HERBICIDE RESISTANT PLANTS

Abstract: From a population of randomly mutated plants, produced by the action of chemical mutagens on pollen or seed, a plant may be isolated by growing the mutant population in the presence of the herbicide clomazone. This plant is resistant to normally lethal doses, that is herbicidally effective doses, of that herbicide. Clomazone may thus be used for weed control in tolerant crops where hitherto the sensitivity of the crop would have precluded such use.

Description: HERBICIDE RESISTANT PLANTS This invention relates to herbicide resistant maize plants. The purpose in providing crop plants which resist the action of a herbicide is to facilitate the destruction of weeds growing between the plants by the overall application of a herbicidally effective concentration of a herbicide which would destroy the crop plant in its normal, that is herbicide sensitive, state. Such resistant plants are also useful for use in a locus of any short term carry-over of herbicide from a previous crop. Methods are known by which populations of plants may be obtained which contain a great number of random mutations. Such methods include tissue culture techniques where spontaneous somaclonal variation occurs in the presence or absence of a utagen. By applying to the cultures some form of selection pressure it is possible to recover cells which resist that pressure. Depending on the plant species it is sometimes possible to regenerate whole plants from the resistant cells. Such tissue culture selection methods have been used in the past to select for resistance to herbicides. Neuffer and Coe [Maydica XXIII (1978) 21-28; pa e 21] have described a procedure for corn (maize) pollen mutagenesis using mutagens suspended in light paraffin oil, followed by pollination of a recipient plant with the mutagenized pollen. As reported, there is no indication that any attempt was ever made to locate and isolate commercially important mutants and, although it is said that the resulting plants were examined for mutants, no details are given as to the properties of any mutants which may have been found, probably by visual inspection. In Plant Breeding Reviews _§_, pages 39 to 180, Bird and Neuffer review the uses of mutagenic processes to produce variation in maize. Pollen mutagenesis is discussed at pages 150 and 151. Pollen mutagenesis gives a relatively high frequency of variation in the M- generation compared with other available procedures. However, no suggestion is made in respect of the use of pollen mutagenesis for generation of mutants which display resistance to herbicide action. Section III (page 149) describes some of the difficulties of the use of mutagenesis as a source of genetic variation. One important aspect is the method by which the population of putative mutants is screened for useful phenotypes. Mutagenesis, of pollen or seed, has previously been used to generate large populations of random mutations in plants. It is generally the case, however, that few mutants of commercial importance have ever been isolated from such population. The technique appears to have been used predominantly to produce mutations for academic study of genetics rather than with any prospect of locating agrono ically useful mutations. Our International Patent Application Number PCT/GB 90/00753 describes and claims maize plants which are resistant to the effects of imidazolinone and/or sulphonyl urea herbicides. These plants were produced by a pollen mutagenesis method and are surprisingly free from deleterious mutations which would be expected from a procedure such as chemical mutagenesis which is generally considered to act randomly. Clomazone, [2-(2-chlorophenyl)methyl-4,4- dimethyl-3-isoxazolidinone] , is a selective herbicide for use on soybean. A single pre-plant application of clomazone controls many grasses and some broad-leafed weeds. This herbicide reduces or stops the accumulation of plastid pigments in susceptible species by inhibiting an enzyme of the terpenoid pathway, resulting in white, yellow or pale green plants. The precise enzymatic site of action of clomazone is not known but some of the suggested sites of action are: (a) isopentenyl pyrophosphate enzyme, (b) prenyl transferaseε, (c) enzymatic phytylation of chlorophyllide, and, (d) some enzyme downstream of geranyl-geranyl- pyrophosphate production in the terpenoid pathway. It is believed that differences in selectivity between tolerant species such as soybean and sensitive species such as maize is not due to to differential absorption, translocation or metabolism but is accounted for by some differences between the species which occur at the site of action. Maize is very sensitive to the action of clomazone and the introduction of tolerance to this herbicide would be beneficial to farmers in allowing greater flexibility in grass and broad—leafed weed control. An object of the present invention is to provide a herbicide resistant maize. According to the present invention there is provided a plant having an increased level of tolerance to a normally lethal dose of a herbicide of the isoxazolidinone class. Preferably the herbicide is clomazone [2-(2- chlorobenzyl)-4,4-dimethyl-l,2- oxazolidin-2-one]. It is further provided by the invention a maize plant having an increased level of tolerance to a normally lethal dose of a herbicide of the isoxazolidinone class. Preferably the maize plant is derived by pollen mutagenesis from the maize inbred line designated UE95. The invention further provides a hybrid maize which is tolerant of normally lethal dose of an isoxazolidinone herbicide, of which at least one of the parents is an isoxazolidinone-tolerant maize plant as hereinabove defined. The invention also provides seed and progeny of the aforesaid plants. The invention further provides seed of the tolerant mutant designated UE95/CLT1, a sample of which has been deposited with the American Type Culture Collection under the terms of the Budapest Treaty, prior to the filing of this application. The resistance trait segregates in the deposited seeds but the tolerant mutants may be obtained by growing the seeds under the conditions specified for screening in Example 2 below. Thus at the lower concentration of herbicide the tolerance exhibits a 3:1 segregation and at the higher concenration at a ratio of 1:2:1 of homozygous tolerant: heterozygous tolerant: homozygous sensitive. Clomazone, a member of the isoxazolidinone family of herbicides,is the active ingredient of a commercial herbicide known as Command (Trade Mark, FMC Corporation): its chemical name is 2-(2-chloro- benzyl)-4,4-dimethyl-l,2-oxazolidin-3-one (C 12 H 14 C1N0 2 : M. .-239.7). It is used as a pre-emergence or pre-planting herbicide, to control broad-leaved and grass weeds principally in soybeans, but is also used in some other crops such as, cassava, maize, oilseed rape, sugar cane and tobacco. Clomazone is known from United States Patent No. 4,405,357. The maize plants of this invention are resistant to clomazone but, as is not uncommon in such cases, it is expected that some degree of cross-resistance will exist in respect of other isoxazolidinone herbicides and other herbicidally active compounds which have similar modes of action. Maize is very sensitive to clomazone. However, although we have demonstrated that genotypic variation for tolerance of clomazone does exist in maize, the level of that tolerance, even in the most tolerant genotype, is well below the field application rate required for efficient herbicidal action by clomazone. Table I classifies 40 commercial maize inbreds according to their degrees of tolerance. TABLE 1 INBRED CORN LINES CLASSIFIED ACCORDING TO THEIR RESPONSE TO CLOMAZONE AT A CONCENTRATION OF 0.2 PPM (97.23 G/HA) The preferred method of creating the clomazone-tolerant plants of this invention is selection from a population of random mutants created via pollen-mutagenesis in the manner described in our International Patent Application Number PCT/GB90/00753. Certain advantages accrue from the use of pollen mutagenesis as a method of creating mutants rather than the more usual method of relying on somaclonal variation to produce the variation. In order that somaclonal variation may occur it is required that a culture of plant tissue be established. This requirement restricts the choice of genotype which may be used as it is not always possible to regenerate whole plants from the cultured tissue. On the other hand, mutagenised pollen may be applied to any recipient maize genotype, including commercially important and well-established elite breeding lines. Also the rate of occurrence of undesirable mutations which somaclonal variation is known to produce is unexpectedly reduced. As described in our International Patent Application No PCT/GB90/00753, selection is carried out at at the M. generation, with the result that only dominant mutations are selected. Also, being carried out on whole plants or on the seed pre-emergence, or both, allows the herbicide concentration to mimic the field conditions more closely than is possible with the application of the selection pressure of the herbicide to a tissue culture. In the tissue culture selection method, whole plants have to be regenerated from the tissue and grown to maturity before any indication of the performance of the progeny under field application rates of the herbicide can be obtained. In our method, selection is made directly on the plants under concentrations of herbicide which are comparable to those which are recommended for normal weed-killing activity in the field. Selection on the M 2 generation, as with the tissue culture method, selects recessive mutations as well as dominant traits. Dominance of a desirable trait is generally viewed as more useful and easier to handle in a breeding programme especially of hybrid crops. We have found, quite surprisingly, that the mutants which we have isolated by the method of the invention are free of deleterious mutations. This was entirely unexpected and the reason for this advantage is not entirely clear. We believe, but do not wish to ' be bound by this explanation, that the degree of control which we are able to exercise over the selection step, using carefully controlled rates of application of the herbicide, for example, giving a good overall and uniform rate of exposure, may be responsible. The invention will now be described by the following summary of the method by which the herbicide- resistant plants of the invention were derived. The Figures which accompany this application are as follows: Figure 1 is a flow-chart showing the derivation of several generations of progeny from plants generated by this invention; Figure 2 shows the chemical structure of clomazone; Figure 3 shows the relative chlorophyll content (measured with a SPAD 502) of (a) control UE95 seedling (b) a wild-type UE95 seedling growing in 1.72 ppm clomazone and (c) the resistant UE95 mutant CLT1 growing in 1.75 ppm clomazone; and. Figure 4 shows the chlorophyll fluorescence transients of (A) control UE95 seedling (B) a wild-type UE95 seedling growing in 1.72 ppm clomazone and (C) the resistant UE95 mutant CLTl growing in 1.75 ppm clomazone Figure 5 shows the visual assessment score of clomazone injury in Arabidopsis thaliana. The invention will now be described, by way of illustration, in the following Examples. EXAMPLE 1 - PRODUCTION OF Ml MAIZE SEED A stock solution of ethyl methane sulphonate (EMS) was made up to contain one millilitre of EMS in 100 ml of light paraffin oil. The stock solution was stored under refrigeration. Fresh pollen grains with anthers were harvested from a total of twenty tassels of field grown maize inbred line UE95. Pollen grains were separated from the anthers using a Glassine (Trade Mark) bag. Around 3 milligrams of pollen were added to 45 millilitres of the EMS stock solution in a 60 ml capacity bottle. The pollen/EMS solution mixture was shaken vigorously for 30 seconds then shaken four or five times every three minutes over a period of 40 minutes, to prevent precipitation of the pollen grains. The treated pollen grains were brushed on to the silks of the detasseled inbred female parent (coded UE95). The plants were grown to maturity and the Ml seeds harvested. EXAMPLE 2 - SCREENING M. seed was sown, approximately 150 seeds in a tray containing no drainage holes, in 7.0 Kg of a 2:1 sand:soil mix (pH 7.0) growing medium. Each tray was then sprayed with 500 ml of a clomazone solution containing 17.5 mg of the active ingredient. A further 3.0 Kg of the aforesaid sand/soil mix was placed on top of the seed. The concentration of clomazone in each tray was approximately 1.75 ppm of active ingredient to sand/soil (by weight). This is roughly equivalent to the 1.5 pints/acre field application of the commercial product Command (Trade Mark, FMC). Every 10 days, 52 seed trays were planted and sprayed as described. The seeds were grown in the glasshouse at 25°C. The chosen application rate was such that germination was close to 100%, but subsequently all susceptible plants were severely affected. However, shortly after emergence, the initial effect of the herbicide became apparent: striped or patchy chlorosis on leaves. After three to four weeks almost all the sensitive plants were completely dead. Unsprayed UE95 plants were always grown in parallel with each screen as a control as it was already known that normal and M. seedlings of UE95 are almost indistinguishable when germinated and grown to maturity without spraying. EXAMPLE 3 - SELECTION AND GROWTH From a screen of 23,400 seeds, a single resistant mutant was obtained. The resistant plant was very green compared with the plants surrounding it. After twelve days growth, the resistant plant was growing well whereas all the others in the seed tray had died. On measurement of the chlorophyll content (Figure 3) and the chlorophyll fluorescence (Figure 4) the resistant plant appeared to demonstrate normal pigment development and • activity. The resistant plant, designated CLT1, was transplanted and grown to maturity. CLT was self-pollinated and backcrossed to unmodified UE95. In addition, CLTl was crossed to the line BD68 to provide F. seed. This F. seed was also grown to maturity and self-pollinated to obtain F- seed for an RFLP linkage analysis, and backcrossed to BD68 again to provide BC^ seed for the conversion of BD68. EXAMPLE 4 - GENETIC ANALYSIS BC^ seed (150) of CLTl were planted and sprayed with clomazone in the manner described above. After 9 days growth the plants were evaluated for tolerance. Out of the 150 seeds only 76 seed germinated and grew. Of these 76, there were 39 green plants and 37 white plants. This is equivalent to a 1:1 segregation, suggesting a single dominant/partially dominant gene for tolerance. The 39 tolerant plants were transplanted to the greenhouse and grown to maturity. The plants were self-pollinated and backcrossed to unmodified UE95. The CLTl gene was then fixed in the UE95 background by the protocol illustrated in Figure 1. BC.S, seed from 19 ears derived from CLTl was rescreened in the above manner. However, 100% silica sand was used to increase the effective dose of herbicide to the plants. After nine days growth the plants were evaluated for tolerance. On this occasion, it was possible to distinguish homozygous tolerant seedlings from heterozygous tolerant seedlings. Seedlings derived from each ear appeared to segregate in a 1:2:1 manner for tolerance to clomazone (Table 2). If homozygous tolerant and heterozygous tolerant seedlings were classed together, the seedlings exhibited a 3:1 segregation (Table 3). Both of these segregation ratios are indicative of a single gene for clomazone tolerance. TABLE 2 SEGREGATION RATIOS AND χ 2 VALUES FOR 19 BClSl EARS DERIVED FROM CLTl (1:2:1) 2 (X must be less than 5.99 for a 1:2:1 segregation) TABLE 3 SEGREGATION RATIOS AND χ 2 VALUES FOR 19 BClSl EARS DERIVED FROM CLTl (3:1) 2 (X must be less than 3.841 for a 3:1 segregation) A total of 164 homozygous tolerant plants were transplanted to the field, gown to maturity and self-pollinated. Plants were selected phenotypically for likeness to UE95 and of the 164 selfed ears only 100 were advanced. Each ear was re-screened in the above manner to confirm non-segregation. EXAMPLE 5 - SEGREGATION STUDY Resistant plants were used in reciprocal back-crosses with homozygous, herbicide sensitive UE95. The frequency of resistant progeny in the M..BC, or M ι BC 2 generations was found by treatment with clomazone and counting the survivors. The ratio of resistant to sensitive plants in the progeny of the backcross was not significantly different from 1:1 for all of the mutants, indicating that resistance is controlled by a single dominant gene. EXAMPLE 6 - PRODUCTION OF FURTHER GENERATIONS Referring to Figure 1, the M.BC, plants were self pollinated to give generation M 1 BC.S. which was again self-pollinated to give M.BC.S,. From that generation it was possible to identify, by the fact that the resistant trait is non-segregating, lines which are homozygous for the trait. These homozygous lines selected for use in the production of F, hybrids which possess resistance to clomazone or for further breeding work. EXAMPLE 7 Arabidopsis thaliana (ecotypes Landsberg erecta and Columbia) M 2 seed derived from EMS-treated M. seed were purchased from Lehle Seeds (Tucson, AZ85718, USA). Seeds were surfaced-sterilised by sequential washing in 95% ethanol (7 minutes), 10% Domestos (Trade Mark) (10 minutes)and autoclaved water (3 washes). Seeds were spread in clear plastic dishes on sterile nutrient medium (half-strength MS salts and vitamins, 10 g/1 sucrose, 8 g/1 Phytagar, pH 5.8) supplemented with herbicide, and sealed with laboratory film. Seeds were allowed to germinate 2 at 25°C under a 16 hour photoperiod (120 /E/m /ε) alternating with 8 hours darkness. The effect of clomazone on wild-type Arabidopsis (Landsberg erecta) was monitored over a concentration range of from zero to lOO M. The amount of pigment in the cotyledons and first leaves of 10-day-old seedlings was visually scored according to a linear index of zero to 5, where zero=no pigment, 5«full amount of pigment. The first leaf proved more sensitive to clomazone than the cotyledon. The first leaf was fully bleached by 5//M clomazone, whereas the cotyledon was incompletely injured at this concentration, (Figure 5) Routine screening for clomazone tolerant mutants was performed on 5//M clomazone, by visually assessing the first leaf for tolerance to bleaching. A total of 175,000 M 2 seed were evaluated by the standard screening procedure. From one parental M. group, four tolerant candidate seedlings were identified (Landsberg erecta). These seedlings retained pigment in the cotyledons, first leaves and emerging second leaves to a greater extent than the background of sensitive seedlings exposed to 5//M clomazone. All four candidate seedlings exhibited partial injury on 5 M clomazone, relative to seedlings grown in the absence of clomazone. The candidate seedlings were transplanted into a 1:1 mix of compost:Perlite and grown to maturity. On flowering each plant was able to self-pollinate. The M 3 progeny derived from one of the selected M 2 seedlings were evaluated for inheritance of the clomazone tolerance trait. 3 seed were germinated on 5//M clomazone under standard culture conditions. All 89 germinated , seedlings exhibited greater tolerance to the bleaching action of clomazone, in comparison with the wild-type seedlings (Landsberg erecta). There was no evidence for segregation of the tolerance trait among the M, population. This demonstrated that the selected M_ parent of these progeny exhibited heritable tolerance to clomazone, and was genetically homozygous for the tolerance trait.

Claims: CLAIMS 1. A plant having an increased level of tolerance to a normally lethal dose of a herbicide of the isoxazolidinone class. 2. A plant as claimed in claim 1 in which the herbicide is clomazone [2-(2- chlorobenzyl) -4,4-dimethyl-l,2- oxazolidin-2-one] . 3. A maize plant having an increased level of tolerance to a normally lethal dose of a herbicide of the isoxazolidinone class. 4. A maize plant as claimed in claim 2 in which the herbicide is clomazone [2-(2- chlorobenzyl)-4,4- dimethyl-1,2- oxazolidin-2-one] . 5. A maize plant as claimed in claim 3, in which the plant is derived by pollen mutagenesis from the maize inbred line designated UE95. 6. A hybrid maize which is tolerant of normally lethal dose of an isoxazolidinone herbicide, of which at least one of the parents is an isoxazolidinone-tolerant maize plant as claimed in claim 3. 7. Seed of the plant claimed in claim 1. 8. Seeds of the maize plant claimed in claim 3. 9. Hybrid maize seed of the plant claimed in claim 6. 10. Herbicide resistant maize mutant designated UE95/CTL1 deposited with the American Typoe Culture Collection prior to the filing of this application.

Inventors: BRIGHT SIMON WILLIAM JONATHAN, CHANG MING TANG, ELLIS JOHN RAYMOND, GREAVES JOHN ANDREW, STURGESS KAREN, BRIGHT, SIMON, WILLIAM, JONATHAN, CHANG, MING, TANG, ELLIS, JOHN, RAYMOND, GREAVES, JOHN, ANDREW, STURGESS, KAREN
Applicants: ICI LTD, IMPERIAL CHEMICAL INDUSTRIES
Application year: 1992
Main classification: A01H5
IPC codes: A01H1, A01H5