JPH0646904B2 - Method for producing plants from calli of grasses - Google Patents

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 a plant from a callus of the family Gramineae, and more particularly to a method for mass-growing rice seedlings utilizing a plant tissue culture technique.

[0002]

2. Description of the Related Art In recent years, the development of tissue culture technology has opened up the possibility of commercialization by mass multiplication in many plants.
Several technologies have been reported for rice that suggest the possibility of mass growth, but it is hard to say that they have reached the point of being commercially viable. There are two possible ways to grow rice seedlings by tissue culture. First, as a first method, there is a method of directly differentiating and inducing adventitious buds or adventitious embryos from rice plant tissue pieces. This technology has already been described in DHLing et al. [Plant Cell Rep.
3)), W. Wernicke et al. (Z. Pflanzen Physiol., 103,361 (1
981), Eur. J. Cell Biol. 24,347 (1981)], DA Stu
art and SG Strickland [International Publication WO 87/027
01] etc., but the differentiation / induction rate of individual or adventitious embryo is very low, and from the viewpoint of proliferation efficiency, it has not reached a commercial mass proliferation technique.

Further, as an example in which embryoid bodies were obtained at a relatively high rate, Matsuno et al. [JP-A-1-256319] mainly used seed roots to give about 5000 embryoid bodies per 1 g root. It has gained. However, the culture period is as long as 14 weeks, and the plant regeneration rate from embryoid bodies is not disclosed. Further, it is difficult to secure the quantitative quantity of the explants, and even when the explants can grow, the growth efficiency is not satisfactory, and it is not suitable as a technique for commercial mass breeding.

As a second method, there is a method of inducing and growing callus from a rice plant tissue piece, inducing and producing adventitious buds or adventitious embryos from the callus, and regenerating them into a plant. Many parts such as ovaries, anthers, immature embryos, mature seeds, juvenile leaves, roots, shoot tips, and panicles are used as explants for callus induction in rice. Mass propagation techniques have not been developed to obtain large numbers of regenerated plants from large amounts of explants.

[0005] As a reported example of inducing somatic embryos at a high rate,
K. Ozawa and A. Komamine 〔Bio Industry 6,343-350
(1989), Theor. Appl. Genet., 77,205-211 (1989))
However, callus induced using immature embryos of "Konansou" as explants was subcultured and maintained every 3 days in liquid growth medium supplemented with proline and casein hydrolyzate. Adventitious embryos are induced from more than 90% of transplanted callus. However, it has been clarified that the callus-inducing explants are limited to immature embryos, and that the technique cannot be applied to other parts when the explants are explants. In addition, when an immature embryo is used as an explant, the supply of the explant is time-limited and requires a considerable amount of labor. Furthermore, even if immature embryos are used as explants, the scope of their application is limited to specific varieties, and it is clear that they cannot be applied to ordinary cultivars.

[0006] Furthermore, as a reported example of regenerating plants at a relatively high rate, T. Abe and Y. Futsuhara [J. Plant P
hysiol., 121, 111-118 (1985)] is "Gaiya Dhan Tosar".
Root-derived callus is subcultured and propagated on MS agar medium containing 2,4-D 3 ppm, and this is redifferentiated medium (Kinetin 1 ppm, C
By transplanting to MS agar medium containing 2000 ppm of asein), 89 plant bodies (corresponding to 200 individuals / g callus) were obtained from the transplanted 60% callus. NV Raghava Ra
m and MW Nabors 〔Plant Cell Tissue Organ Cultur
e, 4, 241-248 (1985)] is a redifferentiation medium (MS agar medium containing 0.5 ppm of BA) in the scutellum-derived callus of "Pokkali".
By setting the density of callus to be placed at 6.5 mg / 1 ml and conditioning the medium (E callus is removed after being placed for 2 weeks), 1 g of callus can be added to 22
We have 0 plants. However, these reports relate to specific varieties, and when the method was applied to ordinary cultivars, sufficient redifferentiation rates were not obtained.

Regarding the induction of somatic embryos and embryoid bodies in a liquid medium, K. Ozawa and A. Komamine [Bio
Industry 6,343-350 (1989), Theor.Appl.Genet., 77,20
5-211 (1989)], T. Yoshida (BRAIN Techno News 13,
1-2 (1988)], Yoshida et al. [Journal of Breeding (Another 2), 140-141 (1
988), ibid (another 1), 62-63 (1989)], T. Abe and Y. Futsu.
hara [Japan. J. Breed .36, 1-6 (1986)], etc.
All of them are applicable to only limited varieties, and when applied to rice plants, their production rates are not always sufficient, and there is no particular disclosure regarding seedling formation from somatic embryoid bodies. According to a report by T. Yoshida, the number of plants obtained from 1 g of the culture is as low as 18 plants.

[0008] In the process of growing callus plants, it is considered that such tissues usually have a low photosynthetic ability and cannot sufficiently produce sugars required for growth by themselves. As a carbon source used for the growth of plants, sucrose, glucose, and other sugars are added to the medium in the range of 10 to 50 g / L, and the medium is adjusted to a certain degree in a culture system in a substantially sterile state. The plant is grown to a size and transplanted to a conditioned medium for acclimation to obtain a seedling.

However, in such a conventional method, since a large amount of saccharides is added to the medium, the medium itself is liable to be contaminated with external bacteria during the culturing period, and when the medium is contaminated with the bacterial contamination, it is resistant. Seedlings that do not have a problem that they easily spoil, in order to prevent this, keep the culture environment such as the culture container and the culture room substantially sterile, and also in the handling thereof. It required particularly strict attention. Moreover, the induced seedlings are
It is considered that the photosynthetic ability is reduced due to the sugar in the medium even though it is irradiated with light. Such plants are 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 sugars 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 the change in energy acquisition caused great stress, which caused a decrease in the survival rate of seedlings and growth Was causing the problem of. In this way, raising seedlings under the sugar-containing condition is considered to cause a very high cost for maintaining sterility.

Yoshida et al. [Breeding magazine (2), 140-141 (19)
88), ibid. (62) -63 (1989)], 40% when seedlings of encapsulated seedlings were aseptically grown in a sugar-containing medium.
However, the rate of seedlings in the open system is as low as 10%. Furthermore, it shows that the growth in this case is extremely poor. Further, even a seedling-growing medium having a low sugar concentration needs to be substantially aseptic, and thus a complete sugar-free medium has been desired.

As described above, there have been no satisfactory reports on commercial mass production of rice seedlings by tissue culture.

[0012]

[Problems to be Solved by the Invention] A stable food supply is an essential issue for people's lives, and there is a strong demand for grasses. An object of the present invention is to produce a large amount of a plant from a callus of a Gramineae plant on a commercial scale stably by utilizing a tissue culture technique, and to have sufficient durability to be acclimatized when transplanted to a seedling medium. Is to create a plant with. Furthermore, even in a sugar-free seedling-growing medium, it is intended to produce a young plant having the ability to grow to the same level as or higher than that in a sugar-containing medium.

[0013]

[Means for Solving the Problems] In order to achieve the above-mentioned object, the present inventors have diligently studied a method for redifferentiating from a callus of the family Gramineae from the viewpoints of the composition of the medium to be used, the culturing process, etc. The present invention has been completed. That is,
The present invention, when the callus of the grass family is cultivated in a liquid medium to regenerate a young plant, after growing in a liquid medium containing no sugar for at least a certain period from induction to regeneration of the young plant, the germinated body is Disclosed and provided is a method for producing a plant from a grass callus, which comprises transplanting to a seedling medium. In the present invention, the term “young plant” refers to a plant having foliage that has been differentiated and regenerated from callus and has not been placed on the floor, and whether rooting or not is not particularly limited.

In the present invention, the growing time and period in the liquid medium containing no saccharides differ depending on the target cultivar or other growing environment, but the growing time is from 2 to 6 weeks after the start of the regeneration of the seedlings. Is usually desirable, and the period is preferably 1 day or longer, more preferably 7 days or longer. Thereby,
The durability of the germinated body is improved, and the acclimation in the subsequent seedling process becomes good.

Furthermore, by giving light and carbon dioxide to the plants in the liquid medium during the growth in the liquid medium containing no saccharide, it becomes possible to obtain a more durable germinated body. . The light intensity may be about 3000 lux, which is used for normal tissue culture, but more intense light may be irradiated. Also, in the seedling regeneration process, usually, the gas environment of the liquid medium is controlled by shaking or aeration, but during the cultivation in the sugar-free liquid medium, shaking or aeration of air may be used, but an oxygen dioxide cylinder By mixing oxygen dioxide with air, etc., from 250ppm to several tens%
It is particularly preferred to ventilate air containing oxygen dioxide up to 1, preferably from 250 to 10,000 ppm.

Further, in the process of regenerating a young plant in the liquid medium, the production effect can be further improved by making the composition of the liquid medium different at least once. The means for varying the composition of the liquid medium may be means for exchanging the components in the liquid medium or means for adding the components. Furthermore, the two means may be used in an appropriate combination.

That is, according to the present invention, after the callus derived from the explant of a grass family plant is grown in, for example, a liquid medium,
Callus was treated with a liquid medium at a high frequency to have a plant regenerating ability stably and regenerated into seedlings in the container by mass culture using a tank culture tank or the like. Enables regeneration of young plants. In particular, the present invention, after growing in a liquid medium that does not contain sugar for at least a certain period leading to the regeneration of seedlings from callus, and transplanting the seedlings into a seedling medium, if necessary, sugar Light and carbon dioxide were given to the plants in the liquid medium during the growth in the liquid medium containing no liquid. As a result, photosynthesis is promoted or the gas environment on the leaf surface is changed, the development of the cuticle layer is promoted and the vitrification is released, and it is resistant to various bacteria and resistant to low humidity environment. It became possible to obtain a germinated body with durability.

The present invention will be described in detail below. 1) Plant used for inducing callus The species of Gramineae applicable in the present invention is not particularly limited, but as for the genus Gramineae, for example, Japonica, Indica, Javanica, African rice and hybrids thereof and the like can be used. Can be mentioned. 2) Explant used for callus induction As an explant for obtaining callus, any explant having a growing point or cambium may be used as root, leaf (leaf blade, leaf sheath),
Stem, seed (brown rice), scutellum, embryoid body, seedling, adventitious bud,
A young ear and the like can be exemplified. In addition, ovules, anthers, etc. having high mitotic activity can also be used as explants. 3) Callus induction The induction medium for inducing callus from explants contains at least an inorganic salt, one or more auxins and sugars as essential components, and an osmotic pressure regulator, vitamins, amino acids,
A casein hydrolyzate, a pH adjuster and the like are added as necessary. Specifically, a basal medium conventionally used for plant tissue culture, for example, MS medium [Murasige a
nd Skoog, Physiol.plant., 15, 473-497 (1962)], N6
Medium (Chu et al., Sci. Sin., 18,658-668 (1975)),
Linsmaier and Skoog White, Ganborg B-5 medium Heller, Kohlenbach Schmidt
A liquid medium or a solid medium prepared by adding auxin to h and Schmidt) can be used. The composition and the like of these conventionally known culture media are described in, for example, “Plant Cell Culture” p390-391 by Harada and Komine, Rigaku Kogyo Co., Ltd., 1984.

Examples of saccharides in the medium include sucrose, glucose, fructose, maltose and the like, and the concentration thereof is preferably 0.1 to 10%, particularly preferably 0.5 to 6%. As auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (I
AA), naphthalene acetic acid (NAA) and the like can be exemplified, and these can be used alone or in combination. If the added concentration is 2,4-D,
It is preferably used at 0.1 to 20 ppm, particularly preferably 2 to 6 ppm. Examples of the osmotic pressure adjusting agent include sorbitol and mannitol, and the concentration is 0.5 to 12% (w / v)
Is preferable, and 1-6% is especially preferable.

Examples of amino acids include L-proline and the like, and it is preferable to add them at a concentration of 1 to 100 mM. The casein hydrolyzate is preferably added at a concentration of 10 to 500 ppm. MES [2- (N-Morpholino) as a pH adjuster
ethanesulfonic acid, monohydrate] and the like, and the pH of the medium is preferably adjusted to 5.4 to 6.4.

As the gelling agent for preparing the solid medium, agar, gellan gum and the like can be exemplified, and the concentration of these is preferably agar 0.8-1% and gellan gum 0.2-0.3%. Then, if callus is to be induced from the explant, the induction medium described above is first prepared and a sterile or sterilized explant is placed. The sterilization of the explant can be carried out using ethyl alcohol, sodium hypochlorite solution or the like according to a conventional method. After the explant is placed, callus can be induced by standing or osmotic culture at a temperature of 15 to 35 ° C (preferably 25 to 32 ° C). 4) Growth of Callus A growth medium for growing callus contains at least inorganic salts, one or more auxins, saccharides, and osmotic pressure adjusting agents as essential components, vitamins, amino acids, casein hydrolyzate, and pH. A regulator or the like is added if necessary.

Specifically, a liquid medium prepared by adding an osmotic pressure adjusting agent to the above induction medium can be exemplified. Examples of the osmotic pressure adjusting agent include sorbitol and mannitol, and the concentration is preferably 1 to 10% (w / v), particularly preferably 1 to 6%. Vitamins, amino acids, casein hydrolysates, pH regulators and the like, which give favorable results when added to the induction medium when inducing callus, have similar results in the growth of callus.

The specific culture conditions of the induced callus are shown below. When callus is induced from the explant by the induction step of 3), the callus is removed from the explant and transplanted to a growth medium, and the callus is grown by culturing under the same conditions as in the induction step. In addition, preferable results can be obtained by subculturing the callus in the growth medium every week. 5) Induction of seedlings from callus A seedling induction medium for inducing seedlings from callus contains at least inorganic salts and saccharides as essential components, and if necessary, plant growth regulators, osmotic pressure regulators, vitamins. It is possible to selectively use both a liquid medium to which a hydrolyzate, an amino acid, a casein hydrolyzate, a pH adjuster and the like is added, and a so-called sugar-free liquid medium obtained by removing saccharides from the liquid medium.

Specific examples of the sugar-containing liquid medium include the above-mentioned MS medium, N6 medium, and Linsmeier-Skoog (Lins).
maier and Skoog) White, Ganborg
borg's B-5 medium Heller, Korenbach
For example, Schmitt (Kohlenbach and Schmidt) and the like can be exemplified, and the inorganic salts thereof are preferably used at a concentration of 1/4 to 4 times, and particularly preferably at a concentration of 1/2 to 2 times. Regarding phosphate, addition of 0.1 mM to 50 mM is preferred
The addition of 0.5 mM to 20 mM is particularly preferred. With respect to the calcium salt, 0.003 mM to 30 mM is preferable, and 0.03 mM to 10 mM is particularly preferable. Trace metals may not be added, but for example M
Even better results are obtained by adding trace metals such as those contained in S.

Regarding the source of inorganic nitrogen (N), nitrate nitrogen and ammonia nitrogen can be supplied, but it is preferable to use nitrate nitrogen alone or a combination of nitrate nitrogen and ammonia nitrogen, and the nitrate nitrogen concentration Is 10 mM ~
200 mM is preferred and 20 mM to 120 mM is particularly preferred. The ammonia nitrogen concentration is preferably 0 mM to 40 mM, particularly preferably 0.1 mM to 10 mM. At this time, the ammonia / nitric acid ratio is 0
It is preferably 2 or more and 2 or less, and particularly preferably 0.001 or more and 1 or less.

When saccharides are used, examples of the saccharides include choucloth, glucose, fructose, maltose, etc., and the concentration thereof is preferably 0.1% to 6%, and 0.5
% To 3% is particularly preferable. Examples of the plant growth regulator include auxin and cytokinin, and these can be used alone, but better results can be obtained by combining them.

NAA, IAA and 2,4-D as auxins
Etc., and the concentration can be NAA and IA.
A 0.01 ppm to 10 ppm and 2,4-D 0.001 ppm to 0.1 ppm are preferable, and NAA 0.04 ppm to 4 ppm is particularly preferable. Further, these can be used alone or in combination. Examples of cytokinins include kinetin, benzyladenine (BA), zeatin and the like, and a concentration of 0.001 ppm to 10 ppm is preferable, and these can be used alone or in combination. The concentration of ABA is preferably 0 to 0.1 ppm.

Examples of the osmotic pressure adjusting agent include sorbitol and mannitol, and the concentration is 0.5 to 12% (w / v).
Is preferred, and sorbitol 1% to 6% is particularly preferred.
Examples of amino acids include L-proline and the like.
It is preferable to add it at a concentration of M 3. The casein hydrolyzate is preferably added at a concentration of 10 to 5000 ppm.
MES is mentioned as a pH adjuster, and the concentration is 1 mM to 40.
mM is preferred.

As the so-called sugar-free liquid medium having no saccharide, specifically, a medium obtained by removing a carbon source from the above-mentioned medium can be mentioned, and it can be appropriately diluted or used in combination with a salt composition. . Further, a rice hydroponic medium can be used. The basal medium of the hydroponic medium of rice may be, for example, the medium of Kasugai, Kimura, Ishizuka, etc., and the composition thereof is, for example, Hideo Okajima, Nutrition Physiology P42-43 of rice.
It is listed in the Rural Fishing Village Cultural Association, etc.

Next, a specific method for inducing seedlings will be described below. The callus obtained in the growth step of 4) is harvested, transplanted to a seedling-inducing medium, and cultured at a temperature of 15 to 35 ° C (preferably 25 to 32 ° C) to induce seedlings. . Then, in the process, it is grown in a liquid medium containing no saccharide for at least a certain period of time, and the culture is continued until it becomes a seedling having a sufficient acclimatizing ability. As specific means for that, addition of a medium from which components have been removed or new components have been added, replacement of the medium with new components,
It can be performed by using both of them together.

By adding and changing the medium and culturing, the rate of seedling regeneration from callus and the germination rate of seedlings can be improved. In addition, the medium addition or exchange can be carried out during the seedling regeneration process. It was possible to obtain more seedlings by carrying out multiple times during the course. Furthermore, in the last step, by growing the seedlings in a liquid medium containing no sugar for at least a certain period of time, the durability of the seedlings was improved and the acclimation when transplanted to the seedling medium was smooth.

As the culturing conditions at that time, it is particularly preferable to irradiate light of 3000 lux or more and aerate air containing 250 ppm or more of oxygen dioxide for culturing. 6) Seedling of seedlings The seedlings of the seedlings obtained in the above steps are transplanted to the following well-known seedling medium and cultured by a conventional method to obtain rice seedlings. be able to.

As the seedling medium, both sugar-containing and sugar-free ones can be used, but a sugar-free medium is particularly preferable. A sugar-containing seedling medium requires at least inorganic salts and sugars, and if necessary, plant growth regulators, osmotic pressure regulators, vitamins, amino acids, casein hydrolysates, pH regulators, etc. are added as necessary. It is a solid medium. Specifically, the above-mentioned MS medium, N6 medium, and Linsmaiera Scoog (Linsmaiera) are used.
nd Skoog) White, Ganborg
Examples of the B-5 medium include Heller, Kohlenbach and Schmidt, and the like.

Examples of saccharides in the medium include sucrose, glucose, fructose, maltose and the like, and the concentration thereof is preferably 0.1% to 10.0%, particularly preferably 0.5% to 6.0%. Auxin and cytokinin can be used alone as the plant growth regulator, but better results can be obtained by combining them. NAA, IAA, 2,4-D and the like can be exemplified as the auxin, and the concentration can be NAA and IAA 0.01.
ppm to 10 ppm and 2,4-D 0.001 ppm to 0.1 ppm are preferable. Further, these can be used alone or in combination.

As the cytokinin, kinetin, benzyladenine (BA), zeatin and the like can be exemplified, and a concentration of 0.001 ppm to 10 ppm is preferable, and these can be used alone or in combination. Examples of the osmotic pressure adjusting agent include sorbitol and mannitol. The concentration is preferably 0.5 to 12% (W / V), and sorbitol is 1% to 6%. Examples of amino acids include L-proline and the like, and it is preferable to add them at a concentration of 1 to 100 mM. The casein hydrolyzate is preferably added at a concentration of 10 to 5000 ppm.

MES is mentioned as a pH adjuster, and the concentration is preferably 1 mM to 40 mM. Examples of the medium support include gelling agents such as sucrose gum and agar. The concentrations thereof are gellite 0.2 to 0.9% and agar 0.8% to 1.
It is preferably used at 8%. It is also possible to use a commercially available medium support such as ceramic fiber or rock wool, which is soaked with the above liquid medium.

Rice seedlings could be produced by placing germinated seedlings on the above seedling medium and culturing at a temperature of 15 to 35 ° C. In addition, a rice plant could be obtained by transplanting the seedling obtained in 5) into a sugar-free seedling medium and culturing by aerating carbon dioxide under light irradiation. The seedling medium is a medium containing at least an inorganic salt as an essential component and, if necessary, a plant growth regulator, amino acids, a pH regulator and the like. Specifically, it is a known medium used for the above-mentioned MS salt, N6 salt and rice hydroponic culture. The composition of a basic medium for rice hydroponics, for example, Kasugai, Kimura, Ishizuka, etc. is described by Hideo Okajima, Nutrition Physiology of Rice P42-43.
It is listed in the Rural Fishing Village Cultural Association.

[0038]

EXAMPLES Hereinafter, the present invention will be described with reference to Examples and Comparative Examples.
This will be specifically described. The results of the examples and comparative examples are summarized in Table 1 below.

[0039]

[Example 1] After peeling the seeds of a rice cultivar Sasanishiki,
The cells were treated with 70% ethanol for 5 seconds, sterilized by treatment with sodium hypochlorite (effective chlorine 10%) for 30 minutes, washed well with sterilized water, and then placed on the following callus induction medium. After culturing at 27 ° C. in the dark for 8 days, the callus generated from the part of 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 every week to fresh growth medium,
The cells were cultivated by swirling at 80 rpm for the duration of time and maintained for passage. About 10 g of callus was placed per 11 liquid media. The increase in callus per week was about 3-4.5 times. [Callus induction medium] 1% shoe cloth, 3% sorbitol, 12 mM proline, 100 ppm casein hydrolyzate, 5 mM
N6 with MES, 4ppm2, 4-D, 0.2% gellite
Solid medium. [Callus growth medium] 1% choux cross, 3% sorbitol, 12 mM proline, 100 ppm casein hydrolyzate, 5 mM
N6 liquid medium containing MES, 4 ppm 2,4-D.

Callus that 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 culture was carried out under 16-day photoperiod (4000 lux). Callus became green. After 3 weeks of culture, the following sugar-free medium-1 was replaced and the culture was continued. As a result, seedlings were formed. After 3 weeks of culture, per 20 mg of callus
20 seedlings were obtained.

The seedlings thus obtained were cultivated in the following seedling medium-1 under static conditions for 16 hours under photoperiod (light period 30 ° C., dark period 25 ° C.) under a completely sterile condition.

[0042]

Example 2 After inducing young plants in the same manner as in Example 1, 1% carbon dioxide was aerated and cultured in the following seedling medium-2. Raising seedlings was not performed under a substantially sterile environment. Comparative Example 1 Except for culturing in a sugar-free medium, culturing was conducted in the same manner as in Example 1 to induce seedlings, and seedlings were similarly sown. That is, the seedlings were cultured in a seedling-inducing medium for 6 weeks to induce the seedlings, and the seedlings were placed in a seedling medium under a completely sterile condition.

Comparative Example 2 Except for culturing in a sugar-free medium, culturing was carried out in the same manner as in Example-2 to induce seedlings to raise seedlings.

[0044]

Example 3 In the same manner as in Example 1, callus was induced, transplanted into a seedling regeneration medium, and cultivated by rotation under a 16-day photoperiod (4000 lux). After 3 weeks, when the medium was replaced with sugar-free medium-1 and cultured, the light intensity was increased to 10,000 lux and 1% carbon dioxide was aerated to perform shaking culture. Seedling medium is seedling medium-
Raised seedlings in 1.

[0045]

[Example 4] In the same manner as in Example 1, callus was induced, transplanted into a seedling regeneration medium, and cultivated by rotation under a 16-hour photoperiod (4000 lux). After 3 weeks, when the medium was replaced with sugar-free medium-1 and cultured, the light intensity was increased to 10,000 lux and 1% carbon dioxide was aerated to perform shaking culture. The seedlings were raised by aeration with 1% carbon dioxide in the seedling medium-2.

[0046]

[Example 5] In the same manner as in Example 1, callus was induced, transplanted to a seedling regeneration medium, and cultivated by rotation under a 16-day photoperiod (4000 lux). After 3 weeks, the medium was replaced with the exchange medium-1 and the cells were cultured for 1 week. Further, the medium was replaced with a sugar-free medium-1 and cultured for 2 weeks.
When culturing in a sugar-free medium, the light intensity was increased to 10,000 lux and 1% carbon dioxide was aerated to perform shaking culture. The obtained seedlings were raised in seedling culture medium-1.

[0047]

Example 6 Seedlings were raised by aerating 1% carbon dioxide using the seedling medium-2 in Example 5. Comparative Example 3 Culture was carried out in the same manner as in Example 5 except that the step of culturing using a sugar-free medium and aerated with carbon dioxide was not used.

Comparative Example 4 Culture was carried out in the same manner as in Example 6 except that the step of culturing with a sugar-free medium and aerated with carbon dioxide was not used. [Seedling induction medium] 1% sucrose, 3% sorbitol, 0.4 ppm NAA, 0.5 ppm kinetin, 2000 ppm casein hydrolyzate, N6 modification containing 12 mM proline, 5 mM MES (MS micrometal element added) liquid medium . [Seedling medium-1] MS solid medium containing 3% sucrose, 2000 ppm casein hydrolyzate, 0.5 ppm kinetin, and 0.3% gellite. [Seedling medium-2] A 0.4% gellite medium prepared by diluting a salt prepared by mixing a N6 salt main component and a fine metal element of MS salt four times. [Exchange medium] N6-modified (containing MS fine metal component) liquid medium containing 1.5% sucrose, 1.5% sorbitol, 1 ppm NAA, 0.5 ppm kinetin, 1000 ppm casein hydrolyzate, and 2.5 mM MES.

[0049]

[Table 1]

[0050]

EFFECTS OF THE INVENTION The clonal multiplication technique by tissue culture does not require a field for seeding and enables industrial production of bio-seedlings of gramineous plants without being affected by natural conditions. According to the present invention, the induction rate of seedlings in a liquid medium from callus cultured and proliferated in a large amount in the liquid medium was significantly improved as compared with the conventional method. In addition, by growing the seedlings in a medium without sugar for at least a certain period in the process from the induction of seedlings to germination, the seedlings are endowed with their durability, thereby improving the acclimatization ability to seedling culture medium. The stable and extremely low rate of regeneration of gramineous plants became extremely stable and possible at a high rate. According to the present invention, all the steps of callus multiplication, seedling induction, and seedling germination can be performed in a liquid medium, and mass production of bio-seedlings of grasses by tank culture or the like has become possible.
In addition, the present invention can be applied not only to fixed varieties but also to unfixed individuals, and hybrid varieties such as hybrid progeny that could not be conventionally varieties can be used as varieties.

Claims (6)

[Claims] 1. When inducing a young plant by culturing a callus of the family Gramineae in a liquid medium, after growing in a liquid medium containing no sugar for at least a certain period from induction to regeneration of the young plant, the young plant A method for producing a plant from a callus of the family Gramineae, which comprises transplanting the body into a seedling medium. 2. The callus of the family Poaceae according to claim 1, characterized in that light and carbon dioxide are given to the plants in the liquid medium at least during the growth in the liquid medium containing no sugar. Plant production method. 3. Production of a plant from a callus of the family Gramineae according to claim 1 or 2, wherein the composition of the liquid medium is varied at least once in the process of regenerating the young plant in the liquid medium. Method. 4. A means for varying the composition of the liquid medium comprises:
The method for producing a plant from a callus of the family Gramineae according to claim 3, wherein the liquid medium is replaced. 5. Means for varying the composition of the liquid medium comprises:
The method for producing a plant from a callus of the family Gramineae according to claim 3, which comprises adding components to the liquid medium. 6. A means for varying the composition of the liquid medium,
The method for producing a plant from a callus of the family Gramineae according to claim 3, wherein the addition of the components to the liquid medium and the exchange of the liquid medium are both performed.