Plant Tissue Culture Protocols

Growth Regulators

 
Preparation and Use
Table for Molar Equivalence and Solution Preparation

 


 

    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. W 3500). 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
Cytokinins
Miscellaneous Plant Growth Regulators
Auxins
Product
Name
Product
Number
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.
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Cytokinins
Product
Name
Product
Number
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 A 5665 135.1 7.40 1.0 HCl Water RT 2-8°C CA 50-250
Adenine hemisulfate Hemisulfate salt A 2545 184.2 5.43 Water RT 2-8°C CA 50-250
6-Benzylaminopurine (BA) B 3408 225.3 4.44 1N NaOH Water RT 2-8°C CA/F 0.1-5.0
6-Benzylaminopurine Hydrochloride B 5920 261.7 3.82 Water RT 2-8°C CA/F 0.1-5.0
6-Benzylaminopurine (BA) B 3274 225.3 4.44 1N NaOH Water RT 2-8°C CA/F 0.1-5.0
N-Benzyl-9-(2-tetrahydropyranyl)adenine (BPA) B 2275 309.4 3.23 EtOH -0°C -0°C CA/F 0.1-5.0
N-(2-Chloro-4-pyridyl)-N'-phenylurea (4-CPPU) C 2791 247.7 4.04 DMSO 2-8°C 2-8°C F 0.001-1.0
6-(gamma,gamma-Dimethylallylamino)purine (2iP) D 7674 203.2 4.92 1N NaOH Water -0°C -0°C CA/F 1.0-30.0
6-(gamma,gamma-Dimethylallylamino)purine (2iP) D 5912 203.2 4.92 1N NaOH Water -0°C -0°C CA/F 1.0-30.0
1,3-Diphenylurea (DPU) D 7535 212.3 4.71 DMSO RT 2-8°C F 0.1-1.0
Kinetin K 0753 215.2 4.65 1N NaOH Water -0°C -0°C CA/F 0.1-5.0
Kinetin K 3378 215.2 4.65 1N NaOH Water -0°C -0°C CA/F 0.1-5.0
Kinetin K 3253 215.2 4.65 1N NaOH Water -0°C -0°C CA/F 0.1-5.0
Kinetin Hydrochloride K 1885 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 P 6186 220.2 4.54 DMSO RT 2-8°C CA/F 0.001-0.05
trans-Zeatin Free base Z 0876 219.2 4.56 1N NaOH Water -0°C -0°C CA/F 0.01-5.0
Zeatin Z 0164 219.2 4.56 1N NaOH Water -0°C -0°C CA/F 0.01-5.0
trans-Zeatin Hydrochloride Z 2753 255.7 3.91 Water -0°C -0°C CA/F 0.01-5.0
trans-Zeatin riboside Z 3541 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.
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Miscellaneous Plant Growth Regulators
Product
Name
Product
Number
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) A 1049 264.3 3.78 1N NaOH Water -0°C -0°C CA/F 0.1-10.0
Ancymidol A 9431 256.3 3.90 DMSO 2-8°C -0°C CA/F 1.0-10.0
Chlorocholine chloride (CCC) C 4049 158.1 6.33 Water RT 2-8°C F up to 500
3,6-Dichloro-o-anisic acid (Dicamba) D 5417 221.0 4.52 EtOH/Water 2-8°C 2-8°C F 0.01-10.0
Gibberellic acid (GA3) G 7645 346.4 2.89 EtOH RT 2-8°C CA/F 0.01-5.0
Gibberellic acid Potassium salt (K-GA3) G 1025 384.5 2.60 Water 2-8°C -0°C CA/F 0.01-5.0
Gibberellin A4 Free acid (GA4) G 7276 332.4 3.01 EtOH -0°C -0°C F 0.01-5.0
(±)-Jasmonic acid J 2500 210.3 4.76 EtOH 2-8°C -0°C F 0.01-100.0
Phloroglucinol P 1178 126.1 7.93 Water RT 2-8°C CA/F up to 162
N-(Phosphonomethyl)glycine (Glyphosate) P 9556 169.1 5.91 1N NaOH Water RT 2-8°C F
Succinic acid 2,2-dimethylhydrazide S 2022 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.

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