Environmental Engineering Reference
In-Depth Information
foreign genes into plant cells, significantly more efficient than purely plant viral based systems
(Azhakanandam et al. 2007).
Agrobacterium
-mediated transformation of plants is a commonly
used approach in which a specific portion of DNA, transfer DNA (T-DNA), on the bacterial
Ti (tumor-inducing) plasmid is transferred to the plant nucleus where it can ultimately be inte-
grated into the plant genome to generate stably transformed plants. However, during the initial
period after agroinfection, the genes on the T-DNA are transiently expressed within 2-3 days in
the plant nucleus. This transient “burst” of expression can lead to higher levels of recombinant
protein compared with levels in stable transgenic plants transformed with the same agrobacte-
ria (Wroblewski et al. 2005). Furthermore, co-infiltration using multiple
Agrobacterium
strains
containing different recombinant Ti plasmids allows coordinated expression of multiple recom-
binant proteins (Voinnet et al. 2003). Recombinant
Agrobacterium
containing appropriate binary
Ti expression vectors can be grown in shake flasks or bioreactors. Scale-up of
Agrobacterium
tumefaciens
cultures up to the 5000-L scale have been reported with final biomass concentra-
tions reaching over 50 g dry weight/L after 96 h of culture (Ha et al. 2007).
A. tumefaciens
can be
grown in Luria-Bertani (LB) media containing appropriate selection antibiotics and supplemented
with approximately 40 M acetosyringone and MES buffer. Before infiltration, the agrobacteria are
centrifuged and resuspended in distilled water containing magnesium chloride (10 mM), aceto-
syringone (150 mM), and a surfactant (e.g., Silwet
®
at 0.01%). Several recombinant agrobacteria
can be mixed to achieve desired relative concentrations depending upon the application. Harvested
leaf biomass (e.g.,
Nicotiana benthamiana
) can be immersed in the agrobacterial solution and a
weak vacuum (~530-610 mm Hg below atmospheric pressure or an absolute pressure of 150-230
mm Hg) applied for a short period of time (10 s to several minutes) and then rapidly released. The
vacuum pulls air trapped in the stomatal cavities out of the plant leaf, and once the vacuum is
released, the agrobacteria solution is infused into the stomata. Alternatively, the entire plant can be
turned upside down and immersed in the
Agrobacterium
solution before applying a vacuum. The
molecular steps involved in the transfer of T-DNA on the Ti plasmid of the
Agrobacterium
to the
plant cell are described in detail by Tzfira and Citovsky (2002), but they basically involve attach-
ment of the agrobacteria to the plant cell wall, excision of a single-stranded portion of the T-DNA
from the Ti plasmid called the T-strand, formation of a channel called the T-pilus that connects the
cytoplasm of the
Agrobacterium
to the plant cytoplasm, and transfer of the T-strand complex into
the plant cytoplasm and ultimately to the plant nucleus where expression of transgenes can take
place. After the agroinfiltration step, the plant leaves are typically removed from solution, allowed
to dry in ambient air for a certain period of time, and incubated in humid air at room temperature
to allow enough time for the agroinfection process and T-DNA transfer to take place. Vacuum
infiltration processes have been scaled up with reports of over 100 kg of wild-type tobacco infil-
trated (Fischer et al. 2004). Vacuum infiltration of harvested biomass in a contained facility is a
promising approach for efficient, scalable, and cost-effective production with high biosafety and
low environmental impact. It is a rapid, low-cost process that does not require aseptic operation or
the generation or deployment of transgenic crops in the field. Although it will be necessary to grow
the agrobacteria in a microbial fermenter, the required fermenter volume is estimated to be signifi-
cantly smaller than would be required for fungal production of enzymes for several reasons. First,
only a single bacterium is needed to transfer the T-DNA to each plant cell (representing a mass
ratio of ~1:10,000) and it is the plant cell's biosynthetic machinery (generated via photosynthesis)
that performs the transcription, translation, folding, post-translational modifications, and secretion
processes. Second, the microbial fermentation is used only to propagate the recombinant agrobac-
teria; resources are not diverted for the high-level production of product. Thus, the approach offers
the benefit of enzyme production in a eukaryotic host grown using photosynthesis without the need
to generate or deploy stably transgenic plants in the environment. In addition, the required fermen-
ter volume is reduced compared with that required for submerged fungal fermentation, and the
agroinfiltration process will be performed in a contained environment but will not require aseptic
operation, which further reduces capital and operating costs.
