tumefaciens strain EHA105. Both the cluster
structures and calli obtained after 4 months cultivation were conducted to co-culture with
Agrobacterium suspension. Results showed that
the preferred type of explant for the transformation of W. arrhiza was cluster structure instead of
calli due to their inefficiency in high necrotization. To promote transformation, plant materials
immersed in Agrobacterium suspension were
shaking first and then drying in an airflow laminar box. Furthermore, the addition of 2 mg/L
2,4-D and BA in the medium during the first 1–
2 weeks and regulator-free after 2 weeks were
found crucial for the successful transformation of
W. arrhiza.
15.2.2 Transient Transformation
of Duckweeds
Although A. tumefaciens has been widely used in
the stable transformation of plants, Agrobacterium-mediated transient transformation has also
attracted substantial attentions in recent years due
to its time-saving property. However, the
host-range restrictions and regeneration problems
were considered to be limiting steps in
Agrobacterium-mediated transformation. Particle
bombardment, by using high-velocity microprojectiles for delivery of foreign DNA into intact
plant tissues has also been demonstrated as an
alternative method for transient transformation
(Klein et al. 1987) and this method has been
successfully applied on W. Columbiana and W.
arrhiza. For W. columbiana, the plasmid
pCAMBIA1301 containing uidA reporter construct under the control of constitutive CaMV35S
promoter was transformed by biolistic approach
(Kruse et al. 2002). When a gold particle with a
size of 0.6 lm was accelerated at 1350 psi with a
target distance of 60 mm, higher efficiency of
transformation was obtained. W. arrhiza was also
transiently transformed by particle bombardment
(Khvatkov et al. 2015a, b). The vector pCamGFP
containing CaMV35S-driven m-gfp5-ER (codonoptimized gfp with localization signal to the
endoplasmic reticulum) was constructed and
introduced into W. arrhiza. In this study, the
optimal parameters of helium pressure and target
distance were 1350 psi and 12 cm, respectively,
showing high level of transient expression as
12%.
Agrobacterium-mediated transient transformation for W. Columbiana was also established
(Boehm et al. 2001). A. tumefaciens strain
LBA4404 harboring a binary vector p35SGUSINT (uidA gene under the control of CaMV35S
promoter) was used for fast screening of transformation results. Since this plant has a compact
structure and few stomata at the upper surface,
other treatments were applied to increase the
infection efficiency. In this study, particle treatment and vacuum infiltration were found to be
more essential in increasing transformation efficiency than Agrobacterium-mediated method.
Though, the average transformation efficiency
was still low with 3.9% of the fronds showing
GUS activity. Currently, only transient expression of marker genes was reported in the genus
Wolffia, including species W. australiana, W.
globosa, W. columbiana and W. arrhiza (Boehm
et al. 2001; Kruse et al. 2002; Pham et al. 2010;
Khvatkov et al. 2015a, b). Among those exogenous gene, the most commonly used reporter
genes in transient transformation are the b-glucuronidase (GUS) gene and the green fluorescent
protein GFP.
15.2.3 Agrobacterium Strains
and Density
The ability of Agrobacterium to transfer T-DNA
into plant genome varied in different strains and
concentrations, therefore, produced different
transformation effects. There are differences in
the susceptibility among species even cultivars
and genotypes of these species (Swarnapiria
2009). Almost all the stable transformation of L.
minor was obtained using Agrobacterium strain
15 Transformation Development in Duckweeds
149
structures and calli obtained after 4 months cultivation were conducted to co-culture with
Agrobacterium suspension. Results showed that
the preferred type of explant for the transformation of W. arrhiza was cluster structure instead of
calli due to their inefficiency in high necrotization. To promote transformation, plant materials
immersed in Agrobacterium suspension were
shaking first and then drying in an airflow laminar box. Furthermore, the addition of 2 mg/L
2,4-D and BA in the medium during the first 1–
2 weeks and regulator-free after 2 weeks were
found crucial for the successful transformation of
W. arrhiza.
15.2.2 Transient Transformation
of Duckweeds
Although A. tumefaciens has been widely used in
the stable transformation of plants, Agrobacterium-mediated transient transformation has also
attracted substantial attentions in recent years due
to its time-saving property. However, the
host-range restrictions and regeneration problems
were considered to be limiting steps in
Agrobacterium-mediated transformation. Particle
bombardment, by using high-velocity microprojectiles for delivery of foreign DNA into intact
plant tissues has also been demonstrated as an
alternative method for transient transformation
(Klein et al. 1987) and this method has been
successfully applied on W. Columbiana and W.
arrhiza. For W. columbiana, the plasmid
pCAMBIA1301 containing uidA reporter construct under the control of constitutive CaMV35S
promoter was transformed by biolistic approach
(Kruse et al. 2002). When a gold particle with a
size of 0.6 lm was accelerated at 1350 psi with a
target distance of 60 mm, higher efficiency of
transformation was obtained. W. arrhiza was also
transiently transformed by particle bombardment
(Khvatkov et al. 2015a, b). The vector pCamGFP
containing CaMV35S-driven m-gfp5-ER (codonoptimized gfp with localization signal to the
endoplasmic reticulum) was constructed and
introduced into W. arrhiza. In this study, the
optimal parameters of helium pressure and target
distance were 1350 psi and 12 cm, respectively,
showing high level of transient expression as
12%.
Agrobacterium-mediated transient transformation for W. Columbiana was also established
(Boehm et al. 2001). A. tumefaciens strain
LBA4404 harboring a binary vector p35SGUSINT (uidA gene under the control of CaMV35S
promoter) was used for fast screening of transformation results. Since this plant has a compact
structure and few stomata at the upper surface,
other treatments were applied to increase the
infection efficiency. In this study, particle treatment and vacuum infiltration were found to be
more essential in increasing transformation efficiency than Agrobacterium-mediated method.
Though, the average transformation efficiency
was still low with 3.9% of the fronds showing
GUS activity. Currently, only transient expression of marker genes was reported in the genus
Wolffia, including species W. australiana, W.
globosa, W. columbiana and W. arrhiza (Boehm
et al. 2001; Kruse et al. 2002; Pham et al. 2010;
Khvatkov et al. 2015a, b). Among those exogenous gene, the most commonly used reporter
genes in transient transformation are the b-glucuronidase (GUS) gene and the green fluorescent
protein GFP.
15.2.3 Agrobacterium Strains
and Density
The ability of Agrobacterium to transfer T-DNA
into plant genome varied in different strains and
concentrations, therefore, produced different
transformation effects. There are differences in
the susceptibility among species even cultivars
and genotypes of these species (Swarnapiria
2009). Almost all the stable transformation of L.
minor was obtained using Agrobacterium strain
15 Transformation Development in Duckweeds
149