JP2008061628A - Method for creating salt-tolerant mutant line of rice - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means enabling the evaluation of salt tolerance in a field-level culture to efficiently create a tolerant mutant line in a short time by increasing the mutation rate and decreasing the number of selection lines. <P>SOLUTION: The method for creating a salt-tolerant mutant line of rice contains the following steps (a) to (c): (a) a step to irradiate rice seeds with heavy ion beam; (b) a step to culture the rice seeds irradiated with heavy ion beam and collect M<SB>2</SB>seeds from the obtained M<SB>1</SB>plant; and (c) a step to culture the collected M<SB>2</SB>seeds on a paddy field added with saline water and select a plant having salt tolerance from the obtained M<SB>2</SB>plants. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for efficiently producing a salt-tolerant rice mutant line by irradiation with a heavy ion beam.

Salt damage, which accumulates salt in the soil and causes serious damage to the soil environment and agriculture, is becoming more serious in other Asian countries such as African countries, Pakistan, and China. In India and the United States, salt damage in arable land due to drought of groundwater is a problem. For this reason, how to prevent growth inhibition due to salt damage has become a major issue, and in recent years, research and development of methods for growing salt-tolerant rice has been desired. In general, environmental resistance is not controlled by one gene but by a plurality of genes, and thus it is considered difficult to cultivate resistant mutant lines. In rice, salt-tolerant varieties (Pokkali, Nona Bokra, IR4630, etc.) and salt-sensitive varieties (I Kong Pao, IR28, etc.) are known, and experiments are carried out to confer resistance to sensitive varieties by crossing and mutation treatment. . On the other hand, there have been few examples of attempts to create and breed salt-tolerant mutant lines by mutagenesis by somatic mutation or EMS or X (X) rays using normal cultivated rice varieties. For example, Zhang et al. (Non-Patent Document 1) have reported the creation of salt-tolerant mutant lines by EMS (ethyl methanesulfonate) treatment. In Zhang literature, callus derived from 1800 cocoons treated with EMS was placed on a medium supplemented with NaCl, salt-tolerant callus was selected, and redifferentiated individuals were cultivated. 22 fertility (R ₀ ) were observed. It was. As a result of seeding the seeds (R ₁ ) obtained from them on soil supplemented with 0.5% NaCl (85.6 mM), nine viable lines were selected as salt-tolerant lines. Guo et al. (Non-patent Document 2) repeated self-breeding of the salt-tolerant line obtained above, and examined salt tolerance in the R ₉ to R ₁₁ generations. It describes that the thousand grain weight was 82% (20.5 / 25.1) compared to the untreated area. Lee et al. (Non-patent Document 3) have reported the production of salt-tolerant mutant lines by gamma (γ) irradiation. In Lee, gamma (γ) rays are irradiated to rice callus, redifferentiated individuals (M ₁ ) are developed in the field, M ₂ seeds are harvested from 200 individuals except those with poor growth, and M ₂ seeds are pots cultivated M ₃ seeds 3000 strains obtained by selfing in submerged state, placed in 10-day flood state, plant height to 3 leaf stage, root length, were selected for flooding tolerance, the number of roots as an index As a result, 64 submergence resistant lines were obtained, and it was described that one of them showed salt tolerance. However, these reports only confirm the salt tolerance in the seedling stage and pot cultivation, and it is not confirmed whether or not the salt tolerance is exhibited in actual field level cultivation. Generally, 1000 to 10,000 lines are required for selection of resistant mutant lines, and cultivation of such many lines requires a large area. Therefore, it is practically difficult to evaluate salt tolerance in cultivation at an actual field level, and there has been no example so far. Therefore, in order to realize the evaluation of salt tolerance in field level cultivation, it is necessary to increase the mutation rate and reduce the number of selected lines.

  In addition, radiation such as gamma (γ) rays and X (X) rays that have been used so far is difficult to accurately irradiate specific tissues of plants. In order to fix it as an inheritance of the whole individual, a great deal of labor is required. In addition, drugs such as EMS induce mutations, damage plant bodies themselves, and reduce germination rate, so it is difficult to obtain a large number of mutants. Furthermore, crossing with a superior line must be repeated in order to eliminate unintended mutations that are expressed in progeny after selection of resistant mutant lines, which requires a long time, labor, and cost. Therefore, there is a demand for means for shortening selection and reducing labor without affecting the traits other than salt tolerance by mutation treatment.

  On the other hand, a new mutagenesis method has been developed to irradiate heavy ion beams (heavy particle beams) in which ion atoms such as carbon are accelerated at high speed using an accelerator. (Patent Documents 1 to 3). Heavy ion beams have been put into practical use as mutagens for flower garden plants, and the appearance of flower color and flower type mutants at that time is as high as several to tens of percent. Furthermore, in order to improve the appearance rate of mutants, there are reports on the examination of heavy ion beam irradiation conditions (type and intensity of irradiated ion beam, pretreatment of irradiation material) (Non-Patent Documents 4 to 7). Heavy ion beam irradiation has stronger energy than gamma (γ-rays) and causes large changes in chromosomes, so it is expected to induce mutations that cannot be obtained with gamma (γ-rays).

Japanese Patent Laid-Open No. 09-28220 JP 2002-12596 A JP 2003-199447 A Zhang G.-Y., Guo Y., Liu F.-H. Chen S.-Y. Chen, S.-L. (1994) RFLP analysis of nine salt tolerant rice mutants, Acta Botanica Sinica, 36 (5) , 345-350 Guo Y., Chen, S.-L., Zhang G.-Y., Chen S.-Y. (1997) Salt-tolerance in rice mutant lines controlled by a major effector gene was obtained by a cell engineering technique, Acta Genet. Sin., 24, 122-126. Lee IS, Kim DS, Hua J. Kang SY, Song HS, Lee SJ, Lim YP, Lee YI (2003) Selection and characterizations of gamma radiation-induced submergence tolerant line in rice, J. Plant Biotechnology, 5 (3) 173 -179 Abe T., et al. (1999) Effective plant-mutation method using heavy-ion beams (III), RIKEN Accel.Prog. Rep., 32, 145 Abe T., et al. (2005) Chlorophyll-deficient mutants of rice induced by C-ion irradiation, RIKEN Accel.Prog. Rep., 38, 132 Abe T., et.al. (2006) Isolation of morphological mutants of rice induced by heavy-ion irradiation, RIKEN Accel.Prog. Rep., 39, 137 Abe T., et al. (2000) Stress-tolerant mutants induced by heavy-ion beams, Gamma Field Symp., 39, 45-54

  Accordingly, an object of the present invention is to provide a means for enabling the evaluation of salt tolerance in field level cultivation by increasing the mutation rate and decreasing the number of selected lines, and for efficiently creating a resistant mutant line in a short period of time. There is to do.

As a result of intensive studies to achieve the above-mentioned problems, the present inventors found that M ₂ seeds collected from M ₁ individuals cultivated rice seeds that had been irradiated with heavy ion beams were subjected to salt-added paddy fields (added with salt water). When cultivated directly in the paddy field), it was found that individuals exhibiting salt tolerance were obtained at a high appearance rate of 1% among the M ₂ individuals. In addition, it was confirmed that individuals exhibiting salt tolerance also exhibited salt tolerance in hydroponic cultivation tests with excess NaCl in the environmental control room. The present invention has been completed based on such findings.

That is, the present invention includes the following inventions.
(1) A method for producing a salt-tolerant rice mutant line comprising the following steps (a) to (c):
(A) A step of irradiating a rice seed with a heavy ion beam (b) A step of cultivating a rice seed irradiated with a heavy ion beam and seeding an M ₂ seed from the obtained M ₁ individual (c) A seeded M ₂ seed A process of cultivating a salt-added paddy field and selecting individuals exhibiting salt tolerance from the obtained M ₂ individuals
(2) The method for producing a salt-tolerant rice mutant strain according to (1), wherein the sodium ion concentration of the brine in the salt-added paddy field is 50 to 100 mM.
(3) Production of a salt-tolerant rice mutant strain according to (1) or (2), wherein the heavy ion beam is a heavy ion beam of 100 MeV / u or higher and the LET is in the range of 20-40 keV / μm. Method.
(4) The method for producing a salt-tolerant rice mutant strain according to any one of (1) to (3), wherein the heavy ion beam is a carbon ion beam or a nitrogen ion beam.
(5) Selection is performed using at least one form or trait selected from the group consisting of the degree of leaf blade mortality, plant height, number of panicles, and panicle weight of M ₂ individuals as an index (1) to (4 ) A method for producing a salt-tolerant rice mutant strain according to any one of the above.
(6) Seed the M ₃ seeds from M ₂ individuals showing salt resistance, the seed was M ₃ seeds were cultivated with brine adding paddy, the step of fixing the salt tolerance trait in M ₃ plants and / or after progeny plants The method for producing a salt-tolerant rice mutant strain according to any one of (1) to (5).
(7) A salt tolerant rice mutant obtained by the method according to any one of (1) to (6).
(8) Salt-tolerant rice mutant line 6-99 (FERM AP-21011).
(9) Salt-tolerant rice mutant line 19-55 (FERM AP-21012).

According to the method of the present invention, it is possible to obtain the individual exhibiting the salt tolerance in M ₂ generation rice seeds mutagenized by heavy ion beam with a high occurrence rate of 1%. That is, by increasing the mutation rate, the number of selected lines for obtaining the target salt-tolerant mutant line can be reduced. As a result, salt-tolerant mutant lines can be efficiently produced in a short time in a salt-added paddy field having a limited cultivation area. Since the salt tolerance mutant produced by the method of the present invention has been evaluated for salt tolerance at the field level, it can exhibit sufficient salt tolerance in the cultivation of actual salt-damaged paddy fields. Further, salt damage resistance traits were selected salt tolerance mutant strain, than that made fixed by M ₂ generations and M ₃ generation, believed to be dominated by a single gene. Therefore, salt tolerance can be imparted to cultivated rice by using these mutant lines as mating parents, and salt damage-tolerant rice breeding that can be cultivated even in cultivated land where salt damage has advanced can be realized.

Hereinafter, the present invention will be described in detail.
A method for producing a salt-tolerant rice mutant line of the present invention, comprising the following steps (a) to (c):
(A) A step of irradiating a rice seed with a heavy ion beam (b) A step of cultivating a rice seed irradiated with a heavy ion beam and seeding M ₂ seed from the obtained M ₁ individual (c) A seeded M ₂ seed A process of cultivating a salt-added paddy field and selecting individuals exhibiting salt tolerance from the obtained M ₂ individuals

  First, in step (a), a heavy ion beam is irradiated to rice seeds. Rice seed varieties that are irradiated with heavy ion beams are not particularly limited as long as they are ordinary varieties except known salt-tolerant varieties. "Sasanishiki", "Akitakomachi", "Kinuhikari" and so on.

  Rice seeds are water-absorbed (soaked) before irradiation with heavy ion beams to form germinated seeds. Water absorption is performed, for example, by immersing in water about twice the amount of seeds at a temperature of 25-30 ° C. for 2-3 days. The germinated seed is a seed germinated by water absorption.

  The type of heavy ion beam is not particularly limited as long as it can induce salt tolerance in rice. For example, carbon (C) ion beam, nitrogen (N) ion beam, argon (Ar) ion beam, neon (Ne) An ion beam, an iron (Fe) ion beam, or the like can be used, but preferred heavy ion beams include a carbon (C) ion beam and a nitrogen (N) ion beam.

  LET (Linear Energy Transfer) in heavy ion beam irradiation is preferably in the range of 20 to 40 keV / μm, and more preferably in the range of 22.6-37.4 keV / μm. The irradiation dose of the heavy ion beam may be determined according to the type of ion beam used, and is not particularly limited as long as it does not damage rice seeds and can induce mutations. In the case of a carbon or nitrogen ion beam (135 MeV / u), the range of 20 to 40 Gray is preferable.

Next, in step (b), and cultivated seeds irradiated with ion beams in the usual fields, from M ₁ individuals obtained to seed progeny seed (M ₂ seeds) in Individual (strain). The seed was M ₂ seeds, cultivated with brine additional paddy adjusting the Na ⁺ concentration of 50 grain increments flooding every system in 50 to 100 mM.

Subsequently, in the step (c), an individual exhibiting salt tolerance is selected from the M ₂ individuals cultivated in the above-described salt water-added paddy field. At this time, normal individuals that do not exhibit salt tolerance are hindered in growth and plant height is shortened, and they die when water drops during harvesting and the salt concentration in the rhizosphere increases. Therefore, selection is performed using the degree of leaf wilt, plant height, number of spikes, panicle weight and the like as indicators. The salt-tolerant mutants are hydroponically cultivated in a culture solution supplemented with NaCl, and the characteristics of the salt-tolerant character are analyzed and confirmed.

Than it selected the salt-tolerant mutant strain to seed the M ₃ seed. M ₃ seeds taken from the salt-tolerant mutant are sown in the same salt-added paddy field as described above, and M ₃ plants are cultivated, or in the same manner, further progeny plants are cultivated, and the stability of the salt-tolerant character is investigated.

  Among the salt-tolerant rice mutant lines obtained by the above method, the seeds of lines 6-99 and 19-55 were patented organisms as of August 29, 2006 (National Institute of Advanced Industrial Science and Technology). Deposited as deposit numbers FERM AP-21011 and FERM AP-21012 at the Deposit Center (IPOD) (1-1-3 East Tsukuba City, Ibaraki Prefecture).

  In addition, the fruiting rate in 6-99 salt water-added paddy field is 78% (67/86) of ordinary rice field, and the grain weight is 88% (18.4 / 20.8). Is 100% of regular rice (93/93) and the weight of 1,000 grains is 102% (20.2 / 19.9).

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the present invention.
(Example 1) Mutation treatment and creation of mutant strain
(1) Creation of mutant line 1 At the ring cyclotron in the RIKEN Nishina Accelerator Research Center facility, after absorbing the Japanese rice cultivar “Nippon Hare” (28 ° C, 3 days), 150 sprouting seeds were charged with carbon ions (per nucleon). 135 MeV, LET 22.6 keV / μm) was irradiated by 40 Gy. The irradiated seeds were cultivated in the field, and M ₂ seeds were collected from each of the M ₁ individuals to obtain M ₂ 128 lines. Of these strains low fertility, the M ₂ 91 strains except for such lines form mutants separated, was used for selection of salt tolerance system.
(2) Creation of mutant line 2 In the ring cyclotron at the Institute of Physical and Chemical Research, Nishina Accelerator Research Center, the Japanese rice cultivar “Nippon Hare” was absorbed (28 ° C, 3 days), then 150 germinated seeds were charged with carbon ions (per nucleon). 135 MeV, LET 22.6 keV / μm) was irradiated by 20 Gy. Irradiation Seeds were grown in the field, and seed the M ₂ seed from M ₁ individuals respectively, to obtain a M ₂ 82 lines. All of these were used to select salt tolerant lines.

(Example 2) Selection method of salt-tolerant lines and fixation of mutation traits Rice seedling culture medium (synthesis) on the second day from the start of water-absorbing germination induction treatment (30 ° C) for the seeds of the total M ₂ 173 lines obtained in Example 1 The seedlings were sown in a plastic pot containing cultivated soil No. 3 (Mitsui East Pressure Fertilizer) (early April) and grown in a greenhouse. In late May, rice that had grown to four-leaf deployment was transplanted at 30 × 15 cm intervals in a test paddy field in the Tohoku University Graduate School of Life Sciences, a submerged ecosystem outdoor experiment facility. The test paddy field was sprayed with a slow-acting chemical fertilizer (manufactured by Coop Chemical Co., Ltd., trade name: Tezuruzu) at a rate of 18.75 kg per 10 a paddy field 7 days before transplanting. This slow-acting fertilizer contains nitrogen, phosphoric acid, and potassium at a component ratio of 16%. For 2 weeks after transplantation, only the agricultural water (Na ⁺ concentration of 10 mM or less) was submerged in all treatment areas. The salt water addition treatment was started from the second week of transplantation. The salt concentration of the brine in the salt water addition treatment zone was adjusted to 50 to 100 mM by pouring salt water having a Na ⁺ concentration of 120 mM and the above-described agricultural water. The Na ⁺ concentration of brine was measured and adjusted every week using a sodium ion electrode (NA-2011, manufactured by Toa Denpa Kogyo Co., Ltd., Tokyo).

As an index for evaluating salt tolerance, the separation ratio and plant height of salt-tolerant individuals and sensitive individuals were observed at the time of harvest in late October. After seeds were dried, ear weights were measured. The salt tolerance of each individual was evaluated using the degree of leaf wilt, plant height, number of ears, and panicle weight as indicators. Mutant strains showing salt resistance is collecting seeds, the M ₃ generation grown with brine additional paddy were confirmation and fixed stability mutation traits. As a result, 6-99 strains from the mutant strain 1 prepared in Example 1 and 19-55 strains from the mutant strain 2 were selected as salt-tolerant rice. The incidence of salt-tolerant strains was 1.1% (6-99 lines) and 1.2% (19-55 lines). In salt-added paddy field, line 6-99 is taller than other individuals and has strong grass momentum, while line 19-55 is not taller than other individuals, but has very strong grass momentum. (Table 1, FIG. 1), fertility was high (Table 2), and the leaf color was green even at the time of harvest. 6-99 strain M ₂ salt-tolerant strains and sensitive strains in generation 1: separated into three, but can be fixed by M ₃ generation. 19-55 system, on the other hand all of the individuals in the M ₂ generation showed salt tolerance.

(Example 3) Salt tolerance test method for salt-tolerant lines In order to evaluate the degree of salt tolerance of selected salt-tolerant lines, hydroponics using a net float, that is, a plastic net is floated on a hydroponic liquid. The method of cultivating plants was used. A net float was created by sandwiching a net cut into 187 x 130 mm with two styrofoam foams (187 x 130 x 5 mm, 187 x 130 x 7 mm) with different thicknesses. Next, a hydroponic apparatus in which the net float was floated on a hydroponic liquid in a polystyrene foam container (220 × 145 × 237 mm) was prepared. Hydroponics used for hydroponics is the literature (Mae, T. and Ohira, K. (1981) The remobilization of nitrogen related to leaf growth and senescence in rice plants ( Oryza sativa L.), Plant and Cell Physiology, 22, 1067-1074). 6-99 strain (M ₅ generations) were seeded approximately 90 grains per vessel on the net in a vessel the germinated seeds were placed 5,000 ml of a water culture medium for 19-55 strain (M ₃ generation). These hydroponic containers were immediately brought into an environmental control device adjusted to a 12-hour day length of 25 ° C.

Five days after sowing, NaCl was applied to rice seedlings at the second leaf development period. As for NaCl, special grade sodium chloride (Wako Pure Chemical Industries) was added so that the Na ⁺ concentration of the hydroponic solution would be 0, 50, 75, and 100 mM, respectively. The pH of the hydroponic solution was adjusted to 5.5 using 0.1 M hydrochloric acid or sodium hydroxide aqueous solution. A pH meter (HM-20P, manufactured by Toa Denpa Kogyo Co., Ltd., Tokyo) was used for pH measurement. In addition, the water culture medium was changed once a day during the cultivation period. The test was repeated at least twice to confirm reproducibility.

Evaluation of salt tolerance was performed 16 to 20 days after sowing, and plant height was measured as an index of salt tolerance. Moreover, after the measurement, the plant body was put in an envelope, dried at 80 ° C. for 5 days, and then the dry weight of the above-ground part was measured. As a result, the relative growth rate of 6-99 and 19-55 strains selected as salt-tolerant rice plants and dry weight above the ground was higher than that of normal strains when NaCl (Na ⁺ concentration 50, 75, 100 mM) was applied. It was kept high in comparison (Tables 3 and 4, FIG. 2). In particular, the relative growth rate of the plant height of 6-99 line under the condition of Na ⁺ concentration of 75 mM was 81%, which was 24% higher than that of Nipponbare. Under the same conditions, the relative growth rate of the above-ground dry weight of the 19-55 line was 97%, which was 16% higher than that of Nipponbare. From these results, it was shown that the 6-99 and 19-55 lines selected in the salt-added paddy field in the field are salt-tolerant lines that are resistant to NaCl application. Furthermore, it was revealed that both lines showed strong tolerance even in salt water with high salt concentration Na ⁺ concentration of 100 mM.

FIG. 1 shows the grass appearance of a salt-tolerant strain (6-99 strain) in a salt-added paddy field. FIG. 1 shows plant heights compared to normal strains of a salt-tolerant strain (6-99 strain) on the 16th day after sowing and a salt-tolerant strain (19-55 strain) on the 20th day after sowing.

Claims (9)

A method for producing a salt-tolerant rice mutant line comprising the following steps (a) to (c):
(A) A step of irradiating a rice seed with a heavy ion beam (b) A step of cultivating a rice seed irradiated with a heavy ion beam and seeding an M ₂ seed from the obtained M ₁ individual (c) A seeded M ₂ seed A process of cultivating a salt-added paddy field and selecting individuals exhibiting salt tolerance from the obtained M ₂ individuals   The method for producing a salt-tolerant rice mutant strain according to claim 1, wherein the sodium ion concentration of the brine in the salt-added paddy field is 50 to 100 mM.   The method for producing a salt-tolerant rice mutant strain according to claim 1 or 2, wherein the heavy ion beam is a heavy ion beam of 100 MeV / u or more and LET is in the range of 20-40 keV / μm.   The method for producing a salt-tolerant rice mutant strain according to any one of claims 1 to 3, wherein the heavy ion beam is a carbon ion beam or a nitrogen ion beam. Selection is about withered leaves only M ₂ individuals, plant height, number of ears, and performs an index of at least 1 or more forms or trait selected from the group consisting of panicle weight, to any one of claims 1 to 4 A method for producing the described salt-tolerant rice mutant line. To seed the M ₃ seeds from M ₂ individuals showing salt resistance, the seed was M ₃ seeds were cultivated with brine additional paddy, further comprising the step of immobilizing the salt tolerance trait in M ₃ plants and / or after generations plants, A method for producing a salt-tolerant rice mutant strain according to any one of claims 1 to 5.   A salt-tolerant rice mutant obtained by the method according to any one of claims 1 to 6.   Salt-tolerant rice mutant line 6-99 (FERM AP-21011).   Salt-tolerant rice mutant line 19-55 (FERM AP-21012).

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