TITLE: Useful methods 3: Media for in vitro-cultivation of duckweed
AUTHORS: Klaus-J. Appenroth

[DESCRIPTION]
This protocol details about various media for in vitro-cultivation of duckweed. It contains protocols from the The International Steering Committee on Duckweed Research and Application (ISCDRA) Newsletter. A complete list of these news letters can be found here. 

[GUIDELINES]
There is a large number of cultivation media in use for duckweed. This simply shows that most duckweed can adapt to a broad range of conditions. This does not mean that physiological responses are always the same. In order to support scientific standards for the duckweed community, we compiled the most useful nutrient media here in form of lab protocols, including the stock solutions used. All protocols are for autotrophic cultivation. If mixotrophic cultivations are required, either glucose (50 millimolar (mM)) or sucrose (25 millimolar (mM)) should be added. We have never added vitamins or other organics as green plants produce it themselves.

Final concentrations are given in molar concentrations – and should be given in scientific publications. Only then different nutrient media can be compared and the data do not depend e.g. on the amount of crystal water of the substances.

Most of the original protocols were published before FeEDTA or FeNaEDTA were available on a commercial basis. In most cases, these substance are much easier to handle than other sources of iron. Only when possible chelating effects may have an influence on the physiological effects under investigation (e.g. flowering) one should take care. All nutrient media should be autoclaved before use. The numbers in brackets are the molecular weight/ mass in g mol-1, which might help in calculations.

[STEPS]
SECTION: Modified Steinberg-Medium
1. 
 

 

 

From stock solution 1 to 3, use 20 mL per litre ready-to-use media. 
              Stock  Compound  Stock   concentration  Final concentration    1  KNO3 (101.1)  173mM  17.50 g/L  3.46 mM      KH2PO4 (136.1)  33mM  4.50 g/L  0.66 mM      K2HPO4x3H2O (228.2)  0.63 g/L  3.6mg/L  72 μM    2  MgSO4x7H2O (246.5)  20.5mM  5.00 g/L  0.41 mM    3  Ca(NO3)2x4H2O (236.1)  62.5mg/L  14.75 g/L  1.25 mM    4  H3BO3 (61.8)  0.388mM  120 mg/L  1.94 μM      ZnSO4x7H2O (287.5)  0.126mM  180 mg/L  0.63 μM      Na2MoO4x2H2O (241.9)  36 μM  44 mg/L  0.18 μM      MnCl2 x 4H2O (197.9)  0.182mM  180 mg/L  0.91 μM    5  FeNaEDTA (367.0)  0.562mM  1.031 g/L  2.81 μM      EDTA-Na2  (372.24)  0.244mM  0.454 g/L  1.22 μM  
 

SECTION: Murashige-Skoog-Medium (1/10 MS-medium)
4. Reference: 
 

 
 
              Stock  Compound  Stock   concentration  Final concentration    1  KNO3 (101.11)  752 mM  76.0 g/L  2.06 mM      NH4NO3 (80.04)  824mM  65.95 g/L  1.88 mM    2  CaCl2x2H2O (147.02)  120mM  17,6 g/L  0.3 mM    3  MgSO4x7H2O (246.48)  60mM  14.79 g/L  0.15 mM    4  KH2PO4 (136.09)  50mM  6.80g/L  125 μM    5  Fe(III)EDTA ( 367.0)  4 mM  1.468 g/L  10 μM    6  H3BO3 (61.83)  4 mM  247 mg/L  10 μM      MnSO4xH2O (169.02)  4 mM  676 mg/L  10 μM      ZnSO4x7H2O (287.5)  1.2 mM  345 mg/L  3 μM      KI (166.0)  0.2 mM  33.2 mg/L  0.5 μM      Na2MoO4x2H2O (241.95)  0.04 mM  9.7 mg/L  0.1 μM      CuSO4x5H2O (249.68)  0.004 mM  1 mg/L  0.01 μM  
 
SECTION: Modified Steinberg-Medium
2. From stock solution 4 and 5, use only 5 mL per litre.

SECTION: Modified Steinberg-Medium
3. Adjust the pH to pH 5.5, e.g. by using 1 % (v/v) HCl.
SECTION: N-Medium
5. Ref: 
 

 
 
              Stock  Compound  stock   concentration   final   concentration    1  KH2PO4 (136.1)  30 mM  4.083 g/L  0.15 mM    2  Ca(NO3)2 . 4H2O (236.2)  0.2 M  47.23 g/L  1 mM    3  KNO3 (101.1)  1.6 M  161.8 g/L  8 mM      H3BO3 (61.83)  1 mM  61.8 mg/L  5 μM      MnCl2.4H2O (197.9)  2.6 mM  514.5 mg/L  13 μM      Na2MoO4.2H2O (241.95)  80 μM  9.4 mg/L  0.4 μM      MgSO4.7H2O (246.48)  0.2 M  49.30 g/L  1 mM    4  Fe(III)EDTA (345.07)  5 mM  1.725 g/L  25 μM    or              FeNaEDTA (MW 367.1)  5 mM  1.835 g/L  25 μ  
 
SECTION: N-Medium
6. Use  5 mL for 1 L from each stock solution nutrient medium. The nutrient medium might be
adjusted to pH 5.5 but this is normally omitted.

SECTION: Schenk-Hildebrand medium (SH-medium)
7. Reference:  
 

 

 
              Stock  Compound  Stock   concentration  Final concentration    1  CaCl2·2H2O (147.02)  136mM  20 g/L  0.68 mM    2  KNO3 (101.1)  2.48 M  250 g/L  12.4 mM      MgSO4·7H2O (246.48)  162 mM  40 g/L  0.8 mM      (NH4)H2PO4 (115.03)  260 mM  30 g/L  1.3 mM    3  MnSO4·H2O (169.02)  5.92 mM  1 g/L  30 μM      H3BO3 (61.83)  8.1 mM  0.5 g/L  40 μM      ZnSO4·7H2O (287.6)  0.35 mM  0.1g/L  1.74 μM      KI (166)  0.6 mM  0.1 g/L  3 μM      CuSO4·5H2O (249.68)  80.1 μM  0.02 g  0.4 μM      Na2MoO4·2H2O (241.95)  41 μM  0.01 g  0.2 μM      CoCl2·6H20 (237.96)  42 μM  0.01 g  0.21 μM    4  FeNaEDTA (367.1)  5.39 mM  1.98 g/L  0.26 mM      Na2EDTA (372.24)  0.55 μM  0.204 g/L  2.75 μM  
 

SECTION: Schenk-Hildebrand medium (SH-medium)
8. Use 5 mL from each stock solution for 1 L ready-to-use nutrient medium.

SECTION: Hoagland medium
9. 
 
 
              Stock  Compound  Stock   concentration  Final concentration    1  KNO3 (101.1)  2 M  202 g/L  10 mM    2  Ca(NO3)2x4H2O (236.15)  0.8 M  189 g/L  4 mM    3  KH2PO4 (136.1)  0.2 M  27.2 g/L  1 mM    4  MgSO4x7H2O (246.5)  0.4 M  98.6 g/L  2 mM      ZnSO4x7H2O (287.5)  0.3 mM  86.2 mg/L  1.5 μM      H3BO3 (61.8)  10 mM  618 mg/L  50 μM      MnCl2x4H2O (197.91)  2 mM  396 mg/L  10 μM      CuSO4x5H2O (249.68)  80 μM  20 mg/L  0.4 μM      Na2MoO4x2H2O* (241.9)  1 mM  242 mg/L  5 μM    5  Iron(III)tartrate** (555.9)  4 mM  2.2 g/L  20 μM  
 
SECTION: Hoagland medium
10. Use 5 mL from each stock solution for 1 L of ready-to-use nutrient medium.

SECTION: Bonner-Devirian medium
11. 
 

 
 
              Stock  Compound  Stock concentration  Final   concentration    1  KNO3 (101.1)  0.168 M  17 g/L  168 mM    2  Ca(NO3)2x4H2O (236.15)  0.2 M  47.2 g/L  1 mM    3  KH2PO4 (136.1)  29.4 mM  4 g/L  0.147 mM      KCl (74.55)  161 mM  12 g/L  0.8 mM    4  MgSO4x7H2O (246.5)  0.4 M  98.6 g/L  2 mM      ZnSO4x7H2O (287.5)  0.3 mM  86.2 mg/L  1.5 μM      H3BO3 (61.8)  10 mM  618 mg/L  50 μM      MnCl2x4H2O (197.91)  2 mM  396 mg/L  10 μM      CuSO4x5H2O (249.68)  80 μM  20 mg/L  0.4 μM      Na2MoO4x2H2O (241.9)  1 mM  242 mg/L  5 μM    5  Iron(III)tartrate* (555.9)  4 mM  2.2 g/L  20 μM  
 
SECTION: Bonner-Devirian medium
12. Use 5 mL from each stock solution for 1 L of ready-to-use nutrient medium.