be treated as an independent factor statistically. The reason for this is that pH can only be changed by adding ions to a solution. Adding ions to "control" pH then confounds the experiment - i.e., is the effect due to pH, due to the specific ions that were added, or some combination? The challenge then for biologists is to design experiments that can separate and capture these effects - and these will vary depending on the measured response. I tried to design such an experiment using NH4 and NO3 ions and attached the paper (BMC Plant Biology).

Here's the BMC Plant Biology paper.

Thanks for the brief explanation. But do you personally think that , if we managed to maintain the pH, we can actually avoid from the metals being reduced ? Because i would like to work on the effect of metals ion on plants, but it seems that when i use metals like Au, the agar turns to become purple in color, which is a good indicator saying that the ions are reduced to Au (0). And as the concentration increases, the pH decrease as well. So that's why i am thinking of maintaining the pH to avoid from the metals to be reduced. Does it make sense?

From a strict experimental design perspective I think of this type of question, if I understand correctly, as follows – Your broader objective seems to be to determine the effect Ag on some set of measures of “plant growth”. However, you think that the pH of MS medium affects Ag ion charge which then affects the color of the medium – which then, in turn, may affect plant growth. To illustrate how I think about it, here are a couple of experiments and the questions they answer –

1) One factor design that varies AgNO3 over a range of, say, 0 to 1 mM. I typically run the metals at log intervals as 0, 0.0001, 0.001, 0.01, 0.1, and 1. What questions would be answered by this approach?

- The level of toxicity using MS medium (most of the metals are toxic at 1 mM).

- The “optimal” level of that metal for each of the measured responses. Again, this would be in the context of MS medium.

What about the issue of the medium turns purple? AgNO3 turning MS medium purple is one of the effects of AgNO3. Because this is one of the effects in MS medium it is irrelevant – because the very purpose of the experiment is to determine the effects on plant growth. Thus, the color or the medium is just another dependent variable like measuring the number of leaves. Does a purple medium always result in poor growth? Maybe or maybe not. This experiment cannot answer this question.

2) An experiment that varies AgNO3 and the amount and ratio of NO3 and NH4. The design cold be constructed as a 3-factor design that varies AgNO3, NH4:K ratio, and NO3 (Note: this is the design structure in the BMC Plant Science paper I attached previously). What questions would be answered by this approach?

- The effect of the amount of NO3 on plant growth and media color change (purpling) from AgNO3.

- The effect of NH4:K on plant growth and media color change (purpling) from AgNO3.

- The effect of AgNO3 on plant growth and purpling.

- The effect of all the interactions between AgNO3, NO3, and NH4:K on plant growth and purpling.

- The “optimal” level of all these factors on plant growth and purpling. Again, this would be in the context of changing NO3, NH4, K, and AgNO3 while keeping all other components of MS medium constant.

3) An experiment that varies AgNO3 and MES. I realize that you mentioned that you do not want to use MES, but I am including it just for context. This would be a 2-factor design that varies both AgNO3 (range as described above) and MES (maybe 0 to 200 mM) over some range. What questions would be answered with this approach?

- The effect of AgNO3 on plant growth and purpling.

- The effect of MES on plant growth and purpling.

- The interaction effect of AgNO3 and MES on plant growth and purpling.

- Optimal levels of AgNO3 and MES for best plant growth.

- Correlation between purpling and plant growth.

Another option would be to fix the amount of AgNO3 but include all the organic acids. I would probably use a screening design - that way all the buffers could be efficiently tested all at once - i.e., more efficient than testing each singly.

As always, my 2 cents - :).