15.2 Transformation Methods
15.2.1 Stable Transformation
of Duckweeds
The first stable transformed duckweed was
obtained by Frey et al. (1980) through incubating
intact plant of Lemna perpusilla with the
Escherichia coli plasmids pMB9 and pBR325
under optimized conditions. This research work
confirmed that intact plant can directly absorb
E. coli plasmids and these plasmids can be used
as vectors for the introduction of exogenous genes into plants. Some factors including plasmid
concentration, incubation time and temperature
could affect the efficiency of transformation. In
case of Lemna DNA, the highest transformation
rate obtained per µg reported in this research was
1.9 Â 10
−8 when 20 µg Lemna DNA was incubated with 57 µg/ml plasmid at 6 °C for 22 h.
However, controversy suggested that exogenous
plasmid may not enter plant cell even nucleus
when using intact plants as receptors. There was
even denial of the obtained transgenic lines.
Agrobacterium-mediated method has been a
preferred technology in creating transgenic plants
due to the crown gall development ability of
Agrobacterium (Tsvetkov et al. 1997). The
crown gall disease could transfer and integrate
the T-DNA from Ti plasmid of the bacterium
into the plant nuclear genome. Moreover, phenolic compounds released by the wound of
dicotyledonous plants were essential for integration of vir genes (Razzaq et al. 2004). However, Agrobacterium-mediated transformation for
monocotyledon plants has been lagging behind
because monocotyledon plants cannot or can
release minute phenolic compounds when
wounded. Therefore, the addition of phenolic
compounds has greatly promoted genetic transformation efficiency of monocotyledon plants
(Cheng et al. 1997; Ali et al. 2007; Khan et al.
2013). Acetosyringone (AS), a phenolic compound, has been widely used in plant genetic
transformation at the period of pre-culture or
co-cultivation for inducing expression of vir
genes and improving the transformation efficiency in monocotyledon plants. Other factors
such as explant type, bacterial density,
Agrobacterium strain and co-culture condition
also affect the efficiency of Agrobacteriummediated transformation (Shrawat and Horst
Lörz 2006).
The stable transformation of Lemna minor,
Lemna gibba, Spirodela punctata, Spirodela
oligorrhiza, Wolffia arrhiza as well as the transient transformation of Spirodela polyrhiza,
Wolffia columbiana have been obtained by
Agrobacterium-mediated method. Yamamoto
et al. (2001) were the first to establish efficient
stable transformation protocols for L. minor and
L. gibba mediated by Agrobacterium tumefaciens C58-Z707. Light green nodules instead of
calli were used as infection receptor in their
study. 100 lM AS played an important role and
it was added in bacterial cultured plate, bacterial
resuspension medium and co-culture medium to
activate A. tumefaciens. Based on the protocol of
Yamamoto et al. (2001), endoglucanase E1 from
Acidothermus cellulolyticus was successfully
expressed in L. minor 8627 without any obvious
observable phenotypic effects on morphology or
the rate of growth (Sun et al. 2007). Although the
expression level of transgenic duckweed (up to
0.24% of total soluble protein) was lower than
several other transgenic expression systems like
tobacco (Ziegelhoffer et al. 2001), it is very
encouraging for us to develop and improve
duckweed expression system. Same transformation method was also successfully used in
expressing monoclonal antibodies (mAbs) (Cox
et al. 2006) as well as Avian influenza hemagglutinin HA gene (Long et al. 2012) in L. minor.
To fully exploit the advantages of duckweed
including rapid multiplication, secretion of
recombinant proteins and high protein yields,
Gasdaska et al. (2003) developed Lemna
Expression System or “LEX SystemSM” for the
production of recombinant proteins. The Lemna
Expression System was also adopted to successfully express H5 hemagglutinin vaccine
antigen by Bertran et al. (2015).
Chhabra et al. (2011) optimized the gene
transformation method of L. minor and established both stable and transient transformation
using A. tumefaciens strain EHA105, and 3.8%
15 Transformation Development in Duckweeds
147
15.2.1 Stable Transformation
of Duckweeds
The first stable transformed duckweed was
obtained by Frey et al. (1980) through incubating
intact plant of Lemna perpusilla with the
Escherichia coli plasmids pMB9 and pBR325
under optimized conditions. This research work
confirmed that intact plant can directly absorb
E. coli plasmids and these plasmids can be used
as vectors for the introduction of exogenous genes into plants. Some factors including plasmid
concentration, incubation time and temperature
could affect the efficiency of transformation. In
case of Lemna DNA, the highest transformation
rate obtained per µg reported in this research was
1.9 Â 10
−8 when 20 µg Lemna DNA was incubated with 57 µg/ml plasmid at 6 °C for 22 h.
However, controversy suggested that exogenous
plasmid may not enter plant cell even nucleus
when using intact plants as receptors. There was
even denial of the obtained transgenic lines.
Agrobacterium-mediated method has been a
preferred technology in creating transgenic plants
due to the crown gall development ability of
Agrobacterium (Tsvetkov et al. 1997). The
crown gall disease could transfer and integrate
the T-DNA from Ti plasmid of the bacterium
into the plant nuclear genome. Moreover, phenolic compounds released by the wound of
dicotyledonous plants were essential for integration of vir genes (Razzaq et al. 2004). However, Agrobacterium-mediated transformation for
monocotyledon plants has been lagging behind
because monocotyledon plants cannot or can
release minute phenolic compounds when
wounded. Therefore, the addition of phenolic
compounds has greatly promoted genetic transformation efficiency of monocotyledon plants
(Cheng et al. 1997; Ali et al. 2007; Khan et al.
2013). Acetosyringone (AS), a phenolic compound, has been widely used in plant genetic
transformation at the period of pre-culture or
co-cultivation for inducing expression of vir
genes and improving the transformation efficiency in monocotyledon plants. Other factors
such as explant type, bacterial density,
Agrobacterium strain and co-culture condition
also affect the efficiency of Agrobacteriummediated transformation (Shrawat and Horst
Lörz 2006).
The stable transformation of Lemna minor,
Lemna gibba, Spirodela punctata, Spirodela
oligorrhiza, Wolffia arrhiza as well as the transient transformation of Spirodela polyrhiza,
Wolffia columbiana have been obtained by
Agrobacterium-mediated method. Yamamoto
et al. (2001) were the first to establish efficient
stable transformation protocols for L. minor and
L. gibba mediated by Agrobacterium tumefaciens C58-Z707. Light green nodules instead of
calli were used as infection receptor in their
study. 100 lM AS played an important role and
it was added in bacterial cultured plate, bacterial
resuspension medium and co-culture medium to
activate A. tumefaciens. Based on the protocol of
Yamamoto et al. (2001), endoglucanase E1 from
Acidothermus cellulolyticus was successfully
expressed in L. minor 8627 without any obvious
observable phenotypic effects on morphology or
the rate of growth (Sun et al. 2007). Although the
expression level of transgenic duckweed (up to
0.24% of total soluble protein) was lower than
several other transgenic expression systems like
tobacco (Ziegelhoffer et al. 2001), it is very
encouraging for us to develop and improve
duckweed expression system. Same transformation method was also successfully used in
expressing monoclonal antibodies (mAbs) (Cox
et al. 2006) as well as Avian influenza hemagglutinin HA gene (Long et al. 2012) in L. minor.
To fully exploit the advantages of duckweed
including rapid multiplication, secretion of
recombinant proteins and high protein yields,
Gasdaska et al. (2003) developed Lemna
Expression System or “LEX SystemSM” for the
production of recombinant proteins. The Lemna
Expression System was also adopted to successfully express H5 hemagglutinin vaccine
antigen by Bertran et al. (2015).
Chhabra et al. (2011) optimized the gene
transformation method of L. minor and established both stable and transient transformation
using A. tumefaciens strain EHA105, and 3.8%
15 Transformation Development in Duckweeds
147