Method for inducing callus of giant miscanthus and method for regenerating plant

Abstract

To provide a stable and efficient callus-inducing method and a method for regenerating a plant body of giant myricaceae. To provide a callus-inducing method and a plant body regenerating method from an easily available plant tissue piece harvestable throughout the year regardless of the season. To provide a callus-inducing method and a plant body-regenerating method which can be carried out inexpensively without using expensive reagents.SOLUTION: A callus induction method for inducing callus by culturing a rhizome of giant miscanthus in a callus induction medium, wherein the callus induction medium contains an auxin and a cytokinin.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for inducing callus from the rhizomes of Giant Miscanthus and a method for regenerating plants. [Background technology]

[0002] Giant miscanthus is a plant of the Miscanthus genus, a natural interspecific hybrid between Miscanthus sinensis and Miscanthus sieboldii. Giant miscanthus exhibits high photosynthetic capacity and high biomass productivity even in the low-temperature environments of cold regions. It also requires only a small amount of fertilizer, so its environmental impact during cultivation is extremely minimal. For these reasons, it is attracting attention as a source of biofuels for power generation and transportation, as well as biomaterials for feed, fertilizer, and building materials.

[0003] Giant Miscanthus is a completely sterile plant. Since completely sterile plants cannot produce seeds, callus production and regeneration are effective methods for propagation and breeding.

[0004] Traditionally, callus induction from immature inflorescences of Giant Miscanthus has been used (see Patent Documents 1 and 2, and Non-Patent Document 1). These documents report a method for inducing callus by culturing immature inflorescences as plant tissue explants in a medium containing 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. However, immature inflorescences can only be harvested in small quantities during a very short growing season. Therefore, callus cannot be produced throughout the year, and there is a quantitative limit, which is a rate-limiting factor in callus production techniques.

[0005] A method for inducing callus from the shoot apex, leaves, roots, and immature inflorescences of Giant Miscanthus has also been reported (see Non-Patent Document 2). This document reports a callus induction method using a callus induction medium consisting of Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid. However, although Non-Patent Document 2 is a technical document published in 1996, no other examples of callus induction using the method described in Reference 2 have been reported, and the method has not yet been established as a technique for reliably producing callus.

[0006] Furthermore, a method for inducing callus from the shoot apical meristem of Giant Miscanthus has been reported (see Non-Patent Document 3). This document describes a callus induction method using a callus induction medium containing a Murashige-Skoog basal salt mixture, vitamins, and sucrose, to which p-chlorophenoxyacetic acid (4-chlorophenoxyacetic acid: pCPA), α-naphthaleneacetic acid (1-naphthaleneacetic acid: NAA), putrescine (Put), 2-aminoindan-2-phosphonic acid (AIP), and other ingredients have been added. However, the method described in Non-Patent Document 3 involves the use of expensive reagents, which increases the cost of callus production.

[0007] Thus, there is currently no established general-purpose technique for producing callus and redifferentiating it that is effective for the propagation and breeding of giant miscanthus. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] China Patent Publication No. 102090339 [Patent Document 2] Korean Patent Publication No. 1020120009653 [Non-patent literature]

[0009] [Non-Patent Document 1] Anthony Trieu et al., “Transformation and gene editing in the bioenergy grass Miscanthus”, Biotechnol. Biofuels. Bioprod. 2022, 15 (1), 148 [Non-patent document 2] Inger Baksted Holme et al., “Callus induction and plant regeneration from different explant types of Miscanthus x ogiformis Honda 'Giganteus',” Plant Cell, Tissue and Organ Culture 1996, 45 (1), 43-52. [Non-patent document 3] Karolina Sobanska et al., “An efficient indirect plant regeneration from shoot apical meristem (SAM) derived embryogenic callus of Miscanthus × giganteus”, Biocatalysis and Agricultural Biotechnology, Volume 47, January 2023, 102576 Summary of the Invention [Problem to be solved by the invention]

[0010] The objective of the present invention is to provide a stable and efficient method for inducing callus and regenerating plants from giant miscanthus. In particular, the objective is to provide a method for inducing callus and regenerating plants from easily available explants of plant tissue that can be harvested year-round, regardless of the season. Another objective is to provide a callus induction method and plant regeneration method that can be performed inexpensively without the use of expensive reagents. [Means for solving the problem]

[0011] As a result of intensive research aimed at solving the above problems, the present inventors have discovered a method for stably and efficiently inducing callus from the rhizomes of Giant Miscanthus. They have also found that the callus induced in this manner can be stably and efficiently regenerated to regenerate plants. Based on these findings, the present invention has been completed.

[0012] The callus induction method according to the present invention is characterized in that it is a callus induction method for inducing callus by culturing the rhizomes of Giant Miscanthus in a callus induction medium, The callus induction medium contains auxin and cytokinin.

[0013] This method allows stable and efficient production of callus from the rhizomes of giant miscanthus, which can be regenerated into plant bodies. In particular, the rhizomes of giant miscanthus are readily available year-round, regardless of the season, making it possible to produce callus without seasonal or quantitative limitations. Furthermore, callus induction does not require expensive reagents, which reduces costs.

[0014] This method allows the callus produced to be stably and efficiently regenerated into plants and propagated, providing a callus production technique that enables the mass production of giant miscanthus seedlings. [Brief explanation of the drawings]

[0015] [Figure 1] Photographs showing the results confirmed in the callus induction experiment of Example 5. These are examples of callus obtained by culturing the rhizomes of Giant Miscanthus on callus induction medium for 7 weeks, with (a) showing compact callus and (b) showing soft callus. [Figure 2]1 is a photograph showing the results confirmed in the callus induction experiment of Example 5. The rhizomes of Giant Miscanthus were cultured on various callus induction media, and the results of callus induction on the various callus induction media were compared. [Figure 3] 1 shows photographs showing an example of callus development, green spot development, shoot development, rooting, and plant regeneration confirmed in the plant regeneration experiment of Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0016] Hereinafter, embodiments of the callus induction method and plant regeneration method according to the present invention will be described. However, the present invention is not limited to the following embodiments and various modifications are possible within the scope of the present invention.

[0017] (Callus induction method according to this embodiment) The callus induction method according to this embodiment involves inducing callus by culturing the rhizomes of Giant Miscanthus in a callus induction medium containing auxin and cytokinin.

[0018] Callus induction refers to the formation of callus from plant cells, and refers to the dedifferentiation of plant cells that have completed functional or morphological differentiation into organs, tissues, etc., or cells that are in the process of differentiating, to form cells or cell masses that have regained pluripotency. Here, dedifferentiation refers to the process by which cells that have completed functional or morphological differentiation into organs, tissues, etc., or cells that are in the process of differentiating, return to an undifferentiated state or a less differentiated state. Such dedifferentiated, undifferentiated or less differentiated cells regain pluripotency and can again functionally or morphologically differentiate into organs, tissues, etc. Furthermore, in many cases, they also regain the ability to divide.

[0019] Therefore, callus refers to a dedifferentiated plant cell or a mass of plant cells that has regained pluripotency, and in many cases, also regained the ability to divide. Callus can be classified into compact callus and soft callus (see, for example, Figure 1). Compact callus refers to a hard callus with a high cell density, in which the cells that make up the callus are tightly packed together, and is generally considered to have a high ability to regenerate into a plant. On the other hand, soft callus refers to a soft callus in which the cells that make up the callus are only loosely associated, and is generally considered to have a low ability to regenerate into a plant. The callus induced by the callus induction method of the present invention is preferably a compact callus.

[0020] The callus induction method of this embodiment can be applied to giant miscanthus (Miscanthus × giganteus). Giant miscanthus is a natural hybrid between Miscanthus sacchariflorus (tetraploid) and Miscanthus sinensis (diploid), and is a plant of the genus Miscanthus in the Poaceae family. As a triploid, it is completely sterile and cannot produce seeds. Because it is completely sterile, callus generation and regeneration are effective methods for its propagation and breeding. Furthermore, callus generation techniques can also be used for improving giant miscanthus varieties using genetic engineering techniques.

[0021] The plant tissue explants used in the callus induction method of this embodiment are rhizomes of Giant Miscanthus. Rhizomes are underground stems and are distinguished from roots by the absence of root caps or root hairs, the presence of leaves or their vestiges, and the arrangement of vascular bundles. There are no particular limitations on the time of collection of rhizomes; they may be harvested at any time of the year, regardless of the season, as long as they are rhizomes. For example, rhizomes can be divided from fully developed rhizomes and grown in a greenhouse until harvest. By using rhizomes as plant tissue explants, Giant Miscanthus-derived callus can be induced without seasonal or quantitative limitations.

[0022] The part of the rhizome that can be used is not particularly limited, but is preferably a region where cell division is actively taking place, such as a region containing a bud that has germinated from the rhizome, and can be harvested by cutting it from the rhizome. A bud is typically a structure consisting of an immature stem and leaves. For example, a region containing an axillary bud of the rhizome is preferred. Either an axillary bud that will grow into an aboveground stem or an axillary bud that will become an underground stem can be used. The size of the rhizome is also not particularly limited, but for example, rhizomes targeted for callus induction can be 0.5 to 5 cm in diameter and 5 to 20 cm in length, preferably 2 to 3 cm in diameter and 8 to 10 cm in length. These can also be cut to an appropriate size for use.

[0023] After harvesting, plant tissue pieces are usually washed, disinfected, sterilized, etc. before use. For disinfection and sterilization, for example, hypochlorous acid solutions such as calcium hypochlorite and sodium hypochlorite solutions, ethanol, hydrogen peroxide, Plant Preservative Mixture (PPM) (Plant Cell Technology), etc. can be used, but are not particularly limited. Furthermore, plant tissue pieces derived from plants cultivated under sterile conditions may also be used, in which case disinfection and sterilization are not necessary.

[0024] The callus induction medium used in the callus induction method of this embodiment refers to a medium prepared to induce callus from plant tissue fragments. The callus induction medium contains a basal medium suitable for the growth of plant cells and plant hormones that contribute to callus induction. Here, plant hormones refer to substances that bind to specific receptors and have the effect of regulating plant growth and responses, and the term is used here to include both naturally occurring and synthetic plant hormones.

[0025] In the callus induction method according to this embodiment, auxin and cytokinin can be used as plant hormones contained in the callus induction medium. Auxin and cytokinin are well-known plant hormones and are easily available.

[0026] Auxin is a plant hormone known to regulate plant cell elongation, cell differentiation, and cell division. The auxin receptor is TIR1. When auxin binds to TIR1, it also binds to Aux / IAA, and ubiquitin is further attached to Aux / IAA. Because ubiquitin serves as a marker for protein degradation, the ubiquitinated Aux / IAA is degraded by the proteasome. This activates ARF, which had been inactivated by Aux / IAA, and auxin-responsive genes are expressed and function. The auxin used in the callus induction method of this embodiment is not particularly limited and may be a naturally occurring auxin or an artificially produced synthetic auxin with auxin-like activity. Examples of auxins include, but are not limited to, 2,4-dichlorophenoxyacetic acid (hereinafter sometimes abbreviated as "2,4-D"), indole-3-acetic acid (IAA), 4-chloroindole-3-acetic acid (4-Cl-IAA), indole-3-butyric acid (IBA), indole-3-propionic acid, 1-naphthaleneacetic acid (NAA), phenylacetic acid (PAA), 2,4,5-trichlorophenoxyacetic acid, and 4-chlorophenoxyacetic acid. Any known substance with auxin-like activity can be used. Auxins may be used alone or in combination of two or more. Preferably, 2,4-dichlorophenoxyacetic acid or its derivatives can be used. These compounds are readily available and inexpensive, thereby reducing the cost of callus induction.

[0027] 2,4-D is a synthetic auxin that exhibits auxin-like activity, a plant hormone. 2,4-D derivatives include compounds derived by substituting any position of 2,4-D with any substituent. Substituents include hydrogen, deuterium, halogen (preferably Cl, F, Br, I, etc.), hydroxyl, alkyl (preferably lower alkyl groups having approximately 1 to 3 carbon atoms, such as methyl, ethyl, and butyl), and carboxyl. Examples include compounds in which any position on the benzene ring of 2,4-D is substituted, such as 2,4,5-trichlorophenoxyacetic acid and 4-chlorophenoxyacetic acid. In addition to the free forms of 2,4-D and compounds derived from 2,4-D, these compounds include pharmaceutically acceptable salts, esters, solvates, hydrates, and other forms. Pharmaceutically acceptable salts refer to salts that are pharmaceutically acceptable and have the desired pharmacological activity of the parent compound, 2,4-D. Examples of salts include, but are not limited to, inorganic metal salts such as sodium, potassium, calcium, magnesium, and zinc salts; inorganic acid salts such as hydrochloride, sulfate, nitrate, and phosphate; organic acid salts such as acetate, trifluoroacetate, oxalate, propionate, succinate, fumarate, lactate, malate, tartrate, citrate, and maleate; and organic base salts such as ammonium salt, triethylamine salt, and triethanolamine salt. 2,4-D derivatives also include compounds that are converted to compounds with 2,4-D activity by the action of intracellular enzymes, such as 2,4-D esters such as methyl 2,4-dichlorophenoxyacetate, ethyl 2,4-dichlorophenoxyacetate, and butyl 2,4-dichlorophenoxyacetate. Hereinafter, 2,4-D and its derivatives may be collectively referred to as "2,4-Ds."

[0028] Cytokinins are plant hormones known to promote cell division in plants. The cytokinin receptor is a sensor histidine kinase localized in the cell membrane. When cytokinin binds to the sensor histidine kinase, phosphates bound to histidines in the kinase moiety inside the cell membrane are transferred sequentially (His-Asp phosphorelay), ultimately to a protein called a response regulator. Response regulators are known to function as transcription factors, promoting the expression of cytokinin-responsive genes. The cytokinins used in the callus induction method of this embodiment are not particularly limited and may be naturally occurring or artificially produced synthetic cytokinins with cytokinin-like activity. Examples of cytokinins include, but are not limited to, 6-benzylaminopurine (hereinafter sometimes abbreviated as "BA"), kinetin, zeatin, zeatin liposide, dihydrozeatin, 2-isopentenyladenine, thidiazuron, and diphenylurea. Any known substance with cytokinin-like activity can be used. Cytokinins may be used singly or in combination with two or more auxins. 6-benzylaminopurine or its derivatives are preferred. These compounds are readily available and inexpensive, which helps reduce the cost of callus induction.

[0029] BA is a synthetic cytokinin that exhibits cytokinin-like activity, a plant hormone. BA derivatives include compounds derived by substituting any position of BA with any substituent. Examples of substituents include hydrogen, deuterium, halogen (preferably Cl, F, Br, I, etc.), hydroxyl, alkyl (preferably lower alkyl groups having approximately 1 to 3 carbon atoms, such as methyl, ethyl, and butyl), and carboxyl. Examples include compounds in which any position on the purine ring of BA is substituted, such as the hydrogen atom on the nitrogen atom at position 9 of the purine ring. These compounds include the free forms of BA and compounds derived from BA, as well as pharmaceutically acceptable salts, esters, solvates, hydrates, and other forms. The same applies to pharmaceutically acceptable salts. BA derivatives also include compounds that are converted to compounds with BA activity by the action of intracellular enzymes. Hereinafter, BA and BA derivatives may be collectively referred to as "BAs."

[0030] The auxin and cytokinin concentrations in the callus induction medium can be appropriately set. When 2,4-Ds are used as auxins, the concentrations can be, for example, 0.1 mg / L to 100 mg / L, preferably 1 mg / L to 10 mg / L, or 2 mg / L to 6 mg / L, and particularly preferably 3 mg / L to 6 mg / L. When BAs are used as cytokinins, the concentrations can be, for example, 0.01 mg / L to 30 mg / L, preferably 0.05 mg / L to 1 mg / L, or 0.1 mg / L to 0.5 mg / L, and particularly preferably 0.1 mg / L to 0.2 mg / L. Auxin and cytokinin can be contained in a callus induction medium in a predetermined ratio. When 2,4-Ds are used as the auxin and BAs as the cytokinin, for example, the 2,4-Ds:BAs can be contained in a mass ratio of 20 to 40:1, preferably 20, 25, 30, 35, or 40:1.

[0031] Furthermore, in addition to the auxins and cytokinins mentioned above, plant growth regulators such as plant hormones may also be added to the callus induction medium. In addition to the auxins and cytokinins mentioned above, known plant hormones include gibberellins, ethylene, abscisic acid, prasinosteroids, jasmonic acid, salicylic acid, and strigolactone. However, if additional plant growth regulators are added, they must be of a type and amount that does not interfere with the callus induction effect of the auxin-cytokinin interaction on Giant Miscanthus-derived rhizomes.

[0032] Gibberellin-based plant hormones include gibberellin A1 to gibberellin A 136 Examples of brassinosteroid plant hormones include brassinolide and castasterone. Examples of strigolactone plant hormones include strigol and orobanchol, but are not limited to these.

[0033] The basal medium is not particularly limited as long as it is a medium used for ordinary plant tissue culture. Therefore, any known medium for plant tissue culture can be used. The basal medium is composed of components necessary for the growth and maintenance of plant cells, such as, but not limited to, a carbon source, inorganic salts, vitamins, a nitrogen source, amino acids, and water.

[0034] Examples of carbon sources that can be used include, but are not limited to, sugars such as sucrose, glucose, trehalose, fructose, lactose, galactose, maltose, and mannitol. Examples of inorganic salts that can be used include, but are not limited to, NH4NO3, NaNO3, KNO3, Ca(NO3), MgCl2, and AlCl4. 3、 KCl, CaCl2, FeCl3, CoCl2, Na2SO4, MgSO4, MnSO4, FeSO4, CuSO4, ZnSO4, KH2PO4, Na2EDTA, H3BO3, MoO 2、Examples of suitable sources of vitamins include, but are not limited to, Na2MoO4, KI, and the like. Commercially available mixed salts can also be used, such as the mixed salts for Chu(N6) medium (Wako Pure Chemical Industries) and the mixed salts for Murashige-Skoog medium (Wako Pure Chemical Industries). Examples of suitable vitamins include, but are not limited to, B vitamins such as thiamine, riboflavin, nicotinic acid, nicotinamide, pantothenic acid, pyridoxine, biotin, folic acid, and inositol, and C vitamins such as ascorbic acid. Examples of suitable nitrogen sources include, but are not limited to, inorganic ammonium salts of (NH4)2SO4 and NH4NO3, amino acids such as glycine, and protein hydrolysates such as casein hydrolysate and peptone. Examples of suitable amino acids include, but are not limited to, glycine, glutamine, glutamic acid, alanine, phenylalanine, cysteine, proline, and lysine.

[0035] The basal medium may be a medium that has traditionally been used as a medium for plant tissue culture, such as Murashige & Skoog medium (MS medium), Linsmaier & Skoog medium (LS medium), Gamborg's B-5 medium, White medium, or Chu(N6) medium, or a medium obtained by modifying such a basal medium, preferably MS medium or a medium obtained by modifying such a medium, but is not particularly limited thereto.

[0036] The pH of the callus induction medium can be, for example, 5.0 to 8.0, 5.0 to 7.0, 5.5 to 6.5, 5.5 to 6.0, 5.6 to 5.9, and preferably 5.7, but is not particularly limited thereto.

[0037] The callus induction medium may be a solid medium, a semi-liquid medium, or a liquid medium, but a solid medium is preferred. Callus induction can be achieved by placing plant tissue fragments on the solid medium. Liquid medium may be used for either static or shaking culture, but static culture is preferred. When using a solid medium for callus induction, the medium can be solidified using a gelling agent such as Gelrite, agar, gellancum, gelatin, or alginate, but this is not particularly limited. For example, these gelling agents are preferably present in the liquid medium at a concentration of 0.1 w / v% to 1.5 w / v%, preferably 0.5 w / v% to 1.0 w / v%, and particularly 0.7 w / v%, based on the total volume of the medium. There are no particular limitations on the containers used for culture, and any known culture vessel can be used.

[0038] In the callus induction method according to the present invention, the culture conditions for culturing on a callus induction medium are not particularly limited as long as callus induction from plant tissue explants is possible, and can be appropriately set depending on the state of the plant tissue explants, the type of medium, etc.

[0039] The light conditions are preferably dark. A dark place refers to an environment without natural or artificial light, and does not necessarily have to be a completely dark environment. Therefore, the culture may be performed in a weak light environment with some light. For example, an environment of 100 lux or less, 10 lux or less, or 1 lux or less may be mentioned.

[0040] The temperature is not limited as long as callus induction is possible, and may be, for example, 15 to 35° C., 20 to 30° C., or 24 to 26° C., preferably 25° C. The culture period may be, for example, 1 day or more, 2 days or more, 3 days or more, 5 days or more, 1 week or more, 2 weeks or more, 3 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, 8 weeks or more, 9 weeks or more, or 10 weeks or more, or may be 20 weeks or less, 15 weeks or less, 12 weeks or less, or 10 weeks or less, preferably 7 weeks.

[0041] The callus induced in this manner can be used for plant regeneration. For example, the induced callus may be transferred to a subculture medium, maintained and propagated, and then transferred to the regeneration medium described below. Alternatively, after callus induction, the callus may be directly transferred to the regeneration medium described below and used for plant regeneration.

[0042] The subculture medium is a medium for maintaining and growing the induced callus in an undifferentiated state, and any conventionally known subculture medium can be used. For example, it may be a basal medium suitable for the growth of plant cells, as described above, to which a plant hormone has been added, or the same medium as the callus induction medium may be used.

[0043] (Plant regeneration method) The plant regeneration method of this embodiment involves regenerating a plant by redifferentiating callus induced by the callus induction method of this embodiment. Plant regeneration refers to regenerating a plant by redifferentiating callus maintained and propagated in an undifferentiated state into organs and tissues that constitute the plant, such as adventitious buds, adventitious embryos, and adventitious roots, or redifferentiating them into a plant. A plant is an organism that contains at least three organs: roots, stems, and leaves. Redifferentiation refers to functional or morphological differentiation of dedifferentiated cells (callus) into cells that perform the functions of organs, tissues, etc.

[0044] The plant regeneration method according to the present embodiment can be carried out based on a conventionally known method, and may include, for example, a redifferentiation step of redifferentiating the callus induced by the callus induction method according to the present embodiment into shoots, etc., and a rooting step of inducing roots from the redifferentiated callus.

[0045] The regeneration step is carried out by culturing the callus induced by the callus induction method on a regeneration medium, and inducing the callus to regenerate into shoots, etc. Here, shoots refer to stems derived from the shoot apical meristem and associated leaves, and also include adventitious buds and somatic embryos.

[0046] The regeneration medium is a medium prepared so that callus can be regenerated into shoots or other tissues, and any conventionally known regeneration medium can be used. For example, a basal medium suitable for the growth of plant cells to which plant hormones have been added may be used. The basal medium and plant hormones are as described above in the "Callus Induction Method."

[0047] The plant hormone may contain the above-mentioned auxin and / or cytokinin. It is known that regeneration from callus into adventitious shoots, somatic embryos, adventitious roots, etc. depends on the ratio of auxin to cytokinin. Generally, adventitious shoots are formed when the cytokinin concentration is higher and the auxin concentration is lower than in the callus induction medium (high cytokinin / auxin ratio). This occurs because cells constituting callus that have regained pluripotency respond to cytokinin to express genes involved in the establishment of the shoot apical meristem, and the shoot apical meristem established by the action of these genes forms a shoot. It is also known that high auxin concentrations induce adventitious root differentiation, while extremely low auxin concentrations induce somatic embryo differentiation.

[0048] For example, the cytokinin concentration can be 0.1 to 50 mg / L, preferably 1 to 10 mg / L, and more preferably 3 to 7 mg / mL, the auxin concentration can be 0.01 to 2.5 mg / L, preferably 0.05 to 1.0 mg / L, and more preferably 0.1 to 0.5 mg / L, and the mass ratio of cytokinin to auxin can be 50 to 5:1, and preferably 40, 30, 25, 20, 15, or 10:1.

[0049] The pH of the regeneration medium can be, for example, 5.0 to 8.0, 5.0 to 7.0, 5.5 to 6.5, 5.5 to 6.0, 5.6 to 5.9, and preferably 5.7, but is not particularly limited.

[0050] The regeneration medium may be a solid medium, a semi-solid medium, or a liquid medium, but is preferably a solid medium.

[0051] The culture conditions for culturing on a regeneration medium are not particularly limited as long as they allow callus regeneration, and can be appropriately set depending on the state of the callus, the type of medium, etc.

[0052] The light conditions may be bright or dark and are not particularly limited, but are preferably performed under a light-dark cycle. A light-dark cycle refers to an environment in which light and dark periods are repeated in a specific cycle. For example, the light period can be 5 to 20 hours, preferably 10 to 18 hours, and more preferably, the light-dark cycle can be 16 hours light and 8 hours dark. The illuminance during the light period is not particularly limited as long as it is possible to induce redifferentiation into shoots, but may be, for example, 500 lux to 50,000 lux or 1,000 lux to 20,000 lux, and the light source may be either natural light or artificial light.

[0053] The temperature is not particularly limited as long as regeneration is possible, and may be, for example, 15 to 35°C, 20 to 30°C, or 24 to 26°C, preferably 25°C. The culture period may be, for example, 1 day or more, 2 days or more, 3 days or more, 5 days or more, 1 week or more, 2 weeks or more, 3 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, 8 weeks or more, 9 weeks or more, or 10 weeks or more, or may be 20 weeks or less, 15 weeks or less, 12 weeks or less, or 10 weeks or less. Preferably, culture is carried out for 4 weeks.

[0054] The rooting step is carried out by culturing the callus redifferentiated in the regeneration step on a rooting medium to induce rooting. Rooting refers to the growth of roots, and refers to the regeneration of roots from callus.

[0055] The rooting medium is a medium prepared to induce rooting from the redifferentiated callus, and any conventional rooting medium can be used. For example, a basal medium suitable for the growth of plant cells can be used, and plant hormones may be added as needed. The basal medium and plant hormones are as described above in the "Callus Induction Method."

[0056] The pH of the rooting medium can be, for example, 5.0 to 8.0, 5.0 to 7.0, 5.5 to 6.5, 5.5 to 6.0, 5.6 to 5.9, and preferably 5.8, but is not particularly limited.

[0057] The rooting medium may be a solid medium, a semi-liquid medium, or a liquid medium, but is preferably a solid medium.

[0058] The culture conditions for culturing on a rooting medium are not particularly limited as long as roots can be induced from the redifferentiated callus, and can be appropriately set depending on the state of the redifferentiated callus, the type of medium, etc.

[0059] The light conditions may be either bright or dark, and are not particularly limited, but are preferably performed under a light-dark cycle. A light-dark cycle refers to an environment in which light and dark periods alternate in a specific cycle. For example, the light period can be 5 to 20 hours, preferably 10 to 18 hours, and more preferably, the light-dark cycle can be 16 hours light and 8 hours dark. The illuminance during the light period is not particularly limited as long as rooting can be induced, but may be, for example, 500 lux to 50,000 lux or 1,000 lux to 20,000 lux, and the light source may be either natural light or artificial light.

[0060] The temperature is not particularly limited as long as rooting is possible, and may be, for example, 15 to 35° C., 20 to 30° C., or 24 to 26° C., preferably 25° C. The culture period may be, for example, 1 day or more, 2 days or more, 3 days or more, 5 days or more, 1 week or more, 2 weeks or more, 3 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, 8 weeks or more, 9 weeks or more, or 10 weeks or more, or may be 20 weeks or less, 15 weeks or less, 12 weeks or less, or 10 weeks or less. Preferably, culture is carried out for 2 weeks.

[0061] After the rooted seedlings are obtained, they are cultured for a certain period of time to allow the roots to develop, and then transplanted into seedling containers or nurseries for growth, thereby obtaining seedlings that can be used for a specific purpose. By raising seedlings in a controlled environment, it is possible to provide a stable and large quantity of high-quality seedlings, regardless of the season or weather. [Example]

[0062] The embodiments of the present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples.

[0063] Example 1: Reagents for preparation of callus induction medium, regeneration medium and rooting medium The callus induction, regeneration, and rooting media used in the following examples were prepared using the following reagents, S-1 to S-13. Note that S-2, S-4, S-8, S-9, and S-13 were prepared in advance with purified water to achieve the compositions or concentrations described.

[0064] The following reagent names, manufacturers (distributors), and catalog numbers are listed. S-1: Mixed salts for Chu (N6) medium (Wako Pure Chemical Industries, Ltd., 391-02021) S-2:N6 Vitamin (x1000) Mixture (Composition) 100 mg / mL myo-inositol (Wako Pure Chemical Industries, Ltd., 096-00285) 0.5 mg / mL nicotinic acid (Wako Pure Chemical Industries, Ltd., 142-01232) 0.5 mg / mL pyridoxine hydrochloride (Wako Pure Chemical Industries, Ltd., 165-05401) 1 mg / mL thiamine hydrochloride (Wako Pure Chemical Industries, Ltd., 201-00852) S-3: Mixed salts for Murashige and Skoog (MS) medium (Wako Pure Chemical Industries, Ltd., 392-00591) S-4: MS Vitamin (x1000) Mixture (Composition) 100 mg / mL myo-inositol (Wako Pure Chemical Industries, Ltd., 096-00285) 0.5 mg / mL nicotinic acid (Wako Pure Chemical Industries, Ltd., 142-01232) 0.5 mg / mL pyridoxine hydrochloride (Wako Pure Chemical Industries, Ltd., 165-05401) 0.1 mg / mL thiamine hydrochloride (Wako Pure Chemical Industries, Ltd., 201-00852) 2 mg / mL glycine (Wako Pure Chemical Industries, Ltd., 077-00735) S-5: Sucrose (Wako Pure Chemical Industries, Ltd., 192-00017) S-6: L-proline (Nacalai Tesque, 29001-42) S-7: Casein hydrolysate (casamino acids) (Wako Pure Chemical Industries, Ltd., 555-35915) S-8: 2.5 mg / mL 2,4-D (Kanto Chemical, 10175-31) S-9: 2.5 mg / mL BA (Sigma, B3408-1G) S-10: Agar (Sigma, A7921-500G) S-11: Gelrite (registered trademark) (Wako Pure Chemical Industries, Ltd., 075-05655) S-12:PPM (Plant Cell Technology, PPM-100) S-13: 1 mg / mL NAA (Sigma, N0640-25G)

[0065] Example 2: Preparation of callus induction medium In this example, callus induction media were prepared for use in the callus induction experiment in Example 5 below. Callus induction media A-1 to A-6 were prepared. Each callus induction medium A-1 to A-6 was prepared to have the composition shown in Table 1 below. R1 to R9 in Table 1 below were added to pure water and dissolved, and the pH was adjusted with potassium hydroxide to a total volume of 1 L. R11 was then added to A-1, and R10 to the others, and dissolved. R12 was then added, and the mixture was autoclaved (121°C, 20 minutes). After autoclaving and allowing to cool to a certain extent, the medium was poured into a petri dish and solidified at room temperature to prepare callus induction media.

[0066] [Table 1]

[0067] Example 3: Preparation of regeneration medium In this example, a regeneration medium was prepared for use in the plant regeneration experiment in Example 6 below. The regeneration medium was prepared to have the composition shown in Table 2 below. R1 to R5 in Table 2 below were added to pure water and dissolved, and the pH was then adjusted with potassium hydroxide to a total volume of 1 L. Next, R6 was added and dissolved, and then R7 was added, and the mixture was autoclaved (121°C, 20 minutes). After autoclaving and allowing to cool to a certain extent, the medium was poured into a petri dish and solidified at room temperature to prepare the regeneration medium.

[0068] [Table 2]

[0069] Example 4: Preparation of rooting medium In this example, a rooting medium was prepared for use in the plant regeneration experiment in Example 6 below. The rooting medium was prepared to have the composition shown in Table 3 below. R1 to R3 in Table 3 below were added to pure water and dissolved, and the pH was adjusted with potassium hydroxide to a total volume of 1 L. Next, R4 was added and dissolved, and then R5 was added, and the mixture was autoclaved (121°C, 20 minutes). After autoclaving and allowing to cool to a certain extent, the medium was poured into a petri dish and solidified at room temperature to prepare the rooting medium.

[0070] [Table 3]

[0071] Example 5: Callus induction The plant tissue explants used for callus induction were buds from the rhizomes of Giant Miscanthus. After washing the rhizomes with tap water, the plant tissue explants for callus induction were harvested using a cutter. The size of the plant tissue explants was 0.5–2.0 cm. The harvested plant tissue explants were washed three times with 70% ethanol, three times with sterile water, three times with hypochlorous acid, and three times with sterile water containing 0.1% (v / v) ppm. In a clean bench, the plant tissue explants were placed on callus induction media A-1–A-6 prepared in Example 3 and placed in an artificial climate chamber (25°C, dark conditions). The plant tissue explants were transferred to fresh callus induction media every two weeks for continued cultivation. Seven weeks after the start of callus induction, some plant tissue explants developed compact calli (Figure 1(a)), which are considered to have a high regeneration rate, and soft calli (Figure 1(b)), which are considered to have a low regeneration rate. The rates of mold occurrence, non-callus occurrence, soft callus occurrence, and compact callus occurrence on various callus induction media are summarized in Table 4 below, and Figure 2 shows photographs showing whether or not callus induction occurred in the rhizomes after 7 weeks of culture.

[0072] [Table 4]

[0073] As shown in Table 4 and Figure 2, soft callus was formed from the explants cultured on callus induction media A-3, A-5, and A-6, and compact callus was formed from the explants cultured on callus induction media A-3 and A-6. Compact callus formation was also confirmed in the A-3 medium supplemented with 2880 mg / L L-proline, 750 mg / L magnesium chloride hexahydrate, and no casein hydrolysate, as well as in the A-3 medium supplemented with 2880 mg / L L-proline, 0.9 mg / L thiamine hydrochloride, and 750 mg / L magnesium chloride hexahydrate. These results confirm that a mass ratio of the plant hormones 2,4-D to BA of approximately 30:1 is important for the formation of compact callus, which is considered to have high regeneration potential.

[0074] Example 6: Plant regeneration The callus induced on callus induction medium A-6, in which compact callus formation was confirmed, was transplanted onto the regeneration medium prepared in Example 3 above and subjected to static culture. Static culture was performed at 25°C under a 16-hour light-dark cycle at 9,250 lux and 8-hour light-dark cycle at 0 lux. After one week, green spot formation was confirmed. Culture was continued in the same manner, and after three weeks, shoot formation was confirmed on the regeneration medium. After four weeks, the callus was transplanted onto the rooting medium prepared in Example 4 above to induce rooting. After two weeks of culture on the rooting medium, root formation was confirmed and continued thereafter. Photographs showing the entire process are shown in Figure 3.

[0075] In the above embodiment, the following configurations are envisioned.

[0076] (1) A callus induction method for inducing callus by culturing the rhizomes of Giant Miscanthus on a callus induction medium, The callus induction method, wherein the callus induction medium contains auxin and cytokinin.

[0077] According to this embodiment, callus that can be regenerated into a plant can be stably and efficiently produced from the rhizomes of Giant Miscanthus. In particular, since the rhizomes of Giant Miscanthus are available continuously throughout the year, regardless of the season, and are easily available, callus can be produced without seasonal or quantitative limitations. Another advantage is that expensive reagents are not required for callus induction, thereby reducing costs.

[0078] The callus produced by this embodiment can be stably and efficiently regenerated into a plant body and propagated, providing a callus production technique that enables the mass production of giant miscanthus seedlings.

[0079] (2) In the callus induction method of (1) above, it is preferable that the auxin is 2,4-dichlorophenoxyacetic acid or a derivative thereof, and the cytokinin is 6-benzylaminopurine or a derivative thereof.

[0080] According to this embodiment, by optimizing the use of 2,4-dichlorophenoxyacetic acid or its derivatives as the auxin and 6-benzylaminopurine or its derivatives as the cytokinin, regenerative callus can be produced more stably and efficiently in plants. In particular, these compounds are easily available and inexpensive, which has the advantage of reducing the cost of callus induction.

[0081] (3) In the callus induction method of (2) above, it is preferable that the 2,4-dichlorophenoxyacetic acid or a derivative thereof and the 6-benzylaminopurine or a derivative thereof are contained in a mass ratio of 20:1 to 40:1.

[0082] According to this embodiment, by optimizing the amounts of 2,4-dichlorophenoxyacetic acid or a derivative thereof, and 6-benzylaminopurine or a derivative thereof added, callus that can be regenerated from a plant body can be produced more stably and efficiently.

[0083] (4) In the callus induction method (1) above, it is preferable that the rhizome includes the region of a germinated bud.

[0084] According to this embodiment, by using a part of the rhizome where cell division is active as a plant tissue piece for callus induction, callus that can be regenerated into a plant body can be produced more stably and efficiently.

[0085] (5) A plant regeneration method for regenerating a plant by redifferentiating a callus induced by any one of the callus induction methods (1) to (4) above.

[0086] According to this embodiment, plant bodies can be regenerated stably and efficiently by redifferentiating callus induced from rhizomes. This allows giant miscanthus to be grown stably and efficiently without time or quantity restrictions, enabling the mass production of giant miscanthus seedlings. Another advantage is that expensive reagents are not required for plant regeneration, thereby reducing costs. [Industrial Applicability]

[0087] The present invention provides a technique for inducing callus from giant miscanthus, which is industrially useful as a biomass crop, and a technique for regenerating plants from the callus. The present invention can be established as a practical seedling raising technique for giant miscanthus.

Claims

1. A callus induction method for inducing callus by culturing the rhizomes of Giant Miscanthus on a callus induction medium, comprising: The callus induction method, wherein the callus induction medium contains auxin and cytokinin.

2. 2. The callus induction method according to claim 1, wherein the auxin is 2,4-dichlorophenoxyacetic acid or a derivative thereof, and the cytokinin is 6-benzylaminopurine or a derivative thereof.

3. 3. The callus induction method according to claim 2, wherein the 2,4-dichlorophenoxyacetic acid or a derivative thereof and the 6-benzylaminopurine or a derivative thereof are contained in a mass ratio of 20:1 to 40:

1.

4. 2. The callus induction method of claim 1, wherein the rhizome comprises a germinated bud region.

5. A method for regenerating a plant, comprising redifferentiating the callus induced by the method for inducing callus according to any one of claims 1 to 4 to regenerate a plant.

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