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
1998-11-04
2002-12-03
Nelson, Amy J. (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
C435S419000, C536S023710, C800S279000, C800S320100
Reexamination Certificate
active
06489542
ABSTRACT:
1.0 BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates generally to transgenic plants having insecticidal capabilities, and to DNA constructs utilized to transfer genes conferring insect resistance into plant genomes. More specifically, the present invention relates to a method of expressing insecticidal proteins in plants transformed with a
B. thuringiensis
&dgr;-endotoxin encoding gene, resulting in effective control of susceptible target pests.
1.2 Description of Related Art
1.2.1 Methods of Controlling Insect Infestation in Plants
The Gram-positive soil bacterium
B. thuringiensis
is well known for its production of proteinaceous parasporal crystals, or &dgr;-endotoxins, that are toxic to a variety of Lepidopteran, Coleopteran, and Dipteran larvae.
B. thuringiensis
produces crystal proteins during sporulation which are specifically toxic to certain species of insects. Many different strains of
B. thuringiensis
have been shown to produce insecticidal crystal proteins. Compositions comprising
B. thuringiensis
strains which produce proteins having insecticidal activity have been used commercially as environmentally-acceptable topical insecticides because of their toxicity to the specific target insect, and non-toxicity to plants and other non-targeted organisms.
&dgr;-endotoxin crystals are toxic to insect larvae by ingestion. Solubilization of the crystal in the midgut of the insect releases the protoxin form of the &dgr;-endotoxin which, in most instances, is subsequently processed to an active toxin by midgut protease. The activated toxins recognize and bind to the brush-border of the insect midgut epithelium through receptor proteins. Several putative crystal protein receptors have been isolated from certain insect larvae (Knight et al., 1995; Gill et al., 1995; Masson et al., 1995). The binding of active toxins is followed by intercalation and aggregation of toxin molecules to form pores within the midgut epithelium. This process leads to osmotic imbalance, swelling, lysis of the cells lining the midgut epithelium, and eventual larvae mortality.
1.2.2 Transgenic
B. thuringiensis
&dgr;-Endotoxins as Biopesticides
Plant resistance and biological control are central tactics of control in the majority of insecticide improvement programs applied to the most diverse crops. With the advent of molecular genetic techniques, various &dgr;-endotoxin genes have been isolated and their DNA sequences determined. These genes have been used to construct certain genetically engineered
B. thuringiensis
products that have been approved for commercial use. Recent developments have seen new &dgr;-endotoxin delivery systems developed, including plants that contain and express genetically engineered &dgr;-endotoxin genes. Expression of
B. thuringiensis
&dgr;-endotoxins in plants holds the potential for effective management of plant pests so long as certain problems can be overcome. These problems include the development of insect resistance to the particular Cry protein expressed in the plant, and development of morphologically abnormal plants because of the presence of the transgene.
Expression of
B. thuringiensis
&dgr;-endotoxins in transgenic cotton, corn, and potatoes has proven to be an effective means of controlling agriculturally important insect pests (Perlak et al., 1990; Koziel et al., 1993; Perlak et al., 1993). Transgenic crops expressing
B. thuringiensis
&dgr;-endotoxins enable growers to significantly reduce the application of costly, toxic, and sometimes ineffective topical chemical insecticides. Use of transgenes encoding
B. thuringiensis
&dgr;-endotoxins is particularly advantageous when insertion of the transgene has no negative effect on the yield of desired product from the transformed plants. Yields from crop plants expressing certain
B. thuringiensis
&dgr;-endotoxins such as Cry1A or Cry3A have been observed to be equivalent or better than otherwise similar non-transgenic commercial plant varieties. This indicates that expression of some
B. thuringiensis
&dgr;-endotoxins does not have a significant negative impact on plant growth or development. This is not the case, however, for all
B. thuringiensis
&dgr;-endotoxins that may be used to transform plants.
The use of topical
B. thuringiensis
-derived insecticides may also result in the development of insect strains resistant to the insecticides. Resistance to Cry1A
B. thuringiensis
&dgr;-endotoxins applied as foliar sprays has evolved in at least one well documented instance (Shelton et al., 1993). It is expected that insects may similarly evolve resistance to
B. thuringiensis
&dgr;-endotoxins expressed in transgenic plants. Such resistance, should it become widespread, would clearly limit the commercial value of corn, cotton, potato, and other germplasm containing genes encoding
B. thuringiensis
&dgr;-endotoxins. One possible way to both increase the effectiveness of the insecticide against target pests and to reduce the development of insecticide-resistant pests would be to ensure that transgenic crops express high levels of
B. thuringiensis
&dgr;-endotoxins (McGaughey and Whalon, 1993; Roush, 1994).
In addition to producing a transgenic plant which expresses
B. thuringiensis
&dgr;-endotoxins at high levels, commercially viable
B. thuringiensis
genes must satisfy several additional criteria. For instance, expression of these genes in transgenic crop plants must not reduce the vigor, viability or fertility of the plants, nor may it affect the normal morphology of the plants. Such detrimental effects have two undesired results: they may interfere with the recovery and propagation of transgenic plants; they may also impede the development of mature plants, or confer unacceptable agronomic characteristics.
There remains a need for compositions and methods useful in producing transgenic plants which express
B. thuringiensis
&dgr;-endotoxins at levels high enough to effectively control target plant insect pests as well as prevent the development of insecticide-resistant pest strains. A method resulting in higher levels of expression of the
B. thuringiensis
&dgr;-endotoxins will also provide the advantages of more frequent attainment of commercially viable transformed plant lines and more effective protection from infestation for the entire growing season.
There also remains a need for a method of increasing the level of expression of
B. thuringiensis
&dgr;-endotoxins which does not simultaneously result in plant morphological changes that interfere with optimal growth and development of desired plant tissues. For example, the method of potentiating expression of the
B. thuringiensis
&dgr;-endotoxins in corn should not result in a corn plant which cannot optimally develop for cultivation. Achievement of these goals such as high expression levels as well as recovery of morphologically normal plants has been elusive, and their pursuit has been ongoing and an important aspect of the long term value of insecticidal plant products.
2.0 SUMMARY OF THE INVENTION
Described are novel methods for expressing Cry2A
B. thuringiensis
&dgr;-endotoxins which lack significant Dipteran inhibiting activity in transformed plants. This method advantageously results in both increased levels of expression of
B. thuringiensis
&dgr;-endotoxins as well as a higher rate of recovery of morphologically-normal plants.
By achieving high rates of expression, the present invention addresses another limitation of the prior art: development of insect resistance. Specifically, the instant invention provides a superior strategy for the delay or elimination of the development of resistance to Cry1A &dgr;-endotoxins, the
B. thuringiensis
proteins most commonly expressed by transgenic lines. The disclosed methods involve expression of the Cry2A class of
B. thuringiensis
&dgr;-endotoxins and particularly those that lack Dipteran-inhibiting activity.
B. thuringiensis
&dgr;-endotoxins of the Cry2A group have no significant homology to Cry1A-type &dgr;-endotoxins and display disti
Corbin David R.
Romano Charles P.
Ball Timothy K.
Hoerner, Jr. Dennis R.
Monsanto Technology LLC
Nelson Amy J.
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