Method for improving saccharification properties of non-edible part of crop, and crop having improved saccharification properties

Abstract

There are developed: a crop in which a cytokinin synthase gene is placed under the control of a promoter for inducing the expression of the cytokinin gene in an aging period, and the cytokinin synthase gene is introduced into an edible crop, whereby the cytokinin is synthesized in a ripening period, and the saccharification properties of a non-edible part of the crop are improved; and a method for producing the crop. The edible part of the edible crop can be utilized directly for eating, and the non-edible part treated as an agricultural residue can be utilized as an efficient bioethanol raw material.

Description

Method for improving saccharification of inedible parts of crops, and crops with improved saccharification

[0001] The present invention relates to a method for improving the saccharification of inedible parts of crops by introducing a cytokinin synthase gene, and in particular to a method for improving the saccharification of rice straw and rice plants with improved saccharification.

[0002] Biorefineries, which produce biofuels, resins, and other products using biomass, a renewable resource, generate carbon dioxide during the process. However, since plants use carbon dioxide to photosynthesize the biomass, the raw material, the technology can be considered carbon-neutral. Biorefineries are attracting attention due to the challenge of achieving carbon neutrality to reduce greenhouse gas emissions, which are a cause of climate change. In addition, the Sustainable Development Goals (SDGs) have become a major global issue, and the use of biomass, a renewable resource, is expected to help build a sustainable society that promotes development while protecting the global environment.

[0003] One way to utilize biomass as energy is through the production and use of bioethanol. Bioethanol produced from biomass was initially produced from biomass used as food, such as sugarcane, corn, and sugar beets. First-generation bioethanol, produced from the molasses and starch extracted from these raw materials, can easily produce ethanol through fermentation. However, because it uses the edible parts of food crops as raw materials, there is a supply limit, and it is thought that an increase in production volume cannot be expected. In addition, competition arises between using the raw material crops for food or for fuel, which has been pointed out as a problem, leading to price hikes. Furthermore, it has been pointed out that land clearing for the production of raw material crops is actually causing ecosystem destruction.

[0004] Therefore, second-generation bioethanol production using biomass other than food crops, such as woody biomass, and cellulosic biomass, such as straw, the inedible parts of food crops, as agricultural residues, has been attracting attention. For example, rice straw is a type of biomass produced in large quantities each year in conjunction with rice production, with 800 to 1 billion tons produced worldwide and approximately 9 million tons produced in Japan. However, most of this is not effectively utilized and is disposed of by composting or incineration. Cellulosic biomass, including rice straw, can be fermented to produce bioenergy if decomposed into sugars and further polymerized to produce chemical products. However, the structural difficulty of cellulose makes it difficult to decompose and convert into sugars, resulting in high production costs. Improving the saccharification properties of rice straw would reduce production costs and make it an excellent bioethanol feedstock.

[0005] Several methods for improving the saccharification of cellulosic biomass have been disclosed. Non-Patent Document 1 discloses that rice was transformed with rice genes encoding three types of cellulase: exoglucanase, endoglucanase, and β-glucosidase, resulting in overexpression of cellulases. Endoglucanase overexpression was not obtained, and β-glucosidase overexpression did not have any particular effect on plant growth. On the other hand, although exoglucanase overexpression exhibited various abnormalities such as leaf blade division, analysis of the entire rice straw revealed that saccharification after maturation was promoted. Patent Document 1 discloses transgenic plants into which lignocellulose-degrading enzymes have been introduced by genetic recombination, demonstrating increased lignocellulose expression and improved glucose yield. Patent Document 2 discloses rice with improved saccharification efficiency that contains a loss-of-function mutation in the lignin biosynthesis-related gene cinnamyl alcohol dehydrogenase (CAD).

[0006] JP-T-2009-528033 A JP-A-2012-191914

[0007] Nigorikawa, M etal., Rice, 2012, 5:14, http: / / www.thericejournal.com / content / 5 / 1 / 14Abe, T. et al., PlantBiotechnology, 2016, 33, 105-110

[0008] However, in the invention described in Non-Patent Document 1, although saccharification was promoted by the expression of exoglucanase, abnormalities in the leaf blades, etc., make it difficult to cultivate and use as a biomass raw material. The invention described in Patent Document 1 also considers application to corn and rice, but at present, only demonstrates increased sugar production in tobacco. Furthermore, it is assumed that both the edible and inedible parts will be used as raw materials for bioethanol, and therefore does not solve the problem of converting food crops. The invention described in Patent Document 2 improves saccharification by increasing acid-soluble lignin, so it is effective when acid treatment is used as a pretreatment, but it is unlikely to improve saccharification efficiency when pretreatment is performed using other methods.

[0009] The present invention aims to provide a method for producing crops, particularly grass crops, with improved saccharification properties so that the edible parts of agricultural crops can be used as they are and the inedible parts, which are currently treated as agricultural residues, can be used to produce bioethanol. Another objective of the present invention is to provide crops, particularly grass crops, with improved saccharification properties, particularly rice, which produces large amounts of biomass. The technology described in this embodiment is a method for modifying rice using genetic modification, but because the edible parts are used as food and feed, it is necessary that the genetic modification does not affect the yield and taste, and that they remain equivalent to those of the parent variety.

[0010] The present invention relates to the following food crops in which the saccharification ability of non-edible parts during the ripening period is improved compared to that of wild-type crops, and a method for producing the same. (1) An food crop in which a construct containing a cytokinin synthase gene under the control of a promoter that induces expression during senescence has been introduced, resulting in improved saccharification ability of non-edible parts during the ripening period compared to that of wild-type crops. By placing the cytokinin synthase gene under the control of a promoter that induces expression during senescence, cytokinin synthase expression during the ripening period was achieved, and improved saccharification ability of the non-edible parts was observed. Improving the saccharification ability of non-edible parts can reduce the production cost of bioethanol. Cytokinin is a type of plant hormone, and its effects have been recognized in plants in general. While rice is examined in the following examples, this technology can be applied not only to rice but also to other grasses, specifically to rice, sorghum, sugarcane, wheat, barley, and corn, as well as to other grasses whose non-edible parts can be used as biomass, such as soybean and rapeseed.

[0011] (2) The food crop according to (1), wherein the food crop is rice, the cytokinin synthase gene is a LOG gene, and the promoter that induces expression during senescence is an SGR promoter, has improved saccharification ability of the inedible portion during the ripening period compared to the wild type. Rice straw is a biomass produced in large quantities each year in conjunction with rice production, with approximately 800 to 1 billion tons produced worldwide, most of which is disposed of as agricultural residue. Improving the saccharification ability of the inedible portion of rice contributes to the efficiency of bioethanol production and leads to the effective use of rice straw that had previously been disposed of as agricultural residue.

[0012] (3) An edible crop described in (1) or (2) in which a gene has been introduced by intragenesis and in which the saccharification ability of the inedible part during the ripening stage is improved compared to the wild type. By introducing a gene by intragenesis, the crop is not considered a genetically modified organism as defined by the Cartagena Protocol and its enforcement regulations, and is not subject to those regulations. Here, rice with improved saccharification ability was produced by intragenesis. However, since cytokinin synthase genes, promoters that induce expression during senescence, and gene mutations that confer resistance to pesticides are known in other plants, it is possible to produce crops with improved saccharification ability by intragenesis using these genes.

[0013] (4) A method for producing food crops with improved saccharification of non-edible parts during ripening by creating a construct in which a cytokinin synthase gene is under the control of a promoter that induces expression during senescence and introducing the construct into food crops. By placing the cytokinin synthase gene under the control of a promoter that induces expression during senescence, cytokinin can be produced during ripening. Improving the saccharification of non-edible parts during ripening leads to increased efficiency in bioethanol production.

[0014] A diagram showing the structure of the introduced gene. A diagram showing the results of confirming by PCR whether the gene has been introduced. The upper row shows the positions of the primers used in the analysis. A diagram showing the results of analyzing the gene of the produced rice by PCR. A diagram showing the results of analyzing the gene of the produced rice by Southern blotting. A diagram showing the results of confirming LOG gene expression in the produced rice. A diagram showing the results of analyzing the saccharification of leaf blades on the day of heading and after heading. A diagram showing the results of analyzing the saccharification of leaf sheaths on the day of heading and after heading. A diagram showing the results of analyzing the saccharification of stems on the day of heading and after heading.

[0015] In the following embodiments, rice is used as an example for explanation, but saccharification can be similarly improved in any other grass family plant, such as sorghum, sugarcane, wheat, rice, barley, or corn, by introducing a construct in which a cytokinin synthase gene is placed under the control of a promoter that induces senescence-specific expression. Furthermore, similar methods can be used to improve the saccharification of inedible parts of dicotyledonous plants such as soybean and rapeseed.

[0016] As shown in the following embodiments, the present inventors have succeeded in suppressing rice senescence and inhibiting the decline in saccharification by expressing a cytokinin synthase gene after senescence has begun, i.e., during the ripening period after heading. Cytokinins are a type of plant hormone, and examples include kinetin, which was isolated from the thermal degradation products of DNA in 1955 as a cell division promoter in plant tissue culture systems, and zeatin, which was isolated from immature maize seeds in 1964 as a natural cytokinin. Cytokinins have been reported to have many other functions in addition to senescence inhibition, such as inducing leaf and bud formation in callus, activating axillary buds, regulating nutrient translocation, regulating the cell cycle, promoting fruit set, and increasing berry size.

[0017] In rice, a comparison of saccharification yields at heading and 40 days after heading has shown that saccharification yields were higher in leaf blades, leaf sheaths, and stems at heading (Non-Patent Document 2). Cytokinins are known to have functions related to saccharification, such as regulating nutrient translocation, but many aspects of their mechanism of action and regulation remain unclear. Furthermore, because many functions of cytokinins have been reported, as mentioned above, it was unclear whether enhancing cytokinin expression could suppress the decline in saccharification after heading. Furthermore, because cytokinins promote fruit set and berry enlargement, constantly enhancing cytokinin synthesis may affect panicle growth and grain maturity.

[0018] Therefore, after extensive investigation, the present inventors decided to analyze whether the saccharification ability of leaf blades and stems could be improved without affecting panicle growth or grain maturity by placing the LOG gene, which encodes a cytokinin synthase, under the control of the SGR promoter, which induces expression specifically during senescence.

[0019] [Vector Construction] The construct shown in Figure 1 was constructed so that LOG would be expressed by the SGR promoter. In Figure 1, SGR pro.: SGR promoter, LOG: LOG transcription region, LOG ter.: LOG terminator, ALS pro.: ALS promoter, ALS(G95A): ALS(G95A) transcription region, and ALS ter.: ALS terminator.

[0020] Each region of the Nipponbare genomic DNA was PCR-amplified by standard methods and incorporated into a cloning vector. The SGR promoter was amplified from the NruI restriction enzyme site upstream of the SGR promoter sequence to a 3129-bp region downstream. The LOG transcription region and terminator were amplified from the LOG translation initiation codon to a 5022-bp region downstream. ALS(G95A) is a rice-derived herbicide resistance gene used as a selection marker. ALS(G95A) encodes a mutant enzyme in which the 95th amino acid of acetolactate synthase (ALS) is replaced by alanine (A) instead of glycine (G), and confers resistance to the herbicide bispyribac-sodium. ALS was amplified from the binary vector pSTARA380RALS, which contained ALS (G95A) (2795 bp), including the promoter and terminator, as a selection marker. The primer sequences used for PCR are shown in Table 1.

[0021]

[0022] Each PCR product was subjected to agarose electrophoresis, stained with CLEAR STAIN Blue (Nippon Gene), and DNA was recovered from the gel. The extracted DNA was cloned into pBluescript SK- using the In-Fusion™ HD Cloning Kit (Takara Bio) (Figure 1, top). The cloned plasmid was purified by standard methods, digested with NruI and KpnI, and then purified to obtain the region from the SGR promoter to the ALS terminator. This DNA fragment does not contain any plasmid sequence and consists only of rice genes (Figure 1, bottom). This DNA fragment is designated SGR:LOG-ALS(G95A).

[0023] All genes used here are derived from rice, and the cloning sites were also incorporated into the vectors using sites present in rice. Therefore, the rice produced by introducing the genes excised from the vectors is intragenic rice. Therefore, the rice produced here does not fall under the category of genetically modified organisms as defined by the Act on the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Genetically Modified Organisms (commonly known as the Cartagena Act) and its Enforcement Regulations, and is not subject to the Cartagena Act regulations. However, it goes without saying that plants with similarly high saccharification properties can be obtained using transgenic plants produced using transgenic technology that combines heterologous promoters and genes.

[0024] [Rice transformation] Callus induction was performed using Taichung No. 65 seeds. After surface sterilization of rice seeds, the seeds were deglazed using a rice huller, and a mixture of DDW, sodium hypochlorite solution, and Triton-X was added in a ratio of 500:500:1 and stirred on a rotator for 30 minutes. The solution was discarded, and the seeds were rinsed 4-5 times with DDW. The rinsed seeds were placed on N6CI medium (Table 2) and cultured at 28°C under light conditions for 7-10 days. The seeds and sprouts were removed from the resulting rice callus and placed on high osmotic pressure medium (Table 2).

[0025] 0.6 mg x (number of shots + 1) of 1.6 μm diameter gold particles were weighed out, 300 μl of 70% ethanol was added, and the mixture was vortexed and centrifuged at 6000 rpm at 4°C for 1 minute. The supernatant was discarded, and 100 μl x number of shots of 99.5% ethanol was added, followed by centrifugation in the same manner. The supernatant was completely discarded, and 2-5 μg of DNA, 2.5 M CaCl ２ A DNA-gold particle solution was prepared by adding 10 μl of 100 μL of 0.1 M spermidine and 4 μl of DDW, totaling 24 μl per shot. This was adjusted to the number of shots. After leaving the solution at room temperature for 30 minutes, rice callus transferred to high-osmolarity medium was bombarded with the DNA-gold particle solution using a particle gun at 1100 psi, two shots at a time. The callus was then incubated in high-osmolarity medium (Table 2) at 28°C in the light. The next day, the callus was transferred to N6CI medium and cultured under the same conditions for one day. The callus was then cultured in N6SE medium (Table 2) for four weeks at 28°C in the light. The proliferated callus was selected and cultured on MSRE medium (Table 2) at 28°C in the light to regenerate shoots. The callus was then subcultured on fresh MSRE medium every two weeks. The resulting regenerated shoots were cultured on MSHF medium (Table 2) at 28°C in the light for two to three weeks to induce root development.

[0026]

[0027] [Rice cultivation] The rice plants regenerated through callus formation were cultivated in a fully artificial light phytotron under controlled conditions of daylight duration and temperature, specifically, under alternating conditions of 10 hours of light at 28°C and 14 hours of darkness at 23°C.

[0028] [Genetic analysis of the produced rice] Genomic DNA was extracted from the produced rice using the CTAB method. Leaf blades were removed, and 500 μl of CTAB buffer (3% CTAB, 1.4 M NaCl, 1% PVP, 0.1 M Tris-HCl (pH 8.0), 20 mM EDTA (pH 8.0)) was added. The leaves were crushed using a pestle and allowed to stand at 60°C for 30 minutes. DNA was then extracted with phenol-chloroform using standard methods, precipitated with an equal volume of isopropanol, and washed with 70% ethanol. The precipitate was dissolved in 20 μl of 1 / 5 TE buffer (2 mM Tris-HCl (pH 8.0), 0.2 mM EDTA) and 0.2 μl of 10 mg / ml RNase A to obtain genomic DNA.

[0029] To confirm the transgene, PCR was performed using the extracted genomic DNA as a template (Figure 2). PCR was performed using three primers: primers that amplify the region between the SGR promoter and LOG (Figure 2(a), SEQ ID NOs: 7 and 8); primers that amplify the region between the LOG terminator and the ALS promoter (Figure 2(b), SEQ ID NOs: 9 and 10); and primers that amplify the region from near the 5' end of the SGR promoter to the LOG terminator (Figure 2(c), SEQ ID NOs: 11 and 4). Numbers 1 to 6 in the figure show the results of amplification using genomic DNA extracted from independent SGR:LOG-ALS (G95A) transgenic lines. As a result, bands were confirmed at the expected positions in all six lines shown as numbers 1 to 6. Additionally, the transgene was detected using the same primers and genomic DNA from a rice plant (Taichung No. 65) that had not been transgenic as a negative control (NC). In the negative control, a band corresponding to the endogenous actin gene was detected, but no band corresponding to the introduced gene was detected, confirming that the bands detected in the six lines that underwent gene transfection were derived from the introduced gene. PC is the positive control, and indicates the plasmid used for transformation (Figures 2(a), (b), and (c)) or the plasmid into which the actin gene was cloned (Figure 2, Actin). The primer sequences used for amplification are shown in Table 3.

[0030]

[0031] Next, we confirmed that rice plants transfected with SGR:LOG-ALS(G95A) did not contain a vector region. PCR was performed using genomic DNA as a template. PCR to amplify the region flanking the vector and SGR promoter, and PCR to amplify the region flanking ALS(G95A) and the vector were performed using primer sets T3-u and SGR-R2, and ALS-F7 and T7-u, respectively (Figure 3A, top panel). No bands were detected in any of the lines, confirming that they did not contain a vector region (Figure 3A, bottom panel). Note that numbers 1 to 6 represent independent transgenic lines, as in Figure 2; WT represents rice plants not transfected with the gene; + represents the plasmid used for transformation as a positive control; and - represents the results of PCR using DDW as a negative control. In all lines, no amplification occurred when one of the primers was located in the vector region, indicating that they did not contain a vector region. The primer sequences used for amplification are shown in Table 4.

[0032]

[0033] Furthermore, Southern blotting was performed using the vector as a probe, confirming that the rice plants into which SGR:LOG-ALS(G95A) was introduced did not contain the vector region (Figure 3B). 1 to 6 indicate the genomic DNA of the six extracted lines, + indicates the plasmid used for transformation, and M indicates the size marker. The vector pBSSK- used for cloning was used as a probe. The results are shown in Figure 3B. No bands were detected in the genomic DNA extracted from all lines 1 to 6, confirming that the resulting lines did not contain the plasmid DNA used as the vector.

[0034] [Confirmation of LOG gene expression] SGR:LOG-ALS (G95A) was introduced, and RNA was extracted from the leaf blades of the next generation self-pollinated from the transformed line at heading day and 20 days after heading, and LOG gene expression was analyzed. Total RNA was extracted from samples of 15-30 mg of the second leaf blade from the flag leaf at heading day and 20 days after heading, which were placed in 1.5 ml tubes and flash-frozen in liquid nitrogen. 500 μl of GT solution (4 M guanidine thiocyanate, 17 mM sodium N-lauroyl sarcosine, 5 mM trisodium citrate) was added and the sample was ground with a pestle. Extraction and purification were then performed by standard phenol-chloroform extraction and dissolved in 30 μl of TE. RNA concentration was measured using a Nano Drop 2000q ultramicrospectrophotometer (Thermo Fisher).

[0035] Five micrograms of extracted RNA was purified using Dynabeads mRNA Purification kit (Thermo Fisher). ＋ RNA was purified and dissolved in 10 μl of 10 mM Tris-HCl (pH 7.5). ＋ RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen). For the negative control, 0.5 μl of DDW was added instead of Superscript III Reverse Transcriptase and the reaction was performed (indicated as RTase- in Figure 4). The synthesized cDNA was diluted with 80 μl of TE, and RT-PCR was performed using primers that detect LOG gene expression. The amplified PCR products were separated by agarose gel electrophoresis to confirm expression. As a result, at the day of heading, slight expression of the LOG gene was observed in five of the six lines into which SGR:LOG-ALS (G95A) had been introduced. On the other hand, no expression was observed in the wild type. At 20 days after heading, expression of the LOG gene was observed in five of the six lines, but no expression was observed in the wild type (Figure 4). This indicates that in at least 5 out of 6 lines, transcription of the LOG gene by the SGR promoter began with aging. The primer sequences used for amplification are shown in Table 5.

[0036]

[0037] [Measurement of Saccharification] The saccharification of rice plants into which the LOG gene had been introduced was measured. The saccharification test was carried out as follows. Rice plants at heading and 40 to 50 days after heading were harvested, and the stem, flag leaf attached to the stem, and the two leaves immediately below it were separated into leaf blades and leaf sheaths, and then dried at high temperature for 5 hours at 105°C. The samples were stored sealed in a box containing silica gel until use. Each sample was chopped into pieces of 5 mm or less, zirconia beads for grinding were added, and the mixture was ground into powder using a Tissue Lyser (Qiagen) at 25 / sec for 15 minutes. The powder was passed through a 75 μm mesh sieve to make the powder size uniform, and 15 mg of the powder that passed through the sieve was weighed into a tube. Two zirconia beads used for milling were added to the tube, along with 500 μl of 0.1 M sodium citrate buffer (pH 4.8), 500 μl of DDW, 0.5 μl of cellulase derived from Trichoderma reesei (Sigma), and 1.5 μl of glucosidase derived from Aspergillus niger (Sigma) (saccharification reaction solution). 0.25 g of glucosidase powder was dissolved in 0.75 ml of DDW. The 1.5 ml tube containing the saccharification reaction solution was subjected to an enzymatic reaction at 50°C for 48 hours while being mixed by inversion using a rotator. The reducing sugar concentration in the reaction solution was measured 48 hours after the start of the reaction. A control sample was also used without the two enzymes.

[0038] The DNS method was used to measure the amount of reducing sugar. The reaction solution was centrifuged at 4°C and 15,000 rpm for 5 minutes, and the supernatant was used for measurement. The 10-fold diluted supernatant was measured using an absorbance spectrometer (PiCOSCOPE Pas-110, Ushio, absorbance 530 nm). The amount of reducing sugar in the sample was calculated by converting it to glucose using the calibration curve. Since the calculated amount of reducing sugar is the sum of endogenous reducing sugar and reducing sugar obtained by the enzyme, the amount of reducing sugar saccharified by the enzyme was calculated by subtracting the amount of reducing sugar in the control without enzyme from the amount of reducing sugar in the enzyme-containing sample. The enzymatic saccharification rate was calculated by dividing the amount of reducing sugar by 15 mg of rice straw powder used in the saccharification reaction. Three samples of each line were measured, and the mean and standard error were calculated. Significance tests were performed using the Dunnett method.

[0039] Saccharification rates of each plant part were examined. At heading, the saccharification rates of SGR:LOG-ALS(G95A)-transfected rice were 2.5-10.8% in the leaf blade, 15.8-27.6% in the leaf sheath, and 42.7-63.5% in the stem. These rates were not significantly different from those of the control (WT rice) at any of the three sites (Fig. 5A-C; the average saccharification rate of the WT is expressed as 1). At 40-50 days after heading, the saccharification rates of the stem of SGR:LOG-ALS(G95A)-transfected rice were 62.6-95.9%, with no significant difference from the control (75.8%) (Fig. 5C). Meanwhile, the saccharification rate of #5 leaf blade (48.6%) was significantly higher than that of the control (14.1%) (Fig. 5A). In the leaf sheaths, the saccharification rates of the SGR:LOG-ALS(G95A)-transfected rice plants ranged from 11.4 to 20.8%, and although there was no significant difference between them and the control (7.4%), all lines showed higher values ​​than the control (Fig. 5B).

[0040] In this study, all rice genes, including promoters that induce expression during senescence and selectable markers, were used, and the rice was produced by intragenesis without using genes from other species. Therefore, the rice does not qualify as a genetically modified organism (GM) as defined by the Cartagena Protocol and its Enforcement Regulations. As of April 2023, the Cartagena Protocol has been ratified by 171 countries, including Japan, the European Union, and Palestine. Under the Cartagena Protocol, the cultivation, import, and distribution of GM organisms, which are subject to environmental release, require review and are subject to certain restrictions. However, the rice shown in the examples is not subject to the Cartagena Protocol because the genes were introduced by intragenesis. While genes can be introduced using either transgenic or intragenic methods, the fact that introducing genes via intragenesis avoids the restrictions of the Cartagena Protocol is advantageous for cultivation and dissemination.

Claims

1. A construct has been introduced in which the cytokinin synthase gene is under the control of a promoter that induces expression during senescence, resulting in an edible crop in which the saccharification ability of the inedible parts during the ripening period is improved compared to the wild type.

2. An edible crop having improved saccharification ability in the inedible parts during the ripening stage compared to the wild type, as described in claim 1, wherein the edible crop is a grass plant.

3. An edible crop according to claim 2, characterized in that the plant of the Poaceae family is rice, in which the saccharification ability of the inedible parts during the ripening stage is improved compared to that of the wild type.

4. An edible crop having improved saccharification ability in the non-edible parts during the ripening period compared to the wild type, as described in claim 3, characterized in that the cytokinin synthase gene is a LOG gene, and the promoter that induces expression during the senescence period is an SGR promoter.

5. An edible crop having improved saccharification ability in the non-edible parts during the ripening stage compared to the wild type, as described in claim 4, characterized in that the rice-derived herbicide resistance gene ALS (G95A) is incorporated into the construct as a selection marker for gene introduction.

6. An edible crop according to any one of claims 1 to 5, in which a gene is introduced by intragenesis, and in which the saccharification ability of the inedible part during the ripening stage is improved compared to that of the wild type.

7. A method for producing food crops with improved saccharification of the inedible parts after ripening by creating a construct in which the cytokinin synthase gene is under the control of a promoter that induces expression during senescence and introducing it into food crops.

8. A method for producing an edible crop with improved saccharification ability in the inedible parts during the ripening stage according to claim 7, characterized in that the edible crop is a grass family plant.

9. A method for producing an edible crop with improved saccharification ability in the inedible parts during the ripening stage according to claim 8, characterized in that the grass plant is rice.

10. A method for producing an edible crop with improved saccharification of non-edible parts during ripening, as described in claim 9, characterized in that the cytokinin synthase gene is a LOG gene, and the promoter that induces expression during the senescence period is an SGR promoter.

11. A method for producing an edible crop with improved saccharification ability in the non-edible parts during the ripening stage as described in claim 10, characterized in that the rice-derived herbicide resistance gene ALS (G95A) is used as a selection marker for gene introduction.

12. A method for producing an edible crop with improved saccharification of non-edible parts during ripening according to any one of claims 7 to 11, in which a construct is created so that no genes other than those of the edible crop are introduced, and the crop is produced by intragenesis.

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