conditions can germinate and form active floating fronds. The turions from each of the S.
polyrhiza clones can be easily harvested and
stored in darkness for several years. However,
we do not know a single laboratory where this is
realized.
All plants in stock collections have to be kept
under axenic (“sterile”) conditions because contamination by bacteria or fungi may inhibit
favourable growth conditions of the plants leading to death of the plants after some time.
Especially dangerous are the contaminations by
green algae as they compete with the duckweeds
for resources. These contaminations can be
removed by surface sterilization of the plants
with a commercially available household bleach
like Eau de Javel or DanKlorix (Sree and
Appenroth 2017). The availability of axenic
cultures allows studies on duckweeds alone
without having any interference from other
organisms (Appenroth et al. 2016). All media
need to be sterilized (autoclaved), and inoculation should be carried out in laminar flow boxes
to avoid any contaminations during the process.
In order to examine and ensure axenic conditions, usually glucose (25 mM or less) is added
to the solidified media. Any microbial contamination can then be easily recognized. However,
this has two disadvantages. 1. If there is a contamination, plants are usually quickly killed in
the presence of sugars as the microbial population explodes. 2. Sugars accelerate senescence
and shorten the time between two subculturing
events. To avoid these disadvantages, the duckweed clones can be kept in both the presence and
absence of glucose, thereby taking advantage of
both cultivation methods.
Cultivation vessels for stock cultivation are
very different in different collections as this
depends on the available space and manpower.
They range from 100-ml Erlenmeyer flasks with
cotton wool stoppers (Fig. 3.3) to Petri dishes of
different diameter (from 35 to 90 mm) closed
with parafilm, to standard glass test tubes
13 Â 100 mm with cotton wool stoppers.
3.5 Species Identification
of the Clones
For a duckweed clone collected from nature to be
successfully integrated into a stock collection,
accurate identification of the species is a prerequisite. Species identification using morphological markers would be the first choice
(Landolt 1980). Based on morphology, it is easy
to identify the duckweed genus to which the
clone belongs. In some cases, it is also easy to
identify the species, e.g. Lemna trisulca or S.
polyrhiza. However, in many cases even experts
of duckweed morphology have the biggest
problems to assign species identity based on
morphological markers. This is partially caused
by the miniaturized phenotype of the duckweeds.
In some cases, the ease of identity might also
depend on the growth conditions. For instance, L.
gibba can be easily recognized when it possesses
the typical gibbous structure. Unfortunately, this
is often not the case in natural environment and
then not many duckweed researchers can distinguish L. gibba from e.g. L. minor. In most cases,
for determining the species identity, integration
of molecular barcoding is unavoidable. One of
the standardized methods includes sequencing of
plastidic fragments that can be used as molecular
markers. It turned out that for different genera,
different sets of molecular markers have to be
used (Bog et al. 2013, 2015, 2018). Unfortunately, even then the results are not always
clear-cut (Borisjuk et al. 2015) and have to be
integrated together with the morphological
markers to eliminate any uncertainty. Several
molecular taxonomic methods are presently in
development but are either still very expensive
(e.g. genotyping by sequencing; Bog et al.
2020b) or need further investigations in order to
expand it to the whole plant family (e.g. polymorphic NB-ARC-related genes; Chu et al.
2018).
3 Worldwide Genetic Resources of Duckweed: Stock Collections
43
polyrhiza clones can be easily harvested and
stored in darkness for several years. However,
we do not know a single laboratory where this is
realized.
All plants in stock collections have to be kept
under axenic (“sterile”) conditions because contamination by bacteria or fungi may inhibit
favourable growth conditions of the plants leading to death of the plants after some time.
Especially dangerous are the contaminations by
green algae as they compete with the duckweeds
for resources. These contaminations can be
removed by surface sterilization of the plants
with a commercially available household bleach
like Eau de Javel or DanKlorix (Sree and
Appenroth 2017). The availability of axenic
cultures allows studies on duckweeds alone
without having any interference from other
organisms (Appenroth et al. 2016). All media
need to be sterilized (autoclaved), and inoculation should be carried out in laminar flow boxes
to avoid any contaminations during the process.
In order to examine and ensure axenic conditions, usually glucose (25 mM or less) is added
to the solidified media. Any microbial contamination can then be easily recognized. However,
this has two disadvantages. 1. If there is a contamination, plants are usually quickly killed in
the presence of sugars as the microbial population explodes. 2. Sugars accelerate senescence
and shorten the time between two subculturing
events. To avoid these disadvantages, the duckweed clones can be kept in both the presence and
absence of glucose, thereby taking advantage of
both cultivation methods.
Cultivation vessels for stock cultivation are
very different in different collections as this
depends on the available space and manpower.
They range from 100-ml Erlenmeyer flasks with
cotton wool stoppers (Fig. 3.3) to Petri dishes of
different diameter (from 35 to 90 mm) closed
with parafilm, to standard glass test tubes
13 Â 100 mm with cotton wool stoppers.
3.5 Species Identification
of the Clones
For a duckweed clone collected from nature to be
successfully integrated into a stock collection,
accurate identification of the species is a prerequisite. Species identification using morphological markers would be the first choice
(Landolt 1980). Based on morphology, it is easy
to identify the duckweed genus to which the
clone belongs. In some cases, it is also easy to
identify the species, e.g. Lemna trisulca or S.
polyrhiza. However, in many cases even experts
of duckweed morphology have the biggest
problems to assign species identity based on
morphological markers. This is partially caused
by the miniaturized phenotype of the duckweeds.
In some cases, the ease of identity might also
depend on the growth conditions. For instance, L.
gibba can be easily recognized when it possesses
the typical gibbous structure. Unfortunately, this
is often not the case in natural environment and
then not many duckweed researchers can distinguish L. gibba from e.g. L. minor. In most cases,
for determining the species identity, integration
of molecular barcoding is unavoidable. One of
the standardized methods includes sequencing of
plastidic fragments that can be used as molecular
markers. It turned out that for different genera,
different sets of molecular markers have to be
used (Bog et al. 2013, 2015, 2018). Unfortunately, even then the results are not always
clear-cut (Borisjuk et al. 2015) and have to be
integrated together with the morphological
markers to eliminate any uncertainty. Several
molecular taxonomic methods are presently in
development but are either still very expensive
(e.g. genotyping by sequencing; Bog et al.
2020b) or need further investigations in order to
expand it to the whole plant family (e.g. polymorphic NB-ARC-related genes; Chu et al.
2018).
3 Worldwide Genetic Resources of Duckweed: Stock Collections
43