Molecular Approaches for the Improvement of Bacillus thuringiensis Against Pests 181
described (Vilas Boas et al. 2000 ). This is very
important from a biotechnological point of view
because many biopesticides can be created. For
example, scientists in Ecogen Corporation (USA)
have developed a broad spectrum Bt biopesticide
foil for controlling lepidopteran, coleopteran and
many plant pests through conjugal transfer and
plasmid curing. An important advantage of the
conjugational approach is that the transconju-
gants are treated as “non-genetically engineered”
and are, thus, subject to relatively simple regula-
tory registration. Strain improvement may also
entail elimination of any undesirable activities
through plasmid curing.
Transformation
Initially, transformation of Bt was only possible
using a protoplast, which yielded low transfor-
mant frequencies. This technique is largely inef-
ficient, complex and can probably not be applied
to most Bt strains. The preferred method of gene
transfer employs the electroporation technique,
for which numerous protocols are available,
where a high voltage electric discharge through
a cell suspension results in a transient increase
in permeability of the cell membrane and hence
DNA enters the cells and transformants can be
obtained within 24 h. Two plasmids can be trans-
formed simultaneously into a recipient using this
technique. The transformation frequencies are in
the range of 10^2 –10^5 colony forming units (cfu)/
μg, depending on the strain or the replicon used.
This technique is useful for the introduction of
cloned toxin genes, in either their native or modi-
fied form, into a variety of host strains, includ-
ing acrystalliferous strains. This allows to the
broadening of the insecticidal activity spectrum
of many strains, which can have biotechnologi-
cal applications against many plant pests. Bao
et al. ( 2009 ) applied Agrobacterium-mediated
genetic transformation to produce transgenic
plants of spinach ( Spinacia oleracea) resistant to
two pest species, Trichoplusia ni (Hubner) and
Autographa nigrisigna. They checked the effect
of Cry1Ac toxin on the development of Autog-
rapha nigrisigna larvae and observed that larvae
fed with non-transgenic plant developed into
pupal stage while the larvae fed with transgenic
plants died within 1 week (Table 1 , Fig. 1 ).
Mehrotra et al. ( 2011 ) modified cry1Ab and
cry1Ac insecticidal genes of Bt under the con-
trol of two different constitutive promoters and
introduced into chickpea ( Cicer arietinum L.)
by Agrobacterium-mediated transformation. One
hundred and eighteen stable transformed T0
plants as independent transformation events were
obtained expressing individual cry1Ab, cry1Ac,
or both pyramided genes for their co-expression
driven by either cauliflower mosaic virus 35S
promoter with duplicated enhancer (CaMV35S)
or synthetic constitutive promoter (Pcec) and
their combinations. Integration and inheritance of
transgenes in T0 and T1 population of transgenic
chickpea plants were determined by PCR, RT-
PCR and Southern hybridization. The performed
insect bioassay with modified transgenic plant
showed relatively higher toxicity for plants ex-
pressing Cry1Ac protein as compared to Cry1Ab
to Helicoverpa armigera (Hubner). Pyramided
transgenic plants with moderate expression levels
(15–20 ng mg−1 of TSP) showed high-level of re-
sistance and protection against pod borer larvae of
H. armigera as compared to high level expression
of a single toxin. These results have shown the
significance of pyramiding and co-expression of
two Cry toxins for efficient protection against lep-
idopteran pests of chickpea. Ibrahim et al. ( 2008 )
studied Cry1Ag-expressing BT 4 Bt strain caused
mortality of S. littoralis larvae only slightly (the
LC 50 was 104 ppm) and observed that in the
presence of only Cry1C, the LC 50 was 64 ppm.
In the presence of Cry1C co-expressed with
Cry1Ag, the LC 50 decreased to 2.2 ppm. Thus,
a combination of the Cry proteins 1C and 1AgInsects n Insect mortality (%)a
Control Cry1Ac
Trichoplusia ni 25 0 100
Autographa nigrisigna 30 10 93.3
a Data were recorded 7 days after feeding. (Bao et al. 2009 )Table 1 Bioassay of
transgenic plant against
Trichoplusia ni and Autog-
rapha nigrisigna