Agriculture Reference
In-Depth Information
can integrate anywhere in any of the plant
chromosomes (Fig. 2.3c). h is is the result of
the plant-mediated illegitimate recombin-
ation process. After the DNA fragment is
transferred to the nucleus, the plant host
enzymes, present to repair double-strand
breaks, recognize the DNA ends of the
bombarded fragment or the T-DNA and of
broken chromosome ends. h ose ends are
then ligated in a non-homologous DNA
end-joining process. When no double-strand
breaks are available, the transferred DNA
cannot integrate. h erefore, many DNA
transfer processes result in transient
expression because the introduced DNA is
not recombined with the plant chromosome
via illegitimate recombination. As a result,
the presence of double-strand breaks and of
the non-homologous end-joining process
determine in part the competence of the
plant cell to be transformed.
It should thus be stressed that the
position of integration of the transgene will
be dif erent in every independently trans-
formed cell. h e integration process is
random, but it is observed that there is a
tendency that genes are inserted in genome
regions that are actively transcribed. In the
(a)
(b)
Oncogenes
Binary vector system
Transgenes
RB
RB
LB
LB
T-DNA
T-DNA
ri
vir
region
vir
Plasmid
ori
ori
vir
Region
vir
region
ori
vir
Region
Ti
Plasmid
Coding sequence
Promoter
3' Terminator
(c)
Plant genome
Plant genome
RB
Selectable marker gene
Gene of interest
LB
T-DNA
Fig. 2.3.
Agrobacterium tumefaciens
-mediated plant transformation. (a) The Ti plasmid contains the
genetic information for transferring DNA to the plant cell in the
vir
region, while the genetic information
that is transferred to the plant cells is present in the T-DNA. The T-DNA is delineated by the left border
(LB) and the right border (RB) and contains the oncogenes, which induce crown galls on wounded
surfaces of plants. (b) The
A. tumefaciens
vector system. The T-DNA is removed from the Ti plasmid,
resulting in a vir plasmid, still containing the information to transfer DNA to plants. In this mutant strain, a
binary vector plasmid is introduced containing a T-DNA in which the transgene construct (horizontally
striped box) and the selectable marker (dotted box) are assembled. On contact with a plant cell, this
A.
tumefaciens
strain will selectively transfer only the T-DNA segment with the gene of interest and
selectable marker to the plant cell. (c) A simple transgene locus in a transformed plant, referred to as a
transformation event, different in every independently obtained transformant. Basically, the inserted
T-DNA segment contains a transgene encoding the trait of interest and a selectable marker gene. Both
genes are assembled to contain a promoter where transcription starts, the coding sequence for the
appropriate protein synthesis and a 3
terminator to stop transcription.
