17
Editing the Genome of Wolffia
australiana
Thomas Reinard, Anke Londenberg, Merlin Brychcy,
Kim Lühmann, Gerrich Behrendt and Maren Wichmann
Abstract
The genus Wolffia has not been the focus of
biotechnological interest so far in contrast to
Lemna and Spirodela. There are only a few
publications on cultivation and regeneration and
even fewer articles about transformation of
species from this genus. However, Wolffia has
great potential, because it can grow both floating
and submerged, which allows for the production
of a larger biomass compared to other genera.
Furthermore, the in planta transformation is
feasible, which may accelerate the development
of new lines, since there is no need for
time-consuming plant regeneration. The groundbreaking technology of genome editing allows
the deletion, substitution, or insertion of genes at
precise, predefined sites in the genome. Here,
we present as proofs of principle the crucial
steps needed to realize Wolffia australiana as a
plant bioreactor. The procedures described
include the cultivation and transformation of
W. australiana, the identification of a suitable
gene for genome editing, which can be used as a
selectable marker as well, and the method of
genome editing of duckweed itself, which
resulted in knock-out Wolffia plants.
17.1 Introduction
Wolffia is one of the five genera of the worldwide
aquatic family Lemnaceae commonly named
duckweed. The genus consists of 11 species (Sree
et al. 2016), categorized by their morphology
and/or molecular barcoding (Table 17.1).
The taxonomic classification of Lemnaceae
was rearranged in the past years. Here, we use
the nomenclature according to Sree et al. (2016).
Among all Lemnaceae, the genus Wolffia is
the most specialized with the simplest level of
organization and the smallest flowering plants.
The plant body of Wolffia, called the frond,
shows an extreme morphological reduction, seen
in the lack of roots and no differentiation into
stem and leaves (Landolt 1986). Reproduction in
Wolffia is one of the fastest among all angiosperms and occurs predominantly vegetatively as
is true for all Lemnaceae. In contrast, sexual
reproduction is very rare (Sree et al. 2015). New
daughter fronds (DFs) bud from one basal cavity
stay attached to the mother frond by a so-called
stipe until maturation (Fig. 17.1a). Within the
budding cavity of the mother frond and the
connected daughter frond, the next generation
will begin developing (Bernard et al. 1990). In
contrast to most other Lemnaceae, Wolffia fronds
are able to live submerged as well as floating
(Thompson 1989), resulting in higher biomass
per area (Fig. 17.1b). To survive unfavorable
conditions like low temperature or starvation, it
T. Reinard (&) Á A. Londenberg Á M. Brychcy Á
K. Lühmann Á G. Behrendt Á M. Wichmann
Faculty of Natural Sciences, Institute of Plant
Genetics, Leibniz University of Hannover,
Herrenhäuser Str. 2, D-30419, Hannover, Germany
e-mail: reinard@genetik.uni-hannover.de
© Springer Nature Switzerland AG 2020
X. H. Cao et al. (eds.), The Duckweed Genomes, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11045-1_17
165
Editing the Genome of Wolffia
australiana
Thomas Reinard, Anke Londenberg, Merlin Brychcy,
Kim Lühmann, Gerrich Behrendt and Maren Wichmann
Abstract
The genus Wolffia has not been the focus of
biotechnological interest so far in contrast to
Lemna and Spirodela. There are only a few
publications on cultivation and regeneration and
even fewer articles about transformation of
species from this genus. However, Wolffia has
great potential, because it can grow both floating
and submerged, which allows for the production
of a larger biomass compared to other genera.
Furthermore, the in planta transformation is
feasible, which may accelerate the development
of new lines, since there is no need for
time-consuming plant regeneration. The groundbreaking technology of genome editing allows
the deletion, substitution, or insertion of genes at
precise, predefined sites in the genome. Here,
we present as proofs of principle the crucial
steps needed to realize Wolffia australiana as a
plant bioreactor. The procedures described
include the cultivation and transformation of
W. australiana, the identification of a suitable
gene for genome editing, which can be used as a
selectable marker as well, and the method of
genome editing of duckweed itself, which
resulted in knock-out Wolffia plants.
17.1 Introduction
Wolffia is one of the five genera of the worldwide
aquatic family Lemnaceae commonly named
duckweed. The genus consists of 11 species (Sree
et al. 2016), categorized by their morphology
and/or molecular barcoding (Table 17.1).
The taxonomic classification of Lemnaceae
was rearranged in the past years. Here, we use
the nomenclature according to Sree et al. (2016).
Among all Lemnaceae, the genus Wolffia is
the most specialized with the simplest level of
organization and the smallest flowering plants.
The plant body of Wolffia, called the frond,
shows an extreme morphological reduction, seen
in the lack of roots and no differentiation into
stem and leaves (Landolt 1986). Reproduction in
Wolffia is one of the fastest among all angiosperms and occurs predominantly vegetatively as
is true for all Lemnaceae. In contrast, sexual
reproduction is very rare (Sree et al. 2015). New
daughter fronds (DFs) bud from one basal cavity
stay attached to the mother frond by a so-called
stipe until maturation (Fig. 17.1a). Within the
budding cavity of the mother frond and the
connected daughter frond, the next generation
will begin developing (Bernard et al. 1990). In
contrast to most other Lemnaceae, Wolffia fronds
are able to live submerged as well as floating
(Thompson 1989), resulting in higher biomass
per area (Fig. 17.1b). To survive unfavorable
conditions like low temperature or starvation, it
T. Reinard (&) Á A. Londenberg Á M. Brychcy Á
K. Lühmann Á G. Behrendt Á M. Wichmann
Faculty of Natural Sciences, Institute of Plant
Genetics, Leibniz University of Hannover,
Herrenhäuser Str. 2, D-30419, Hannover, Germany
e-mail: reinard@genetik.uni-hannover.de
© Springer Nature Switzerland AG 2020
X. H. Cao et al. (eds.), The Duckweed Genomes, Compendium of Plant Genomes,
https://doi.org/10.1007/978-3-030-11045-1_17
165