Method for promoting flowering of plants deficient in flowering regulatory genes, method for advancing timing of seed formation, and plant

Abstract

To provide a method for promoting the flowering of plants deficient in flowering regulatory genes, a method for advancing the timing of seed formation, and a plant.SOLUTION: A method for promoting the flowering of plants deficient in flowering regulatory genes includes performing grafting using a plant that overexpresses a flowering regulatory gene as the rootstock and using a plant deficient in the flowering regulatory gene as the scion. Also provided are a method for advancing the timing of seed formation, and a plant.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a method for promoting flowering in a plant lacking a flowering control gene, a method for accelerating seed formation, and the plant. [Background technology]

[0002] In crop cultivation, the flowering time is closely related to the amount of growth and is the main factor that determines the harvest time. From the viewpoint of ensuring stable production volume and optimizing the harvest time, appropriate control of the flowering time is an important issue.

[0003] To date, genes involved in flower bud formation have been searched for and identified for the purpose of genetic engineering control of flowering time, and one example of such an identified gene is the FT (Flowering Locus T) gene (Non-Patent Document 1). When a plant senses that the day length conditions are suitable for flowering, the expression of the FT gene is induced or enhanced in the leaves, and the FT protein is synthesized in the vascular phloem. Flower bud formation is induced by the action of the FT protein transported to the shoot apex through the sieve tube. In the model plant Arabidopsis thaliana, in a strain that overexpresses the FT gene, the period required for flowering is significantly shorter than that of the wild type, regardless of whether the day is long or short. On the other hand, it has been reported that in a strain in which the function of the FT gene is defective, flowering is delayed even under suitable day length conditions.

[0004] Based on the above, artificially deleting the function of flowering control genes in crops can delay flower bud formation and increase growth by prolonging vegetative growth, which can then lead to increased crop yields.

[0005] In order to control flower bud formation in plants, it is believed that temperature control, in addition to day length control by light irradiation or shading, can be effective (Non-Patent Documents 2 and 3).

[0006] Grafting techniques for joining different plants together are known. Grafting is a technique for joining a part of a different plant (scion) to a base plant (rootstock) (Non-Patent Document 4). Grafting makes it possible to obtain a plant that is a chimeric individual in which the vascular bundles of the rootstock and scion are connected. Grafting is performed for the purpose of, for example, avoiding soil-borne diseases in the scion, imparting low-temperature elongation properties, adapting to long-term nutrient solution cultivation, and clonal propagation. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 309, 1052-1056 (2005) [Non-Patent Document 2] Guide to selecting and installing light sources for chrysanthemum lighting, March 2014, Ministry of Agriculture, Forestry and Fisheries commissioned project "Revolutionary low-cost realization project for domestic agricultural products" "Light Flower Consortium" (https: / / www.naro.go.jp / publicity_report / publication / files / light_source_guidance_201403.pdf) [Non-Patent Document 3] Early spring harvest of turnip "Haruno Kagayaki" by vernalization treatment, Tohoku Agricultural Research Center, FY1996 research results information (https: / / www.naro.affrc.go.jp / org / tarc / seika / jyouhou / H08 / tnaes96085.html) [Non-Patent Document 4] Current status and issues of grafted vegetable cultivation, Vegetable and Tea Industry Research Institute Research Material No. 7, February 2011 (https: / / www.naro.go.jp / publicity_report / publication / archive / files / naro-se / yacha_siryou7.pdf) Summary of the Invention [Problem to be solved by the invention]

[0008] In plants lacking flowering control genes, even when day length and temperature conditions are appropriately controlled, flower bud formation is inhibited or significantly delayed, and in some cases seeds may not form, resulting in a long period of time required for seed production. This is problematic from these perspectives, as it significantly reduces the efficiency of genotype fixation and seed multiplication, and leads to delays in the development of new varieties of the plants produced. The present disclosure has been made to solve the above-mentioned problems, and aims to provide a method for promoting flowering in a plant lacking a flowering control gene, a method for accelerating seed formation, and a plant body. [Means for solving the problem]

[0009] One aspect of the present disclosure is a method for promoting flowering of a plant lacking a flowering control gene, the method comprising grafting a plant that overexpresses the flowering control gene as a rootstock and a plant lacking the flowering control gene as a scion.

[0010] One aspect of the present disclosure is a method for accelerating seed formation in a plant lacking a flowering control gene, the method comprising grafting a plant that overexpresses the flowering control gene as a rootstock and a plant lacking the flowering control gene as a scion.

[0011] One aspect of the present disclosure is a grafted plant body comprising a plant that overexpresses a flowering control gene as a rootstock and a plant lacking the flowering control gene as a scion. Effect of the Invention

[0012] According to the present disclosure, it is possible to provide a method for promoting flowering in a plant lacking a flowering control gene, a method for accelerating seed formation, and a plant body. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a diagram showing the grafting procedure in a method for promoting flowering and accelerating seed formation in a plant lacking a flowering control gene according to an embodiment. [Diagram 2] FIG. 2 is a diagram showing flowering and seed formation in a wild-type strain, an FT-overexpressing strain, a grafted strain of an embodiment, and an FT-deficient strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, non-limiting embodiments of the present disclosure will be described. The present disclosure is not limited to the examples in the following embodiments.

[0015] <How to promote flowering>

[0016] In one embodiment, a method for promoting flowering of a plant lacking a flowering control gene is provided, which comprises grafting a plant that overexpresses the flowering control gene as a rootstock and a plant lacking the flowering control gene as a scion.

[0017] In an embodiment, the flowering control gene may be a gene that controls the flowering of a plant, and more specifically, may be a gene that encodes a flowering control protein. The flowering control gene of the embodiment may be a protein that controls flowering non-cell autonomously, in particular, a flowering control protein synthesized in leaves. Furthermore, the flowering control gene of the embodiment may be a gene that controls flowering non-cell autonomously or controls the expression of a gene that encodes a flowering control protein synthesized in leaves. The control of flowering by the flowering control gene is usually not flowering-repressive control but flowering-promoting control.

[0018] Examples of flowering control genes include, but are not limited to, the FLOWERING LOCUS T (FT), CONSTANTS (CO), FLOWERING LOCUS D (FD), APETALA1 (AP1), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) genes. In this disclosure, these are described by conventional gene or protein names in the model plant Arabidopsis thaliana, but it will be understood by those of skill in the art that, for example, structurally and functionally corresponding genes or proteins in other plants may have different names. Those of skill in the art can recognize genes or proteins that have different names but are structurally and functionally corresponding to known genes or proteins in Arabidopsis. For example, the FT gene is called Hd3a in rice. Therefore, the use of a specific gene or protein name identifies a specific gene or protein known to those skilled in the art, but does not mean to limit the plant to a specific species (e.g., Arabidopsis thaliana), or to limit the gene or protein to those derived from Arabidopsis thaliana.The flowering control gene of the embodiment is preferably the FT gene.The term FT gene encompasses the corresponding gene (e.g., ortholog) in other plants of the FT gene identified in Arabidopsis thaliana.

[0019] In the embodiment, the type of plant is not particularly limited as long as the feasibility of grafting is known to those skilled in the art, and may include terrestrial plants, aquatic plants, epiphytes, and rock plants. The plant of the embodiment may be, for example, any of Malvaceae, Chenopodiaceae, Rubiaceae, Brassicaceae, Poaceae, Cucurbitaceae, Asteraceae, Mulberry Family, Pesamiaceae, Araceae, Solanaceae, Papaya Family, Rosaceae, Fabaceae, Rutaceae, Oleaceae, and Liliaceae, but is not limited thereto. It is also known that grafting is possible between different species, and those skilled in the art can appropriately select a combination of rootstock and grafting based on their general knowledge.

[0020] A plant lacking a flowering control gene of the embodiment is a plant in which the functional expression of an endogenous flowering control gene is reduced or eliminated to such an extent that the flowering phenotype of the plant is altered. A plant lacking a flowering control gene may be, for example, a plant in which the flowering control gene is deleted in the genome, a plant having a mutation that prevents its normal transcription, or a plant having a mutation that prevents the translation of the full-length normal protein that it encodes. Thus, the term "lacking a flowering control gene" encompasses not only the absence of the gene, but also the presence of the gene but the lack of expression of its function. A plant lacking a flowering control gene may exhibit reduced or eliminated expression at the transcription level, translation level, or both.

[0021] The method by which the plant lacking the flowering control gene of the embodiment is produced is not particularly limited. Specifically, for example, the flowering control gene may be deleted by any of genome editing techniques known to those skilled in the art, including homologous recombination, mutagenesis, or the CRISPR / CAS9 method, or the expression of the flowering control gene may be post-transcriptionally suppressed by expressing or introducing siRNA, dsRNA, or the like into the plant. In addition, the gene may be destroyed by inserting a foreign nucleic acid sequence into the flowering control gene on the genome using transposon or knock-in technology.

[0022] In the embodiment, the grafting is performed using a plant that overexpresses a flowering control gene as a rootstock. As will be understood by those skilled in the art, the rootstock refers to the lower part (i.e., the part containing the roots) of two plant individuals that are to be grafted together at the joining surface by the grafting.

[0023] The plant that overexpresses the flowering control gene used as a rootstock in the grafting of the embodiment may be a plant that overexpresses an endogenous flowering control gene, or a recombinant plant that overexpresses an exogenous flowering control gene. The exogenous gene may be artificially introduced into the plant individual, for example, by transformation. It may also be a recombinant plant in which overexpression is achieved by introducing an additional copy of the endogenous flowering control gene.

[0024] In the present disclosure, transformation includes introducing a gene from outside to increase the expression of the gene compared to a corresponding non-transformed individual. Transformation can be performed by various techniques known to those skilled in the art, including various genome editing techniques such as the Agrobacterium method, the gene gun (particle gun) method, the electroporation method, the PEG method, homologous recombination, and the CRISPR / CAS9 method.

[0025] In the rootstock of the embodiment, the flowering control gene may be a gene operably linked to a functional promoter. The functional promoter is not particularly limited as long as it can induce the expression of the flowering control gene in a plant individual, and a suitable example includes the 35S promoter of cauliflower mosaic virus (CaMV) and its variants. The flowering control gene linked to a functional promoter may be included in any vector suitable for transformation. The flowering control gene may also include a non-translated region (UTR) in addition to the coding region. Overexpression refers to increased expression in a plant that has been modified or mutated so that the expression of the flowering control gene is significantly increased compared to a corresponding wild-type plant. For example, a plant that overexpresses a flowering control gene may have 1.5 times or more, 2 times or more, 5 times or more, or 10 times or more of the flowering control gene product (i.e., flowering control protein) per dry weight of the leaf compared to a corresponding wild-type plant under the same conditions.

[0026] In the embodiment, the grafting is performed using a plant lacking a flowering control gene as a scion. As will be understood by those skilled in the art, the scion here refers to the upper part (i.e., the part not including the roots) of two plant individuals to be grafted together at the joining surface by grafting.

[0027] The plant lacking a flowering control gene that is used as a scion in the grafting embodiment is a plant lacking the flowering control gene for which it is desired to promote flowering (or to advance the seed formation period, as described below).

[0028] The grafting of the embodiment may be carried out, for example, by the procedure described below with reference to FIG.

[0029] For any plant, a flowering control gene function-deficient strain 1 (e.g., an FT-deficient strain) and a flowering control gene overexpression strain 2 (e.g., an FT overexpression strain) are prepared by genetic manipulation such as genome editing technology, mutagen treatment, or genetic recombination.

[0030] For both plants, plants at the growth stage where multiple leaves are developed are selected. Grafting techniques are known to those skilled in the art, and it is within the ordinary skill of those skilled in the art to select specific individuals suitable for grafting. For example, it is known to those skilled in the art that it is preferable to select individuals whose stems are approximately the same thickness.

[0031] For the flowering control gene overexpression strain 2, a part of the plant body including the growing point of the shoot apex (the part not including the roots) is cut off, for example, by cutting with a sharp blade such as a razor, and the remaining plant body (the part including the roots) is used as rootstock 2'.

[0032] For the flowering control gene function-deficient strain 1, a part of the plant body including the growing point of the shoot apex (the part not including the roots) is cut off, for example by cutting with a sharp blade such as a razor, and this cut part is used as scion 1'.

[0033] The cut surfaces of the rootstock and scion are glued together, and the glued state is maintained using a grafting clip 3 or the like. Grafting clips, grafting tapes, and other tools for the same purpose are known to those skilled in the art, and the specific type of tool is not limited as long as it can maintain the glued state between the rootstock 2' and the scion 1' without substantially damaging the plant body.

[0034] Until the junction develops into callus and the rootstock 2' and scion 1' are established, it is preferable to cover the chimeric plant body 4 with a bag 5 or the like having excellent moisture retention properties, and maintain a high humidity atmosphere.

[0035] After the rootstock 2' and scion 1' have taken root, the clip 3 is removed, and the plant body 4 can be cultivated under photoperiod and temperature conditions suitable for the flowering of the target plant.

[0036] The method for promoting flowering according to the embodiment can promote flowering in a strain lacking the function of a flowering control gene, which would not flower or flower late in the absence of grafting (Figure 2). The term "promoting flowering" can include not only accelerating flowering, but also causing a plant that would not normally flower within a given period to flower within that given period. In the grafted plant obtained by the above-mentioned method, a large amount of the flowering control protein biosynthesized in the leaves of the rootstock can be transported to the shoot apex of the scion through the joined phloem tube. As a result, even in a plant lacking the function of a flowering control gene, the process including flower bud formation to flowering can be completed in a shorter period of time than not only the non-grafted plant lacking the function, but also the wild-type strain (Figure 2).

[0037] <Method to accelerate seed formation>

[0038] In one embodiment, there is provided a method for accelerating seed formation in a plant lacking a flowering control gene, comprising grafting a plant that overexpresses the flowering control gene as a rootstock onto a plant lacking the flowering control gene as a scion.

[0039] Seed formation occurs after flowering, and promoting the flowering of a plant is directly related to accelerating the seed formation time. Therefore, it should be understood that the explanation provided for the embodiment of the method for promoting flowering can also be applied to the embodiment of the method for accelerating the seed formation time, and vice versa. The method for accelerating the seed formation time of a plant lacking a flowering control gene of the embodiment can include grafting using the same plant line, rootstock, scion and procedure as described in the section of the method for promoting flowering. The flowering control gene in the embodiment is preferably the FT gene.

[0040] The method of accelerating the seed formation time of a plant lacking a flowering control gene of the embodiment may further include harvesting seeds from the scion. The seed harvesting may be performed by a standard method known by a person skilled in the art. The time from the grafting step to the seed harvesting step may be shortened by, for example, at least one day, at least two days, at least one week, or at least one month compared to a corresponding non-grafted defective plant. Alternatively, the method of accelerating the seed formation time of a plant may include forming seeds or harvesting seeds within a predetermined period using a defective plant that was not able to form seeds or harvest seeds within the predetermined period.

[0041] The method of accelerating seed formation in an embodiment can promote seed formation in a strain lacking a flowering control gene that would not form seeds or would form seeds late in normal cases without grafting (Figure 2). In a grafted plant obtained by the above-mentioned technique, a large amount of the flowering control protein biosynthesized in the leaves of the rootstock can be transported to the shoot apex of the scion through the joined phloem tube. As a result, even in a plant lacking the function of a flowering control gene, the reproductive growth process, including flower bud formation, flowering, and seed formation, can be completed in a shorter time than not only a non-grafted plant lacking the function, but also a wild-type strain (Figure 2).

[0042] <Plant body>

[0043] In one aspect, a grafted plant is provided, comprising a plant that overexpresses a flowering control gene as a rootstock and a plant lacking the flowering control gene as a scion.

[0044] It should be understood that the plant of the present embodiment can be produced by the grafting procedure described in the above method embodiment. Therefore, the description provided in the above method embodiment can also be applied to the plant embodiment. The plant of the embodiment can be produced by grafting using the same plant line, rootstock, scion, and procedure as described in the sections <Method of promoting flowering> and <Method of accelerating seed formation>. The flowering control gene in the embodiment is preferably the FT gene.

[0045] Although the present disclosure has been described with reference to the above several embodiments, the present disclosure is not limited to the above several embodiments. Various modifications can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0046] <Additional Notes> (Appendix 1) A method for promoting flowering in a plant lacking a flowering control gene, comprising: Grafting a plant that overexpresses the flowering control gene as a rootstock and a plant lacking the flowering control gene as a scion. The method includes: (Appendix 2) The method described in Appendix 1, wherein the plant that overexpresses the flowering control gene is a recombinant plant into which the flowering control gene has been introduced. (Appendix 3) 3. The method of claim 1 or 2, wherein the flowering control gene is a FT gene. (Appendix 4) A method for accelerating seed formation in a plant lacking a flowering control gene, comprising: Grafting a plant that overexpresses the flowering control gene as a rootstock and a plant lacking the flowering control gene as a scion. The method includes: (Appendix 5) The method described in Appendix 4, wherein the plant that overexpresses the flowering control gene is a recombinant plant into which the flowering control gene has been introduced. (Appendix 6) 6. The method of claim 4 or 5, wherein the flowering control gene is a FT gene. (Appendix 7) The method according to any one of claims 4 to 6, further comprising harvesting seeds from the scion portion after the grafting. (Appendix 8) A grafted plant body comprising a plant which overexpresses a flowering control gene as a rootstock and a plant lacking said flowering control gene as a scion. [Explanation of symbols]

[0047] 1. Flowering control gene knockout strains 1' Scion 2. Flowering control gene overexpression strains 2' Rootstock 3 Clips 4 Grafted plants 5 bags

Claims

1. A method for promoting flowering in a plant lacking a flowering control gene, comprising: Grafting a plant that overexpresses the flowering control gene as a rootstock and a plant lacking the flowering control gene as a scion. The method includes:

2. The method according to claim 1 , wherein the plant that overexpresses the flowering control gene is a recombinant plant into which the flowering control gene has been introduced.

3. The method according to claim 1 or 2, wherein the flowering control gene is the FT gene.

4. A method for accelerating seed formation in a plant lacking a flowering control gene, comprising: Grafting a plant that overexpresses the flowering control gene as a rootstock and a plant lacking the flowering control gene as a scion. The method includes:

5. The method according to claim 4 , wherein the plant that overexpresses the flowering control gene is a recombinant plant into which the flowering control gene has been introduced.

6. The method according to claim 4 or 5, wherein the flowering control gene is the FT gene.

7. 6. The method of claim 4 or 5, further comprising harvesting seeds from said scion portion after said grafting.

8. A grafted plant body comprising a plant which overexpresses a flowering control gene as a rootstock and a plant lacking said flowering control gene as a scion.

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