Method for estimating the rate of white immature grains in rice and method for determining cultivation conditions

Abstract

To provide a method for enabling quick estimation of a white immature grain rate after grain-filling in an early grain-filling period of rice.SOLUTION: A white immature grain rate estimation method includes an estimation of a white immature grain rate after grain-filling of rice on the basis of a nuclear phase (nuclear DNA copy number) in an endosperm of rice in an early grain-filling period.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for estimating the rate of white immature grains of rice and a method for determining cultivation conditions. [Background technology]

[0002] Due to the effects of global warming in recent years, high temperature damage to rice has become a problem. A typical symptom of high temperature damage is the clouding of brown rice (white immature grains) due to poor ripening, and an increase in white immature grains leads to a decrease in grade, which has a significant impact on farmers' income. Therefore, producers and others are very interested in predicting the rate of white immature grains before harvest.

[0003] A device that can predict the occurrence of white immature grains before harvest is the White Immature Grain Occurrence Predictor (manufactured by Kett Electric Laboratory Co., Ltd.). This device cuts brown rice about 10 days before harvest and predicts the occurrence of milky white grains (a type of white immature grain) through image analysis of the cut surface. However, to make predictions using this device, brown rice samples close to harvest are required. In other words, the occurrence of white immature grains cannot be predicted until close to harvest.

[0004] As a prediction method that can be implemented at an earlier stage, there is a report on a method in which the expression of a marker gene 10 days after flowering is measured to predict the incidence of white immature kernels (Patent Document 1). However, the drawback is that it takes several hours to analyze gene expression. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2019-058103 A Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method capable of estimating the rate of white immature grains after ripening in a short period of time during the early stage of ripening of rice. [Means for solving the problem]

[0007] As a result of extensive research, the inventors have discovered a new finding that there is a negative correlation between the nuclear phase (nuclear DNA copy number) in rice endosperm tissue at the early stage of ripening and the percentage of white immature grains at the maturity stage, and have completed the present invention based on this finding.

[0008] That is, the present invention includes, for example, the following inventions. [1] A method for estimating the percentage of white immature grains in rice, the method comprising estimating the percentage of white immature grains after ripening of the rice based on the nuclear phase in the endosperm of the rice at an early stage of ripening. [2] The method according to [1], further comprising harvesting rice plants at an early stage of ripening and measuring the nuclear phase. [3] The method according to [1] or [2], wherein estimating the rate of white immature grains after ripening of the rice based on the nuclear phase in the endosperm of the rice at the early stage of ripening includes estimating that the rate of white immature grains after ripening of the rice is a predetermined value or less when the nuclear phase in the endosperm of the rice at the early stage of ripening is a predetermined value or more. [4] The method according to any one of [1] to [3], wherein the rice plant in the early ripening stage is rice plant at a time point when the accumulated temperature after flowering is 170°C to 240°C, or rice plant 6 to 10 days after flowering. [5] A method for determining cultivation conditions for rice, the method including determining cultivation conditions such that the nuclear phase in the endosperm of rice in the early ripening stage is equal to or greater than a predetermined value. [6] The method according to [5], wherein a nuclear phase in the endosperm of rice at an early stage of ripening that is equal to or greater than a predetermined value indicates that the cultivation conditions are suitable. [7] The method according to [5] or [6], wherein the cultivation conditions are cultivation temperatures up to 10 days after flowering. [8] The method according to any one of [5] to [7], wherein the rice plant in the early ripening stage is rice plant at a time point when the accumulated temperature after flowering is 170°C to 240°C, or rice plant 6 to 10 days after flowering. [9] A method for cultivating rice, comprising determining cultivation conditions by any of the methods according to [5] to [8], and cultivating rice under the determined cultivation conditions. Effect of the Invention

[0009] According to the present invention, a method for estimating the rate of white immature grains after ripening in a short time at the early stage of ripening of rice can be provided. By predicting the rate of white immature grains at the early stage of ripening for rice grains that head and flower early in the field, and implementing appropriate cultivation management measures based on the predicted value, it becomes possible to suppress the occurrence of white immature grains in rice grains that head and flower late and reduce the rate of white immature grains in the entire field. [Brief description of the drawings]

[0010] [Figure 1] 1 is a photograph showing the grains sampled in Example 1. Of the grains bearing seeds on the 3rd, 4th, and 5th upper primary rachis branches of each panicle, the 4th, 5th, and 6th grains from the tip were used (a total of 9 grains, including round 4, round 5, and round 6). [Diagram 2] This shows a histogram of nuclear DNA content in brown rice obtained using a flow cytometer. [Diagram 3] This is a graph showing the results of an analysis of the effects of temperature and panicle fertilizer on the nuclear phase of the endosperm (variety: Hinohikari). [Figure 4] This is a graph showing the results of an analysis of the relationship between the nuclear phase of the endosperm in the early ripening stage and the kernel weight at maturity, and the relationship between the nuclear phase and the percentage of white immature kernels (variety: Hinohikari). [Diagram 5] This is a graph showing the results of an analysis of the effect of changing the temperature 10 days after flowering on the percentage of white immature grains (variety: Hinohikari). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] As one embodiment, the present invention provides a method for estimating a rate of white immature grains of rice, the method comprising estimating a rate of white immature grains after ripening of rice based on a nuclear phase in endosperm of the rice at an early stage of ripening.

[0012] Rice is a plant of the genus Oryza in the family Poaceae, and the variety is not particularly limited, and may be Oryza sativa, and may be selected from the japonica, indica, and javanica species. Colored rice such as red rice can also be the subject of the method of this embodiment because the endosperm is white. In addition, it may be non-glutinous rice or glutinous rice. In addition, it may be, for example, Hinohikari, Koshihikari, Koganebare, Hitomebore, Akitakomachi, Kirara 397, Haenuki, Sasanishiki, Kinuhikari, Himenomochi, Yamada Nishiki, Hokuriku 193, and varieties derived therefrom. In addition, the method of this embodiment can be applied to feed rice, but it is preferable to apply it to staple rice from the viewpoint that the white immature grain rate is important.

[0013] Generally, the growth stages of rice after heading are described by the terms "heading stage," "milky ripe stage," "paste ripe stage," "yellow ripe stage," and "fully ripe stage." "Heading" refers to the emergence of the tip of the ear from the flag leaf.

[0014] The estimation method of the present embodiment can estimate the rate of white immature grains after ripening of rice based on the nuclear phase in the endosperm of rice in the early ripening stage. "Early ripening stage" refers to the period from the heading stage to the milky stage (about 0 to about 12 days after heading).

[0015] Furthermore, in order to estimate the white immature grain rate more accurately, the estimation method of this embodiment may be based on the nuclear phase of rice endosperm in the "early milk ripe stage" (about 6 to about 12 days after heading), which is the stage when the dry weight of brown rice becomes 25 to 35% of the dry weight at the mature stage.

[0016] Furthermore, in order to estimate the white immature grain rate more accurately, the estimation method of this embodiment may be based on the nuclear phase of the endosperm of rice 6 to 10 days after flowering, or may be based on the nuclear phase of the endosperm of rice 7 to 9 days after flowering. The number of days from flowering is based on the day when the rice grains under investigation actually flowered. In the case of rice, the flower closes 2 to 3 hours after flowering, so the number of days from flowering can also be the number of days from pollination.

[0017] Furthermore, the estimation method of the present embodiment may be based on the nuclear phase of the endosperm of rice when the accumulated temperature after flowering is 170° C. to 240° C., may be based on the nuclear phase of the endosperm of rice when the accumulated temperature after flowering is 170° C. to 220° C., may be based on the nuclear phase of the endosperm of rice when the accumulated temperature after flowering is 190° C. to 230° C., or may be based on the nuclear phase of the endosperm of rice when the accumulated temperature after flowering is 200° C. to 220° C. The growth rate of rice varies depending on the temperature, and the higher the temperature, the faster the growth. Therefore, using the accumulated temperature after flowering (the sum of the daily average temperatures from the flowering date to the survey date) rather than the number of days after flowering allows for comparison at the same growth stage, and the white immature grain rate can be estimated more accurately.

[0018] The term "ripened rice" refers to rice whose ears are mature, and the term "ripened brown rice" refers to mature grains.

[0019] The estimation method of this embodiment estimates the rate of white immature grains based on the nuclear phase (nuclear DNA copy number) in rice endosperm. In endosperm cells, which make up the majority of rice, a phenomenon called "endoreduplication" occurs in which nuclear DNA continues to be replicated without dividing, increasing the nuclear phase. The estimation method of this embodiment uses the nuclear phase of endosperm cells as an index showing the degree of progress of endoreduplication to diagnose the developmental status of rice.

[0020] The nuclear phase of the endosperm cells may be the average value of the nuclear phase in the entire endosperm tissue. When multiple brown rice samples are used, the average value obtained by dividing the sum of the nuclear phases in the endosperm of each brown rice sample by the number of brown rice samples used for the measurement may also be used. In addition, as described below, when rice plants from multiple different positions in a rice plant group of the same variety under the same cultivation conditions are used, the average value obtained by dividing the sum of the nuclear phases in the endosperm of each brown rice sample by the number of brown rice samples taken from the multiple rice plants and used for the measurement may also be used.

[0021] The nuclear phase of rice endosperm can be measured using brown rice. Therefore, the estimation method of the present embodiment may further include collecting rice unhulled grains or brown rice in an early ripening stage and measuring the nuclear phase.

[0022] Harvesting rice in the early ripening stage may be harvesting unhulled rice in the early ripening stage or harvesting brown rice. The number of unhulled rice or brown rice harvested is not particularly limited, but the estimation method of the present embodiment can be carried out even if the number of unhulled rice or brown rice harvested is small. For example, 1 to 12 grains, 2 grains or more, or 2 to 9 grains may be harvested from one rice plant or one ear. The harvested unhulled rice or brown rice may be harvested from, for example, the primary rachis branch of the ear, or may be harvested from the tip of the primary rachis branch of the ear. In terms of the incidence rate of sterility and the progress rate of ripening, it is preferable to use the primary rachis branch rather than the secondary or tertiary rachis branch, since it allows for more stable investigation. It is not necessary to use all of the harvested unhulled rice or brown rice for measurement and analysis of the nuclear phase. For example, when using a flow cytometer or a fluorescent microscope described later, 2 to 3 grains are sufficient.

[0023] The measurement and analysis of the nuclear phase can be carried out, for example, using a flow cytometer or a fluorescence microscope. The collected unhulled rice or brown rice may be crushed or cut before use. Crushing or cutting may be carried out using a scalpel, a pestle, or the like, or using a solution such as Galbraith, LB01, Otto, Tris.MgCl 2 (For nuclear extracts, see, for example, Table 2 in Louriro et al., Annals of Botany (2006) 98:679-689.) The size of the fragments or cut pieces is appropriately adjusted according to the method of measurement and analysis of the nuclear phase. For example, the average size of the fragments or cut pieces is 1 mm. 2The rice grains or brown rice may be crushed or cut to the following size. When the rice grains or brown rice are crushed or cut, it is preferable to remove the residue using a mesh filter, a centrifuge, or the like. When a mesh filter is used, for example, a filter with a mesh of 40 μm to 60 μm or 50 μm to 70 μm may be used. After crushing or cutting the rice grains or brown rice, preferably after removing the residue, a fluorescent reagent (e.g., propidium iodide or 4',6-diamidino-2-phenylindole (DAPI)) is added to the crushed or cut rice grains or brown rice, so that the nuclear phase can be confirmed by a flow cytometer or a fluorescent microscope. Here, when the crushed or cut rice grains or brown rice are contained in a solution, a fluorescent reagent is added to the solution (in the state of suspension). In the case of a flow cytometer, first, the amount of DNA in the nuclei in the suspension is investigated, and a histogram (exemplified in FIG. 2) is created. Next, the average value of the nuclear phase in the entire endosperm tissue is calculated from the obtained histogram. When a flow cytometer is used, the entire process of crushing or cutting the rice or brown rice, removing the residue, adding the fluorescent reagent, investigating the amount of DNA in the nuclei and creating a histogram, and calculating the average value of the nuclear phase for the entire endosperm tissue can be completed in about 10 minutes.When a fluorescence microscope is used, image analysis is performed using image software such as ImageJ (NIH), and the average value of the nuclear phase for the entire endosperm tissue can be calculated in the same way, and in this case too, the analysis can be completed in about 90 minutes.

[0024] "White immature grain" refers to "rice that is partially or entirely cloudy" and is a general term for "milky white grains," "white-core grains," "white-belly immature grains," "white-back grains," and "immature base grains" among the "immature grains" stipulated in the quality regulations for agricultural products (Ministry of Agriculture, Forestry and Fisheries Notification No. 244, February 28, 2001). "White immature grain rate" refers to the ratio of white immature grains to the total number or weight of brown rice surveyed.

[0025] The white immature grain rate can be investigated, for example, using a grain classifier (RGQI10B, manufactured by Satake Corporation). The grain classifier (RGQI10B, manufactured by Satake Corporation) displays "milky white grains + white-core grains" as "milky white grains" and "white-belly immature grains + white-back grains" as "white-belly immature grains." In this case, the "milky white grains," "base immature grains," and "white-belly immature grains" are collectively referred to as "white immature grains," and the white immature grain rate can be calculated as a percentage of the total brown rice.

[0026] In the estimation method of this embodiment, estimating the rate of white immature grains after ripening of the rice based on the nuclear phase in the endosperm of the rice in the early stage of ripening may include estimating that the rate of white immature grains after ripening of the rice is a predetermined value or less when the nuclear phase in the endosperm of the rice in the early stage of ripening is a predetermined value or more.

[0027] The specified value for the nuclear phase in the endosperm of rice in the early ripening stage may be, for example, a value of 6.3 or more, a value of 6.4 or more, or a value of 6.5 or more, a value of 7.5 or less, a value of 7.4 or less, or a value of 7.3 or less, a value between 6.0 and 7.5, a value between 6.4 and 7.0, or a value between 6.5 and 6.8, or may be 6.4, 6.5, 6.6, 6.7, or 6.8.

[0028] The specified value for the white immature grain rate may be, for example, a value of 40% or less, a value of 35% or less, a value of 30% or less, a value of 25% or less, a value of 20% or less, a value of 25% or less, a value of 22% or less, a value of 20% or less, a value of 15% or less, or a value of 10% or less, a value of 0% or more, a value greater than 0%, a value of 1% or more, a value of 5% or more, or a value of 7% or more, a value between 2% and 30%, a value between 5% and 15%, or a value between 7% and 12%.

[0029] It is considered that the relationship (negative correlation) between the nuclear phase of the endosperm and the white immature grain rate does not change between varieties. The larger the nuclear phase in the endosperm in the early ripening stage, the smaller the white immature grain rate. Therefore, the predetermined value of the nuclear phase in the endosperm in the early ripening stage of rice and the predetermined value of the white immature grain rate can be appropriately determined by a person skilled in the art through testing. For example, a certain variety is cultivated, brown rice (e.g., caryopsis attached to the primary rachis branch) is collected in the early ripening stage, and the nuclear phase in the endosperm is investigated. Thereafter, cultivation is continued until the ears mature, caryopsis at the sampling target positions are collected, and the white immature grain rate is calculated. This makes it possible to determine the predetermined value of the nuclear phase of the endosperm and the predetermined value of the white immature grain rate. For example, it is possible to determine that the predetermined value of the nuclear phase of the endosperm is 6.5, and when the nuclear phase is 6.5 or more, it can be estimated that the white immature grain rate is 20% or less.

[0030] The white immature grain rate estimated by the estimation method of this embodiment can be applied to a group of rice plants of the same variety under the same cultivation conditions. Also, for example, in a group of rice plants of the same variety under the same cultivation conditions, the white immature grain rate may be measured using rice plants at multiple different positions in the group, and the average of the measured white immature grain rates may be used as the white immature grain rate of the group of rice. For example, the white immature grain rate may be measured for rice plants at multiple different positions in a field (3 to 5 points), and the average of the measured white immature grain rates may be used as the white immature grain rate of the field. For example, the white immature grain rate may be measured using rice plants at multiple different positions in a field (3 to 5 points), and the average of the measured white immature grain rates may be used as the white immature grain rate of the field. 2 ~500m 2 1 rice plant or 100m 2 ~200m 2 It may be acceptable to survey one rice plant per field.

[0031] The method of this embodiment makes it possible to predict the white immature grain rate in the early stage of rice ripening, and by implementing appropriate cultivation management measures based on the predicted value of the white immature grain rate of rice grains that flowered early in the field, it becomes possible to suppress the occurrence of white immature grains in rice grains that flower late and reduce the white immature grain rate in the entire field. Examples of such cultivation management measures include run-off irrigation and nitrogen fertilization.

[0032] The present inventors have found that, in addition to the relationship that the larger the nuclear phase in the endosperm in the early ripening stage, the smaller the white immature grain rate, the nuclear phase in the endosperm in the early ripening stage is larger under optimal temperature conditions than under high temperature conditions, and larger when panicle fertilization is applied than when panicle fertilization is not applied. The present inventors have also found that, while the cultivation temperature up to the early ripening stage has a large effect on the white immature grain rate, the cultivation temperature after the early ripening stage (e.g., after 10 days after flowering) has a small effect on the white immature grain rate. Based on these findings, the present invention also provides, as one embodiment, a method for determining cultivation conditions for rice, which includes determining cultivation conditions such that the nuclear phase in the endosperm of rice in the early ripening stage is a predetermined value or more. By determining cultivation conditions such that the nuclear phase in the endosperm of rice in the early ripening stage is a predetermined value or more, rice can be cultivated so that the white immature grain rate is a predetermined value or less.

[0033] Examples of the cultivation conditions for rice include the cultivation temperature in the early ripening stage (for example, up to 7 to 10 days after flowering, for example, up to 10 days after flowering). For example, by setting the cultivation temperature in the early ripening stage to an appropriate temperature, for example, 22 to 26°C, 23 to 24°C, 23°C to 25°C on average, or 24°C on average, the nuclear phase in the endosperm of rice in the early ripening stage can be increased. Although the influence of the cultivation temperature after the early ripening stage (for example, 10 days after flowering) on ​​the white immature grain rate is small, the cultivation temperature after the early ripening stage may be, for example, 20 to 35°C, 22 to 32°C, or 23°C to 25°C. The cultivation temperature may be changed between daytime and nighttime, and may be set, for example, 1 to 3°C or 1 to 2°C higher during the daytime than at night.

[0034] In addition, the cultivation conditions for rice include fertilization. Examples of the fertilizer include fertilizers selected from nitrogen, phosphoric acid, potassium, and combinations thereof. When using a mixed fertilizer containing two or more of nitrogen, phosphoric acid, and potassium, the ratio of nitrogen, phosphoric acid, and potassium can be appropriately set by a person skilled in the art depending on the purpose, but for example, a fertilizer containing nitrogen, phosphoric acid, and potassium in approximately equal amounts may be used as a basal fertilizer, and for example, a fertilizer containing nitrogen, phosphoric acid, and potassium in approximately 1:2:2, or ammonium sulfate may be used as a top dressing.

[0035] In addition, the cultivation conditions for rice include, for example, the presence or absence of ear fertilizer. Ear fertilizer is a fertilizer intended to enrich the ear and the grains in the ear, and is applied immediately before ear emergence (for example, about 15 to 25 days or about 18 days before ear emergence). The appropriate type and amount of ear fertilizer can be appropriately determined by those skilled in the art, but for example, nitrogen at 1 to 3 kg / 10a, or 2 to 5 g / m 2 By applying ear fertilizer, the kernel phase in the endosperm at the early stage of ripening can be enlarged.

[0036] The method for determining the predetermined value of the nuclear phase in the endosperm of rice in the early ripening stage and the predetermined value of the white immature grain rate is as described above. The lower the predetermined value of the white immature grain rate, the more preferable it is, and it may be, for example, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 25% or less, 22% or less, 20% or less, 15% or less, or 10% or less, or 0% or more, more than 0%, 1% or more, 5% or more, or 7% or more, or it may be a value between 2% and 30%, a value between 5% and 15%, or a value between 7% and 12%.

[0037] The predetermined value of the nuclear phase in the endosperm of rice in the early ripening stage may be set to a value at which the white immature grain rate is equal to or less than a predetermined value, and may be, for example, 6.3 or more, 6.4 or more, or 6.5 or more, 7.5 or less, 7.4 or less, or 7.3 or less, between 6.0 and 7.5, between 6.4 and 7.0, or between 6.5 and 6.8, or may be 6.4, 6.5, 6.6, 6.7, or 6.8. For example, in Hinohikari, by determining the cultivation conditions so that the nuclear phase (average value) is 6.8 or more, it is possible to make the white immature grain rate 10% or less. As the cultivation conditions in this case, for example, the air temperature until 10 days after flowering is set to an appropriate temperature (for example, 22 to 26°C, for example, 24°C on average).

[0038] In this embodiment, the nuclear phase in the endosperm of rice in the early ripening stage may be a predetermined value or more, which may indicate that the cultivation conditions are suitable. Suitable cultivation conditions are conditions that make the white immature grain rate less than or equal to a desired value. For example, in the case of Hinohikari, by determining the cultivation conditions so that the nuclear phase (average value) is 6.8 or more, it is possible to make the white immature grain rate 10% or less. Therefore, when it is desired to make the white immature grain rate 10% or less, it indicates that the nuclear phase in the endosperm of rice in the early ripening stage is 6.8 or more, which indicates that the cultivation conditions are suitable. For example, examples of cultivation conditions for making the nuclear phase in the endosperm of rice in the early ripening stage 6.8 or more include a cultivation temperature of 22 to 25°C until 7 to 10 days after flowering, and optional ear fertilization.

[0039] As described above, the rice in the early ripening stage may be, for example, rice at a time when the accumulated temperature after flowering is 170° C. to 240° C., or may be rice 6 to 10 days after flowering.

[0040] The specific aspects of the method of this embodiment can be the same as those described above without any limitations.

[0041] As one embodiment, the present invention also provides a method for cultivating rice, which includes determining cultivation conditions by the above-mentioned method for determining cultivation conditions for rice, and cultivating rice under the determined cultivation conditions.

[0042] Cultivating rice under the determined cultivation conditions can be carried out by those skilled in the art using standard methods for rice cultivation, for example, as described in the cultivation standards and guidelines of each prefecture.

[0043] The specific aspects of the method of this embodiment can be the same as those described above without any limitations. EXAMPLES

[0044] [Example 1] The experiment was carried out at the West Japan Agricultural Research Center (Fukuyama, Hiroshima Prefecture) for three years from 2014 to 2016. Seeds were sown in seedling boxes, and the seedlings of the rice variety "Hinohikari" were transplanted into 1 / 5000 Wagner pots, 16 plants at a time. The rice was then grown outdoors until the heading stage, and tillers were removed as appropriate to leave only the main stem. Fertilization consisted of basal fertilization with nitrogen, phosphorus, and potassium at 0.6g per pot, and top dressing with nitrogen, phosphorus, and potassium at 0.28g, 0.42g, and 0.42g, respectively, four weeks after transplantation. In addition, 0 or 0.32g of nitrogen was applied as panicle fertilization approximately 18 days before heading. Each pot was moved to two natural light greenhouses with different temperatures at the beginning of heading. The temperature conditions were moderate and high, with the average temperature in the moderate temperature zone being 25°C during the day (12 hours) and 23°C at night (12 hours), and the average temperature in the high temperature zone being 30.5°C during the day and 28.5°C at night. The temperature conditions assigned to each greenhouse were switched in 2015.

[0045] The caryopsis sampled were the 4th, 5th, and 6th caryopsis from the top of the 3rd, 4th, and 5th primary rachis branches of each panicle (a total of 9 caryopsis, circle 4, circle 5, and circle 6 in Fig. 1). The flowering date of each caryopsis was investigated, and sampling was performed on the day when the accumulated temperature after flowering reached 200-220°C (optimum temperature area: 9 days after flowering, high temperature area: 7 days after flowering). Five pots were used for sampling, and two caryopsis that were at the sampling positions were collected for the flow cytometer, and after removing the husk, they were frozen and stored at -80°C. The remaining caryopsis at the sampling positions were dried at 60°C for 3 days and the dry weight was measured. 10 days after flowering, the greenhouses for some of the pots were changed to change the temperature conditions. Cultivation was then continued until the panicles matured, and the caryopsis at the sampling positions were collected to investigate the dry weight and the percentage of white immature grains. The white immature grain rate was investigated using a grain classifier (RGQI10B, Satake Corporation). “Milky white grains,” “base immature grains,” and “white belly grains” were collectively classified as “white immature grains,” and the rate was calculated as a percentage of the total brown rice.

[0046] Analysis by flow cytometer was performed according to the method of Galbraith et al., Science (1983) 220: 1049-1051. The extraction solution (chopping buffer (pH 7.0)) was a mixture of 45 mM magnesium chloride, 30 mM sodium citrate, 20 mM MOPS (4-morpholinepropane sulfonate), and 1 mg / ml Triton X-100. Frozen caryopsis was thawed in the extraction solution, chopped for 2 minutes and 30 seconds using a scalpel, and filtered through a 70 μm nylon mesh. Propidium iodide (Dojindo Laboratories) was added to the suspension to a concentration of 30 μM, and a histogram was created and analyzed using a flow cytometer (guava easyCyte 6-2L, Merck Millipore, Billerica, MA). The histogram is shown in Figure 2. 10,000 nuclei were used to create the histogram.

[0047] From the obtained histogram, the average value of the nuclear phase for the entire endosperm tissue was calculated.

[0048] Statistical analysis was performed using statistical software (JMP (registered trademark) 11, manufactured by SAS Institute Inc.). Each year was considered as a replication, and analysis was performed using a three-replica split-plot method with temperature as the main plot and fertilization conditions as the secondary plot.

[0049] The results of analyzing the effects of temperature and panicle fertilization on the nuclear phase of endosperm are shown in Figure 3. "a," "b," and "ab" in Figure 3 show the results of statistical analysis, and the same alphabet indicates that there was no significant difference between treatments. It was found that the average nuclear phase value obtained by this method varied depending on the environmental conditions, and was smaller under high temperature conditions (average 29°C) than under optimal temperature conditions (average 24°C), and was also smaller than when panicle fertilization was not applied.

[0050] In addition, the results of analyzing the relationship between the nuclear phase of the endosperm in the early ripening stage and the kernel weight at maturity (a), and the relationship between the nuclear phase and the rate of white immature kernels (b) are shown in Figure 4. Fluctuations in the nuclear phase and the rate of white immature kernels are linked, and a significant negative correlation was found between the average nuclear phase value of endosperm cells at accumulated temperatures after flowering of 200-220°C and the rate of white immature kernels in brown rice at maturity.

[0051] Figure 5 shows the results of an analysis of the effect of changing the temperature 10 days after anthesis on the percentage of white immature grains. "a," "b," and "ab" in Figure 5 show the results of statistical analysis, and the same alphabet indicates that there is no significant difference between treatments. It was found that the effect of temperature from 10 days after anthesis onwards on the percentage of white immature grains is small, and that even if the temperature conditions are changed 10 days after anthesis, no significant difference is observed in the percentage of white immature grains in brown rice.

[0052] It was considered possible to estimate the percentage of white immature kernels in mature kernels by investigating endoreduplication during the early ripening stage when the accumulated temperature was 200-220℃.

[0053] [Example 2] We investigated the differences in the growth rate of brown rice varieties. The test was carried out at the West Japan Agricultural Research Center (Fukuyama, Hiroshima Prefecture) in 2014. Five japonica varieties (Akitakomachi, Kinuhikari, Koshihikari, Nipponbare, Sasanishiki) and five indica varieties (Habataki, Hokuriku 193, IR64, Kasalath, Takanari) were sown and raised in seedling boxes, and transplanted to an outdoor test field on June 24th. As basal fertilizer, 28.5g / m of advanced chemical fertilizer (nitrogen, phosphorus and potassium content of 14% each) was applied. 2 As a top dressing, ammonium sulfate was applied at 9.5g / m2 one month after transplanting. 2 The flowering dates of the 4th, 5th and 6th caryopsis from the tip of the grains that had borne grains on the 1st and 2nd upper primary rachis branches of the panicle were investigated, and some of these were harvested 7-8 days after flowering and ventilated and dried at 60°C for 72 hours to determine their dry weight. The remaining caryopsis were harvested at maturity and ventilated and dried at 60°C for 72 hours to determine their dry weight. As an index of the developmental stage of brown rice at the time of investigation, the percentage of the dry weight of brown rice 7-8 days after flowering to the dry weight of brown rice at maturity was calculated.

[0054] The results are shown in Table 1.

[0055] [Table 1]

[0056] It was found that for all varieties, when the accumulated temperature was between 170 and 240 degrees Celsius, the growth stage was reached where the dry weight of brown rice was 25 to 35% of the dry weight at maturity.

Claims

1. A method for estimating the rate of white immature grains of rice, comprising estimating the rate of white immature grains after ripening of the rice based on the nuclear phase in the endosperm of the rice at an early stage of ripening.

2. The method according to claim 1, further comprising harvesting rice plants at an early stage of ripening and measuring the nuclear phase.

3. 3. The method according to claim 1 or 2, wherein estimating the rate of white immature grains after ripening of rice based on the nuclear phase of the endosperm of the rice in the early ripening stage includes estimating that the rate of white immature grains after ripening of the rice is a predetermined value or less when the nuclear phase of the endosperm of the rice in the early ripening stage is a predetermined value or more.

4. The method according to any one of claims 1 to 3, wherein the rice plant in the early ripening stage is rice plant at a time point when the accumulated temperature after flowering is 170°C to 240°C, or rice plant 6 to 10 days after flowering.

5. When the nuclear phase in the endosperm of rice in the early stage of ripening is equal to or greater than a predetermined value, it is estimated that the rate of white immature grains after ripening of the rice is equal to or less than a predetermined value; A method for determining cultivation conditions for rice, comprising determining cultivation conditions such that the nuclear phase in the endosperm of rice in the early ripening stage is equal to or greater than a predetermined value.

6. The method according to claim 5 , wherein a nuclear phase in the endosperm of rice at an early stage of ripening that is equal to or greater than a predetermined value indicates that the cultivation conditions are suitable.

7. The method according to claim 5 or 6, wherein the cultivation conditions are cultivation temperatures up to 10 days after flowering.

8. The method according to any one of claims 5 to 7, wherein the rice plant in the early ripening stage is rice plant at a time point when the accumulated temperature after flowering is 170°C to 240°C, or rice plant 6 to 10 days after flowering.

9. A method for cultivating rice, comprising: determining cultivation conditions by the method according to any one of claims 5 to 8; and cultivating rice under the determined cultivation conditions.

Images