JP3290013B2 - Method for inducing and germinating somatic embryos - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to mass propagation of plants by adventitious embryo formation using plant tissue culture technology.

[0002]

2. Description of the Related Art There have been many studies on plant tissue culture for the purpose of multiplying vegetatively propagating plants, plants that are difficult to genetically fix, or plants that are difficult to seed, in large quantities. For example, in asparagus Chee-kok Chin (H
ortscience 17 (4): 590-591, 1982), Takayama et al.
In garlic, S. Bhojwani (Scienti
a Horticulturae, 13: 47-52, 1980), and many other reports on carnations, such as Fujino et al. (Abstracts of Papers Presented at the Spring Meeting of the Japanese Society of Horticultural Science, 1971, p.302-303). Especially in recent years,
Numerous studies have been made on techniques for mass propagation of plants using somatic embryogenesis. For example, in carrots, Kamata (chemical regulation of plants, Vol.15 (2): 62-78,1980), and in asparagus, T. Saito et al. (Plant Cell Reports 10: 230-234,199).
1), Komura et al. (Abstracts of Papers Presented at the Spring Meeting of the Horticultural Society of Japan, 1988, p.232-233), Hirata et al. (P.230-231).

[0003] The method of propagation using this somatic embryo formation is as follows: 1. The growth rate is faster than the conventional tissue culture method. It has advantages such as easy labor saving by scale-up and mechanization, and significant cost reduction compared to the conventional tissue culture method, which mostly involves manual work.

[0004] Although the mass propagation method using somatic embryo formation has the above advantages, there are some problems in practical use. It is: 1. The efficiency of somatic embryo induction and somatic embryo germination is low in many plant species. For systematization of mass culture using somatic embryo formation,
It is desirable to induce synchronized somatic embryos and germinate somatic embryos, but in many plants, induction of somatic embryos or germination of somatic embryos is poor.

[0005] In mass propagation utilizing somatic embryo formation, it is possible to reduce costs by mechanizing seedling production by artificial seeding or the like. When mechanization of somatic embryo production is performed, the size of the somatic embryo must be uniform. When using somatic embryos of non-uniform size,
The possibility that an adventitious embryo becomes clogged in the adventitious embryo production system increases. One of the causes that hinders the uniformity of the somatic embryo is that a new somatic embryo is secondarily formed from the induced somatic embryo itself. Since the secondary somatic embryo thus formed remains bound to the original somatic embryo, when secondary somatic embryogenesis occurs, the size or shape of the somatic embryo becomes uneven.

[0006] Furthermore, the adventitious embryo may germinate but die during the seedling process, or the culture may take a long time due to the slow growth, which may reduce the cost of the step of seeding the adventitious embryo-derived seedling. It is a big problem in raising.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to suppress the formation of secondary somatic embryos from induced somatic embryos and to provide compact and uniform-sized somatic embryos. Another object of the present invention is to improve the seedling establishment rate of a young plant germinated from a somatic embryo.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, have found that the above object can be achieved by applying nitrilotriacetic acid to somatic embryogenesis. This nitrilotriacetic acid is known as a chelating agent and is known to promote the activity of indoletriacetic acid (IAA) oxidase, which is known as one of endogenous hormones in plants (R. Pressey, 1989, Plant Physiolog
y 93: 798-804, 1990). In the adventitious embryo-inducing medium to which nitrilotriacetic acid has been added, the ratio of the adventitious embryos that have become individual ones has increased, and when added to the germination medium, the somatic embryos germinate and the plants obtained grow vigorously. This has led to the present invention. In the present invention, each somatic embryo is referred to as a single embryo. The ratio of single embryos to all somatic embryos induced is referred to as a single embryo rate.

That is, the first of the present invention is a method for inducing somatic embryos, which comprises inducing somatic embryos by culturing plant tissue in a medium containing nitrilotriacetic acid. Here, as a plant tissue, asparagus ( Asparagu
s officinalis L. ) can be used.
A second aspect of the present invention provides a medium containing nitrilotriacetic acid,
This is a method for germinating somatic embryos, which comprises germinating somatic embryos by culturing somatic embryos. Here, as the somatic embryo, an somatic embryo derived from asparagus ( Asparagus officinalis L. ) can be used.

A third aspect of the present invention is an adventitious embryo-inducing medium containing nitrilotriacetic acid. A fourth aspect of the present invention is a somatic embryo germination medium containing nitrilotriacetic acid. Hereinafter, a specific method for carrying out the invention will be described. (1) Preparation of somatic embryo-derived material (1-1) Preparation of plant tissue as explant The plant species used as the material of the present invention can be used not only for dicotyledonous plants and monocotyledonous plants, but for example, dicotyledonous plants Examples include Apiaceae, Leguminosae, Solanaceae, Brassicaceae, Cucurbitaceae, Rutaceae, and Rosaceae.Specifically, carrots, alfalfa,
Cellulose, spinach, cyclamen, wasabi, tomato, etc. can be used, and monocotyledonous plants include lilies, gramineae, orchids, etc.Specifically, asparagus, rice, corn, garlic, leek, onion, etc. are used it can. In the present invention, plant tissues used for inducing somatic embryos include true leaves, cotyledons, hypocotyls, false leaves, stems, seeds, seed embryos, immature embryos, roots cut from greenhouse plants or in vitro sterile plants. Explants are available. When using a greenhouse plant as a material, it is necessary to perform a treatment such as surface sterilization to make it sterile. For the surface sterilization of the plant material, it is also possible to soak the plant material in a 70% ethanol solution for 30 seconds and then soak in a 1% sodium hypochlorite solution for 10 minutes.

[0011] Furthermore, the plant tissue used for inducing somatic embryos is not limited to explants cut from plants. That is, in addition to the already differentiated tissue, either dedifferentiated callus or a cell mass having an epidermal system (see JP-A-2-513159) may be used as long as it has an adventitious embryo-forming ability. Examples of the preparation of callus having the ability to form somatic embryos include, for example, a review of Amirato (Amirato, PV, 1982. Handbook of pla
nt cell culture, vol.1, (Evans, DA et al.) 82-113.
Macmilian Publishing Company, New York.) And a review by Nishimura et al. (Bioholty, Vol. 5, pp. 9-15). (1-2) Callus as a material for inducing somatic embryos
Explants cut from the above-mentioned plants can be used for induction and callus induction. Explants of plant bodies, at least inorganic salts,
Callus is induced by culturing in a medium containing a carbon source and auxin as essential components and adding vitamins and amino acids as necessary. Examples of the basic medium include Murashige and Skoog's medium (MS medium), Rinsmeyer and Skoog's medium (LS medium), Gamborg's B5 medium (B5 medium), and white medium. A basal medium can be used. As a carbon source, 5-100 g / l of sucrose, glucose, fructose, maltose, galactose and the like can be used. As hormones, auxins include 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), IA
A, indole-3-butyric acid (IBA), picloram, p-chlorophenoxyacetic acid (pCPA) and dicamba (DICAMBA) can be used, but 2,4-D and NAA are preferred. The concentration of auxins in the medium may be from 0.01 to 50 mg / l, but is more preferably from 0.1 to 20 mg / l. Callus induction medium is agar,
Gellite, even a solid medium containing agarose, etc.
A liquid medium containing no gelling agent may be used. The concentration of the gelling agent is 0.4-3.0% for agar and 0.05-2.0 for gellite.
%. Callus induction medium with pH 4.0-
After adjusting to 7.0, preferably 5.0-6.5, it can be sterilized by high-pressure steam sterilization or filter sterilization.

As the physical conditions in the callus induction step, it is possible to perform shaking culture using a liquid medium or static culture using a solid medium. The shaking method may be either reciprocating or rotating. Shake culture amplitude 60-1
60 rpm is available, but 70-100 rpm is more preferred. The culture conditions for callus induction may be either light or dark.

The callus induced by the above method can be grown in the same manner and medium as in callus induction. Also, lowering the auxin concentration in the callus growth medium compared to the callus induction medium may provide favorable results. (1-3) Cell mass as a material for inducing somatic embryos
Roots cut from the above-mentioned plants can be used as explants for induction and induction of proliferating cell mass. The cell mass is induced by culturing the root in a liquid medium containing at least an inorganic salt, a carbon source, and auxin as essential components, and adding vitamins, amino acids, and the like as necessary. As the basic medium, various basic media used for plant tissue culture such as MS medium, LS medium, B5 medium, and white medium can be used. As a carbon source, 5-100 g / l of sucrose, glucose, fructose, maltose, galactose and the like can be used. As hormones, auxins include 2,4-D, NAA, IAA, IBA, picloram, pCPA, DICAMBA
Can be used, but 2,4-D and NAA are preferred. The concentration of auxins in the medium may be from 0.01 to 50 mg / l, but is more preferably from 0.1 to 20 mg / l. After adjusting the pH to 4.0-7.0, preferably 5.0-6.5, the callus induction medium can be sterilized by high-pressure steam sterilization or filter sterilization.

[0014] In the above medium, 5-15
By adding 0 g / l of sugar alcohol such as mannitol or sorbitol, more preferable results may be obtained. The step of inducing the cell mass requires culturing using a liquid medium. As a physical condition at the time of culturing, a stationary state is possible, but shaking is preferable. The shaking method may be either reciprocating or rotating. The amplitude number of the shaking culture can be 60-160 rpm, but 70-100 rpm is more preferable.

The culture conditions for inducing the cell mass may be a light place or a dark place. The cell mass induced by the above method can be grown in the same medium as the cell mass induction. Also,
Lowering the auxin concentration in the cell mass growth medium compared to the cell mass induction medium may produce favorable results. (2) Induction of somatic embryos In the present invention, somatic embryos are induced by culturing plant tissues in an adventitious embryo induction medium containing nitrilotriacetic acid.

The somatic embryo induction medium described below is characterized by containing nitrilotriacetic acid. Nitrilotriacetic acid can be autoclaved after being added to the medium, but preferably sterilized by a filter and then added aseptically to the medium after autoclaving. When a high concentration of nitrilotriacetic acid is added to the medium, it is desirable to adjust the pH of the nitrilotriacetic acid solution to the pH of the medium to be added before adding the medium. (2-1) When Callus is Used as a Material for Inducing an Adventitious Embryo The details of the adventitious embryo induction step will be described below, taking the case where callus is used as a material as an example. Calli having the ability to form somatic embryos can be referred to the methods described in the reviews of Amirat and the reviews of Nishimura et al. The somatic embryo induction medium has at least inorganic salts and a carbon source as essential components,
Vitamins, amino acids, and hormones are added as needed. For example, as a basal medium,
Various basic media used for plant tissue culture, such as an MS medium, an LS medium, a B5 medium, and a white medium, can be used. As a carbon source, 5-100 g / l of sucrose, glucose, fructose, maltose, galactose and the like can be used. Induction of somatic embryos from callus is possible without using auxins or cytokinins, but good results may be obtained by using auxins and cytokinins alone or in combination. Hormones include 2,4-D, NAA, and IA as auxins
A, IBA, picloram, pCPA, DICAMBA are available,
Preferably, NAA, IAA, and 2,4-D are used. Cytokinins include kinetin, benzyladenine (BA), zeatin, isopentenyladenine, 4-phenylurea (4-phenylurea
PU) can be used, but zeatin, kinetin and BA are preferred. In addition, by adding gibberellin, ancimidol, or the like, more somatic embryos can be induced in some cases. The somatic embryo induction medium may be a solid medium containing agar, gellite, or the like, or a liquid medium containing no gelling agent. The concentration of the gelling agent is 0.
It can be used at 4-3.0% and 0.05-2.0% for gellite. The somatic embryo induction medium has a pH of 4.0-7.0, preferably 5.0
After adjusting to -6.5, it can be sterilized by high-pressure steam sterilization or filter sterilization.

As physical conditions in the somatic embryo induction step, shaking culture using a liquid medium or static culture using a solid medium is possible. When a liquid medium is used, the shaking method may be either reciprocating or rotating. The shaking culture can be used at an amplitude of 60-160 rpm,
100 rpm is more preferred. The present invention is characterized in that by adding nitrilotriacetic acid to a somatic embryo induction medium, the rate of single somatic embryos to be induced is increased. Nitrilotriacetic acid can be autoclaved after being added to the medium, but preferably sterilized by a filter and then added aseptically to the medium after autoclaving. When a high concentration of nitrilotriacetic acid is added to the medium, it is desirable to adjust the pH of the nitrilotriacetic acid solution to the pH of the medium to be added before adding the medium. The nitrilotriacetic acid to be added to the somatic embryo induction medium can use 0.1-1000 μM. Nitrilotriacetic acid can be added to the somatic embryo induction medium from the beginning, and in some cases, between the start of somatic embryo induction and the recovery of somatic embryos, many somatic embryos with a high single embryo rate can be obtained. May be obtained. When nitrilotriacetic acid is added to the medium from the beginning at the time of somatic embryo induction, the preferred concentration is 0.1-
200 μM. When nitrilotriacetic acid is added after initiating somatic embryo induction, the preferred concentration is 0.5-500 μM.
Spherical embryos are formed from callus in the somatic embryo induction medium, and by adding nitrilotriacetic acid while progressing from the spherical embryos to more differentiated somatic embryos, more somatic embryos with a high single embryo rate can be obtained. (2-2) Using an explant of plant tissue as a material for somatic embryo induction
When inducing somatic embryos using plant explants as a material,
B. Tisserat's review (Plant cell culture, a practical a
pproach; RADixon, 95-96, 1985, IPLPress, W
ashington DC) or the method described by Shimizu et al. (Plant Cell Engineering, Vol. 2, No. 5, pp. 659-663, 1990) can be used to induce somatic embryos. In that case, the above-mentioned somatic embryo induction medium can be used. Also in this case, the rate of single embryos of somatic embryos can be increased by adding nitrilotriacetic acid. (2-3) When a cell mass is used as a material for somatic embryo induction The details of the somatic embryo induction step are described below, taking a case where a cell mass is used as a material as an example. When the cell mass is used as a material for inducing somatic embryos, the method described in Japanese Patent Application No. 2-513159 can be referred to. The somatic embryo induction medium contains at least inorganic salts and a carbon source as essential components, and is supplemented with vitamins, amino acids, and hormones as necessary. As the basal medium, various basal media used for plant tissue culture can be used. For example, various basic media used for plant tissue culture such as the above-described MS medium, LS medium, B5 medium, and white medium can be used. As the carbon source, 5-100 g / l of sucrose, glucose, fructose, maltose, galactose and the like can be used.
Although somatic embryos can be induced from cell masses without using auxins and cytokinins, good results may be obtained by using auxins and cytokinins alone or in combination. As hormones, auxins include 2,4-D, NAA, IAA, IBA, picloram, pCPA, DICAMBA, preferably NA
A, IAA, 2,4-D are good. Cytokinins include kinetin, BA, zeatin, isopentenyl adenine,
PU can be used, but preferably zeatin, kinetin,
BA is good. Preferably, a sugar alcohol such as mannitol or sorbitol at 5-150 g / l can be added to the medium as an osmotic pressure regulator. In addition, more adventitious embryos may be induced by adding gibberellin, ancimidol, or the like. The pH of the somatic embryo induction medium is 4.0-7.
After adjusting to 0, preferably 5.0-6.5, it can be sterilized by high-pressure steam sterilization or filter sterilization.

The adventitious embryo-inducing medium may be a solid medium containing agar, gellite or the like, or a liquid medium containing no gelling agent. The gelling agent can be used at a concentration of 0.4-3.0% for agar and 0.05-2.0% for gellite. As physical conditions in the somatic embryo induction step, shaking culture is preferably performed using a liquid medium, but static culture can also be performed using a solid medium.
When a liquid medium is used, the shaking method may be either reciprocating or rotating. The amplitude number of the shaking culture can be 60-160 rpm, but 70-100 rpm is more preferable.

The present invention is characterized in that the rate of single embryos to be induced is increased by adding nitrilotriacetic acid to the somatic embryo induction medium. Nitrilotriacetic acid can be autoclaved after being added to the medium, but preferably sterilized by a filter and then added aseptically to the medium after autoclaving. If a high concentration of nitrilotriacetic acid is added to the medium, p
It is desirable that H be adjusted to the pH of the medium to be added in advance. The amount of nitrilotriacetic acid added to the somatic embryo induction medium is 0.
1-1000 μM is available. Nitrilotriacetic acid can be added to the somatic embryo induction medium from the beginning, and in some cases, between the start of somatic embryo induction and the recovery of somatic embryos, many somatic embryos with a high single embryo rate can be obtained. May be obtained. When nitrilotriacetic acid is added to the culture medium from the beginning when somatic embryos are induced, the preferred concentration is 0.1-200 μM. When nitrilotriacetic acid is added after initiating somatic embryo induction, the preferred concentration is 0.5-500 μM. By adding nitrilotriacetic acid before the differentiation of the cell mass into the somatic embryo in the somatic embryo induction medium, more somatic embryos with a high single embryo rate can be obtained.

By inducing somatic embryos with such a medium for inducing somatic embryos containing nitrilotriacetic acid, it is possible to obtain somatic embryos with a higher single embryo rate than in the case of inducing with somatic embryo-free medium. . (3) Germination of Adventitious Embryo In the present invention, the somatic embryo is cultivated in an adventitious embryo germination medium containing nitrilotriacetic acid to improve the seedling rate of seedlings germinated from the adventitious embryo, ie, germination from the adventitious embryo. It is possible to promote an increase in the number of stems of the young plant body. Somatic embryos used in the germination method of the present invention are somatic embryos directly derived from explants,
Any adventitious embryo derived from callus or a somatic cell can be used. As the somatic embryo used in the germination method of the present invention, not only somatic embryos obtained by culturing in the somatic embryo induction medium to which nitrilotriacetic acid is added, but also somatic embryos created by other methods may be used. Can be.

The somatic embryo germination medium is characterized by containing nitrilotriacetic acid. Nitrilotriacetic acid can be sterilized by the same method as described in the step of inducing the somatic embryo and added to the somatic embryo germination medium. For the adventitious embryo germination medium on which nitrilotriacetic acid is added, for example, the method described in JP-A-2-513159 can be referred to. The somatic embryo germination medium contains at least inorganic salts and a carbon source as essential components, and is supplemented with vitamins and amino acids as necessary. As the basal medium, various basal media used for plant tissue culture can be used. For example, MS medium, LS medium, B5 medium, white medium and the like can be used. As a carbon source, 5-100 g / l of sucrose, glucose, fructose, maltose, galactose and the like can be used. The concentration of the carbon source is preferably 10-60 g / l. As the somatic embryo germination medium, either a liquid medium or a solid medium can be used. As a solid medium support, agar, gellite, carrageenan,
A gelling agent such as agarose can be used. When using a solid medium, the concentration of the gelling agent is 0.4-3.0 on agar.
%, And for Gellite, 0.05-2.0%.
After adjusting the pH to 4.0-7.0, preferably 5.0-6.5, the somatic embryo germination medium can be sterilized by high-pressure steam sterilization or filter sterilization.

Culture conditions for germination of the somatic embryo may be either a light place or a dark place, but a bright place is preferred. When culturing in a light place, the illuminance is preferably 500-15000 lux, more preferably 2000-8000 lux. The somatic embryos placed on the somatic embryo germination medium germinated 1-2 months after implantation and became seedlings. Seedlings generated from adventitious embryos cultured in an adventitious embryo germination medium containing nitrilotriacetic acid have a higher number of stems per adventitious embryo than those germinated in a medium not containing nitrilotriacetic acid. Seedlings also grew vigorously.

[0023]

Embodiments of the present invention will be described below. Example 1 Induction and Propagation of Cell Cluster Asparagus variety UC157 was used as a material for cell cluster induction and proliferation. Sterile asparagus seedlings were obtained from asparagus individuals grown in a greenhouse. About 50 mm was cut from the tip of a young stem of asparagus, the surface was sterilized with 70% ethanol and 1% sodium hypochlorite solution, and the tip and the growth point of the lateral bud were cut out. This growing point portion was made up of 30 g / l sucrose, 0.2 mg / l ansimidol, 1 g / l morpholinoethanesulfonic acid (MES),
By culturing on a solid medium containing an MS medium containing g / l of gellite, a sterile asparagus seedling was obtained. The roots of this sterile plant were used for cell mass induction. The medium used to induce cell mass was 40 g / l sucrose, 30 g / l mannitol, 12 mM proline, 0.2 g / l casein hydrolyzate, 2 mg / l 2,4-D, and 1 g / l 5 is an MS liquid medium containing MES. 100 ml of the cell mass induction medium was dispensed into 500 ml Erlenmeyer flasks. Two to three roots of a sterile plant extending to 10 to 50 mm were placed on each Erlenmeyer flask and cultured for 1-2 months to induce a cell mass. The cell mass thus induced was added to a cell mass growth medium (30 g / l sucrose, 30 g / l sorbitol, 0.2 g / l casein hydrolyzate, 0.5 mg / l 2,4-D, 0.2 g / l). (MS liquid medium containing mg / l kinetin and 1 g / l MES). Dispense 100 ml of cell mass growth medium into 500 ml Erlenmeyer flask,
Cell clumps (0.5 g) were placed. The cell mass was cultured under shaking at 80 rpm at 25 ° C. in the dark. Cell clumps were subcultured every three weeks to cell clump growth medium. [Example 2] Induction of somatic embryos (Effects of Nito on the number of somatic embryos and the rate of single embryos in somatic embryos)
Effect of Lilotriacetate) An adventitious embryo induction medium (10 g / l sucrose, 30 g) was added to the cell mass (0.02 g) on day 21 after the subculture, and nitrilotriacetic acid was added to a concentration of 10 μM to 100 μM. / l sorbitol,
An MS liquid medium containing 2 g / l casein hydrolyzate and 1 g / l MES) was transferred to a 500 ml Erlenmeyer flask in which 50 ml was dispensed, and somatic embryos were induced. Cell mass, culture environment, illuminance about
Shaking culture was performed at 5,000 lux and 30 ° C. at 80 revolutions per minute. One month later, the somatic embryos were collected, and after removing excess water for 1 week in a petri dish covered with sterile filter paper, the number of somatic embryos was measured. In the measurement of the number of somatic embryos, those in which secondary somatic embryos were formed and a plurality of somatic embryos remained attached were counted as one somatic embryo. The single embryo rate indicates the ratio of the number of single embryos to the total number of somatic embryos (Table 1).

When induced in a somatic embryo induction medium containing 10 μM of nitrilotriacetic acid, the number of somatic embryos was not affected. When somatic embryos were induced in the somatic embryo induction medium containing 100 μM of nitrilotriacetic acid, the number of somatic embryos decreased compared to the control group,
There was no statistically significant difference between the control plot and each test plot at the 5% level. As shown in Table 1, the rate of single embryos was found to be higher when nitrilotriacetic acid was added at 100 μM than in the control group, as shown in Table 1. This difference was statistically significant at the 1% level. Table 1 Effect of nitrilotriacetic acid (NTA) on the number of somatic embryos and the rate of single embryos ＮＴ NTA concentration (μM) Somatic embryo count ^* Single embryo rate ^** (%) ─────────────────────────── {0 (control) 22720 36.0 10 23930 37.5 100 17485 63.0} ──────── *, **: Numerical values are the average of values in 5 compartments. [Example 3] Induction of somatic embryos (Effect of timing of addition of nitrilotriacetic acid to culture medium )
Result) As shown in Example 2, the rate of single embryos of somatic embryos induced by the somatic embryo induction medium containing 100 μM of nitrilotriacetic acid could be increased. Therefore, a test was performed in which the timing of adding nitrilotriacetic acid to the somatic embryo induction medium was changed.

The adventitious embryo was induced in the same manner as in Example 2. The cell mass (about 0.02 g) on the 21st day after the subculture was placed on a somatic embryo induction medium. Nitrilotriacetic acid was added to the somatic embryo induction medium one to two weeks after the start of somatic embryo induction. The number of somatic embryos thus obtained and the rate of single embryos were determined in the same manner as in Example 2 (Table 2).

As shown in Table 2, addition of nitrilotriacetic acid to the adventitious embryo induction medium was carried out one week or two weeks after the start of the adventitious embryo culture. It was found to increase. With respect to the single embryo rate,
The difference between the test group to which nitrilotriacetic acid was added one week later and the test group to which nitrilotriacetic acid was added two weeks later was statistically significant at the 1% level.

Regarding the number of somatic embryos obtained, there was no statistically significant difference at the 5% level between the control section and each test section. Table 2 Effect of the time of addition of nitrilotriacetic acid on the rate of single embryos in somatic embryos ─── addition timing Tanhairitsu (%) ^* somatic embryos / number plated product gram ^** ───────────────────────────── 1 week after the start of culture 52.9 23125 2 weeks after the start of culture 49.3 30450 No addition (control) 41.1 25930 ───────────────── *, **: Numerical value is the average value of the values of 5 sections [Example 4] Germination of somatic embryos (the number of stems per somatic embryo three
Effect of acetic acid) was then investigated the effect of nitrilotriacetic acid on somatic embryo germination. The somatic embryo obtained by the method of the control in Example 2 was used for the somatic embryo germination test. The medium used to germinate somatic embryos was 30
g / l sucrose, 0.2 g / l casein hydrolyzate, 1 g / l ME
S, MS medium containing 2 g / l gellite. Nitrilotriacetic acid was added at 1 μM, 10 μM and
It was added to a concentration of 100 μM and dispensed in 50 ml portions into a plastic container (60 mm × 60 mm × 97 mm). The adventitious embryos were placed on the culture medium 16 per culture vessel. Each test group performed three tests. The somatic embryos were cultured under the conditions of 25 ° C. and an illuminance of about 5000 lux. . One and a half months after the start of the culture, the number of stems was examined for the seedlings germinated from the somatic embryo (Table 3). The numerical values were expressed by calculating an average value per somatic embryo from the total number of stems of seedlings germinated from 48 somatic embryos.

As shown in Table 3, it was found that the addition of nitrilotriacetic acid increased the number of stems obtained per somatic embryo. The difference between each test group to which nitrilotriacetic acid was added and the control group was statistically significant at the 1% level. Table 3 Effect of nitrilotriacetic acid (NTA) on somatic embryos during germination of somatic embryos ────────────────────── NTA concentration (μM) Number of stems / undetermined Embryo １ 1 3.7 10 3.0 100 2.8 Control 1.4 ────────── [Example 5] Growth of seedlings germinated from somatic embryos Fig. 1, Fig. 2, Fig. 3 Shown in the figure. FIG. 1, FIG. 2, and FIG. 3 show seedlings germinated in an adventitious embryo germination medium supplemented with 1, 10, and 100 μM of nitrilotriacetic acid, respectively. The upper row of each figure shows the seedlings germinated in a medium to which nitrilotriacetic acid was added, and the lower row shows the seedlings germinated in a medium not containing nitrilotriacetic acid (control). In all test plots, after somatic embryos are placed on
Day after showed the results. The seedlings germinated in the adventitious embryo germination medium to which nitrilotriacetic acid was added as compared with the control were
The number of stems per book increased. Since the number of stems per seedling increased, a large number of foliage, which are photosynthetic organs, was obtained. As a result, good growth was exhibited. This trend is
It was observed in each of the media supplemented with 1, 10, and 100 μM nitrilotriacetic acid.

These young plants were raised in pots and grown in a greenhouse. Seedlings obtained from the test plot to which nitrilotriacetic acid was added, due to the large number of stems per seedling,
It was possible to prevent withering after potting. As a result, in the nitrilotriacetic acid-added group, the efficiency of seedling transformation of seedlings (the number of final produced seedlings / the number of seedlings planted in pots) could be improved in the nitrilotriacetic acid-added group. Thus, by culturing the somatic embryo in the somatic embryo germination medium containing nitrilotriacetic acid, germination can be promoted from the somatic embryo and the number of stems per somatic embryo can be increased. By increasing the number of stems, somatic embryos can prevent death when they are raised and increase the seedling conversion rate.
Obtain good quality seedlings.

[0030]

Industrial Applicability The present invention provides a large amount of compact and uniform somatic embryos suitable for tissue culture.
In addition, it promotes the germination of somatic embryos and greatly improves the seedling conversion rate of young plants. Therefore, the present invention relates to the production of cultured seedlings using somatic embryo formation, which enables mechanization to reduce costs and brings great benefits.

[Brief description of the drawings]

FIG. 1 is a photograph showing the morphology of seedlings germinated in a somatic embryo germination medium containing 1 μM nitrilotriacetic acid.

FIG. 2 is a photograph showing the morphology of seedlings germinated in an adventitious embryo germination medium containing 10 μM nitrilotriacetic acid.

FIG. 3 is a photograph showing the morphology of seedlings germinated in an adventitious embryo germination medium containing 100 μM of nitrilotriacetic acid.

Claims (6)

(57) [Claims] 1. A method for inducing somatic embryos, comprising inducing somatic embryos by culturing a plant tissue in a medium containing nitrilotriacetic acid. 2. A plant tissue, asparagus (Asparagus
officinalis L. ), the method for inducing somatic embryos according to claim 1. 3. A method for germinating somatic embryos, comprising germinating somatic embryos by culturing somatic embryos in a medium containing nitrilotriacetic acid. 4. The somatic embryo is an asparagus of Asparagus of Asparagus.
The method for germinating somatic embryos according to claim 3, wherein the somatic embryos are derived from ficinalis L. ). 5. An adventitious embryo induction medium comprising nitrilotriacetic acid. 6. A somatic embryo germination medium comprising nitrilotriacetic acid.