JPH04293437A - Preparation of plant material from gramineous callus - Google Patents

Abstract

PURPOSE:To multiply a large amount of seedlings of rice plant by culturing a gramineous callus in a liquid medium, growing the callus in a liquid medium not containing a saccharide in a fixed period from induction to regeneration of young plant material. CONSTITUTION:In culturing a gramineous callus and inducing the gramineous callus into a young plant material, at least in a fixed period from induction to regeneration of young plant material, the gramineous callus is grown in a liquid medium not containing a saccharide and the young plant material is transplanted to a seedling forming medium to prepare a plant material from the gramineous callus. At least during growth of the young plant material in the liquid medium not containing a saccharide, preferably the plant material is provided with light rays and carbon dioxide.

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 plants from callus of the Poaceae family, and in particular to a method for mass propagating rice seeds and seedlings using plant tissue culture technology.

0002

BACKGROUND OF THE INVENTION In recent years, the development of tissue culture technology has opened up the possibility of commercialization through mass propagation of many plants. Several technologies have been reported that suggest the possibility of mass propagation of rice, but it is difficult to say that the technology has reached a commercially viable level. When trying to propagate rice seedlings by tissue culture, the following two methods are possible. The first method is to directly differentiate and induce adventitious buds or embryos from rice plant tissue pieces. This technology has already been described by D. H. Ling et al. [Plant Cell
Rep. , 2, 172 (1983)], W. Were
Nicke et al. [Z. Pflanzen Physiol
．． , 103, 361 (1981), Eur. J.
Cell Biol. 24, 347 (1981)]
,D. A. Stuart and S. G. S
trickland [International Publication Publication WO 87/
[02701], etc., but the differentiation/induction rate of individuals or somatic embryos is very low, and from the point of view of proliferation efficiency, commercial mass proliferation technology has not yet been achieved.

[0003] Another example of obtaining embryoid bodies at a relatively high rate is that Matsuno et al. It has gained. However, the culture period is as long as 14 weeks, and the rate of plant regeneration from embryoid bodies is not particularly disclosed. Furthermore, it is difficult to secure a quantity of explants, and even if the explants can be propagated, the propagation efficiency is unsatisfactory, making it unsuitable as a commercial mass propagation technique.

[0004] The second method is to induce and propagate callus from rice plant tissue pieces, induce and produce adventitious buds or embryos from the callus, and regenerate them into plants. Many parts such as ovaries, anthers, immature embryos, ripe seeds, young leaves, roots, shoot apices, and panicles are used as explants for callus induction in rice, but all of them have low redifferentiation rates and are small. Mass propagation techniques have not been developed to obtain large numbers of regenerated plants from a large amount of explants.

[0005] Reported cases of inducing somatic embryos at a high rate include:
K. Ozawa and A. Komamine
Bio Industry 6, 343-350 (1
989), Theor. Appl. Genet. ，
77, 205-211 (1989)] used immature embryos of ``Konansou'' as explants and subcultured them every 3 days in a liquid growth medium supplemented with proline and casein hydrolyzate. Using a regeneration medium, somatic embryos have been induced from up to 90% or more of the transplanted callus. However, the explants for inducing callus are limited to immature embryos, and it has become clear that this technique cannot be applied to explants from other parts. Furthermore, when immature embryos are used as explants, the supply of explants is subject to time constraints and requires a considerable amount of labor. Furthermore, even though immature embryos are used as explants,
It has become clear that the range of application is limited to specific varieties and cannot be applied to ordinary cultivated varieties.

Furthermore, as a reported example of regenerating plants at a relatively high rate, T. Abe and Y. Futs
uhara [J. Plant Physiol. ，
121, 111-118 (1985)] is “Gaiy
aDhan Tosar” root-derived calli were subcultured and grown on MS agar medium containing 3 ppm of 2,4-D.
This was mixed with regeneration medium (Kinetin 1ppm, Ca
89 plants (equivalent to 200 individuals/1 g of callus) were obtained from 60% of the transplanted calli (MS agar medium containing 2000 ppm of sein). N. V. Raghava Ram and M.
W. Nabors〔Plant Cell Tis
sue Organ Culture, 4, 241-2
48 (1985)] used callus derived from the scutellum of "Pokkali" and set the density of callus on a regeneration medium (MS agar medium containing 0.5 ppm BA) at 6.5 mg/1.
ml, and by conditioning the medium (removing the E callus after leaving it on the plate for 2 weeks), we calculated that 220 plants were obtained from 1 g of callus. However, these reports concern specific cultivars, and when the method was applied to conventional cultivars, sufficient regeneration rates were not obtained.

[0007] Regarding the induction of somatic embryos and embryoid bodies in a liquid medium, the above-mentioned K. Ozawa and A.
Komamine〔Bio Industry 6
, 343-350 (1989), Theor. Appl
．． Genet. , 77, 205-211 (1989)
], T. Yoshida [BRAIN Techno News 13, 1-2 (1988)], Yoshida et al.
-63 (1989)], T. Abe and Y. Fu
tsuhara [Japan. J. Breed
．． 36, 1-6 (1986)], but all of them have a limited number of applicable varieties, and when applied to rice plants, the yield rate is not necessarily sufficient, and somatic embryoid bodies. There is no particular disclosure regarding the production of seedlings from the plant. T. According to a report by Yoshida, the number of plants obtained from 1 g of culture is as low as 18 individuals.

[0008] In addition, during the callus plant growth process, such tissue usually has a low photosynthetic ability and is thought to be unable to sufficiently produce sugars required for growth by itself, and therefore, it is usually difficult to raise seedlings. A culture medium prepared by adding saccharides such as sucrose and glucose in a range of 10 to 50 g/L as a carbon source used for the growth of plants is cultured at a constant rate in a substantially sterile culture system. The plants are grown to a suitable size, transplanted to a conditioned medium, acclimatized, and used as seedlings.

However, in such conventional methods, since a large amount of sugar is added to the medium, the medium itself is easily contaminated by bacteria from the outside during the culture period, and if the medium is contaminated with bacteria, the resistance to the medium becomes weak. There is a problem that seedlings without sterilization easily rot and die, and in order to prevent this, the culture environment such as culture containers and culture rooms must be maintained in a virtually sterile state, and care must be taken when handling them. Special attention was required. Moreover, the induced seedlings are
It is thought that the photosynthetic ability is reduced due to the sugar in the medium, despite being irradiated with light. Such plants are thought to be able to grow sufficiently in a seedling medium containing sugars, but in the normal seedling raising process, they are grown in an environment in which they were grown in a sterile culture system using only sugars as a carbon source without photosynthesis. Suddenly, plants are forced to carry out photosynthesis in a natural environment where they are exposed to various bacteria, and changes in the external environment and energy acquisition create great stress, leading to a decline in the survival rate of seedlings and slow growth. This was causing problems. However, raising seedlings under sugary conditions is considered to result in extremely high costs due to maintaining sterility.

[0010] Yoshida et al.
41 (1988), same (Separate 1) 62-63 (1989
)], when encapsulated seedlings are grown in a sugar-containing medium under sterile conditions, a seedling formation rate of 40% is obtained, but in an open system, the seedling formation rate is as low as 10%. It has become. Furthermore, the growth in this case shows that it is extremely poor. In addition, even a seedling growing medium with a low sugar concentration requires substantially sterility, so a completely sugar-free medium has been desired.

[0011] Thus far, no satisfactory report has been made regarding the commercial mass production of rice seedlings by tissue culture.

0012

[Problem to be solved by the invention] A stable supply of food is an essential issue for people's lives, and this demand is particularly strong for grasses. The purpose of the present invention is to utilize tissue culture technology to stably produce plants from grass callus on a commercial scale in large quantities, and to have sufficient durability to acclimatize when transplanted to a seedling medium. The goal is to create plants that have the following properties. Furthermore, the aim is to produce seedlings that have the same or better ability to grow in a sugar-free seedling growth medium than in a sugar-containing medium.

[0013]

[Means for Solving the Problems] In order to achieve the above object, the present inventors have conducted intensive studies on the regeneration method from Poaceae callus from the viewpoint of the medium composition to be used, the culture process, etc. The present invention has now been completed. That is,
In the present invention, when culturing grass callus in a liquid medium to regenerate seedlings, the germinated plant is grown in a liquid medium free of sugars for at least a certain period of time from induction to regeneration of the seedling. A method for producing a plant from a grass callus, which is characterized by transplanting to a seedling medium, is disclosed and provided. In addition, when referring to "young plant" in the present invention,
It refers to a plant with stems and leaves differentiated and regenerated from callus, which is not placed in a bed, and whether or not it has roots does not matter.

In the present invention, the time and period of growing in a sugar-free liquid medium differs depending on the target variety or other growing environment, but the growing time is 2 to 6 weeks after the start of regeneration of the seedlings. is usually desirable, and the period is preferably 1 day or more, particularly 7 days or more. Thereby,
The durability of the germinated body is improved, and the acclimatization in the subsequent seedling process is improved.

[0015] Furthermore, by providing light and carbon dioxide to the plants in the liquid medium during growth in a liquid medium that does not contain sugars, it becomes possible to obtain germinated plants with even more durability. . The intensity of the light may be about 3000 lux, which is used for normal tissue culture, but even stronger light may be used. In addition, during the regeneration process of seedlings, the gas environment of the liquid medium is usually controlled by shaking and aeration, but during cultivation in sugar-free liquid medium, shaking and aeration of air may be used, but oxygen cylinders may be used. etc., by mixing air with carbon dioxide and adding 250p
pm to several tens of percent, preferably from 250 to 100
Particular preference is given to venting with air containing up to 0.00 ppm of oxygen dioxide.

[0016] Furthermore, the production effect can be further improved by changing the composition of the liquid medium at least once during the process of regenerating the seedlings in the liquid medium. The means for varying the composition of the liquid medium may be a means for exchanging components to the liquid medium, or may be a means for adding components to the liquid medium. Furthermore, the two means may be used in combination as appropriate.

That is, in the present invention, after propagating callus derived from an explant of a gramineous plant, for example, in a liquid medium,
Callus is treated in a liquid medium so that it has the ability to regenerate plants stably at high frequency, and then regenerated into young plants in the container. Enables regeneration of young plants. In particular, the present invention involves growing seedlings from callus in a liquid medium that does not contain sugars for at least a certain period of time until regeneration, and then transplanting the seedlings to a seedling medium and, if necessary, adding sugars to the seedlings. During growth in a liquid medium containing no water, light and carbon dioxide were provided to the plants in the liquid medium. This promotes photosynthesis or changes the gas environment on the leaf surface, which promotes the development of the cuticle layer and releases vitrification, making it more resistant to bacteria and resistant to low humidity environments. It became possible to obtain durable germinated bodies.

The present invention will be explained in detail below. 1) Plants used for callus induction Species of the Poaceae family that can be applied in the present invention are not particularly limited, but for the genus Poaceae, for example, japonica, indica, Javanica, African rice, and hybrids thereof. can be mentioned. 2) Explants used for callus induction Explants for obtaining callus may include roots, leaves (leaf blades, leaf sheaths), stems, seeds (brown rice), embryos, as long as they have growing points or cambium. Examples include discs, embryoid bodies, seedlings, adventitious buds, and young panicles. Furthermore, ovules, anthers, etc. with high mitotic activity can also be used as explants. 3) Induction of callus The induction medium for inducing callus from explants contains at least an inorganic salt, one or more types of auxin, and sugars as essential components, as well as osmotic pressure regulators, vitamins, amino acids,
A casein hydrolyzate, a pH adjuster, etc. are added as necessary. Specifically, basic media conventionally used for plant tissue culture, such as MS medium [Muras
ige and Skoog, Physiol. pla
nt. , 15, 473-497 (1962)], N6
Medium [Chu et al. , Sci. Sin. ，
18, 658-668 (1975)], Linsmaier and Skoo
g) White, Ga
B-5 medium of Heller (nborg)
), Kohlenbach-Schmidt (Kohlenbach
A liquid medium or a solid medium prepared by adding auxin to a culture medium (e.g., H. and Schmidt) can be used. The composition of these conventionally known culture media can be found, for example, in "Plant Cell Culture" by Harada and Komamine, p. 390-39.
1, Rikogakusha, 1984.

Examples of sugars in the medium include sucrose, glucose, fructose, maltose, etc., and the concentration thereof is preferably 0.1 to 10%, preferably 0.5 to 6%.
It is particularly preferable to use it. As auxin, 2
, 4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA)
For example, these can be used alone, but they can also be used in combination. In the case of 2,4-D, the concentration thereof is preferably 0.1 to 20 ppm, particularly preferably 2 to 6 ppm. Examples of the osmotic pressure regulator include sorbitol, mannitol, etc., and the concentration is preferably 0.5 to 12% (w/v).
Particularly preferred is 1 to 6%.

Examples of amino acids include L-proline, which is preferably added 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-Morphorino)ethanesul
fonic acid, monohydrate], etc., and the pH of the medium is preferably adjusted to 5.4 to 6.4.

[0021] Further, as the gelling agent for preparing the solid medium, agar, gellan gum, etc. can be exemplified, and the concentration of these is 0.8 to 1% for agar and 0.2 to 0 for gellan gum.
．． 3% is preferred. Next, when trying to induce callus from the explant, first prepare the above-mentioned induction medium,
Place the sterile or sterile explant. The explant can be sterilized using ethyl alcohol, sodium hypochlorite solution, etc. according to a conventional method. After placing the explant, 15
Callus can be induced by standing or permeating culture at a temperature of ~35°C (preferably 25-32°C). 4) Growth of callus The growth medium for growing callus contains at least inorganic salts, one or more types of auxin, sugars, and osmotic pressure regulators as essential components, vitamins, amino acids, casein hydrolyzate, and pH. A regulator and the like are added as necessary.

[0022] A specific example is a liquid medium obtained by adding an osmotic pressure regulator to the above-mentioned induction medium. Examples of the osmotic pressure regulator include sorbitol and mannitol, and the concentration thereof is preferably 1 to 10% (w/v), particularly preferably 1 to 6%. When inducing callus, vitamins, amino acids, casein hydrolysates, pH regulators, and the like that produce favorable results when added to the induction medium have similar results in callus proliferation.

[0023] Specific culture conditions for the induced callus are shown below. When callus is induced from the explant in the induction step of 3), the callus is removed from the explant, transplanted to a growth medium, and cultured under the same conditions as in the induction step to proliferate the callus. Also, favorable results can be obtained by subculturing the callus in the growth medium every week. 5) Induction of seedlings from callus The seedling induction medium for inducing seedlings from callus contains at least inorganic salts and sugars as essential components, and optionally contains plant growth regulators, osmotic pressure regulators, and vitamins. It is possible to selectively use both a liquid medium to which saccharides, amino acids, casein hydrolyzate, a pH regulator, etc. have been added, and a so-called sugar-free liquid medium obtained by removing sugars from the liquid medium.

[0024] Specific examples of the sugar-containing liquid medium include the above-mentioned MS medium, N6 medium, and Linsmeyer-Skoog (Li
nsmaier and Skoog) White (
White), Ganborg's B
-5 medium Heller, Kohlenbach and Schm
idt) etc., and the inorganic salts thereof are 1/
It is preferable to use it at a concentration of 4 to 4 times, and 1/2 to 2 times the concentration.
It is particularly preferred to use double the concentration. Regarding phosphate, it is preferable to add 0.1mM to 50mM, preferably 0.5mM.
Additions of ~20mM are particularly preferred. Regarding calcium salts, 0.003mM to 30mM is preferable.
Particularly preferred is between mM and 10mM. Although it is not necessary to add trace metals, better results can be obtained by adding trace metals such as those contained in MS.

As for the inorganic nitrogen (N) source, 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, so that the nitrate nitrogen concentration is 10m
M to 200mM is preferred, and 20mM to 120mM is particularly preferred. Ammonia nitrogen concentration is 0mM to 40m
M is preferred, and 0.1mM to 10mM is particularly preferred. At this time, the ratio of ammonia/nitric acid is preferably 0 or more and 2 or less, and particularly preferably 0.001 or more and 1 or less.

[0026] When using saccharides, examples of the saccharides include sucrose, glucose, fructose, maltose, etc., and the concentration thereof is preferably 0.1% to 6%.
It is particularly preferred to use 0.5% to 3%. Auxin and cytokinin can be exemplified as plant growth regulators, and although these can be used alone, better results can be obtained by combining them.

[0027]As auxin, NAA, IAA, 2,4
-D, etc., and the concentration is preferably 0.01 ppm to 10 ppm for NAA and IAA, 0.001 ppm to 0.1 ppm for 2,4-D, and
Particularly preferred is A0.04ppm to 4ppm. Moreover, although these can be used alone, they can also be used in combination. Examples of cytokinin include kinetin, benzyladenine (BA), zeatin, etc., and a concentration of 0.001 ppm to 10 ppm is preferred, and these can be used alone or in combination. ABA is 0-0.1p
A concentration of pm is preferred.

[0028] Examples of the osmotic pressure regulator include sorbitol, mannitol, etc., and the concentration is 0.5 to 12%.
(w/v) is preferred, and 1% to 6% sorbitol is particularly preferred. Examples of amino acids include L-proline, etc. 1 to 10
Preferably, it is added at a concentration of 0mM. The casein hydrolyzate is preferably added at a concentration of 10 to 5000 ppm. Examples of pH regulators include MES, with concentrations ranging from 1mM to
40mM is preferred.

[0029] The so-called sugar-free liquid medium, which does not contain sugars, can specifically be a medium from which the carbon source has been removed from the above-mentioned medium, and it can also be used by diluting it appropriately or by combining the salt composition. . It can also include a rice hydroponic medium. Basic media for rice hydroponic culture include, for example, Kasugai, Kimura, and Ishizuka media, and their compositions are described, for example, in Hideo Okajima, Nutritional Physiology of Rice, P42-4.
3 (Foundation) Rural Fishing Village Culture Association, etc.

Next, a specific method for inducing seedlings will be described below. The callus obtained in the multiplication step of 4) is harvested, transplanted to a seedling induction medium, and cultured at a temperature of 15 to 35°C (preferably 25 to 32°C) to induce seedlings. . During this process, the plants are grown in a liquid medium that does not contain sugars for at least a certain period of time, and the culture is continued until they become seedlings that have sufficient acclimatization ability. Specific means for this include adding a medium from which the components have been removed or adding new components, replacing the medium with a medium containing new components, or a combination of both.

[0031] By culturing after adding or exchanging the medium, it is possible to improve the regeneration rate of seedlings from callus and the germination rate of the seedlings. By repeating the process multiple times during the process, more seedlings could be obtained. Furthermore, by growing the seedlings in a sugar-free liquid medium for at least a certain period of time during the final process, the durability of the seedlings was improved and acclimatization when transplanted to the seedling medium was smooth.

[0032] The culture conditions at that time were 3000 lu
It is particularly preferable to culture by irradiating with light of x or more and aerating air containing 250 ppm or more of oxygen dioxide. 6) Seedling generation of seedlings The seedlings obtained in the above steps are transplanted to a conventionally known seedling medium as described below and cultured in a conventional manner to obtain rice seedlings. be able to.

[0033] Although both sugar-containing and sugar-free media can be used for the seedling formation medium, a sugar-free medium is particularly preferred. Sugar seedling medium requires at least inorganic salts and sugars, and 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 aforementioned MS medium, N6 medium, Linsmeyer-Skoog (Linsm
aierand Skoog) White
e), Gamborg's B-5 medium Heller, Kohlenbach and Schmidt
) etc. can be exemplified.

Examples of sugars in the medium include sucrose, glucose, fructose, maltose, etc., and the concentration thereof is preferably 0.1% to 10.0%, preferably 0.5% to
Particularly preferred is use at 6.0%. Auxin and cytokinin can be used alone as plant growth regulators, but better results can be obtained by combining them. NAA, IAA as auxin
, 2,4-D, etc., and the concentration is N
AA and IAA 0.01ppm to 10ppm,
2,4-D is preferably 0.001 ppm to 0.1 ppm. Moreover, although these can be used alone, they can also be used in combination.

Examples of the cytokinin include kinetin, benzyladenine (BA), zeatin, etc., and the concentration is preferably 0.001 ppm to 10 ppm.
These can be used alone or in combination. Examples of osmotic pressure regulators include sorbitol and mannitol, with a concentration of 0.5
~12% (W/V) is preferred, sorbitol ~1%
6% is particularly preferred. Examples of amino acids include L-proline, which is preferably added at a concentration of 1 to 100 mM. Casein hydrolyzate is 10-5000pp
Preferably, it is added at a concentration of m.

[0036] Examples of the pH regulator include MES, and its concentration is preferably 1mM to 40mM. Examples of the support for the culture medium include gelling agents such as shlan gum and agar, and the concentration thereof is 0.2 to 0.9%.
, agar is preferably used at 0.8% to 1.8%. It is also possible to use a commercially available medium support such as ceramic fiber or rock wool impregnated with the liquid medium.

[0037] Rice seedlings were produced by placing germinated seedlings on the above-mentioned seedling medium and culturing them at a temperature of 15 to 35°C. In addition, rice plants could be obtained by transplanting the seedlings obtained in 5) to a seedling medium containing no sugars and culturing them under light irradiation while aerating carbon dioxide. The seedling growth medium is a medium containing at least inorganic salts as an essential component and optionally containing plant growth regulators, amino acids, pH regulators, and the like. Specifically, the above-mentioned MS salts, N6 salts, and known media used in rice hydroponic culture are used. The composition of the basic culture medium for rice hydroponics, such as Kasugai, Kimura, Ishizuka, etc., can be found, for example, in Hideo Okajima, Nutritional Physiology of Rice, P42-
43 (Foundation) Rural Fishing Village Culture Association, etc.

[0038]

[Examples] Hereinafter, the present invention will be explained with reference to Examples and Comparative Examples.
I will explain in detail. The results of both Examples and Comparative Examples are summarized in Table 1 below.

[0039]

[Example 1] After peeling the seeds of the rice variety Sasanishiki,
The cells were sterilized by treatment with 70% ethanol for 5 seconds and sodium hypochlorite (available chlorine 10%) for 30 minutes, thoroughly washed with sterilized water, and then placed on the callus induction medium described below. The callus was cultured at 27° C. in the dark for 8 days, and the callus generated from the scutellum was removed and transplanted into the same medium as the callus induction medium. The culture was continued for an additional 14 days, and the callus was transplanted into the growth medium described below. The callus was transplanted into a fresh growth medium every week and maintained for subculture by rotating culture at 80 rpm under a 16-hour photoperiod. Approximately 10 g of callus was placed on each 11 liquid medium. The increase in callus per week is approximately 3-4.5
It was double that. [Callus induction medium] 1% sucrose, 3% sorbitol, 12mM proline, 100ppm casein hydrolyzate, 5mM MES, 4ppm2, 4-D, 0
．． N6 solid medium containing 2% Gelrite. [Callus growth medium] N6 liquid medium containing 1% sucrose, 3% sorbitol, 12mM proline, 100ppm casein hydrolyzate, 5mM MES, 4ppm2,4-D.

[0040] Calli that had been subcultured every week in a liquid medium were transplanted to the following seedling induction medium in the first week of subculture and cultured in rotation under a 16-hour photoperiod (4000 lux). The callus turned green. After 3 weeks of culture, the medium was replaced with sugar-free medium-1 described below and culture was continued, and seedlings were formed. In the third week of culture, 20 calluses were placed on the bed.
20 plantlets per mg were obtained.

Further, the obtained seedlings were statically cultured in the following seedling medium-1 under photoperiod (light period: 30° C., dark period: 25° C.) for 16 hours under completely sterile conditions.

[0042]

[Example 2] After seedlings were induced in the same manner as in Example 1, they were cultured in the following seedling medium-2 with 1% carbon dioxide aerated. Seedling cultivation was not performed in a substantially sterile environment. Comparative Example 1 Plantlets were induced by culturing in the same manner as in Example 1, except that they were cultured in a sugar-free medium, and were made to grow into seedlings in the same manner. That is, the plants were cultured for 6 weeks on a seedling induction medium to induce seedlings, and placed on a seedling medium under completely sterile conditions.

Comparative Example 2 The plants were cultured in the same manner as in Example 2, except that they were cultured in a sugar-free medium, and seedlings were induced and seedlings were raised.

[0044]

[Example 3] Callus was induced in the same manner as in Example 1, and transplanted to a seedling regeneration medium with a photoperiod of 16 hours (4000 lux).
). After 3 weeks, the medium was changed to sugar-free medium-1, and when cultured, the light intensity was increased to 10,000 lux, and 1% carbon dioxide was aerated for shaking culture. The seedlings were grown in seedling medium-1.

[0045]

[Example 4] Callus was induced in the same manner as in Example 1, and transplanted to a seedling regeneration medium with a photoperiod of 16 hours (4000 lux).
). After 3 weeks, the medium was changed to sugar-free medium-1, and when cultured, the light intensity was increased to 10,000 lux, and 1% carbon dioxide was aerated for shaking culture. The seedlings were grown in seedling medium-2 by aerating 1% carbon dioxide.

[0046]

[Example 5] Callus was induced in the same manner as in Example 1 and transplanted to a seedling regeneration medium with a photoperiod of 16 hours (4000 lux).
). After 3 weeks, the medium was replaced with replacement medium-1 and cultured for 1 week. Furthermore, the medium was changed to 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 for shaking culture. The obtained seedlings were grown in seedling growing medium-1.

[0047]

[Example 6] In Example 5, seedlings were raised using the seedling growing medium-2 by aerating 1% carbon dioxide. Comparative Example 3 Culture was carried out in the same manner as in Example 5, except that a sugar-free medium was used and the step of culturing while aerating carbon dioxide was not used.

Comparative Example 4 Culture was carried out in the same manner as in Example 6, except that a sugar-free medium was used and the step of culturing while aerating carbon dioxide was not used. [Sapling induction medium] N6 modification containing 1% sucrose, 3% sorbitol, 0.4ppmNAA, 0.5ppm kinetin, 2000ppm casein hydrolyzate, 12mM proline, 5mM MES (added micrometallic elements of MS) liquid medium. [Seedling medium-1] 3% sucrose, 2000 ppm
casein hydrolyzate, 0.5 ppm kinetin, 0.
MS solid medium containing 3% Gelrite. [Seedling medium-2] A 0.4% Gelrite medium containing a mixture of the main component of N6 salt and the fine metal elements of MS salt, diluted 4 times. [Replacement medium] 1.5% sucrose, 1.5% sorbitol, 1ppm NAA, 0.5ppm kinetin, 1000ppm casein hydrolyzate, 2.5mM
N6 modified (MS micrometal component added) liquid medium containing MES.

[0049]

[Table 1]

[0050]

[Effects of the Invention] Clone propagation technology using tissue culture does not require a field for seed collection and is not influenced by natural conditions, making it possible to industrially produce bio-saplings of gramineous plants. According to the present invention, the induction rate of seedlings in a liquid medium from calli grown in large quantities in a liquid medium has been significantly improved compared to conventional methods. In addition, growing the seedlings in a sugar-free medium for at least a certain period of time during the process from induction to germination gives the seedlings durability, which improves their ability to acclimatize to the seedling medium. The regeneration of gramineous plants, which used to be stable and at an extremely low rate, has now become possible at an extremely stable and high rate. The present invention has made it possible to carry out all of the steps of callus propagation, seedling induction, and seedling germination in a liquid medium, making it possible to mass-produce bio-based seedlings of grasses by tank culture or the like. Furthermore, the present invention is applicable not only to fixed breeds but also to unfixed individuals, and it is possible to make hybrid individuals, etc. of cross-breeding progeny, which could not be made into breeds conventionally, into breeds.

Claims (6)

[Claims] Claim 1: When culturing grass callus in a liquid medium to induce seedlings, the seedlings are grown in a sugar-free liquid medium for at least a certain period of time from induction to regeneration of the seedlings. A method for producing a plant body from a callus of the Poaceae family, which comprises transplanting the body to a seedling medium. 2. From the Poaceae callus according to claim 1, wherein light and carbon dioxide are provided to the plant body in the liquid medium at least during growth in the liquid medium not containing sugars. A method for producing plants. 3. Plant production from Poaceae callus according to claim 1 or 2, wherein the composition of the liquid medium is varied at least once during the process of regenerating the plantlet in the liquid medium. Method. 4. The method for producing a plant from Poaceae callus according to claim 3, wherein the means for varying the composition of the liquid medium is exchange of the liquid medium. 5. The method for producing a plant from Poaceae callus according to claim 3, wherein the means for varying the composition of the liquid medium is the addition of components to the liquid medium. 6. Plant production from Poaceae callus according to claim 3, wherein the means for varying the composition of the liquid medium includes both addition of components to the liquid medium and replacement of the liquid medium. Method.