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(12) EUROPEAN PATENT APPLICATIONpublished in accordance with Art. 153�(4) EPC

(43) Date of publication: 23.09.2009 Bulletin 2009/39

(21) Application number: 07850363.8

(22) Date of filing: 10.12.2007

(51) Int Cl.: �A01N 37/20 (2006.01) A01G 1/00 (2006.01)

A01G 7/00 (2006.01) A01G 7/06 (2006.01)

A01N 63/00 (2006.01) A01P 21/00 (2006.01)

(86) International application number: PCT/JP2007/073795

(87) International publication number: WO 2008/072602 (19.06.2008 Gazette 2008/25) �

(84) Designated Contracting States: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

(30) Priority: 11.12.2006 JP 2006333635

(71) Applicant: Japan Science and Technology AgencyKawaguchi-�shiSaitama 332-0012 (JP)�

(72) Inventors: • OGAWA, Kenichi

600-8853 Kyoto (JP) �• HENMI, Kenji

701-0111 Okayama (JP) �

(74) Representative: Bittner, Thomas L. et alForrester & Boehmert Pettenkoferstrasse 20-2280336 München (DE) �

(54) PLANT GROWTH REGULATOR AND USE THEREOF

(57) A plant growth regulator containing glutathione allows increasing harvest index. This provides a technique forspecifying a control factor for a plant, thereby effectively controlling germination, growth, anthesis etc. of the plant.

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Description

Technical Field

�[0001] The present invention relates to a plant growthregulator for regulating growth of a plant and a techniquefor use thereof. To be specific, the present invention re-lates to a plant growth regulator capable of increasingharvest index by use of glutathione, and a technique foruse of the plant growth regulator.

Background Art

�[0002] Conventionally, plants have been deeply in-volved with human as foods, ornaments, industrial ma-terials such as paper and chemicals, and fuels. Further,recently, plants have been spotlighted as biomass ener-gy that will substitute fossil fuel.�[0003] Although plants have been used in such variousfields, their mechanisms such as budding, growth, andanthesis have not yet been clarified in many regards.Consequently, cultivation of plants has been mainlybased on experiences and intuition, and harvest of theplants has been greatly influenced by natural conditionssuch as weather. Therefore, clarification of plants’ mech-anisms such as budding, growth, and anthesis and reg-ulating and controlling the mechanisms are very impor-tant not only for increasing yields of ornamental plantsand food plants such as grains and vegetables, but alsofor growing woods in forests and biomass energy.�[0004] In order to regulate growth of plants, there havebeen made attempts such as regulation of anthesis byartificial environments such as a conservatory, and pro-motion of growth by use of chemicals such as ethylene.However, most of these conventional attempts are reg-ulations of growth of plants based on experiences andintuition, and are not based on data that allows scientificevaluation of growth of plants.�[0005] In view of the above, the inventors of the presentinvention have researched on the plants’ mechanisms ofbudding, growth, and anthesis. Consequently, the inven-tors have shown that reactive oxygen species (ROS) isessential not only as a substrate for biosynthesis but alsoas a factor for controlling growth of plants (see PatentLiterature 1). Specifically, Patent Literature 1 describesa regulator that contains a redox state regulation sub-stance for cells and that regulates differentiation of cellsor organs, a method for controlling differentiation andmorphogenesis of a organism, and a organism thus ob-tained.�[0006] Further, Patent Literature 2 discloses an adju-vant for regulating growth of plants and a method for pre-paring redifferentiated plants by used of the adjuvant.Specifically, a callus induced from a part of a plant suchas rice and eustoma is cultivated in a redifferentiatingculture medium containing glutathione, preferably oxi-dized glutathione (which may be hereinafter referred toas GSSG) so as to promote rhizogenesis, effectively ob-

taining a redifferentiated body from the callus in a shorttime.

Citation List

�[0007]

Patent Literature 1International Application Publication No.WO01/080638 (Publication Date: July 22, 2003)Patent Literature 2Japanese Patent Application Publication, Tokukai,No. 2004-352679 A (Publication Date: December16, 2004)

Summary of Invention

�[0008] Patent Literature 1 discloses a technique forcontrolling differentiation and morphogenesis of plantsby use of a substance that regulates a redox state of theplants. However, the control mechanism is not yet suffi-ciently clarified and it is not known what substance servesas a factor for controlling growth of the plants. Further,although Patent Literature 2 discloses a technique forpreparing a redifferentiated plant body, the technique isnot sufficient. A new technique for controlling the amountof biomass, the yield of seeds, the quality of next-�gener-ation seeds etc is required.�[0009] Scientifically understanding the process ofgrowth of plants, scientifically predicting anthesis, andregulating them are very important not only to ornamentalflowers and plants for foods, but also to forests and plantresources for biomass energy. Therefore, there has beena strong request for developing a technique for specifyinga control factor for plants and effectively controlling bud-ding, growth, anthesis etc. of plants.�[0010] The present invention was made in view of theforegoing problems. An object of the present inventionis to provide a technique for specifying a control factorfor plants and effectively controlling budding, growth, an-thesis etc. of plants.�[0011] In order to solve the foregoing problems, theinventors of the present invention have diligently studiedand found that cultivation of a plant by use of glutathioneallows greatly increasing the number of seeds and thenumber of flowers of the plant. Further, the inventors havefound that when a plant having mutation in its functionfor synthesizing a plant hormone (e.g. gibberellin) or re-sponding to a plant hormone is cultivated by use of glu-tathione, it is possible to greatly increase the number oflateral shoots (axillary buds), and accordingly to increasethe number of flowers (sheaths). The inventors havecompleted the present invention based on these findings.The present invention has been completed based onthese new findings and includes the following subjectmatters. �

(1) A plant growth regulator for increasing harvest

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index, comprising glutathione.(2) The plant growth regulator as set forth in (1),wherein the glutathione is oxidized glutathione.(3) The plant growth regulator as set forth in (1) or(2), for increasing the number of seeds and/or flow-ers of a plant.(4) The plant growth regulator as set forth in (1) or(2), for increasing the number of lateral shoots and/ortillers of a plant.(5) A method for cultivating a plant, comprising thestep of cultivating a plant by use of glutathione so asto increase harvest index of the plant.(6) The method as set forth in (5), wherein the glu-tathione is oxidized glutathione.(7) The method as set forth in (5) or (6), wherein theglutathione is supplied intermittently.(8) The method as set forth in any one of (5)-(7),wherein the glutathione is supplied at around a timeof transition from vegetative to reproductive devel-opment.(9) A method for increasing the number of seedsand/or flowers of a plant by use of glutathione.(10) The method as set forth in (9), wherein the glu-tathione is oxidized glutathione.(11) A method for increasing the number of lateralshoots and/or tillers of a plant by use of glutathione.(12) The method as set forth in (11), wherein theglutathione is oxidized glutathione.(13) The method as set forth in (11) or (12), whereinthe plant has mutation in a function for synthesizinga plant hormone and/or a function for responding toa plant hormone.(14) The method as set forth in (13), wherein theplant hormone is gibberellin.(15) A plant obtained by a method as set forth in anyone of (5)-(14), having increased harvest index.

�[0012] For a fuller understanding of the nature and ad-vantages of the invention, reference should be made tothe ensuing detailed description taken in conjunction withthe accompanying drawings.

Brief Description of Drawings

�[0013]

Fig. 1Fig. 1 is a drawing illustrating states of Arabidopsistreated with water, a GSSG solution, or an H2O2 so-lution, observed 3 or 4 weeks after sowing.Fig. 2Fig. 2 is a drawing illustrating states of Arabidopsistreated with water, a GSSG solution, or an H2O2 so-lution, observed 6 weeks after sowing.Fig. 3Fig. 3 is a drawing illustrating states of Arabidopsistreated with water, a GSSG solution, or an H2O2 so-lution, observed 7 weeks after sowing.

Fig. 4Fig. 4 is a drawing illustrating states of Arabidopsistreated with water, a GSSG solution, or an H2O2 so-lution, observed 8 weeks after sowing.Fig. 5Fig. 5 is a drawing illustrating yields and states ofseeds obtained from Arabidopsis treated with water,a GSSG solution,� or an H2O2 solution.Fig. 6Fig. 6 is a drawing illustrating the result of examina-tion on the influence of concentration of oxidized glu-tathione on growth of Arabidopsis.Fig. 7Fig. 7 is a drawing illustrating the result of examina-tion on the influence of concentration of oxidized glu-tathione on growth of Arabidopsis.Fig. 8Fig. 8 is a drawing illustrating the result of examina-tion on the influence of oxidized glutathione on seedsof Arabidopsis.Fig. 9Fig. 9 is a drawing illustrating the result of examina-tion on the influence of oxidized glutathione on a gib-berellin synthesis mutant of Arabidopsis.Fig. 10Fig. 10 is a drawing illustrating the result of exami-nation on the influence of a treatment time of oxidizedglutathione and treatment concentration of oxidizedglutathione on the seed weight of Arabidopsis.Fig. 11Fig. 11 is a drawing illustrating the result of exami-nation on the influence of a treatment time of oxidizedglutathione and treatment concentration of oxidizedglutathione on the seed weight of Arabidopsis.Fig. 12Fig. 12 is a drawing illustrating the result of exami-nation on the influence of a treatment time of oxidizedglutathione and treatment concentration of oxidizedglutathione on the seed weight of Arabidopsis.Fig. 13Fig. 13 is a drawing illustrating the result of exami-nation on the influence of a treatment time of oxidizedglutathione on the seed weight of Arabidopsis.Fig. 14Fig. 14 is a drawing illustrating the result of exami-nation on the influence of a treatment time of oxidizedglutathione on the seed weight of Arabidopsis.Fig. 15Fig. 15 is a drawing illustrating the result of exami-nation on the influence of a treatment time of oxidizedglutathione on the seed weight of Arabidopsis.Fig. 16Fig. 16 is a drawing illustrating the result of exami-nation on the influence of a treatment time of oxidizedglutathione and treatment concentration of oxidizedglutathione on the seed weight, the dry weight, andthe harvest index of Arabidopsis.Fig. 17

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Fig. 17 is a drawing illustrating the result of exami-nation on the influence of oxidized glutathione ongrowth of a rose (breed; Patiohit alicante).Fig. 18Fig. 18 is a drawing illustrating the result of exami-nation on the influence of oxidized glutathione ongrowth of a rose (breed; English rose).Fig. 19Fig. 19 is a drawing illustrating the result of exami-nation on the influence of oxidized glutathione ongrowth of tsai-�hsin that is a kind of coleseed.Fig. 20Fig. 20 is a drawing illustrating positions of individualsoybeans in an agricultural field test in which theinfluence of oxidized glutathione on growth of soy-bean is examined.Fig. 21Fig. 21 is a drawing illustrating how to supply oxidizedglutathione in the agricultural field test in which theinfluence of oxidized glutathione on growth of soy-bean is examined.Fig. 22Fig. 22 is a drawing illustrating the results of exam-inations on the weight of seeds, the whole amountof biomass, and the harvest index in the agriculturalfield test in which the influence of oxidized glutath-ione on growth of soybean is examinedFig. 23Fig. 23 is a drawing illustrating the results of exam-ination on the influence of oxidized glutathione onformation of floral buds of corn.Fig. 24Fig. 24 is a drawing illustrating the results of exam-ination on the influence of oxidized glutathione onthe crop yields of corn.Fig. 25Fig. 25 is a drawing illustrating the results of exam-inations on the influence of oxidized glutathione onthe amount of biomass and the harvest index of fruits,ground parts, and portions other than the fruits ofindividual corns.Fig. 26Fig. 26 is a drawing illustrating the results of exam-inations on the influence of a treatment time of oxi-dized glutathione on the amount of biomass and theharvest index of fruits, ground parts, and portionsother than the fruits of individual corns.Fig. 27Fig. 27 is a drawing illustrating the results of exam-inations on the influence of a treatment method ofoxidized glutathione on the amount of biomass andthe harvest index of fruits, ground parts, and portionsother than the fruits of individual corns.Fig. 28Fig. 28 is a drawing illustrating the results of exam-inations on the influence of a treatment method ofoxidized glutathione and a treatment time of oxidizedglutathione on the harvest index of corn.

Fig. 29Fig. 29 is a drawing illustrating positions of individualcorn in an agricultural field test in which the influenceof oxidized glutathione on growth of corn is exam-ined.Fig. 30Fig. 30 is a drawing illustrating the results of exam-inations on the influence of a treatment time of oxi-dized glutathione on the whole amount of biomassper area, the crop yields of pistils per area, and theharvest index in an agricultural field test in which theinfluence of oxidized glutathione on growth of cornis examined.Fig. 31Fig. 31 is a drawing illustrating the results of exam-inations on the influence of a treatment time of oxi-dized glutathione on the amounts of biomass offruits, ground parts, and portions other than the fruitsof individual corns in an agricultural field test in whichthe influence of oxidized glutathione on growth ofcorn is examined.Fig. 32Fig. 32 is a drawing illustrating the results of exam-inations on the influence of oxidized glutathione onthe crop yields of corn under a nitrogen- �deficient con-dition.Fig. 33Fig. 33 is a drawing illustrating the results of exam-inations on the influence of oxidized glutathione onthe amounts of biomass and the harvest index offruits, ground parts, and portions other than the fruitsof individual corns under a nitrogen- �deficient condi-tion.Fig. 34Fig. 34 is a drawing illustrating the result of exami-nation on the influence of oxidized glutathione ongrowth of sprout and anthesis of a rose (breed; Pur-ple rose).Fig. 35Fig. 35 is a drawing illustrating the result of exami-nation on the influence of oxidized glutathione ongrowth of sprout and anthesis of a rose (breed; JJscarlet and JJ apricot).Fig. 36Fig. 36 is a drawing illustrating the results of exam-inations on the influences of oxidized glutathione andreduced glutathione on growth of roots of eustoma.Fig. 37Fig. 37 is a drawing illustrating the results of exam-inations on the influence of oxidized glutathione oninduction of floral buds of a rose.Fig. 38Fig. 38 is a drawing illustrating the results of exam-inations on the influence of oxidized glutathione oninduction of floral buds of a rose.Fig. 39Fig. 39 is a drawing illustrating the results of exam-inations on the influences of oxidized glutathione and

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reduced glutathione on growth and multiplication(number of runners) of strawberry (Eminent gardenseries Yokubari-�ichigo kurenai (SUMIKA)).Fig. 40Fig. 40 is a drawing illustrating positions of individualplants in cultivation and a time of supplying oxidizedglutathione in a test in which the influence of oxidizedglutathione on growth of transformants of Arabidop-sis to which a gFBA gene is introduced is examined.Fig. 41Fig. 41 is a drawing illustrating the results of exam-inations on concentration of ammonium nitrate atwhich the effects of increasing the harvest index, thewhole amount of biomass, and the seed weight ofwild-�type Arabidopsis and transformants of Arabi-dopsis to which a gFBA1 gene is introduced are sat-urated.Fig. 42Fig. 42 is a drawing illustrating the results of exam-inations on the influence of oxidized glutathione onthe harvest index, the whole amount of biomass, andthe seed weight of transformants to which a gFBAgene is introduced, under a condition where the ef-fect of nitrogen fertilization is saturated.Fig. 43Fig. 43 is a drawing illustrating the results of exam-inations on the influences of oxidized glutathione,reduced glutathione, and ammonium sulfate on theharvest index, the whole amount of biomass, andthe seed weight of transformants of Arabidopsis towhich a gFBA gene is introduced, under a conditionwhere the effect of nitrogen fertilization is saturated.Fig. 44Fig. 44 is a drawing illustrating the result of exami-nation on the influence of concentration of GSSG asa fertilizer on the yields of seeds of transformants ofArabidopsis to which a gFBA gene is introduced andwild-�type Arabidopsis.Fig. 45Fig. 45 is a drawing illustrating the result of exami-nation on the influence of concentration of GSSG asa fertilizer on the harvest index of transformants ofArabidopsis to which a gFBA gene is introduced andwild-�type Arabidopsis.Fig. 46Fig. 46 is a drawing illustrating the results of exam-inations on the influences of fertilization with GSSG,GSH, and ammonium sulfate as sulfate sources onthe yield of seeds of transformants of Arabidopsis towhich a gFBA gene is introduced.Fig. 47Fig. 47 is a drawing illustrating the results of exam-inations on the influences of fertilization with GSSG,GSH, and ammonium sulfate as sulfate sources onthe harvest index of transformants of Arabidopsis towhich a gFBA gene is introduced.

Description of Embodiments

�[0014] One embodiment of the present invention is de-scribed below with reference to the attached drawings.Note that the present invention is not limited to the em-bodiment.�[0015] A plant growth regulator of the present inventionis not particularly limited as long as it contains glutath-ione, and specific factors of the plant growth regulatorsuch as density and other components etc. are not par-ticularly limited. The glutathione may be reduced glutath-ione (which may be hereinafter referred to as "GSH") ormay be an oxidized glutathione (which may be hereinaf-ter referred to as "GSSG"), but GSSG is preferable.�[0016] As is well known by a person skilled in the art,GSH has a property of easy oxidization. Consequently,when GSH is added as glutathione to the plant growthregulator of the present invention, the plant growth reg-ulator generally contains not a small amount of GSSG.That is, the plant growth regulator of the present inventionmay contain, as glutathione, GSH and GSSG in a mixedstate.�[0017] The plant growth regulator of the present inven-tion may be arranged so as to contain GSH as glutathioneand the GSH is oxidized to be GSSG when the plantgrowth regulator is stored or used. Further, GSH may beoxidized to be GSSG after the plant growth regulator wassupplied to a plant.�[0018] A method for oxidizing GSH to be GSSG is notparticularly limited. For example, GSH can be easilychanged to GSSG by air oxidization. Alternatively, GSHmay be changed to GSSG by any conventional artificialmethod that has been publicly known so far.�[0019] "Oxidized glutathione" in the present inventionis a substance that is well known by a person skilled inthe art and does not require any special explanation. Forexample, "oxidized glutathione" may be defined as a mol-ecule obtained by disulfide-�bonding two-�molecular re-duced glutathiones.�[0020] In general, it is known that most (98% or more)of glutathione in cells of an organism is reduced glutath-ione. Consequently, a person skilled in art would con-ceive reduced glutathione as glutathione, and thereforeuse of oxidized glutathione is not general. Further, a per-son skilled in the art has an impression that oxidized glu-tathione worsens growth of plants. Therefore, a personskilled in the art would not have been motivated to useoxidized glutathione for cultivation of plants.�[0021] Under such a situation, the inventors havefound that functions of fructose-�1,6-�bisphosphate aldo-lase that is an enzyme for Calvin cycle are controlled bybonding with glutathione. The inventors have added ox-idized glutathione necessary for the bonding from out-side, succeeding in greatly increasing the productivity ofbiomass and the yield.�[0022] That is, the inventors of the present inventionhave found that cultivation of a plant by use of "oxidizedglutathione (GSSG)" that has not been generally used in

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cultivation of plants allows greatly increasing the numberof seeds of the plant and the number of flowers of theplant, and thus completed the present invention. There-fore, the present invention has a significant originality.�[0023] The plant growth regulator of the present inven-tion contains glutathione and increases harvest index ofplants.�[0024] In the present specification, "harvest index" in-dicates a ratio of the weight of harvest to the weight of aplant as a whole. In other words, "harvest index" indicatesa ratio of the amount of biomass of the harvest to theamount of all biomass of individual plants.�[0025] In the present specification, "harvest" indicatesa portion of a plant to be eaten. For example, in a caseof a plant whose fruit is to be eaten, the "harvest" is thefruit. In a case of a plant whose seed is to be eaten, the"harvest" is the seed. In a case of a plant whose stem isto be eaten, the "harvest" is the stem. In a case of a plantwhose root is to be eaten, the "harvest" is the root. In acase of a plant whose flower is to be eaten, the "harvest"is the flower. In a case of a plant whose leaf is to be eaten,the "harvest" is the leaf. Further, the "harvest" indicatesa portion that is not edible but contains a product targetedin cultivating a plant. Specifically, in a case of ornamentalplants, examples of the "harvest" include flowers, stems,leaves, roots, seeds etc. each of which is to be appreci-ated.�[0026] Further, in the present invention, "increasingharvest index" indicates an effect of increasing harvestindex compared with a condition under which the plantgrowth regulator of the present invention is not supplied,and indicates that a ratio of the amount of biomass of theharvest to the amount of all biomass can be increasedunder a conventional and standard fertilization conditionthat is optimized to obtain the maximum amount of yieldper unit area. Although the amount of yield per unit areaincreases as a planting rate increases, this effect getssaturated at a certain planting rate. "Increasing harvestindex" in the present specification indicates that a ratioof the amount of biomass of harvest to the amount of allbiomass can be increased even under such a plantingcondition.�[0027] Since the plant growth regulator of the presentinvention allows increasing harvest index of a plant, it ispossible not only to increase the amount of foods or bi-omass resources produced per unit area but also togreatly contribute to increased production of industriallyapplicable plants and harvests obtained therefrom.�[0028] Further, it is preferable that the plant growth reg-ulator of the present invention increases the number ofseeds of a plant and/or the number of flowers of the plant.As described in later- �mentioned Examples, it is clearlydemonstrated that use of the plant growth regulator ofthe present invention allows increasing the number ofseeds and the number of flowers. Further, it is also dem-onstrated that other performance of the plant growth reg-ulator of the present invention is lengthening of the lifeof a plant, causing a leaf to be more round and larger,

shortening the plant length, and thickening a stem of theplant.�[0029] This allows increasing the yield of seeds for ex-ample. Therefore, industrial applicability of the plantgrowth regulator is quite high not only in a case of sellingseeds themselves but also in a case of seeds containingfats and oils and other effective components since theyields of such fats and oils etc. also increase. Further,such increased yields are also useful for biomass mate-rial production.�[0030] When the plant growth regulator is applied toornamental plants or shade trees, the performance oflengthening the life of a plant allows extending the intervalof exchanging plants due to withering of the plants. Thisreduces work burden on maintenance of the ornamentalplants or the shade trees. Further, the performance ofcausing a leaf to be more round and larger is applicableto production of unique ornamental plants having unusualappearances. Further, the performance of shortening theplant length and thickening a stem of the plant is appli-cable to production of ornamental plants and to increas-ing durability of crops against strong winds.�[0031] Further, it is preferable that the plant growth reg-ulator of the present invention increases the number oflateral shoots and/or tillers of plants. As explained in later-mentioned Examples, this invention is made on a newfinding that when a plant having mutation in function forsynthesizing plant hormone (gibberellin) or function forresponding to plant hormone is cultivated by use of oxi-dized glutathione, the number of lateral shoots increasesgreatly. As the number of lateral shoots and/or tillers in-creases, the number of flowers (sheaths) increases.�[0032] Therefore, when the plant growth regulator ofthe present invention is applied to a plant such asgramineae whose tillers have a great influence on theyield, it is possible to increase the yield of seeds.�[0033] The target plant to which the plant growth reg-ulator is to be applied is preferably a plant having muta-tion in function for synthesizing plant hormone or re-sponding to plant hormone. This is because applicationof glutathione, preferably oxidized glutathione to the mu-tant or a transformant having the same function as themutant allows further exerting the performance of the ox-idized glutathione.�[0034] Herein, "a plant having mutation in function forsynthesizing plant hormone or responding to plant hor-mone" indicates a plant which has mutation in at leastone of an enzyme of a biosynthesis system of plant hor-mone, a receptor of plant hormone, a biological sub-stance of a communication system of plant hormone etc.and which is a plant whose function concerning planthormone does not work compared with a wild- �type plantor which is a plant highly susceptive (acquired) to planthormone due to mutation. In particular, it is preferable touse a mutant whose function concerning plant hormoneis lower than that of a wild- �type or whose function con-cerning plant hormone is substantially lost.�[0035] An example of the plant is, as explained in later-

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mentioned Examples, a mutant in which a DNA fragmentsuch as T- �DNA is inserted into a gene encoding an en-zyme of a biosynthesis system of plant hormone.�[0036] The plant hormone is preferably gibberellin. Itis considered that oxidized glutathione functions at thedownstream of plant hormone such as gibberellin or incorporation with plant hormone.�[0037] Further, it is preferable that the plant growth reg-ulator of the present invention promotes growth of asprout, induction of a floral bud, and/or anthesis. As ex-plained in the later- �mentioned Examples, it is clearlydemonstrated that the plant growth regulator of thepresent invention promotes growth of a sprout, inductionof a floral bud, and/or anthesis.�[0038] This allows shortening the length of cultivatinga plant and increasing productivity of the plant. In a caseof a plant used as foods, this performance contributes toincreasing production of foods. Further, since the plantgrowth regulator allows controlling anthesis or growth ofa plant, application of the plant growth regulator allowseffective production of a plant.� This allows regulating sup-ply of the plant to the market in response to demand ofthe market.�[0039] Further, it is preferable that the plant growth reg-ulator of the present invention promotes growth of rootsof a plant. As explained in the later- �mentioned Examples,it is clearly demonstrated that use of the plant growthregulator of the present invention promotes growth ofroots of a plant.�[0040] This allows obtaining a large number of har-vests from a plant whose roots are harvests in a shorttime. Therefore, application of the plant growth regulatorto a plant whose roots are edible allows increasing pro-duction of foods.�[0041] Further, it is preferable that the plant growth reg-ulator of the present invention prevents deterioration ofgrowth due to lack of nitrogen. It is known that shortageor lack of a nitrogen source generally reduces growth ofa plant. However, as explained in the later-�mentionedExamples, even when a plant is cultivated under a nitro-gen-�deficient condition, application of the plant growthregulator of the present invention allows preventing de-teriorated growth due to lack of a nitrogen source.�[0042] Therefore, even when a plant suffers from de-teriorated growth due to lack of nitrogen, application ofthe plant growth regulator to the plant allows promotionof the growth of the plant.�[0043] In a case where the plant growth regulator con-tains oxidized glutathione, the amount of glutathione isnot particularly limited. In a case of Arabidopsis, theamount of glutathione is preferably 10PM- �20mM, morepreferably 0.2mM- �5mM, and further more preferably0.5mM-�2mM.�[0044] On the other hand, in a case where the plantgrowth regulator contains reduced glutathione, theamount of reduced glutathione is preferably larger thanthat of oxidized glutathione to be contained in the plantgrowth regulator. Specifically, in a case of Arabidopsis,

the amount of reduced glutathione is preferably 100uM-40mM, more preferably 0.4mM-�20mM, and further morepreferably 4mM-�10mM.�[0045] In the case where the plant growth regulatorcontains reduced glutathione in the above range, when50% of reduced glutathione is oxidized while preservingor using the plant growth regulator, the concentration ofoxidized glutathione in the plant growth regulator rangesat least from 1mM to 2.5mM. This yields substantially thesame effect as when the plant growth regulator contains1mM-�2.5mM of oxidized glutathione. Oxidization of 50%of reduced glutathione in the plant growth regulator easilyoccurs because of properties of the reduced glutathione.This can be easily understood by a person skilled in theart.�[0046] In a case of supplying a specific amount of so-lution as explained in the later-�mentioned Examples,when the amount of oxidized glutathione or reduce glu-tathione is in the above range, it is possible to controlgrowth of a plant appropriately. Note that the above con-centration range is a range in a case of supplying a spe-cific amount of solution to Arabidopsis. Change of theamount to be supplied or change of the kinds of plants(e.g. tree etc.) may allow oxidized glutathione or reducedglutathione with higher concentration to be supplied. Insome cases, it is possible to realize the performance ofthe plant growth regulator of the present invention withoxidized glutathione or reduced glutathione with lowerconcentration.�[0047] The feature of the present invention is basedon the finding that oxidized glutathione increases thenumber of seeds and/or the number of flowers of a plant,lengths the life of the plant, causes a leaf to be moreround and larger, increases the number of lateral shootsand/or tillers, and increases the number of flowers(sheaths) in accordance with an increase in the numberof lateral shoots etc. so as to increase the yield of seeds,and other limitation is not intended. Therefore, the con-centration range in the present invention is not limited tothe above range.�[0048] How to supply the plant growth regulator of thepresent invention to a plant is not particularly limited, andthe plant growth regulator of the present invention maybe used in the same manner as a conventional and pub-licly known plant growth regulator. For example, in a casewhere the plant growth regulator of the present inventionis in the form of a liquid or emulsion, the plant growthregulator may be sprayed to, dropped on, or applied tonot only a vegetative point but also a part of or all of aplant such as a stem and a leaf. In a case where the plantgrowth regulator of the present invention is in the formof a solid agent or a powder agent, the plant growth reg-ulator may be absorbed into a root via the earth. In a casewhere a plant is a water plant such as a floating grass,the plant growth regulator of the present invention maybe absorbed as an aquarium additive into a root or theplant growth regulator in the form of the solid agent maybe dissolved gradually in water. In particular, in a case

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where the plant growth regulator of the present inventionis used for a terrestrial plant, it is preferable that the plantis subjected to a solution culture by use of the plant growthregulator in the form of an aqueous solution.�[0049] As long as the plant growth regulator of thepresent invention contains glutathione (GSH and/orGSSG), other specific components of the plant growthregulator are not particularly limited. For example, in acase where the plant growth regulator is in the form ofan aquarium additive or a solid agent, carrier componentsare solid carriers, examples of which include inorganicmaterials such as talc, clay, vermiculite, � diatomite, kao-line, calcium carbonate, calcium hydroxide, white clay,and silica gel, and flour and starch. In a case where theplant growth regulator is in the form of a liquid agent,carrier components are liquid carriers, examples of whichinclude: water; aromatic hydrocarbons such as xylene;alcohols such as ethanol and ethyleneglycol; ketonessuch as acetone; ethers such as dioxane and tetrahy-drofuran; dimethylformamide; dimethylsulfoxide; and ac-etonitrile.�[0050] Further, the plant growth regulator of thepresent invention may contain other adjuvant if neces-sary. Examples of the adjuvant include: negative ion sur-factants such as ester alkylsulfates, alkyl sulfonate, alky-laryl sulfonate, and dialkyl sulfosuccinate; positive ionsurfactants such as salts of higher aliphatic amine; non-ion surfactants such as polyoxyethyleneglycol alkylether,polyoxyethyleneglycol acylester, polyoxyethyleneglycolmultivalent alcohol acylester, and cellulose derivative;and a thickener such as gelatin, casein, and gum Arabic;a filler; and a binder.�[0051] If necessary, other plant growth regulator suchas benzoic acid, nicotine acid, nicotine acid amide, andpipecolic acid may be added to a product in such anamount that does not prevent an intended effect of thepresent invention. Further, a conventional and well-known fertilizer may be added to the product.�[0052] A plant to which the plant growth regulator ofthe present invention is to be supplied is not particularlylimited, and the plant growth regulator may be suppliedto every kinds of plants such as monocotyledons, dicot-yledons, and trees. Examples of monocotyledons in-clude: Lemnaoideae including Spirodela (floating grass)and Lemna (L. perpusilla and L. trisulca); Orchidaceaeincluding Cattleya, Cymbidium, Dendrobium, Pha-laenopsis, Vanda, Paphiopedilum, and Oncidium;� Ty-phaceae; Sparganiaceae; Potamogetonaceae; Naja-daceae; Scheuchzeriaceae; Alismataceae, Hydrochari-taceae; Triuridaceae; Poaceae; Cyperaceae; Arecace-ae; Araceae; Eriocaulaceae; Commelinaceae; Pontede-riaceae; Juncaceae; Stemonaceae; Liliaceae; Amarylli-daceae; Dioscoreaceae; Iridaceae; Musaceae; Zingiber-aceae; Cannaceae; and Burmanniaceae.�[0053] Examples of dicotyledonous include: Convol-vulaceae including Pharbitis (morning glory), Calystegia(Calystegia japonica, Calystegia hederacea, and Calys-tegia soldanella), Ipomoea (Ipomoea pes-�caprae, Ipo-

moea batatas), and Cuscuta (Cuscuta japonica, Cuscutaaustralis); Caryophyllaceae including Dianthus (Dian-thus caryophyllus etc.), Stellaria, Minuartia, Cerastium,Sagina, Arenaria, Moehringia, Pseudostellaria, Honck-enya, Spergula, Spergularia salina, Silene, Lychnis, Me-landryum, and Cucubalus; Casuarinaceae; Saururace-ae; Piperaceae; Chloranthaceae; Salicaceae; Myricace-ae; Juglandaceae; Betulaceae; Fagaceae; Ulmaceae;Moraceae; Urticaceae; Podostemaceae; Proteaceae;Olacaceae; Santalaceae; Viscum album; Aristolochiace-ae; Mitrastemonaceae; Balanophoraceae; Polygonace-ae; Chenopodiaceae; Amaranthaceae; Nyctaginaceae;Theligonaceae; Phytolaccaceae; Tetragoniaceae; Por-tulacaceae; Magnoliaceae; Trochodendraceae; Cercidi-phyllaceae; Nymphaeaceae; Ceratophyllaceae; Ranun-culaceae; Lardizabalaceae; Berberidaceae; Menisper-maceae; Calycanthaceae; Lauraceae; Papaveraceae;Capparaceae; Brassicaceae; Droseraceae;Nepenthaceae; Crassulaceae; Saxifragaceae; Pittospo-raceae; Hamamelidaceae; Platanaceae; Rosaceae; Fa-baceae; Oxalidaceae; Geraniaceae; Linaceae; Zygo-phyllaceae; Rutaceae; Simaroubaceae; Meliaceae; Po-lygalaceae; Euphorbiaceae; Callitrichaceae; Buxaceae;Empetraceae; Coriariaceae; Anacardiaceae; Aquifo-liaceae; Celastraceae; Staphyleaceae; Icacinaceae; Ac-eraceae; Hippocastanaceae; Sapindaceae; Sabiaceae;Balsaminaceae; Rhamnaceae; Vitaceae; Elaeaocar-paceae; Tiliaceae; Malvaceae; Sterculiaceae; Actinidiaarguta; Theaceae; Clusiaceae; Elatinaceae; Tamari-caceae; Violaceae; Flacourtiaceae; Stachyuraceae;Passifloraceae; Begoniaceae; Cactaceae; Thymelae-aceae; Elaeagnaceae; Lythraceae; Punica granatum;Rhizophoraceae; Alangiaceae; Melastomataceae; Tra-paceae; Onagraceae; Haloragaceae; Hippuridaceae;Araliaceae; Apiaceae; Cornaceae; Diapensiaceae;Clethraceae; Pyrolaceae; Ericaceae; Myrsinaceae;Primulaceae; Plumbaginaceae; Ebenaceae; Symplo-caceae; Styracaceae; Oleaceae; Buddlejaceae; Gen-tianaceae; Apocynaceae; Asclepiadaceae; Polem-oniaceae; Boraginaceae; Verbenaceae; Lamiaceae;Solanaceae; Scrophulariaceae; Bignoniaceae; Pe-daliaceae; Orobanchaceae; Gesneriaceae; Lentibular-iaceae; Acanthaceae; Myoporaceae; Phrymaceae;Plantaginaceae; Rubiaceae; Caprifoliaceae; Adoxace-ae; Valerianaceae; Dipsacaceae; Cucurbitaceae; Cam-panulaceae; and Asteraceae.�[0054] The plant to which the plant growth regulator issupplied may be a mutant or a transformant of the aboveplant as well as a wild-�type of the above plant. As ex-plained in the later-�mentioned Examples, application ofthe plant growth regulator of the present invention to atransformant plant to which a specific gene is introducedincreases the effect of the plant growth regulator of thepresent invention (in other words, application of the plantgrowth regulator of the present invention to such trans-formant yields a greater effect than application of theplant growth regulator of the present invention to a wild-type plant).

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�[0055] Therefore, such transformant plant may be re-garded as an object to which the plant growth regulatorof the present invention is preferably applied.�[0056] A specific example of such transformant is atransformant plant to which a gene for encoding glutath-ione- �binding plastid-�type fructose-�1,6-�bisphosphate al-dolase (which may be hereinafter referred to as "gFBA")is introduced.�[0057] As explained in the later-�mentioned Examples,it is demonstrated that application of the plant growthregulator of the present invention to a transformant plantto which a gFBA gene is introduced allows the plantgrowth regulator to further enhance the effect of increas-ing harvest index.�[0058] A transformant plant to which the gFBA geneis introduced and a method for producing the transform-ant plant are described in International Application Pub-lication No. WO 2007/091634A1 (published on August16, 2007) etc. Therefore, the contents of the InternationalApplication Publication serve as a reference for thepresent specification.�[0059] The plant growth regulator of the present inven-tion may be applied to various organisms, organs, tis-sues, or cells by a method suitable for the form of theplant growth regulator.�[0060] Further, a seed obtained from a plant treatedwith the plant growth regulator of the present inventionis industrially useful and is encompassed in the presentinvention. As explained in the later-�mentioned Examples,analysis of ripeness (germination rate) of such seedshowed that such seed germinated faster than a normalseed. Therefore, it may be considered that a seed ob-tained from a plant cultivated with glutathione, preferablyoxidized glutathione, has a higher ripeness.�[0061] A treatment with the plant growth regulator ofthe present invention may be performed in such a mannerthat the plant growth regulator with appropriate concen-tration is used before and/or while normally cultivating aseed or callus of a target plant. Normally, it is effectiveto use the plant growth regulator in a treatment suitablefor the nature (such as long- �day property, short-�day prop-erty) of the target plant. Since such treatment is wellknown to a person skilled in the art, detailed explanationsthereof are omitted here. For example, in a case of arelative long-�day plant, it is effective to use the plantgrowth regulator of the present invention while irradiatinglight with predetermined intensity or more.�[0062] Therefore, the present invention encompassesuse of the plant growth regulator in a method of growingany plant (method of producing a plant) that is normallyused in the field to which the present invention pertains.�[0063] The plant growth regulator of the present inven-tion may be made of only glutathione that is an effectivecomponent. However, it is preferable that the plantgrowth regulator of the present invention is used in theform applicable to individual plants, such as a liquidagent, a solid agent, a powder agent, an emulsion, andan aquarium additive. Such agent may be produced

through a conventional method by appropriately adding,to glutathione that is an effective component, a publiclyknown carrier and an adjuvant etc. that are pharmaceu-tically usable in individual fields in such an amount thatdoes not impair the effect of the plant growth regulatorof the present invention.�[0064] Further, the present invention encompasses amethod of cultivating a plant by use of the plant growthregulator. That is, the method of cultivating a plant inaccordance with the present invention is a method ofcultivating a plant by use of glutathione so as to increaseharvest index of the plant, and other specific steps andconditions etc. of the method are not particularly limited.In the method, glutathione may be GSH and/or GSSG,but it is preferable that glutathione contains GSSG.�[0065] One embodiment of a method of cultivating aplant in accordance with the present invention is ex-plained below. Note that the present invention is not lim-ited to the following embodiment.�[0066] In the method of cultivating a plant in accord-ance with the present invention, glutathione may be sup-plied to a plant under a condition that allows the plant toalways absorb glutathione, or glutathione may be sup-plied to a plant under a condition that allows a plant tointermittently absorb glutathione during the cultivation(e.g. a condition that glutathione is supplied with an in-terval, once a week or twice a week). Further, glutathionemay be supplied during a specific time, i.e., during a spe-cific growth time.�[0067] Intermittently supplying glutathione allows re-ducing the amount of glutathione to be supplied. Thisreduces costs for cultivating a plant. In the case of inter-mittently supplying glutathione, it is preferable to supplyglutathione at a constant interval. Alternatively, glutath-ione may be supplied at an inconstant interval.�[0068] The interval at which glutathione is supplied isnot particularly limited, and may be determined in accord-ance with concentration of glutathione to be supplied, aplant to which glutathione is to be supplied, and a time(more specifically, a growth time) when glutathione is tobe supplied. Generally, in a case where a plant to whichglutathione is to be supplied is a herbaceous plant, it ispreferable that glutathione is supplied once a week ortwice a week or supplied at the same time as the time ofadditional fertilization.�[0069] In the case of supplying glutathione at a specifictime, it is preferable to supply glutathione around at thetime of transition from vegetative to reproductive devel-opment (including the time of transition from vegetativeto reproductive development) or at the time of forming afloral bed after the transition from vegetative to reproduc-tive development or at the time of translocation to a targetharvest. This allows effectively obtaining the effect yield-ed by supplying glutathione. Further, since glutathioneis supplied only at a specific time, it is possible to reducecosts for cultivating a plant.�[0070] In the case of supplying glutathione only at aspecific time, glutathione may be supplied to a plant un-

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der a condition that allows the plant to always absorbglutathione during a predetermined period in the specifictime, or glutathione may be supplied to a plant under acondition that allows the plant to intermittently absorbglutathione during a predetermined period in the specifictime. Intermittently supplying glutathione during a prede-termined period in the specific time allows further reduc-ing costs for cultivating a plant.�[0071] Further, the present invention encompasses amethod of increasing the number of seeds of a plantand/or the number of flowers of the plant. In the method,too, glutathione may be oxidized glutathione or reducedglutathione. It is preferable that glutathione includes ox-idized glutathione.�[0072] In the method, a time at which glutathione issupplied to a plant and the amount of glutathione to besupplied to the plant etc. is not particularly limited. It ispreferable that glutathione is supplied under the conditionexplained in the method of cultivating a plant.�[0073] Further, the present invention encompasses aplant obtained by the above method. The plant in accord-ance with the present invention shows a higher harvestindex. Since the plant in accordance with the presentinvention shows a higher harvest index than a plant cul-tivated under a normally recommended condition, meas-urement of the harvest index allows clearly distinguishingthe plant in accordance with the present invention froma plant obtained by a method other than the method ofthe present invention.�[0074] Further, such a plant can be easily and clearlydistinguished from a plant obtained by a method otherthan the method of the present invention by examiningthe amount or the rate of oxidized glutathione in the plant.Other than the method of examining the amount and con-centration of oxidized glutathione in a plant, it is possibleto distinguish the plant in accordance with the presentinvention from a plant obtained by a method other thanthe method of the present invention by comparing geneexpression patterns by use of a DNA micro allay etc.Specifically, the gene expression pattern of a plant cul-tivated with oxidized glutathione is examined in advance,and is compared with that of a plant cultivated by a meth-od other than the method of the present invention so asto specify an expression pattern specific to a case of sup-plying oxidized glutathione (GSSG expression pattern).The expression pattern of a plant to be examined is ex-amined, and is compared with the GSSG expression pat-tern. Thus, it is possible to easily determine whether theplant to be examined is a plant in accordance with thepresent invention or not.�[0075] Further, such a plant can be clearly distin-guished from a plant cultivated by a method other thanthe method of the present invention by measuring theharvest index of the plant.�[0076] The methods as explained above (i.e. the meth-ods for specifying a plant of the present invention) maybe carried out singularly or in combination. Carrying outthe methods in combination allows further clearly distin-

guishing the plant of the present invention from a plantcultivated by a method other than the method of thepresent invention.�[0077] The following more details the present Embod-iment with reference to Examples. Note that the presentinvention is not limited to the following Examples, andvarious modifications are possible with respect to detailsof the present invention. Further, the present inventionis not limited to the description of the embodimentsabove, but may be altered by a skilled person within thescope of the claims. An embodiment based on a propercombination of technical means disclosed in different em-bodiments is encompassed in the technical scope of thepresent invention.

[Examples]

<1. Influence of oxidized glutathione on growth of Arabi-dopsis>

�[0078] Arabidopsis was cultivated with light of100PE/m2 at 22°C under a day length condition that alight period is 16 hours and a dark period is 8 hours, byuse of a culture medium with two parts of vermiculite(Asahi Kogyo, Inc.) in a lower layer, one part of The Kure-ha Ikubyou Baido soil (Kureha) in a middle layer, and onepart of vermiculite (Asahi Kogyo, Inc.) in an upper layer.Normally, Arabidopsis cultivated under these conditionsdoes not exhibit the symptom of lacking nitrogen withoutadditional fertilization.�[0079] In the present test, the states of the growingplants were observed while the plants were treated withonly water, a 1mM oxidized glutathione (GSSG) solution,or a 5mM H2O2 solution. Specifically, the plants werecultivated with two or three plants in one pod of approx-imately 65 in width, 65 in depth, and 50 in height, and anappropriate amount of a treatment solution was supplied.�[0080] The effect of the treatment was evaluated withrespect to the number of rosette leaves, the speed of agrowing flower stalk,� the number of flowers, and thenumber of seeds. The results are shown in Figs. 1-5.�[0081] As shown in Fig. 1, it was found that 3-4 weeksafter sowing, leaves of the plant cultivated with the 1mMGSSG solution grew larger and rounder than leaves ofthe plant cultivated with only water and than leaves ofthe plant cultivated with the 5mM H2O2 solution.�[0082] Further, as shown in (a) and (b) of Fig. 2, it wasfound that 6 weeks after the sowing, the plant cultivatedwith the 1mM GSSG solution grew to have a shorter plantlength, a thicker stem, and larger leaves than the plantcultivated with only water and the plant cultivated withthe 5mM H2O2 solution. (a) and (b) of Fig. 2 are photo-graphs of the same sample taken in different angles.�[0083] Further, as shown in Fig. 3, it was found that 7weeks after the sowing, the number of flowers and thearea of leaves of the plant cultivated with the 1mM GSSGsolution were greatly larger than those of the plant culti-vated with only water and the plant cultivated with the

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5mM H2O2 solution.�[0084] Further, as shown in Fig. 4, it was found that 8weeks after the sowing, the plant cultivated with only wa-ter and the plant cultivated with the 5mM H2O2 solutionwere withering, whereas the plant cultivated with the1mM GSSG solution showed much amount of greenleaves. This shows that the plant cultivated with the 1mMGSSG solution elongated its life.�[0085] Further, as shown in Fig. 5, it was found thatthe yield of seeds of the plant cultivated with the 1mMGSSG solution was greatly larger than the yield of seedsof the plant cultivated with water and than the yield ofseeds of the plant cultivated with the 5mM H2O2 solution.The result of the measurement showed that the effectivenumber of seeds per plant of the plant cultivated with the1mM GSSG solution was approximately three or fourtimes as large as the effective number of seeds per plantof the plant cultivated with only water and the plant cul-tivated with the 5mM H2O2 solution (see the upper pho-tograph in Fig. 5).�[0086] Further, it was examined whether any differenc-es in the shape of a seed and the size of a seed wereobserved. The result of the examination was shown bythe lower photographs in Fig. 5. As shown in Fig. 5, seedsobtained from the plant cultivated with the 1mM GSSGsolution had substantially the same shape and the samesize as those obtained from the plant cultivated with onlywater. It was found that seeds obtained from the plantcultivated with the 5mM H2O2 solution were a bit larger.�[0087] The above results clearly show that cultivatinga plant by use of oxidized glutathione increases thenumber of seeds and/or the number of flowers.

<2. Effect of concentration of oxidized glutathione on growth of Arabidopsis>

�[0088] The influence of concentration of oxidized glu-tathione on growth of Arabidopsis was examined. Spe-cifically, soil filled in a pod of approximately 65 in width,65 in depth, and 50 in height was immersed in 0mM-,0.01 mM-, 0.2mM-, 1mM-, 2mM-, and 5mM-�GSSG solu-tions. Seeds of Arabidopsis were sown in such a mannerthat approximately three seeds existed in each pod, andthe seeds were observed chronologically 3 weeks afterthe sowing.�[0089] The results of the observation are shown inFigs. 6 and 7. As shown in the drawings, performance ofmaking a leaf rounder and performance of lengtheningthe life of a plant were clearly observed at concentrationof 0.2-2mM, and performance of making a stem thickerwas observed at concentration of 1-2mM. In a case whereconcentration of GSSG was 0.01mM, almost no differ-ence was observed between Arabidopsis cultivated withGSSG and Arabidopsis cultivated with water (0mM). Ina case where concentration of GSSG was 5mM, thegrowth of Arabidopsis was greatly prevented and muchof Arabidopsis withered.

<3. Influence of oxidized glutathione on Arabidopsis seeds>

�[0090] Ripeness of seeds obtained from Arabidopsiscultivated with oxidized glutathione was examined. Spe-cifically, seeds obtained from plants cultivated with wateror a GSSG solution were sown in 1/2 MS culture mediaand germination rates of the seeds were examinedchronologically.�[0091] The results of the examination are shown in (a)and (b) of Fig. 8. As shown in the drawing, seeds of plantscultivated with the GSSG solution germinated faster thanseeds of plants cultivated normally. In particular, the ger-mination rate of the seeds of the plants cultivated withthe GSSG solution was significantly high 2 days after thesowing. However, 7 days after the sowing, almost nodifference was observed between the seeds of the plantscultivated with the GSSG solution and the seeds of theplants cultivated normally.

<4. Effect of oxidized glutathione on gibberellin synthesis mutant>

�[0092] The effect of oxidized glutathione on gibberellin(GA) synthesis mutant was examined. Specifically, Ara-bidopsis GA synthesis mutants ga20ox1 were cultivatedwith water or GSSG (1mM) from the time when they weresown, and the state of their growth was observed.�[0093] Fig. 9 shows the state of plants 8 weeks afterthe sowing. The upper photograph of Fig. 9 shows plantsobserved from the front side, and the lower photographof Fig. 9 shows the plants observed from the upper ob-lique direction. In the drawing, "Col" indicates a wild-�typeColumbia, and "ga20ox1" indicates a mutant in which T-DNA is inserted into a GA20 oxidase gene that codes anenzyme of a GA biosynthesis. "ga20ox1-1" and"ga20ox1-2" are independent mutants in which T-�DNAare inserted into different portions.�[0094] As illustrated in the drawing, GA mutantga20ox1 cultivated with GSSG showed significantly in-creased number of lateral shoots than plants cultivatedwith water. In accordance with the increase in the numberof lateral shoots, the number of flowers (sheaths) alsoincreased. Further, as shown in the right side of Fig. 9,the seed weight significantly increased, too.�[0095] Therefore, this method allows increasing theyield of seeds by applying GSSG to a mutant having mu-tation in synthesis of a plant hormone and reaction to aplant hormone. In particular, the method is effective fora plant such as gramineae whose yield is greatly depend-ent on tillers. This is evident from the fact that when rice(Akita 63) was cultivated in hydroponics with the standardamount of fertilization being 5kgN/�10a and additional fer-tilization of 2kgN/ �10a was made at the panicle formationstage and the meiotic stage, application of GSSG in thestandard amount of 0.2gN to each test location (0.1m2)in additional fertilization made the number of ears ap-proximately 1.4 times larger than the number of ears of

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the plant cultivated with only water.�[0096] The plant growth regulator of the present inven-tion contains oxidized glutathione and therefore can pro-mote growth of a plant. For example, the plant growthregulator of the present invention can increase thenumber of seeds of a plant and/or the number of flowers(sheaths) of a plant.�[0097] Further, cultivating with oxidized glutathione amutant having mutation in synthesis of a plant hormone(e.g. gibberellin) or response to a plant hormone allowsgreatly increasing lateral shoots, and accordingly allowsincreasing the number of flowers (sheaths). Consequent-ly, application of the plant growth regulator of the presentinvention to a plant such as gramineae whose tillersgreatly influence the yield allows increasing the numberof yield of seeds.

<5. Influence 1 of treatment condition of oxidized glutath-ione on seeds of Arabidopsis>

�[0098] Seeds of Arabidopsis were sown in pods filledwith soil immersed in 0mM-, 0.01mM-, 0.2mM-, 1mM-,2mM-, and 5mM-�GSSG. The pods were transferred totrays with water containing no GSSG 2 days after, 1 weekafter, 2 weeks after, 3 weeks after, or 4 weeks after thesowing.�[0099] Arabidopsis was cultivated under the sameconditions as those described in <1. Influence of oxidizedglutathione on growth of Arabidopsis> except for theabove condition.�[0100] The seed weight per one pod, obtained fromArabidopsis thus cultivated (three plants in each pod),was measured. The results of the measurement areshown in Figs. 10-12. n is the number of plants that canbe finally harvested.�[0101] In Fig. 10, "Ratio" stands for a ratio of the seedweight of the plant thus cultivated to the seed weight (con-sidered as 1) of a plant cultivated in a tray filled with watercontaining no GSSG.�[0102] As shown in Figs. 10-12, it was found that sus-ceptibility of Arabidopsis to GSSG varied depending onthe amount of GSSG (concentration of GSSG) to be sup-plied and the time of supplying GSSG. It was found thatin a case of supplying GSSG for a long time, GSSG withlow concentration is more effective than GSSG with highconcentration, and in a case of supplying GSSG for ashort time, GSSG with high concentration is more desir-able.


�[0103] Seeds of Arabidopsis were sown in pods filledwith soil immersed in water. The pods were transferredto trays with water containing 1mM-�GSSG 2 days after,1 week after, 2 weeks after, 3 weeks after, 4 weeks after,5 weeks after, 6 weeks after, or 7 weeks after the sowing.Further, there was prepared a pod that kept in a tray filled

with water containing no GSSG, without being trans-ferred into water containing 1mM- �GSSG.�[0104] Arabidopsis was cultivated under the sameconditions as those described in <1. Influence of oxidizedglutathione on growth of Arabidopsis> except for theabove condition.�[0105] The seed weight per one pod, obtained fromArabidopsis thus cultivated (three plants in each pod),was measured.�[0106] The result of the measurement showed that asshown in Fig. 13, even when concentration of GSSG tobe supplied was the same among the pods, there existeda great difference in the amount of obtained seeds de-pending on the time of treating the plants with GSSG,indicating that there was a time most suitable for supply-ing GSSG. On the other hand, regardless of the time oftreating the plants with GSSG, the treatment with GSSGallowed obtaining more seed weight than the case of notreatment with the GSSG.


�[0107] Seeds of Arabidopsis were sown in pods filledwith soil immersed in water. The pods were transferredto trays filled with water containing 1mM-�GSSG and cul-tivated there only for one week that is the first week (0th-7th day), the second week (8th- 4th day), the third week(15th-�21st day), the fourth week (22nd- �28th day), the fifthweek (29th-�35th day), the sixth week (36th- �42nd day),and the seventh week (43rd- �49th day) after the sowing.That is, Arabidopsis was treated with 1mM-�GSSG onlyfor one week in a specific growth period.�[0108] There were prepared a pod that was kept in atray filled with water containing no GSSG and that wasnot transferred into water containing 1mM-�GSSG con-sistently from the sowing and a pod that was kept in atray filled with water containing 1mM- �GSSG consistentlyfrom the sowing.�[0109] Arabidopsis was cultivated under the sameconditions as those described in <1. Influence of oxidizedglutathione on growth of Arabidopsis> except for theabove condition.�[0110] The seed weight per one pod, obtained fromArabidopsis thus cultivated (three plants in each pod),was measured.�[0111] The result of the measurement showed that asshown in Fig. 14, application of GSSG to Arabidopsisonly for one week in a specific growth period significantlyincreased the seed weight compared with the seedweight of Arabidopsis having not been treated withGSSG, although the effect yielded by applying GSSG toArabidopsis only for one week in a specific growth periodwas smaller than the effect yielded by continuously ap-plying GSSG to Arabidopsis.�[0112] In this case, too, there existed a great differencein the amount of obtained seeds depending on the timeof treating the plants with GSSG. In particular, application

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of oxidized glutathione 4 weeks after the sowing yieldedthe maximum seed weight obtained. 4 weeks after thesowing corresponds to around a time of bolting.


�[0113] Seeds of Arabidopsis were sown in pods filledwith soil immersed in water. The pods were transferredto trays filled with water containing 1mM-�GSSG and cul-tivated there only for two weeks that are the first andsecond weeks (0th-�14th day), the second and thirdweeks (8th-�21st day), the third and fourth weeks (15th-28th day), the fourth and fifth weeks (22nd-�35th day), thefifth and sixth weeks (29th-�42nd day), or the sixth andseventh weeks (36th-�49th day) after the sowing. That is,Arabidopsis were treated with 1mM-�GSSG only for twoweeks in a specific growth period.�[0114] There were prepared a pod that was kept in atray filled with water containing no GSSG and that wasnot transferred into water containing 1mM- �GSSG con-sistently from the sowing and a pod that was kept in atray filled with water containing 1mM- �GSSG consistentlyfrom the sowing.�[0115] Arabidopsis was cultivated under the sameconditions as those described in <1. Influence of oxidizedglutathione on growth of Arabidopsis> except for theabove condition.�[0116] The seed weight per one pod, obtained fromArabidopsis thus cultivated (three plants in each pod),was measured.�[0117] The result of the measurement showed that asshown in Fig. 15, application of oxidized glutathione toArabidopsis only for two weeks in a specific growth periodsignificantly increased the seed weight compared withthe seed weight of Arabidopsis having not been treatedwith GSSG, although the effect yielded by applying oxi-dized glutathione to Arabidopsis only for two weeks in aspecific growth period was smaller than the effect yieldedby continuously applying oxidized glutathione to Arabi-dopsis.�[0118] In this case, too, there existed a great differencein the amount of obtained seeds depending on the timeof treating the plants with GSSG.


�[0119] Seeds of Arabidopsis were sown in pods filledwith soil immersed in water. The pods were transferredto trays filled with water containing 0.2mM-�GSSG or1mM-�GSSG and cultivated there only for two weeks thatwere the first and second weeks (0th-�14th day), the thirdand fourth weeks (15th-�28th day), the fifth and sixthweeks (29th-�42nd day), or the seventh and eighth weeks(43rd- �56th day) after the sowing. That is, Arabidopsiswere treated with 0.2mM-�GSSG or 1mM-�GSSG only fortwo weeks in a specific growth period.

�[0120] There were prepared a pod that was kept in atray filled with water containing no GSSG and that wasnot transferred into water containing 0.2mM-�GSSG or1mM-�GSSG consistently from the sowing and a pod thatwas kept in a tray filled with water containing 0.2mM-GSSG or 1mM-�GSSG consistently from the sowing.�[0121] Arabidopsis was cultivated under the sameconditions as those described in <1. Influence of oxidizedglutathione on growth of Arabidopsis> except for theabove condition.�[0122] The seed weight, dry weight, and harvest indexof Arabidopsis thus cultivated (three plants in each pod)were measured and an average of three pods was cal-culated.�[0123] As shown in Fig. 16, the result of the calculationshowed that when Arabidopsis was treated with 0.2mM-GSSG, the dry weight increased little compared with acase where Arabidopsis was not treated with GSSG,while the seed weight increased, which increased har-vest index.�[0124] On the other hand, when Arabidopsis was treat-ed with 1mM-�GSSG, the seed weight, dry weight, andharvest index evidently increased compared with a casewhere Arabidopsis was not treated with GSSG, exceptfor harvest index in a case where Arabidopsis was treatedwith GSSG 1 or 2 weeks after the sowing.�[0125] In Fig. 16, asterisk indicates a great differencebetween a normal cultivation (water) and a GSSG treat-ment in the t-�test (*P<0.05, **<0.01).�[0126] As described above, the results of <5. Influenceof treatment condition of oxidized glutathione on seedsof Arabidopsis 1 > - <9. Influence of treatment conditionof oxidized glutathione on seeds of Arabidopsis 5>showed that susceptibility of Arabidopsis to oxidized glu-tathione was different depending on the growth time ofArabidopsis. To be specific, in a case where Arabidopsiswas treated with oxidized glutathione for 1 or 2 weeks,when Arabidopsis was treated at 4 weeks to 5 weeksafter the sowing, the yield of seeds increased effectively.Under the present growth condition, the time of around2 weeks after the sowing corresponds to the time of tran-sition from vegetative to reproductive development, andthe time of 4 to 5 weeks after the sowing corresponds toaround the time of bolting of Arabidopsis. That is, it wasfound that treatment of Arabidopsis with oxidized glutath-ione at a time ranging from the time of transition fromvegetative to reproductive development to around thetime of bolting allows effectively increasing the yield ofseeds.

<10. Influence of oxidized glutathione on growth of rose (breed; Patiohit alicante) �>

�[0127] Rose (breed; Patiohit alicante) was fertilizedwith 50mL of 0.5mM-�GSSG solution two times a weekfor four months and then pruned completely and cultivat-ed. In addition to GSSG, the rose was additionally ferti-lized with 2g of S604 per 2 weeks.

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�[0128] Consequently, as shown in Fig. 17, it was foundthat the plant treated with GSSG (plant positioned in theleft-�side tray in the drawing) exhibited significant promo-tion of growth of new floral buds compared with the plantthat was not treated with GSSG (plant positioned in theright-�side tray in the drawing).

< 11. Influence of oxidized glutathione on growth of rose (breed; English rose)�>

�[0129] Rose (breed; English rose) was fertilized with50mL of a 0.5mM-�GSSG solution two times a week forfour months and then pruned completely and cultivated.In addition to GSSG, the rose was additionally fertilizedwith 2g of S604 every two weeks.�[0130] Consequently, as shown in Fig. 18, it was foundthat the plant treated with GSSG (plant positioned in theupper- �side in the drawing) exhibited significantly earliergermination and significant promotion of growth of newbuds compared with the plant that was not treated withGSSG (plant positioned in the lower- �side in the drawing).

<12. Influence of oxidized glutathione on growth of tsai-�hsin (Sakata Seed Co.) 1>

�[0131] Tsai-�hsin was cultivated for two weeks, andthen transferred to an agricultural field and grown with orwithout the treatment with GSSG. Tsai-�hsin was fertilizedwith a nitrogen fertilizer that was KUMIAI RIN RYUANKA-RI S604 in a ratio of 20kgN per 10 are, and was addition-ally fertilized with 5kgN 3 weeks later.�[0132] In a GSSG-�treated management zone, 0.5mM-or 5mM-�GSSG solution was sprayed to surfaces ofleaves of the plant two times a week in such a mannerthat 1L of the GSSG solution was sprayed per 1 zone(3m2).�[0133] Consequently, as shown in Fig. 19, the plant inthe GSSG- �treated management zone exhibited signifi-cant increase in the amount of growth than the plant ina normal nitrogen management zone (not treated withGSSG).

< 13. Influence of oxidized glutathione on growth and yield of seeds of soybean 2>

�[0134] Soybean (breed: Tsurumusume) and soybean(breed; Toyomusume) were cultivated for two weeks, andthen transferred to an agricultural field in such a manneras to be positioned as shown in Fig. 20, and grown withor without the treatment with GSSG. The soybean wasfertilized with a nitrogen fertilizer that was KUMIAI RINRYUANKARI S604 in a ratio of 20kgN per 10 are, andwas additionally fertilized with 5kgN 3 weeks later.�[0135] Further, in a GSSG-�treated management zone,a 0.5mM-�GSSG solution was given to the base of a stem(see the arrow in Fig. 21) in the amount of 50mL per oneindividual two times a week in such a manner that thesolution flowed along the stem.

�[0136] In a case where it was likely to rain, the GSSGsolution was supplied to the plant after the rain. Similarly,in a case of watering, the GSSG solution was suppliedto the plant after the watering. When the harvest time ofthe plant of the control (i.e. normal nitrogen managementzone) was coming, supply of the GSSG solution to theplant in the GSSG-�treated management zone wasstopped.�[0137] The seed weight, the amount of biomass, andthe harvest index of the plant thus obtained in the GSSG-treated management zone, each relative to that of theplant in the normal nitrogen management zone, weremeasured.�[0138] Consequently, as shown in Fig. 22, with respectto each breed, the plant in the GSSG-�treated manage-ment zone exhibited a higher seed weight, a higheramount of biomass, and a higher harvest index thanthose of the plant in the normal nitrogen managementzone.

< 14. Effect 1 of oxidized glutathione on productivity of corn>

�[0139] Sweet corn (Canberra 90, TAKII & CO., LTD.)was sown, and then transferred to a hydroponics pot(1/2000 are) filled with a culture soil (6L of vermiculite asthe lower layer, 3L of The Kureha Ikubyou Baido soil asthe middle layer, and 3L of vermiculite as the upper layer)two weeks later and additionally fertilized with 3g of KU-MIAI RIN RYUANKARI S604 four weeks later and sixweeks later. Further, the GSSG-�treated plant was given200mL of 0.5mM-�GSSG two times a week for 12 weeksafter germination at its root. As for the time of the treat-ment with GSSG, see (c) of Fig. 25. The time indicatedby the hatched part in (c) of Fig. 25 was the time of thetreatment with GSSG.�[0140] Consequently, as shown in Fig. 23, the planttreated with GSSG (left-�side (a) in Fig. 23) exhibited pro-motion of formation of floral buds compared with the plantthat was not treated with GSSG (right-�side (b) in Fig. 23).�[0141] Further, as shown in Fig. 24, fruits of the planttreated with GSSG (upper- �left panel (a) in Fig. 24) werelarger than pistils of the plant that was not treated withGSSG (upper- �right panel (b) in Fig. 24) and had morenumber of edible seeds than the plant that was not treatedwith GSSG.�[0142] Further, seeds of the plant treated with GSSG(lower-�left panel (c) in Fig. 24) were larger than seeds ofthe plant that was not treated with GSSG (lower- �rightpanel (d) in Fig. 24).�[0143] This clearly shows that treating corn with GSSGincreases the crop yields of the corn.�[0144] Subsequently, the amounts of biomass of fruits,ground parts, and portions other than the fruits of theplant treated with GSSG and the plant that was not treat-ed with GSSG were measured. As shown in (a) of Fig.25, the result of the measurement showed that there wasno great difference in the amount of biomass of the por-

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tions other than the fruits between the plant treated withGSSG and the plant that was not treated with GSSG,whereas the amounts of biomass of the fruits and groundparts of the plant treated with GSSG were significantlyhigh.�[0145] In view of the above, harvest index was calcu-lated. The result of the calculation showed that as shownin (b) of Fig. 25, the plant treated with GSSG exhibitedsignificantly high harvest index than the plant that wasnot treated with GSSG.


�[0146] Effect of the time of the treatment with oxidizedglutathione on productivity of corn was examined.�[0147] Sweet corn (Canberra 90, TAKII & CO., LTD.)was sown, and then transferred to a hydroponics pot(1/2000 are) filled with a culture soil (6L of vermiculite asthe lower layer, 3L of The Kureha Ikubyou Baido soil asthe middle layer, and 3L of vermiculite as the upper layer)two weeks later and additionally fertilized with 3g of KU-MIAI RIN RYUANKARI S604 four weeks later and sixweeks later.�[0148] Further, the GSSG- �treated plant was given50mL of 0.2mM-�GSSG solution four times in 2 weeks(two times a week) after 2 weeks, 4 weeks, or 6 weekshad elapsed from the sowing. Further, there were pre-pared a management zone to which 0.2mM-�GSSG wasgiven 22 times in total for 11 weeks after 2 weeks hadelapsed from the sowing and a management zone towhich 0.2mM-�GSSG was not given. As for the time ofthe treatment with GSSG, see (c) of Fig. 26. The timeindicated by the hatched part in Fig. (c) of 26 was thetime of the treatment with GSSG.�[0149] The amount of fruits and the amount of biomassof ground parts of each plant were measured and harvestindex was calculated.�[0150] Consequently, as shown in (a) of Fig. 26, theplant treated with GSSG for two weeks after four weekshad elapsed from the sowing and for two weeks after sixweeks had elapsed from the sowing and the plant treatedwith GSSG for eleven weeks after two weeks had elapsedfrom the sowing exhibited larger harvest index than theplant that was not treated with GSSG. In particular, theplant treated with GSSG for two weeks after four weekshad elapsed from the sowing and the plant treated withGSSG for eleven weeks after two weeks had elapsedfrom the sowing exhibited a great increase in harvestindex.�[0151] As shown in (b) of Fig. 26, the plant treated withGSSG for two weeks after four weeks had elapsed fromthe sowing and the plant treated with GSSG for elevenweeks after two weeks had elapsed from the sowing hada larger amount of fruits and a larger amount of biomassof ground parts per each plant than the plant that wasnot treated with GSSG. However, the plant treated withGSSG for two weeks after six weeks had elapsed from

the sowing had a smaller amount of fruits and a smalleramount of biomass of ground parts per each plant thanthe plant that was not treated with GSSG.

<16. Effect 3 of oxidized glutathione on productivity of corn>

�[0152] Sweet corn was cultivated under the same con-ditions as those of < 15. Effect of oxidized glutathione onproductivity of corn 2> except that GSSG was sprayedto a stem and leaves four times in two weeks after sixweeks had elapsed from the sowing, the zone wheresweet corn was cultivated was considered as a GSSG-treated zone, and GSSG was foliar-�sprayed. See the timeof treatment with GSSG in (c) of Fig. 27. The time indi-cated by the hatched part in (c) of Fig. 27 is the time oftreatment with GSSG.�[0153] The amount of fruits and the amount of biomassof ground parts of the plant thus obtained were measuredwith respect to each plant and harvest index was calcu-lated.�[0154] Consequently, as shown in (a) and (b) of Fig.27, even in a case of spraying GSSG to a stem andleaves, the treatment with GSSG increased the amountof fruits, the amount of biomass of ground parts, and theharvest index of each plant.

<17. Effect 4 of oxidized glutathione on productivity of corn>

�[0155] Sweet corn was cultivated under the same con-ditions as those of <15. Effect of oxidized glutathione onproductivity of corn 2> except that GSSG was suppliedto roots in the form of a solution or sprayed to a stem andleaves for two weeks after two weeks had elapsed fromthe sowing, or for eleven weeks after two weeks hadelapsed from the sowing, or for two weeks after fourweeks had elapsed from the sowing, or for two weeksafter six weeks had elapsed from the sowing, and 20mLof 0.2mM-�GSSG was supplied each time. See the timeof treatment with GSSG in (b) of Fig. 28. The time indi-cated by the hatched part in (b) of Fig. 28 is the time oftreatment with GSSG.�[0156] Harvest index of the plant thus obtained wasmeasured. Consequently, as shown in (a) of Fig. 28, har-vest index significantly increased under any of the aboveconditions compared with the case of the plant that wasnot treated with GSSG.


�[0157] Sweet corn was cultivated in an agricultural fieldin such a manner that the sweet corn was positioned asshown in Fig. 29 (density: approximately 12000 individ-uals/ 10a).�[0158] There were prepared plants treated with GSSGas follows: plants whose roots were sprayed with GSSG

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(20mL of 0.5mM-�GSSG per each time) two times a weekfor two weeks after two weeks had elapsed from the sow-ing, for two weeks after three weeks had elapsed fromthe sowing, for two weeks after four weeks had elapsedfrom the sowing, for two weeks after five weeks hadelapsed from the sowing, for two weeks after six weekshad elapsed from the sowing, and for seven weeks aftertwo weeks had elapsed from the sowing; plants that werenot treated with GSSG; and plants whose stem andleaves were sprayed with GSSG (20mL of 0.5mM- �GSSGper each time with respect to each plant) two times aweek for seven weeks after two weeks had elapsed fromthe sowing. See the time of treatment with GSSG in (b)of Fig. 30. The time indicated by the hatched part in (b)of Fig. 30 is the time of the treatment with GSSG.�[0159] The whole amount of biomass per area and theyield of pistils (fruits) per area of plants having growninside a colony out of the plants thus obtained were meas-ured and harvest index was calculated.�[0160] Consequently, as shown in (a), (c), and (d) ofFig. 30, any of the conditions for treatment with GSSGdid not exhibit a significant difference in the whole amountof biomass per area from the case of the plants that werenot treated with GSSG, but exhibited higher yield of pistils(fruits) and higher harvest index.�[0161] Further, the amounts of biomass of fruits,ground parts, and portions other than the fruits of eachof the plants were measured. As shown in Fig. 31, theresults of the measurement showed that any of the con-ditions for treatment with GSSG exhibited higher amountof biomass of fruits (i.e. amount of fruits) than the caseof the plants that were not treated with GSSG.�[0162] However, any of the conditions for treatmentwith GSSG exhibited a lower amount of biomass of theportions other than fruits than the case of the plants thatwere not treated with GSSG. Further, any of the condi-tions for treatment with GSSG exhibited substantially thesame or lower amount of biomass of ground parts thanthe case of the plants that were not treated with GSSG.In particular, the plant whose stem and leaves weresprayed with GSSG for seven weeks after two weekshad elapsed from the sowing exhibited greatly loweramounts of biomass of the portions other than fruits andground parts than the case of the plants that were nottreated with GSSG.�[0163] The above results show that oxidized glutath-ione increases the amount of fruits that were harvests ofcorn and thus increases harvest index, without increasingthe amount of biomass of ground parts.

< 19. Effect of oxidized glutathione on productivity of corn under nitrogen-�deficient condition>

�[0164] Sweet corn was sown and cultivated for 84 days(7 weeks) after the sowing without supplying a nitrogensource to the sweet corn. Thereafter, the sweet corn wascultivated with 30kgN/ �10a of a nitrogen source. Treat-ment with GSSG was performed in such a manner that

GSSG solutions with 0mM-, 0.2mM-, 0.5mM, and1.0mM-�concentrations were supplied in the amount of50mL per one time, two times a week, 8 weeks after thesowing (i.e. after the supply of the nitrogen source be-gan). See the cultivation conditions in (c) of Fig. 33.�[0165] As shown in (a) and (b) of Fig. 32, the plantsthus obtained exhibited a larger amount of fruits than theplants that were not treated with GSSG in (c) of Fig. 32.�[0166] In order to examine the amount of productionof the plants in more detail, the amounts of biomass offruits, ground parts, and portions other than the fruitswere measured, and harvest index was calculated.�[0167] As shown in (a) and (b) of Fig. 33, the resultsof the measurement and the calculation showed that theplants treated with GSSG exhibited larger amounts ofbiomass of fruits, ground parts, and portions other thanthe fruits and harvest index than the plants that were nottreated with GSSG.�[0168] The above results clearly shows that whengrowth of a plant is restricted due to deficiency in nitrogenin vegetative development, the yield of fruits normallydrops, but the treatment with GSSG allows preventingthe yield from dropping even after the growth of the plantwas restricted due to the deficiency in nitrogen.

<20. Effect 1 of oxidized glutathione on promoting growth of sprout and floral bud of rose>

�[0169] Rose saplings (breed; Purple rose) were pur-chased at a hardware store and cultivated in pods forrice hydroponics. The rose saplings were fertilized with50mL of a 0.5mM-�GSSG solution in the form of a liquidfertilizer two times a week. In addition to GSSG, the rosesaplings were additionally fertilized with 2g of S604 everytwo weeks.�[0170] Consequently, the plants treated with GSSG(plants surrounded by full circles of Fig. 34) exhibitedpromoted growth of sprout at one month from the fertili-zation compared with the plants that were not treatedwith GSSG (plants surrounded by broken circles of Fig.34).�[0171] The above result clearly shows that the treat-ment with GSSG promotes growth of sprouts and floralbuds of roses (breed; Purple rose).

<21. Effect 2 of oxidized glutathione on promoting growth of sprout and floral bud of rose>

�[0172] Rose saplings (breed; JJ scarlet and JJ apricot)were purchased at a hardware store and cultivated inpods for rice hydroponics. The rose saplings were ferti-lized with 50mL of a 0.5mM-�GSSG solution in the formof a liquid fertilizer two times a week. In addition to GSSG,the rose saplings were additionally fertilized with 2g ofS604 every two weeks.�[0173] Consequently, the plants treated with GSSG((a) and (c) of Fig. 35) exhibited promoted growth ofsprout at fourth and eighth day from the fertilization com-

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pared with the plants that were not treated with GSSG((b) and (d) of Fig. 35).�[0174] In (a)-(d) of Fig. 35, the breeds of three plantsare JJ scarlet, JJ apricot, and JJ scarlet, respectivelyfrom the left.

<22. Effect of oxidized glutathione on promoting growth of roots of eustoma>

�[0175] Seeds of eustoma were germinated in an MSculture medium containing 1mM-�GSSG or GSH, weregrown for 1 month, and transferred to a planter. In theplanter, 2 parts of vermiculite, 1 part of The Kureha Iku-byou Baido soil, and 1 part of vermiculite were layeredas a lower layer, a middle layer, and an upper layer, re-spectively, and the above plants were transferred there-to.�[0176] Consequently, as shown in Fig. 36, the plantstreated with GSSG exhibited significant growth of rootscompared with the plants that were not treated withGSSG. On the other hand, the plants treated with GSHexhibited subdued growth of roots compared with theplants that were not treated with GSSG.

<23. Effect 1 of oxidized glutathione on promotion of in-duction of floral bud of rose>

�[0177] Roses (breed; Patiohit alicante) were pur-chased at a hardware store and fertilized with 50mL of a0.5mM-�GSSG solution in the form of a liquid fertilizer twotimes a week. Approximately three and a half monthslater, plants in a GSSG-�treated zone and plants in a non-GSSG- �treated zone were compared with each other.�[0178] Consequently, the plants treated with GSSG((a) of Fig. 37 and (a) of Fig. 38) exhibited greatly largernumber of bloomed flowers than the plants that were nottreated with GSSG ((b) of Fig. 37 and (b) of Fig. 38).�[0179] The above results clearly show that oxidizedglutathione promotes induction of floral buds of roses.

<24. Effect of oxidized glutathione on growth of straw-berry>

�[0180] A sapling of strawberry (breed; Eminent gardenseries, � Yokubari-�ichigo kurenai (SUMIKA)) was pur-chased at a hardware store and cultivated. The straw-berry was fertilized with 50mL of a 0.5mM-�GSSG solutionin the form of a liquid fertilizer two times a week. In ad-dition to GSSG, The strawberry was additionally fertilizedwith 2g of S604 every two weeks.�[0181] Consequently, as shown in Fig. 39, four monthsafter the fertilization, a Cont zone (non- �GSSG- �treatedzone) exhibited two or three runners per one stock,whereas a GSSG- �treated zone exhibited a greatly largenumber of runners, i.e., several ten runners per onestock.�[0182] On the other hand, although a GSH-�treatedzone exhibited more number of runners per one stock

than a Cont zone, the increase in the number of runnersof the GSH-�treated zone was approximately one third ofthe increase in the number of runners of the GSSG-�treat-ed zone.�[0183] The above results show that oxidized glutath-ione promotes growth of strawberry and increases thenumber of runners, promoting proliferation of stocks.

<25. Influence 1 of oxidized glutathione on growth of transformant Arabidopsis to which a gene encoding gF-BA is introduced>

�[0184] Initially, transformants of Arabidopsis to whicha gone encoding glutathione-�binding plastid- �type fruc-tose- �1,6-�bisphosphate aldolase (which may be hereinaf-ter referred to as "gFBA") was introduced and wild-�typeArabidopsis (Columbia; Col) were cultivated under thesame conditions as those in <1. Influence of oxidizedglutathione on growth of Arabidopsis> except that thetransformants of Arabidopsis and the wild-�type Arabidop-sis were fertilized with a liquid of 0mM-, 3mM, 9mM-, or18mM-�ammonium nitrate in the amount of 25mL perthree individuals one time a week.�[0185] The fertilization was performed in such a man-ner that a pot was put in a scale and an ammonium nitratesolution was poured in the scale. As for conditions forcultivating a plant, see Fig. 40.�[0186] The whole amount of biomass and the seedweight of the plants thus obtained were measured andharvest index was calculated. Consequently, as shownin Fig. 41, in any of the plants, the whole amount of bio-mass and the seed weight increased as concentration ofammonium nitrate used as a fertilizer increased, but theincrease slowed down at 9mM, and the effect was satu-rated at 18mM.�[0187] On the other hand, in any of the plants, when3mM- and 9mM-�ammonium nitrate were used as fertiliz-ers, the harvest index increased. However, when 18mM-ammonium nitrate was used as a fertilizer, the harvestindex dropped lower than the harvest index in a case ofusing no fertilizer. Such drop in harvest index was gen-erally seen when nitrogen was excessively used as afertilizer. In this case, as a fertilization amount N getsfurther higher, the drop in harvest is observed. In a caseof crops, in order to avoid reduction of crop yields due toexcessive fertilization, agricultural experimental stationsand nursery companies disclose information on a stand-ard fertilization amount N most suitable for crop yields.�[0188] Under fertilization conditions (18mM-�ammoni-um nitrate) where the effect of increasing crop yields wassaturated with respect to the fertilization amount N, theeffect of supplying GSSG was compared with the caseof supplying no GSSG. The transformants of Arabidopsisto which a gene encoding gFBA was introduced and thewild- �type Arabidopsis (Columbia; Col) were fertilized withonly 1mM-�GSSG, or only 18mM-�ammonium nitrate, orboth 18mM-�ammonium nitrate and 1mM-�GSSG in theform of a liquid fertilizer in the same manner as fertiliza-

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tion with ammonium nitrate in Fig. 41, and cultivated.�[0189] The whole amount of biomass and the seedweight of the plants thus obtained were measured andharvest index was calculated. Consequently, as shownin Fig. 42, fertilization with only oxidized glutathione in-creased any of the harvest index, the whole amount ofbiomass, and the seed weight, and the effect of oxidizedglutathione was observed under conditions where theeffect of ammonium nitrate on growth of the seed weightwas saturated.�[0190] Combination of oxidized glutathione and am-monium nitrate resulted in a greater effect in the trans-formants to which the gene encoding gFBA was intro-duced than in the wild- �type.�[0191] The above results show that supplying combi-nation of oxidized glutathione and ammonium nitrate tothe transformants to which the gene encoding gFBA wasintroduced allows further enhancing the effect of GSSGon increasing the harvest index, the whole amount ofbiomass, and the seed weight.

<26. Influence 2 of oxidized glutathione and reduced glu-tathione on growth of transformant Arabidopsis to which a gene encoding gFBA is introduced>

�[0192] Under the conditions in Fig. 42, instead of 1mM-GSSG, GSH or ammonium sulfate was supplied in thesame amount in terms of the N amount, and the effectof GSH or ammonium sulfate was compared with theeffect of GSSG. Transformants of Arabidopsis to whicha gene encoding gFBA was introduced and wild-�type Ara-bidopsis (Columbia; Col) were cultivated in the samemanner as in Figs. 41 and 42 except for a condition con-cerning a liquid fertilizer. As the liquid fertilizer, 18mM-ammonium nitrate, 18mM-�ammonium nitrate + 1mM-GSSG, 18mM-�ammonium nitrate + 2mM-�GSH, or 18mM-ammonium nitrate + 2mM-�ammonium sulfate was used.�[0193] The fertilization was performed in such a man-ner that a pot was put in a scale and the above liquidfertilizer was poured in the scale. As for a condition forcultivating a plant, see Fig. 40.�[0194] The whole amount of biomass and the seedweight of the plants thus obtained were measured andharvest index was calculated. Consequently, as shownin Fig. 43, supplying combination of oxidized glutathioneand ammonium nitrate to the transformants to which thegFBA gene was introduced allows greatly increasing theharvest index, the whole amount of biomass, and theseed weight, compared with a case of treating the trans-formants in other manner.�[0195] As for the harvest index, although combinationof reduced glutathione and ammonium nitrate resultedin an effect that was not so great as the effect of combi-nation of oxidized glutathione and ammonium nitrate, thecombination of reduced glutathione and ammonium ni-trate greatly increased the harvest index compared witha case of using no fertilizer or a case of using only am-monium nitrate as a fertilizer.

�[0196] The above results showed that both of oxidizedglutathione and reduced glutathione allow greatly in-creasing the harvest index and significantly increasingcrop yields when combined with ammonium nitrate. Fur-ther, the above results showed that the effect increasedwhen the combination was supplied to plants to whichthe gFBA gene was introduced. Further, the above re-sults showed that oxidized glutathione had a higher abilityto increase the harvest index and to significantly increasecrop yields than reduced glutathione.

<27. Influence 3 of oxidized glutathione on growth of transformant of Arabidopsis to which gFBA is intro-duced>

�[0197] Under the conditions similar to those in Figs.41-43 except that only GSSG was supplied as a liquidfertilizer, transformants of Arabidopsis to which the gFBAgene was introduced and wild-�type Arabidopsis (Colum-bia; Col) were cultivated. Concentration of GSSG sup-plied as the liquid fertilizer varied from 0mM to 5mM.�[0198] The whole amount of biomass and the seedweight of the plants thus obtained were measured andharvest index was calculated. The result of the seedweight was shown in Fig. 44 and the result of the harvestindex was shown in Fig. 45. The seed weight increasedas concentration of GSSG increased, and the effect wasnot saturated at the tested concentration. This tendencywas more clearly observed in the transformants of Ara-bidopsis to which the gFBA gene was introduced.�[0199] On the other hand, the harvest index increasedas concentration of GSSG increased, and the effect wassaturated when the concentration of GSSG was approx-imately 2mM. The effect of increasing the seed weightand the effect of increasing the harvest index that wereyielded by the treatment with GSSG were greatly largerthan the maximum effects yielded by fertilization with ni-trogen resulting from ammonium nitrate.�[0200] The above results showed that the effect of in-creasing the seed weight and the effect of increasing theharvest index that were yielded by GSSG are greaterthan the effects yielded by ammonium nitrate and en-hancing the gFBA gene allows increasing the effects.

<28. Comparisons of influences of oxidized glutathione, reduced glutathione, and ammonium sulfate as sulfur sources on growth>

�[0201] Under the conditions similar to those in Figs.41-45 except that any of GSSG, GSH, and ammoniumsulfate was supplied as a liquid fertilizer, the effects ofthe liquid fertilizers on growth of transformants to whichthe gFBA gene was introduced were compared. Theamounts of respective liquid fertilizers were set so thatconcentrations of the liquid fertilizers were the same interms of an S amount. The seed weight and the wholeamount of biomass of the plants thus obtained weremeasured and harvest index was calculated. The result

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of the seed weight was shown in Fig. 46 and the resultof the harvest index was shown in Fig. 47.�[0202] The effect of ammonium sulfate generally usedas a nitrogen fertilizer on increasing the seed weight wassaturated at a value similar to that of the effect of ammo-nium nitrate. On the other hand, the effects of GSSG andGSH were not saturated at tested concentrations, indi-cating that GSSG and GSH yield higher effects of in-creasing the seed weight than ammonium sulfate. Onthe other hand, in any of the liquid fertilizers, the effectof increasing the harvest index reached its maximumwhen the N amount was 12mM. Among the three liquidfertilizers, GSSG yielded the highest effect.�[0203] The above results showed that the effect of glu-tathione is higher than that of a conventional fertilizer andGSSG yields a higher effect than GSH.�[0204] The embodiments and concrete examples ofimplementation discussed in the foregoing detailed ex-planation serve solely to illustrate the technical details ofthe present invention, which should not be narrowly in-terpreted within the limits of such embodiments and con-crete examples, but rather may be applied in many var-iations within the spirit of the present invention, providedsuch variations do not exceed the scope of the patentclaims set forth below.

Industrial Applicability

�[0205] The present invention allows increasing harvestindex of a plant. Further, the present invention allowsincreasing the number of seeds or flowers of the plant.Further, the present invention allows increasing lateralshoots and tillers and increasing the yield of seeds.Therefore, the present invention allows increasing thenumber of flowers and yields not only in ornamental flow-ering grasses and plants to be eaten but also in forestsand plant resources for biomass energy. Therefore, thepresent invention has wide industrial applicability not onlyin agriculture but also food industries and energy indus-tries.

Claims

1. A plant growth regulator for increasing harvest index,comprising glutathione.

2. The plant growth regulator as set forth in claim 1,wherein the glutathione is oxidized glutathione.

3. The plant growth regulator as set forth in claim 1 or2, for increasing the number of seeds and/or flowersof a plant.

4. The plant growth regulator as set forth in claim 1 or2, for increasing the number of lateral shoots and/ortillers of a plant.

5. A method for cultivating a plant, comprising the stepof cultivating a plant by use of glutathione so as toincrease harvest index of the plant.

6. The method for cultivating a plant as set forth in claim5, wherein the glutathione is oxidized glutathione.

7. The method for cultivating a plant as set forth in claim5 or 6, wherein the glutathione is supplied intermit-tently.

8. The method for cultivating a plant as set forth in anyone of claims 5-7, wherein the glutathione is suppliedat around a time of transition from vegetative to re-productive development.

9. A method for increasing the number of seeds and/orflowers of a plant by use of glutathione.

10. The method as set forth in claim 9, wherein the glu-tathione is oxidized glutathione.

11. A method for increasing the number of lateral shootsand/or tillers of a plant by use of glutathione.

12. The method as set forth in claim 11, wherein theglutathione is oxidized glutathione.

13. The method as set forth in claim 11 or 12, whereinthe plant has mutation in a function for synthesizinga plant hormone and/or a function for responding toa plant hormone.

14. The method as set forth in claim 13, wherein theplant hormone is gibberellin.

15. A plant obtained by a method as set forth in any oneof claims 5-14, having increased harvest index.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the Europeanpatent document. Even though great care has been taken in compiling the references, errors or omissions cannot beexcluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• WO 01080638 A [0007]• JP 2004352679 A [0007]

• WO 2007091634 A1 [0058]

(19) - JST

Documents

Transcript of (19) - JST