JPH03285624A - Cultivation of plant tissue and artificial seed - Google Patents

Abstract

PURPOSE:To effectively promote the cuntivation and proliferation of plant tissue, etc., and to improve the germination ratio of artificial seeds by using a filtrate or extract of the cultivation product of the heterocyst of cyanobacteria. CONSTITUTION:Tissue or organ of a plant, a part of the tissue, etc., or a cultured cell is cultured in a medium containing the filtrate and/or extract of the cultivation product of the heterocyst of cyanobacteria. An artificial seed is produced by embedding a plant tissue with an artificial endosperm or an artificial membrane and including the filtrate and/or extract of the cultivation product of the heterocyst of cyanobacteria in an artificial endosperm of the artificial seed.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to a plant tissue culture method and artificial seeds for regenerating a plant body by culturing plant tissues, organs, parts thereof, or cultured cells. .

(Conventional technology and its challenges) Regenerating plants using plant tissue culture technology is
For example, it is becoming increasingly important with recent advances in plant biotechnology, as seen in the development of artificial seeds.

Plant regeneration methods using this plant tissue culture technique can be broadly divided into two types, dedifferentiation systems and differentiation systems, depending on the starting material.

The dedifferentiation system is a method of regenerating a plant body through a dedifferentiated state such as callus or liquid cultured cells. Specifically, a large number of shoes 1- are generated from cluster callus, and each of the generated shoes 1- There are two methods: one is to cause roots to form and regenerate seedlings, and the other is to form somatic embryos (somatic embryos) directly in cells and regenerate them into seedlings.

In the differentiation system, starting materials include shoot apices, somatic buds, lateral buds, embryos, and seeds that contain growing points, and hypocotyls, cotyledons, and stems that do not contain growing points are also used. In this system, a continuous shoot production system was established in which multiple shoots 1 to 1 were formed in the above-mentioned plant tissues, and then these were excised. There is a method of regenerating young plants by rooting them from shoots.

Regardless of whether these methods, dedifferentiation or differentiation, are used, various conditions have been studied in order to increase the regeneration efficiency. For example, physical conditions such as temperature, light intensity, and photosynchronization, chemical conditions of the medium such as inorganic salts, vitamins, and sugars, plant hormone conditions such as the amount ratio of cytokinin/auxin, and addition of gibberellins and abscisic acids, etc. , conditions related to the age and tissue of the plants used, and whether solid culture or liquid culture (static culture, rotation culture, shaking culture, tank culture, etc.) is used.

However, a plant tissue culture method that effectively proliferates or differentiates plant tissues, cultured cells, etc., or efficiently promotes plant regeneration from plant tissues, cultured cells, etc., has not yet been established. In fact, in plant regeneration using a dedifferentiated system, there are many plant species that cannot induce somatic embryo formation or the formation of shoots or roots from callus. Although the reason is unclear, there is a phenomenon in which somatic embryos stop growing once they grow to a torpedo-shaped embryo, and the rate of redifferentiation into a plant body is extremely low. Furthermore, even with plant regeneration in a differentiated system, some plants and tissues still do not form shoots or roots.

Furthermore, in both dedifferentiation and differentiation systems, problems such as inhibition of proliferation and differentiation occur depending on the type of plant hormone and the amount added.

Also, artificial seeds with a high germination rate have not been perfected.

There is a need for a method to increase the rate of somatic embryo formation from various plant tissues, organs, or cultured cells, and to effectively promote the growth of somatic embryos and their regeneration into plants.

(Means for Solving the Problem) The present inventors have conducted various studies focusing on photosynthetic prokaryotic microorganisms in order to solve the two-legged problem. The present invention was completed based on the finding that the following method is particularly effective. That is, the present invention provides for culturing plant tissues, organs, parts thereof, or cultured cells in a medium containing a culture filtrate and/or extract of cyanobacteria that have formed heterosis 1. Characteristic plant tissue culture method.

(2) An artificial seed formed by embedding a plant tissue in an artificial endosperm and an artificial membrane, characterized in that the artificial endosperm contains a culture filtrate and/or an extract of cyanobacteria that have formed heterocysts. seed.

The gist is:

The details will be explained below.

Among photosynthetic prokaryotic microorganisms, some species of cyanobacteria have been recognized that exhibit a type of cell differentiation.

R, Rjppka et al. (Journal of Gene
ral Microbiology.

This corresponds to the strains belonging to groups ■ and ■ according to the classification of 111.1-61 (1979)). As strains belonging to group ■, for example, Anabaen spp.
a), Nodularia, Cylindrospermum
, Nostoc , Sitnema spp.
cytonema), Calo Rix m (Calo
thrj, x). In addition, as strains belonging to group
orogloeopsjs), Fischerella sp. (F
sherel,]-a). Specific examples include Anabaena sp. A, T C
C29211, Nodularia (Nodularj, a
sp,) ATCC29167, Cylindrospermum sp,) A
T CC29204, female 1 Nostoc
sp,) A T CC27896, Shi1 Henema (
Cytonema sp,) AT CC291
71, Calothrj, x sp.
,) ATCC29F, 56, Talologleophysis and Fritsii (Chl, orogloeopsj,
s frjtschN) ATCC27181, Fsherella sp. A, TCC2
9114 and the like. In addition, these variants and different species, as well as other marine and freshwater strains isolated from nature, also exist.

These have a filamentous structure and form spores called AkjneteS as a form of cell differentiation. Heterocyst
st) is one of these cells that are formed and is an atypical cell. It has been revealed that these heterocyst cells are differentiated and formed when a nitrogen source is severely depleted, and that the formed heterocyst cells perform nitrogen fixation.

Therefore, for example, although duplicate strains are usually cultured in a medium containing a nitrogen source such as ammonium salts or nitrates, heterocysts can be formed by culturing in a medium that does not contain this nitrogen source. The culture filtrate or extract of the cyanobacteria that formed this heterocyst is used. Here, the light irradiation conditions for culturing are the same as those for normal culturing, but preferably about several hundred to 10,000 lux.

In addition, culture can usually be carried out by tank culture or outdoor open culture using sunlight, but if the bacteria exist in a certain amount in abundance in nature, seawater or fresh water in which the bacteria can grow may be used as the culture medium. can.

The culture filtrate is obtained, for example, by centrifuging or filtering the culture solution of one or more types of bacterial cells as appropriate. However, if the obtained culture filtrate has low biological activity, it may be concentrated by vacuum concentration, etc. You may also use it as however,
At this time, if the concentration ratio increases and the salt concentration increases, it may have an adverse effect on the plant tissue, so it is desirable to desalinate it by electrodialysis or the like until the plant tissue is no longer adversely affected.

Although it depends on culture conditions and bacterial strains, heterocyst cells are usually scattered in cultured cyanobacteria at a ratio of one cell to every few cells. Extracts are usually obtained by contacting appropriately crushed microbial cells with a heated solvent as necessary, but in order to obtain an extract with strong biological activity, for example, Normal cells can be destroyed by mechanical treatment such as a French press or enzyme treatment using lysotium, and only heterocyst cells can be separated and then extracted.

The solvent to be used is one or a combination of two or more, as appropriate.
Generally, aqueous ones are preferred. For example, it is a solution containing water, an acid, a base, a salt, an organic solvent, and the like. Also,
It is also possible to perform extraction with an organic solvent such as methanol, ethanol, ethyl acetate, or ether, followed by extraction with an aqueous solvent.

Furthermore, the culture filtrate and extract can be treated with, for example, a polymer fraction, etc.
It can be divided into two parts as appropriate.

The polymer fraction can be obtained from the culture filtrate or extract by a general fractionation method based on molecular weight, such as dialysis, gel filtration, ultrafiltration, etc. Molecular weight is approximately 8,000
This is both of the above.

The polymer fraction thus obtained can also be used after being appropriately concentrated or diluted. Furthermore, powdered products obtained by drying these fractions by vacuum drying, freeze drying, spray drying, etc. can also be used.

The amount of such culture filtrate and/or extract, which has been appropriately fractionated as necessary, to be added to the basic medium is difficult to define, depending on conditions such as the bacteria used, culture conditions, and heterocyst formation rate. However, the effective addition amount can be easily determined by experiment. For example, Anabaena sp. ATC
When using C29211, 1 for the culture filtrate.
50 times concentrated 0.1-5% of basic medium
(v/v), and as an extract, 3 microbial cells of heterocyst cells extracted with 100 ml of extraction solution can be added to the basic medium at a concentration of 5 to 1000 ppm. Incidentally, if the concentration added is too high, the effect may decrease. Note that the basic medium and culture method used for plant tissue culture are the same as those for normal plant tissue culture. That is, as a basic medium, Murashige & Skoog
) medium (1962) (hereinafter abbreviated as "MS medium") is mentioned as a typical example, but other various media suitable for plant tissue culture or modified media thereof can be selected and used as appropriate. You can also. Furthermore, plant hormones, coconut milk, casein decomposition products, yeast extracts, etc. used in normal culture may also be added depending on the purpose. Furthermore, there is no particular restriction on the type of plant to be cultured, as long as it is totipotent and tissue culture is possible, and any plant can be used. For these, plant tissues, organs, or parts thereof, or cultured cells can be cultured, and both primary culture and subculture can be cultured. Therefore, when forming somatic embryos, regenerating plants, and regenerating plants,
It is possible to culture callus, somatic embryos, and protocomoid spheres. In addition, by including a culture filtrate and/or extract, which is appropriately fractionated as necessary, in the artificial endosperm, artificial seeds with a high germination rate can be obtained.

(Example) Below, we will explain what to use first, and then:
An example will be given using a typical carrot as a sample, but the present invention is not limited thereto.

(1) Heterocyst formation of cyanobacteria Anabaena ATCC 29211 was cultured in BG 11 medium (A, TCC designated medium) without a nitrogen source. 25
This is an aerated culture under light irradiation (12 hour light/dark cycle) at 2000 lux at °C.

(H) Preparation of culture filtrate and extract <i> Preparation of culture filtrate The culture obtained in Bipod (T) was centrifugally filtered to obtain a filtrate, which was concentrated 100 times using an evaporator.

This concentrated solution was transferred to a mosaic charged membrane desalter (Desarton DS-
403: Tosoh Corporation) and filtered using a 0.45 μm membrane filter.

<jl> Preparation of extract" Collect bacterial cells from the culture obtained in step (I), and after washing,
Lysotium treatment (concentration 1■/ml, 37°C 130 minutes)
The normal cells were then destroyed by sonication.

1 2 This was centrifuged to obtain heterocyst cells, which were then freeze-dried and suspended in water to a concentration of 3%.
Extract with hot water for 60 minutes at ℃, centrifuge and collect the supernatant at 0.4
It was filtered using a 5 μm membrane filter.

<iii> Preparation of culture filtrate for comparative example A culture was obtained using the BG11 medium in (1) above without removing the nitrogen source, and this was treated in the same manner as in (11) <i> above.

<iv> Preparation of Extract for Comparative Example In step (1), BGII medium without the nitrogen source was used to obtain a culture, which was treated in the same manner as in (IT) (jj) above.

(Ill) Preparation of high molecular fraction 50ml of the extract obtained in (H) <ii> above was added to Viscose (
Distilled water I using cellulose tube for dialysis (product name: cellulose tube 30/32) manufactured by VISKASE
Dialysis was performed against Q to obtain a dialysate solution, which was freeze-dried.

(IV) Culture of Carrot Somatic Embryos Germinated sterile carrot seeds with hypocotyls that have grown to about 10 centimeters are cut into approximately 1 cm pieces and placed in the following medium for 25 minutes.
The cells were cultured at ℃ in the dark. MS medium was used as the basic medium, to which 3% sucrose and 1 mg/ff of 2,4-D (2,4-dichlorophenoxyacetic acid), an auxin plant hormone, were added to adjust the pH to 5,5-. The pH was adjusted to 5.7. After about 1 month of culture, the 2,4D concentration in the medium was changed to 0, 1. The cells were transplanted into a medium reduced to 'J, mg/Q, and cultured with shaking using a reciprocating shaker at a shaking rate of 90 times/min. After that, once a week,
2,4. -D was subcultured into a medium containing 0.11 μ/Q to obtain cultured carrot cells.

Then, the cultured cells subcultured by such liquid culture were transplanted to a basic medium not containing 2.4-D and cultured to form somatic embryos.

(Stream side 1) Implantation from carrot somatic embryos.
Somatic embryos with a size of 425] 3 4 to 800 pm were selected using a μm mesh and used. The obtained somatic embryos ranging from heart-shaped to torpedo-shaped were cultured with liquid shaking in a plant hormone-free MS medium to which 1.5% of the culture filtrate prepared in (U)<i> was added.

In Example 1, 1.5% of the culture filtrate prepared in IT (i) was added to MS medium containing no plant hormones.
The same procedure as in Example j was carried out except that 1.50 ppm of the extract prepared in Kitaki (n) <ij> was added instead of adding the extract.

In Example 1, instead of adding 1.5% of the culture filtrate prepared in (II) < j) above to the MS@ground that does not contain plant hormones, Everything was the same as in Example 1 except that 50 ppm of the molecular fraction was added.

In "Example for using salmon in the mouth", 1.5% of the culture filtrate prepared in (II) <i> above was added to MS medium containing no plant hormones.
Everything was the same as in Example 1-, except that 1.5% of the culture filtrate prepared in (Tl) (iii) above was used instead of adding.

(Comparative Example 2) In Example 1 above, instead of adding 1.5% of the culture filtrate prepared in (II) <j> to the MS medium that does not contain plant hormones, "IT"<iv> The same procedure as in Example 1 was repeated except that 50 ppm of i was added to the extract prepared in Example 1.

All examples were the same except that in Example 1, the culture filtrate prepared in (TI)<i> above was used without adding 1.5% to the MS medium containing no plant hormones. I did the same as J.

Above, for each side, under 25°C1 light condition (20
Table 1 shows the results of culturing for 7 days under 0.00 lux and 16 hours of illumination, and examining the plant regeneration rate (rate of somatic embryos showing greening, germination and rooting).

5-6- Table-1 Carrot somatic embryos were obtained from 200 somatic embryos obtained above (TV).
Only somatic embryos that had grown to a size of 200 to 425 μm were selected and used using 71, rn and a 425 μm nylon mesh. Most of the selected somatic embryos were globular to early heart-shaped somatic embryos.

The somatic embryo thus obtained was suspended in 25 ml of MS medium, and the above suspension was mixed with 75 ml of MS medium containing 3% by weight of sodium alginate as an embedding agent, and the mixture was mixed]○Oml. Obtained. At this time, the above mixed liquid Loo
The culture filtrate prepared in (TI)<i> above was added to the culture filtrate at a concentration of 1.5%. The obtained final mixed liquid was heated to 50 m
By dropping M into a calcium chloride solution, spherical artificial seeds having an artificial membrane made of calcium alginate were obtained.

(Example 5) In Example 4, the above (IT
) Instead of adding the culture filtrate prepared in <i> at a concentration of 1.5%, the extract prepared in (IT) <ji> above was added at 1.5% concentration.
Everything was the same as in Example 4 except that 0 ppm was added.

(Example 6) In Example 4, two feet (I
I) Instead of adding the culture filtrate prepared in <j> at a concentration of 1-35%, add 50% of the polymer fraction prepared in (m) above.
The same procedure as in Example 4 was carried out except that ppm was added.

8 All procedures were carried out except that instead of adding the culture filtrate prepared in (H) <i> at a concentration of 1.5%, the culture filtrate prepared in (II) <1ii) above was added at 1.5%. Example 4
I did the same thing.

1 - Comparison 11 In Murakami Example 4, the above (TI
) Instead of adding the culture filtrate prepared in <i> at a concentration of 1.5%, the extract prepared in step (n)<jV) was added to
Everything was the same as in Example 4 except that 0 ppm was added.

In Example 4, the above (TI
) Everything was carried out in the same manner as in Example 4 except that the culture filtrate prepared in <j> was used without addition.

The artificial seeds on each side were cultured aseptically at 25° C. under light conditions (2000 lux, 16 hours of illumination) for 25 days, and the germination rates were examined. Table 2 shows the results.

Table 2 (Effects of the invention) According to the plant tissue culture method of the present invention, which involves culturing in a medium containing the culture filtrate and/or extract of cyanobacteria that have formed heterosis 1~, plant tissues, organs, and other It can effectively promote the cultivation and proliferation of cells or cultured cells, and can promote the formation of somatic embryos and the regeneration of plants.

Furthermore, the artificial seeds of the present invention, which contain the culture filtrate and/or extract of cyanobacteria that have formed heterocysts in the artificial endosperm, can have a high germination rate.

Procedural amendment (1'l'ilj, 1. Indication of the case θn. FEJ'-737 Heisei Patent Application No. (filed on March 30, 1990) 2. Title of the invention Plant tissue culture method and artificial seeds 3. Relationship with the case of the person making the amendment Patent applicant (',) 5],) Name Pentel Co., Ltd. 1 tablet wedge (, 9 4. Detailed description of the invention in the specification subject to amendment) 30th creation 4M
5. Contents of amendment (1) Specification box 1, page 4, line 6 from the bottom, r2, 4. -D
Although it says, "Basic medium that does not contain 2,4D," it has been corrected to read, "A 1/4 concentration basic medium that does not contain 2,4D."

(2) The ninth line from the bottom of page 1-7 of the specification box says "200 μm and 425 μm", but "425 μm and 800 μm"
” he corrected.

(3) 1200-425 on page 17 of the specification, line 8 from the bottom
Although it says "μm", it is corrected to "425 to 800 μm".

(4) On page 17 of the specification, from the second line from the bottom to the bottom line, it says that 75 ml of MS medium is mixed with the turbid liquid indicated in -, but it should be corrected to ``mix with 75 ml of solution.'' .

(5) Statement Box" - The second line from the bottom of page 9 says "25th," but it is corrected to "14th j.

(C2 Exhibit 1B Concern and Inbound.

2

Claims (2)

[Claims] (1) A plant tissue culture method characterized by culturing plant tissues, organs, or parts thereof, or cultured cells in a medium containing a culture filtrate and/or extract of cyanobacteria that have formed heterocysts. (2) An artificial seed formed by embedding a plant tissue in an artificial endosperm and an artificial membrane, characterized in that the artificial endosperm contains a culture filtrate and/or an extract of cyanobacteria that have formed heterocysts. seed.