WO1991003157A1

WO1991003157A1 - Monocotyledon crop plants resistant to cyclohexanedione and aryloxy(amino)-phenoxypropionate herbicides

Info

Publication number: WO1991003157A1
Application number: PCT/AU1990/000389
Authority: WO
Inventors: Prem Bhalla; Anne Campbell
Original assignee: Ici Australia Operations Proprietary Limited
Priority date: 1989-08-31
Filing date: 1990-08-31
Publication date: 1991-03-21
Also published as: ZA906806B

Abstract

Herbicide resistant monocotyledon crop plant or seed thereof selected from wheat, barley, sorghum, rice, oats, rye, millet and other cereal crops. The monocotyledon crop plants are resistant to herbicides selected from the cyclohexanedione and aryloxy(amino)phenoxy propionate classes.

Description

M-trøDmEDON CROP PLANTS RESISTANT TO CraLOEXANEDICNE AND AEYLQXy (A_l__NO)-_?HENCiXyPEDPIONAIE HERBICIEES

This invention relates to herbicide-resistant plants, plant tissues and seeds. In particular, the invention relates to agronomically important monocotyledon crops which are resistant to herbicides.

Herbicides have been used for decades for controlling weeds or unwanted plants in crops. Present day herbicides used singly or in so-called tank mixes require efficient management to be effective. The time and method of application and stage of weed plant development are critical to the achievement of weed control with herbicides. Some weed species are resistant to present day herbicides and this has led to much research into the development of new herbicides which are capable of more effective weed control. Application of large doses of moderately effective herbicides on resistant weeds can result in a commitment to grow the same crop in subsequent years because of chemical persistence in the soil which prevents crop rotation with a crop sensitive to that herbicide. Herbicides with great potency, a broad weed spectrum of activity and more rapid degradation in the soil would have a significant impact on these problems, however such compounds tend to kill all plants. Crop hybrids or varieties with resistance to such herbicides provide an attractive solution by allowing such herbicides to be used without risk of damage to the crop.

Several workers in the art have produced plants by cell and tissue culture techniques in dicotyledon plants (Yamada, 1977, in Plant Cell, Tissue and Organ Culture eds. Rinert and Bajaj, ppl44-159, Springer-Verlag, Berlin) yet relatively few other workers have studied or produced monocotyledon plants using cell and tissue culture techniques.

PCT Application No. WO 88/09612 describes a method for producing rice plants and seeds which are resistant to the herbicide bensulfuron methyl. This application teaches the use of cell culture techniques in obtaining bensulfuron-resistant rice plants. AU 39507/85 teaches that cell culture techniques utilising suspension cultures of maize cells have been employed in generating maize which is resistant to herbicidal sulphonamides and i idazol inones or derivatives thereof. The site of action of these latter two herbicides is thought to be the enzyme acetohydroxyacid synthase (AHAS) and that application describes that the enzyme in the so-generated maize plant is not inhibited by the application of herbicide at concentrations which normally inhibit that enzyme's activity. A further problem arises in growing crops of monocotyledon plants when the weeds themselves are monocotyledons, such as grasses and the like. Invading grasses grow quickly and compete vigorously with the crop plant for space.

Therefore an object of the present invention is to provide a monocotyledon crop plant resistant to herbicides. A further object is to generate a herbicide-resistant monocotyledon plant utilising plant cell culture techniques. While plant cell culture techniques have been used to generate herbicide-resistant crop strains before, as mentioned above, it is not possible to predict that any plant cell culture may be manipulated to produce any herbicide-resistant plant by simple addition of herbicide to cell culture since in practice only some plant cells in culture will enable generation of herbicide-resistant plants.

Examples of failure to generate herbicide resistance in plants abound in the literature. For instance, regeneration of herbicide-resistant variants in, for example, alfalfa, soybean and rapeseed were not obtained using the herbicides, asula , paraquat and atrazine (Meredith C.P. et al (1982) Herbicide Resistance in Plant Cell Cultures, in: Herbicide Resistance in Plants, (HM Lebaron and J. Gressel, eds, pp 275-291 Wiley, New York). The cyclohexanedione or aryloxy(amino)phenoxypropionate classes of herbicides are particularly potent classes of monocotyledon plant herbicides which are thought to act on a key enzyme involved in lipid synthesis, namely acetyl CoA carboxylase. This enzyme is found inter alia in the plant chloroplast and is known to be essential for life. What we have surprisingly found is that we have been able to generate monocotyledon crop plants from plant tissue culture which are resistant or tolerant to the cyclohexanedione and/or aryloxy(amino)phenoxy propionate classes of herbicides. This is surprising because these two classes of herbicide are very efficient at killing all types of monocotyledons and the ability to confer herbicide resistance to monocotyledons in respect of these classes of herbicides did not appear to be generally practicable. However as discussed hereinabove we have now been able to demonstrate that conferring of herbicide resistance to monocotyledon plants is practicable.

Accordingly, the present invention provides a herbicide-resistant monocotyledon crop plant or seed thereof selected from wheat, barley, sorghum, rice, oats, rye, millet and other cereal crops wherein growth and development are substantially unaffected by the application of monocotyledon-selective herbicides selected from the cyclohexanedione and aryloxy(amino)phenoxy propionate classes.

Preferably, the monocotyledon crop plant is selected from wheat, oats or barley and more preferably is selected from the wheat family, and most preferably is of the "Millewa" variety.

A "herbicide-resistant monocotyledon plant" is one in which growth and development parameters are not significantly affected by the application of herbicide to it. Without the intention of being bound by theory, one way in which herbicide resistance is thought to be conferred on plants is through spontaneous genetic mutation which gives rise to subtle differences in genetic coding for protein products. Such protein products may be altered through transcriptional and translational process steps thereby causing variations which may affect the product protein's function. Thus if the conformational or functional shape of an enzyme essential for life is altered, say one involved in lipid synthesis in the chloroplast in plants, for example acetyl CoA carboxylase, then lipid synthesis may be impeded or arrested and the plant would die. Such a change in conformational shape may provide a possible reason why a potent cyclohexanedione herbicide fatal to a plant, would have no significant effect on a tolerant plant: the conformational shape of the herbicide may be unable to interfere with, say, the catalytic function of the enzyme by competing with a substrate.

Alternatively, herbicide resistance may be conferred by over-expression of an enzyme to such a level that an amount of applied herbicide may not be sufficient to occupy all the binding sites on the available enzyme.

A further mechanism by which herbicide resistance may be conferred is through biochemical degradation of the herbicide before it can affect a target enzyme. It is also possible that herbicides may affect genes which act to switch-on or switch-off gene expression which may give rise to cellular products which are able to directly or indirectly attack herbicides or block their action. Herbicide resistance may also be as a result of the simple physical inability of the herbicide to invade plant tissue.

Typically, the dose rate of the applied herbicide is at any dose rate which may be applied in the field, though it is noted that for the purposes of the present invention, the dose rates employed may be at concentrations in excess of those applied in the field. "Monocotyledon selective herbicide of the cyclohexanedione or aryloxy(ami no)phenoxy propionate classes" may be any member from those classes, however preferably the herbicide is selected from tralkoxydim or 2-Cl-(ethoxyimino)propyl ]-3- hydroxy-5-(3- butyrylmesityl )cyclohex-2-enone. Most preferably the herbicide is 2-Cl-(ethoxyimino) propyll-3-hydroxy-5-(3-butyrylmesityl )cyclohex- 2-enone. In a preferred embodiment of the present invention there is provided a herbicide-resistant monocotyledon crop plant or seed thereof selected from wheat, barley, sorghum, rice, oats, rye, millet and other cereal crops wherein the biochemical and physiological mechanisms of the said crop plant are capable of responding to an applied monocotyledon - selective herbicide selected from the cyclohexane dione or aryloxy(amino)phenoxy propionate classes such that the herbicidal action of the said monocotyledon -selective herbicide is at least substantially reduced.

Typically, the physiological and biochemical mechanisms act so as to degrade herbicide to substantially harmless components. In a further preferred embodiment of the present invention, there is provided a herbicide-resistant monocotyledon crop plant selected from wheat, barley, sorghum, rice, oats, rye, millet and other cereal crops wherein the physiological function of an enzyme involved in lipid synthesis is substantially unimpaired by the action of a monocotyledon-selective herbicide of the cyclohexanedione or aryloxy(amino)phenoxy propionate class. Preferably, the enzyme is one essential for life, and most preferably the enzyme is acetyl CoA carboxylase. In a further preferred embodiment of the present invention, there is provided a herbicide- resistant monocotyledon crop plant or seed thereof selected from wheat, barley, sorghum, rice, oats, rye, millet and other cereal crops wherein growth and development are substantially unaffected by the action of monocotyledon selective herbicides selected from the cyclohexane dione or aryloxy(amino)phenoxy propionate classes which plant or seed is generated via plant cell tissue culturing techniques.

In yet a further preferred embodiment of the present invention there is provided a method for producing a herbicide-resistant monocotyledon crop plant selected from wheat, barley, sorghum, rice, oats, rye, millet and other cereal crops wherein growth and development are substantially uninhibited by the action of a monocotyledon-selective herbicide of the cyclohexanedione or aryloxy(amino)phenoxy propionate classes which comprises

(A) selection of embryos from developing monocotyledon seeds

(B) cultivation of embryos in dedifferentiation tissue culture medium to induce callus formation (C) application of herbicide

(D) selection of callus exhibiting somaclonal variation

(E) regeneration of cells to produce shoots and roots

The generation of plants from cell culture is generally known in the art and the method of producing an herbicide-resistant monocotyledon crop plant as described herein above provides a general illustration only. It is to be understood that the following simplified method is not to be viewed as representing any limitation of the invention.

Young embryos of about 13 to 15 days old obtained from developing seeds are used to induce callus formation in a dedifferentiation tissue culture medium in vitro. A callus is a pale coloured mass of dedifferentiated cells and is a source from which somaclonal variants may be obtained. Somaclonal variants arise from somatic cells under tissue culture conditions, however the cause and mechanism of somaclonal variation is not at present understood. After a period of time, typically in the order of about four weeks, calli are subjected to concentrations of cyclohexanedione herbicide or aryloxy(amino)phenoxy propionate and those calli which are not killed are propagated further under recovery conditions, that is in a dedifferentiation medium in the absence of herbicide. After a period the calli may be subjected to a further herbicide challenge or challenges. Typically, the herbicide concentrations are of the same order of concentration at each application however this is not necessarily the case. For example, a relatively low concentration may be used on a first herbicide challenge and on a subsequent challenge the herbicide concentration may be elevated or depressed. The surviving calli are believed to have undergone a somaclonal change which gives rise to a somaclonal variant which is tolerant to the cyclohexanedione or aryloxy(amino)phenoxy propionate classes of herbicides. The somaclonal variants thus obtained are regenerated in a differentiation medium to form shoots and further regenerated in a modified differentiation medium to form roots. The regeneration media permit differentiation of selected somaclonal variants to proceed to a level from which whole plants may be generated therefrom. The whole plants are then permitted to grow to maturity and fruit. Seeds are then harvested from the plants and herbicide tolerance is confirmed by for example, spraying seedlings derived from the above mentioned seed with herbicide or by pre-emergence herbicide treatment of seeds. Comparisons of the response of sensitive plants may be made with tolerant plants using the same rates of herbicide application.

A "dedifferentiation culture medium" refers to a culture medium in which a callus is able to be maintained in an undifferentiated state. Typically, the culture medium contains an appropriate combination of components suitable for the aforesaid purpose. As exemplary components of a dedifferentiation culture medium there are included the following components:-

Ammonium nitrate, NH. NO-

Potassium nitrate, KN0₃

Hydrated Magnesium sulphate, MgS0..7HpO

Potassium dihydrogen phosphate, KH PO.

Ferric EDTA Calcium chloride, CaCl₂.2H₂0

L-asparagine

A synthetic auxin such as, 2,4-D

Micronutrient

Potassium Iodide, KI

Thiamine - HC1

The above mentioned components would be present in appropriate proportions, the relative proportions being well within the skilled artisans level of knowledge. The herbicide is selected from the cyclohexanedione or aryloxy(amino)phenoxy propionate classes. Preferably the herbicide is selected from tralkoxydi or 2-Cl-(ethoxyimino)propyl 1-3 -hydroxy-5-(3- butyryl esityl )cyclohex-2-enone. Most preferably, the herbicide is 2-C1- (ethoxyimino)propyll-3-hydroxy -5-(3- butyrylmesityl )eyelohex-2-enone.

Regeneration of cells may be performed in differentiation medium. Typically, the differentiation medium comprises a modified version of the dedifferentiation medium.

There may be two types of modified differentiation medium which are designed so as to permit a callus to develop shoots and roots.

A first modified medium typically comprises, with the exception of a plant growth hormone or synthetic auxin similar components to those in the dedifferentiation medium and further includes such components as indole acetic acid (IAA) and benzyl adenine purine (BAP). A second modified medium comprises similar ingredients to the said first modified medium save for the inclusion of IAA and BAP. In the case of the modified differentiation media and referring to the relative proportions of medium components contained therein, the actual amount of each component will be sufficient to enable differentiation of the cell mass to proceed in the appropriate direction. Thus, if shoots are required, these will develop in physiological response to a particular environment, and similarly, roots will develop in physiological response to another different environment. There now follow examples, Tables and figures relating to the invention as described hereinabove. It is to be understood that the disclosures in the examples, accompanying tables and figures are not intended to restrict the scope of the invention in any way.

TABLE 1

Critical dose rate of herbicide using embryo germination test under tissue culture conditions.

TABLE 2

Evaluation of herbicide stability under tissue culture conditions. 19-20 day old embryos were used. Stability of herbicide in tissue culture medium was determined after storage at 4°C and 20°C. Tests were done weekly and followed for at least 6 weeks .

TABLE 3

Critical dose rate in glasshouse screening using non-herbicide-resistant wheat.

TABLE 4

Glasshouse screening of herbicide tolerant plants. FIGURE 1A: Effect of herbicide on callus. Four week old calli were exposed to herbicide for four weeks (0.002 uM to 2 uM) and effect was measured on fresh weight and dry weight of calli.

FIGURE IB: Effect of herbicide on callus. Four week old calli were exposed to herbicide for four weeks (10 uM to 500 uM) and effect was measured on fresh weight and dry weight of calli.

TABLE 1

TABLE 2

Examp l e 1

Wheat (variety "Millewa") was grown under glasshouse conditions. Ears were bagged at the anthesis stage to determine the age of embryos. Critical kill dose of herbicide was evaluated via the embryo germination test. Critical kill dose (Table 1) is the minimum lethal dose of herbicide required to arrest germination. Herbicide dose tested ranged from 0.005 uM to 1 mM. Stability of herbicide under tissue culture conditions was also determined via embryo germination tests. Herbicide stability was evaluated at two temperatures 4°C and 20°C (Table 2). Dose of herbicide tested ranged from 0.005 uM to 1 mM. Developing embryos (from 10 days to 22 days after pollination) were evaluated for callus production and it was found that 14 to 15 day old embryos produced good healthy callus. Maximisation of callus production was achieved by studying different levels of 2,4D in combination to dark and light conditions. It was found that 2,4D at a rate of 1 mg/L gave the best result when incubated in the dark. Callus produced this way was more suitable for selection since no differentiation was observed. The effect of herbicide on callus was also studied in terms of fresh weight and dry weight. Four week old callus was exposed to varying levels (0.002 uM to 500 uM) of herbicide. A marked reduction in callus growth was observed at 0.002 uM to 0.2 uM dose of herbicide and thereafter (from 0.2 uM to 500 uM) callus growth did not occur, the callus became enfeebled indicating that they were dying. Different selection cycles were evaluated by varying herbicide concentration (critical kill or above critical kill) and time of exposure to callus. A typical selection cycle involved culturing 14-15 days old embryos on callus induction medium (i.e. dedifferentiation medium) under sterile conditions to produce callus. Four week old callus was exposed to herbicide (1-1.5 uM) for a period of two weeks. As a result of herbicide treatment, non-tolerant cells become enfeebled and die. The herbicide treatment 1s followed by a recovery period of two weeks. CalU were again subjected to selection pressure by exposure to herbicide (1-1.5 uM) for two weeks. Most of the calli (cells) had died by the end of the second round of herbicide treatment, except for the tolerant cells. The second round of herbicide treatment 1s followed by a recovery period of two weeks. Recovery medium 1s callus medium minus added herbicide. Surviving cells are termed tolerant cells because they are able to grow 1n the presence of herbicide concentrations which normally kill calli.

The Hvlng/tolerant cells were placed on shoot Induction medium to regenerate shoots. The tolerant cells are thus cultured over a period of 16 weeks, with four week subculture intervals, on shoot induction medium. Regenerated shoots were placed on root Induction medium for four weeks to regenerate roots. Using the above entloned selection cycle 2,200 calli were exposed to herbicide selection pressure and 176 plants were regenerated from them. These plants were grown under glasshouse conditions to maturity and 4068 seeds were collected. Herbldde tolerance was confirmed by growing these seeds to seedling stage (three leaf) and spraying with herbicide or evaluating via the seed germination test.

Example 2

Using the selection cycle as described in Example 1 an additional 1185 calli were exposed to herbicide (1-1.5 uM) and 148 plants were regenerated. These plants were grown to maturity under glasshouse conditions and seeds were col lected.

Example 3

Glasshouse screening to confirm herbicide tolerance: Seeds regenerated as described in Examples 1 and 2 were grown under glasshouse conditions to seedling stage. They were sprayed with herbicide at the 3-leaf stage. Several screening trials were performed using non-herb1dde resistant wheat in order to find the critical dose rate (i.e. the lowest rate of herbicide application which results in the death of all non-herbidde resistant plants). Table 3 shows the result of a typical experiment using non-resistant wheat. Concentration of herbicide used to spray herbicide tolerant/resistant plants (seedlings at 3-leaf stage) was higher than critical dose rate (approx 2 times). Out of 17,071 seeds screened 42 survived the glasshouse screening trial confirming herbicide tolerance (Table 4). These plants were grown to maturity and seeds were col lected. TABLE 3: Critical Dose Rate in Glasshouse Screening (Experiment using non-resistant wheat)

TABLE 4: Glasshouse Screening of Herbicide Tolerant Plants

Claims

The claims defining the Invention are as fol lows :

1. A herbicide-resistant monocotyledon crop plant or seed thereof selected from wheat, barley, sorghum, rice, oats, rye, millet and other cereal crops wherein growth and development are substantially unaffected by the application of monocotyledon-selective herbicides selected from the cyclohexanedione and aryloxy(amino)phenoxypropionate classes .

2. A crop plant or seed thereof according to claim 1 wherein the monocotyledon crop plant is selected from wheat, oats or barley.

3. A crop plant according to claim 1 or claim 2 wherein the monocotyledon crop plant is wheat.

4. A crop plant according to claim 3 wherein the physiological function of an enzyme involved in lipid synthesis is substantially unimpaired by the action of a monocotyledon-selective herbicide of the cyclohexanedione or aryloxy(amino)phenoxy propionate class.

5. A crop plant according to claim 4 wherein the enzyme is acetyl CoA carboxylase.

6. A crop plant according to any one of the preceding claims wherein the herbicide is selected from tralkoxydim and 2-Cl-(ethoxyimino)propyl]-3- hydroxy-5-(3-butyrylmesityl )cyclohex-2-enone.

7. A crop plant according to any one of the preceding claims wherein the herbicide is 2-C1- (ethoxyim1no)propyl]-3-hydroxy-5-(3-butyrylmesityl )- cyclohex-2-enone.

8. A method of generating a herbicide resistant monocotyledon plant or seed thereof using plant cell tissue culturlng techniques wherein the plant cell tissue is selected from wheat, barley, sorghum, rice, oats, rye, millet and other cereal crops wherein growth and development are substantially unaffected by the action of herbicides selected from the cyclohexane dlone or aryloxy(amino)phenoxy propionate classes.

9. A method according to claim 8 for producing a herbicide-resistant monocotyledon crop plant selected from wheat, barley, sorghum, rice, oats, rye, millet and other cereal crops wherein growth and development are substantially uninhibited by the action of a monocotyledon-selective herbicide of the cyclohexanedione or aryloxy(am1no)phenoxy propionate classes which comprises

(A) selection of embryos from developing monocotyledon seeds

(B) cultivation of embryos 1n dedifferentiation tissue culture medium to Induce callus formation

(C) application of herbicide (D) selection of callus exhibiting somaclonal variation

(E) regeneration of cells to produce shoots and roots.

10. A method according to claim 8 or claim 9 wherein the herbicide-resistant monocotyledon crop plant is wheat.

11. A method according to any one of claims 8 to 10 wherein the applied herbicide is tralkoxydim or 2-Cl-(ethoxyimino)propyl]-3-hydroxy-5-(3-butyry1- mes ityl )cyclohex-2-enone.

12. A herbicide-resistant monocotyledon crop plant according to claim 1 and with reference to any one of Examples 1 to 3.

13. A method according to claim 8 with reference to the accompanying Tables and Figures.

DATED this day of 1990

ICI AUSTRALIA OPERATIONS PROPRIETARY LIMITED

LRS P24.1