WO2022079927A1 - Method for culturing plant cell - Google Patents

Abstract

The present invention relates to a method for culturing a plant cell and a method for reproducing a plant body.　The method for culturing a plant cell according to the present invention comprises culturing a cell of a plant belonging to the family Gramineae using a culture medium containing trehalose. The method for reproducing a plant body according to the present invention is a method for reproducing a plant body from a cell of a plant belonging to the family Gramineae, and comprises (1) culturing a cell of a plant belonging to the family Gramineae using a culture medium containing trehalose and (2) culturing the resultant product in a redifferentiation culture medium.

Description

Plant cell culture method

The present invention relates to a method for culturing plant cells and a method for regenerating plants.

(1) Culturing of plant cells and regeneration of plants There have been reports of cases in which immature or ripe embryos were isolated and cultured in grasses such as rice, corn, and wheat, and the plants were regenerated. There are large differences in culture efficiency between species and varieties, and many of them cannot be cultivated. In particular, among major crops, many varieties that are difficult to cultivate in wheat and corn are known. To date, its efficiency has been gradually improved by various ingenuity. There are mainly two means for this: modification of the culture medium and pretreatment of plant tissue pieces.

1) Culture medium Various improvements in culture medium have been tried in the transformation of maize with Agrobacterium. Selection of transformed cells in N6 basal medium (Non-Patent Document 1), addition of AgNO3 and carbenicillin to medium (Non-Patent Document 1, Non-Patent Document 2), addition of cysteine to coexisting medium (Non-Patent Document 3) As a result, the culture efficiency was improved. It has also been reported that the transformation efficiency in indicine is enhanced by modifying the composition of the co-culture medium and the gelling agent (Non-Patent Document 4).

2) Pretreatment of plant tissue pieces Hiei et al. (2006) (Non-Patent Document 5) shows that the transformation efficiency is increased by heat-treating or centrifuging immature embryos of rice and maize before inoculation with Agrobacterium, and even varieties that could not be transformed so far. It was reported that a transformant was obtained. Further, Ishida and Hiei (WO2011 / 013764) (Patent Document 1) found that transformed plants can be obtained with high efficiency by removing hypocotyls from immature wheat embryos co-cultured with Agrobacterium.

In this way, by devising measures such as modifying the medium composition and pretreating the plant tissue pieces used as materials, the culture method for transforming rice, corn and wheat with Agrobacterium has significantly improved efficiency and expanded the number of applicable varieties. Has been made.

As other methods, in recent years, as a method for producing a transformant, there are a method using a fertilized egg (Non-Patent Document 6) and an implanter particle gun method (Non-Patent Document 7).

(2) Trehalose Small animals such as Polypedilum vanderplanki and plants such as Selaginella can sleep and survive in extremely dry environments such as deserts because trehalose is accumulated in the living body at a high concentration. It has been known. In addition, it has been reported that trehalose functions even during dry dormancy and imparts various stress-tolerant functionality to animals and plants (Non-Patent Document 8).

However, general plants do not contain as high a concentration of trehalose as the above-mentioned animals and plants that survive in extreme environments. In addition, trehalose is not used as a carbon source in the primary metabolism of plants. In fact, it has been revealed that the concentration of trehalose is lower than that of sucrose in plants. For example, in white sucrose, the content of sucrose in the leaves is 9 mg / g (dry weight), the content of trehalose is 0 mg / g (dry weight), and the content of sucrose in the stem is 7.5 mg. Although it is / g (dry weight), the content of trehalose is 0.1 mg / g (dry weight) (Non-Patent Document 9).

In culturing artificial plant tissues, sucrose and / or maltose are used as carbon sources as a conventional method. On the other hand, trehalose is rarely used in plant tissue culture. However, as an exception, the addition of trehalose has been shown to be effective in the formation of protocomb-like spheres (PLBs) of Cymbidium finlaysonianum, a tropical epiphytic cymbidium. PLB formation was promoted at concentrations of 5-40 g / l of sucrose and 10-80 g / l of trehalose, with concentrations of 20 g / l each being the most effective. Rooting from shoots was observed only in the trehalose-added group, and trehalose has been reported to be effective for rooting from shoots (Non-Patent Document 10).

On the other hand, in wheat, there is a study in which an attempt was made to regenerate a plant by tissue culture of microspores using trehalose, but it is reported that trehalose had no effect (Non-Patent Document 11). Further, trehalose is known to inhibit the metabolism and growth of plants (Non-Patent Document 12, Non-Patent Document 13). In barley, trehalose has been reported to interfere with the regulation of carbohydrate-related genes (Non-Patent Document 14, Non-Patent Document 15). As a result, trehalose was not used for culturing gramineous plants in place of sucrose and / or maltose.

WO2011 / 013764 WO2017 / 171092 WO2018 / 143480 WO2014 / 157541

An object of the present invention is to provide a method for culturing plant cells and a method for regenerating plants.

As a result of diligent research to solve the above problems, the present inventors cultivate immature wheat embryos in a medium containing trehalose instead of the carbon source (sucrose or maltose) used in the conventional method. As a result, it was found that the plant regeneration rate was remarkably improved as compared with the conventional medium, and the present invention was completed. By using this method, it has become possible to efficiently produce transformed crops and genome-edited crops.

The present invention includes, but is not limited to, the following aspects.
[Aspect 1]
A method for culturing plant cells, which comprises culturing cells of a gramineous plant using a culture medium containing trehalose.
[Aspect 2]
The method according to aspect 1, wherein the plant cells of the Gramineae family are cultured to obtain a plant cell group and / or a regenerated plant.
[Aspect 3]
The method according to aspect 1 or 2, wherein the gramineous plant is selected from the group consisting of rice, corn, wheat, barley, rye, and sorghum.
[Aspect 4]
The method according to any one of aspects 1-3, wherein the gramineous plant is wheat or corn.
[Aspect 5]
The method according to any one of aspects 1-4, wherein the cells of a gramineous plant are immature embryos, ripe seeds, fertilized eggs, callus, protoplasts, or a cell group derived from them.
[Aspect 6]
The method according to any one of aspects 1-5, wherein the cells of a gramineous plant are immature embryos or a cell group derived from the immature embryos.
[Aspect 7]
The method according to any one of aspects 1-6, comprising the step of introducing a substance selected from the group consisting of nucleic acid, protein and nucleic acid protein complex into cells of a gramineous plant.
[Aspect 8]
The method according to aspect 7, wherein transformation or genome editing is performed by introducing a substance.
[Aspect 9]
The method according to aspect 7 or 8, wherein the substance is introduced by a particle gun method or an Agrobacterium method.
[Aspect 10]
The method according to any one of aspects 1-9, wherein the culture efficiency of plant cells is improved.
[Aspect 11]
The method according to any one of aspects 7-9, wherein the introduction efficiency of the substance is improved.
[Aspect 12]
It is a method of regenerating a plant from the cells of a gramineous plant.
(1) The cells of Gramineae plants are cultured using a culture medium containing trehalose, and then
(2) Culturing in redifferentiation medium,
How to regenerate a plant, including that.

It has become possible to easily cultivate gramineous plants, which are said to be difficult to cultivate in the past, and to efficiently obtain regenerated plants from cultured cells.

1. 1. Plant Cell Culturing Method The present invention relates to a plant culturing method. The method of the present invention comprises culturing grass cells using a culture medium containing trehalose.

Plant The "plant" in the above method is a gramineous plant. As the gramineous plant, any plant belonging to the gramineous family (Poaceae) can be used. In one aspect, the gramineous plant is selected from the group consisting of rice, corn, wheat, barley, rye, and sorghum, without limitation. (In the present specification, the term "one aspect" is intended to be non-limiting.) The above culture method can be used particularly for plants or varieties that are considered to be "difficult to culture". Is. "Difficult-to-culture" means that it is difficult to culture, specifically, for example, it is difficult to culture cells or tissues isolated from a plant, formation of callus by treatment such as dedifferentiation, or a plant from callus. It means that it is difficult to redifferentiate into.

As one aspect, the gramineous plant preferably belongs to the genus Wheat (Triticum) or the genus Corn (Zea). In the present specification, "wheat" and "corn" mean plants of the genus Wheat and the genus Corn, respectively, unless otherwise specified.

As the genus Wheat, for example, as a one-grain wheat, T.I. aegilopodes, T.I. thaudar and T.I. Using monococcum (1 grain wheat) as a 2-grain wheat, T.I. dicoccoides, T.I. dicoccum (2 grains of wheat, emmer wheat), T.I. pyromidale, T.I. Orientale (Khorasan wheat), T.I. durum (durum wheat, macaroni wheat), T.I. turgidium (rivet wheat), T.I. Polishum (Polish wheat), and T.I. Persicum (Persian wheat), as well as three-grain wheat, T.I. aestivum (ordinary wheat, bread wheat), T.I. spelled, T.I. compactum (Club Wheat, Mitsuho Wheat), T.I. sphereococcum (Indian dwarf wheat), T.I. maha, and T.I. Vavilovii can be mentioned. In the present invention, varieties belonging to bread wheat (T. aestivum) or macaroni wheat (T. durum) are suitable, and in particular, T.I. Varieties belonging to aestivum are suitable.

As the genus Corn, five species are known: Zea mays, Zea diploperennis, Zea luxurians, Zea nicaraguensis and Zea perennis. Many cultivars have been developed for Zea mays. The seeds and varieties of corn used in the present invention are not particularly limited, but are preferably varieties belonging to Zea mays.

Cell of Gramineae Plants Plant materials include, but are not limited to, immature embryos, ripe seeds, fertilized eggs, callus, protoplasts, or cell groups derived from them. Immature embryos or ripe seeds are preferred. As used herein, the term "immature embryo" refers to an embryo of an immature seed that is in the process of ripening after pollination. The stage (ripening stage) of the immature embryos used in the method of the present invention is not particularly limited and may be collected at any time after pollination, but those 7 to 21 days after pollination are used. preferable. "Ripe seeds" are those that have completed the ripening process after pollination and are fully ripe as seeds. "Fertilized egg" means a cell in which a sperm cell and an egg cell are fused. A "plant fertilized egg" is a fertilized egg cell isolated from a tissue containing a plant embryo sac, that is, a fertilized egg that has been fertilized and fertilized at the stage of a plant and isolated from the plant. May be good. Alternatively, an egg cell and a sperm cell may be isolated from a plant before pollination and fertilization, and then fused to produce and obtain a fertilized egg cell. A fertilized egg may be prepared and obtained by electrical fusion. Further, it may be a fertilized egg obtained by fusing egg cells and sperm cells of different kinds of plants. A "protoplast" is a cell from which a cell wall has been removed from a plant cell.

Culture Medium Containing Trehalose The method for culturing a plant includes culturing cells of a gramineous plant using a culture medium containing trehalose. Limited, in one embodiment, the culture of plant cells is carried out in the step of regenerating the plant from the immature embryo.

Hereinafter, an example of the mode of the method for culturing plant cells will be described. The culture medium for plant cells may be one that is usually used for culturing plant cells, and is not particularly limited. Non-limiting examples thereof include WLS medium, WLS-RES medium, MS medium, LS medium, N6 medium, B5 medium, R2 medium, and medium based on CC medium. These media may be concentrated from the basic concentration or diluted. The amount is preferably 1/10 to 2 times, more preferably 1/5 to 1 times.

In one embodiment, the basic medium is a WLS medium or a WLS-RES medium. These media have the following composition.

WLS medium: 1 x LS major inorganic salt, 100 μM FeEDTA, 1 x LS minor inorganic salt, 1 x MS vitamin, 0.5 mg / L 2,4-D, 2.2 mg / L picrolam, 4% maltose monohydrate , 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl _2, 0.195% MES, 57 μM ascorbic acid, 5 μM AgNO ₃ , 5 g / L agarose, pH 5.8.

WLS-RES medium (medium in which 2.2 mg / l picrolam of WLS medium is replaced with 2.5 mg / l dicamba): 1 × LS major inorganic salt, 100 μM FeEDTA, 1 × LS minor inorganic salt, 1 × MS vitamin, 0 .5 mg / L 2,4-D, 2.5 mg / L medium, 4% maltose monohydrate, 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl _2, 0.195% MES, 57 μM ascorbic acid, 5 μM AgNO ₃ , 5 g / L agarose, pH 5.8.

"Trehalose" is a type of disaccharide in which glucose is 1,1-glycosidic bonded. Trehalose is added to the appropriately prepared culture medium. The concentration of trehalose added to the culture medium is not particularly limited. In one embodiment, the concentration of trehalose in the culture medium is 20 mM or more, 50 mM or more, 75 mM or more, 100 mM or more, 125 mM or more, 150 mM or more, 180 mM or more, 200 mM or more, 220 mM or more, 240 mM or more, 250 mM or more. In one embodiment, the trehalose concentration in the culture medium is 1000 mM or less, 800 mM or less, 600 mM or less, 500 mM or less, 450 mM or less, 400 mM or less, 350 mM or less, 300 mM or less, 280 mM or less. Not limited, the concentration of trehalose in the culture medium is 20 mM or more and 1000 mM or less, 50 mM or more and 800 mM or less, 100 mM or more and 600 mM or less, and 150 mM or more and 400 mM or less.

In one embodiment, the culture medium containing trehalose does not contain a carbon source (for example, sucrose or maltose) used in a conventional method, or may have a reduced content, instead of containing trehalose. In one embodiment, the culture medium contains no carbon source other than trehalose. The carbon source means sucrose, maltose, glucose and the like. In one aspect, the carbon source means maltose. However, the case where the only sugar in the medium is trehalose cannot be used for a long period of time. In other words, in one embodiment, long-term cultures on carbon sources containing no other than trehalose are excluded. Non-limitingly long-term culture means, for example, 6 days or more, 7 days or more, 8 days or more, 10 days or more, 17 days or more.

The concentration of trehalose can be grasped as the content per unit volume of the medium when the trehalose is not always uniformly contained in the whole medium, for example, when the medium is a solid.

In the culture step, the time when trehalose is added to the medium is not particularly limited. The addition time may be at or after the start of culturing. In one embodiment, without limitation, the culture medium containing trehalose is used at any time of the step of culturing the plant cells until the time when the plant cells have the ability to regenerate and differentiate. In one embodiment, the culture medium containing trehalose is used for a short period of time after the immature embryos are prepared, for example, within 16 days, within 8 days, within 5 days, within 3 days, or within 2 days. In one embodiment, the culture medium containing trehalose is used for the period from the preparation of immature embryos to the introduction of the substance into the plant. During the culture period, trehalose may be added to the culture medium at one time or in divided doses (for example, several times). In one embodiment, in a culture involving substance introduction with a particle gun, an immature embryo and a cell group derived from the immature embryo are placed and cultured on a culture medium containing trehalose from 1 day or 2 days before the particle gun treatment. Is good. In addition, in one embodiment, in a culture involving introduction of a substance with agrobacterium, when a cell group derived from an immature embryo is used as a material, the day before the agrobacterium inoculation treatment, one day before, or two days after the inoculation. It is better to place them on a culture medium containing trehalose and cultivate them. It is good to place it on the bed and inoculate it.

In one embodiment, the medium may include, but is not limited to, a plant growth regulator. Plant growth regulators are auxins. Specifically, for example, as auxins, indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), picrolam, daikamba, naphthalene acetic acid (NAA), naphthoxyacetic acid, phenylacetic acid, for example. , 2,4,5-Trichlorophenoxyacetic acid or other auxins can be added. In one embodiment, the auxins are plant hormones selected from the group consisting of 2,4-D, IAA, NAA. Alternatively, other plant growth regulators such as kinetin and cytokinins such as 4PU can be added. These plant growth regulators may be used alone or in admixture of a plurality at different concentrations.

In a uniform state, feeder (nurse) cells may be added to the culture medium. The type of feeder cell is not particularly limited. For example, liquid cultured cells of rice, liquid cultured cells of wheat, and the like can be used. It is possible to use known feeder cells and plant explants. Examples thereof include rice suspension cell culture (Oc, RIKEN BioResource Research Center) and wheat suspension culture cells (HT3-1). Examples of the plant explants include ovaries. The timing of adding the feeder cells is not limited, and may be before, at the start, or after the start of culturing the plant cells.

The culture temperature can be appropriately selected, preferably 18 ° C.-35 ° C., more preferably 20-30 ° C., and most preferably 23-28 ° C. Further, the culture in this step is preferably performed in a dark place or a dim place, but the culture is not limited to this.

In one embodiment, the above-mentioned culturing method includes culturing plant cells of the Gramineae family to obtain a plant cell group and / or a regenerated plant.

The cell group may be an embryoid body, a spherical embryo, a callus, or the like. The process of inducing division of a fertilized egg and causing cell proliferation to form an embryoid body, a spherical embryo, a callus, or the like is not particularly limited because the optimum conditions differ depending on the plant.

"Obtaining a plant regeneration body" will be described in detail in "2. Plant regeneration method".

The present invention is also used in a culture medium containing trehalose for culturing grass cells, use of trehalose in a culture medium for grass cells, and a culture medium for grass cells. Also includes trehalose.

In one embodiment, the above-mentioned culturing method improves the culturing efficiency of plant cells as compared with the case of culturing in a medium containing no trehalose. The culture efficiency of plant cells is not limited, and means, for example, the callus formation rate in culture.

Non-limitingly, the culture efficiency (for example, callus formation rate) is improved to, for example, 1.1 times or more, 2 times or more, 3 times or more, 5 times or more, 10 times or more, and 30 times or more. "Improved culture efficiency" includes the case where callus formation occurs in Gramineae varieties in which callus was not formed when cultured in a medium containing no trehalose.

2. 2. Method for Regenerating Plants The present invention relates to a method for regenerating plants. The regeneration method of the present invention is a method for regenerating a plant from cells of a gramineous plant.
(1) The cells of Gramineae plants are cultured using a culture medium containing trehalose, and then
(2) Culturing in redifferentiation medium,
Including that.

The meanings of terms such as "plant", "grass", "cells (of grass)", "culture medium", and "trehalose" are as described in "1. Method for culturing plant cells". ..

The redifferentiation step, that is, the culture step in the redifferentiation medium is not particularly limited. The cultured cell group is transferred to an arbitrary medium, for example, a redifferentiation medium consisting of the LSF medium described in the examples of the present specification (for example, LSFCuP5 (—C) medium), and cultured. Non-limitingly, the redifferentiation step may be performed by irradiating with light. The redifferentiation medium may be, for example, LSF medium, MS medium, B5 medium, N6 medium, and may be a liquid medium containing no solid medium using a solidifying material such as agarose, agar, gellan gum, and gellite.

A plant growth regulator containing auxin and cytokinin may be added to the redifferentiation medium. In one embodiment, the redifferentiation medium comprises auxins. The meanings of terms such as "auxin" and "cytokinin" are as described in "1. Plant cell culture method".

By transplanting an individual whose roots, shoots, etc. have been redifferentiated by the redifferentiation step into a pot containing soil, it can be grown as a normal plant individual.

Gramineae plants (regenerated plants) regenerated by the above plant regeneration method are also provided. The regenerated plants include plants regenerated by culturing immature embryos and redifferentiation, cells, tissues, etc. obtained from the plants, and "T0 generation" which is the redifferentiation generation obtained by the above-mentioned plant regeneration method. Includes progeny plants such as "T1 generation" derived from the self-fertilized seeds of T0 generation plants, hybrid plants crossed with them as one parent, and progeny plants.

Prior to the present invention, it was not possible to efficiently cultivate species and varieties of Gramineae plants, which are considered to be "difficult to culture", and it was difficult or impossible to obtain regenerated plants. The above-mentioned plant regeneration method has made it possible to efficiently cultivate such plants and varieties by a simple method and obtain regenerated plants.

3. 3. Introduction of substances into plants The genetic recombination technology for plants, especially monocotyledonous plants, has rapidly become widespread since the development of methods using Agrobacterium in rice and maize in the 1990s. To date, various transformation methods have been developed.

In recent years, it has become possible to efficiently edit the genome, but the fact that the ease of tissue culture differs depending on the crop species and varieties has a great influence on the efficiency of genome editing (genome editing efficiency). Therefore, it hinders practical use.

According to the method for culturing plant cells and the method for regenerating plants of the present invention, it has become possible to stably and efficiently obtain regenerated plants from the cell group obtained by culturing cells of grasses. By introducing a substance into a plant cell at any stage of the steps of the method for culturing the plant cell and the method for regenerating the plant cell, the regenerated plant into which the substance is introduced can be stably and efficiently obtained. In one embodiment, the transformant is introduced into the immature embryo.

In one embodiment, the plant cells of the above-mentioned culture method and plant regeneration method are plant cells into which a substance has been introduced. In one embodiment, the plant cells of the culture method and the plant regeneration method of the present invention include a step of introducing a substance selected from the group consisting of nucleic acid, protein and nucleic acid protein complex into cells of grasses.

The substance to be introduced is selected from the group consisting of nucleic acid, protein and nucleic acid-protein complex without limitation. The nucleic acid is not particularly limited, and may be DNA, RNA, a combination of both, or a mixture. It is preferably a circular DNA such as a vector, a linear DNA, a circular RNA or a linear RNA. Any length can be used depending on the transformation method used. Protein is a general term for molecules in which various amino acids are linked in a fixed order by amide bonds (also referred to as "peptide bonds"). The protein contains a nuclease such as Cas9 nuclease for genome editing, a modifying enzyme, an antibody and the like. A nucleic acid-protein complex is a complex formed by a nucleic acid and a protein. For example, deoxyribonuclear protein (complex of DNA and VirD2 protein, etc.), ribonuclear protein (complex of guide RNA and Cas9 protein, etc.) and the like are included.

Two or more types of nucleic acids, proteins and nucleic acid-protein complexes may be introduced. The nucleic acid may be two or more types of DNA or RNA, or a combination of DNA and RNA. Different types of substances such as nucleic acids and proteins may be introduced.

The method for introducing a substance into a plant is not particularly limited as long as it is a known method capable of introducing a desired substance into a plant, and can be appropriately selected depending on the type of plant. For example, a physicochemical method (direct DNA introduction method) such as a particle gun method, a polyethylene glycol method (PEG method), an electroporation method, a microinjection method, a whisker method, or a biological method such as an Agrobacterium method (agrobacterium method). The method of indirect introduction of DNA) can be preferably used. In one embodiment, the substance is introduced by the particle gun method or the Agrobacterium method.

The introduction of the substance into the plant can be performed, for example, by the method described in International Publication WO2017 / 171092 (Patent Document 2) and International Publication WO2018 / 143480 (Patent Document 3) without limitation.

In one embodiment, transformation or genome editing may be performed by introducing a substance.
Genome editing is a technique for modifying a target gene as desired by using a site-specific nuclease. Known site-specific nucleases include ZFN (Zinc finger nuclease), TALEN, and CRISPR / Cas9 (Crisper Cas9), which were developed and discovered after 2005.

Tissue culture of gramineous plants, for example, wheat and production of regenerated bodies are necessary techniques in the development of wheat varieties. In particular, when producing recombinant wheat or genome-editing wheat, for example, immature embryos or cultured cells that have been gene-introduced by the Agrobacterium method or the particle gun method are tissue-cultured, and regenerated bodies are produced from callus. The method of acquisition is the standard method. In the genome editing production, instead of introducing DNA, immature embryos or cultured cells into which ribonucleoprotein (RNP) has been introduced may be cultured to obtain a regenerated body.

In one embodiment, the above-mentioned culturing method improves the efficiency of introducing a substance as compared with the case of culturing in a medium containing no trehalose. The introduction efficiency of a substance means, for example, a callus formation rate, a transformation rate (transformation frequency), a genome editing rate (genome editing frequency), and the like in culture without limitation.

In the present specification, "high efficiency of substance introduction" means that the target substance is introduced into plant cells with high efficiency, callus is induced with high efficiency from immature embryos, etc., and it is high from transformed callus. It is a concept that includes the fact that redifferentiation occurs with efficiency. Further, in the present specification, "improvement of introduction efficiency of a substance" means improvement of introduction efficiency of a target substance into plant cells, improvement of callus induction rate from immature embryos, etc., and improvement of callus induction rate from transformed callus. It is a concept that includes improving the efficiency of redifferentiation.

Non-limitingly, the introduction efficiency (for example, transformation efficiency) of a substance is, for example, 1.3 times or more, 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 10 times or more, 25 times. It will be improved to the above. "Improvement of substance introduction efficiency" includes cases where transformation is not possible when cultured in a medium containing no trehalose, or these can be achieved in grass varieties that cannot be genome-edited.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify or modify the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.

Example 1: Callus induction from immature wheat embryos (1) Preparation and culture of immature embryos Part of the methods of WO2011 / 013764 (Patent Document 1) and Ishida (2015) (Non-Patent Document 16) are modified to produce wheat. Immature embryos were prepared. Nine varieties of Fielder, Claire, Mace, JTX001, Cadenza, Paragon, JTX002, Cronox and Jugger were tested.

Immature wheat seeds about 14 days after flowering were demineralized, surface sterilized in 70% ethanol for 45 seconds, and then surface sterilized with 1% sodium hypochlorite containing 1 drop of Tween 20 for 10 minutes. The treated immature seeds were washed 4 times with sterile water, immature embryos were collected under a stereomicroscope and recovered in WLS-liq medium. The immature embryos were washed once with WLS-liq medium and then centrifuged at 5,000 × g at 4 ° C. for 10 minutes.

Twenty-five immature embryos are transferred to a petri dish, the hypocotyl is removed with a scalpel, and then placed on the following "WLS90-RES medium" or "WLS0 / 90-RES" with the scutellum side facing up at 25 ° C. The cells were cultured in the dark for 1 day. The obtained immature embryos were used for the particle gun treatment of (3).

The composition of each medium is as follows.
WLS-liq medium: 0.1 × LS major inorganic salt, 10 μM FeEDTA, 0.1 × LS minor inorganic salt, 0.1 × MS vitamin, 1% glucose, 0.05% MES, pH 5.8

WLS90-RES medium: 1 x LS major inorganic salt, 100 μM FeEDTA, 1 x LS minor inorganic salt, 1 x MS vitamin, 0.5 mg / L 2,4-D, 2.5 mg / L daikamba, 9% maltose water. Japanese product (250 mM), 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl _2, 0.195% MES, 57 μM ascorbic acid, 5 μM 5 g / L agarose, pH 5.8.

WLS0 / 90-RES medium: A medium in which maltose monohydrate of WLS90-RES medium is changed to trehalose dihydrate (250 mM) having a concentration of 9.46%.

(2) Construction and preparation of plasmids for the bar gene, Cas9 gene and sgRNA gene A plasmid containing the bar gene, Cas9 gene and sgRNA gene was constructed. The base sequence of the bar gene is shown in SEQ ID NO: 1. The bar gene was controlled by the 35S promoter, and Nos was used as the terminator. These were inserted into the pUC19 vector.

The nucleotide sequence of the SpCas9 (Cas9 derived from Streptococcus pyogenes) gene is shown in SEQ ID NO: 2. SEQ ID NO: 2 is Svitashev et al. It is quoted from the report (Non-Patent Document 17) of (2015). SpCas9 is inserted between the corn ubiquitin promoter with the first intron of corn ubiquitin and the Nos and 35S terminators, and in addition, the SV40-derived nuclear localization signal (9 amino acids) and carboxyl terminus (C) on the amino-terminal (N-terminal) side. A plasmid inserted into the pUC19 vector was constructed so that the (terminal) side contained a sequence encoding a virD2-derived nuclear localization signal (18 amino acids). The base sequence of the sgRNA transcription unit is shown in SEQ ID NO: 3. A plasmid was constructed by inserting the sgRNA gene sequence containing the promoter and terminator of wheat U6 polymerase III into the pUC18 vector. The target sequence of the wheat LOX2 gene was used as the sgRNA gene sequence (Non-Patent Document 18).

(3) DNA introduction by particle gun and subsequent culture The method of introducing DNA by verticle gun into wheat is basically Barcelo and Lazzeri (1995) (Non-Patent Document 19) and Rasco-Gaunt (2001) (Non-patent). The method of Document 20) was referred to.

15 mg of gold particles (manufactured by BIO-RAD, particle size 0.6 μm) were separated into 1.5 ml tubes. 0.5 ml of 70% ethanol was added to the above-mentioned tube, ultrasonic waves were performed for 90 seconds, and then shaking was performed for 15 minutes. Centrifuge for 5 seconds and discard the supernatant. It was suspended in 0.5 ml of sterile water, vortexed for 1 minute, and the supernatant was removed after centrifugation. The above process was repeated 3 times. After suspension in 0.375 ml of 50% glycerol (40 mg / ml), it was stored as a gold particle suspension at 4 ° C.

The prepared gold particle suspension (40 mg / ml) was vortexed for 5 minutes until it became uniform. The plasmid DNA was coated on gold particles and adhered to microcarriers. After mixing 20 μl (0.8 mg) of gold particles and the plasmid DNA (bar gene 680 fmol, Cas9 gene 204 fmol and sgRNA gene 204 fmol) prepared in (2), water was added to make a total of 40 μl, and then TransIT-. 2020 (registered trademark) (0.8 μl) was added. Then, after vortexing continuously for 2 to 3 minutes, the tube was allowed to stand still for 1 minute. After flush (2s) centrifugation, the supernatant was discarded and then washed with 56 μl of 70% ethanol. After flush (2s) centrifugation, the supernatant was discarded and washed with 56 μl of 100% ethanol. After centrifugation by flash (2s), the supernatant was discarded and then resuspended in 34 μl of ethanol (100%). After gently suspending by tapping, it was vortexed (2-3 seconds). Then, it was treated with an ultrasonic cleaner for 30 seconds.

For 25 immature embryos prepared as described in (1), a gene was used with a particle gun (BioLISTIC PDS1000 / He, Bio-Rad) according to the Biolistic PDS-1000 / He Particure Delivery System instruction manual (BIO-RAD). Introduced. 5 μl of gold particle suspension was used per shot. The target distance was 5 cm from the stopping plate, and the shooting pressure was 650 psi. After the particle gun treatment, immature embryos on the plate medium were cultured at 28 ° C. for 1 day (dark place) and then at 25 ° C.

The table below summarizes the number of days, medium, and experimental content (process) of each process from immature embryo preparation to leaf sampling.

Composition of WLS60-P5: 1 × LS major inorganic salt, 100 μM FeEDTA, 1 × LS minor inorganic salt, 1 × MS vitamin, 0.5 mg / L 2,4-D, 2.2 mg / L picrolam, 6% maltose 1 Hydrate, 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl ₂ , 0.195% MES, 57 μM ascorbic acid, 5 μM AgNO ₃ , 5 mg / L phosphinotricin, 5 g / L agarose, pH 5 8.8.

Composition of WLS-P10: 1 × LS major inorganic salt, 100 μM FeEDTA, 1 × LS minor inorganic salt, 1 × MS vitamin, 0.5 mg / L 2,4-D, 2.2 mg / L picrolam, 4% maltose 1 Hydrate, 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl _2, 0.195% MES, 57 μM ascorbic acid, 5 μM AgNO ₃ , 10 mg / L phosphinotricin, 5 g / L agarose, pH 5 8.8.

Composition of LSCFCuP5 (-C): 1 x LS major inorganic salt, 100 μM FeEDTA, 1 x LS minor inorganic salt, 1 x LS vitamin, 0.2 mg / L IBA, 10 μM CuSO ₄ , 1.5% sucrose, 0.05 % MES, 5 mg / L phosphinotricine, 3 g / L gelite, pH 5.8.

(4) Results (i) Effect of trehalose-added medium on callus formation rate and transformation rate In (3), the callus formation rate and transformation rate of immature embryos introduced with DNA by a particle gun were examined.

The callus formation rate was determined by (number of callus generated / number of immature embryos under test) × 100 (%) in step 6 of Table 1. For the transformation rate, in step 7 of Table 1, callus was replanted in a medium containing phosphinotricin for selection by the selection marker bar gene and cultured (number of premature embryos tested in which regenerated individuals were generated /). It was determined by the number of immature embryos to be tested) × 100 (%). The results are shown in Table 2.

As shown in Table 2, in the nine varieties tested, Fielder, Claire, Mace, JTX001, Cadenza, Paragon, JTX002, Cronox and Jagger, in which DNA was introduced by a particle gun in (3), from the standard medium containing maltose. However, the callus formation rate (%) was significantly improved in the trehalose medium. Furthermore, in 7 varieties, Claire, Mace, JTX001, Cadenza, Paragon, JTX002 and Cronox, the transformation rate (%) was significantly improved in the trehalose medium as compared with the standard medium containing maltose.

(Ii) Effect of trehalose-added medium on genome editing rate In (3), the genome editing rate was investigated for immature embryos that had undergone DNA introduction by a particle gun. The genome editing rate was investigated as follows, and was determined by (number of immature embryos tested / number of immature embryos tested) x 100 (%).

Approximately 85 days after the particle gun treatment, about 5 to 10 mg of leaves that had grown to about 10 cm to 15 cm were cut out and sampled in the plant box. The leaf sample was frozen in liquid nitrogen, and the frozen leaf sample was crushed using TissueLyser II (QIAGEN). Then, according to the protocol of QIAcubeHT (QIAGEN), DNA was extracted by QIAcubeHT. PCR / RE analysis was performed on the TaLOX2 genes of the A, B, and D genomes targeted for genome editing, and mutations due to genome editing were detected.

For the PCR reaction, 10 × ExTaq Buffer 2 μl, dNTP Mixture (2.5 mM each) 1.6 μl, primer LOX2-F (10 μM) 0.4 μl, primer LOX2-R (10 μM) 0.4 μl, in 20 μl reaction solution, ExTaqHS 0.1 μl and genomic DNA 4 μl were mixed, and 98 ° C. for 10 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute were performed 30 times. As the PCR primer, the primer of Zhang et al. (Non-Patent Document 18) was used. Take 5 μl of the obtained PCR product, prepare a 20 μl solution containing 2 μl of 10 × NEB CutSmart buffer and 1 μl of the restriction enzyme SacI-HF (20 U / μl), incubate at 37 ° C. for 3 hours, and then 1.5% TypeII. Analysis was performed by electrophoresis using an agarose gel. Fielder's genomic DNA was used for the negative control. When the PCR product was cleaved by the restriction enzyme SacI, it was determined that the genome was not edited, and when it was not cleaved, it was determined that the genome was edited.

The results are shown in Table 3.

As shown in Table 3, in the five test varieties, Claire, Mace, JTX001, Paragon and JTX002, the genome editing rate was significantly improved in the trehalose medium as compared with the standard medium containing maltose.

Example 2: Effect of maltose / trehalose ratio on callus induction from wheat immature embryos (1) Preparation and culture of immature embryos Immature embryos of wheat (variety: Fielder) were prepared in the same manner as in Example 1. As the initial culture medium, WLS90-RES medium, or various trehalose-modified WLS media (WLS72 / 18-RES medium, WLS60 / 30-RES medium, WLS30 /) in which all or part of the maltose of WLS-RES medium is replaced with trehalose. 60-RES medium, WLS18 / 72-RES medium and WLS0 / 90-RES medium) were used to culture immature embryos as shown in Table 4.

WLS90-RES medium: 1 x LS major inorganic salt, 100 μM FeEDTA, 1 x LS minor inorganic salt, 1 x MS vitamin, 0.5 mg / L 2,4-D, 2.5 mg / L daikamba, 9% maltose hydrate Japanese product (250 mM), 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl ₂ , 0.195% MES, 57 μM ascorbic acid, 5 μM 5 g / L agarose, pH 5.8 (maltose: trehalose = 10) : 0).

WLS72 / 18-RES medium: Change maltose monohydrate in WLS90-RES medium to 7.2% maltose monohydrate (200 mM) and 1.89% trehalose dihydrate (50 mM). Medium (maltose: trehalose = 8: 2).

WLS60 / 30-RES medium: A medium in which the maltose monohydrate of the WLS90-RES medium is changed to a maltose monohydrate (167 mM) having a concentration of 6% and a trehalose dihydrate (83 mM) having a concentration of 3.14%. (Maltose: trehalose = 6.7: 3.3).

WLS30 / 60-RES medium: A medium in which the maltose monohydrate of the WLS90-RES medium is changed to a maltose monohydrate (83 mM) having a concentration of 3% and a trehalose dihydrate (167 mM) having a concentration of 6.32%. (Maltose: trehalose = 3.3: 6.7).

WLS18 / 72-RES medium: Change maltose monohydrate in WLS90-RES medium to 1.8% maltose monohydrate (50 mM) and 7.57% trehalose dihydrate (200 mM). Medium (maltose: trehalose = 2: 8).

WLS0 / 90-RES medium: A medium in which the maltose monohydrate of the WLS90-RES medium is changed to trehalose dihydrate (250 mM) having a concentration of 9.46% (maltose: trehalose = 0:10).

The table below summarizes the number of days, medium, and experimental content (steps) of each step of preparing immature embryos, inducing callus, and counting callus.

(2) Effect of maltose / trehalose ratio on callus formation rate and plant regeneration rate In the same manner as in Example 1 (3), the callus formation rate was examined for immature embryos that had been DNA-introduced by a particle gun. The callus formation rate was determined by (number of callus generated / number of immature embryos under test) × 100 (%) in step 6 of Table 4.

As shown in Table 5, among the 6 varieties tested, 4 varieties of Mace, JTX001, Paragon and Cronox had a callus formation rate in the WLS0 / 90-RES medium rather than the standard medium containing maltose (WLS90-RES medium). (%) Has improved significantly. Furthermore, in the WLS30 / 60-RES medium and WLS18 / 72-RES medium of the cultivar Mace, a slight improvement in the callus formation rate was observed. Improvements in callus formation were also observed in Cronox's WLS60 / 30-RES medium and WLS30 / 60-RES medium.

Example 3: Effect of trehalose on callus induction and seedling formation from immature wheat embryos (1) Preparation and culture of immature embryos In the same manner as in Example 1, immature embryos of wheat (variety: Fielder) were prepared. As the initial culture medium, WLS40-RES medium, WLS90-RES medium, or WLS0 / 40-RES medium and WLS0 / 90-RES medium as trehalose-modified WLS medium were used for culturing.

WLS40-RES medium: 1 x LS major inorganic salt, 100 μM FeEDTA, 1 x LS minor inorganic salt, 1 x MS vitamin, 0.5 mg / L 2,4-D, 2.5 mg / L Daikamba, 4% maltose water Japanese product (111 mM), 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl2, 0.195% MES, 57 μM ascorbic acid, 5 μM 5 g / L agarose, pH 5.8.

WLS90-RES medium: 1 x LS major inorganic salt, 100 μM FeEDTA, 1 x LS minor inorganic salt, 1 x MS vitamin, 0.5 mg / L 2,4-D, 2.5 mg / L Daikamba, 9% maltose water Japanese product (250 mM), 500 mg / L glutamine, 100 mg / L casein hydrolyzate, 3.7 mM MgCl2, 0.195% MES, 57 μM ascorbic acid, 5 μM 5 g / L agarose, pH 5.8.

WLS0 / 40-RES medium: A medium in which maltose monohydrate of WLS40-RES medium is changed to trehalose dihydrate (111 mM) having a concentration of 4.2%.

WLS0 / 90-RES medium: A medium in which maltose monohydrate of WLS90-RES medium is changed to trehalose dihydrate (250 mM) having a concentration of 9.46%.

The table below summarizes the number of days, medium, and experimental content (steps) of each step of preparing immature embryos, inducing callus, forming regenerated bodies, and counting.

The callus formation rate was determined by (number of callus generated / number of immature embryos under test) × 100 (%) in step 4 of Table 6. The seedling rate was determined by counting the regenerated seedlings in step 6 of Table 6 (number of premature embryos tested / number of immature embryos tested) × 100 (%).

Table 7 shows the effect of the trehalose-added medium on the callus formation rate, and Table 8 shows the effect of the trehalose-added medium on the seedling rate.

As shown in Tables 7 and 8, in WLS0 / 40-RES medium, when the culture period was 1 day rather than 0 days (that is, when WLS0 / 40-RES medium was not used), the callus formation rate and The seedling rate was significantly improved. However, when cultured for 5 days or 16 days, the callus formation rate and the seedling formation rate were lower than those for 0 days. In the WLS0 / 90-RES medium, the callus formation rate and the seedling rate were significantly improved when the culture period was 0 days (that is, when the WLS0 / 90-RES medium was not used) and the culture period was 5 days.

Example 4: Callus induction from immature maize embryos The immature embryos of maize inbread B104 were aseptically collected, and Ishida et al. (2007) Agrobacterium tumefaciens was inoculated by the method of (Non-Patent Document 21). In order to increase the transformation efficiency before inoculation, heat treatment at 46 ° C. and centrifugation at 20,000 × g were performed (Non-Patent Document 4). In the agrobacterium strain, the T-DNA region of pLC41GWH of WO2014 / 157541 (Patent Document 4) was regulated by the corn ubiquitin promoter and intron, and the GUS gene (Pubi-Iubi-Icat GUS-) mediated by the catalase intron of Hima. Tonos), and LBA4404 (pLC41 GUS-bar) having a binary vector modified to T-DNA having a Bar gene (P35S-bar-T35S) controlled by the cauliflower mosaic virus 35S promoter, and WO2014 / 157541 (Patent Document 4). ) LBA4404 (pLC41 GUS-Bar :: pVGW9) having a booster vector was used.

In addition, the GUS gene (P35S-Icat GUS-Tnos) regulated by the cauliflower mosaic virus 35S promoter in the T-DNA region and mediated by the catalase intron of Hima, and the Bar gene (P35S-bar) regulated by the cauliflower mosaic virus 35S promoter. LBA4404 (pSB131) (Non-Patent Document 22) having a super binary vector having -Tnos) was also subjected to the test.

The immature embryos after inoculation were placed on the LS-AS medium and cultured at 25 ° C. in the dark for 7 days. For the trehalose-added medium, 2% sucrose in the LS-AS medium was reduced to 1.5%, 1% glucose was reduced to 0.75%, and 1.08% trehalose dihydrate was added.

After culturing in selective media LSD1.5A and LSD1.5B containing phospinothricin at 25 ° C. in the dark for about 1 month, immature embryos forming compact type I callus were counted. Table 9 shows the effects of the trehalose-added medium on the callus formation rate and the transformation rate.

In any of the tests 1 to 4, immature embryos were cultured in a coexisting medium to which trehalose was added and then selected and cultured as compared with immature embryos which were cultured in a control coexisting medium to which no trehalose was added and then subjected to selective culture. Embryos formed transformed callus that showed drug resistance with higher efficiency. From this, it was clarified that the effect of improving the transformation efficiency by trehalose can be seen even in maize subjected to gene transfer treatment by the Agrobacterium method.

According to the present invention, it has become possible to facilitate the cultivation of gramineous plants, which are conventionally said to be difficult to culture, and to efficiently obtain plant regenerated cells from cultured cells. By introducing a substance into a plant cell at any stage of the steps of the method for culturing the plant cell and the method for regenerating the plant cell, it is possible to stably and efficiently obtain the regenerated plant into which the substance has been introduced. By using this method, it becomes possible to efficiently produce transformed crops of grasses and genome-edited crops, which have been difficult in the past.

SEQ ID NO: 1 is the base sequence of the bar gene.
SEQ ID NO: 2 is the base sequence of the SpCas9 gene.
SEQ ID NO: 3 is the base sequence of the sequence of the sgRNA expression unit.

Claims (12)

1. A method for culturing plant cells, which comprises culturing cells of grasses using a culture medium containing trehalose.
2. The method according to claim 1, wherein the plant cells of the Gramineae family are cultured to obtain a plant cell group and / or a regenerated plant.
3. The method according to claim 1 or 2, wherein the gramineous plant is selected from the group consisting of rice, corn, wheat, barley, rye, and sorghum.
4. The method according to any one of claims 1-3, wherein the gramineous plant is wheat or corn.
5. The method according to any one of claims 1-4, wherein the cells of a gramineous plant are immature embryos, ripe seeds, fertilized eggs, callus, protoplasts, or a cell group derived from them.
6. The method according to any one of claims 1-5, wherein the cells of a gramineous plant are immature embryos or a cell group derived from the immature embryos.
7. The method according to any one of claims 1-6, comprising the step of introducing a substance selected from the group consisting of nucleic acid, protein and nucleic acid-protein complex into cells of Gramineae plants.
8. The method according to claim 7, wherein transformation or genome editing is performed by introducing a substance.
9. The method according to claim 7 or 8, wherein the substance is introduced by the particle gun method or the Agrobacterium method.
10. The method according to any one of claims 1-9, which improves the efficiency of culturing plant cells.
11. The method according to any one of claims 7-9, which improves the efficiency of introducing a substance.
12. It is a method of regenerating a plant from the cells of a gramineous plant.
    (1) The cells of Gramineae plants are cultured using a culture medium containing trehalose, and then
    (2) Culturing in redifferentiation medium,
    How to regenerate a plant, including that.