JP7463045B2 - Method for producing seedlings of Brassicaceae intergeneric hybrids and method for inhibiting waterlogging of adventitious shoots of Brassicaceae intergeneric hybrids - Google Patents

Description

The present invention relates to a method for producing seedlings of intergeneric hybrids of the Brassicaceae family and a method for inhibiting waterlogging of adventitious shoots of intergeneric hybrids of the Brassicaceae family.

In recent years, what is being demanded in the market is vegetables with new value. Patent Document 1 discloses new shoots of plants in the Cruciferae family, such as broccoli sprouts. These shoots contain a lot of sulforaphane glucosinolate (hereinafter referred to as "SGS"). When SGS is metabolized, it becomes sulforaphane. Patent Document 2 discloses "Hiperl" (registered trademark, Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 20555), a type of kale. This kale contains a lot of glucosinolates. As mentioned above, glucosinolates are precursors of sulforaphane. Patent Document 3 discloses the effects of sulforaphane, specifically its detoxifying and antioxidant effects.

When developing new vegetables, one of the candidates is the Brassica vegetables. The reason why Brassica vegetables are one of the candidates is because there are many vegetables that people have eaten before. Brassica vegetables include Brassica vegetables (e.g., Chinese cabbage, turnip, cabbage, broccoli, kale, etc.), Radish vegetables (e.g., radish, etc.), Watercress vegetables (e.g., watercress, etc.), Atractylodes genus vegetables (e.g., arugula, etc.), and Wasabi vegetables (e.g., wasabi, etc.).

There are various methods for developing Cruciferous vegetables, including interspecific and intergeneric hybrids. Interspecific hybrids of the Brassicaceae family are widely used, such as rapeseed. For example, Patent Document 4 discloses the interspecific hybrid "Petit Vert" of the Brassicaceae family. On the other hand, intergeneric hybrids of the Brassicaceae family are not widely used.

Intergeneric hybrids of the Cruciferae family are not widespread because they are sterile. It is difficult to propagate plants that are sterile. One method to solve this problem is tissue culture. Tissue culture of cruciferous vegetables is rare. This is because many of the cruciferous vegetables that have been developed are interspecific hybrids. In other words, the general method of propagating cruciferous vegetables is seed propagation. To give an example of tissue culture, Patent Document 5 discloses a method of propagating wasabi seedlings. Patent Document 6 also discloses a method of culturing wasabi.

International Publication WO97/09889 International Publication WO2015/163442 JP 2009-148240 A JP 2009-213451 A JP 5-130815 A Patent No. 2632631

The problem that this invention aims to solve is to suppress waterlogging of adventitious buds of intergeneric hybrids of the Brassicaceae family. When propagating plants by tissue culture to produce seedlings, it is extremely important to suppress waterlogging of adventitious buds. This is because waterlogged adventitious buds stop growing and cannot be made into seedlings. In other words, waterlogging of adventitious buds reduces the proliferation efficiency of adventitious buds. A decrease in the proliferation efficiency of adventitious buds directly leads to a decrease in the production efficiency of seedlings.

Based on the above, the inventors of the present application have conducted extensive research and discovered that adjusting the concentration of cytokinins can suppress the waterlogging of adventitious buds. In other words, increasing the concentration of cytokinins increases the proportion of adventitious buds that become waterlogged, but waterlogged adventitious buds do not grow, which reduces proliferation efficiency. From this perspective, the present invention is defined as follows.

The method for producing seedlings of intergeneric Brassicaceae hybrids according to the present invention comprises at least the adjustment step. In the adjustment step, the concentration of cytokinins in the medium is adjusted, thereby suppressing the waterlogging of the cultivated adventitious shoots.

The method for producing seedlings of an intergeneric Brassicaceae hybrid according to the present invention comprises at least subculture. In the subculture, adventitious buds are placed on the medium and the concentration of cytokinins in the subculture medium is adjusted, thereby suppressing the adventitious buds from becoming waterlogged.

The method for inhibiting water-soaked adventitious buds of intergeneric hybrids of the Brassicaceae family according to the present invention comprises at least subculture. In the subculture, adventitious buds are placed on a substrate, and the concentration of cytokinins in the subculture medium is adjusted.

The present invention makes it possible to suppress the waterlogging of adventitious shoots of intergeneric hybrids of the Brassicaceae family. In other words, it makes it possible to increase the proliferation efficiency of adventitious shoots of intergeneric hybrids of the Brassicaceae family.

Flow diagram of the production method according to the present embodiment

<Brassicaceae intergeneric hybrids>
Brassicaceae intergeneric hybrid refers to a plant belonging to the Brassicaceae family, which is a plant that is produced by crossing plants belonging to different genera. Examples of plants belonging to the Brassicaceae family include Brassica plants (Chinese cabbage, turnip, bok choy, komatsuna, takana, mizuna, cabbage, broccoli, cauliflower, Brussels sprouts, kohlrabi, kale, etc.), Radish plants (radish, etc.), Nasturtium plants (watercress, etc.), Scutellaria plants (white mustard, etc.), Aleucanthemum plants (arugula, etc.), Horseradish plants (horseradish, etc.), and Wasabi plants (wasabi, etc.). Preferably, it is an intergeneric hybrid between a Brassica plant and a Radish plant, and more preferably, it is an intergeneric hybrid between kale and radish. Most preferred is Ministry of Agriculture, Forestry and Fisheries Variety Registration Application No. 33291 (name of application variety: Sainte Vert 48).

<Multiple shoots>
A multiple shoot is a plant tissue having multiple buds. Examples of the means for obtaining multiple shoots include cutting and culturing a terminal bud that grows at the tip of a plant stem, an axillary bud that grows in the axil of a plant leaf, or an adventitious bud that grows in another location.

＜Adventitious buds＞
The adventitious bud is a bud that grows in a place other than the tip of the stem or the axil of a plant. In the present invention, it particularly means an individual bud of a multiple shoot, because the multiple shoot obtained by culture does not have a stem or axil of a plant.

<Production method of seedlings>
Figure 1 shows the flow of the seedling production method (hereinafter referred to as "this production method"). This production method mainly consists of growing point extraction (S10), primary culture (S20), subculture (S30), and seedling production (S40). Here, the contents of "Vegetable Tissue and Cell Culture and Multiplication" (edited by the Latest Biotechnology Encyclopedia Editorial Committee) are appropriately incorporated for reference in this specification.

<Growth point extraction (S10)>
The growing point is extracted from the tissue containing the growing point. The purpose of extracting the growing point is to simplify the formation of multiple shoots. Since the growing point contains tissue with active cell division, multiple shoots can be easily formed. The growing point is preferably a shoot apical meristem. The tissue containing the growing point is not particularly limited. Examples of tissue containing the growing point include a terminal bud formed at the apex of the stem, an axillary bud (also called a lateral bud) formed at the leaf axil, and an adventitious bud possessed by multiple shoots. The tissue containing the growing point is sterilized by a known method as necessary. An example of a specific method of sterilization is immersion in a bactericide such as ethanol or hypochlorous acid. The concentration of the bactericide and the immersion time are appropriately set so that the tissue containing the growing point is appropriately sterilized. The bactericide may be used alone. A plurality of types of bactericide may be used in combination. When sterilizing the tissue containing the growing point, it is preferable to wash the tissue with sterile water or the like after sterilization. The extraction of the growing point is preferably performed under aseptic conditions. The size of the tissue to be excised may be any size as long as it includes the growing point, and the size of the tissue to be excised is preferably 0.1 mm to 1.0 mm.

<Primary culture (S20)>
The excised growing point is placed on a primary medium under sterile conditions and cultured. Multiple shoots are formed from the cultured growing point. The purpose of the primary culture is to obtain multiple shoots from the growing point. The primary medium may be a known medium for plant culture. Examples include MS medium, B5 medium, white medium, LS medium, etc. The primary medium may be a known medium composition for plant culture as is. The primary medium may be a known medium for plant culture that is appropriately diluted to change the concentration. The primary medium may be a solid medium or a liquid medium. The preferred is as follows. The primary medium contains at least sugars, a solidifying agent, and a plant hormone. More preferred is as follows. The sugars are sucrose, and the concentration is 10 g/L to 100 g/L. The solidifying agent is agar, and the concentration is 5 g/L to 15 g/L. The plant hormones are cytokinins and/or auxins, the concentration of cytokinins is 0.016 mg/L to 12,000 mg/L, and the concentration of auxins is 2,000 mg/L or less. Auxins may not be included. The culture conditions are sufficient as long as multiple shoots are formed. Preferably, the temperature is 20° C. to 30° C., the illuminance is 2,000 lux to 15,000 lux, and the day length is 12 hours to 16 hours. The adventitious shoots of the multiple shoots are excised under sterile conditions.

<Subculture (S30)>
The excised adventitious buds are placed on a subculture medium under sterile conditions and cultured. Multiple shoots are formed from the cultured adventitious buds. The purpose of subculture is to proliferate the adventitious buds. The subculture medium may be a known medium for plant culture. Examples include MS medium, B5 medium, white medium, LS medium, etc. The subculture medium may have the same composition as a known medium for plant culture. The subculture medium may be appropriately diluted to change the concentration. The subculture medium may be a solid medium or a liquid medium. The subculture medium contains cytokinins.

What is adjusted here is the concentration of cytokinins in the subculture medium. The purpose is to suppress the water-soaked adventitious buds. The concentration of cytokinins may be adjusted within a range in which the water-soaked adventitious buds are suppressed. The concentration is preferably 0.016 mg/L to 12,000 mg/L. More preferably, it is 0.016 mg/L to 6,000 mg/L. Even more preferably, it is 0.016 mg/L to 4,000 mg/L. Most preferably, it is 0.100 mg/L to 2,500 mg/L.

A second adjustment may also be made here. The size of the adventitious buds to be placed in the bed is adjusted in the second adjustment. The purpose is to prevent the adventitious buds from becoming waterlogged. The size of the adventitious buds may be adjusted within a range in which the adventitious buds are prevented from becoming waterlogged. The size is preferably 2.5 cm or less. More preferably, the size is 0.5 cm or more and 2.5 cm or less.

The subculture medium may contain, in addition to cytokinins, sugars, solidifying agents, and plant hormones other than cytokinins. Preferably, they are as follows. The sugars are sucrose, and the concentration is 10 g/L to 100 g/L. The solidifying agent is agar, and the concentration is 5 g/L to 15 g/L. The plant hormones other than cytokinins are auxins, and the concentration is 2,000 mg/L or less. Auxins may not be included. The culture conditions are sufficient as long as multiple shoots are formed. Preferably, the temperature is 20°C to 30°C, the illuminance is 2,000 lux to 15,000 lux, and the day length is 12 hours to 16 hours. The adventitious shoots of the multiple shoots are cut out under sterile conditions. The subculture process may be repeated until the required number of adventitious shoots are obtained.

<Waterlogging rate>
The waterlogging rate is the percentage of adventitious shoots that have become waterlogged and stopped growing among those placed on the subculture medium. Waterlogging (vitrification) is a phenomenon in which tissues become semi-transparent (also called vitrification). Waterlogged tissues often cause poor growth due to the lack of tissues that are normally found in tissues. Once waterlogged tissues are cultured, they rarely return to a normal state. In the propagation of plants by tissue culture, it is particularly important to know how to suppress this waterlogging.

In the present invention, it is preferable that the water-submersion rate is less than 30%. If the water-submersion rate is less than 30%, practical application is expected. In other words, water-submersion is suppressed, meaning that the water-submersion rate is less than 30%.

<Growth efficiency>
The proliferation efficiency is the value obtained by dividing the number of adventitious buds possessed by multiple shoots formed by subculture by the number of adventitious buds placed on the subculture medium. In other words, it indicates how many times one adventitious bud has proliferated in subculture. It is preferable that the proliferation efficiency is 2 times or more. If the proliferation efficiency is 2 times or more, practical application is expected. In other words, high proliferation efficiency means that the proliferation efficiency is 2 times or more.

<Seedling production (S40)>
The excised adventitious shoots are grown into seedlings. The purpose of the seedling process is to grow the shoots to a state where they can grow in an external environment. The seedling process may be a one-step process. The seedling process may be two or more steps. The preferred process is as follows:

First, the excised adventitious buds are cultured in a seedling medium under sterile conditions. The cultured adventitious buds are allowed to grow to a certain size. The seedling medium may be a known medium for plant culture. Examples include MS medium, B5 medium, white medium, LS medium, etc. The seedling medium may have the composition of a known medium for plant culture as is. The seedling medium may be appropriately diluted to change the concentration of a known medium for plant culture. The seedling medium may be a solid medium or a liquid medium. The following is preferable. The seedling medium contains at least sugars, a solidifying material, and a plant hormone. The following is more preferable. The sugars are sucrose, and the concentration is 10 g/L to 100 g/L. The solidifying material is agar, and the concentration is 5 g/L to 15 g/L. The plant hormones are auxins, and the concentration is 2.000 mg/L or less. Auxins may not be included. The culture conditions should be such that adventitious shoots can grow. Preferably, the temperature is 20°C to 30°C, the illuminance is 2000 lux to 15000 lux, and the day length is 12 hours to 16 hours. Alternatively, adventitious shoots may be rooted in a seedling medium.

Next, the adventitious shoots are removed from the seedling culture medium. The removed adventitious shoots are transplanted into a cultivation container filled with cultivation soil and allowed to grow. The cultivation soil may be organic or inorganic. For example, akadama soil, black soil, baked akadama soil, vermiculite, zeolite, montmorillonite, etc. These cultivation soils may be used alone or in combination. The means for growing the shoots may be a known method. For example, the temperature, humidity, light intensity, etc. may be appropriately adjusted, and fertilizer may be applied as necessary. The adventitious shoots are allowed to grow until they can grow in the external environment.

<Inorganic components>
Inorganic components are necessary for plant growth. Inorganic components are mixed into the medium. Examples of inorganic components include nitrogen, phosphorus, potassium, sodium, calcium, magnesium, boron, manganese, zinc, iodine, molybdenum, copper, cobalt, iron, chlorine, and sulfur. Inorganic components may be mixed and used. Commercially available mixed salts may be used.

<Sugars>
Sugars are components necessary for plant growth. Sugars are blended into the medium. Examples of sugars include sucrose, glucose, fructose, maltose, trehalose, lactose, galactose, xylose, mannitol, sorbitol, xylitose, erythritol, and the like. Sucrose, glucose, fructose, and maltose are preferred, and sucrose is more preferred. A single sugar may be used. A combination of multiple sugars may be used. Fruit juice, vegetable juice, and extracts containing these sugars may also be used.

<Solidification material>
The solidifying material is mixed into the medium to solidify the medium. Examples of the solidifying material include agar, gellan gum, and gellite. Agar is preferable. A single solidifying material may be used. A combination of a plurality of solidifying materials may be used. The solidifying material does not necessarily have to be mixed.

<Plant Hormones>
Plant hormones refer to natural or synthetic components that have the function of regulating plant growth. Examples include cytokinins and auxins. Examples of cytokinins include benzylaminopurine (benzyladenine), kinetin, zeatin, isopentenylaminopurine, thidiazuron, isopentenyladenine, zeatin riboside, and dihydrozeatin. Benzylaminopurine, kinetin, and zeatin are preferred, and benzylaminopurine and kinetin are more preferred. Examples of auxins include indole-3-acetic acid, indole-3-butyric acid, 1-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, indolepropionic acid, chlorophenoxyacetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4,5-trichlorophenoxyacetic acid, parachlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, 4-fluorophenoxyacetic acid, 2-methoxy-3,6-dichlorobenzoic acid, 2-phenylacetic acid, picloram, picolinic acid, etc. Preferred are indole-3-acetic acid, indole-3-butyric acid, 1-naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid, and more preferred is 1-naphthaleneacetic acid.

<pH>
The pH of the medium used in the present production method is a pH at which plants can grow, preferably 4.0 to 10.0, and more preferably 5.0 to 7.0.

<Relationship between adventitious bud size and waterlogging rate>
<Growth point removal>
A terminal bud was collected from a plant of an intergeneric hybrid (Japan Variety Registration Application No. 33291, application variety name: Sainte Vert 48) with kale as the pollen parent and radish as the stigma parent. To sterilize the terminal bud, the collected terminal bud was immersed in 70% ethanol for 5 seconds. Then, it was immersed in 0.2% sodium hypochlorite solution for 5 minutes. In a clean bench, about 0.5 mm of the tip (growth point) of the sterilized terminal bud was extracted.

<Primary culture>
The excised growth point was placed on a primary medium. The growth point placed on the primary medium was cultured under sterile conditions. The composition of the primary medium was MS medium containing 60 g/L sucrose, 8 g/L agar, 1.000 mg/L benzylaminopurine, and 0.020 mg/L naphthalene acetic acid at pH 5.8. The culture conditions were a temperature of 25°C, an illumination of 12,500 lux, and a day length of 16 hours. The growth point was cultured for one month to form multiple shoots. Adventitious shoots were excised one by one from the multiple shoots under sterile conditions.

<Subculture>
The excised adventitious buds were divided into those with a size of 2.5 cm or less and those with a size of 2.6 cm or more, and each was placed on a subculture medium. The adventitious buds placed on the subculture medium were cultured under sterile conditions. The composition of the subculture medium was MS medium containing 20 g/L sucrose, 12 g/L agar, and 0.050 mg/L naphthalene acetic acid at a pH of 5.8. The concentration of cytokinins in the subculture medium was 0.500 mg/L. The culture conditions were a temperature of 25°C, an illumination of 12,500 lux, and a day length of 16 hours. The adventitious buds were cultured for 3 weeks to form multiple shoots.

<Size of adventitious buds and waterlogging rate>
The relationship between the size of adventitious buds and the rate of water soaking is shown in Table 1. What can be seen from Table 1 is that the rate of water soaking changes depending on the size of the adventitious buds placed on the subculture medium. In other words, larger adventitious buds have a higher rate of water soaking, while smaller adventitious buds have a lower rate of water soaking. In other words, by adjusting the size of the adventitious buds placed on the subculture medium, it is possible to suppress the water soaking of adventitious buds.

<Relationship between cytokinin concentration and waterlogging rate>
<Primary culture>
This was carried out in the same manner as described above.

<Subculture>
Among the excised adventitious buds, those with a size of 2.5 cm or less were placed on a subculture medium. The adventitious buds placed on the subculture medium were cultured under sterile conditions. The composition of the subculture medium was MS medium containing 20 g/L sucrose, 12 g/L agar, and 0.050 mg/L naphthalene acetic acid at a pH of 5.8. The concentrations of cytokinins in sections 1 to 9 were as shown in Table 2. The culture conditions were a temperature of 25°C, an illuminance of 12,500 lux, and a day length of 16 hours. The adventitious buds were cultured for 3 weeks to form multiple shoots. From the multiple shoots, one adventitious bud was excised one by one under sterile conditions.

<Waterlogging rate and propagation efficiency>
Table 2 shows the water-soaking rate and proliferation efficiency for each concentration of cytokinins. It can be seen from Table 2 that if the concentration of cytokinins is 12,000 mg/L or less, the water-soaking rate is low. Conversely, if the concentration of cytokinins exceeds 12,000 mg/L, the water-soaking rate increases sharply, resulting in a low proliferation efficiency. Also, if the concentration of cytokinins is 0.016 mg/L or more, the proliferation efficiency is high. On the other hand, if the concentration of cytokinins is 0.002 mg/L or less, the water-soaking rate is low, but the number of adventitious buds possessed by the multiple shoots formed by subculture is reduced, resulting in a low proliferation efficiency.

From the above, by setting the concentration of cytokinins to 0.016 mg/L or more and 12 m.000 g/L or less during subculture, it is possible to increase the proliferation efficiency of adventitious shoots, leading to efficient seedling production.

<Repeated subculture>
The adventitious buds subcultured in sections 3 to 5 were subcultured two more times (three times in total). The culture conditions for each section were the same as those for the first subculture. The waterlogging rate and proliferation efficiency for the sections where subculture was repeated are shown in Table 3. It can be seen from Table 3 that waterlogging can be suppressed even with repeated subculture.

<Kinetin>
In sections 10 to 12, kinetin was used as the cytokinin. For sections 10 to 12, the waterlogging rate and proliferation efficiency were confirmed when subculture was repeated three times. The kinetin concentration in each section is shown in Table 3. Primary culture was performed in the same manner as described above. Subculture was performed under the same conditions as sections 1 to 9, except for the type and concentration of cytokinins. The waterlogging rate and proliferation efficiency in sections 10 to 12 are shown in Table 3. It can be seen from Table 3 that waterlogging can be suppressed regardless of the type of cytokinin.

<Seedling production>
Some of the adventitious buds were placed on a seedling medium. The adventitious buds placed on the seedling medium were cultured under sterile conditions. The composition of the seedling medium was MS medium containing 30 g/L sucrose, 10 g/L agar, and 0.500 mg/L naphthalene acetic acid at pH 5.8. The culture conditions were a temperature of 25°C, an illuminance of 12,500 lux, and a day length of 16 hours. The adventitious buds were cultured for two weeks to allow them to root. The rooted adventitious buds were transplanted into a cell tray filled with vermiculite. The cell tray to which the adventitious buds were transplanted was appropriately fertilized with diluted Hyponex to allow them to grow in a greenhouse. By allowing them to grow for one month, it was possible to produce seedlings that could be planted.

The field in which this invention is useful is seed production.

Claims (3)

A method for producing seedlings of intergeneric Brassicaceae hybrids, comprising at least the following steps:
Adjustment: what is adjusted here is the concentration of cytokinins in the medium, what is cultured in the medium are adventitious buds (excluding those generated from callus), the adjustment is carried out at the time of subculture, and what is thereby suppressed is the water-soaking of the cultured adventitious buds,
The concentration is adjusted in a range of 0.016 mg/L to 12.000 mg/L;
Second adjustment: what is adjusted here is the size of the adventitious buds, and the result of the adjustment is the adventitious buds to be used for culture;
The size adjustment range is 2.5 cm or less;
The Brassicaceae intergeneric hybrid is Sainte Vert 48 (Ministry of Agriculture, Forestry and Fisheries Variety Registration Application No. 33291). A method for producing seedlings of intergeneric Brassicaceae hybrids, comprising at least the following steps:
Subculture: In this method, adventitious buds (excluding those generated from callus) are placed on the subculture medium, and the concentration of cytokinins in the subculture medium is adjusted, thereby suppressing the water-soaking of the adventitious buds.
The concentration is adjusted in a range of 0.016 mg/L to 12.000 mg/L;
In the subculture, the size of the adventitious bud is further adjusted, and the result of the adjustment is the adventitious bud to be used for culture.
The size adjustment range is 2.5 cm or less;
The Brassicaceae intergeneric hybrid is Sainte Vert 48 (Ministry of Agriculture, Forestry and Fisheries Variety Registration Application No. 33291). A method for inhibiting waterlogging of adventitious shoots of intergeneric hybrids of the Brassicaceae family, comprising at least the following steps:
Subculture: In this method, adventitious shoots (excluding those generated from callus) are placed on the subculture medium, and the concentration of cytokinins in the subculture medium is adjusted.
The concentration range is 0.016 mg/L to 12.000 mg/L;
In the subculture, the size of the adventitious bud is further adjusted, and the result of the adjustment is the adventitious bud to be used for culture.
The size adjustment range is 2.5 cm or less,
The Brassicaceae intergeneric hybrid is Sainte Vert 48 (Ministry of Agriculture, Forestry and Fisheries Variety Registration Application No. 33291).

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