JPH089811A - Storage of young rice plant and raising seedling of stored young rice plant - Google Patents

Abstract

PURPOSE:To provide a liquid for the storage of a young rice plant, composed of an inorganic salt, having an osmotic pressure adjusted to a specific level and effective for the long-term storage of a young rice plant redifferentiated by a tissue-culture technique to obtain a seedling suitable for the transplantation to a paddy field. CONSTITUTION:This storage liquid is composed of an inorganic salt and has an osmotic pressure adjusted to 5-120mOSMOL/kg. The storage liquid is preferably a culture medium residue left after the redifferentiation treatment of rice plant. The liquid is preferably incorporated with a plant growth regulator having a spindly growth suppressing action such as ancymidol. In the case of storing a young rice plant, it is preferable to culture the plant in an inorganic salt liquid medium free from sugar and containing ancymidol and an agent having root-taking and growthpromoting actions under light irradiation and aeration with air enriched with carbon dioxide gas and store the cultured plant in the storage liquid at 15-20 deg.C, a photoperiod of 3-24hr and an illumination intensity of 200-30,000Lux.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for storing rice seedlings and a method for raising seedlings of rice after storage. Although plant tissue culture technology has made remarkable progress, liquid regeneration technology has also been developed in the field of tissue culture of rice in recent years, and it has been developed from rice callus to redifferentiate cloned seedlings (hereinafter referred to as “biolarvae”).
Say. ) Is in a state where a large amount can be obtained. In addition, these bio-larvae can be acclimated and reared to grow into seedlings that can be planted in the field (hereinafter referred to as "bio-seedlings"). Further, bio seedlings can be cultivated to produce rice.

In recent years, there have been many technical problems to be solved in Japanese rice farming circumstances, such as reduction of rice production cost, international competitiveness, and difficulty in succession. If the required amount of cloned seedlings of various varieties can be produced in a timely and inexpensive manner by the rice tissue culture technology, it will help improve the situation of rice cultivation in Japan.

By the way, in order to produce a large amount of bio-larvae and acclimate and grow them using the current seedling raising technology system (current seedling raising center system), it is necessary to supply bio seedlings in a large amount, at low cost and in a proper time. , It becomes necessary to store the bio-cub. In other words, if it becomes possible to store the bio-larvae for a long time, it is not necessary to adapt the bio-seedling production time to the current rice planting time, and the production period can be extended. As a result, the operating rate of the redifferentiation culture device and the like is dramatically improved, and the required amount can be planned and produced on a commercial scale in a timely and inexpensive manner. Therefore, the technique of the present invention can be used in a rice seedling production site where it is required to inexpensively and systematically supply the required number of rice seedlings of different varieties at low cost.

On the other hand, the technique of the present invention provides a technique which can be directly or indirectly applied to or applied to the storage of redifferentiated seedlings other than rice and seedlings grown by shoot apical culture other than rice. did. Furthermore, we have given the basic and basic knowledge to the development of the storage technology for seedlings other than rice.

[0005]

[Prior Art] When acclimatizing and growing bio-seedlings into bio-seedlings, the number of seedlings (required seedlings) required according to the rice planting time (specific time) has not been available because there was no conventional storage technology.
Expensive production equipment equipped with a large number of redifferentiation culture tanks for supplying water (among the production equipment in the non-storage technology generation, the redifferentiation culture tanks are all equipments, technically and economically Was very high). Here, if the number of redifferentiation culture tanks required to produce the required number of seedlings is N,
N operates only at a specific time and is idle at other times, so the operation rate of production equipment in the storageless technology generation is extremely low (1 turn), and the production cost of bio seedlings is lower than that of seed-derived seedlings. In comparison, it was extremely high, which was the main cause of delaying the spread of bio-seedlings.

[0006]

[Problems to be Solved by the Invention] Production facilities in the storageless technology generation will operate only for a very limited period,
It provided the main reason for making the production cost of bio-seedling more than the cost of seed-derived seedling. Here, if long-term storage of bio-larvae is possible, bio-seedling production equipment can be repeatedly used. Therefore, the operation rate is improved, the initial capital investment is reduced, the final production cost of bio-seedling is reduced, and bio-seedlings of useful varieties utilizing excellent tissue culture technology can be widely supplied.

[0007] Here, the effect when the long-term storage of bio juveniles becomes possible will be specifically described. The liquid regeneration culture period to regenerate the bio youngs from callus and T _R Week
If the storage period of bio-larvae is T _S week, T _S / T _R
(= R) indicates a multiple (R) of the storage period of the biolarvae with respect to the liquid regeneration culture period. In other words, R
Is a scale showing the number of times the redifferentiation culture tank has been used (the number of times of repeated use), and the exact number of times of use can be represented by R + 1. In order to simplify the explanation, assuming that the survival rate is 100% by ignoring the biolarvae lost during the storage period, the number N of initial redifferentiation culture tanks required in the non-storage technology generation is N.
Is the number of culture tanks N [R / (R +
1)] becomes unnecessary (in other words, the required number of tanks is N [1 /
(R + 1)], and it is possible to significantly reduce N. For example, if a long-term storage technology such that R = 2 is established, 2 / 3N becomes unnecessary and 1 / 3N can be produced. Even if R = 1, 1/2 of N can be reduced, and 2-turn production with 1 / 2N of tanks is possible, which can significantly reduce the initial investment amount.

[0008] As described above, the problems in the storageless technology generation (large initial capital investment, low annual operating rate due to short production period with high seasonal dependency, high bio-seedling production cost,
Etc.) and that the long-term storage technology has a very large industrial and economic advantage in the established generation. An object of the present invention is to establish a long-term storage technology and solve the above-mentioned problems in the non-storage technology generation.

[0009]

Means for Solving the Problems The inventors of the present invention have made extensive studies as to means for improving the storability of rice seedlings, and as a result, by adjusting the osmotic pressure of the stock solution to a predetermined value, the storability is improved. Have found a dramatic improvement. Furthermore, it was found that the seedling yield can be improved by culturing the seedlings after storage in a seedling medium containing a predetermined drug, and the present invention based on these findings was completed.

That is, the first aspect of the present invention is a stock solution of rice seedlings comprising at least an inorganic salt and having an osmotic pressure of 5 to 120 mOSMOL / Kg. A second aspect of the present invention is the above-described rice seedling stock solution, wherein the stock solution is a residual medium solution after the rice redifferentiation culture is completed. The third aspect of the present invention is the above-mentioned rice seedling stock solution, which contains a plant growth regulator having an elongation growth inhibitory action.

A fourth aspect of the present invention is that the plant growth regulator having an elongation growth inhibitory effect is ancimidol, uniconazole, inabenfide, abscisic acid, or choline chloride. Is a storage solution of. A fifth aspect of the present invention is a method for storing rice seedlings, which comprises storing the rice seedlings in the above-mentioned storage solution.

A sixth aspect of the present invention is that the temperature condition during storage is 10 to
The rice seedling storage method described above is characterized in that the temperature is 20 ° C., the light condition is an illuminance of 200 to 30,000 Lux, and the photoperiod day length is 3 to 24 hours. A seventh aspect of the present invention is characterized in that, before storage, rice seedlings are placed in a sugar-free inorganic salt liquid medium and subjected to culture treatment under light irradiation / carbon dioxide-enriched aeration conditions. This is a storage method for rice seedlings.

An eighth aspect of the present invention is the method for storing rice seedlings as described above, wherein the sugar-free inorganic salt liquid medium contains ansimidol. In a ninth aspect of the present invention, the rice seedlings stored by the above-described rice seedling storage method are placed in a sugar-free inorganic salt medium, and seedling is formed under light irradiation / carbon dioxide-enriched aeration conditions. This is a method for raising seedlings of rice seedlings after storage, which is characterized by:

The tenth aspect of the present invention is the method for raising seedlings of rice seedlings after storage as described above, characterized in that the sugar-free inorganic salt medium contains an agent having a rooting and growth promoting action. The eleventh aspect of the present invention is an agent having a survival and growth promoting action, 1,6-diaminohexane, 1,7-diaminoheptane,
Characterized by being 1,8-diaminooctane, 1,9-diaminononane, α-carotene, β-carotene, γ-carotene, cefotaxime, amphotericin B, α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin The method for raising seedlings of rice seedlings after storage as described above.

The present invention will be described in detail below. The seedlings to be stored in the present invention are not particularly limited as long as they are seedlings regenerated from callus (bio seedlings), but those having a bud length of 1 to 30 mm are preferable. Regeneration from callus can be performed according to a known method, for example, a two-step regeneration method [Japanese Journal of Bree] which is a medium exchange method for efficiently regenerating seedlings from callus.
ding, 42 (3), 583-594 (1992)] and the like. That is, callus was induced by the same method as in the above-mentioned document, and this was propagated and subcultured, and a certain amount of it (callus mass number 20
[About 20 mg (FW)] / 20 mL liquid regeneration medium} is inoculated into the above liquid regeneration medium (preculture medium) and cultured for 3 weeks (preculture). Then replace the old medium with a fresh liquid medium (late medium)
Are replaced (replaced) with each other and cultured for 3 more weeks (late culture). The early medium and late medium used here may be the same as each other, but the early medium is diluted to about 1/2 and used as the late medium, and this is exchanged (replaced) with twice the amount of the early medium.
A higher regeneration rate can be obtained by using (for example,
The volume of the early medium is 20 mL and the volume of the late medium is 40 mL). The redifferentiation culture is performed under illumination (illuminance: about 2000 Lux, day length: 12 hours or longer) at 27 to 32 ° C. while being subjected to rotary shaking (120 rpm for a 100 mL Erlenmeyer flask).

The method for preparing the stock solution used in the present invention is roughly classified into the following two. One is an artificial composition method in which an osmotic pressure of at least an inorganic salt is adjusted to 5 to 120 mOSMOL / Kg,
The other is used for regeneration culture and its osmotic pressure is 5-12.
This is a method of reusing the medium residual solution after the completion of culture, which is 0 mOSMOL / Kg (or adjusted to 5-120 mOSMOL / Kg). The former artificial composition method is used to adjust the stock solution by demineralizing pure water (desalted / distilled osmotic pressure 0) of various media commonly used in plant tissue culture experiments or various culture solutions commonly used in hydroponics. Water), or by adjusting the salt concentration of these media / culture solutions from the time of preparation, so that the osmotic pressure is 5 to 120 mOSMOL / Kg.

As an example of the medium for the plant tissue culture experiment, MS is used.
Medium [Murasige and Skoog, Physiol. Plant., 15, 473
-497 (1962)], N6 medium [Chu et al., Sci. Sin., 18,
658-668 (1975)], Rinsmeier Scoog (Linsmai
er and Skoog LS medium, White medium, Ganborg B5 medium, Heller medium, Kohlenbach and Schm
idt) medium, etc. [These medium compositions are described in Masayuki Takeuchi, Tetsuo Nakajima, Riki Furuya, “Technology of Plant Tissue Culture” (pp. 218-22)
1, Asakura Shoten, 1986 edition)] can be mentioned. When used as a stock solution, the inorganic salts are essential in these media, and the concentration thereof can be 1/20 to 1.2 times, but particularly 1/8 to
4/5 times is preferable (however, the pH is adjusted to 5.8 before use). As a matter of course, the osmotic pressure of this concentration is 5 to 120 m
It falls within the range of OSMOL / Kg. The optimum concentration (molar concentration ratio) of various inorganic salts such as phosphate and calcium and the molar concentration ratio of ammonia nitrogen / nitrate nitrogen is the same as that of the regeneration medium described later. However, the major difference from the regeneration medium described later is that the inorganic salt is essential as described above, but other components (carbon source, plant growth regulator, osmotic pressure regulator, amino acids, casein hydrolyzate, Vitamins
The pH adjustor) is not essential and may not be added. If other components are added in a tissue culture experiment in a usual use range (a range in which a specific component does not become excessive) and the osmotic pressure is adjusted to an optimum range, it is not harmful and can be used as a stock solution. However, these other components are more expensive than the inorganic salts, and the additional storage advantages obtained are hardly recognized. Another method of adjusting the artificial composition liquid is the Hoagland hydroponics solution used in hydroponics, gravel cultivation, and sand culture, and the balanced culture solution for gravels cultivation by Yamazaki & Hori et al. Arnon) et al.
The stock solution having an osmotic pressure of 5 to 120 mOSMOL / Kg used in the present invention can be arbitrarily adjusted by adjusting only the concentration without changing the combination concentration ratio of the respective salt compositions used. The composition of these culture solutions is as follows: Experimental Agricultural Chemistry Vol. 1 [Agricultural Chemistry Department, The University of Tokyo, published by Asakura Shoten Co., Ltd. (April 30, 1964), pages 100 to 105], and facility horticulture. Cultivation technology) [Toshitaka Itaki, Ltd.
Published by Seibundo Shinkosha (February 10, 1987), pages 408-4
17].

Another method for reusing a redifferentiation medium (storage solution storage method), which is an osmotic pressure of 5 to 120 mOSMOL / Kg, will be described. As the stock solution, the residual solution (osmotic pressure 5 to 120 mOSMOL / Kg) after the completion of the redifferentiation culture used during the redifferentiation is most suitable as the stock solution. This is because it is used as a regeneration medium once, and this residual culture solution is reused, which is economically, resourceally, and economically advantageous compared to the artificial composition method described above in which the stock solution is newly adjusted. Is large. Component composition of the liquid regeneration medium used here, at least inorganic salts, carbon source is essential, plant growth regulator, osmotic pressure regulator, if necessary,
It is a medium to which vitamins and amino acids, casein hydrolyzate, pH adjuster and the like are added. Specifically, MS medium usually used in the above-mentioned plant tissue culture experiment,
N6 medium, Rinsmeier-Skoog LS medium, white medium, Gamborg's B5 medium, Heller's medium, Korenbach-Schmidt medium, etc. can be mentioned, and the inorganic salts have a concentration of 1/4 to 4 times. However, a concentration of 1/2 to 2 times is particularly preferable. The phosphate may be used at a concentration of 0.1 to 60 [mM], but 0.5 to 20 [mM] is particularly preferable. As a calcium salt, 0.003 to 30 (mM) is good, but especially 0.03 to 10 (m
M] addition is preferred. As the trace metals, more preferable results can be obtained by adding the trace metals contained in the MS medium. As the inorganic nitrogen (N) source, it is preferable to use a combination of nitrate nitrogen and ammonia nitrogen, and the former combination of 20 to 120 [mM] and the latter 0.1 to 10 [mM] are particularly preferable. However, it is preferable that the molar concentration ratio of ammonia nitrogen / nitrate nitrogen is 0.001-1. A carbon source must be added to the above-mentioned inorganic salts, and plant growth regulators (auxins and cytokinins), osmolarity regulators, amino acids (particularly L-proline), casein hydrolysates, vitamins, and pH as necessary. A liquid medium prepared by adding a regulator and the like. Examples of the carbon source of the medium include sucrose, glucose, fructose, and the like,
The concentration is preferably 0.1 to 10%, particularly preferably 0.5 to 6%. As a plant growth regulator, auxin and cytokinin are used alone or in combination. 0.1-10 of α-naphthalene acetic acid (α-NAA) as auxin.
0ppm, or indole-3-acetic acid (IAA) 0.001-0.1
Examples include ppm and the like. As cytokinins, kinetin and zeatin (trans-or ribosyl-zeatin)
Examples are 0.001 to 5 ppm. As an osmotic pressure regulator, 1-6% of sorbitol and 0.1-3 of mannitol are used.
%, Etc. can be illustrated. Examples of the amino acid include L-glutamine, L-asparagine, and L-proline, and it is particularly preferable to add 0.02 to 5 g / L of L-proline. Further, when the casein hydrolyzate is added, 0.01 to 5 g / L is preferable. MES (4-morpholinoethane) is used as a pH adjuster.
sulphonic acid) 0.2 to 7 g / L can be exemplified, and the pH of the medium
Is adjusted to 5.8 before use. In addition, vitamins include myo-inositol and nicotinic acid (nicoti).
nic acid), pyridoxine (pyridoxine), thiamine (thiamine) 50-200, 0.05-3, 0.1-
2, 0.5-1 mg / L is preferably added. In addition, the above 2
When redifferentiation is carried out by the stepwise liquid redifferentiation method, the culture residual liquid of each of the above and after the end of the late culture can be used as the stock solution of the present invention. As a stock solution, when the culture residual solution and the late culture residual solution are compared, the late culture residual solution gives a more preferable storability result. The osmotic pressure of the stock solution is preferably 5 to 120 mOSMOL / Kg. Osmotic pressure is 5mOSMOL / K
It may be g or less, but the storage period is limited to a relatively short period. On the other hand, above 120 mOSMOL / Kg, the storage properties of seedlings (definition of this term and its explanation will be described later) are easily influenced by the physiological state of the seedlings, and stable storage properties cannot be obtained. There are cases.

The stock solution of the present invention has a growth inhibitory action.
It is preferable to add a plant growth regulator having the same. here
As a plant growth regulator having a growth inhibitory effect on
Ansimi d'or [ancymidol (FW, 256.3): α-cyclopropy
l-α- (4-methoxyphenyl) -5-pyrimidinemethanol],
Nabenfide [inabenfide (FW, 338.6): 4'-chloro-2 '
-(α-hydroxybenzyl) -isonicotinanilide], Unicona
Sol [uniconazole (FW, 291.7): (E)-(RS) -1- (4-chlor
ophenyl) -4,4-dimethyl-2- (1H-1,2,4-triazol-1-yl) -1-
penten-3-ol], abscisic acid (FW, 26
4.3): 5- (1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohe
xen-1-yl) -3-methyl-2,4-pentadienoic acid], choline
Chloride [choline chloride (FW, 139.6): 2- (hydroxy
ethyl) trimethylammonium chloride], etc.
However, as long as it has a growth inhibitory effect,
It is not limited to these. The effective concentrations of these drugs are
10 for simidol^-8~Ten^-6[M], 1 for Inabenfide
0^-9~Ten^-7[M], 10 for uniconazole^-Ten~Ten
^-8[M], 2 × 10 for abscisic acid^-7~ 2 x 10
^-Five[M], 10 for choline chloride^-Four~Ten^-2[M], and
It In addition, the optimal effective concentration of the above drug is
0.5 × 10^-7~ 5.0 x 10^-7[M], Inabenfide
0.7 x 10^-8~ 7.0 x 10^-8[M], 0.8 × for uniconazole
Ten^-9~ 8.0 x 10^-9[M], 7.0 × 10 for abscisic acid^-7~ 1
0^-6[M], 0.5 × 10 for choline chloride^-3~ 5.0 x 10 ^-3
It is [M].

In the storage method of the present invention, the storage temperature is 10
C. to 25.degree. C. is preferable, and 15.degree. C. to 20.degree. C. is particularly preferable. The light conditions during the storage period are preferably such that the photoperiod day length is 3 to 24 hours and the illuminance is 200 to 30000 Lux.

In the storage method of the present invention, it is preferable that before storage, the seedlings are cultured for a certain period of time in a sugar-free inorganic salt liquid medium with or without addition of ancimidol. This pre-storage culture treatment is preferably carried out under a carbon dioxide-enriched aeration condition by maintaining the temperature at 27 to 30 ° C. and irradiating light with an illuminance of 1000 to 30000 Lux for a day length of 3 hours or more. Here, as the inorganic salt liquid medium containing ancimidol and containing no sugar, for example, a medium containing MS inorganic salt at a concentration of 1/4 and ancimidol can be mentioned. A sufficient effect can be obtained if the concentration of ancimidol is 10 ⁻⁸ to 10 ⁻⁷ [M], but it is particularly preferably 0.5 × 10 ^{−7 to} 5.0 × 10 ⁻⁷ [M]. Aeration conditions are 5-10% carbon dioxide
It is preferable to aerate the carbon dioxide-enriched air containing it at 0.01 to 0.1 vvm. The culture period is preferably about 1 to 2 weeks.

The seedlings stored by the storage method of the present invention are placed in a sugar-free inorganic salt medium and seedled under light irradiation and aeration conditions enriched with carbon dioxide gas. It is preferable to add an agent having an activity of promoting survival and growth to the activated medium. As the seedling medium, a known medium can be used, and for example, a solid medium containing 1/4 concentration of MS inorganic salt, gellan gum and the like can be used. Examples of the agent having the activity of promoting survival and growth include α, ω-diamino- n- alkane, α, ω-diamino- n- alkane, cephotaxime, and amphotericin B.
cin B), α-, β- or γ-cyclodextrin, α-, β- or γ
-Carotene (α-, β- or γ-carotene) and the like can be exemplified, but not limited to these as long as they have an activity of promoting rooting and growth. The effective concentration range of these drugs is α,
ω- diamino - n - 10 ^-6 to 10 ^-3 in an alkane (M), 10 ^-6 to 10 ^-3 in cefotaxime (M), the amphotericin B 10 ^-7 to 10 ^-3 [M], alpha-, beta -Or γ-cyclodextrin is 10 ^-6 to 10 ^-4 [M], α-, β- or γ-
For carotene, it is 10 ^-7 to 10 ^-4 [M]. The optimum concentration range is 10 ^-5 to 10 for α, ω-diamino- n -alkanes.
^-4 [M], the cefotaxime ^{2 × 10 -5 ~2 × 10 -} 4 [M]
Amphotericin B is 10 ^-5 to 10 ^-4 [M], and α-, β- or γ-cyclodextrin is 10 ^-6 to 10 ^-5 [M],
It is 10 ⁻⁵ to 10 ⁻⁴ [M] for α-, β- or γ-carotene.

Next, the storage technology level evaluation judgment method in the present invention will be explained. What is the "storability" of a young bio?
How much survival rate, average plant height (growth rate), and solidity are affected by the length of the storage period due to the storage of the bio-larvae, conversely, how long the storage period can be extended, This is a concept related to storage, which means how much the survival rate, growth rate, and solidity change when the rice is extended. In terms of technology development, it is the storage technology evaluation judgment value used for the development of storage technology. That is, the biological juveniles are stored for a certain period of time, and then the seedlings are raised for 3 weeks by the following method, and the following (1), (2), (3) and (4) are obtained, and these 4 persons are stored ( This is a comprehensive consideration by comparing the values of the bio-larvae just before storage with the bio-larval test section.

(1) Storage period (unit, week). (2) Survival rate (survival rate after storage, expressed in%) = [Number of surviving biolarvae / 20 (Number of bed biolarvae at the start of the experiment)] × 100.
The survival rate in the original meaning is (survival rate after storage / survival rate without storage) × 100, but in the present invention, the survival rate was simplified to be the survival rate. In addition, since the bio-larvae are multibuds, they may be displayed on the basis of the number. In that case, the number of biolarvae after storage [number of living cells after storage, absolute number display (S)] is represented. In this case, the reference value is the number of living seedlings of non-stored biolarvae [absolute number display (P)] (different for each test). Here, if (S / P) × 100 (%), the survival rate is obtained. However, in the present invention, it is not displayed on the basis of the number, but is displayed by the survival rate (survival rate after storage,%). (3) Average plant height (in mm or cm) = sum of above-ground longest plant height per individual / total number of biolarvae. (4) Solidity [mg (DW) / cm] = total dry weight (DW, mg) of above-ground part (leaf blade / sheath) / total above-ground part (leaf blade / sheath) (cm).

A seedling raising method for converting bio-larvae into bio-seedlings will be described. As a seedling-growing medium, a sugar-free medium in which the salt concentration of the above-mentioned medium of the two-step redifferentiation method is set to 1/4 and sucrose and sorbitol are not added is used. The seedling growing medium used here may be either solid or liquid, and when used as a solid medium, gellan gum or agar which is a gelling agent in the liquid medium is 0.2 to 0.8%, respectively 0.8 to
Add 1.2%.

Since the seedling raising is carried out in order to evaluate the storability of bio-larvae, the seedling raising conditions are kept constant. That is, the area density of bio-larvae was set to 1/3 cm ² , the volume density was set to 1/2 cm ³ (amount of seedling-growing medium), the photoperiod was 12 hours, the illuminance was 5000 Lux, and the aeration rate was 5% carbon dioxide enriched. 0.0
Raise seedlings as 1 vvm for 3 weeks. After the seedlings are raised, investigate (2), (3) and (4) (described above) for each storage period [(1)].

Next, the seedling raising property in the present invention will be described. It is considered that the stored biological juveniles lose their vitality due to long-term storage and suffer physiological damage.
At the seedling raising stage, it is required to develop a seedling raising method for recovering the storage damage of the bio-larvae after storage as quickly as possible. The present inventor, in exactly the same manner as in the case of obtaining the above-mentioned storage evaluation value, an agent having a large number of rooting and growth promoting effects on the seedling raising properties (sustaining rate / growth rate / solidity) of stored biolarvae I investigated the effect of. Here, the seedling raising property is used in the same concept as the above-mentioned storability. That is, when the stored bio-larvae are added under certain conditions, for example, an agent having a survival and growth promoting action is added, survival rate (survival rate), average plant height (growth rate),
It is a concept that means how much the degree of fulfillment is affected. The specific evaluation value for raising seedlings is expressed by the survival rate, plant height (growth rate), and degree of solidification, like the evaluation value for storage quality.

Hereinafter, the storage technique of the present invention will be specifically described with reference to Test Examples and Examples. Before that, the medium compositions used in the Test Examples and Examples will be listed.

[0029]

[Table 1]

[0030]

[Table 2]

[0031]

[Table 3]

Hereinafter, the culture medium used is indicated by the above abbreviations.

[0033]

[Test example]

Test Example 1 Selection / Selection of Storage Solution Selecting and selecting a storage solution for biolarvae is the most basic initial condition in establishing storage technology. The storage test was repeated from various points of view, considering the influencing factors of the storage solution on the storability of bio juveniles. As a result, it was found that the most influential factor was osmotic pressure. Liquids having different osmotic pressures such as (a) plant tissue culture liquid, (b) hydroponic liquid, or (c) culture residual liquid after redifferentiation culture, which are commonly used, are prepared and used to prepare biolarvae. The optimum osmotic pressure range for each stock solution, which affects the storability of, was determined. The basic concentration composition table of the artificial medium used at that time is shown in Table 1. By appropriately changing the basic concentrations shown in this table, artificial composition liquids having different osmotic pressures can be arbitrarily adjusted. Fine adjustment of the osmotic pressure can be performed by dropping pure water (desalted and distilled water having an osmotic pressure of 0). For example, the osmotic pressure of the #RAM medium in Table 1 above is around 184 mOSMOL / Kg immediately after the medium is adjusted. Therefore, by adding an appropriate amount of pure water to this medium, Table 4 (selection of stock solution
A test solution having an osmotic pressure (selection test) can be prepared.

[0035]

[Table 4]

Various artificial composition culture solutions were prepared according to Table 1, and test solutions having the osmotic pressure of each level shown in Table 4 were prepared. Bio-seedlings (bud length 5 to 10 mm) prepared in the same manner as in [Example 1] described below were stored in these test solutions under the conditions of a temperature of 15 ° C and an illuminance (day length) of 10,000 Lux (12 hours). It was stored for 8 weeks by the method. Then, the seedlings were raised for 3 weeks according to the above-mentioned storage technology level evaluation and determination method, and the survival rate (%), which is the storage property evaluation value, was determined. As shown in Table 4, the survival rate (suspension rate) of the bio-larvae after storage for 8 weeks was determined by the type of artificial culture solution (MS salt composition solution, balanced culture solution, modified Hoagland solution,
#RAM), and it was confirmed that they were almost the same. Further, the survival rate in storage in these artificial medium composition liquids was not significantly different from the survival rate in #CRAM liquid storage, which is the residual liquid after the end of culture using the #RAM medium for culture. That is, it was found that #CRAM, which is the residual culture solution of the #RAM medium, can be used as the stock solution of the biolarvae.

In the subsequent storage experiments, the artificial medium composition liquid was not used as the storage liquid, and the #CRA after the redifferentiation culture was completed.
I decided to use M. This is because it is used as a regeneration medium once, and since this residual liquid after culturing is reused, it is economically, resource-wise, and labor-saving compared to the above-mentioned artificial medium composition liquid in which the stock solution is newly prepared. This is because the advantage is great.

[Test Example 2] Optimization of storage environment conditions The storage environment conditions of biolarvae are optimized by selecting biolarvae with a constant bud length (for example, 10 mm) under different environmental conditions. Store for a certain period of time (for example, 8 weeks), and immediately after storage for each (1) of the above-mentioned storage technology level evaluation judgment method.
It was carried out by the method of obtaining the storability evaluation value in (2), (3) and (4).

[0039] Illuminance during storage is about 5000 Lux (12 days long)
And the effect of storage temperature was investigated. When the storage temperature was 10 ° C or lower, the survival rate became extremely low after storage for 8 weeks or longer, and it was often observed that the biolarvae died. On the other hand, when the storage temperature was above 25 ℃, the bio-larva continued to grow and became weak during the storage period, and the quality of the bio-seedling deteriorated. In addition, the bud length distribution tended to expand during the storage period,
This was contrary to the purpose of seeking uniform bud length of bio seedlings. On the other hand, storage at 15 to 20 ° C showed almost no bud growth during storage, and the survival rate was 80%, 50%, 30% or more after storage for 6, 8 and 10 weeks, respectively. (Survival rate after storage,%)
Admitted. The survival rate of the control group (non-stored biolarvae) (the survival rate of biolarvae immediately before storage,%) was 90% or more.

Maintaining the storage temperature at 15 or 20 ° C.,
The effect of irradiation intensity (Lux) was investigated. When the day length is 12 hours, when stored in the range of 1000 to 20000Lux for 6/8/10 weeks, the survival rate for each storage period is 80% ・ 40% ・
It was 20%. Day length 24 hours (continuous lighting), 200 to 300
The survival rate when stored for 6/8/10 weeks in the range of 0 Lux is
The survival rate was almost the same as in the case of the 12-day length above.
When the day length was set to 3 hours, a survival rate almost equal to the result obtained by the above-mentioned day length of 12 hours and continuous illumination was observed in the range of 10000 to 30000 Lux. In addition, darkness-light irradiation (0-
At 50Lux), the survival rate after storage for more than 8 weeks was almost zero, regardless of the length of day.

[Test Example 3] Long-term storage method The following two methods were examined in order to prolong the storage period of biolarvae and improve the survival rate.

First, various plant growth regulators (hereinafter referred to as "PGS") having an inhibitory effect on elongation and growth are added to #CRAM so that the median value of the optimum concentration is reached.
Put a 10 mm bio juvenile, storage temperature 15 ℃, light illuminance 10000L
Stored as ux (length 12 hours) for 8/10/12 weeks. The results are shown in Table 5 (PGS addition method).

The second is #PAM medium containing 2.3 × 10 ^-7 [M] of antimidol, and #PAM medium not containing ansimidol.
Biolarvae with a bud length of 10 mm were placed in a PAM medium and precultured for 1 week for storage. Culture treatment is light irradiation (10000Lux.
Under a temperature of 27 ° C under a day length of 12 hours)
% CO ₂ enriched air (0.02 vvm)]. Then, the bio-larvae after the culture treatment were cultured for 8/10/12 weeks. The results are shown in Table 5 (pretreatment method for improving storability).

In Table 5, "storage solution storage method" means that the storage solution is #CRAM and stored therein (the same applies hereinafter).

[0045]

[Table 5]

As shown in Table 5, the greatest feature of the above-mentioned two methods for long-term storage of biolarvae is that the storability (survival rate, solidity) is improved as the storage period becomes longer. . That is, the survival rate (solidity) was 12 (0.201) when stored in a storage solution containing no PGS for 12 weeks.
When stored for 12 weeks in a stock solution containing GS, the survival rate (solidity) is as high as 37 to 17 (0.236 to 0.212), and when cultured in a sugar-free medium containing no ancimidol before storage. The survival rate (solidity) was 45 (0.286), and the survival rate (solidity) was 50 (0.300) when cultured in a sugar-free medium containing ansimidol. In short,
By the storage treatment culture treatment method shown in Table 5, a survival rate (survival rate) of about 1/2 could be secured even when the storage period was 12 weeks. [Test Example 4] Method for promoting survival / growth of bio-larvae after storage The bud length of the bio-larvae at the start of storage was adjusted to 5 to 10 mm, and the temperature was 15 ° C and the photoperiod was 12 hours (15000 Lux) under environmental conditions. Stored in #CRAM for 6/8/10 weeks below. After that, the biolarvae are divided into two, one of which is a solid medium for raising seedlings #PAMG (each drug is added so that the median optimum concentration is obtained) to which an agent having a survival and growth promoting action is added, and the other is not added The seedlings were placed on a solid medium #PAMG.
Then, seedlings were raised for 3 weeks by the same method as the seedling raising method shown in the above storage technology level evaluation and determination method, and the effect of addition of the above-mentioned agents on the raising ability was examined, and the results are generalized and shown in Table 6. It was

[0047]

[Table 6]

[0048]

【Example】

[Example 1] (Preparation of bio-larvae to be tested and preparation of stock solution of bio-larvae) Callus was induced from the scutellum of mature rice seeds, and this callus was proliferated and passaged to give a seedling. Regeneration method (two-stage liquid regeneration method for rice) [Japanese Journal of Breeding , 4
2 ( 3), 583-594 (1992)] (the culture vessel used is a 100 ml Erlenmeyer flask, the culture period is 3 weeks in the first half, and the third period in the second half is 3 weeks). #
RIM) was inoculated, and in the middle of the culture, the medium was replaced with a fresh medium (late redifferentiation medium, #RAM) to continue the culture to obtain rice redifferentiated seedlings (biolarvae). At the same time, the regenerated seedlings and the callus mass and other cultures were removed, and then #R after the culture was completed.
A residual solution of AM medium was obtained. Next, measure the osmotic pressure of this #RAM medium and confirm that it is in the range of 5 to 120 mOSMOL / Kg.
This solution is used as a storage solution #CRAM for bio-larvae in a cold room (5
(° C). When necessary, the stored #CRAM was taken out and used as a stock solution for seedlings.

In order to confirm that the storage effect is exhibited when the osmotic pressure of the stock solution is within the range of 5 to 120 mOSMOL / Kg, a storage test was conducted using #CRAM having different osmotic pressures, and the results were The results are shown in Table 7. In the rice two-step liquid regeneration method described above, the osmotic pressure is adjusted by culturing in the #RAM medium for 3 weeks in the latter half after the first 3 weeks in culturing. By shortening the basic culture period of the latter 3 weeks, the #RAM residual liquid having an arbitrary osmotic pressure can be obtained. For example, in the latter two and three weeks of culture, 80-100 and 50-70 mOSMOL / Kg, respectively.
#CRAM is obtained. For fine adjustment of osmotic pressure, use #CRA
Pure water (desalted and distilled water having an osmotic pressure of 0) was appropriately added to M. In this test, the bud length of the test biolarvae was 5 to 10 mm, the storage temperature was 15 ° C, and the illuminance / day length during storage was 10 mm.
It was stored at 000Lux for 12 hours and stored for 3, 6 or 8 weeks by the storage solution storage method. After that, seedlings were raised for 3 weeks according to the above-mentioned storage technology level evaluation and determination method, and the storability evaluation value was obtained. The results are shown in Table 7.

When a biolarva with a long bud length is required, it can be prepared by using a fermenter (1 or 2 L volume) as a redifferentiation culture vessel and extending the culture period.

[0052]

[Table 7]

As shown in the above table, the survival rate (survival rate) of the biolarvae after storage decreases as the storage period increases, but when the osmotic pressure is less than 5, it is stored for 6 weeks, and when it is 120 or more, it is 8
With the weekly storage, it became zero. Similarly, pure water or #
Even if stored in RAM for 6 weeks or longer, the survival rate became zero.
On the other hand, in #CRAM with an osmotic pressure of 5 to 120, 8
The survival rate was observed even after weekly storage, and it was reconfirmed that this solution is effective as a storage solution for biolarvae.

[Example 2] (Necessity of light irradiation during storage) The effects of light irradiation intensity and photoperiod on the storability of biolarvae were examined by the following method. The bud length of the test biolarvae is 5-10
mm, the storage solution was #CRAM, the storage temperature was 20 ° C., the illuminance during storage was changed, and the storage solution was stored for 8 weeks. Then, the seedlings were raised for 3 weeks according to the above-mentioned storage technology level evaluation judgment method, and the storage property evaluation values shown in Table 8 were obtained.

[0055]

[Table 8]

As shown in the above table, light irradiation is indispensable for the storage of bio juveniles. The required illuminance is determined in relation to the photoperiod. That is, relatively low illuminance (about 200 to 3000 Lux) is required for continuous illumination, relatively medium illuminance (about 1000 to 20000 Lux) for 12 hours illumination, and relatively high illuminance (about 10000 Lux) for 3 hours illumination. ~ 20000Lux) is suitable.

It was confirmed that #CRAM was suitable as a storage solution for bio-larvae, but depending on the physiological condition of the bio-larvae, the survival rate of about 20% or less was rarely obtained after long-term storage for 8 weeks or longer. Was also recognized, and technological development was needed to further improve stability. Then, PGS was added to the storage solution #CRAM for the purpose of increasing the storability of the biolarvae, particularly the survival rate after storage for 8 weeks or more, and the effect on the storability of the biolarvae was investigated.

[Example 3] (Storage method for adding PGS storage fluid) [0058] Using a regeneration tank as a fermenter (1 L), the bud length was increased.
A 10-15 mm biolarva was prepared. Put the prepared larvae in #CRAM containing various PGS in a predetermined concentration,
8 at storage temperature 15 ℃, illuminance (day length) 10000Lux (12 hours)
・ Stored for 10 to 12 weeks. After that, according to the seedling raising method for storage quality investigation shown in the above-mentioned storage technology level evaluation judgment method, 3
The seedlings were raised for a week and the results shown in Table 9 were obtained.

[0059]

[Table 9]

From the above table, it can be seen that although the survival rate can be obtained by storing #CRAM alone, a higher survival rate can be obtained by storing PGS in #CRAM. In addition, it is clear that there is no storage effect when stored in pure water or #RAM. The same results as in the above table were observed even when the temperature and illuminance during storage were changed.

[Example 4] (Stored liquid storage method after storability-enhancing culture treatment) The storability of the biolarvae is affected by the physiological condition of the biolarvae itself in addition to the storage environmental conditions. Therefore, the redifferentiated seedlings were cultured in a sugar-free inorganic salt liquid medium and then stored in #CRAM (storage solution storage method after storability-enhancing culture treatment).

The test was conducted as follows. First, #RI
By the two-stage liquid regeneration method of M and #RAM, the bud length is 8-12mm
I got a bio-nanny. Next, this was divided into three equal parts and the following treatment (1), (2) or (3) was performed.

(1) The biolarvae were transferred to fresh #RAM (container, 500 mL Erlenmeyer flask) and shake-cultured (40 rpm, illuminance 200 Lux, temperature 27 ° C.) for 1 week in a non-aerated state. (2) Transfer the larvae of biotechnology to a fermenter (1 L volume) containing liquid #PAM containing no ancimidol, and illuminance (day length) 5000 Lux (12 hours), temperature 27 ° C, carbon dioxide rich The cells were cultured for 1 week under the condition that the aeration rate was 0.2 vvm. (3) 5 × 10 ^-8 [M] of bio-juvenile and ansimidol
The liquid #PAM was added to the fermenter (1 L volume) and the illuminance (day length) 5000 Lux (12 L).
Cultivation was carried out for 1 week under the conditions of temperature) 27 ° C., carbon dioxide enriched aeration rate of 0.2 vvm.

By the above three culture treatments, the bud lengths of the biolarvae were both 12 to 17 mm. Each of these 3 kinds of bio juveniles prepared here was put in the storage solution #CRAM, and the illuminance (day length) 10000Lux (12 hours) and temperature 15 ° C.
It was stored for 8/10/12 weeks. After storage, it was placed on #PAMG, and seedlings were raised for 3 weeks according to the above-mentioned storage technology level evaluation and determination method, and a storage property evaluation value was obtained, which is shown in Table 10.

[0065]

[Table 10]

As is clear from the above table, by storing the biolarvae after the storage-enhancing culture treatment, a considerably high survival rate was obtained even after long-term storage for 8 weeks or longer.

[Example 5] (Test of method for promoting survival and growth promotion of bio-larvae after storage) A bio-larvae having a bud length of 10 ± 2 mm was placed in #CRAM and the temperature was increased.
10 under the condition of 15 ℃ and illuminance (day length) 5000 Lux (24 hours)
Stored for a week. The stored biolarvae were placed on #PAMG and seedlings were raised for 3 weeks. At this time, #PAMG
A drug presumed to promote survival / growth was added in advance, and an effective drug was searched for. Table 11 summarizes the results.

[0068]

[Table 11]

From the above table, at the stage of raising seedlings of bio-young plants after long-term storage for 10 weeks to prepare bio-seedlings, by adding a rooting / growth promoting agent to the seedling-growing medium, the storage failure due to long-term storage can be accelerated. It can be recovered and at the same time the yield after long-term storage can be improved.

[0070]

INDUSTRIAL APPLICABILITY According to the present invention, seedlings of rice that have been redifferentiated by the tissue culture technique can be stored for a long period of time and used as seedlings that can be transplanted to paddy fields after storage. Compared to the non-storage technology generation, the operating rate of the redifferentiation device has been dramatically increased, and as a result, a cheaper and systematic supply of redifferentiated rice seedlings has been realized. Further, the storage technology of the present invention provided the principle and basic knowledge that can be directly or indirectly applied / applied to the storage of seedlings other than rice grown or redifferentiated by tissue culture.

Claims (11)

[Claims] 1. A storage solution for rice seedlings, which comprises at least an inorganic salt and has an osmotic pressure of 5 to 120 mOSMOL / Kg. 2. The stock solution of rice seedlings according to claim 1, wherein the stock solution is a residual medium solution after completion of the rice redifferentiation culture. 3. The stock solution of rice seedlings according to claim 1 or 2, which contains a plant growth regulator having a growth inhibitory effect. 4. The stock solution of rice seedlings according to claim 3, wherein the plant growth regulator having a growth inhibitory effect is ansimidol, uniconazole, inabenfide, abscisic acid, or choline chloride. . 5. A method for storing rice seedlings, which comprises storing rice seedlings in the storage solution according to any one of claims 1 to 4. 6. The rice seedling according to claim 5, wherein the temperature condition during storage is 10 to 20 ° C., the light condition is illuminance of 200 to 30,000 Lux, and the photoperiod day length is 3 to 24 hours. How to store the body. 7. The rice seedlings are placed in a sugar-free inorganic salt liquid medium prior to storage and subjected to a culture treatment under light irradiation / carbon dioxide enriched aeration conditions. The method for storing rice seedlings described. 8. The method for storing rice seedlings according to claim 7, wherein the sugar-free inorganic salt liquid medium contains ansimidol. 9. The rice seedlings stored by the method for storing rice seedlings according to claim 5 are placed in a sugar-free inorganic salt medium and subjected to light irradiation and carbon dioxide enriched aeration conditions. A method for raising seedlings of rice seedlings after storage, which is characterized by forming seedlings. 10. The method for raising seedlings of post-storage rice seedlings according to claim 9, wherein the sugar-free inorganic salt medium contains an agent having a rooting and growth promoting action. 11. An agent having a survival and growth promoting action,
1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane, 1,9-diaminononane, α-carotene,
The method for raising seedlings of rice seedlings after storage according to claim 10, which is β-carotene, γ-carotene, cefotaxime, amphotericin B, α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin.