182 A. Patel et al.
could result in effective insect control. With this
approach, a combination of Cry proteins can be
designed rather than discovered (Fig. 2 , Table 2 ).
Genetic Recombination
To avoid segregation or structural instability of
plasmids in the recombinant strain, strategies for
the integration of genes into resident plasmids or
into the chromosome of the desired strain by in
vivo homologous recombination were developed
with the help of integration vectors. Since 1991,
genetic recombination has been used as a tech-
nique to improve Bt strains. The integration via
single or double crossover can occur by homolo-
gous recombination between the resident plasmid
and the Bt subsp. israelensis. DNA fragments
flanking the erm gene harbored in the integration
plasmid. A very low frequency of recombination
was observed for this event; this is not surprising,
since the integration of the nonreplicative plas-
mid is the result of both a transformation and a
recombination event between two different plas-
mids. Due to low frequency, the insecticidal host
range of Bt, transformation and recombination
were uncoupled using a thermo sensitive plasmid
as integrative vector.
Another tool for the recombination in Bt
strains was the deployment of a site-specific re-
combination (SSR) system, to selectively delete
ancillary or foreign DNA elements from recom-
binant plasmids after their introduction into a Bt
host. The SSR system is useful for engineering
strains with unique combinations of cry genes,
resulting in new active ingredients with improved
insecticidal properties. This system consists in
Tn5401, which is a transposable element indig-
enous to the Bt subsp. morrisoni strain EG2158.
This transposon encodes a transposase protein
(TnpA), a recombinase protein (TnpI) and a site-
specific recombination site, or internal resolution
site (IRS) which is required for Tn5401 transposi-
tion in Bt (Baum et al. 1999 ). Transposon Tn4430
was used to eliminate in vivo unwanted DNA se-
quences from transforming vectors harboring two
IRSs. The transposon vector pEG922, containing
transposon Tn5401 was used to disrupt the spo0F
gene. In addition, a native Bt plasmid replicon
was combined with an indigenous site specific re-
combination system that allowed for the selective
removal of foreign DNA from the recombinant
bacterium after introduction of the Cry-encoding
plasmid vector. In this way, a coleopteran-active
strain, approved as the native ingredient for Raven
OF bioinsecticide, was constructed. The lethal
concentration 50 % (LC 50 ) of G033A against S.
exigua, P. xylostella, and H. amigera was 4.26,
0.86, and 1.76 μg/ml, respectively (Table 3 ).Fig. 1 Effect of Cry1Ac toxin on development of Autographa nigrisigna larvae. A larva developed into pupal stage
when fed with leaves of control non-transgenic plant (a), whereas the larvae fed with leaves of transgenic plants died
within 1 week of feeding (b). Arrows indicated dying larvae. (Bao et al. 2009 )