16.2.1 Agrobacterium-Mediated Stable Transformation
Scientists have exploited the ability ofA. tumefaciensto transform plant cells by
creating binary vectors and‘disarmed’Agrobacteriumstrains (Hellens et al. 2000 ).
TheA. tumefaciensT-DNA region contains genes responsible for the tumorigenic
crown gall disease in host plants (Chilton et al. 1977 ; Escobar and Dandekar 2003 ).
In disarmed strains, the disease-encoding genes contained within the T-DNA region
are removed from the Ti-plasmid but thevirgenes are kept to mediate T-DNA
transfer. To simplify the cloning process, a gene of interest is cloned into an
artificial T-DNA region placed on a separate plasmid, the binary vector (Hoekema
et al. 1983 ). The binary vector, containing the gene of interest, is then introduced
into a disarmedAgrobacteriumstrain. Upon infection of a plant cell, products of the
virgenes act intranson the T-DNA region of the binary vector to mediate transfer
of the gene of interest to the host plant cell (Gelvin 2003 ).
A. tumefaciensis now regularly used in laboratories to transfer genes of interest
into host plant cells to generate stable, transgenic plant lines with heritable traits.
Despite its widespread use, this method of plant transformation presents some
challenges (Altpeter et al. 2016 ). Stable transformation requires that the T-DNA be
integrated into the plant genome. However, integration is not an efficient process
and non-integrated T-DNA copies are transiently present in the nucleus, as will be
discussed below (Kapila et al. 1997 ; Altpeter et al. 2016 ). Furthermore, a prereq-
uisite to generate stable transformed plants is the ability to regenerate whole plants
from transformed cells (Altpeter et al. 2016 ). If successful, the entire process can
take several months to achieve (Wroblewski et al. 2005 ). However, recalcitrance to
tissue culture and transformation is an unfortunate reality for many crops, including
Cannabis, which limits the potential for this technology.
16.2.2 Transient Gene Expression by Agroinfiltration
Agroinfiltration has become a popular method to transiently express genes in plants.
It can be used to evaluate gene expression, study protein localization,
protein-protein interactions, biochemical analyses (Sparkes et al. 2006 ) and for the
accumulation of high-value proteins (Menassa et al. 2004 ;O’Keefe et al. 2009 ).
The method involves introducingA. tumefacienscarrying a gene of interest directly
into leaves using syringe injection (Sparkes et al. 2006 ) or vacuum infiltration
(Kapila et al. 1997 ). Once inside the leaf intercellular spaces, the bacteria transfer
the gene of interest from the T-DNA region of the binary vector into mesophyll
cells. Those T-DNAs that are not integrated into the host chromosome are tran-
scriptionally active and expression is usually detected after 2–5 days (Kapila et al.
1997 ; Joensuu et al. 2010 ).Nicotiana tabacumandN. benthamianaare model
plants used for this purpose (Conley et al. 2011 ) though the technique works well
with other host plants (Wroblewski et al. 2005 ). Agroinfiltration does not require
346 M. Feeney and Z.K. Punja