Highly efficient generation of nutritional stress mutants by heavy ion beam irradiation

Abstract

To provide a method for efficiently producing nutritional stress tolerant mutants by inducing targeted mutations in plant nutrition.SOLUTION: According to the present invention, there is provided a method for producing a nutritional stress tolerant mutant, which comprises irradiating a plant with a heavy ion beam and, after the irradiation, cultivating the plant under nutritional stress conditions.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing a nutritional stress tolerant mutant, which comprises irradiating a plant with a heavy ion beam and cultivating the plant under nutritional stress conditions after the irradiation. [Background technology]

[0002] Plant breeding has traditionally been carried out using mutation induction techniques involving heavy ion beam irradiation. Mutation induction by heavy ion beam irradiation is more efficient than mutation induction using X-rays, gamma rays, or chemical mutagens. For example, in the breeding of flowering plants, heavy ion beam irradiation has been used to successfully produce flower color mutants in many plant species (Non-Patent Document 1).

[0003] Conventional methods for producing plant mutants require a great deal of time and effort because selection is based on mutations that are accidentally induced. Mutation induction by heavy ion beam irradiation also involves accidental mutation induction when plant material is irradiated, and the production of useful mutants requires a great deal of time and effort. For example, Non-Patent Document 2 reports the production of three low-cadmium-accumulating rice mutants from 2,592 strains irradiated with heavy ion beams.

[0004] Furthermore, in order to respond to abnormal weather caused by climate change and food shortages due to population growth, there is also the challenge of developing vegetables and grains that are resistant to environmental and nutritional stress and increasing their yields. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Abe Tomoko, Hirano Tomoya: Agricultural Technology System, Flowers, Vol. 5 Supplement, No. 15, 2013, p. 124. [Non-patent document 2] Ishikawa et al. Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice. 19166-19171, PNAS, November 20, 2012, vol. 109, no. 47. Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present invention is to provide a method for efficiently producing nutritional stress-tolerant mutants by inducing mutations in line with plant nutritional targets. [Means for solving the problem]

[0007] As a result of extensive research aimed at solving the above problems, the present inventors discovered that irradiating plants under nutritional stress conditions with heavy ion beams can induce targeted mutations and produce useful mutants with high efficiency, leading to the completion of the present invention.

[0008] According to the present invention, the following inventions are provided. [1] A method for producing a mutant that is resistant to nutritional stress, comprising irradiating a plant with a heavy ion beam and cultivating the plant under nutritional stress conditions after irradiation. [2] The method according to [1], wherein heavy ion beam irradiation is carried out on plants grown under nutritional stress conditions. [3] The method according to [1] or [2], wherein the heavy ion beam irradiation is carried out on plant seedlings grown under nutritional stress conditions. [4] The method according to [1] or [2], wherein the heavy ion beam is selected from the group consisting of carbon (C), neon (Ne), argon (Ar), and iron (Fe) ion beams. [5] The method according to [1] or [2], wherein the nutritional stress condition is an excess or deficiency of an essential element or an excess of a harmful element. [6] A nutritional stress-tolerant mutant plant produced by the method described in [1] or [2]. [Effects of the Invention]

[0009] The present invention makes it possible to efficiently create nutritional stress-tolerant mutants that exhibit desired phenotypes. This technology will enable the efficient creation of crops that are useful not only academically but also agriculturally. Furthermore, this technology is thought to be applicable to the creation of plants that are tolerant to other abiotic stresses, such as low temperature and drought stress, in addition to nutritional stress. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows the first selection (M2), second selection (M3), and third selection (M3) plants of mutants exhibiting significant nickel (Ni) excess tolerance. "0 Gy" represents unirradiated, wild-type Arabidopsis thaliana. The mutant "60 GyNi10" is a mutant derived from seedlings grown under Ni excess conditions. The control for the third selection (M3) is an individual grown in Ni-free medium. [Figure 2] FIG. 2 is a graph showing a comparison of root length between the mutant "60GyNi10" grown under Ni-excess conditions and the wild type "WT." [Figure 3] FIG. 3 is a graph showing the aboveground and belowground fresh weights of the mutant "60GyNi10" grown under Ni-excess conditions and the wild type "WT." DETAILED DESCRIPTION OF THE INVENTION

[0011] The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0012] The present invention provides a method for producing a nutritional stress tolerant mutant, which comprises irradiating a plant with a heavy ion beam and cultivating the plant under nutritional stress conditions after the irradiation.

[0013] As used herein, "nutritional stress" refers to the phenomenon in which plant growth is inhibited due to a deficiency or excess of essential elements necessary for plant growth, or the phenomenon in which plant growth is inhibited due to the excess of harmful elements. Plants require 17 essential elements for growth, namely, the three major elements of nitrogen (N), phosphorus (P), and potassium (K), as well as calcium (Ca), oxygen (O), hydrogen (H), carbon (C), magnesium (Mg), sulfur (S), iron (Fe), manganese (Mn), boron (B), zinc (Zn), molybdenum (Mo), copper (Cu), chlorine (Cl), and nickel (Ni). Of these, nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), oxygen (O), hydrogen (H), carbon (C), magnesium (Mg), and sulfur (S) are essential macroelements, while iron (Fe), manganese (Mn), boron (B), zinc (Zn), molybdenum (Mo), copper (Cu), chlorine (Cl), and nickel (Ni) are essential trace elements. For example, iron (Fe) deficiency causes new leaves to show chlorosis and, in severe cases, plant death. Excess nickel (Ni) inhibits root growth and causes leaves to show chlorosis. Sodium (Na), cadmium (Cd), and aluminum (Al) are known as toxic elements and inhibit plant growth.

[0014] The "plants" targeted by the present invention may be any type of fern, gymnosperm, or angiosperm. In certain embodiments, the "plants" targeted by the present invention may be agricultural crops, grains, or vegetables used for food or feed. The "nutritional stress-tolerant mutants" of the present invention are mutant plants that have improved resistance to nutritional stress, such as deficiency or excess of essential elements or excess of harmful elements, compared to wild-type plants. Nutritional stress-tolerant mutant plants can be cultivated in environments or regions unsuitable for the cultivation of conventional varieties, for example, and are therefore thought to contribute to improved yields.

[0015] Mutation breeding using heavy ion beam irradiation involves irradiating plants with a heavy ion beam to damage DNA and thereby induce mutations. In the present invention, the plant material irradiated with a heavy ion beam can be at various plant parts and growth stages, such as leaves, petals, seedlings, seeds, and cultured cells. In certain embodiments of the present invention, seedlings are preferably irradiated with a heavy ion beam. Heavy ion beam irradiation can be performed, for example, by placing the plant material in a petri dish, plastic case, culture vessel, or centrifuge tube. In certain embodiments, seedlings are aseptically cultured in a petri dish and then irradiated within the dish. The nuclide of the heavy ion beam is not limited, but carbon (C), neon (Ne), argon (Ar), and iron (Fe) are preferred, with neon (Ne) being more preferred. It is preferable to select a nuclide with an optimal LET (linear energy transfer) that can induce mutations suitable for breeding. The LET can be selected, for example, from 100 to 500 keV / μm, but is not limited to this range. Heavy ion beams can be irradiated at doses ranging from, for example, 0.1 to 500 Gy, 1 to 300 Gy, or 1 to 100 Gy, but are not limited to these. The optimal dose for inducing mutations suitable for breeding can be determined appropriately depending on the plant species, plant part, and nuclide. Irradiation accelerator facilities available in Japan include HIMAC (Chiba City), TIARA (Takasaki City), RIBF (Wako City), W-MAST (Tsuruga City), and SPring-8 (Sayo District).

[0016] In the present invention, the plant is grown under nutritional stress conditions after at least heavy ion beam irradiation. The nutritional stress conditions in the present invention refer to conditions in which at least one element selected from the group consisting of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), oxygen (O), hydrogen (H), carbon (C), magnesium (Mg), sulfur (S), iron (Fe), manganese (Mn), boron (B), zinc (Zn), molybdenum (Mo), copper (Cu), chlorine (Cl), and nickel (Ni) is deficient or present in excess, or conditions in which at least one element selected from the group consisting of sodium (Na), cadmium (Cd), and aluminum (Al) is present in excess.

[0017] Deficiency stress of essential elements can be inflicted on plants by growing them in a medium that does not contain at least one essential element or that contains at least one essential element at a concentration less than the minimum required. Those skilled in the art can appropriately determine the minimum required concentration of an essential element by analyzing the growth of plants in media containing gradually varying amounts of one essential element, taking into account factors such as the plant species and growth stage, the amount of element contained in the seeds, and the pH of the medium. For example, to inflict deficiency stress on plants in the essential trace elements iron (Fe), manganese (Mn), boron (B), zinc (Zn), molybdenum (Mo), copper (Cu), chlorine (Cl), or nickel (Ni), it is sufficient to omit the addition of one of the essential trace elements to the medium. Some essential trace elements are difficult to deplete due to contamination by reagents or agar used in the medium; in such cases, they can be removed from the medium by adding a strong chelator.

[0018] When applying excess stress from essential or harmful elements to plants, the plants can be grown in a medium containing an excess concentration of at least one of the essential or harmful elements. The excess concentration of an essential or harmful element is a concentration exceeding the concentration suitable for normal plant growth. Those skilled in the art can appropriately determine the excess concentration by analyzing the growth of plants in media containing gradually varying amounts of one essential element, taking into account the plant species, growth stage, medium pH, etc., as needed. The essential or harmful element can be added to the medium, for example, as an inorganic salt. When applying excess Ni stress to Arabidopsis seedlings, the plants can be grown in a medium containing NiSO4 at a concentration of 50 μmol / L to 400 μmol / L. The concentration at which excess stress is applied varies depending on the plant species, growth stage, and medium pH, but seedlings of plants other than Arabidopsis thaliana can also be subjected to excess Ni stress by growing them in a medium containing 50 μmol / L or more of NiSO4.

[0019] Manganese (Mn) excess stress can be induced by growing plants in a medium containing 1200 μmol / L or more of MnSO4. Zinc (Zn) excess stress can be induced by growing plants in a medium containing 600 μmol / L or more of ZnSO4. Iron (Fe) excess stress can be induced by growing plants in a medium containing 300 μmol / L or more of Fe-EDTA. Copper (Cu) excess stress can be induced by growing plants in a medium containing 50 μmol / L or more of CuSO4. Cadmium (Cd) excess stress can be induced by growing plants in a medium containing 100 μmol / L or more of CdCl2. These excess element concentrations are examples and can be adjusted appropriately depending on the plant species, growth stage, and medium pH. It is thought that exposure to nutritional stress after heavy ion beam irradiation may prevent plants from repairing advantageous mutations that allow them to survive under nutritional stress conditions. In one embodiment of the present invention, the nutritional stress condition can be a deficiency or excess stress of the essential trace elements iron (Fe), manganese (Mn), boron (B), zinc (Zn), molybdenum (Mo), copper (Cu), chlorine (Cl), or nickel (Ni).

[0020] In a specific embodiment, heavy ion beam irradiation is performed on a plant grown under nutritional stress conditions. It is believed that irradiating the plant with a heavy ion beam while the expression of the target genes is enhanced by nutritional stress will enable a high probability of mutation induction in the target genes. In a further specific embodiment, heavy ion beam irradiation is performed on plant seedlings grown under nutritional stress conditions. The plant seedlings are young plants several days after sowing. In a specific embodiment, heavy ion beam irradiation is preferably performed on seedlings 1 to 11 days after sowing, 1 to 7 days after sowing, or 1 to 3 days after sowing. It is believed that irradiating plant seedlings with a heavy ion beam will increase the possibility of introducing targeted mutations.

[0021] In certain embodiments, plants are exposed to nutritional stress conditions before and after heavy ion beam irradiation. In another embodiment, plants are exposed to nutritional stress conditions throughout the entire process, before, during, and after heavy ion beam irradiation. When the plant material is a seedling, seeds sown in a medium under nutritional stress conditions can be germinated, and the seedlings grown in the medium under nutritional stress conditions can be irradiated. The seedlings irradiated with heavy ion beams are preferably grown under nutritional stress conditions for 1 to 16 days, 1 to 8 days, or 1 to 3 days after irradiation. As shown in the Examples below, the nutritional stress treatment increased the frequency of the production of nutritional stress-resistant mutants, which is thought to be due to the increased probability of mutations being introduced into genes that respond to nutritional stress by heavy ion beam irradiation during nutritional stress treatment. It is possible that growing plants under nutritional stress conditions after irradiation may not repair mutations that are advantageous for surviving nutritional stress conditions. [Example]

[0022] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0023] Example 1: Creation of Ni-tolerant Plants Experimental methods and materials Arabidopsis thaliana (Arabidopsis thaliana, ecotype, Columbia (col-0)) was used as the material. All cultivation using sterile 1 / 2 concentration MS (Murashige & Scoog) solid medium was carried out in an artificial climate chamber at 23°C with a 16-hour photoperiod. Plants for seed collection were grown in an artificial climate chamber at 22°C with a 16-hour photoperiod. Ion beam irradiation was carried out using the HIMAC heavy ion cancer therapy device at the National Institutes for Quantum and Radiological Science and Technology. The optimal dose for Arabidopsis seedlings 5 ​​days after sowing on 1 / 2 concentration MS solid medium was investigated based on the plant growth rate after each ion beam irradiation. The height of the flower stalks was measured four weeks after irradiation with three types of heavy ion beams (acceleration energy per nucleon: neon (Ne) 400 Mev / u (hereafter referred to as Ne400), argon (Ar) 500 Mev / u (hereafter referred to as Ar500), and iron (Fe) 500 Mev / u (hereafter referred to as Fe500); irradiation field size was 10 cm for all), and the growth rate at each irradiation dose was calculated, assuming the height of the unirradiated plant as 100%. The optimal dose was determined to be around the irradiation dose at which the growth rate reached 50%.

[0024] The mutants were generated as follows. Five days after sowing on 1 / 2-strength MS solid medium containing 100 μM NiSO4 (a mild Ni-excess stress condition), seedlings were irradiated with appropriate doses of Ne, Ar, and Fe and then cultivated in a greenhouse for 16 days. They were then transplanted onto rock wool and cultivated with a 1000-fold diluted Hyponex concentrate (Hyponex Japan Co., Ltd.), and seeds (M2) were harvested. In this example, plants were exposed to Ni-excess stress throughout the entire process, before, during, and after irradiation. 16 days after irradiation, they were transplanted to normal conditions for seed collection.

[0025] Ni-excess-tolerant mutants were selected by visually selecting lines that showed better growth than unirradiated Arabidopsis (wild-type, WT) plants after 2 weeks of self-pollination of irradiated M1 plants onto 1 / 2-concentration MS solid medium containing 400 μM NiSO4 or 200 μM NiSO4 (Ni-excess conditions) (primary selection). The M3 lines obtained by self-pollination of the selected M2 plants were then grown under Ni-excess conditions for 2 weeks, and lines showing better growth than unirradiated Arabidopsis plants were visually selected (secondary selection). To quantify and compare the Ni-excess tolerance of the mutants, a third selection was performed as follows. Five days after sowing on 1 / 2-concentration MS solid medium, 15 WT and mutant seedlings were transplanted onto 1 / 2-concentration MS solid medium (without NiSO4) (control condition) or 1 / 2-concentration MS solid medium containing 200 μM NiSO4 (Ni-excess condition) and grown for 2 weeks. After cultivation, the root length, aboveground fresh weight, and underground fresh weight of each individual were measured. Root length (%) was calculated as a percentage of the root length under control conditions, which was set at 100%.

[0026] dose Based on the results of growth measurements of Arabidopsis seedlings under control conditions irradiated with Ne400, Ar500, and Fe500, it was suggested that the optimal doses of Ne400 irradiation for Arabidopsis seedlings were 30-60 Gy, 25-35 Gy for Ar500, and 10-20 Gy for Fe500. Arabidopsis seedlings sown 5 days after sowing under Ni-excess conditions (100 μM NiSO4) were irradiated with appropriate doses of neon, argon, or iron ions (Ne400: 30 Gy, 40 Gy, 50 Gy, 60 Gy; Ar500: 25 Gy, 30 Gy, 35 Gy; Fe500: 10 Gy, 15 Gy, 20 Gy). M1 plants were then selfed to generate M2 plants. M2 plants were then selected under Ni-excess conditions (400 μM NiSO4). Finally, mutants exhibiting significant Ni-excess tolerance were obtained only from seedlings irradiated with 60 Gy of Ne400 (Table 1). These results suggest that 60 Gy of Ne400 irradiation is appropriate for generating Ni-excess-tolerant mutants in Arabidopsis. [Table 1]

[0027] Ni overstress treatment To investigate the effect of Ni excess stress on the frequency of Ni excess-tolerant mutants, Arabidopsis seedlings 5 ​​days after sowing under control and Ni excess conditions (100 μM NiSO4) were irradiated with 60 Gy of Ne and then subjected to the three selection rounds (200 μM NiSO4) described above. This experiment was repeated twice. Figure 1 shows the appearance of plants after the three selection rounds, using a mutant exhibiting significant Ni excess tolerance as an example. The mutant "60GyNi10" was derived from seedlings exposed to Ni excess. Under Ni excess conditions, the roots of the mutant "60GyNi10" were significantly longer than those of the WT (Fig. 2), and the aboveground and belowground fresh weights were significantly heavier than those of the WT (Fig. 3).

[0028] Table 2 shows the number of M2 plant lines and the results of the third selection. In Table 2, lines that were significantly higher than WT in root length, aboveground fresh weight, and underground fresh weight after two weeks of cultivation under Ni excess conditions were designated as Ni excess-tolerant mutants. As a result, the frequency of Ni excess-tolerant mutants was an average of 0.2% under control conditions and 1.0% under Ni excess conditions. This suggests that Ni excess stress treatment increases the frequency of Ni excess-tolerant mutants. [Table 2]

[0029] The increased frequency of Ni excess stress treatment in the production of Ni excess-tolerant mutants is presumably due to the increased probability of introducing mutations into genes that respond to Ni excess stress when exposed to heavy ion beams during Ni excess treatment. In this study, plants were transplanted to normal conditions for seed collection 16 days after irradiation, so they were exposed to Ni excess stress throughout the entire process, before, during, and after irradiation. It is possible that plants continue to be exposed to Ni excess stress after irradiation, preventing the repair of advantageous mutations for survival under Ni excess conditions. One gene whose expression is induced by Ni excess stress is nicotianamine synthase. Nicotianamine has the ability to bind to Ni and detoxify excess Ni within plant cells. Therefore, Arabidopsis plants with enhanced nicotianamine synthesis exhibited significant nickel excess tolerance. Irradiation of seedlings under Ni excess conditions may highly likely introduce mutations into genes involved in such Ni excess-induced tolerance.

[0030] Example 2: Creation of plants tolerant to Fe deficiency Experimental methods and materials Rice (Oryza sativa L. cv. Koshihikari) was used as the material. All cultivation using sterile solid media was carried out in an artificial climate chamber at 28°C with a 16-hour light period. Plants for seed collection were grown in a greenhouse at 30°C under natural light. Heavy ion beam irradiation was carried out at HIMAC, National Institutes for Quantum and Radiological Science and Technology.

[0031] The mature seeds were irradiated with 300 Gy of Ne400 (accidental mutagenesis). The irradiated seeds were sown on MS solid medium and then cultivated in a greenhouse for 14 days. They were then transplanted to normal conditions (nursery soil: Bonsol No. 1 synthetic granular soil, manufactured by Sumitomo Chemical Co., Ltd.) and cultivated there. Seeds (M2) were then harvested.

[0032] Heavy ion beam irradiation (targeted mutation induction) was performed on rice seedlings 11 days after sowing on iron-free MS solid medium by irradiating them with 25 Gy of Ne400. The irradiated seedlings were then grown in iron-deficient medium for 3 days, after which they were transplanted to normal conditions (Bonsol No. 1 seedling soil) and grown, and seeds (M2) were harvested.

[0033] Selection of iron deficiency tolerant rice was carried out by sowing seeds directly into alkaline soil, applying iron-free Kasugai solution (Kasugai Shinichiro, Research on Hydroponic Methods, Japanese Journal of Soil Science and Plant Nutrition 13 669-822 (1939)) and cultivating for four weeks, followed by visually selecting the plants with the best growth.

[0034] result Table 3 shows the frequency of iron-deficiency-tolerant rice mutants. In an experiment in which mature seeds were irradiated with 300 Gy of Ne, the frequency of iron-deficiency-tolerant rice mutants was 0.7%. On the other hand, in an experiment in which rice seedlings under iron-deficiency stress conditions were irradiated with 25 Gy, the frequency of iron-deficiency-tolerant rice mutants was 6.5%. This suggests that targeted mutation introduction can increase the frequency of producing iron-deficiency-tolerant rice mutants. [Table 3]

[0035] The results of this example suggest that this targeted mutagenesis can also be applied to cereals, and that it increases the frequency of appearance of the desired mutant compared to accidental mutagenesis.

Claims

1. A method for producing a mutant resistant to nutritional stress, comprising irradiating a plant with a heavy ion beam and cultivating the plant under nutritional stress conditions after the irradiation.

2. The method of claim 1, wherein the heavy ion beam irradiation is carried out on a plant grown under nutritional stress conditions.

3. 3. The method according to claim 1, wherein the heavy ion beam irradiation is carried out on plant seedlings grown under nutritional stress conditions.

4. 3. The method of claim 1, wherein the heavy ion beam is selected from carbon (C), neon (Ne), argon (Ar), and iron (Fe) ion beams.

5. 3. The method of claim 1 or 2, wherein the nutritional stress condition is an excess or deficiency of an essential element or an excess of a harmful element.

6. A nutritional stress-tolerant mutant plant produced by the method of claim 1 or 2.

Images