Growth Regulators – Plant Tissue Culture Protocol

The importance of plant growth regulators in plant tissue culture is well documented. Sigma offers a broad range of plant growth regulators specifically tested for plant cell culture. Each product is assayed for physical and chemical characteristics, then is biologically tested following the criteria established for powdered media. Each auxin is tested for enhancement of callus growth and/or root initiation in vitro. Each cytokinin is tested for stimulation of shoot production.

The plant growth regulator solutions are sterile filtered through a double 0.2 µm filter unit into sterile bottles. Each lot of plant growth regulator solution is tested for sterility according to specifications established by U.S. Pharmacopeia Vol. XXI, as well as biological testing in plant cell culture using criteria identical to those established for our plant tissue culture media.

FOR LABORATORY USE, PLANT TISSUE CULTURE MEDIA PREPARATION AND PLANT RESEARCH PURPOSES ONLY. NOT FOR USE AS A PLANT GROWTH REGULATOR ON DEVELOPED PLANTS. NOT FOR DRUG OR HOUSEHOLD USES.

Preparation and Use

To prepare a 1 mg/ml stock solution: Add 100 mg of the plant growth regulator to a 100 ml volumetric flask or other glass container. Add 2-5 ml of solvent to dissolve the powder. Once completely dissolved, bring to volume with double processed water (Product No. W3500). Stirring the solution while adding water may be required to keep the material in solution. Store the stock solution as recommended in the tables. Add 1.0 ml of the stock solution to 1 liter of medium to obtain a final concentration of 1.0 mg/L of the plant growth regulator in the culture medium. (See Table below.)

Volume of Stock Solution = (Desired Hormone Conc. X Medium Volume) / Stock Solution Conc.

Plant Cell Culture Tested Auxins are generally used in plant cell culture at a concentration range of 0.01-10.0 mg/L. When added in appropriate concentrations, they may regulate cell elongation, tissue swelling, cell division, formation of adventitious roots, inhibition of adventitious and axillary shoot formation, callus initiation and growth, and induction of embryogenesis.

Plant Cell Culture Tested Cytokinins are generally used in plant cell culture at a concentration range of 0.1-10.0 mg/L. When added in appropriate concentrations, they may regulate cell division, stimulate axillary and adventitious shoot proliferation, regulate differentiation, inhibit root formation, activate RNA synthesis and stimulate protein and enzyme activity.

Table for Molar Equivalence and Solution Preparation

Auxins

Product Name Product No. Molar Equivalence Solution Preparation
Mol. Wt. µM for 1mg/L Solvent Diluent Powder Storage Liquid Storage Steriliz-
ation*
Working Conc. (mg/L)
p-Chlorophenoxyacetic acid (4-CPA) C0413 186.6 5.36 EtOH RT 2-8°C CA 0.1-10.0
2,4-Dichlorophenoxyacetic acid D7299 221 4.53 RT 2-8°C CA 0.01-6.0
2,4-Dichlorophenoxyacetic acid Sodium salt D6679 243 4.12 Water RT 2-8°C CA 0.01-6.0
Indole-3-acetic acid Free acid (IAA) I2886 175.2 5.71 EtOH/1N NaOH Water -0°C -0°C CA/F 0.01-3.0
Indole-3-acetic acid Sodium salt I5148 197.2 5.07 Water Water 2-8°C -0°C CA/F 0.01-3.0
Indole-3-acetic acid methyl ester I9770 189.2 5.29 2-8°C 2-8°C
Indole-3-acetyl-L-aspartic acid I9387 290.3 3.45 0.5N NaOH Water -0°C -0°C F 0.01-5.0
Indole-3-butyric acid (IBA) I5386 203.2 4.90 EtOH/1N NaOH Water 2-8°C -0°C CA/F 0.1-10.0
Indole-3-butyric acid Potassium salt (K-IBA) I7512 241.3 4.14 Water 2-8°C -0°C CA/F 0.1-10.0
alpha-Naphthaleneacetic acid Free acid (NAA) N0640 186.2 5.37 1N NaOH Water RT 2-8°C CA 0.1-10.0
beta-Naphthoxyacetic acid Free acid (NOA) N3019 202.2 4.95 1N NaOH Water RT 2-8°C CA 0.1-10.0
Phenylacetic acid (PAA) P6061 136.2 7.34 EtOH RT 2-8°C CA/F 0.1-50.0
Picloram P5575 241.5 4.14 DMSO RT 2-8°C CA 0.01-10.0
2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) T5785 255.5 3.91 EtOH RT 2-8°C CA 0.01-5.0
2,3,5-Triiodobenzoic acid Free acid (TIBA) T5910 499.8 2.00 1N NaOH Water -0°C -0°C F 0.05-5.0
*CA = coautoclavable with other media components. F = filter sterlize. CA/F = coautoclavable with other media components, however, some loss of activity may occur. This can be compensated for by increasing component concentration. Component may be filter sterilized.

 

Cytokinins

Product Name Product No. Molar Equivalence Solution Preparation
Mol. Wt. µM for 1mg/L Solvent Diluent Powder Storage Liquid Storage Steriliz-
ation*
Working Conc. (mg/L)
Adenine Free base A5665 135.1 7.40 1.0 HCl Water RT 2-8°C CA 50-250
Adenine hemisulfate Hemisulfate salt A2545 184.2 5.43 Water RT 2-8°C CA 50-250
6-Benzylaminopurine (BA) B3408 225.3 4.44 1N NaOH Water RT 2-8°C CA/F 0.1-5.0
6-Benzylaminopurine Hydrochloride B5920 261.7 3.82 Water RT 2-8°C CA/F 0.1-5.0
6-Benzylaminopurine (BA) B3274 225.3 4.44 1N NaOH Water RT 2-8°C CA/F 0.1-5.0
N-Benzyl-9-(2-tetrahydropyranyl)adenine (BPA) B2275 309.4 3.23 EtOH -0°C -0°C CA/F 0.1-5.0
N-(2-Chloro-4-pyridyl)-N'-phenylurea (4-CPPU) C2791 247.7 4.04 DMSO 2-8°C 2-8°C F 0.001-1.0
6-(gamma,gamma-Dimethylallylamino)purine (2iP) D7674 203.2 4.92 1N NaOH Water -0°C -0°C CA/F 1.0-30.0
6-(gamma,gamma-Dimethylallylamino)purine (2iP) D5912 203.2 4.92 1N NaOH Water -0°C -0°C CA/F 1.0-30.0
1,3-Diphenylurea (DPU) D7535 212.3 4.71 DMSO RT 2-8°C F 0.1-1.0
Kinetin K0753 215.2 4.65 1N NaOH Water -0°C -0°C CA/F 0.1-5.0
Kinetin K3378 215.2 4.65 1N NaOH Water -0°C -0°C CA/F 0.1-5.0
Kinetin K3253 215.2 4.65 1N NaOH Water -0°C -0°C CA/F 0.1-5.0
Kinetin Hydrochloride K1885 251.7 3.97 Water -0°C -0°C CA/F 0.1-5.0
1-Phenyl-3-(1,2,3-thiadiazol-5-yl)urea P6186 220.2 4.54 DMSO RT 2-8°C CA/F 0.001-0.05
trans-Zeatin Free base Z0876 219.2 4.56 1N NaOH Water -0°C -0°C CA/F 0.01-5.0
Zeatin Z0164 219.2 4.56 1N NaOH Water -0°C -0°C CA/F 0.01-5.0
trans-Zeatin Hydrochloride Z2753 255.7 3.91 Water -0°C -0°C CA/F 0.01-5.0
trans-Zeatin riboside Z3541 351.4 2.85 1N NaOH Water -0°C -0°C F 0.01-5.0
*CA = coautoclavable with other media components. F = filter sterlize. CA/F = coautoclavable with other media components, however, some loss of activity may occur. This can be compensated for by increasing component concentration. Component may be filter sterilized.

 

Miscellaneous Plant Growth Regulators

Product Name Product No. Molar Equivalence Solution Preparation
Mol. Wt. µM for 1mg/L Solvent Diluent Powder Storage Liquid Storage Steriliz-
ation*
Working Conc. (mg/L)
(±)-cis,trans-Abscisic acid (ABA) A1049 264.3 3.78 1N NaOH Water -0°C -0°C CA/F 0.1-10.0
Ancymidol A9431 256.3 3.90 DMSO 2-8°C -0°C CA/F 1.0-10.0
Chlorocholine chloride (CCC) C4049 158.1 6.33 Water RT 2-8°C F up to 500
3,6-Dichloro-o-anisic acid (Dicamba) D5417 221.0 4.52 EtOH/Water 2-8°C 2-8°C F 0.01-10.0
Gibberellic acid (GA3) G7645 346.4 2.89 EtOH RT 2-8°C CA/F 0.01-5.0
Gibberellic acid Potassium salt (K-GA3) G1025 384.5 2.60 Water 2-8°C -0°C CA/F 0.01-5.0
Gibberellin A4 Free acid (GA4) G7276 332.4 3.01 EtOH -0°C -0°C F 0.01-5.0
(±)-Jasmonic acid J2500 210.3 4.76 EtOH 2-8°C -0°C F 0.01-100.0
Phloroglucinol P1178 126.1 7.93 Water RT 2-8°C CA/F up to 162
N-(Phosphonomethyl)glycine (Glyphosate) P9556 169.1 5.91 1N NaOH Water RT 2-8°C F
Succinic acid 2,2-dimethylhydrazide S2022 160.2 6.24 Water 2-8°C 2-8°C CA/F 0.1-10.0
*CA = coautoclavable with other media components. F = filter sterlize. CA/F = coautoclavable with other media components, however, some loss of activity may occur. This can be compensated for by increasing component concentration. Component may be filter sterilized.