Adapted from an excellent review by K. Sowjanya Sree and Klaus-J. Appenroth (see the 12th issue of Duckweed Forum)
Several liquid media have been developed for the optimal cultivation of duckweeds at high growth rates (see page on growth medium). For long-term cultivation of stocks such high growth rates are not desired because aliquots of the cultures would have to be transferred into fresh nutrient medium between every two weeks to perhaps two months, depending on clone and species. Cultivation of stocks thus means, at first, to reduce the rate of growth.
The first measure is to reduce the temperature. The optimal growth rates are measured under standardized temperature, at 25°C. For stock cultivation we reduce the temperature to 18°C. All the duckweed species can deal with this temperature, of course with a dramatically reduced growth rate. Even 15°C is possible. The late Elias Landolt mentioned that clones collected from tropical climates cannot tolerate 5°C for a longer time.
The next important environmental factor is the light intensity. The light intensity is reduced from 100 µmol m-2 s-1 continuous white light (standardized for optimal growth rate) to ca. 30 µmol m-2 s-1. We observed that duckweeds are able to deal with low light intensity only when the temperature is also reduced. At 25°C several species (e.g. from the genus Wolffiella) die at such low light intensities. This light intensity seems to be close to the photon compensation point, i.e., the light intensity where the rate of photosynthesis exactly matches the rate of cellular respiration.
It is also very useful to reduce the water availability. This can be done by replacing the liquid medium by an Agar-based medium. Instead of Agar also a kind of synthetic Agar, GELRITE, can be used. These semisolid media have to be prepared with a defined nutrient medium to supply the macro- and micro-nutrients to the plants. We normally use N-medium. The concentration of KH2PO4 is increased to 1 mM. At the low concentration used in liquid medium (e.g. 60 µM), the survival of plants on Agar would be for a very short duration. However, also other media are possible. Often MS-medium is used because this medium is commercially available as a ready-to-use mixture. Solid Agar is added to the nutrient medium, normally at a concentration of 0.9 %. For sensitive clones, addition of as low as 0.7 % Agar is possible. Gelrite is used at a concentration of ca. 0.45 %. Normally one litre Agar suspension is prepared, heated in a microwave oven to 80°C, and 50 – 75 ml of it is poured into each of the 100 ml-Erlenmeyer flasks secured with cotton wool stoppers. The flasks are then autoclaved. It is indeed a very large amount of Agar in each flask having the advantage that the life time of stock cultures is very long, ca. 4 to 5 or even 6 months. In other stock collections standard glass test tubes are used, filled with 5 ml Agar medium and also closed with cotton wool stoppers. Alternatively, sterile plastic Petri dishes could be used. The diameter of the Petri dishes might be 9 cm (standard quality, very cheap) or much smaller, down to a diameter of 3 cm in order to spare medium and space. In these cases, the Agar medium is first autoclaved, normally in 1 L flasks, and then poured into sterile Petri dishes in a laminar flow box. After inoculating the plants, the Petri dishes are closed by parafilm to prevent faster rate of drying of the Agar layer.
In order to control the sterility (in terms of pure, uncontaminated cultures) of plants it is useful to add low molecular weight carbohydrates. Glucose (50 mM) or sucrose (25 mM) are commonly used. We learned recently, that in some cases (e.g. Wolffia) after sterilization, 50 mM glucose is too high for their regeneration and plants often die. In this case, but perhaps also in general, 25 mM glucose or 25 mM sucrose are better used in Agar or gelrite media. Fructose is not recommended.
Species of the genus Spirodela (S. polyrhiza, S. intermedia) tend to grow in several layers, one above the other. As a consequence, the younger fronds in the upper most layers lose their contact to water supplying Agar and die thereafter. Therefore, for these two species, we use liquid medium without sugar. The disadvantage is that the contaminations cannot be recognized and in each case a sterility test is necessary before using these plants. When sugar is added, the survival of the fronds is too short.
For S. polyrhiza there exists another opportunity for their long-term preservation. This species forms turions, survival organs that sink to the bottom of the flasks and can easily be harvested. Turions can be stored in a fridge (5°C, darkness) for several years. Turion formation can be easily initiated by inoculating plants in a liquid medium with low phosphate concentrations, e.g. 60 µM. The inoculated plants and their emerging daughters use the available phosphate for vegetative growth and after a certain time period, the drastically reduced phosphate concentration (e.g. to 2 µM) induces turion formation. Addition of glucose (50 mM) is recommended to accelerate this process and to increase the turion yield.
Adapted from an excellent review by K. Sowjanya Sree and Klaus-J. Appenroth (see the 12th issue of Duckweed Forum).