15.3.1.2 Reporter Genes
Reporter genes are used to demonstrate the transient or stable transformation of
plant material. Some reporter genes encode enzymes whose substrate is not normally found in plants, such as the uidA gene coding for b-glucuronidase enzyme
which produces a blue color to degrade the substrate X-gluc in the transformant
tissue (Jefferson et al. 1987). Green fluorescent protein (GFP) from the jellyfish
Aequorea victoria has also been used as a selection gene and allows visualization
of cells and transformed plastids by light excitation and without supplying the
plant substrate (Chiu et al. 1996). This category also has Southern blot analyses to
determine how many copies of the gene of interest have been inserted into the
plant genome (Birch 1997).
15.3.2 Transfer of Multiple Genes Through Improved
Vectors
The transformation with multiple genes simultaneously allows researchers to study
and manipulate an entire metabolic pathway to produce secondary metabolites and
proteins of interest. However, there are several barriers for transformation with
multiple genes because the first plant transformation methods were developed to
introduce only one or two genes. As more genes are introduced is lower the probability that all of them will be integrated and expressed (Naqvi et al. 2010) in plantlets.
The transfer of multiple genes can be achieved using conventional methods like
crossing homozygous transgenic lines or through sequential transformation of a
unique transgenic line; however, these methods are time-consuming and laborintensive and the transgenic plants produced can segregate independently and lose
their phenotype. This problem has been partially resolved with co-transformation
and design vectors that enable the insertion of multiple genes in a single transformation event (Naqvi et al. 2010).
The transfer of multiple genes can be mediated by A. tumefaciens using binary
vectors, wherein a vector has the T-DNA region with the gene of interest and the
second vector contains the vir region. Ti binary vectors have a replication origin
for E. coli that allows its multiplication, and the other has a replication origin for
A. tumefaciens. This method is effective when the size of the DNA to be transferred is less than 50 kb (Valderrama et al. 2005). With conventional transfer
vectors the introduction of multiple genes in a single DNA segment is possible,
and it is efficient to introduce segments of 50–80 kb.
Another transformation system of higher potential is the use of artificial
chromosomes with A. tumefaciens (BIBACs) as transformation vector. This system is similar to the bacterial artificial chromosomes or BACs. This vector was
developed based on the knowledge generated from BACs, but it also contains an
origin of replication for E. coli and one for A. tumefaciens allowing the
428
L. A. Cruz-Ramírez et al.
Reporter genes are used to demonstrate the transient or stable transformation of
plant material. Some reporter genes encode enzymes whose substrate is not normally found in plants, such as the uidA gene coding for b-glucuronidase enzyme
which produces a blue color to degrade the substrate X-gluc in the transformant
tissue (Jefferson et al. 1987). Green fluorescent protein (GFP) from the jellyfish
Aequorea victoria has also been used as a selection gene and allows visualization
of cells and transformed plastids by light excitation and without supplying the
plant substrate (Chiu et al. 1996). This category also has Southern blot analyses to
determine how many copies of the gene of interest have been inserted into the
plant genome (Birch 1997).
15.3.2 Transfer of Multiple Genes Through Improved
Vectors
The transformation with multiple genes simultaneously allows researchers to study
and manipulate an entire metabolic pathway to produce secondary metabolites and
proteins of interest. However, there are several barriers for transformation with
multiple genes because the first plant transformation methods were developed to
introduce only one or two genes. As more genes are introduced is lower the probability that all of them will be integrated and expressed (Naqvi et al. 2010) in plantlets.
The transfer of multiple genes can be achieved using conventional methods like
crossing homozygous transgenic lines or through sequential transformation of a
unique transgenic line; however, these methods are time-consuming and laborintensive and the transgenic plants produced can segregate independently and lose
their phenotype. This problem has been partially resolved with co-transformation
and design vectors that enable the insertion of multiple genes in a single transformation event (Naqvi et al. 2010).
The transfer of multiple genes can be mediated by A. tumefaciens using binary
vectors, wherein a vector has the T-DNA region with the gene of interest and the
second vector contains the vir region. Ti binary vectors have a replication origin
for E. coli that allows its multiplication, and the other has a replication origin for
A. tumefaciens. This method is effective when the size of the DNA to be transferred is less than 50 kb (Valderrama et al. 2005). With conventional transfer
vectors the introduction of multiple genes in a single DNA segment is possible,
and it is efficient to introduce segments of 50–80 kb.
Another transformation system of higher potential is the use of artificial
chromosomes with A. tumefaciens (BIBACs) as transformation vector. This system is similar to the bacterial artificial chromosomes or BACs. This vector was
developed based on the knowledge generated from BACs, but it also contains an
origin of replication for E. coli and one for A. tumefaciens allowing the
428
L. A. Cruz-Ramírez et al.