Title of Invention


Abstract Expression and stability of desirable proteins in transgenic plants are promoted and maintained by treatment with a protein transport enhancer. Preferably, the transgenic plant is a commodity crop that has been modified to express pesticidally effective protein proteins.
Full Text FORM 2
THE PATENT ACT 1970 (39 of 1970)
& The Patents Rules, 2003
(See section 10 and rule 13)
The following specification particularly describes the invention and the manner in which it is to be performed.

The invention relates to a method for the treatment of transgenic plants, especially crop plants that are designed to express pesticidally effective proteins.
Transgenic plants have had a significant impact on commercial agriculture, with promising benefits but raising new pest management issues. Notably, agricultural crops, based on plants, that have been modified with pesticidally effective crystal protein genes to express pesticidally effective proteins hold the promise of selectively targeting pests by making their food source (the plant tissues) toxic thereby reducing or eliminating the amount of chemical insecticides that are used to control pest populations.
Certain members of the gram positive bacteria belonging to the genus Bacillus produce proteins that are insecticidal. The most well characterized are those of Bacillus thuringiensis (BT), where the insecticidal proteins are found as crystalline bodies with sporulating bacteria. The insecticidal crystal proteins are characterised by their potency and specificity towards specific insect pests, many of which are agronemieaMy important; and their relative safety to non-target insect species and vertebrates, particularly humans. They have enjoyed a long history of use in horticultural industries where the mixture of crystals and spores are sprayed just like a chemical pesticide, but they have not been used with much success on broad-acre field crops.
The insecticidal crystals are composed of a large protein that is essentially inactive. When a caterpillar ingests some of the insecticidal crystals, the alkaline reducing conditions of the insects midgut cause the crystals to dissociate and release the crystal protein. At this stage the protein toxin is inactive, but specific proteases within the gastric juices of the insect chop the protein down to its protease resistant core that is now fully active. This activated insecticidal protein then binds to a specific

receptor on the brush border membranes of the cells lining the midgut and inserts itself into the cells membrane. When about eight of these aggregate together, they form a pore or channel through the membrane, and allow the cell contents to leak out causing the death of the cells essential for nutrient absorption. The insects rapidly stop feeding and eventually starve to death or die from secondary bacterial infections \vithin about 24 hours. The processes of crystal solublization, proteolytic processing to an active insecticidal protein and the binding to a specific receptor, all make the BT proteins highly specific and very desirable from an environmental perspective.
Many thousands of different isolates of B. thuringiensis have been collected and their insecticidal protein content and activity spectrums determined. A large number of BT-insecticidal protein genes have also now been cloned and sequenced. To avoid any confusion researchers have proposed a uniform naming system for the crystal protein genes (Cry genes) based on their protein sequence and the types of insects for which they were toxic.
Table 1

J Gene Type j Insect Host J Size(kD)
CrylA Caterpillars 133.2
Cry IB Caterpillars 138.0
CrylC Caterpillars 134.8
Cry HA Caterpillars/ 70.9
CryllB Fly larvae 70.8
CrylHA Beetle larvae 73.1
CrylVA Fly larvae 134-4

CryIVB Fly larvae 127.8
CryIVC Fly larvae 77.8
CiylVD Fly larvae 72.4
The proteins encoded by the Cry I genes are all similar in protein sequence and are toxic only to caterpillars, ie. the larvae of moths and butterflies (Lepidoptera); the Cry II genes encode proteins toxic to Lepidoptera and/or Diptera (flies and mosquitoes) while"the Cry IV proteins are only active against Diptera. Cry III genes produce proteins active against beetle (Coleoptera) larvae. Within these major groupings smaller divisions have been made by considering the similarities or differences between the different protein sequences. The Cry I group, for example was originally divided into Cry IA, IB and IC (although it is now up to Cry IG) where the different sub-groups may only be 50% identical at the protein sequence level. Finer sub-divisions have also been made and Cry IA now consists of Cry IA (a), IA (b) and IA(c).
A number of methods have been developed for introducing desired genes
into crop plant cells and growing fertile plants therefrom. See, US Patent No.
6,329,574 and attp;//www.eotton,picsiro the contents of
which are herein incorporated by reference. The method of choice depends in part upon tike target species, but cotton modification often rely on a natural gene transfer agent that has evolved its own method of plant genetic engineering. The disease called Crown Gall is a plant tumor disease caused by the soil-borne bacterial pathogen, Agrobacterium tumefaciens. In the early 70"s it was recognized that the bacterium caused the disease by transferring some of its own genetic material into the DNA of the plant cells that it infected. These parasitic genes subverted the normal biochemical machinery of the infected cells and caused them to make novel compounds that only

the bacterium could utilize. This process of genetic colonization by the bacterium was just what genetic engineers were looking for, provided that they could stop the bacterium causing the disease symptoms. After further study scientists were able to identify which genes caused the disease and because bacteria are much simpler organisms to genetically manipulate, they were able to replace the disease-causing genes with the novel genes they were constructing from parts of potentially useful genes. The bacterium could then be used to piggy-back genes from the test-tube into plant cells. However not all plant cells exposed to the bacterium eventually receive the novel genes. Removal of unmodified plant cells from modified cells uses antibiotic purification.
Plant cells are sensitive to many of the antibiotics that are used to control bacterial infections in animals and humans. If a gene could be isolated that gave the plant cells tolerance to one of these toxic antibiotics then if physically linked to some desirable gene and inserted into the Agrobacterium, it would provide a useful selection system to kill off those cells that don"t receive the genes during the "infection" process. Genes have been known in bacteria for many years that give the bacteria resistance to antibiotics by producing,enzymes~thatbreakdown or chemically modify the antibiotic so mat it is no longer toxic. Using the techniques of gene splicing described above researchers have been able to modify a bacterial gene that encodes an enzyme that detoxifies the antibiotic kanamycin and have produced a new hybrid gene that causes the production of this enzyme in plant cells and prevents their death in the presence of potentially lethal doses of kanamycin. Combining this antibiotic selection system with plant tissue culture procedures it has been possible to use Agrobacterium to deliver genes into a wide variety of plants from petunias to cottons.
Cotton is a crop of particular interest. Commercially available forms of transgenic cotton use the CrylAc (BOLLGARD™ by Monsanto) or a combination of CrylAc with Cry2Ab (BOLLGARD™ II by Monsanto) genes to express the endotoxin

protein of B. thuringiensis. Field efficacy reports indicate a 50-70% reduction in the amount of applied pesticide needed to control the pests Helicoverpa armigera and H. punctigera. Also of interest are crop plants modofied with the B.. thuringiensis crystal toxin genes designated cryET33 and cryET34 which encode the colepteran-toxic crystal proteins, CryET33 (29-kDa) crystal protein, and the cryET34 gene encodes the 14-kDa CryET34 crystal protein. The CryET33 and CryET34 crystal proteins are toxic to red flour beetle larvae and Japanese beetle larvae. (See, US Patent No. 6,399,330.)
The use of transgenic crop plants raises new issues in the ongoing struggle towards integrated pest management. Some of these issues concern a reduction in the amount of expressed endotoxin as the plants mature which leads to a loss of efficacy in the latter stages of the growing season (the last 1/3 of the cotton growing season) and me increased probability of surviving pests that can develop immunity to the endotoxin. Such drawbacks have lead to the development of pest control strategies that dictate a planting "window" relative to the development cycle of local pests and designated pest population minimum threshold values for pesticide application.
Physiological stress and physical damage to the transgenic plants can also result in a reduction of expressed endotoxin protein with a corresponding drop in pest control efficacy. Thus, an extended drought and/or high temperatures can reduce the endotoxin expression rate in the transgenic crop and provide a significant drop in pest protection that can dictate the need for pesticide spraying
The specific reasons for the drop in endotoxin protein expression are not well understood. In BT cotton, it is theorized that expression of the CrylAc gene drops because the CMV35S promoter concentration declines, the gene is "silenced, or other post-transcription events. It is also thought that the CrylAc protein is reduced due to increased turnover, sequestration within the plant, or dilution due to growth and aging. It is understood that CrylAc transription levels are unstable in both immature and mature BT cotton plants.

It would be desirable to have a system for treating transgenic plants designed to express pesticidally effective proteins that would promote the expression of these proteins despite increasing plant maturity, physiological stress, and physical damage.
It is an objective of the invention to provide a method for treating transgenic plants, preferably transgenic crops that express pesticidai proteins, and especially for transgenic crops that express insecticidal proteins.
It is anpther objective of the invention to provide a method for extending the period over which expressed proteins are present in sufficient quantity to control pest insect populations feeding on the treated plants.
m accordance with these and other objectives that will become apparent from the description herein, a method for treating transgenic crop plants according to the invention comprises applying to foliage of transgenic plants that are designed to express pesticdally effective proteins a protein transport enhancer that promotes the expression and/or stability of pesticidally effective proteins within the treated plants.
Although not wishing to be bound by any particular theory of operation, it is thought that the proteinjransport enhancer, acts in one or more of several wa vs-fo^ as a form of protective water substitute for cellular membranes during times of water deprivation stress, (b) as a protein stabilizer for the desired pesticidai protein, and/or (c) as a binder for proteins that facilitates movement via intiaplant transport mechanisms. The result is that transgenic crop plants treated according to the invention express and move pesticidally effective proteins into fruit tissues despite physiological stress from water shortage and plant damage. It is thought that the treatment according to the invention will also continue to express effective levels of pesticidai protein through plant growth and maturity.

Transgenic plants are treated, according to the invention, with a protein transport enhancer that stimulates and/or protects cellular expression and intraplant transport mechanisms sufficiently that desired levels of pesticidal protein proteins are maintained in plant tissues, fruits, and seeds despite water deprivation, physical damage to plant tissues, growth, and plant maturity. Maintenance of desired protein expression levels and concentrations of protein proteins within transgenic plant tissues help to maintain efficacy levels for better pest control, further reductions in amounts of applied pesticides now required to counteract reductions in efficacy, and should help to prevent survival of exposed pests and the development of resistant pest populations.
It will be understood that all percentages identified herein are by weight-with
respect to the total weight of product, unless otherwise noted.
Suitable protein transport enhancers for use in the present invention include one or more compounds and agrichemically acceptable salts of compounds according to the structure in Formula 1:
- . I-

In particular, moiety Y can be methyl, ethyl, propyl, butyl, iso-butyl, pentyl, hexyl, methoxy, ethoxy, propoxy, butoxy, iso-tutoxy, pentoxy„and hexoxy.
Preferably protein transport enhancers used in the present invention include one or more compounds and salts of compounds according to the above structure in

which X is a nitro group at the ortho or para positions relative to the hydroxy group, Y is hydrogen or a C1C3 alkyloxy, and Z is a carbon atom. Suitable salts include water soluble alkali metal salts (especially sodium and potassium salts), ammonium salts, and other water soluble salts that are not phytotoxic or of environmental concern.
The most preferred protein transport enhancer includes a combination of the
! sodium salts of p-nitrophenolate (A), o-nitrophenolate (B), and 2-methoxy-5-
i nitrophenolate (C). It is particularly preferred that the protein transport enhancer
contain a mixture of these salts in a range of ratios within the range of A: B: C of (0.1 -
10) :( 0.1-10):l. A commercial source of these salts is available under the name
ATONIK® Asahi Chemical Mfg. Co., Ltd. at a ratio of A: B: C of 2:3:1.
Protein transport enhancers according to the invention are applied at a rate generally less than 20 grams of each active ingredient per acre of treated field (gAI/ac). Preferably, these enhancers are applied at a rate within the range of 1-20 gAI/ac and most preferably at a rate within the range of 3-18 gAI/ac. It is especially preferred when salts of the protein transport enhancers are used within the range of 0.01 -5 wt% based on total weight and applied at a rate (combined) within the range of 0.5-20 fluid ounces per acre (oz/ac).
Protein transport enhancers according to the invention can be applied in combination with one or more active ingredients, spray aids, spreading agents, or additional- agents --suitable for agricultural use on the target plant Exemplary active ingredients that can be applied with the protein transport enhancer of the invention include herbicides, plant growth enhancing agents, plant growth stunting agents, foliar fertilizers, fungicides (external and systemic), and insecticides (external and systemic).
Herbicides that can be used include the trlazines (e.g., atrazine), the ureas, glyphosate, sulfosate, glyfosinate, and sethoxydim.
Suitable plant growth enhancing agents for the present invention include plant growth hormones such as at least one of the 84 identified gibberillins with GA3,

GA4, GA5, GA7 and GA9 being preferred; cytokinins (e.g., zeatin, kinetin, benzyladenine, dihydrozeatin, and isopentenyl adenine); auxins (e.g., indolacetic acid (IAA), indolebutyric acid (IBA), and naphthalenacetic acid (NAA)); and polyhydroxycarboxylic acids of 2, 4, 5, and 6 carbon structures; ethephon; and fertilizers.
Suitable plant growth stunting agents useful in the invention include chlormequat chloride, mepiquat chloride, as well as maleic hydrazide and its esters. Such plant growth regulators affect and alter plant metabolic processes to enhance or retard plant growth. All such agents can be used according to the appUcation rates and timing specified by the manufacturer on the product label.
Systemic fungicides that will benefit from the invention include tridemorph, metalaxyl, iprodione, fosetyl-aluminum, thiophanate, benomyl, triadimefon, carboxin, oxycarboxin, carbendazim, thiabendazole, thiophanate, ethirimol, bupirimate, and dimethirimol.
Suitable systemic insecticides include aldicarb, acephate, carbofuranj _ dimethoate, phorate, and terbufos.
The specific mechanisms by which the expression and transport mechanisms are effected are not well known. For example, tests on unmodified wheat with radiolabeled nitrophenolate salts have tracked the treatment agent to proteins transported to the seed kernel in wheat plants. Another study suggests that phenolic agents act as hydration agents for cellular membranes.
In cotton, it is recognized that cotton seed requires high amounts of protein, and seeds produce lint from carbohydrates within the plant system. Tests with ATONIK® on normal (i.e., not genetically modified or otherwise altered to express pesticidal proteins as a normal part of plant metabolism function) show an increased yield of seed and lint. Such an effect is consistent with action as a plant growth regulator, but does not necessarily suggest anything regarding the effect of treating

plants that have been genetically modified to produce a designated protein as part of the normal plant function.
Transgenic plants that can be treated according to the invention are generally those that have been modified from wild type to contain a gene that expresses a desired pesticidally effective protein sequence. Preferably, the transgenic plant includes genes that express pesticidally effective proteins that are effective to provide resistance against attacks or infections by insects, bacteria, fungus, mildew, mold, mites, and the like.
Two genes of particular effectiveness are the CrylAc gene (driven by the CMV35S promoter) and the Cry2Ab gene. These genes can be inserted individually or in combination into cotton, corn, wheat, sorghum, soybeans, and similar agricultural commodity crops to provide pesticidally effective protection against a variety of pest insects. Of special interest is cotton stock that has been modified to express pesticidally effective protein proteins (e.g., BT cotton). Seed stock for BT cotton that can be treated according to the invention is commercially available from several sources under the tradename BOLLGARD™ or BOLLGARD™ II (Monsanto Co.).
Proteintransport enhancers according to the invention are applied to transgenic plants at rates and times in the plant"s growing cycle or season so as to maintain pesticide protein expression and stabilization within plant tissues. The table below identifies preferred transgenic plants to be treated and the amount of protein transport enhancer to be applied within generally useful, preferred, and more preferred application rates.
Table 1

Bt cotton 1-100 1-50 5-30

Btcorn 1-100 1-50 5-30
Transgenic Bt cotton (Deltapine Nucotn B) and unmodified cotton plants
in two liter pots in a controlled environment were sprayed with ATONIK®, a
commercially available product containing a mixture of agents thought to be general
growth regulating agentsjmt which appears to actwell as protein transport enhancers
for genetically modified commodity crop plants. ATONIK® contains the sodium salts
of p-nitrophenolate (0.3%), o-nitrophenolate (0.2%), and 5-nitroguaiacolate (0.1%).
Cotton plants were treated at at 7th true leaf (TL) and sampled 10 days later for the
upper expanded main stem leaf. The temperature was maintained within the range of
76-86°F with adequate watering. After sampling (7th TL +10 days), the plants were
again sprayed with ATONIK® and subjected to stress from an elevated temperature
(86-102°F) and inadequate water supply (drought conditions) until they were sampled
5 days later (7th TL +15 days) for expanded upper main stem leaf. Five days thereafter
(7th TL +20 days) under the same conditions, the plants were sampled for leaf and
squares. t
After each sampling, tissue samples were placed in sealed bag and immediately taken to a facility for bollworm feeding tests. Bollworm mortality was measured at 24, 48, 72 and 96 hrs from initiation of feeding for the first spray application samples and at 72 and 96 hrs for the 2nd spray application samples.
Once the mortality study was completed, a leaf profile from representative cotton plants was taken for protein analysis. Leaves were collected at nodes 2, 6, 8, and 10 (counting from the top) and stored at -80° F.
The bollworm mortality for collected leaves in the first collected sample, (7th TL +10 days) are shown in Table 2.
Table 2 12

The bollworm mortality from squares collected at 7 TL + 20 days (2" treatment) spraying are reported in Table 4.

The results show that treatment with the protein transport enhancer according to the invention resulted in higher bollworm mortality under both pptimum growth conditions (Table 2) and under stress conditions from both temperature and

1 A method for promoting the pesticidai efficacy of transgenic plants that include a
gene which expresses a pesticidally effective protein, said method comprising:
treating said transgenic plants with a mixture of a protein transport enhancer comprising a mixture of phenolate and nitroguaiacolate salts and at least one selected from the group consisting of plant growth enhancing agents, herbicides, plant growth stunting agents, foliar fertilizers, fungicides and insecticides to stabilize the transport of pesticidally effective proteins within said plant.
2. A method according to claim 1 wherein said transgenic plant is cotton or corn.
3. A method according to claim 1 wherein said transgenic plant is cotton that has been modified to express B. thuringiensis protein.
4. A method according to claim 1 wherein said protein transport enhancer comprises a mixture of sodium salts of p-nitrophenolate, o-nitrophenolate, and 2-methoxy-5-nitrophenolate.
5. A method according to claim 4 wherein said protein transport enhancer is applied to said plants at a rate within the range of 1-100 oz/acre.
6. A method according to claim 5 wherein said protein transport enhancer is applied to said plants at a rate within the range of 1-50 oz/acre.
7. A method according to claim 6 wherein said protein transport enhancer is applied to said plants at a rate within the range of 5-30 oz/acre.
8. A method according to claim 1 wherein said protein transport enhancer comprises polyols obtained from reduction of aldo- and keto- groups in a carbohydrate.

9. t A method according to claim 1, wherein said protein transport enhancer is a
mixture of the sodium salt of p-nitrophenolate, o-nitrophenolate, and 2-methoxy-5-nitrophenolate in a ratio of (0.1-10):(0.1-10):1.

Dated this 28m day of January 2005.





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Patent Number 206570
Indian Patent Application Number 89/MUMNP/2005
PG Journal Number 30/2007
Publication Date 27-Jul-2007
Grant Date 03-May-2007
Date of Filing 31-Jan-2005
Applicant Address 4748 COUNTRY ROAD 3667, PORTLAND, TX 78374. U.S.A.
# Inventor's Name Inventor's Address
1 LACKEY, James P.O.BOX 193, TAFT, TX 78390. U.S.A.
PCT International Classification Number A01N33/22
PCT International Application Number PCT/US2003/023823
PCT International Filing date 2003-07-31
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10/429,974 2003-05-06 U.S.A.
2 60/399,716 2002-08-01 U.S.A.