How to make, store and trace plant growth regulator stock solutions

Why it matters

Small additions that decide what a medium does

Growth regulators are the smallest additions to a batch of medium and often the ones that decide what it does. PhytoTechnology Laboratories gives general working ranges of 0.01 to 10.0 mg/L for auxins and 0.1 to 10.0 mg/L for cytokinins. At those amounts the dose comes out of a stock bottle made days or weeks earlier, often by someone else.

That bottle carries a lot of trust. A 2016 study of cytokinin stability by Hart and colleagues, published in In Vitro Cellular and Developmental Biology, points out that cytokinins are used at very low concentrations, that those concentrations can be affected by how the concentrated stock was prepared and stored, and that stock solutions are made with protocols that vary from laboratory to laboratory.

So a stock solution deserves the same treatment as any other reagent in the media room: a written method, a label that says exactly what is in it, and a line on the batch record that says which bottle went in.

Preparation

Dissolve first, then dilute

Most growth regulators are not simply stirred into water. The usual method is to dissolve the powder in a small volume of solvent, then bring it to volume with water. PhytoTechnology's method for a 1 mg/mL stock is to put 100 mg of the regulator in a 100 mL volumetric flask, add 3 to 5 mL of solvent, and once it has completely dissolved, bring it to volume with distilled or deionized water, stirring while the water goes in to keep the material in solution.

The solvent depends on the compound. PhytoTechnology's handling table lists KOH for IAA, IBA, NAA, BA and kinetin, ethanol for GA3, DMSO for thidiazuron and picloram, and 1 N HCl for adenine, while the potassium salt forms of IBA and NAA dissolve in water. The table's footnote recommends 1 N KOH. Other protocols choose differently: a banana media SOP from the International Institute of Tropical Agriculture (IITA) dissolves BAP in 1 to 5 mL of 1 M NaOH before topping up to 100 mL, and NAA in a few drops of absolute ethanol.

The choice of alkali is not neutral. A 2022 paper in Frontiers in Plant Science by Doungous, Al-Khayri and Kouassi notes that NaOH, used both for pH adjustment and for regulator stocks, adds sodium to media that are mostly formulated without it, and argues that KOH may be the better choice. That is an argument rather than a settled result, but it is reason enough to record which alkali a stock was made with.

Strength matters as well. Hart and colleagues found BA, kinetin, 2iP and meta-topolin at 1.0 mg/mL in 0.05 N KOH showed no significant change over 90 days of storage at any temperature they tested. Trans-zeatin at 50 mg/mL in 0.5 N KOH was a different story: the authors estimate a loss of 20 to 50 percent over 90 days, and they note that protocols calling for 1.0 N KOH are not uncommon. Their advice when moving a protocol from one lab to another is to consider the exact dissolution method along with storage time and temperature.

Units

Milligrams per liter or micromolar

Protocols give regulator concentrations in two ways. Milligrams per liter is a weight. Micromolar counts molecules. Because molecular weights differ, the same mg/L of two regulators is not the same number of molecules.

The conversion is simple: divide the mg/L figure by the molecular weight and multiply by 1,000 to get micromolar. PhytoTechnology's table works it through: 1.0 mg/L of BA, molecular weight 225.3, is 4.4 µM. The same 1.0 mg/L of IAA, at 175.2, is 5.7 µM, and of GA3, at 346.4, is 2.9 µM.

Salt forms are where this goes wrong quietly. The same table gives IBA a molecular weight of 203.2 and its potassium salt 241.3, so 1.0 mg/L works out to 4.9 µM of one and 4.1 µM of the other. A recipe written for the free acid and made up with the salt at the same milligrams is a lower dose.

In PhytoTechnology's example, 1 mL of a 1 mg/mL stock in a liter of medium gives 1.0 mg/L. Write the stock's concentration on the label in the same units the recipe uses, so nobody converts at the bench.

Storage

Cold, dated and checked before use

Storage advice differs by compound and by form. In PhytoTechnology's table, stock solutions of BA, NAA, GA3 and thidiazuron go in the refrigerator at 2 to 6 °C, while IAA, IBA, kinetin and zeatin stocks go in the freezer at -20 to 0 °C. The IITA SOP keeps its BAP stock in the refrigerator if it will be used soon, or splits it into tubes for the freezer if it is not used regularly.

There is no single shelf life to borrow. The 90-day results above apply to particular compounds, concentrations and solvents, not to every stock in your refrigerator. Set a use-by period per compound in the SOP, based on the supplier's guidance and your protocol, and put the date on the label.

Look before you pipette. Hart and colleagues saw BA and kinetin crystallize from solution now and then, for example when an autoclaved solution went into 2 to 6 °C storage before it had cooled to 25 °C. Material sitting at the bottom of the bottle is not in the milliliter you draw off, so a stock with crystals or cloudiness is not delivering the concentration on its label. That study kept its samples in amber glass, and the IITA SOP keeps its iron stock in an amber or foil-wrapped bottle. Dark containers are a cheap precaution for regulator stocks too.

The IITA SOP asks staff to label every stock with the date and the stock name. A fuller label costs little more: the compound and its form, the concentration with units, the solvent and its strength, the date made, the initials of the person who made it, the use-by date, the supplier's lot number for the powder, and a lot code for the stock itself.

Sterilizing

Autoclave it or filter it

PhytoTechnology's table sorts regulators into three groups. Some, such as BA and NAA, can be autoclaved along with the rest of the medium. Others, including GA3, IAA, IBA, kinetin, zeatin and thidiazuron, can be co-autoclaved, but the table warns that some loss of activity may occur. A third group, which includes zeatin riboside and meta-topolin, is marked for filter sterilization, a label the table uses for compounds that are heat labile or have no heat stability information available.

GA3 is the usual example of a heat-sensitive regulator. A 2008 paper in the Journal of Applied Biosciences by Msogoya and colleagues at Sokoine University of Agriculture lists GA3 among the components that become inactive when autoclaved, and describes filtering such components through a 0.22 µm membrane and adding them to the sterilized medium after it has cooled. The sources differ in how much loss they expect, so the protocol should state which route your lab uses for each regulator, and the batch record should show which route was actually taken.

Hart and colleagues found that the cytokinins they tested came through an autoclave cycle at 121 °C for 30 minutes in 0.05 N KOH with no significant degradation, and they note that most of the literature on regulator heat stability is anecdotal. Where a compound has no data at all, treating it as filter-only is the conservative reading of PhytoTechnology's table.

Verify this

Before a stock goes into a batch

Check that the label names the compound, its form and its concentration in the recipe's units, and that the use-by date has not passed.

Look for crystals, cloudiness or growth, and confirm whether the protocol adds it before or after autoclaving. Write the stock's lot code on the batch record as you add it.

Records

Tracing a stock to a batch

When a batch of medium behaves differently, with fewer shoots, slower rooting or more callus than usual, the regulator stock is one of the first things to rule in or out. That only works if you know which bottle went in.

Give every stock a lot code when it is made and record that code on the media batch record next to the volume added. The IITA SOP already has staff tick each stock off a checklist as it goes into the medium. Writing the stock code beside the tick is a small step that turns the checklist into a trace.

With that in place, a problem can be followed both ways: from a batch back to the stock and the powder lot it was made from, and from a suspect stock forward to every batch and every vessel that used it. Keeping those links per batch and per lot is what lab software like xPlant Pro is for, though a bound notebook does the job if every line is filled in.