JPH05304848A - Method for raising young plant body derived from culture - Google Patents

Abstract

PURPOSE:To obtain a method for creating a plant seedling culturable without requiring a special germ-free space by transplanting a young plant body induced from a callus to a culture medium without containing a saccharide. CONSTITUTION:The objective method for creating a plant seedling is characterized by transplanting a young plant body to a culture medium without containing a saccharide and culturing the young plant body therein while aerating carbon dioxide at >=1vol./vol.% concentration in a method for culturing the young plant body induced from a callus in a vapor-phase space and creating the plant seedling.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing plant seedlings, more specifically, plant seedlings capable of growing large numbers of callus-derived seedlings easily without requiring a special sterile space. Regarding the production method of.

[0002]

2. Description of the Related Art In recent years, with the development of tissue culture technology, it has become possible to culture a large number of various kinds of plants, and a concrete method therefor is being sought. In particular, a method of inducing and multiplying callus from a plant tissue piece, inducing a young plant from the callus, and producing a plant seedling from them is attracting attention as a method capable of efficiently providing a plant seedling. ..

However, in general, callus-derived seedlings have a low photosynthetic ability and cannot themselves sufficiently produce the carbohydrates required for the growth of plant seedlings. Usually, as a carbon source used for the growth of plants, sucrose, glucose and other sugars are added to the seedling culture medium in a large amount in the range of 10 to 50 g / l, and the medium is substantially sterile. Plants of a certain size are grown in this culture system, transplanted to a conditioned medium and acclimated to obtain seedlings. In the conventional method of adding a large amount of sugars to such a medium, the medium itself is susceptible to contamination by external bacteria during the culture period, and when the medium is contaminated with the contamination, seedlings that are not resistant are easy to There is a problem of spoilage, and in order to prevent this, the culture environment such as the culture container and the culture room is maintained substantially sterile,
In addition, it has been required to be particularly careful when handling the same.

Moreover, it is considered that the photosynthetic ability of the induced seedlings is lowered due to the sugar in the medium, even though they are irradiated with light. Such a plant is considered to grow sufficiently in a seedling medium containing sugar, but in the normal seedling raising process, without photosynthesis, from an environment grown in a sterile culture system using only sugar as a carbon source. Suddenly, we were forced to perform photosynthesis in a natural environment exposed to various bacteria, and this change in the external environment and change in energy acquisition became a great stress, resulting in a decrease in the survival rate of seedlings and a growth bug. Was causing the problem of. As described above, raising seedlings under the sugar-containing condition is very costly for maintaining sterility.

In response to this, Yoshida et al.
2), 140-141 (1988), ibid. (Another 1) 62-63 (1989)], obtained 40% seedling rate when seedlings of encapsulated seedlings were aseptically grown in a medium containing sugar. However, the seedling rate in the open system is 10
It is as low as%. Furthermore, the growth in this case is
It also shows that it is extremely bad. Further, even a low-sugar-concentration seedling-growing medium substantially requires aseptic conditions, and therefore, establishment of a seedling-growing method using a completely sugar-free medium has been earnestly desired.

[0006] Furthermore, it is attempted to produce plant seedlings in an environment with good ventilation, and the concentration of carbon dioxide is adjusted to the atmospheric temperature (300 ppm).
Attempts have been made to increase the photosynthetic efficiency of plant seedlings by increasing the concentration to about 10 times higher than the The addition of sugar into itself is still necessary. Further, even a low-sugar-concentration seedling-growing medium usually requires an aseptic state, so that the above problems in terms of cost and the like remain substantially unsolved.

[0007]

Therefore, according to the present invention, seedlings derived from callus are cultivated in a completely sugar-free medium, which does not require a special sterile space and is a plant in a gas phase space in general. The challenge is to establish a method that makes it possible to provide a large amount of seedlings.

[0008]

Means for Solving the Problems As a result of intensive studies on the above problems, the present inventor has found that the problems can be solved by aeration of a large amount of carbon dioxide in the process of culturing the seedlings. That is, the present invention provides the following inventions. (1) In a method of cultivating a callus-induced seedling in a vapor phase space to produce a plant seedling, the seedling is transplanted into a sugar-free medium to obtain 1 v / v% or more of carbon dioxide. A method for producing plant seedlings, which comprises culturing under aeration of. (2) In a method of cultivating callus-induced seedlings in a gas phase space to produce plant seedlings, the washed seedlings are transplanted to a sugar-free medium to give a concentration of 1 v / v% or more. A method for producing plant seedlings, which comprises culturing under aeration of carbon dioxide.

The present invention will be described in detail below. A. The present invention is premised on culturing seedlings derived from plant-derived callus in a gas phase space. The type of the target plant is not particularly limited as long as it is possible to induce a callus and produce a plant seedling from the callus through a seedling.

For example, grasses such as rice and wheat; turfgrass such as bentgrass; fruit vegetables such as melon and tomato; flowering plants such as statice and lily; seedlings of trees such as birch and eucalyptus are widely used. In particular, rice is cultivated on about 40% of Japan's cultivated area, and is a major grain that is produced about 300 million a year worldwide, and it is an industrially inexpensive and large-scale seedling production method in tissue culture. Occupies an important position among the above-mentioned plants in that the establishment of the above is still required. When rice is selected, it does not matter what kind of rice it is. For example, Japonica, Indica, Javanica, African rice, or hybrids thereof can be used.

As a method for inducing callus from such a plant and a method for inducing a seedling from callus, a conventional method corresponding to the type of the plant can be used. The induction method in general is described in, for example, “Harada, Komamine et al., Encyclopedia of Plant Biotechnology (Asakura Shoten), 1990”. The rice induction method is described in detail in Japanese Patent Application No. 3-57333. In the case of inducing an industrial scale by tank culture plantlets of rice, after tank culture volumetric oxygen transfer coefficient (k _L a) as 0.01~1.00 (1 / min), the seedlings wherein the porting to Naeka medium, is a particular k _L a in the culture initial 0.01 to 1.00
(1 / min) were cultured in the k _L a culture early late culture 1 /
It is preferable to adopt a method of culturing with a reduction to 6 to 4/5.

In the present application, the term "young plant" means
Plants that have stems and leaves that have been differentiated and regenerated from callus and have not been placed on the floor, with or without rooting. Further, in the present invention, the "gas phase space" is a gas space with which the seedlings other than the medium into which the seedlings are transplanted are in contact, and it is intended to actively grow the seedlings, not to mention the atmosphere. Includes regulated gas space. Further, it is a major feature of the present invention that the seedlings can be suitably grown even in the atmosphere where the disinfection treatment is not performed. B. Next, the present invention is characterized in that the above-mentioned seedlings are transplanted to a sugar-free medium and cultured in a gas phase space with aeration of carbon dioxide at a high concentration of 1 v / v% or more.

The "sugar-free medium" used for cultivating seedlings in the present invention is a medium that does not contain any saccharide as a carbon source. Specifically, it does not include sugars such as sucrose, glucose, fructose, and maltose, which are usually added to a medium used for plant tissue culture, as well as sugar alcohols such as sorbitol and mannitol, as an osmotic pressure adjusting agent. It is a medium.

The "sugar-free medium" has the same composition as that of a medium usually used for plant tissue culture, and contains at least inorganic salts. Specific components such as the composition of the inorganic salts can be adopted according to a known medium used for plant culture. For example, Murashige-Scoog (M
S) medium, N6 medium, Rinsmeier Scoog (Lins
Use a medium having a composition according to the inorganic salt composition such as maier and Skoog medium, White medium, Ganborg B5 medium, Heller medium, and Kohlenbach and Schmidt medium. You can

Further, trace components can be appropriately added to the above-mentioned medium composition containing only inorganic salts to be used for planting seedlings. As a minor component to be added, a plant growth regulator,
Examples include osmotic pressure regulators, amino acids, pH regulators, media supports and the like. As a plant growth regulator,
Examples of suppuration include auxin and cytokinin, and these may be added in combination.

As auxins, NAA, IAA,
2,4-D etc. are used, NAA or IAA is 0.01ppm
~ 10 ppm and 2,4-D can be added at a concentration of 0.001 ppm to 0.1 ppm, respectively. As the cytokinin, kinetin, benzyladenine (BA), zeatin and the like are used, and usually, they can be added individually or in combination at a concentration of 0.01 ppm to 10 ppm.

As the osmotic pressure regulator, it is necessary to use a regulator which is not itself catabolized as a carbon source in the plant. Specifically, polyethylene glycol and the like can be mentioned as preferable ones. Further, such polyethylene glycol can be usually added to the medium in an amount of 1 to 6 wt%. "Sugar-free medium" used in the present invention
As described above, it is unsuitable to add sugar alcohols such as sorbitol and mannitol as osmotic pressure regulators, since the sugar alcohol can be a carbon source of the plant.

As the amino acids, for example, L-proline, casein hydrolyzate and the like can be added. Usually, L-proline is added at a concentration of about 1 to 100 mM, and the casein hydrolyzate is added at a concentration of about 10 to 500 ppm. Examples of the pH regulator include MES [2- (N-Morpholino) et ha
nesulfonic acid monohydrate] can be added. The amount to be added can be determined according to the pH of the intended medium.

Examples of the support of the medium include gelling agents such as shlan gum and agar. It is usually preferable to use gellite at a concentration of 0.2 to 0.9 wt% and agar at 0.8% to 1.8 wt%. Also, ceramic fiber,
It is also possible to use a commercially available culture medium support such as rock wool impregnated with the above liquid culture medium. Callus-induced seedlings are placed on the above-mentioned seedling medium to produce plant seedlings under the following conditions.

First, the callus-derived seedlings are
It can be placed on the seedling medium as it is, but in the sense of preventing contamination of the plant seedling production environment as much as possible,
It is preferable to wash the seedlings in advance. The washing method is not particularly limited as long as it is effective for eliminating the above-mentioned contamination, and it is possible to wash with pure water, ethyl alcohol, sodium hypochlorite solution, etc. by a conventional method, and use tap water as it is. It is also possible to wash it. From the viewpoint of cleaning cost and the like, it is preferable to use the method of using tap water as it is. In such a case, it is sufficient to expose the seedlings to running water for about 30 minutes. A reference example to be described later shows an aspect of the cleaning device used for cleaning with running water, but the cleaning device is not necessarily limited to this.

Next, the seedlings placed in the above-mentioned sugar-free seedling medium are cultivated under a gas phase condition in which carbon dioxide at a concentration of 1 v / v% or more is aerated. The concentration of carbon dioxide to be aerated is not particularly limited as long as the seedlings can grow as long as it is 1 v / v% or more, and it is particularly preferable that the concentration is 5 v / v% or more because the plant seedling has a high survival rate and seedling formation. It is preferable in that the rate can be obtained. The aeration method is not particularly limited, and for example, carbon dioxide is mixed with air using a carbon dioxide cylinder or the like, and the mixed gas is aerated to perform the ventilation.

The culture conditions other than the above medium composition and carbon dioxide concentration are generally in accordance with the usual culture conditions according to the kind of plant to be cultured. That is, a light intensity of about 4000 lux is sufficient, but more intense light can be emitted. Specifically, the strength is preferably 10,000 lux or more. Irradiation time tends to be the most efficient for 24-hour irradiation as compared with the case where time is limited.

As the culture temperature, it is possible to adopt an optimum temperature corresponding to the type of plant. For example, in the case of rice, culturing is usually performed at 15 to 35 ° C.

[0024]

EXAMPLES The present invention will be described in detail below with reference to examples and examples. [Reference Example 1] Seedling body washer Fig. 1 shows one embodiment of a seedling body washer used for carrying out the method of the present invention. The cleaning pipe 1 is fixed by the support fifth. The water supply pipe 2 is fixed to the upper portion of the washing pipe 1, and the siphon pipe 3 is fixed to the lower portion of the washing pipe 1 at the position where the seedling trap 4 is attached. In addition, 6 is a removable lid part.

The shape and material of the washing tube 1 are not particularly limited, but it is preferably a tubular body from the viewpoint of easy handling and the like, and transparent glass or a transparent glass for easily grasping the state of the fixed seedlings, Plastics and the like are preferable. The seedling trap 4 is not particularly limited as long as it can pass the washing liquid. For example, a net made of metal or plastics can be used as the seedling trap. When using a metal assembly, it is preferable to use a material that is as resistant to corrosion as possible, such as stainless steel.
Further, the seedling-fixing net 4 may be fixed to a washing device or may be detachable, but if the washed seedlings are easily handled, they can be detached. It is preferably possible. The water supply pipe 2 needs to be provided above the curved portion of the siphon pipe 3 in the cleaning pipe 1, and the discharge portion of the siphon pipe 3 extends below the mounting position of the siphon pipe 3 in the cleaning pipe 1. It is necessary to. The lid 6 is not always necessary, and its material and shape are not particularly limited.

Such a seedling washer is introduced into the washing pipe directly through the water supply pipe 2, and if a certain amount of tap water is stored in the washing pipe, the seedling washing device is put outside through the siphon pipe 3. Is discharged. By repeating this washing step, the young plant can be effectively washed. The arrows in the figure indicate the directions of inflow and outflow of tap water in the washer.

[Reference Example 2] Induction of rice seedlings After peeling ripe seeds of rice variety Sasanishiki, they were treated with 70% ethanol for 5 seconds, and sodium hypochlorite (effective chlorine 10
%) For 30 minutes for sterilization and thorough washing with sterilized water, and then placed on the following callus induction medium. 27 ° C,
After culturing in the dark for 8 days, the callus generated from the scutellum was excised, transferred to the same medium as the callus induction medium, further cultured for 14 days, and then transferred to the growth medium described below. Callus was transplanted to fresh growth medium every week, and under 16-day photoperiod,
Rotation culture was performed at 80 rpm and the cells were subcultured and maintained. Approximately 10 g of callus was placed per 11 liquid media. The callus increase per week was about 3-4.5 times. [Callus induction medium] 1 wt% sucrose, 3 wt% sorbitol, 12 mM proline, 100 ppm casein hydrolyzate, 5
mM MES, 4ppm 2, 4-D, N containing 0.2% gellite
6 solid medium. [Callus growth medium] 1 wt% shoe cloth, 3 wt% sorbitol, 12 mM proline, 100 ppm casein hydrolyzate, 5
N6 solid medium containing mM MES, 4 ppm 2, 4-D.

Callus which had been subcultured every one week in a liquid medium was transplanted to the following seedling induction medium at the first week of subculture and swirling-cultured under 16-day photoperiod (4000 lux). Callus became green. After 3 weeks of culture, the medium was replaced with the following sugar-free medium-1 and the culture was continued. As a result, seedlings were formed. After 3 weeks of culture, 20 seedlings were obtained per 20 mg of callus.

[0029]

Example 1 The seedlings obtained in Reference Example 2 were washed with tap water in the seedling washer described in Reference Example 1 for one hour, then transplanted to the following seedling-growing medium (1) and humidified. Three
Air that has not been sterilized containing v / v% carbon dioxide is aerated in a culture device that can be aerated, and while changing the illuminance and light irradiation time of the white fluorescent lamp, seedlings at 28 ° C for 3 weeks The body was raised. That is, (1) Illuminance 8000lux
・ 24 hours irradiation, (2) 4000lux ・ 24 hours irradiation, (3) 8000lux ・
The growth states of the three groups, which were irradiated for 12 hours, were compared.

[Seedling culture medium (1)] MgSO ₄ 7H ₂ O 46 mg / l KH ₂ PO ₄ 100 mg / l (NH ₄ ) ₂ SO ₄ 116 mg / l KNO ₃ 708 mg / l CaCl ₂ 2H ₂ O 42 mg / l MnSO ₄・ 6H ₂ O 1.1 mg / l ZnSO ₄・ 7H ₂ O 0.4 mg / l H ₃ BO ₃ 0.4 mg / l KI 0.2 mg / l Fe-Na-EDTA 10.5 mg / l Gellite 4 g / l [Seedling raising medium (2)] Sucrose was added to the above raising medium (1) at a rate of 30 g / l.

[0031]

[Table 1]

As a result, the survival rate, seedling rate, and degree of fulfillment are
At the same carbon dioxide concentration, it was found that the growth result was better when the illuminance was stronger and the light irradiation time was longer.
The system in which the seedlings were not washed before transplantation was also examined, but the seedlings after transplanting tended to rot more strongly than in the case of washing.

[0033]

[Example 2] The seedlings obtained in Reference Example 2 were washed with tap water for 1 hour using the seedling washer described in Reference Example 1, and then transplanted to the seedling growth medium used in Example 1, Carbon dioxide of various concentrations was mixed with humidified, non-sterilized air and irradiated at an illuminance of 8000 lux for 24 hours and grown at 28 ° C. for 3 weeks. The results are shown in Table 2.

[0034]

[Table 2]

As a result, it was found that the system having a carbon dioxide concentration of 1 v / v% or more markedly improved both the survival rate and the seedling conversion rate as compared with the system having 0.2 v / v%. More specifically, it was found that raising the carbon dioxide concentration to at least 5 v / v% markedly improved the survival rate and seedling conversion rate, and that even at 5 v / v% or higher, the seedling integrity was improved. did.

[0036]

INDUSTRIAL APPLICABILITY The present invention provides a method for producing plant seedlings by transplanting a callus-derived seedling into a sugar-free medium and culturing it without requiring a special sterile space. ..

[Brief description of drawings]

FIG. 1 is a schematic view of the seedling washer described in Reference Example 1.

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[Procedure amendment]

[Submission date] August 19, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0012

[Correction method] Change

[Correction content]

In the present invention , the term "young plant" refers to a plant having foliage differentiated and regenerated from callus and having no bed, and whether rooting or not is not particularly limited. Further, in the present invention, the “gas phase space” means
It is a gas space with which the relevant seedlings other than the medium into which the seedlings are transplanted come into contact, and includes, of course, the atmosphere, as well as the gas space adjusted for the active growth of the seedlings. Further, it is a major feature of the present invention that the seedlings can be suitably grown even in the atmosphere where the disinfection treatment is not performed. B. Next, the present invention is characterized in that the above-mentioned seedlings are transplanted to a sugar-free medium and cultured in a gas phase space with aeration of carbon dioxide at a high concentration of 1 v / v% or more.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0015

[Correction method] Change

[Correction content]

Further, trace components can be appropriately added to the above-mentioned medium composition containing only inorganic salts to be used for planting seedlings. As a minor component to be added, a plant growth regulator,
Examples include osmotic pressure regulators, amino acids, pH regulators, media supports and the like. As a plant growth regulator,
Auxin, it is possible to include cytokinin like, may also be added in combination.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction content]

As the amino acids, for example, L-proline, casein hydrolyzate and the like can be added. Usually, L-proline is added at a concentration of about 1 to 100 mM, and casein hydrolyzate is added at a concentration of about 10 to 500 ppm. Examples of pH regulators include MES [ 2- (N-Morpholino) ethan
esulfonic acid monohydrate ] can be added. The amount to be added can be determined depending on the intended pH of the medium.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction content]

Callus which had been subcultured every one week in a liquid medium was transplanted to the following seedling induction medium at the first week of subculture and swirling-cultured under 16-day photoperiod (4000 lux). Callus became green. After 3 weeks of culture, change to the following medium.
When the culture was replaced and the culture was continued, 20
Individual seedlings were obtained . [Seedling induction medium] 1 wt% sucrose, 3 wt% sow
Rubitol, 0.4ppm NAA, 0.5ppm kinetin, 2000pp
Contains m-casein hydrolyzate, 12mM proline, 5mM MES
N6 modified liquid (with trace metal elements of MS medium added)
Culture medium. [Exchange medium] 1.5wt% sucrose, 1.5wt% sorbit
, 1ppm NAA, 0.5ppm kinetin, 1000ppm cold
In hydrolyzate hydrolyzate, N6 modification containing 2.5 mM MES (M
Liquid medium supplemented with trace metal elements of S medium).

Claims (2)

[Claims] 1. A method for producing a plant seedling by culturing callus-induced seedlings in a gas phase space, wherein the seedlings are transplanted to a sugar-free medium to obtain 1 v / v% or more. A method for producing plant seedlings, which comprises culturing under aeration of carbon dioxide. 2. A method for producing plant seedlings by culturing callus-induced seedlings in a gas phase space, wherein the washed seedlings are transplanted to a sugar-free medium to give 1 v / v%.
A method for producing a plant seedling, which comprises culturing under the aeration of carbon dioxide as described above.