Apr 14, 2023

Public workspaceUseful methods 3: Media for in vitro-cultivation of duckweed

This protocol is a draft, published without a DOI.
  • Klaus-J. Appenroth1
  • 1University of Jena, Plant Physiology, Dornburger Str. 159, 07743 Jena, German
Chris Carlson
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Protocol Citation: Klaus-J. Appenroth 2023. Useful methods 3: Media for in vitro-cultivation of duckweed. protocols.io https://protocols.io/view/useful-methods-3-media-for-in-vitro-cultivation-of-b559q896
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 10, 2022
Last Modified: April 14, 2023
Protocol Integer ID: 59297
Keywords: duckweed, Steinberg-Medium, Murashige-Skoog-Medium, N-Medium, Schenk-Hildebrand medium, Hoagland medium, Bonner-Devirian medium
Abstract
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.
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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 (Concentration50 millimolar (mM) ) or sucrose (Concentration25 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.
Materials
Materials

  • KNO3
  • KH2PO4
  • K2HPO4x3H2O
  • MgSO4x7H2O
  • Ca(NO3)2x4H2O
  • H3BO3
  • ZnSO4x7H2O
  • Na2MoO4x2H2O
  • MnCl2 x 4H2O
  • FeNaEDTA
  • EDTA-Na2
  • NH4NO3
  • CaCl2x2H2O
  • Fe(III)EDTA
  • MnSO4xH2O
  • KI
  • CuSO4x5H2O
  • KNO3
  • CoCl2x6H20
  • Iron(III)tartrate
  • KCl
Modified Steinberg-Medium
Modified Steinberg-Medium

CITATION
R. A. Steinberg. Mineral requirements of Lemna minor. Plant Physiology.
LINK
https://doi.org/10.1104/pp.21.1.42

Note
Modified 1998 by R. Altenburger, Dept. of Chemical Ecotoxicology, UFZ Centre for Environmental
Research, Leipzig. Germany. Described in ISO standard (ISO 20079).

Note
This nutrient medium is recommended for investigating the effects of substances that might be chelated, e. g. heavy metals. Therefore, the concentration of chelators is so low that the optimal growth of duckweed is not possible. If you want to use this medium but want to have higher growth rates, the FeEDTA and EDTANa2 concentrations should be increased fivefold. EDTANa2 was added to keep the same ration of chelated and free EDTA as in the original protocol.

From stock solution 1 to 3, use Amount20 mL per litre ready-to-use media.
ABCDE
StockCompoundStock concentrationFinal concentration
1KNO3 (101.1)173 mM17.50 g/L3.46 mM
KH2PO4 (136.1)33 mM4.50 g/L0.66 mM
K2HPO4 x 3H2O (228.2)0.63 g/L3.6 mg/L72 μM
2MgSO4 x 7H2O (246.5)20.5 mM5.00 g/L0.41 mM
3Ca(NO3)2 x 4H2O (236.1)62.5 mg/L14.75 g/L1.25 mM
4H3BO3 (61.8)0.388 mM120 mg/L1.94 μM
ZnSO4 x 7H2O (287.5)0.126 mM180 mg/L0.63 μM
Na2MoO4 x 2H2O (241.9)36 μM44 mg/L0.18 μM
MnCl2 x 4H2O (197.9)0.182 mM180 mg/L0.91 μM
5FeNaEDTA (367.0)0.562 mM1.031 g/L2.81 μM
EDTA-Na2 (372.24)0.244 mM0.454 g/L1.22 μM

From stock solution 4 and 5, use only Amount5 mL per litre.

Adjust the pH to Ph5.5 , e.g. by using 1 % (v/v) HCl.
Murashige-Skoog-Medium (1/10 MS-medium)
Murashige-Skoog-Medium (1/10 MS-medium)
Reference:
CITATION
Toshio Murashige, Folke Skoog. A revised medium for rapid growth and bio assay with tobacco tissue cultures. Physiologia Plantarum.
LINK
https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

Note
This medium is especially popular for stock cultivations. Several of the components in stock solution 6 might not be required at all, e.g. KI. Landolt and Kandeler (1987) cited this version of the MS-medium according to Jacobs (1947) and mentioned that it is very rich of nitrate and potassium. They also criticized the complete lack of sodium and the low concentrations of trace elements.
ABCDE
StockCompoundStock concentrationFinal concentration
1KNO3 (101.11)752 mM76.0 g/L2.06 mM
NH4NO3 (80.04)824 mM65.95 g/L1.88 mM
2CaCl2 x 2H2O (147.02)120 mM17.6 g/L0.3 mM
3MgSO4 x 7H2O (246.48)60 mM14.79 g/L0.15 mM
4KH2PO4 (136.09)50 mM6.80 g/L125 μM
5Fe(III)EDTA ( 367.0)4 mM1.468 g/L10 μM
6H3BO3 (61.83)4 mM247 mg/L10 μM
MnSO4 x H2O (169.02)4 mM676 mg/L10 μM
ZnSO4 x 7H2O (287.5)1.2 mM345 mg/L3 μM
KI (166.0)0.2 mM33.2 mg/L0.5 μM
Na2MoO4 x 2H2O (241.95)0.04 mM9.7 mg/L0.1 μM
CuSO4 x 5H2O (249.68)0.004 mM1 mg/L0.01 μM
From each stock solution 2.5 ml per litre final medium.
N-Medium
N-Medium
Ref:
CITATION
K.-J. Appenroth, S. Teller, M. Horn (1996). Photophysiology of turion formation and germination in Spirodela polyrhiza.. Biologia Plantarum.
LINK
10.1007/BF02879642

Note
This medium was developed in our Institute and used for several decades for all duckweed species. The original low concentration of phosphate (Concentration60 micromolar (µM) ) was used to induce turions formation. As this is rather a special case, we increased the phosphate concentration to Concentration150 micromolar (µM) as given in the table. We never found nutrient media that supported a faster growth of duckweed than this one. When this nutrient medium should be used for stock cultivation, we increase the KH2PO4 concentration to even Concentration1 millimolar (mM) , add Concentration50 millimolar (mM) glucose and solidify by 0.9 % agar. Even after several decades of cultivation, the plants do not miss the trace elements that are not supplied. Most probably, even in chemicals of p.A.- quality, these elements are present as contaminations.

ABCDE
StockCompoundStock concentration Final concentration
1KH2PO4 (136.1)30 mM4.083 g/L0.15 mM
2Ca(NO3)2 . 4H2O (236.2)0.2 M47.23 g/L1 mM
3KNO3 (101.1)1.6 M161.8 g/L8 mM
H3BO3 (61.83)1 mM61.8 mg/L5 μM
MnCl2.4H2O (197.9)2.6 mM514.5 mg/L13 μM
Na2MoO4.2H2O (241.95)80 μM9.4 mg/L0.4 μM
MgSO4.7H2O (246.48)0.2 M49.30 g/L1 mM
4Fe(III)EDTA (345.07)5 mM1.725 g/L25 μM
or
FeNaEDTA (MW 367.1)5 mM1.835 g/L25 μM
Use Amount5 mL for Amount1 L from each stock solution nutrient medium. The nutrient medium might be
adjusted to Ph5.5 but this is normally omitted.

Schenk-Hildebrand medium (SH-medium)
Schenk-Hildebrand medium (SH-medium)
Reference:
CITATION
Schenk, R. U. and A. C. Hildebrandt (1972). Medium and Techniques for Induction and Growth of Monocotyledonous and Dicotyledonous Plant Cell Cultures.. Canadian Journal of Botany.
LINK
https://doi.org/10.1139/b72-026

Note
SH-Medium is not even described in Landolt and Kandeler (1987). However, following
recommendation by Dr. Ann Stomp, USA Elias Landolt introduced this medium in his lab and was very satisfied with it for stock cultivation. This medium is also commercially available. As in many other cases, several of the trace elements (stock solution 3) might have no function for duckweed cultivation.

ABCDE
StockCompoundStock concentrationFinal concentration
1CaCl2·2H2O (147.02)136 mM20 g/L0.68 mM
2KNO3 (101.1)2.48 M250 g/L12.4 mM
MgSO4·7H2O (246.48)162 mM40 g/L0.8 mM
(NH4)H2PO4 (115.03)260 mM30 g/L1.3 mM
3MnSO4·H2O (169.02)5.92 mM1 g/L30 μM
H3BO3 (61.83)8.1 mM0.5 g/L40 μM
ZnSO4·7H2O (287.6)0.35 mM0.1g/L1.74 μM
KI (166)0.6 mM0.1 g/L3 μM
CuSO4·5H2O (249.68)80.1 μM0.02 g0.4 μM
Na2MoO4·2H2O (241.95)41 μM0.01 g0.2 μM
CoCl2·6H20 (237.96)42 μM0.01 g0.21 μM
4FeNaEDTA (367.1)5.39 mM1.98 g/L0.26 mM
Na2EDTA (372.24)0.55 μM0.204 g/L2.75 μM

Use Amount5 mL from each stock solution for Amount1 L ready-to-use nutrient medium.

Hoagland medium
Hoagland medium

Note
Reference: We describe the nutrient medium as given by Landolt and Kandeler (1987), p. 60-62 as the original paper was published already 1920. This might be the reason why so many modifications of this medium exists, e.g. Venkataraman et al. (1970).
ABCDE
StockCompoundStock concentrationFinal concentration
1KNO3 (101.1)2 M202 g/L10 mM
2Ca(NO3)2 x 4H2O (236.15)0.8 M189 g/L4 mM
3KH2PO4 (136.1)0.2 M27.2 g/L1 mM
4MgSO4 x 7H2O (246.5)0.4 M98.6 g/L2 mM
ZnSO4 x 7H2O (287.5)0.3 mM86.2 mg/L1.5 μM
H3BO3 (61.8)10 mM618 mg/L50 μM
MnCl2 x 4H2O (197.91)2 mM396 mg/L10 μM
CuSO4 x 5H2O (249.68)80 μM20 mg/L0.4 μM
Na2MoO4 x 2H2O* (241.9)1 mM242 mg/L5 μM
5Iron(III)tartrate** (555.9)4 mM2.2 g/L20 μM
* Used instead of MoO3
** This substance is difficult to solve. Heat shortly and stir over night. Venkataraman et al. (1970) replaced this substance by Iron(III)citrate and often added Na2EDTA, 10 μM to the final medium.
Use Amount5 mL from each stock solution for Amount1 L of ready-to-use nutrient medium.

Bonner-Devirian medium
Bonner-Devirian medium

Note
Reference: Bonner, J., and P. S. Devirian: Growth factor requirements of four species of isolated roots. Amer. J. Bot. 26: 661-665 (1939)


Note
This nutrient medium has rather low concentrations. We have only good experiences with this
nutrient medium cultivating Lemna trisulca.
ABCDE
StockCompoundStock concentrationFinal concentration
1KNO3 (101.1)0.168 M17 g/L168 mM
2Ca(NO3)2 x 4H2O (236.15)0.2 M47.2 g/L1 mM
3KH2PO4 (136.1)29.4 mM4 g/L0.147 mM
KCl (74.55)161 mM12 g/L0.8 mM
4MgSO4 x 7H2O (246.5)0.4 M98.6 g/L2 mM
ZnSO4 x 7H2O (287.5)0.3 mM86.2 mg/L1.5 μM
H3BO3 (61.8)10 mM618 mg/L50 μM
MnCl2 x 4H2O (197.91)2 mM396 mg/L10 μM
CuSO4 x 5H2O (249.68)80 μM20 mg/L0.4 μM
Na2MoO4 x 2H2O (241.9)1 mM242 mg/L5 μM
5Iron(III)tartrate* (555.9)4 mM2.2 g/L20 μM
* This substance is difficult to solve. Heat shortly and stir over night. Venkataraman et al. (1970) replaced this substance by Iron (III) citrate and often added Na2EDTA, 100 μM to the final medium to study flower induction of Wolffia microscopica.
Use Amount5 mL from each stock solution for Amount1 L of ready-to-use nutrient medium.
Citations
Step 1
R. A. Steinberg. Mineral requirements of Lemna minor
https://doi.org/10.1104/pp.21.1.42
Step 4
Toshio Murashige, Folke Skoog. A revised medium for rapid growth and bio assay with tobacco tissue cultures
https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Step 5
K.-J. Appenroth, S. Teller, M. Horn. Photophysiology of turion formation and germination in Spirodela polyrhiza.
10.1007/BF02879642
Step 7
Schenk, R. U. and A. C. Hildebrandt. Medium and Techniques for Induction and Growth of Monocotyledonous and Dicotyledonous Plant Cell Cultures.
https://doi.org/10.1139/b72-026