Apr 14, 2023
Useful Methods 2: Sterilization of Duckweed
- Klaus-J. Appenroth1
- 1University of Jena, Plant Physiology, Dornburger Str. 159, 07743 Jena, Germany
Protocol Citation: Klaus-J. Appenroth 2023. Useful Methods 2: Sterilization of Duckweed. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lynwzrvx9/v1
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 11, 2022
Last Modified: April 14, 2023
Protocol Integer ID: 59347
Keywords: Sterilization of duckweed, Landoltia, Lemna, Wolffia, Wolffiella, Spirodela
Abstract
This protocol details about sterilization 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.
Attachments
377-833.pdf
51KB
Guidelines
Sterilization of duckweed plants is unavoidable in the lab because many physiological or toxicological properties were influence in an unknown way by microorganisms, be it bacteria or fungi. Thus, the first step isolating clones from natural populations is surface sterilization. Moreover, it happens again and again that an already sterile clone gets infected during handling, even in a safety hood. Wherever axenic clones were kept under in vitro cultivation conditions, sterilization belongs to the routine techniques.
Different species (genera of species) have different sensitivity toward bleaching agents usually used for sterilization. Most sensitive are Wolffiella species followed by Wolffia species. Lemna is much more resistant and Landoltia and Spirodela are the most resistant.
I learned this method from the late Elias Landolt, ETH Zurich during one of my visits in his lab. Before that we kept each clone in 5 copies excluding this way that the whole clone with all copies will be infected. Now we keep usually only two copies from each clone.
Materials
Materials
Eau de Javel
NaOCl
glucose
sucrose
70 % ethanol
Erlenmeyer flasks
Agar
Danklorix (CP GABA, Hamburg, Germany)
Petri dishes
For surface disinfection
For surface disinfection
Put the plants in a plastic tube (Falcon), depending on the size of the plants having a volume of 15 mL or 50 mL .
Dilute the commercial available “Eau de Javel” in water for different concentrations and treat the fronds for different periods by gentle shaking:
Wolffia, Wolffiella: 1 – 3%, 2, 3, 4, and 5 min.
Lemna: 3 – 5%, 3, 4, 5, and 6 min.
Landoltia, Spirodela: 5 – 10%, 3, 5, 7, and 9 min.
Note
“Eau de Javel” is available at least in Switzerland and Germany (producer Floreal Haagen, Wadgassen, Germany). It contains 2.4% NaOCl.
Sterilize Landoltia fronds in a 0.24% solution of NaOCl using “Eau de Javel".
Transfer the plants into Erlenmeyer flasks with nutrient medium containing sugar (50 millimolar (mM) glucose or 25 millimolar (mM) sucrose) (e.g. 180 mL per flask) without further washing in sterile water.
After a few days, better after 14 days, the medium remains clear when all bacteria and fungi are killed.
Wash the plants in 70% ethanol before bathing them in “Eau de Javel”. However this does not improve the rate of success.
Treat the 4 Erlenmeyer flasks with fronds at different times in the bleaching bath.
In the cultivation room at 25 °C follow the development of the plants in the Erlenmeyer flasks.
Transfer the sterilized plants on Agar in Petri dishes.
Note
The Agar-layer also has to contain sugar to control the success of the sterilization. In this case, some colonies might be unsterile without infecting sterile colonies in the same Petri dish. If this method is used, washing the fronds in sterile water after sterilization is indispensable in order to remove traces of the beaching solution.
We also use other commercially available preparations like “Danklorix” (CP GABA, Hamburg, Germany), available at least in Austria and Germany. This product contains 2.8% NaOCl and can be used in the same way as “Eau de Javel”.
Limited experience exists with commercial available sodium hypochlorit (NaOCl) solution. However, the concentration is much higher (typically 12%) and dilute it accordingly.
Moreover, as it is free of surfactants, it is useful to add a small amount of a mild detergent like 0.5% of Triton X-100 to get the surface of the whole plant body wetted.
There are other beaching agents like “Lizol” (Reckitt Benckisier, Parwanno, Himachel Pradesh, India) having e.g. 0.6% NaOCl. However in this case, a very high concentration of surfactants is included. We were not able to wash the surfactants properly away from the surface of the plants and the recovering rates were very low.