Principles of Plant Genetics and Breeding

of genes of known functions. Three genes with on/off
switches are strategically engineered into the plant to
interact in a predetermined sequence, the last gene
becoming activated very late in seed development. At
this stage, the gene is turned on by an inducer, causing a
toxin to be produced that kills the embryo. The three
proposed approaches to accomplishing seed sterility are
described in Figure 14.2:
1 Transfection of target plant cells with three different
transgenes. Three different but functionally
related transcriptional units are used – a repressor
gene, a recombinase gene, and a toxin gene (Figure
14.2). The repressor gene codes for a protein called
recombinase inhibitory protein (RIP). A DNA fragment
that is a binding site to the repressor gene is
BIOTECHNOLOGY IN PLANT BREEDING 245
Transgenes Reaction for normal seed Reaction for sterilization
Transgene 1: Repressor system
Blocking sequence
5′ 3′
LEA RIP
LoxP LoxP
Transgene 2: Recombinase gene
5′ 3′
35S/3TetO
derepressible
CRE protein
cDNA
Transgene 3: Toxin gene
5′ 3′
Plant-active
promoter
TetR cDNA
TetR protein binds
Tet operators
CRE expression prevented
RIP not expressed
Seed is fertile
Seed treated with tet (tetracycline)
before selling to farmer
Inducers interfere with tetR
Recombinase (CRE) expression
LEA RIP
Figure 14.2 A diagrammatic presentation of how the technology protection system (TPS) works as described by Marvin
Oliver and colleagues. LEA (late embryogenesis abundant) promoter is derived from cotton. It is active only during the
late stages of embryogenesis. RIP (recombinase inhibitory protein) cDNA is derived from soapwort. It inhibits cellular
translation, resulting in cell death. Its expression is inhibited by the blocking sequence. LoxP sites are derived from
bacteriophage P1. It comprises direct repeats recognized by CRE (recombinase gene) protein and mediates the removal of
the blocker sequence. Column 1 shows three alternative genetic systems, while columns 2 and 3 show the application of
only one genetic system – the repressor system – in normal seed and in inducing seed sterility.
CRE
Blocker is excised
Floxing reaction
Embryo is killed before
seed matures
located between the promoter and the recombinase
gene. In the absence of an exogenous inducer, the
repressor binds to the binding site to prevent the
plant from producing the recombinase protein. The
toxin gene is controlled by a late promoter. A blocker
sequence is located between this promoter and the
toxin gene to interfere with the ability of the promoter
to turn the gene on. An inducer is needed to
release the recombinase enzyme that has the capacity
to snip out the blocker gene to allow the late promoter
to turn on the toxin gene.
2 Creation of a sterile hybrid. In this approach, two
fertile transgenic plants (A × B) containing two different
sets of transgenes are developed. Plant A has a
transcriptional unit made of the LEA (late embryogenesis
abundant) promoter, LoxP sequences, blocking