Methods for introducing male sterility into true dicotyledonous plants

Abstract

A male sterility inducer for eudicotyledonous plants is provided, which comprises trifluoromethanesulfonamide (TFMSA) as an active ingredient. Also provided is a method for inducing male sterility in eudicotyledonous plants, which comprises administering TFMSA to the eudicotyledonous plant.

Description

[Technical field]

[0001] The present disclosure relates to methods for introducing male sterility into eudicotyledonous plants, which can be useful, for example, in crop breeding and efficient production of hybrid seeds. [Background technology]

[0002] Hybridization is essential for plant breeding. Hybrid seeds produced by hybridization play a vital role in the agricultural industry. In this context, hybridization means artificial pollination between one individual and another. In self-pollinating plants, where pollination occurs within a single plant, emasculation, i.e., the removal or sterilization of the stamens, is necessary for crossing to avoid self-pollination. Conventional emasculation techniques include physical removal of the stamens by hand, hot water emasculation, and the use of cytoplasmic male sterile lines. However, these techniques have major limitations, such as being laborious and time-consuming, and being available only for some plants. For example, cytoplasmic male sterility depends on the presence of a specific genetic background, and hot water emasculation is not practical for legumes, although it can be used in Poaceae. Mendel also used manual removal of stamens in his famous breeding experiments, but it is said that he damaged his eyesight by continuing to do so (Chapter 7, "An Explanation of Mendel: The Story of the Priest Who Opened the Door to Genetics," by Nakamura Chiharu, Kobe University Faculty of Agriculture, Intergenomics Research Group, 2016), and it is by no means an efficient method, especially from an industrial point of view.

[0003] In monoecious plant species, such as maize, pollination control is relatively easy because the pistil and stamen flowers are separate on the plant. In contrast, in legumes, for example, which have hermaphrodite flowers and are cleistogamic or have co-located pistils and stamens, pollination occurs within each flower even before it opens, so self-pollination is almost guaranteed. Pollination control in such species is particularly difficult, and the only practical means available so far has been the inefficient manual removal of stamens.

[0004] Legumes are consumed as a staple food worldwide and are also valuable as livestock feed. Cowpea, in particular, is an agricultural product that can be grown in arid regions and may be a key to improving food security in arid regions such as sub-Saharan Africa. The use of soybeans in the developing alternative meat industry has also become increasingly important in recent years. If a method could be developed to efficiently emasculate and control pollination in legumes, it could be a groundbreaking innovation in legume breeding and have a positive impact on global food issues. Due to the current lack of availability of genetic male sterility tools for legumes such as cowpea and the difficulty of physically controlling self-pollination as mentioned above, there is a long-felt but unsolved need to efficiently induce male sterility, especially in legumes.

[0005] For some agricultural crops, including rice, wheat, corn, cotton, and sunflower, a method of emasculation using chemicals (chemical emasculation method) is also known (Non-Patent Document 1). The chemical emasculation method has the advantage that it allows large-scale plant treatment and can emasculate many plants in a short period of time. The chemical substance that is the active ingredient used in the chemical emasculation method is called a CHA (chemical hybridizing agent). The history of CHA development is long, having already begun in the 1950s. As a result, quite a number of types of CHA have become known. However, each CHA is usually discovered and described for a specific plant group, and it is not easy to identify a substance that can effectively function as a CHA for a desired plant species from among the known CHAs. For example, Non-Patent Document 1 describes how maleic hydrazide (MH), whose potential to induce male sterility in corn was first discovered in 1950, sparked a frenzy of testing MH on many crop species thereafter, but the usefulness of MH as a CHA was never demonstrated in any crop, mainly due to insufficient male / female selectivity.

[0006] As described in the aforementioned Non-Patent Document 1, most of the substances that are potential CHA candidates have some form of toxicity or inhibitory activity against plant tissues, and therefore often cause not only male sterility but also female sterility (i.e., low male / female selectivity), and furthermore, often have adverse effects on plant survival processes such as germination. The diverse plant groups that exist in nature and are used in the agricultural industry have very different structures and mechanisms of reproduction, so it is a difficult task to discover an effective CHA that can exert male / female selectivity for each of them.

[0007] Patent Document 1 describes the use of sulfonylurea derivatives having a characteristic urea group and a heterocyclic six-membered ring as gametocides. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] McRae, D. (1985) Plant Breed. Rev. 3: 169-192. [Patent documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 64-68305 Summary of the Invention

[0010] The present inventors have found that trifluoromethanesulfonamide (TFMSA) is a male sterility inducer, or CHA, that is effective in a wide range of eudicots. TFMSA was able to induce male sterility in legume eudicots without substantial phytotoxicity or female sterility. Similar results were obtained in other eudicots. Loussaert D (2004) Sexual Plant Reproduction 16(6): 299-307 reports that TFMSA acts as a male reproductive inhibitor in maize, a monocot grass. Hodnett GL, Rooney WL (2018) Canadian Journal of Plant Science 98(5): 1102-1108 reports that TFMSA was also able to induce male sterility in sorghum (also known as sorghum, guinea corn, etc.), a grass plant closely related to maize. The paper states that TFMSA may have applicability in sorghum due to the similarity between sorghum and maize. However, it was an unexpected and significant discovery that TFMSA is an effective male sterility agent in eudicots, which diverged from the group including monocots approximately 123 to 147 million years ago (Bell et al. (2010) American Journal of Botany 97(8): 1296-1303) and are significantly different from the Gramineae in terms of phylogenetic evolution, development, plant structure, and even molecular biology (e.g., receptors and transporters).

[0011] For example, monocots and non-eudicots have a shape called "single grooved grains" that has only one hole (germinal pore) through which the pollen tube extends, whereas eudicots have evolved independently to produce triple grooved grains, i.e., three-hole pollen. Regarding this point, Kurata (Ecological Environment Research, 26(1):53-66, 2020) writes: "The more germ pores there are, the more advantageous it is. For example, in the case of a single grooved grain, if the surface attached to the stigma is opposite the germ pore, it is difficult for the pollen tube to germinate and extend, but if there are multiple holes, the pollen tube can extend from one of the holes. This must have been a strategy advantageous for reproduction acquired during the evolutionary process." As mentioned above, this clearly illustrates that eudicot pollen cannot be considered the same as that of grasses in terms of phylogenetic evolution, structure, development, etc., and supports the non-obviousness of the invention disclosed in this application. Furthermore, it is well known that there is considerable diversity within eudicotyledonous plants themselves, and it was unexpected that TFMSA acts as a male sterility agent in multiple distantly related eudicotyledonous plants.

[0012] Proline is known to be a key amino acid required for plant pollen development. For example, Schwacke et al. (1999) Plant Cell, Vol. 11, 377-391 reported that proline accumulates in tomato pollen, accounting for more than 70% of the total free amino acids. The work of Loussaert (supra) teaches that the inhibition of proline transport required for proline accumulation is the mechanism of action of TFMSA as a male sterility inducer in maize. Loussaert experimentally demonstrated that TFMSA dose-dependently reduces proline accumulation in maize anthers (pollen-forming organs), that changes in proline content are the first metabolic abnormality detected before the appearance of pollen failure after TFMSA administration, and that TFMSA administration actually reduces proline transport in anthers, and wrote: "TFMSA is presumed to induce male sterility by interfering with the transport of proline from the site of synthesis to the site of accumulation, which results in feedback inhibition of proline biosynthesis and ultimately depletes developing anthers of proline." Hodnett et al. (supra) also wrote: "TFMSA may induce male sterility in maize and sorghum by reducing anther proline in pollen."

[0013] Schwacke et al. (supra) identified a proline transporter protein LeProT1 (GenBank accession number AF014808) that is specifically and predominantly expressed in the stamens / anthers / pollen of tomato (a eudicot plant). The most similar sequence to this maize proline transporter homolog appears to be XP_008651898, but the maize homolog shares a high amino acid sequence identity of about 92% with the most similar sorghum homolog, whereas the maize homolog shares only 61% amino acid sequence identity with the tomato homolog. There was a scientific rationale for applying TFMSA to sorghum, a close relative of maize, to obtain male sterility, as expressed by Hodnett et al.'s statement that "Given the interest in transient sterility in sorghum systems and the similarity between sorghum and maize, TFMSA or a sister compound may have applicability in sorghum." However, since Loussaert taught that the mechanism of action of TFMSA in maize is inhibition of proline transport, it was not reasonable to infer that TFMSA has a male sterility effect in eudicotyledonous plants, whose proline transporter structure as well as the organ structure around the pollen are different from those of maize.

[0014] The present disclosure includes the following embodiments. [1] A male sterility inducer for eudicotyledonous plants containing trifluoromethanesulfonamide (TFMSA) as the active ingredient. [2] The male sterility inducer described in [1], wherein the eudicotyledonous plant is a plant of the family Fabaceae, Brassicaceae, or Solanaceae. [3] The male sterility inducer described in [1], wherein the eudicotyledonous plant is a legume plant. [4] The male sterility inducer described in [3], wherein the legume is cowpea. [5] The male sterility inducer according to any one of [1] to [4], which is in the form of an aqueous solution. [6] The male sterility inducer according to any one of [1] to [5], which is administered via the roots of the plant. [7] A method for inducing male sterility in eudicotyledonous plants comprising administering trifluoromethanesulfonamide (TFMSA) to the eudicotyledonous plant. [8] The method according to [7], wherein the eudicotyledonous plant is a plant of the family Fabaceae, Brassicaceae, or Solanaceae. [9] The method according to [7], wherein the eudicotyledonous plant is a legume.

[10] The method according to [9], wherein the legume is cowpea.

[11] The method according to any of [7] to

[10] , wherein the TFMSA is administered in the form of an aqueous solution.

[12] The method according to any one of [7] to

[11] , wherein the TFMSA is administered via the roots of the plant. [Brief description of the drawings]

[0015] [Figure 1] 13A-13C are photomicrographs showing Alexander staining of pollen from cowpea treated with TFMSA doses of 30 mg (left, "TFMSA") or 0 mg (right, "Control") per plant, taken 7 days after treatment. [Diagram 2] Micrographs comparing Alexander staining of cowpea anthers treated with different doses of TFMSA per plant, indicated by the numbers (mg) on ​​the far left: (a) line IT86D-1010 and (b) line IT97K-499-35. [Diagram 3] The experimental results show that the more frequently 30mg TFMSA treatments were applied, the stronger the inhibition of cowpea growth. "5 times" refers to plants that were treated with TFMSA five times at one-week intervals starting five weeks before flowering, while "1 time" refers to plants that were treated with TFMSA only once one week before flowering. [Figure 4](a) Photograph of cowpea line IT97K-499-35 with white seed coat. This line was rendered male sterile by TFMSA treatment and used as the female parent for artificial pollination. (b) Photograph of cowpea line 74826 with brown seed coat. This line was not treated with TFMSA and used as the male parent to provide pollen for artificial pollination. Photographs of the seeds are shown in the insets of (a, b). (c) Hybrid F1 plant of IT97K-499-35 × 74826. (d) F2 seeds from the plant shown in c, all of which have black seed coats. The white scale bar indicates 5 cm, and the black scale bar indicates 3 cm. [Diagram 5] (a) shows Arabidopsis plants 21 days after the second TFMSA treatment with different doses. The right side is a control plant treated with 0 mg dose, and the left side is a plant treated with 10 mg dose. (b) shows Alexander staining of anthers of the plant in (a) 7 days after the second TFMSA treatment. (c) is a partially enlarged photograph of (b). (d) shows tobacco plants similarly 21 days after the second TFMSA treatment with different doses. (e) shows Alexander staining of anthers of the plant in (d) 7 days after the second TFMSA treatment. (f) is a partially enlarged photograph of (e). [Figure 6] (a) Photographs of Micro-Tom tomato plants (Solanaceae) at the fruiting stage after treatment with TFMSA doses of 0, 0.63, 1.3, or 2.5 mg per plant. (b) Graph corresponding to the plants in (a) showing the number of fruits produced. Fruit production decreased in a concentration-dependent manner, with no fruit produced at all in the case of the 2.5 mg dose. [Figure 7] A liquid solution containing different doses of TFMSA was applied to Arabidopsis thaliana via the roots. The right side shows a control plant treated with 0 mg dose, while the left side shows plants treated with different doses of TFMSA ranging from 0.2 to 10 mg per plant. When 0.5 mg of TFMSA was absorbed via the roots, sufficient male sterility occurred and no seeds were formed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] In one aspect, the present disclosure provides a male sterility inducer for eudicot plants comprising trifluoromethanesulfonamide (TFMSA).

[0017] The structural formula of trifluoromethanesulfonamide (TFMSA) is shown below. [ka]

[0018] The eudicots in this embodiment may be, for example, plants belonging to the core eudicots, more specifically, but not limited to, the order Fabidae, e.g., Fabales, or the order Malvidae, e.g., Brassicales, or the order Lamiids, e.g., Solanales. Non-limiting examples of suitable eudicots are the order Fabatae, the order Brassicaceae, and the order Solanaceae.

[0019] The eudicot plant in this embodiment may be, for example, Fabaceae, Brassicaceae, or Solanaceae. Fabaceae is particularly preferred. Leguminous plants have traditionally been difficult to efficiently control pollination (ironically, since they are a symbol of Mendelian genetics), no practically effective CHAs have been known, and there has been a long-felt but unsolved need for efficiently inducing male sterility, but this embodiment provides a novel solution in the Fabaceae. Fabaceae flowers can be characterized by the co-positioning of pistils and stamens, and it was surprising that the chemical TFMSA, administered indiscriminately, was able to selectively act on male and female organs that are so physically close to each other. Non-limiting examples of leguminous plants include cowpea, soybean, broad bean, pea, kidney bean, chickpea, adzuki bean, lentil, mung bean, peanut, and rooibos. Other non-limiting examples of plants in the legume family include Jack bean, African yam bean, Bambara groundnut, grass pea, Lablab, Rice bean, Sword bean, Velvet bean, Winged bean, and Black gram.

[0020] The male sterility inducer of the present embodiment may typically be in the form of an aqueous solution. An aqueous solution refers to a solution in which water is the main solvent (50% or more, for example 70% or more, 90% or more, or 100% by weight of the solvent). A relatively small amount of other solvents, such as ethanol, may be added. As with common pharmaceuticals used in the agricultural field, the aqueous solution may contain components other than TFMSA, such as one or more of surfactants, thickeners, wetting agents, colorants, nutrients, and other active ingredients. The other active ingredients may be sterilizing active ingredients, or may be active ingredients that are not directly related to sterilizing activity, such as insecticidal activity, bactericidal activity, and fungicidal activity. The male sterility inducer of a particular embodiment may be provided in a dosage form other than an aqueous solution, such as a dust, granule, microcapsule, tablet, or emulsion, and may be used, for example, by spraying the powder as it is, or by dissolving the granules in water immediately before application.

[0021] The concentration of the active ingredient TFMSA in the male sterility inducer (e.g., in the form of an aqueous solution) may be, for example, 100 to 2000 mg / l, or 500 to 1200 mg / l, but a person skilled in the art can adjust this concentration appropriately based on this disclosure and common knowledge, and depending on the individual application.

[0022] In another aspect corresponding to the above aspect, the present disclosure provides a method for inducing male sterility in eudicotyledonous plants, the method comprising administering TFMSA to the eudicotyledonous plant. In the present disclosure, administering means attaching or absorbing the active ingredient TFMSA to the eudicotyledonous plant (e.g., its leaves or roots). It should be understood that the TFMSA may be administered in the form of the male sterility inducer described above. In other words, the present disclosure provides TFMSA or a male sterility inducer or composition containing it for use in inducing male sterility in eudicotyledonous plants. The male sterility inducer aspect and the method aspect of the present disclosure correspond to each other, and therefore the description provided above regarding the target eudicotyledonous plant, TFMSA dosage form, etc. may also be applied to the method embodiment. In the following description, reference to TFMSA may be replaced with a TFMSA-containing composition or a male sterility inducer.

[0023] Those skilled in the art can predict with a certain degree of accuracy when flowering or normal pollination will occur based on sowing timing, cultivation conditions, and experience. In this embodiment, TFMSA is administered before flowering, preferably 5 to 20 days before. After confirming that the plant has bloomed and removing the flowers and sheaths, TFMSA may be administered to act on subsequent flowers. It was observed that male fertility was restored more than two months after administration of TFMSA. In that sense, the male sterility induction of eudicotyledonous plants by TFMSA may be reversible. It has been found that TFMSA may have a certain degree of growth inhibitory effect in eudicotyledonous plants. Those skilled in the art who refer to the present disclosure may adjust the dose of TFMSA in one administration depending on the type of plant or the size of the biomass. The dose may typically be within the range of 1.5 to 50 mg per plant individual, or for example, 0.5 to 3 mg for small plants. For example, in the case of cowpea, 20 to 40 mg per individual, particularly 30 mg, is preferred. Multiple doses can also be administered, for example with 5-10 days between doses.

[0024] In one embodiment, the present disclosure provides a schedule for inducing male sterility in cowpea. The method according to this schedule includes administering two doses of TFMSA of 20-40 mg (e.g., 30 mg) per cowpea plant, 5-10 days (e.g., 7 days) apart, prior to flowering. The second dose can be 5-10 days prior to flowering. This administration schedule can maintain female fertility and ensure complete male sterility, while sufficiently avoiding plant growth inhibition, regardless of the difference in cowpea lineage.

[0025] TFMSA is typically administered to the above-ground parts of plants, including the leaves. For example, it is preferable to administer TFMSA or a solution containing it so that it adheres to the surface of all leaves found at the time of administration. As illustrated in the following examples, the possibility that TFMSA applied to soil is absorbed through the roots and reaches the above-ground parts to act is not excluded. The effectiveness of administering TFMSA through the roots has been confirmed not only in dicotyledonous plants but also in monocotyledonous plants such as pearl millet. Thus, in one aspect, the present disclosure provides a method for inducing male sterility in plants, which includes absorbing TFMSA into the plant through the roots, and also provides a TFMSA-containing male sterility inducer for use in such a method. The plant in this case may be a dicotyledonous plant, particularly a eudicotyledonous plant, or a monocotyledonous plant. Regardless of whether the administration location is above ground or below ground, the method of administration can be appropriately determined by one skilled in the art, and examples include, but are not limited to, spraying with a sprayer, scattering with a watering can or dropper, applying with a brush or sponge, adding to a nutrient solution, or applying a liquid agent or a solid agent such as a tablet or granule on the surface and / or underground. EXAMPLES

[0026] The present invention will be described in more detail below with reference to examples, but these examples merely describe typical experiments for illustrative purposes, and the present invention is not limited to these specific embodiments. For example, similar results can be obtained with eudicotyledonous plants other than the specific species described below.

[0027] Materials and Methods Dozens of cowpea seeds were sown simultaneously at the experimental farm of the Arid Land Research Center, Tottori University, between May and October. Two more lines (IT86D-1010 and IT97K-499-35) were grown in a greenhouse (26°C for 16 h daytime / 18°C for 8 h nighttime). Cowpea (Vigna unguiculata (L.) Walp.) is a eudicot plant in the legume family.

[0028] Trifluoromethanesulfonamide (TFMSA; Tokyo Chemical Industry Co., Ltd.) was dissolved in an aqueous solution containing Approach BI (0.1% v / v, Maruwa Biochemical Co., Ltd.), a commercially available agrochemical spreading agent (polyoxyethylene hexatane fatty acid ester surfactant). In this way, TFMSA solutions with different concentrations between 0 and 1000 mg / l (w / v) were prepared. 0 mg / l is the control. Starting 1 to 5 weeks before flowering, the above TFMSA solution was spray-applied to cowpea plants once a week. It was confirmed that all leaf surfaces were covered with the solution.

[0029] For detailed analysis of pollen fertility, anthers were harvested 1 week after the last TFMSA treatment and approximately 24 hours before anthesis, fixed in 6:3:1 (v / v / v) ethanol / chloroform / glacial acetic acid at room temperature for 3 days, and then stained with Alexander (Alexander M (1969) Stain Technology. 44(3): 117-122). Alexander stain was prepared with the following composition: 10 ml 95% ethanol, 1 ml malachite green (1% solution in 95% ethanol), 50 ml distilled water, 25 ml glycerol, 5 ml acid fuchsin (1% solution in water), 0.5 ml orange G (1% solution in water), and 4 ml glacial acetic acid. Fixed anthers were added to a 1.5 ml tube containing 200 μl of Alexander stain and placed on a heat block at 95°C for 5 minutes. The anthers were then transferred onto a glass slide and either crushed between the glass slide and a cover slip or cut open with a needle to disperse the pollen grains inside, and microscopic images were taken. Fertile pollen grains are stained magenta and sterile pollen grains are stained blue by Alexander staining. The number of fertile and sterile pollen grains was counted using the "Pixel Classification + Object Classification" workflow of the machine learning software "ilastik" (Berg S, et al (2019) Nature Methods 16(12): 1226-1232). For that purpose, one flower was taken from each plant individual, two anthers from each flower were cut open, and the total number of fertile and sterile pollen grains in the two anthers was counted.

[0030] To confirm female function, the following experiment was performed. Cowpea of ​​IT97K-499-35 line (white seed coat) was treated twice with 30 mg TFMSA per plant, and then hand-pollinated with pollen of cowpea of ​​another 29 line (brown seed coat). Two hybrid F1 seeds from each cross were sown in the field, and F2 seeds were also obtained. True hybridity, i.e., the presence of normal female function and the absence of male function in TFMSA-treated individuals, was confirmed by observing the color of the seed coat of the F2 seeds.

[0031] Essentially similar experiments to those described above for cowpea were also performed for other eudicots, including diploid and tetraploid Arabidopsis thaliana (Brassicaceae) and tobacco (Nicotiana benthamiana) and tomato (Solanum lycopersicum) (Solanaceae). Arabidopsis, tobacco, and tomato were treated with TFMSA when the first flower buds were observed. Diploid Arabidopsis was used for the root treatment experiment. Specifically, 100 ml of an aqueous solution containing 0 to 10 mg of TFMSA per plant with 0.1% Approach BI was poured once into a water tray, and the plant was allowed to grow while the roots absorbed TFMSA by placing the pot on the water tray.

[0032] [Example 1] In cowpea anthers treated twice with TFMSA at a dose of 30 mg per plant, Alexander staining showed a predominance of blue-stained pollen grains, indicating a high degree of pollen sterility, i.e. male sterility. This result was in contrast to the control cowpea pollen grains, which were stained entirely with magenta-red, indicating fertility. The two can also be distinguished by the fact that the normal fertile pollen grains are generally round and large, whereas the blue-stained sterile pollen grains are small and relatively indistinctly outlined. Figure 1 shows the experimental results for line IT86D-1010. Different cowpea lines and lower doses of TFMSA treatment were also tested, but in all lines a high degree of pollen sterility was induced (Figure 2). For example, in line IT97K-499-35, significant pollen sterility was observed following treatment with 7.5 mg of TFMSA per plant, and even at doses as low as 1.9 or 3.8 mg, numerous non-functional (blue-stained) pollen grains were observed.

[0033] Table 1 summarizes the detailed counts of fertile / sterile pollen grains after two treatments of different doses of TFMSA on IT86D-1010 lines grown in greenhouse. The percentage of fertile pollen grains in TFMSA-treated cowpea was significantly different from that in the control-treated cowpea. Pollen sterility was 99% with a treatment dose of 30 mg TFMSA per plant. [Table 1]

[0034] [Example 2] In field trials using TFMSA at 30 mg per plant in cowpea, complete pollen sterility was observed in 38 lines, one line had partially functional pollen, and three lines appeared to lack significant effect, however, retesting these three lines with two treatments was able to induce male sterility.

[0035] [Example 3] Field-grown cowpea IT97K-499-35 was treated with TFMSA at a dose of 30 mg per plant for different times (1-week intervals) before flowering, from 0 to 5 times, and a delayed growth was observed with increasing number of treatments (Fig. 3). Thus, plants treated every week for 5 weeks, i.e. 5 times, were smaller than plants treated only once during the last week of the 5-week period. Treated plants produced significantly fewer pods and seeds on average per plant than untreated controls. Seed number was significantly reduced after only one treatment, and few seeds were formed in plants treated 2 to 5 times.

[0036] [Example 4] When cowpeas with white seed coats (IT97K-499-35 line) that had been treated twice with TFMSA to make them male sterile were crossed with cowpeas with brown seed coats (29 line) that had not been treated with TFMSA, the former as female and the latter as male, respectively, the F1 seeds were obtained, which germinated normally and gave rise to normal F2 seeds, and the F2 seeds were black (Figure 4). The black seed coat indicates that the F1 plants are true hybrids (Herniter IA et al (2019) Frontiers in Plant Science 10). This demonstrates that the optimal TFMSA treatment causing pollen sterility does not have a significant adverse effect on female reproductive function.

[0037] [Example 5] Similar results to those described above can also be observed in eudicots other than legumes. For example, a lack of functional pollen formation was observed after TFMSA treatment at doses of 1.3 mg per plant or more in diploid Arabidopsis and 2.5 mg per plant or more in tobacco (Figure 5). Both Arabidopsis and tobacco tended to have growth inhibition as seen in the reduction of plant height by TFMSA treatment. Arabidopsis, which is a plant with a relatively small biomass to begin with, was more susceptible to growth inhibition by TFMSA treatment, and treatment with TFMSA doses of 5 mg per plant or more not only reduced plant height to less than 1 / 4 of the control, but also inhibited the formation of flowers themselves. However, in both species, the number of branches was not observed to decrease in a TFMSA dose-dependent manner. Interestingly, tetraploid Arabidopsis was more resistant to growth inhibition by TFMSA than diploids, maintaining a high plant height up to the maximum dose tested, 10 mg, and forming flowers at doses of 5 mg to 10 mg (not shown). A similar effect on male sterility was also observed in tomatoes, with a concentration-dependent decrease in fruit set, and no fruit set at all when 2.5 mg was applied (Figure 6).

[0038] Although the above examples show the application of TFMSA solution to above-ground parts, especially leaves, it is also possible to induce male sterility by, for example, administering TFMSA to soil or nutrient solution in hydroponic culture and absorbing TFMSA through the roots. As shown in Figure 7, the number of seed-containing pods was reduced in a concentration-dependent manner by treating diploid Arabidopsis thaliana only through the roots (self-pollination experiment is shown). Treatment with 0.5 mg TFMSA caused significant male sterility, significantly reducing the number of seed-containing pods compared to the control, and almost no seeds were formed. When applied to the leaves of diploid Arabidopsis thaliana, male sterility caused by the 0.5 mg dose was still limited (Figure 5), so administration via the roots may be more efficient than administration via the leaves. No flowers were formed when TFMSA was treated with 5 mg or 10 mg through the roots. Although not shown, the effectiveness of the TFMSA male sterility inducer administered via the roots was confirmed not only in dicotyledonous plants but also in monocotyledonous plants such as pearl millet.

[0039] Overall, these experiments in model eudicotyledonous plants indicate that although TFMSA may have the side effect of inhibiting growth in a dose-dependent manner, it is possible to determine the optimal TFMSA dose for each plant that induces male sterility while avoiding severe growth inhibition and female sterility.

[0040] In summary, these studies analyzed the utility of TFMSA as an effective male sterility inducer in eudicots, including cowpea, Arabidopsis, tobacco, and tomato. The lack of ability of treated plants to form self-pollinated pods, detailed analysis of pollen sterility by Alexander staining in various genotypes and species, and evaluation of the effects of TFMSA administration under different conditions confirmed the robust efficacy of this technique in eudicots. For example, virtually complete male sterility was achieved in cowpea with TFMSA treatment at 30 mg / plant × 2, and male sterility was also observed in other model eudicots, often at relatively low doses. Administration via the roots was also effective. Although a clear tendency for TFMSA to have a dose-dependent growth-inhibiting side effect on eudicotyledonous plants was observed, with reference to the examples disclosed above, it appears possible to determine for a given eudicotyledonous plant species a suitable dose for inducing male sterility while preserving female fertility and a sufficient degree of growth.

Claims

1. A male sterility inducer for eudicotyledonous plants, comprising trifluoromethanesulfonamide (TFMSA) as an active ingredient, wherein the eudicotyledonous plant is a plant of the family Fabaceae, Brassicaceae, or Solanaceae.

2. The male sterility inducer according to claim 1 , wherein the legume is cowpea.

3. The male sterility inducer of claim 1 , which is in the form of an aqueous solution.

4. The male sterility inducer of claim 1 , which is administered via the roots of the plant.

5. 1. A method of inducing male sterility in an eudicot plant comprising administering trifluoromethanesulfonamide (TFMSA) to the eudicot plant, wherein the eudicot plant is a plant of the family Fabaceae, Brassicaceae, or Solanaceae.

6. The method of claim 5, wherein the legume is cowpea.

7. The method of claim 5 , wherein the TFMSA is administered in the form of an aqueous solution.

8. The method of claim 5 , wherein the TFMSA is administered via the roots of the plant.

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