c58-z707 followed by EHA105 (Yamamoto et al.
2001; Cox et al. 2006; Sun et al. 2007; Chhabra
et al. 2011; Ko et al. 2011; Long et al. 2012;
Yang et al. 2013). Other strains including
GV3101 and CBE21 have also made considerable transformation effect in transferring the
exogenous gene into L. minor genome (CantóPastor et al. 2015; Firsov et al. 2015). The above
revealed that L. minor specie was relatively easy
to be transformed therefore it has more advantages in expressing some beneficial traits over
other duckweed species. Furthermore, Agrobacterium strains EHA105 was found to respond
best in the transformation experiments of S.
punctata (Balaji et al. 2015), S. oligorrhiza
(Rival et al. 2008; Vunsh et al. 2007) and W.
arrhiza (Khvatkov et al. 2015a, b). While transgenic S. polyrhiza was only acquired under the
effective infection of Agrobacterium strain
AGL1 (Thu et al. 2015). In general, Agrobacterium strain EHA105 has made good effect in
the stable transformation of monocotyledon plant
of duckweeds.
High density of bacterial cells could cause the
death of plants, meanwhile, low density of bacterial cells could lead to ineffective transformation. The effect of bacterial densities on
transformation efficiency of duckweeds has not
been reported in detail. Generally, the adoptive
bacterial density (presented by OD600 value) in
L. minor transformation mediated by Agrobacterium strains EHA105, C58-Z707 and GV3101
was about 1.0 (Yamamoto et al. 2001; Cox et al.
2006; Sun et al. 2007; Chhabra et al. 2011; Long
et al. 2012; Yang et al. 2013; Cantó-Pastor et al.
2015). However, the setting value of OD600 in
transformation experiment of L. minor mediated
by Agrobacterium strain CBE21 was only 0.2
indicating distinctly difference in infecting
capability of different Agrobacterium strains
(Firsov et al. 2015). For three Spirodela species,
the values of OD600 of Agrobacterium strain
EHA105 varied from 0.5 to 1.5 (Vunsh et al.
2007; Thu et al. 2015; Balaji et al. 2015).
Although the same Agrobacterium strain was
used, the value of OD600 adopted in the transformation of W. arrhiza was only 0.4–0.6
(Khvatkov et al. 2015a, b).
15.3 Regeneration and Selection
of Transformed Plants
A complete process of gene transformation
includes explants cultivation, regeneration and
selection of transgenic plants, as well as cultivation of transformed plants. Therefore, the
successful establishment of genetic transformation system especially Agrobacterium-mediated
and microprojectile bombardment method
depends on one efficient and stable plant regeneration system. Almost all of the transformation
experiments of duckweed species were conducted using calli, nodules or cluster structures as
infected objects. Published researches on callus
induction and regeneration of Spirodela, Lemna
and Wolffia species laid the foundation for their
gene transformation experiments (Stefaniak et al.
2002; Li et al. 2004; Wang 2016; Khvatkov et al.
2015a, b).
The regeneration and selection of transformed
plants were very critical for obtaining single lines
and always carried out simultaneously. Efficient
selection depends on the kind of antibiotics
employed which was determined by selectable
marker genes and concentration applied (Rani
et al. 2013). The most effective antibiotics are
those which either inhibit regeneration of
untransformed plants or slowly kill the untransformed plants while transformed plants survive
in good conditions (Swarnapiria 2009). The
neomycin phosphotransferase (nptII) gene
encoding for kanamycin, neomycin, geneticin
(G418) and paromomycin, and the hygromycin
phosphotransferase (hpt) gene encoding for
hygromycin have been used extensively in plant
gene transformation. Different concentrations of
kanamycin have been adopted in almost all the
stable transformation of L. minor, L. gibba, S.
oligorrhiza ranging from 10 to 200 mg/L
(Yamamoto et al. 2001; Cox et al. 2006; Sun
et al. 2007; Chhabra et al. 2011; Ko et al. 2011;
Long et al. 2012; Bertran et al. 2015; Firsov et al.
2015). 10 mg/L phosphinothricin (PPT) was
added in frond regeneration medium in the
transformation experiment of L. minor by CantóPastor et al. (2015), because the construct contained a selectable marker conferring resistance
150
J. Yang et al.
2001; Cox et al. 2006; Sun et al. 2007; Chhabra
et al. 2011; Ko et al. 2011; Long et al. 2012;
Yang et al. 2013). Other strains including
GV3101 and CBE21 have also made considerable transformation effect in transferring the
exogenous gene into L. minor genome (CantóPastor et al. 2015; Firsov et al. 2015). The above
revealed that L. minor specie was relatively easy
to be transformed therefore it has more advantages in expressing some beneficial traits over
other duckweed species. Furthermore, Agrobacterium strains EHA105 was found to respond
best in the transformation experiments of S.
punctata (Balaji et al. 2015), S. oligorrhiza
(Rival et al. 2008; Vunsh et al. 2007) and W.
arrhiza (Khvatkov et al. 2015a, b). While transgenic S. polyrhiza was only acquired under the
effective infection of Agrobacterium strain
AGL1 (Thu et al. 2015). In general, Agrobacterium strain EHA105 has made good effect in
the stable transformation of monocotyledon plant
of duckweeds.
High density of bacterial cells could cause the
death of plants, meanwhile, low density of bacterial cells could lead to ineffective transformation. The effect of bacterial densities on
transformation efficiency of duckweeds has not
been reported in detail. Generally, the adoptive
bacterial density (presented by OD600 value) in
L. minor transformation mediated by Agrobacterium strains EHA105, C58-Z707 and GV3101
was about 1.0 (Yamamoto et al. 2001; Cox et al.
2006; Sun et al. 2007; Chhabra et al. 2011; Long
et al. 2012; Yang et al. 2013; Cantó-Pastor et al.
2015). However, the setting value of OD600 in
transformation experiment of L. minor mediated
by Agrobacterium strain CBE21 was only 0.2
indicating distinctly difference in infecting
capability of different Agrobacterium strains
(Firsov et al. 2015). For three Spirodela species,
the values of OD600 of Agrobacterium strain
EHA105 varied from 0.5 to 1.5 (Vunsh et al.
2007; Thu et al. 2015; Balaji et al. 2015).
Although the same Agrobacterium strain was
used, the value of OD600 adopted in the transformation of W. arrhiza was only 0.4–0.6
(Khvatkov et al. 2015a, b).
15.3 Regeneration and Selection
of Transformed Plants
A complete process of gene transformation
includes explants cultivation, regeneration and
selection of transgenic plants, as well as cultivation of transformed plants. Therefore, the
successful establishment of genetic transformation system especially Agrobacterium-mediated
and microprojectile bombardment method
depends on one efficient and stable plant regeneration system. Almost all of the transformation
experiments of duckweed species were conducted using calli, nodules or cluster structures as
infected objects. Published researches on callus
induction and regeneration of Spirodela, Lemna
and Wolffia species laid the foundation for their
gene transformation experiments (Stefaniak et al.
2002; Li et al. 2004; Wang 2016; Khvatkov et al.
2015a, b).
The regeneration and selection of transformed
plants were very critical for obtaining single lines
and always carried out simultaneously. Efficient
selection depends on the kind of antibiotics
employed which was determined by selectable
marker genes and concentration applied (Rani
et al. 2013). The most effective antibiotics are
those which either inhibit regeneration of
untransformed plants or slowly kill the untransformed plants while transformed plants survive
in good conditions (Swarnapiria 2009). The
neomycin phosphotransferase (nptII) gene
encoding for kanamycin, neomycin, geneticin
(G418) and paromomycin, and the hygromycin
phosphotransferase (hpt) gene encoding for
hygromycin have been used extensively in plant
gene transformation. Different concentrations of
kanamycin have been adopted in almost all the
stable transformation of L. minor, L. gibba, S.
oligorrhiza ranging from 10 to 200 mg/L
(Yamamoto et al. 2001; Cox et al. 2006; Sun
et al. 2007; Chhabra et al. 2011; Ko et al. 2011;
Long et al. 2012; Bertran et al. 2015; Firsov et al.
2015). 10 mg/L phosphinothricin (PPT) was
added in frond regeneration medium in the
transformation experiment of L. minor by CantóPastor et al. (2015), because the construct contained a selectable marker conferring resistance
150
J. Yang et al.