medium no. 57: Amino Acid medium, 0.02 µM 2,4-dichlorophenoxyacetic acid, 2 µM N-(2-chloro-4-pyridyl)-N′-phenylurea, pH 6.2

Plant cell lines

• rpc00087: Bruguiera sexangula BsLs cell suspension culture

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

• Sucrose

• CaCl₂ (0.6 M)

  Chemical	Concentration (mg/mL)
  CaCl2·2H2O	88

• AA_macro

  Chemical	Concentration (mg/mL)
  KCl	149
  MgSO4·7H2O	37
  KH2PO4	17

• MS_micro_1

  Chemical	Concentration (mg/mL)
  H3BO3	0.62
  MnSO4·5H2O	2.41
  ZnSO4·7H2O	0.86
  KI	0.083
  Na2MoO4·2H2O	0.025
  CuSO4·5H2O	0.0025
  CoCl2·6H2O	0.0025

• Fe(III)-EDTA (20 mM)

  Chemical	Concentration (mg/mL)
  Fe(III)-EDTA	8.422

  Ethylenediamine-N,N,N′,N′-tetraacetic acid, iron (III), sodium salt, trihydrate, FUJIFILM Wako Pure Chemical Corporation (#343-01241)

• AA_5

  Chemical	Concentration (mg/mL)
  L-Glutamine	8.77
  L-Aspartic acid	2.66
  L-Arginine	2.28
  Glycine	0.75

  Store at −20°C

• AA_VT

  Chemical	Concentration (mg/mL)
  Nicotinic acid	0.05
  Pyridoxine·HCl	0.05
  Thiamine·HCl	0.01
  Glycine	0.16

  Store at −20°C

• 2,4-D (1 mM)

  Chemical	Concentration (mg/mL)
  2,4-D sodium monohydrate	0.261

  (2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich (D6679)

• 4-CPPU (2 mM)

  Chemical	Concentration (mg/mL)
  4-CPPU	0.495

  N-(2-Chloro-4-pyridyl)-N′-phenylurea, Sigma-Aldrich (C2791); Dissolve 4-CPPU in ethanol and store at −20°C

• NaOH (1 N)

Medium preparation

1. Dissolve the following chemicals in approximately 800 mL of distilled water.

   Chemical	Amount
   Sucrose	30 g

2. Add following stock solutions, and fill up to approximately 950 mL with distilled water.

   Stock solution	Volume (mL)
   CaCl2 (0.6 M)	5
   AA_macro	10
   MS_micro_1	10
   Fe(III)-EDTA (20 mM)	5
   AA_5	100
   AA_VT	10
   2,4-D (1 mM)	0.02
   4-CPPU (2 mM)	1

3. Adjust the pH of the solution to 6.2 with NaOH (1 N), and fill up to 1 L with distilled water.

4. Autoclave the medium at 121°C for 20 min.

Formulation

Chemical	Concentration (mg/L)
KCl	1490
L-Glutamine	877
L-Aspartic acid	266
L-Arginine	228
CaCl2·2H2O	440
MgSO4·7H2O	370
KH2PO4	170
H3BO3	6.2
MnSO4·5H2O	24.1
ZnSO4·7H2O	8.6
KI	0.83
Na2MoO4·2H2O	0.25
CuSO4·5H2O	0.025
CoCl2·6H2O	0.025
Fe(III)-EDTA	42.11
Nicotinic acid	0.5
Pyridoxine·HCl	0.5
Thiamine·HCl	0.1
Glycine	76.6
Sucrose	30000
2,4-D sodium monohydrate	0.00522
4-CPPU	0.495