Growth Regulators – Plant Tissue Culture Protocol

• Preparation and Use
  
• Table for Molar Equivalence and Solution Preparation

The importance of plant growth regulators in plant tissue culture is well documented. We offer 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
  
• Cytokinins
  
• Miscellaneous Plant Growth Regulators

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.