nitrate and the accumulation of heavy metals
(Lewis 1995; Dushenkov et al. 1995; Dhir et al.
2009). Finally, they are useful in the production
of biofuels due to their high content of starch
(Cheng and Stomp 2009; Cui and Cheng 2015;
Xu et al. 2011).
Wolffia australiana was raised to the status of
a species in 1972. Before this year, it was
incorrectly regarded as a variety of W. arrhiza
(den Hartog and van der Plas 1972). W. australiana originated from surfaces of calm waters
of the temperate region of southern Australia and
New Zealand (Landolt 1994). With a frond size
of about 0.5–0.95 mm long, 0.30–0.52 mm
wide, and 0.79–1.32 mm high, W. australiana
represents one of the larger species among the
genus Wolffia (den Hartog and van der Plas 1972;
Landolt 1994). Figure 17.1a shows the elliptical
dorsal surface and a bright green appearance, due
to the high concentration of chloroplasts in the
upper cell layer. The ventral bulge with its lighter
green appearance is composed of larger, highly
vacuolated cells with a lower concentration of
chloroplasts. Usually, W. australiana appears as
a two-plant colony consisting of one mother
frond attached to a daughter frond. The average
lifespan of one plant is about 17 days, during
which approximately eleven daughter fronds
develop by budding (Bernard et al. 1990). With
doubling times of around 24–48 h, the growth
rate is remarkably high (Ziegler et al. 2015).
Because Wolffia can be grown on solid and on
liquid media while both floating and submerged
(Fig. 17.1c), the cultivation can be adapted to
nearly all requirements (Kruse et al. 2002;
Thompson 1989). Its cultivation in liquid can
result in a higher biomass production compared
to other duckweed species, which are limited to
floating modus vivendi.
17.2 Culture and Transformation
As summarized by Khvatkov (Khvatkov et al.
2015a), different media have been used for the
various duckweed species. We found Schenk and
Hildebrandt (SH) medium adjusted to pH 6.0 to
be the most suitable for W. australiana
(Rechmann et al. 2007; Khvatkov et al. 2015a),
but according to the literature, MS media is used
as well (Boehm et al. 2001; Kruse et al. 2002). In
our experiments, in vitro culture of the fronds
occurred at 20 °C at a light intensity of 60–
80 µmol m
−2 s
−1 during a 16-h day photoperiod
(PAR-region of 400–700 nm). Solid media was
used before and following the actual transformation of W. australiana with A. tumefaciens, as
well as for long-term cultivation (Fig. 17.1b).
Several transformation approaches to obtain
transgenic Lemnaceae have been described
(Table 17.2). Most approaches rely on the
transformation of induced structures like calli.
For the genus, Lemna and Spirodela stable
transformations have been shown by various
groups, whereas all but one approach for Wolffia
has been transient up to now.
We would like to quote that there are some
patents as well (Edelman et al. 1998; Stomp and
Rajbhandari 2000; Spencer et al. 2011) and two
unpublished PhD theses from Bonn University
(Friedrich 2005; Becker 2006), which are not
listed in Table 17.2. Some groups used the
transformation systems listed in Table 17.2 for
their appropriate biotechnological applications of
Lemna without further modification of the protocols (e.g., Woodard et al. 2009; Bertran et al.
2015).
For Lemna minor, Lemna gibba, as well as
Spirodela oligorrhiza (Table 17.2), transformation protocols usually depend on the use of callus
—respective nodule—culture as it was initially
described by (Stomp and Rajbhandari 2000;
Yamamoto et al. 2001). In contrast to Spirodela
and Lemna, only very few approaches for culture
and/or transformation of members of the genus
Wolffia have been published. Kruse and
co-worker used particle bombardment for the
transient transformation of Wolffia (Kruse et al.
2002).
Another induced structure, obtained for
Wolffia arrhiza, is the so-called cluster culture
(Khvatkov et al. 2015a). The cluster structure
develops directly from complete W. arrhiza
fronds. Despite the cluster culture’s success in
the creation of a stable callus culture and
regeneration of whole plants, only the novel
17 Editing the Genome of Wolffia australiana
167
(Lewis 1995; Dushenkov et al. 1995; Dhir et al.
2009). Finally, they are useful in the production
of biofuels due to their high content of starch
(Cheng and Stomp 2009; Cui and Cheng 2015;
Xu et al. 2011).
Wolffia australiana was raised to the status of
a species in 1972. Before this year, it was
incorrectly regarded as a variety of W. arrhiza
(den Hartog and van der Plas 1972). W. australiana originated from surfaces of calm waters
of the temperate region of southern Australia and
New Zealand (Landolt 1994). With a frond size
of about 0.5–0.95 mm long, 0.30–0.52 mm
wide, and 0.79–1.32 mm high, W. australiana
represents one of the larger species among the
genus Wolffia (den Hartog and van der Plas 1972;
Landolt 1994). Figure 17.1a shows the elliptical
dorsal surface and a bright green appearance, due
to the high concentration of chloroplasts in the
upper cell layer. The ventral bulge with its lighter
green appearance is composed of larger, highly
vacuolated cells with a lower concentration of
chloroplasts. Usually, W. australiana appears as
a two-plant colony consisting of one mother
frond attached to a daughter frond. The average
lifespan of one plant is about 17 days, during
which approximately eleven daughter fronds
develop by budding (Bernard et al. 1990). With
doubling times of around 24–48 h, the growth
rate is remarkably high (Ziegler et al. 2015).
Because Wolffia can be grown on solid and on
liquid media while both floating and submerged
(Fig. 17.1c), the cultivation can be adapted to
nearly all requirements (Kruse et al. 2002;
Thompson 1989). Its cultivation in liquid can
result in a higher biomass production compared
to other duckweed species, which are limited to
floating modus vivendi.
17.2 Culture and Transformation
As summarized by Khvatkov (Khvatkov et al.
2015a), different media have been used for the
various duckweed species. We found Schenk and
Hildebrandt (SH) medium adjusted to pH 6.0 to
be the most suitable for W. australiana
(Rechmann et al. 2007; Khvatkov et al. 2015a),
but according to the literature, MS media is used
as well (Boehm et al. 2001; Kruse et al. 2002). In
our experiments, in vitro culture of the fronds
occurred at 20 °C at a light intensity of 60–
80 µmol m
−2 s
−1 during a 16-h day photoperiod
(PAR-region of 400–700 nm). Solid media was
used before and following the actual transformation of W. australiana with A. tumefaciens, as
well as for long-term cultivation (Fig. 17.1b).
Several transformation approaches to obtain
transgenic Lemnaceae have been described
(Table 17.2). Most approaches rely on the
transformation of induced structures like calli.
For the genus, Lemna and Spirodela stable
transformations have been shown by various
groups, whereas all but one approach for Wolffia
has been transient up to now.
We would like to quote that there are some
patents as well (Edelman et al. 1998; Stomp and
Rajbhandari 2000; Spencer et al. 2011) and two
unpublished PhD theses from Bonn University
(Friedrich 2005; Becker 2006), which are not
listed in Table 17.2. Some groups used the
transformation systems listed in Table 17.2 for
their appropriate biotechnological applications of
Lemna without further modification of the protocols (e.g., Woodard et al. 2009; Bertran et al.
2015).
For Lemna minor, Lemna gibba, as well as
Spirodela oligorrhiza (Table 17.2), transformation protocols usually depend on the use of callus
—respective nodule—culture as it was initially
described by (Stomp and Rajbhandari 2000;
Yamamoto et al. 2001). In contrast to Spirodela
and Lemna, only very few approaches for culture
and/or transformation of members of the genus
Wolffia have been published. Kruse and
co-worker used particle bombardment for the
transient transformation of Wolffia (Kruse et al.
2002).
Another induced structure, obtained for
Wolffia arrhiza, is the so-called cluster culture
(Khvatkov et al. 2015a). The cluster structure
develops directly from complete W. arrhiza
fronds. Despite the cluster culture’s success in
the creation of a stable callus culture and
regeneration of whole plants, only the novel
17 Editing the Genome of Wolffia australiana
167