Principles of Plant Genetics and Breeding

pathogen to give resistance), was cloned in 1993 by
Greg Martin.
Engineering insect resistance
There are two basic approaches to genetic engineering
of insect resistance in plants:
1 The use of protein toxins of bacterial origin.
2 The use of insecticidal proteins of plant origin.
Protein toxins from Bacillus thuringiensis (Bt)
The Bacillus thuringiensis (Bt) endotoxin is a crystalline
protein. It was first identified in 1911 when it was
observed to kill larvae of the flour moth. It was registered
as a biopesticide in the USA in 1961. Bt is very
selective in action, that is, one strain of the bacterium
kills only certain insects. Formulations of whole sporulated
bacteria are widely used as biopesticide sprays for
biological pest control in organic farming. There are
several major varieties of the species that produce spores
for certain target pests: B. thuringiensis var. kurstaki
(for controlling lepidopteran pests of forests and agriculture),
var. brliner (wax moth), and var. israelensis
(dipteran vectors of human disease). The most commercially
important type of the crystalline proteinaceous
inclusion bodies are called δ-endotoxins. To become
toxic, these endotoxins, which are predominantly protoxins,
need to be proteolytically activated in the midgut
of the susceptible insect to become toxic to the insect.
These endotoxins act by collapsing the cells of the lining
of the gut regions.
Bt resistance development has been targeted especially
at the European corn borer, which causes significant
losses to corn in production. Previous efforts
developed resistance in tobacco, cotton, tomato, and
other crops. The effort in corn was more challenging
because it required the use of synthetic versions of the
gene (rather than microbial Bt per se) to be created.
Two genes, cryB1 and cryB2, were isolated from B.
thuringiensis subsp. kurstaki HD-1. These genes were
cloned and sequenced. The genes differed in toxin
specificities, the cryB1 gene product being toxic to both
dipteran (Aedes aegypti) and lepidoteran (Manduca
sexta) larvae, while cryB2 affects only the latter. The Bt
toxin is believed to be environmentally safe as an insecticide.
In engineering Bt resistance in plants, scientists
basically link the toxin to a constitutive (unregulated)
promoter that will express the toxin systemically (i.e., in
all tissues).
BREEDING FOR RESISTANCE TO DISEASES AND INSECT PESTS 377
Transgenic plants expressing the δ-endotoxin gene
have been developed. The first attempt involved the
fusion of the Bt endotoxin to a gene for kanamycin
resistance to aid in selecting plants (conducted by a
Belgian biotechnology company, Plant Genetic Systems,
in 1987). Later, Monsanto Company researchers expressed
a truncated Bt gene in tomato directly by using
the CaMV 35S promoter. Agracetus Company followed
with the expression of the Bt endotoxin in tobacco with
the CaMV 35S promoter linked to an alfalfa mosaic virus
(AMV) leader sequence. Since these initial attempts were
made, modifications to the protocols have increased
expression of the toxin in transgenic plants by several
hundred-fold. Transformation for expressing the chimeric
Bt genes was Agrobacterium-mediated, using the TR2′
promoter. This promoter directs the expression of
manopine synthase in plant cells transformed with the
TR-DNA of plasmid pTiA6.
The original Bt coding sequence has since been
modified to achieve insecticidal efficacy. The complete
genes failed to be fully expressed. Consequently,
truncated (comprising the toxic parts) genes of Bt var.
kurstaki HD-1 (cry1A[b]) and HD-73 (cry1A[c]) were
expressed in cotton against lepidopteran pests. In truncating
the gene, the N-terminal half of the protein was
kept intact. For improved expression, various promoters,
fusion proteins, and leader sequences have been used.
The toxin protein usually accounts for about 0.1% of the
total protein of any tissue, but this concentration is all
that is needed to confer resistance against the insect pest.
Genetically engineered Bt resistance for field application
is variable. For example, Ciba Seed Company has
developed three versions of synthetic Bt genes capable
of selective expression in plants. One is expressed only in
pollen, another in green tissue, and the third in other
parts of the plant. This selectivity is desirable for several
reasons. The European corn borer infestation is unpredictable
from year to year. The life cycle of the insect
impacts the specific control tactic used. The insect attack
occurs in broods or generations. The Bt genes with
specific switches (pollen and green tissue) produce the
Bt endotoxin in the plant parts that are targets of attack
at specific times (i.e., first and second broods). This
way, the expression of the endotoxin in the seed and
other parts of the plant where protection is not critical
is minimized. Monsanto’s “YieldGard” corn produces
Bt endotoxin throughout the plant, and protects against
both first and second broods of the pest. The commercially
available Bt corn cultivars were developed by
different transformation events, each with a different
promoter.