Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1998-10-22
2001-01-09
Nelson, Amy (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S279000
Reexamination Certificate
active
06172281
ABSTRACT:
This invention relates to plant cells and plants, the genomes of which are transformed to contain at least two genes, each coding for a different non-competitively binding
Bacillus thuringiensis
(“
B.thuringiensis
” or “Bt”) insecticidal crystal protein (“ICP”) for a specific target insect species, preferably belonging to the order of Lepidoptera or Coleoptera. Such transformed plants have advantages over plants transformed with a single
B. thuringiensis
ICP gene, especially with respect to the prevention of resistance development in the target insect species against the at least two
B. thuringiensis
ICPs, expressed in such plants.
This invention also relates to a process for the production of such transgenic plants, taking into account the competitive and non-competitive binding properties of the at least two
B. thuringiensis
ICPs in the target insect species' midgut. Simultaneous expression in plants of the at least two genes, each coding for a different non-competitively binding
B. thuringiensis
ICP in plants, is particularly useful to prevent or delay resistance development of insects against the at least two
B. thuringiensis
ICPs expressed in the plants.
This invention further relates to a process for the construction of novel plant expression vectors and to the novel plant expression vectors themselves, which contain the at least two
B. thuringiensis
ICP genes encoding the at least two non-competitively binding
B. thuringiensis
ICPs. Such vectors allow integration and coordinate expression of the at least two
B. thuringiensis
ICP genes in plants.
BACKGROUND OF THE INVENTION
Since the development and the widespread use of chemical insecticides, the occurrence of resistant insect strains has been an important problem. Development of insecticide resistance is a phenomenon dependent on biochemical, physiological, genetic and ecological mechanisms. Currently, insect resistance has been reported against all major classes of chemical insecticides including chlorinated hydrocarbons, organophosphates, carbamates, and pyrethroid compounds (Brattsten et al., 1986).
In contrast to the rapid development of insect resistance to synthetic insecticides, development of insect resistance to bacterial insecticides such as
B. thuringiensis
sprays has evolved slowly despite many years of use (Brattsten et al., 1986). The spore forming gram-positive bacterium B. thuringiensis produces a parasporal crystal which is composed of crystal proteins (ICPs) having insecticidal activity. Important factors decreasing the probability of emergence of resistant insect strains in the field against
B. thuringiensis
sprays are: firstly the short half-life of
B. thuringiensis
sprays after foliar application; secondly the fact that commercial
B. thuringiensis
preparations often consist of a mixture of several insecticidal factors including spores, ICPs and eventually beta-exotoxins (Shields, 1987); and thirdly the transitory nature of plant-pest interactions. Many successful field trials have shown that commercial preparations of a
B. thuringiensis
containing its spore-crystal complex, effectively control lepidopterous pests in agriculture and forestry (Krieg and Langenbruch, 1981).
B. thuringiensis
is at present the most widely used pathogen for microbial control of insect pests.
Various laboratory studies, in which selection against
B. thuringiensis
was applied over several generations of insects, have confirmed that resistance against
B. thuringiensis
is seldom obtained. However, it should be emphasized that the laboratory conditions represented rather low selection pressure conditions.
For example, Goldman et al. (1986) have applied selection with
B. thuringiensis israelensis
toxin over 14 generations of
Aedes aegypti
and found only a marginal decrease in sensitivity. The lack of any observable trend toward decreasing susceptibility in the selected strains may be a reflection of the low selection pressure (LC
50
) carried out over a limited number of generations. However, it should be pointed out that Georghiou et al. (In: Insecticide Resistance in Mosquitoes: Research on new chemicals and techniques for management. In “Mosquito Control Research, Annual Report 1983, University of California.”) with
Culex guinguefasciatus
obtained an 11-fold increase in resistance to
B. thuringiensis israelensis
after 32 generations at LC
95
selection presssure.
McGaughey (1985) reported that the grain storage pest
Plodia interpunctella
developed resistance to the spore-crystal complex of
B. thuringiensis
; after 15 generations of selection with the Indian meal moth,
Plodia interpunctella,
using a commercial
B. thuringiensis
HD-1 preparation (“Dipel”, Abbott Laboratories, North Chicago, Ill. 60064, USA), a 100-fold decrease in
B. thuringiensis
sensitivity was reported. Each of the colonies was cultured for several generations on a diet treated with a constant
B. thuringiensis
dosage which was expected to produce 70-90% larval mortality. Under these high selection presssure conditions, insect resistance to
B. thuringiensis
increased rapidly. More recently, development of resistance against
B. thuringiensis
is also reported for the almond moth,
Cadra cautella
(McGaughey and Beeman, 1988). Resistance was stable when selection was discontinued and was inherited as a recessive trait (McGaughey and Beeman, 1988). The mechanism of insect resistance to
B. thuringiensis
toxins of
Plodia interpunctella
and
Cadra cautella
has not been elucidated.
The main cause of
B. thuringiensis
resistance development in both reported cases involving grain storage was the environmental conditions prevailing during the grain storage. Under the conditions in both cases, the environment was relatively stable, so
B. thuringiensis
degradation was slow and permitted successive generations of the pest to breed in the continuous presence of the microbial insecticide. The speed at which Plodia developed resistance to
B. thuringiensis
in one study suggests that it could do so within one single storage season in the bins of treated grain.
Although insect resistance development against
B. thuringiensis
has mostly been observed in laboratory and pilot scale studies, very recent indications of
B. thuringiensis
resistance development in
Plutella xlostella
populations in the (cabbage) field have been reported (Kirsch and Schmutterer, 1988). A number of factors have led to a continuous exposure of
P. xlostella
to
B. thuringiensis
in a relatively small geographic area. This and the short generation cycle of
P. xylostella
have seemingly led to an enormous selection pressure resulting in decreased susceptibility and increased resistance to
B. thuringiensis.
A procedure for expressing a
B. thuringiensis
ICP gene in plants in order to render the plants insect-resistant (European patent publication (“EP”) 0193259 [which is incorporated herein by reference]; Vaeck et al., 1987; Barton et al., 1987; Fischhoff et al., 1987) provides an entirely new approach to insect control in agriculture which is at the same time safe, environmentally attractive and cost-effective. An important determinant for the success of this approach will be whether insects will be able to develop resistance to
B. thuringiensis
ICPs expressed in transgenic plants (Vaeck et al., 1987; Barton et al., 1987; Fischhoff et al., 1987). In contrast with a foliar application, after which
B. thuringiensis
ICPs are rapidly degraded, the transgenic plants will exert a continuous selection pressure. It is clear from laboratory selection experiments that a continuous selection pressure has led to adaptation to
B. thuringiensis
and its components in several insect species. In this regard, it should be pointed out that the conditions in the laboratory which resulted in the development of insect-resistance to
B. thuringiensis
are very similar to the situation with transgenic plants which produce
B. thuringiensis
ICPs and provide a continuous selection pressure on insect populations feeding on the plants. M
Botterman Johan
Joos Henk
Van Mellaert Herman
Van Rie Jeroen
Aventis CropScience N.V.
Burns Doane Swecker & Mathis L.L.P.
Nelson Amy
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