JP7331343B2 - Shoot rooting method - Google Patents

Description

The present invention relates to a method for rooting shoots.

Natural rubber (a type of polyisoprenoid) currently used in industrial rubber products is produced by cultivating rubber-producing plants such as Hevea brasiliensis of the Euphorbiaceae family and Ficus elastica of the mulberry family. It is obtained by biosynthesizing natural rubber in mammary duct cells of the plant and manually collecting the natural rubber from the plant.

Currently, Hevea brasiliensis is almost the only source of natural rubber for industrial use. It is also widely used in large quantities in various applications as a main raw material for rubber products. However, Hevea brasiliensis is a plant that can grow only in limited regions such as Southeast Asia and South America. Furthermore, the Hevea brasiliensis requires about seven years from planting until it becomes a mature tree from which rubber can be harvested, and the seasons in which rubber can be harvested are sometimes limited. Also, the period during which natural rubber can be collected from mature trees is limited to 20 to 30 years.

Demand for natural rubber is expected to increase in the future, mainly in developing countries, and there is concern about the depletion of natural rubber resources.

Under these circumstances, there is a movement to increase the production of natural rubber by Hevea brasiliensis. Hevea brasiliensis is grown by sowing seedlings, growing them, and then using them as rootstocks.

Also, grafting, a clonal propagation technique obtained from conventional cloned seedlings, can carry diseases carried by the original tree with it, and can propagate diseased seedlings.

Furthermore, scions are not true clones because they may be affected by the rootstock.

On the other hand, there is micropropagation as a method of propagating cloned seedlings using tissue culture. The micropropagation technique propagates seedlings in sterile tissue culture. Specifically, it is necessary to induce shoots by culturing tissues such as buds and stem ends collected from individual plants to be propagated, and finally root the shoots. In any tissue culture, a step of rooting shoots is required to produce cloned seedlings. Therefore, for plant species with a low rooting rate, such as woody plants, the low rooting rate poses a serious problem when attempting to commercially use cloned seedlings. Therefore, it is very important to improve the rooting rate of shoots.

For example, in Patent Literature 1, the rooting rate is improved by co-cultivating cultured tissue and roots, but it is desired to provide other methods.

JP 2005-102516 A

SUMMARY OF THE INVENTION It is an object of the present invention to solve the above problems and to provide a method for rooting shoots that can obtain a good rooting rate.

Rooting in tissue culture is generally said to require auxin-based plant hormones, and it has been common technical knowledge to culture shoots in a medium containing auxin-based plant hormones for a long period of time. However, the present inventors focused on the fact that the rooting ability may change depending on the method and period of addition of plant hormones, and investigated methods for efficiently rooting from shoots.
Therefore, in order to induce rooting from shoots, we investigated a method of efficiently supplying auxin-based plant hormones. As a result of intensive studies, the present inventors found that rooting induction efficiency is improved by culturing in a plant culture medium after immersing shoots in a solution containing auxin plant hormones, and completed the present invention. .

That is, the present invention provides a method for producing shoots, comprising a soaking treatment step of soaking shoots in an auxin solution containing an auxin-based plant hormone, and a culturing step of culturing the shoots soaked in the soaking treatment step in a rooting-inducing medium. Regarding the root method.

It is preferable to carry out the immersion treatment step for 24 to 168 hours.

The auxin plant hormones are preferably indole-3-butyric acid and 1-naphthaleneacetic acid.

The auxin plant hormone concentration in the auxin solution is preferably 15 mg/L or less.

Preferably, the auxin solution contains glutathione.

The glutathione is preferably reduced glutathione.

The concentration of glutathione in the auxin solution is preferably 10-500 μmol/L.

It is preferable that the shoot subjected to the dipping treatment step has a length of 10 to 100 mm.

The concentration of the plant hormone in the rooting induction medium is preferably 0.1 mg/L or less.

Preferably, the shoots are those of a woody plant.

Preferably, the shoot is a shoot of a plant belonging to the genus Hevea.

Preferably, the shoots are Hevea brasiliensis shoots.

The method for rooting shoots of the present invention comprises a soaking treatment step of soaking the shoots in an auxin solution containing an auxin-based plant hormone, and a culturing step of culturing the shoots soaked in the soaking treatment step in a rooting-inducing medium. Contains a good rooting rate.

The method for rooting shoots of the present invention comprises a soaking treatment step of soaking the shoots in an auxin solution containing an auxin-based plant hormone, and a culturing step of culturing the shoots soaked in the soaking treatment step in a rooting-inducing medium. include.
As a result, the rooting rate of shoots can be improved, so that, for example, cloned seedlings can be produced efficiently. In particular, it is possible to efficiently create cloned seedlings even for plants with a low rooting rate, such as woody plants.

As used herein, the term "shoot" refers to apical buds, axillary buds, adventitious buds, as well as buds differentiated from multiple buds or shoot primordia, and those in the elongated state of these buds.

Plants (shoot-derived plants) to which the method of the present invention can be applied are not particularly limited, but are preferably woody plants.
The woody plant is not particularly limited, and includes a wide range of types and varieties of deciduous trees and evergreen trees. In particular, rubber trees from which rubber can be collected as a resource are preferable, and Hevea brasiliensis ( Ficus carica, Ficus elastica, Ficus pumila L., Ficus erecta Thumb., Ficus ampelas Burm.f. ), Genus Ficus such as Ficus benguetensis Merr., Ficus irisana Elm., Ficus microcarpa L.f., Ficus septica Burm.f., Ficus benghalensis; Le ( Parhenium argentatum) is more preferred. Plants belonging to the family Euphorbiaceae, such as plants belonging to the genus Hevea, are more preferred, and plants belonging to the genus Hevea are particularly preferred. Among them, Para rubber tree (Hevea brasiliensis) is most preferable.

Materials for inducing the shoot include plant tissues such as petioles, leaf discs, hypocotyls of somatic embryos, nodes, axillary buds, and apical buds. Among these, tissues containing nodes, axillary buds, or apical buds are preferable because they can stably induce shoots. Specifically, the tissue derived from an adult tree, a young tree, a seedling, a cloned seedling, or a sterile seedling grown from a seedling in a test tube (a sterile seedling) can be used.

When using the above tissues derived from mature trees, young trees, saplings, or cloned seedlings, they can be used by sterilizing or sterilizing the surface after cutting them to the required size. When using the tissue derived from a sterile seedling grown from a seedling (sterile seedling), it can be used after being appropriately cut into a required size.

When using the above tissue derived from an adult tree, a young tree, a seedling, or a cloned seedling, the surface of the tissue is first washed before culturing in the induction medium described later. For example, it may be washed with polishing powder or washed with a soft sponge, but washing with running water is preferable. The washing water may contain about 0.1% by weight of a surfactant.

The tissue is then disinfected or sterilized. Sterilization or sterilization can be performed using well-known germicides and sterilizers, but ethanol, benzalkonium chloride, and aqueous sodium hypochlorite solution are preferred. After sterilization or sterilization treatment, it may be washed with sterilized water.

Specific examples of the cleaning, sterilization, or sterilization include the following procedures. After washing the surface of the tissue with running water, wash it with ethanol. Next, sterilize with an aqueous sodium hypochlorite solution while stirring as necessary. Then wash with sterile water.

Although the method of inducing shoots is not particularly limited, an example of the inducing step of inducing shoots from the tissue or the like will be described.

(Induction process)
In the induction step, the tissue is cultured in an induction medium containing plant hormones and a carbon source to induce and form shoots. The induction medium may be either liquid or solid, but solid culture is preferred because it facilitates the induction of shoots by inserting the tissue into the medium and culturing it. Also, when the induction medium is a liquid medium, static culture may be performed, or shaking culture may be performed.
In addition, when a tissue that has been sterilized or sterilized is used, it is preferable to cut the cut end and use it for culture in order to eliminate the influence of the sterilizing agent and the sterilizing agent.

Plant hormones (plant growth hormones) include, for example, auxin plant hormones and/or cytokinin plant hormones. Among them, it is preferable to use cytokinin plant hormones.

Auxin plant hormones include 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, indole-3-butyric acid, indole-3-acetic 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-phenylic acid, picloram, picolinic acid, etc. mentioned. Among them, 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid and indole-3-butyric acid are preferred, and 2,4-dichlorophenoxyacetic acid and 1-naphthaleneacetic acid are more preferred.

Cytokinin plant hormones include benzyladenine, kinetin, zeatin, benzylaminopurine, isopentenylaminopurine, thidiazuron, isopentenyl adenine, zeatin riboside, dihydrozeatin and the like. Among them, benzyladenine, kinetin, and zeatin are preferred, benzyladenine and kinetin are more preferred, and benzyladenine is even more preferred.

Carbon sources are not particularly limited, and include sucrose, glucose, trehalose, fructose, lactose, galactose, xylose, allose, talose, gulose, altrose, mannose, idose, arabinose, apiose, mannitol, sorbitol, xylitol, erythritol, maltose. and other sugars. Among them, sucrose is preferred.

Preferably, the induction medium further contains activated charcoal to prevent growth inhibitors from accumulating in the tissue. In addition, it is preferable to further contain silver nitrate in order to promote the formation of shoots. Additionally, coconut water (coconut milk) may be included to promote shoot formation.

Examples of the induction medium include White medium (described in Introduction to Plant Cell Engineering (Gakkai Shuppan Center) p20-p36), Heller's medium (Heller R, Bot.Biol.Veg.Paris 14 1-223 (1953)), SH medium (Schenk and Hildebrandt's medium), MS medium (Murashige and Skoog's medium) (described in Introduction to Plant Cell Engineering (Gakkai Publishing Center) p20-p36), LS medium (Linsmaier and Skoog's medium) (Introduction to Plant Cell Engineering (Gakkai Publication Center) Described on p20-p36), Gamborg medium, B5 medium (Introduction to plant cell engineering (Gakkai Publishing Center) Described on p20-p36), MB medium (Biotechnology in Agriculture and Forestry volume 5 (TreesII) Described on p222-245) A basal medium such as WP medium (Woody Plant: for woody plants) or a modified basal medium obtained by modifying the composition of the basal medium to which a plant hormone is added may be used. Among them, MS medium, B5 medium, WP medium, and MB medium to which plant hormones are added are preferable, and MS medium, MS-modified medium whose composition has been modified, MB medium, or MB-modified medium whose composition has been modified. A medium containing a plant hormone is more preferable.

When the induction medium is a solid medium, a solidifying agent may be used to solidify the medium. The solidifying agent is not particularly limited, and includes agar, gellan gum, agarose, gelrite, agar, phytagel and the like.

The composition and culture conditions of a suitable induction medium differ depending on the plant species and whether the medium is a liquid medium or a solid medium, but usually have the following composition (particularly in the case of rubber trees).

The concentration of the carbon source in the induction medium is preferably 0.1% by mass or more, more preferably 1.0% by mass or more, and even more preferably 3.0% by mass or more. The concentration of the carbon source is preferably 10% by mass or less, more preferably 9.0% by mass or less, and even more preferably 5.0% by mass or less. In this specification, the concentration of carbon source means the concentration of saccharides.

It is preferred that substantially no auxin-based plant hormone is added to the induction medium. Specifically, the concentration of the auxin-based plant hormone in the induction medium is preferably 1.0 mg/L or less, more preferably 0.1 mg. /L or less, more preferably 0.05 mg/L or less, particularly preferably 0.01 mg/L or less.

When the cytokinin plant hormone is added to the induction medium, the concentration of the cytokinin plant hormone in the induction medium is preferably 0.01 mg/L or more, more preferably 0.1 mg/L or more, and still more preferably 0.5 mg. /L or more, particularly preferably 0.8 mg/L or more, most preferably 3.0 mg/L or more. The concentration of the cytokinin plant hormone is preferably 8.0 mg/L or less, more preferably 7.0 mg/L or less, still more preferably 6.0 mg/L or less.
In particular, when benzyladenine is used as the cytokinin plant hormone, the concentration of benzyladenine is preferably 4.0 to 6.0 mg/L, most preferably 5.0 mg/L. On the other hand, when kinetin is used as the cytokinin plant hormone, the concentration of kinetin is preferably 0.8-1.2 mg/L, most preferably 1.0 mg/L.

The concentration of activated carbon in the induction medium is preferably 0.01 wt% or more, more preferably 0.03 wt% or more. The concentration of the activated carbon is preferably 1.0% by mass or less, more preferably 0.1% by mass or less.

The concentration of silver nitrate in the induction medium is preferably 0.1 mg/L or higher, more preferably 0.3 mg/L or higher, even more preferably 0.5 mg/L or higher. The silver nitrate concentration is preferably 5.0 mg/L or less, more preferably 3.0 mg/L or less.

The pH of the induction medium is preferably 4.0 to 10.0, more preferably 5.0 to 6.5, even more preferably 5.5 to 6.0.
In this specification, the pH of the solid medium means the pH of the medium to which all components except the solidifying agent have been added.

The induction step is usually performed under a controlled environment in which culture conditions such as temperature and lighting time are controlled. Cultivation conditions can be appropriately set. For example, the culture temperature is preferably 0 to 40.degree. C., more preferably 20 to 40.degree. C., and even more preferably 25 to 35.degree. Cultivation may be performed in a dark place or in a bright place. Light conditions include, for example, 12.5 μmol/m ² /s illumination and 14 to 16 hours of light. be done. Cultivation time is not particularly limited, but culturing for 1 to 10 weeks is preferable, and 3 to 5 weeks is more preferable.

In the case of a solid medium, the concentration of the solidifying agent in the induction medium is preferably 0.1% by mass or more, more preferably 0.2% by mass or more. The concentration of the solidifying agent is preferably 2.0% by mass or less, more preferably 1.1% by mass or less, and even more preferably 0.8% by mass or less.

Among the above conditions, the plant hormone is a cytokinin plant hormone (especially benzyladenine or kinetin), its concentration is 3.0 to 8.0 mg / L, and the culture temperature is 25 to 35 ° C. is particularly preferred.

As described above, it is possible to induce and form shoots by culturing the tissue in the induction medium.

(Immersion treatment step)
In the immersion treatment step, the shoots are immersed in an auxin solution containing auxin plant hormones.
Specifically, shoots (for example, about 2 cm shoot sections) obtained by the induction step or the like may be immersed in an auxin solution.
When the shoots are immersed in the auxin solution, it is preferable that the ends of the shoot segments, ie, the cut ends of the shoots, are immersed in the auxin solution.
In addition, when the shoots are immersed in the auxin solution, the shoots may be left standing or shaken.

The time for the immersion treatment step is preferably 24 hours or longer, more preferably 40 hours or longer, still more preferably 60 hours or longer, particularly preferably 70 hours or longer, preferably 168 hours or shorter, more preferably 150 hours or shorter. , more preferably 130 hours or less, particularly preferably 100 hours or less, most preferably 90 hours or less, and most preferably 80 hours or less. This gives a better rooting rate.

The immersion treatment process is preferably performed under a controlled environment in which temperature, illumination time, etc. are controlled. For example, the temperature at which the immersion treatment step is performed is preferably 0 to 40°C, more preferably 20 to 40°C, and even more preferably 25 to 35°C. The immersion treatment step may be performed in a dark place or in a bright place, but the light conditions include, for example, 5 to 20 μmol/m ² /s illumination and 14 to 16 hours of light. is mentioned.

The shoot to be subjected to the immersion treatment step is not particularly limited, and any shoot formed by any method can be used.
Also, the shoots to be subjected to the immersion treatment step are preferably pieces of shoots. For example, shoot segments cut from tissues such as axillary buds used as materials for inducing shoots are preferred.
It is also preferable to use shoots that have been cultured in an induction medium for 6 months or less.

The length of the shoot (shoot section) subjected to the immersion treatment step is preferably 10 mm or more, more preferably 15 mm or more, still more preferably 20 mm or more, preferably 100 mm or less, more preferably 80 mm or less, and even more preferably. is 50 mm or less. This gives a better rooting rate.

As the auxin-based plant hormone contained in the auxin solution, the same auxin-based plant hormone used in the induction medium can be used. Among them, indole-3-butyric acid and 1-naphthaleneacetic acid are preferred, and it is more preferred to use indole-3-butyric acid and 1-naphthaleneacetic acid in combination.

The concentration of the auxin plant hormone in the auxin solution is preferably 15 mg/L or less, more preferably 12 mg/L or less, preferably 1.0 mg/L or more, more preferably 3.0 mg/L or more, and even more preferably. is 5.0 mg/L or more, particularly preferably 8.0 mg/L or more. This gives a better rooting rate.
Here, when using a plurality of auxin-based plant hormones, the concentration of auxin-based plant hormones means the total concentration of auxin-based plant hormones.

When indole-3-butyric acid and 1-naphthaleneacetic acid are used together as auxin plant hormones,
The concentration of indole-3-butyric acid in the auxin solution is preferably 10 mg/L or less, more preferably 7.5 mg/L or less, still more preferably 6.0 mg/L or less, and preferably 1.0 mg/L or more. , more preferably 2.5 mg/L or more, still more preferably 3.0 mg/L or more, particularly preferably 4.0 mg/L or more,
The concentration of 1-naphthaleneacetic acid in the auxin solution is preferably 10 mg/L or less, more preferably 7.5 mg/L or less, still more preferably 6.0 mg/L or less, preferably 1.0 mg/L or more, More preferably 2.5 mg/L or more, still more preferably 3.0 mg/L or more, and particularly preferably 4.0 mg/L or more. This gives a better rooting rate.

The auxin solution may contain an auxin-based plant hormone, and the dispersion medium for dissolving the auxin-based plant hormone is not particularly limited. . Examples of isotonic solutions include liquids made to 0.01 to 7M, preferably 0.5 to 2M by adding inorganic salts such as KCl, NaCl, CaCl ₂ and MgCl ₂ . Buffers include phosphate buffers, Tris buffers, MES buffers, and the like. Examples of tissue culture media include the media described above. Among them, water is preferable because the effect can be obtained more preferably. That is, the auxin solution is preferably an aqueous solution obtained by dissolving the auxin plant hormone in water.

In addition to the auxin-based plant hormones, ingredients that can be added to the auxin solution are not particularly limited, but glutathione is preferred. This gives a better rooting rate.
Glutathione is a tripeptide composed of glutamic acid, cysteine and glycine as constituent amino acids, and may be reduced glutathione, oxidized glutathione (glutathione disulfide) or a mixture thereof, preferably reduced glutathione.

The concentration of glutathione in the auxin solution is preferably 10 μmol/L or more, more preferably 30 μmol/L or more, still more preferably 40 μmol/L or more, particularly preferably 60 μmol/L or more, most preferably 80 μmol/L or more, It is preferably 500 μmol/L or less, more preferably 400 μmol/L or less, still more preferably 300 μmol/L or less, particularly preferably 200 μmol/L or less, and most preferably 150 μmol/L or less. This gives a better rooting rate.

It is also possible to use plant hormones other than auxin plant hormones in the auxin solution. As the cytokinin plant hormone that can be used in the auxin solution, the same cytokinin plant hormone used in the induction medium can be used, but the auxin solution should not contain any plant hormone other than the auxin plant hormone. preferable.

The concentration of plant hormones other than auxin plant hormones in the auxin solution is preferably 2.0 mg/L or less, more preferably 1.0 mg/L or less, even more preferably 0.1 mg/L or less, and particularly preferably 0.1 mg/L or less. 08 mg/L or less, most preferably 0 mg/L. This gives a better rooting rate.

(Culturing process)
In the culturing step, the shoots immersed in the immersion treatment step are cultured in a rooting-inducing medium for rooting.
The rooting-inducing medium may be either liquid or solid, but solid culture is preferred because rooting is facilitated by inserting shoots into the medium and culturing. Moreover, when the rooting-inducing medium is a liquid medium, static culture may be performed, or shaking culture may be performed.

Shoots to be subjected to the culturing step are not particularly limited as long as they are immersed in the above immersion treatment step. Among them, shoots in which a mass of tissue is formed at the base of the shoots by soaking the shoots in the above soaking treatment step are preferable. Furthermore, it is more preferable to use shoots in which a tissue mass has already been formed at the base of the shoots for the dipping treatment step.

A rooting induction medium contains a carbon source.

The plant hormones used in the rooting induction medium are not particularly limited, and the same plant hormones (auxin plant hormones, cytokinin plant hormones) used in the induction medium can be used.

The carbon source used in the rooting induction medium is not particularly limited, and the same carbon sources as those used in the above induction medium can be used, but sucrose is particularly preferred. This gives a better rooting rate.

The rooting induction medium preferably further contains activated charcoal and silver nitrate in the same manner as the above induction medium. This gives a better rooting rate.

The rooting induction medium preferably further contains glutathione as well as the auxin solution. This gives a better rooting rate.
Glutathione may be reduced glutathione, oxidized glutathione, or a mixture thereof, but reduced glutathione is preferred.

As the rooting-inducing medium, the basal medium used as the above-mentioned inducing medium, or the same medium obtained by adding a carbon source to the base medium, such as a modified basal medium obtained by modifying the composition of the basal medium, can be used. However, among these, MS medium, B5 medium, WP medium, and MB medium to which a carbon source has been added are preferable, and MS medium, MS modified medium whose composition has been changed, MB medium, or its composition has been changed. A modified MB medium to which a carbon source has been added is more preferred, and a MB medium or a modified MB medium whose composition has been modified to which a carbon source has been added is even more preferred.

When the rooting-inducing medium is a solid medium, a solidifying agent may be used to solidify the medium. The solidifying agent is not particularly limited, and includes agar, gellan gum, agarose, gelrite, agar, phytagel and the like.

The composition and culture conditions of a suitable rooting induction medium differ depending on the plant species and whether the medium is a liquid medium or a solid medium. be.

The carbon source concentration in the rooting induction medium is preferably 0.1% by mass or more, more preferably 1.0% by mass or more. The concentration of the carbon source is preferably 10% by mass or less, more preferably 6.0% by mass or less.

The plant hormone concentration in the rooting induction medium is preferably 2.0 mg/L or less, more preferably 1.0 mg/L or less, still more preferably 0.1 mg/L or less, and particularly preferably 0.08 mg/L or less. , most preferably 0 mg/L. This gives a better rooting rate.

The concentration of activated carbon in the rooting induction medium is preferably 0.005% by mass or more, more preferably 0.008% by mass or more. The concentration of the activated carbon is preferably 1.0% by mass or less, more preferably 0.1% by mass or less.

The concentration of silver nitrate in the rooting induction medium is preferably 0.1 mg/L or more, more preferably 0.3 mg/L or more, still more preferably 0.5 mg/L or more. The silver nitrate concentration is preferably 5.0 mg/L or less, more preferably 3.0 mg/L or less.

The concentration of glutathione in the rooting induction medium is preferably 10 μmol/L or more, more preferably 30 μmol/L or more, still more preferably 40 μmol/L or more, particularly preferably 60 μmol/L or more, most preferably 80 μmol/L or more. preferably 500 μmol/L or less, more preferably 400 μmol/L or less, still more preferably 300 μmol/L or less, particularly preferably 200 μmol/L or less, and most preferably 150 μmol/L or less.

The pH of the rooting induction medium is preferably 4.0 to 10.0, more preferably 5.0 to 6.5, even more preferably 5.5 to 6.0.

The culturing step is usually carried out in a controlled environment in which culture conditions such as temperature and lighting time are controlled. Cultivation conditions can be appropriately set. For example, the culture temperature is preferably 0 to 40.degree. C., more preferably 20 to 40.degree. C., and even more preferably 25 to 35.degree. Cultivation may be performed in a dark place or in a bright place. Light conditions include, for example, 5 to 20 μmol/m ² /s illumination and 14 to 16 hours of light. be done. Cultivation time is not particularly limited, but culturing for 1 to 10 weeks is preferable, and 4 to 8 weeks is more preferable.

In the present specification, the culture time of the culture step is defined as the start of culture (0 hours) when the shoots (shoot segments) immersed in the rooting induction medium are transplanted, and the culture period is 3. Week 9 means 504 hours after the start of culture, and Week 9 means 1512 hours after the start of culture. The culture period is cumulatively added.

In the case of a solid medium, the concentration of the solidifying agent in the rooting induction medium is preferably 0.1% by mass or more, more preferably 0.2% by mass or more. The concentration of the solidifying agent is preferably 2.0% by mass or less, more preferably 1.1% by mass or less, and even more preferably 0.75% by mass or less.

Among the above conditions, it is preferable that the plant hormone concentration is low (substantially free) and glutathione is contained, and the plant hormone concentration is low (substantially free) and glutathione is contained. Containing is more preferable.

As described above, by culturing the shoots immersed in the immersion treatment step in the rooting-inducing medium, it is possible to root, and rooted shoots (herein, rooted shoots (also referred to as "seedlings") are obtained, and clonal seedlings, which are complete plants, are formed. The young plant may be directly transplanted into soil, or may be transplanted into soil after acclimatization. The method of acclimation is not particularly limited.
By repeatedly performing the induction step, immersion treatment step, and culture step using the cloned seedlings formed as described above, it is also possible to stably produce a large amount of cloned seedlings of excellent varieties.

EXAMPLES The present invention will be specifically described based on Examples, but the present invention is not limited to these.

Various chemicals used in the examples are collectively described below.
BA: benzyl adenine KI: kinetin silver nitrate: silver nitrate from Merck Co. Gelling agent (solidifying agent): Phytagel from Sigma-Aldrich
Disinfectant: Pyrad-Povidone (trade name) (povidone-iodine solution) manufactured by The United Drug

<Induction process>
Tissues containing axillary buds were collected from Hevea brasiliensis seedlings.
Next, the tissue containing the axillary bud collected from the seedling is washed with running water, further washed with 70% by mass ethanol, sterilized with an aqueous solution of sodium hypochlorite diluted to about 5 to 10% by volume, and washed with sterilized water. did.

Next, the sterilized tissue was inserted into an induction medium (solid medium) and cultured (induction step). The induction medium was prepared by adding 5.0 mg/L of benzyladenine, 1.0 mg/L of silver nitrate, 3.0% by mass of sucrose, and 0.05% by mass of activated carbon to MB medium, and adjusting the pH of the medium to 5.7. Thereafter, a gelling agent was added to 0.275% by mass, sterilized in an autoclave (121° C., 20 minutes), and cooled in a clean bench.

The Hevea brasiliensis tissue was inserted into an induction medium (solid medium) and cultured at a culture temperature of 28° C. under illumination of 12.5 μmol/m ² /s for 16 hours of light to induce shoots. In addition, the cells were transferred to an induction medium having the same composition every 4 weeks.
The shoots induced in the induction step were cut into pieces of about 20 mm, and all leaves were cut off to prepare shoot sections, which were used in the following Examples and Comparative Examples.
In addition, each example was implemented 10 times, respectively.

(Example 1)
The cut end of the shoot was immersed in an auxin solution (5.0 mg/L 1-naphthaleneacetic acid, 5.0 mg/L indole-3-butyric acid, 100 μmol/L reduced glutathione) for 72 hours (immersion treatment step, temperature: 28°C, 16 hours photoperiod under illumination of 12.5 μmol/m ² /s). Next, a hormone-free (phytohormone (concentration of 0 mg/L)) and cultured by inserting the cut surface of the shoot that had been immersed in the immersion treatment step into a solid medium (pH 5.7) (culture step). The culture was cultivated for 8 weeks at a temperature of 25-28° C. under illumination of 12.5 μmol/m ² /s, 16-hour photoperiod. Two weeks after the start of culture in the hormone-free medium, there were individuals in which roots could be confirmed, and they elongated by the eighth week, and well-formed lateral roots. After that, the plant was replanted in a pot containing potting soil, and acclimatization was performed, and acclimatization was also successful.
The auxin solution was prepared by dissolving the above components in distilled water.
In addition, the medium is prepared by adding each of the above components except the solidifying agent to the basal medium, adjusting the pH of the medium to 5.7, and then adding the solidifying agent to 0.275% by mass. It was prepared by sterilizing in an autoclave (121° C., 20 minutes) and cooling in a clean bench.
In addition, in the culturing step, subculture was performed by transplanting to a medium having the same composition every 3 weeks. Observations and photographs were taken every 1 week (168 hours) during the culture to confirm the state of root formation, root elongation, stem and sprout elongation, and the presence or absence of withered leaves. The growth condition and root formation condition were evaluated.

[Evaluation of rooting rate]
In the culture step, the number of individuals whose rooting was confirmed was counted from the 0th week to the 8th week after the start of culture.
Rooting rate = (number of individuals confirmed to have rooted at 0 to 8 weeks)/total number of individuals x 100

[Evaluation of habituation rate]
Of the individuals in which rooting was observed, acclimation treatment was started when the number of root tips of roots reached 5 or more and the total length of roots (total length of all roots) reached 10 cm or more.
From the start of the acclimatization treatment, the acclimatization was completed when the new leaves developed and turned from copper to green.
Habituation rate = (number of individuals for which acclimatization was completed)/(number of individuals for which acclimatization was started) x 100

〔comprehensive evaluation〕
Considering the rooting rate, the subsequent acclimatization rate, and the growth state of the above-ground part at the completion of acclimatization, it was judged whether or not the conditions were comprehensively suitable for rooting.
◎: 80% or more rooting induction rate, 75% or more acclimatization rate, good growth ○: 60% or more rooting induction rate, 60% or more acclimatization rate, good growth ×: No specified rooting induction rate, less than 10% acclimatization rate

(Example 2)
Same as Example 1, except that the auxin solution of Example 1 was changed to an auxin solution (5.0 mg/L 1-naphthaleneacetic acid, 5.0 mg/L indole-3-butyric acid, 50 μmol/L oxidized glutathione). did the same. As a result, there were individuals in which roots could be confirmed from 2 weeks after the start of culture in the hormone-free medium, and they elongated by the 8th week, and the formation of lateral roots was also observed. After that, the plant was replanted in a pot containing potting soil, and acclimatization was performed, and acclimatization was also successful.

(Example 3)
The procedure of Example 1 was repeated except that the auxin solution of Example 1 was changed to an auxin solution (5.0 mg/L 1-naphthaleneacetic acid, 5.0 mg/L indole-3-butyric acid). As a result, there were individuals in which roots could be confirmed from 2 weeks after the start of culture in the hormone-free medium, and they elongated by the 8th week, and the formation of lateral roots was also observed. After that, the plant was replanted in a pot containing potting soil, and acclimatization was performed, and acclimatization was also successful.

(Comparative example 1)
Solid medium containing 3.0 wt% sucrose, 0.01 wt% activated carbon, 1.0 mg/L silver nitrate, 0.275 wt% solidifying agent in MB basal medium containing 1.0 mg/L indole-3-butyric acid ( (pH 5.7) and cultured by inserting the cut surface of the shoot. The culture was cultivated for 8 weeks at a temperature of 25-28° C. under illumination of 12.5 μmol/m ² /s, 16-hour photoperiod. When root formation was confirmed in some individuals, the plants were transferred to a hormone-free medium and culture was continued. The plant with rooting was then acclimatized, but died.

(Comparative example 2)
MB basal medium containing 1.0 mg/L indole-3-butyric acid, 0.5 mg/l benzyladenine, 3.0% by mass sucrose, 0.01% by mass activated carbon, 1.0 mg/L silver nitrate, 0.275% by mass The cut surface of the shoot was inserted into a solid medium (pH 5.7) containing a solidifying agent and cultured. The culture was cultivated for 8 weeks at a temperature of 25-28° C. under illumination of 12.5 μmol/m ² /s, 16-hour photoperiod. When root formation was confirmed in some individuals, the plants were transferred to a hormone-free medium and culture was continued. The plant with rooting was then acclimatized, but died.

(Comparative Example 3)
Solid medium containing 3.0 wt% sucrose, 0.01 wt% activated carbon, 1.0 mg/L silver nitrate, 0.275 wt% solidifying agent in MB basal medium containing 5.0 mg/L indole-3-butyric acid ( (pH 5.7) and cultured by inserting the cut surface of the shoot. The culture was cultivated for 8 weeks at a temperature of 25-28° C. under illumination of 12.5 μmol/m ² /s, 16-hour photoperiod. When root formation was confirmed in some individuals, the plants were transferred to a hormone-free medium and culture was continued. The plant with rooting was then acclimatized, but died.

Table 1 shows evaluation results and the like in Examples and Comparative Examples.
In Table 1, plant age means the period of culture in the induction medium.

From Table 1, it can be seen that the examples including the immersion treatment step of immersing the shoots in an auxin solution containing an auxin-based plant hormone and the culturing step of culturing the shoots immersed in the immersion treatment step in a rooting-inducing medium were It was found that the root rate could be improved.
Further, by including a immersion treatment step of immersing the shoots in an auxin solution containing an auxin-based plant hormone, and a culture step of culturing the shoots immersed in the immersion treatment step in a rooting induction medium, the subsequent acclimation treatment It was also found that the success rate of

Claims (12)

an induction step of inducing and forming shoots by culturing tissue of a plant belonging to the genus Hevea in an induction medium containing a plant hormone and a carbon source;
an immersion treatment step of immersing the shoots obtained in the induction step in an auxin solution containing an auxin plant hormone;
a culturing step of culturing the shoots immersed in the immersion treatment step in a rooting induction medium;
A method for rooting a shoot of a plant belonging to the genus Hevea, wherein the concentration of auxin-based plant hormone in the auxin solution is 3.0 mg/L or more. 2. The method of rooting shoots according to claim 1, wherein said soaking treatment step is carried out for 24 to 168 hours. 3. The shoot rooting method according to claim 1 or 2, wherein the auxin plant hormones are indole-3-butyric acid and 1-naphthaleneacetic acid. 4. The method for rooting shoots according to any one of claims 1 to 3, wherein the concentration of the auxin plant hormone in the auxin solution is 15 mg/L or less. The method for rooting shoots according to any one of claims 1 to 4, wherein the auxin solution contains glutathione. 6. The method for rooting shoots according to claim 5, wherein the glutathione is reduced glutathione. 7. The method for rooting shoots according to claim 5 or 6, wherein the concentration of glutathione in said auxin solution is 10 to 500 μmol/L. The shoot rooting method according to any one of claims 1 to 7, wherein the length of the shoots subjected to the soaking treatment step is 10 to 100 mm. The method for rooting shoots according to any one of claims 1 to 8, wherein the concentration of the plant hormone in the rooting induction medium is 0.1 mg/L or less. 10. The method of rooting shoots according to any one of claims 1 to 9, wherein when the shoots are immersed in the auxin solution, the ends of the shoot segments are immersed in the auxin solution. The method for rooting shoots according to any one of claims 1 to 10, wherein the shoots are shoots of Hevea brasiliensis. The concentration of indole-3-butyric acid in the auxin solution is 1.0 mg/L to 10 mg/L, and the concentration of 1-naphthaleneacetic acid in the auxin solution is 1.0 mg/L to 10 mg/L. 12. The shoot rooting method according to any one of 11.