stable transformation efficiency was obtained for
the first time. In comparison with Yamamoto
et al. (2001), the effect of different concentrations
of A. tumefaciens, co-culture conditions and
application of nonionic surfactants was studied.
Agrobacterium at 10
7 cell ml
−1 concentration,
the presence of 100 lM AS in co-culture medium at pH 5.2, nodular calli inoculated with
bacterial suspension for 60 min and co-culturing
for 3 days at 25 °C under 16 h light/8 h dark
photoperiod were significant for the transformation of L. minor and gave the highest percentage
of calli showing GUS activity. In addition, nonionic surfactant (Tween 20) adopted in this study
with relatively low concentration, i.e., 0.2%
significantly elevated transient GUS expression.
Following the transformation protocol described
by Chhabra et al. (2011), an Arabidopsis photorespiratory pathway gene serine: glyoxylate
aminotransferase (SGAT), named as AtAGT1,
was successfully overexpressed in L. minor
which provided an effective way to promote salt
tolerance in duckweeds and solve the freshwater
salinity problems (Yang et al. 2013).
Both of Firsov et al. (2015) and Cantó-Pastor
et al. (2015) have established the stable transformation of L. minor using calli instead of
nodular as infected object mediated by A. tumefaciens strains GV3101 and CBE21, respectively. Without AS application in their protocol,
Firsov et al. (2015) obtained 20 different lines of
duckweed with confirmed transgenic status.
Unlike Firsov et al. (2015), 200 lM AS was
added in Agrobacterium resuspension in the
study of Cantó-Pastor et al. (2015). Furthermore,
the selection and regeneration procedures were
executed simultaneously in liquid media to
reduce the overall transformation duration from
6–7 weeks to 5 weeks. 59% of GFP expressing
was obtained in this study which was significantly higher than previous studies.
Rival et al. (2008) and Vunsh et al. (2007)
successfully established the stable transformation
of S. oligorrhiza using calli as infected object to
express high levels of protein. Calli wounded
with DNA-free tungsten particles using a
PDS-1000/He System was adopted in the study
of Rival et al. (2008). Then the wounded calli
were co-cultured with Agrobacterium suspension
adding 100 lM AS and a highly stable GFP
expression level over 25% was obtained. The
stable transformation of S. polyrhiza was established using the cut fronds co-cultured with
prepared A. tumefaciens strain AGL1 suspension
which has been shaken for 4–6 h with 200 lM
AS. The above mixture was centrifuged and
vacuumed before cultured for 3 days with photoperiod 12/24 h to promote the infection of
Agrobacterium (Thu et al. 2015).
Balaji et al. (2015) put forward different
opinions on the effect of the wound to improve
transformation efficiency of duckweeds.
Two-week old S. punctata fronds were used in
plant transformation mediated by A. tumefaciens
strain EHA105. Results showed that 95% of
fronds without any wound were successfully
transformed, whereas all of the wounded fronds
gradually died during selection indicating that
intact plants can be used for efficient transformation. Callus induction and regeneration of
duckweeds always need a long period (Stefaniak
et al. 2002; Li et al. 2004; Wang 2016), thus in
plant transformation mediated by A. tumefaciens
would be a good choice for saving time in stable
transformation. The ability of the fronds of
duckweeds to be transformed has also been
found in other duckweed species (Ko et al.
2011). In the research of Ko et al. (2011), fronds
of L. minor were immersed with the bacterial
suspension of A. tumefaciens strain EHA105
harboring the PEDV spike protein 1 gene for
30 min after wounded with a pair of forceps and
scalpel. The difference from previous studies was
that a comparatively high concentration
(200 mg/L) of kanamycin was used to select
kanamycin-resistant fronds. The reason may be
different sensitivity between fronds and calli to
antibiotics as well as various geographic isolates
of Lemna species. In addition, 100 lM AS was
only added in prepared bacterial cells.
Comparatively, the stable transformation of
Wolffia was indeed a time-consuming procedure
because the period for inducing infected materials was at least 4 months (Khvatkov et al. 2015a,
b). Only the stable transformation of W. arrhiza
has been successfully established mediated by A.
148
J. Yang et al.
the first time. In comparison with Yamamoto
et al. (2001), the effect of different concentrations
of A. tumefaciens, co-culture conditions and
application of nonionic surfactants was studied.
Agrobacterium at 10
7 cell ml
−1 concentration,
the presence of 100 lM AS in co-culture medium at pH 5.2, nodular calli inoculated with
bacterial suspension for 60 min and co-culturing
for 3 days at 25 °C under 16 h light/8 h dark
photoperiod were significant for the transformation of L. minor and gave the highest percentage
of calli showing GUS activity. In addition, nonionic surfactant (Tween 20) adopted in this study
with relatively low concentration, i.e., 0.2%
significantly elevated transient GUS expression.
Following the transformation protocol described
by Chhabra et al. (2011), an Arabidopsis photorespiratory pathway gene serine: glyoxylate
aminotransferase (SGAT), named as AtAGT1,
was successfully overexpressed in L. minor
which provided an effective way to promote salt
tolerance in duckweeds and solve the freshwater
salinity problems (Yang et al. 2013).
Both of Firsov et al. (2015) and Cantó-Pastor
et al. (2015) have established the stable transformation of L. minor using calli instead of
nodular as infected object mediated by A. tumefaciens strains GV3101 and CBE21, respectively. Without AS application in their protocol,
Firsov et al. (2015) obtained 20 different lines of
duckweed with confirmed transgenic status.
Unlike Firsov et al. (2015), 200 lM AS was
added in Agrobacterium resuspension in the
study of Cantó-Pastor et al. (2015). Furthermore,
the selection and regeneration procedures were
executed simultaneously in liquid media to
reduce the overall transformation duration from
6–7 weeks to 5 weeks. 59% of GFP expressing
was obtained in this study which was significantly higher than previous studies.
Rival et al. (2008) and Vunsh et al. (2007)
successfully established the stable transformation
of S. oligorrhiza using calli as infected object to
express high levels of protein. Calli wounded
with DNA-free tungsten particles using a
PDS-1000/He System was adopted in the study
of Rival et al. (2008). Then the wounded calli
were co-cultured with Agrobacterium suspension
adding 100 lM AS and a highly stable GFP
expression level over 25% was obtained. The
stable transformation of S. polyrhiza was established using the cut fronds co-cultured with
prepared A. tumefaciens strain AGL1 suspension
which has been shaken for 4–6 h with 200 lM
AS. The above mixture was centrifuged and
vacuumed before cultured for 3 days with photoperiod 12/24 h to promote the infection of
Agrobacterium (Thu et al. 2015).
Balaji et al. (2015) put forward different
opinions on the effect of the wound to improve
transformation efficiency of duckweeds.
Two-week old S. punctata fronds were used in
plant transformation mediated by A. tumefaciens
strain EHA105. Results showed that 95% of
fronds without any wound were successfully
transformed, whereas all of the wounded fronds
gradually died during selection indicating that
intact plants can be used for efficient transformation. Callus induction and regeneration of
duckweeds always need a long period (Stefaniak
et al. 2002; Li et al. 2004; Wang 2016), thus in
plant transformation mediated by A. tumefaciens
would be a good choice for saving time in stable
transformation. The ability of the fronds of
duckweeds to be transformed has also been
found in other duckweed species (Ko et al.
2011). In the research of Ko et al. (2011), fronds
of L. minor were immersed with the bacterial
suspension of A. tumefaciens strain EHA105
harboring the PEDV spike protein 1 gene for
30 min after wounded with a pair of forceps and
scalpel. The difference from previous studies was
that a comparatively high concentration
(200 mg/L) of kanamycin was used to select
kanamycin-resistant fronds. The reason may be
different sensitivity between fronds and calli to
antibiotics as well as various geographic isolates
of Lemna species. In addition, 100 lM AS was
only added in prepared bacterial cells.
Comparatively, the stable transformation of
Wolffia was indeed a time-consuming procedure
because the period for inducing infected materials was at least 4 months (Khvatkov et al. 2015a,
b). Only the stable transformation of W. arrhiza
has been successfully established mediated by A.
148
J. Yang et al.