JPH02142467A - Process for plant tissue culture - Google Patents

Abstract

PURPOSE:To promote the proliferation and differentiation of cell and to culture a plant under a definite condition by carrying out the tissue culture of a plant using a medium containing extract of microalgae, etc. CONSTITUTION:Tissue culture of a plant is carried out by using a medium containing an extract of microalgae (e.g., red algae, green algae, yellowish green algae or diatom) and/or filtrate of the culture liquid of microalgae.

Description

[Detailed description of the invention] [Industrial application field] TECHNICAL FIELD The present invention relates to a plant tissue culture method.

[Prior Art] In general, in plant tissue culture, part or all of the plant body is treated with inorganic salts necessary for plant growth.

In addition to vitamins and sugars, plant hormones (auxins and cytokinins) are added as a medium to induce callus formation (this is called callus induction or dedifferentiation), and the callus is then transplanted and cultured. Useful substances are obtained or plants are regenerated (restored) from the callus.

In the regeneration of a plant, the plant goes through a somatic embryo from a callus, or from a plant rod group m (within the shell there is an active part called a growth point) through a protocorm-like sphere, as in the case of a garden plant orchid. It is also being played back.

[Problem to be Solved by the Invention] In the above-mentioned plant tissue culture, various auxins such as indoleacetic acid, naphtholacetic acid, and 2,4-dichlorophenoxyacetic acid, and rhinocerosinids such as kinetin, benzyladenine, and zeatin are used. Callus growth and differentiation (morphological differentiation accompanied by regeneration of the plant, metabolic differentiation producing useful substances) is achieved by adding plant hormones in various proportions, casein decomposition products, yeast extract, etc. Attempts are being made to control the however.

In reality, it is difficult to control proliferation and differentiation, and plant hormones in particular have the problem of inhibiting proliferation and differentiation depending on the amount added.

[Means for Solving the Problems] The present invention has been made in view of the above-mentioned problems, and is characterized in that plant tissues are cultured using a medium containing a microalgae extract and/or a microalgae culture filtrate. The main focus is on tissue culture methods.

Here, plant tissue culture includes (1) plant culture (whole culture), (2) embryo culture, (3) organ culture, (4) tissue culture, callus culture, and (5) cell culture.

Examples of microalgae that can be used in the present invention include red algae, green algae, yellow-green algae, diatoms, xanthoflagellates, and dinoflagellates. Brachiomonas (Brachi) is a green algae.
oIllonas) genus, Chlamydomonas (Chla@
ydomonas), Chlorella (Chlorella)
a) i, Lobomonas horse, Nephelochlamys (Nep) + r.

chlamys), Nephrodella (Nephrod)
iella) genus, Protosiphon (Protosiph)
on) genus, Prototheca fi,
The genus Scenedesmus and the genus Selenastrum are included, and a specific example is Brachiomonas submarina (Ilra
Chiomonas submarina) AT
CC30597, Chlamydomonas torsoventralis (Chlamydom.

nas dorsoventralis) AT C
C30594, Chlamydomonas augametus (Chl
amydomonas eugametos) ATCC
30401, Chlamydomonas monoica (Chlamy
domonas monoica) AT CC30
629, Chlamydomonas pseudogloe (ChlaI
IlydoIIlonas pseudogloe)
ATCC12235.

Chlorella eritusoidea (Chlorella e
Chlorella luteoviridis (Chlorella luteoviridis) ATCC 11466
viridis) AT CC30406, Chlorella miniata (Chlorella miniata) AT
CC30546, Chlorella satsurikilophylla (Chlo
rella 5accharophila var, 5
accharophila) AT CC30408
, Chlorella sp.
CC11469, Chlorella variegata
lla variegata) AT CC3040
9. Chlorella vulgaris (Chlorella vu
lgaris) ATCCl 1468, Chlorella xanthella
A T CC30411, Lobomonas piriformis A
T CC30403, Nephrochlamys subsolitaria (Nephrochlamys 5ubso1ita)
ria) AT CC30433, Nephelodeera
Nephrodiella brevis
AT CC30440, Protosiphon botryoides
es) A T CC30436, Prototheca stagnora A
TCC16528, Scenedesmus bijugatous (S
cenedesmus bijugatus) A T
CC11462, Scenedesmus quadrifolius (
Scenedesmus quadricauda)
5 Yellow-green algae, including A T CC30428, include Boteiridium (13otrydium) genus,
Mischococcus, liE
, the genus Monodus, and the genus Ophiocytium. Specific examples include the genus Monodus.

Botrydium pekerianium
becherianum) ATCC30602, Botrydium cystosum
tosum) AT CC30589, Mischococcus sphaerocephalus
5phaerocaphalus) AT CC3
0592, Monodus Septeraneus
5ubterraneus) ATCC30593, Ophiocytium majus
n+ajus) AT CC30601, etc. As a yellow flagellate, Ochromonas (Och
romonas), and a specific example is Ochromonas dani.
ca) AT CC30004, Ochromonas malha+ne
nsis) ATCC11532. Further, microalgae are not limited to the above-mentioned bacterial cells or their variants and mutants, but also include marine and freshwater microalgae isolated from nature. Furthermore, there is no problem in using one or more of these image tones in combination. Cultivation of microalgae can normally be carried out using a medium containing inorganic salts, etc., by tank culture or outdoor open culture using sunlight. If so, seawater or fresh water in which the bacterial cells are grown can be used as the culture medium. Microalgae culture filtrate is obtained by subjecting the culture solution obtained by the above-mentioned culture method to centrifugation or filtration, but if the desired culture filtrate has low biological activity, the culture filtrate can be obtained by vacuum concentration, etc. It may be used after being concentrated.In this case, if the concentration ratio increases and the salt concentration increases, it may have an adverse effect on plant tissue, so it should be desalted by electrodialysis etc. until there is no longer any adverse effect on the plant tissue. is desirable. In addition, microalgae extract can be obtained by contacting the bacterial cells obtained as described above or appropriately crushed bacterial cells with an appropriate solvent at room temperature or heating, but the solvent used here is teeth,
Depending on the bacterial cell, various solvents may be used alone or in combination; however, an aqueous solvent is preferable in the shell.For example, the aqueous solvent may be water alone or a solution containing an acid, base, salt, or organic solvent. and so on. Alternatively, after extraction with an organic solvent such as methanol, ethanol, ethyl acetate, or ether, the organic solvent may be removed and the product may be dissolved in water. The extract that can be used in the present invention is an extract obtained by the method described above.

Alternatively, they can be used after being concentrated or diluted as appropriate. Furthermore, these extracts and fractions can be dried by vacuum drying, freeze drying, spray drying, etc. and used as powder. The amount of microalgae culture filtrate and/or microalgae extract to be added to the basic medium can be appropriately selected from 0.01 to 70% depending on the purpose and method of use, but is preferably 0.01 to 2.
It is 5%.

As mentioned above, the microalgae extract and/or culture filtrate is added to the basic medium and the plant tissue is cultured using the medium, but the basic medium, culture method, etc. are the same as those for normal plant tissue culture. .

That is, as a basic medium, Murashige
ge) & Skoog (1962).

Basic synthetic media containing trace components, vitamins, etc., or various modified media suitable for plant tissue culture can be appropriately selected and used. Furthermore, plant hormones, coconut milk, casein decomposition products, yeast extracts, etc. used in normal culture may also be added depending on the purpose.

Furthermore, it is known that plants to be cultured have monopotency. That is, explants (whole plants or parts thereof) can be cultured.

In addition, primary cultures or subcultures of whole plants or parts thereof can also be cultured. Particularly preferred are the shoot apex, cambium, young hypocotyls, and the like.

[Example] The present invention will be explained in more detail below using examples.

Example 1 (1) Preparation of cultured carrot cells Sprouted carrot sterile seeds with hypocotyls grown to about 10 openings were cut to about 101st position and cultured in the following medium for 25 minutes.
The cells were cultured at ℃ in the dark. The medium is M u as a basic medium.
Rashige K Skoog medium was used, and 3% of 2.4-D 1/Ω (auxin) was added thereto to adjust the pH to 15.5 to 5.7.

After about 1 month of culture, the 2,4-D concentration in the medium was reduced to 0°11.
The cells were transplanted into a medium reduced to 1/Ω and cultured with shaking using a reciprocating shaker at a shaking rate of 90 times/min. After that, once a week, 2・4-D 0.11■/
The cells are subcultured into a medium containing Q to obtain cultured carrot cells that grow rapidly.

(■) Preparation of microalgae culture filtrate and microalgae extract As microalgae, Chlorella vulgaris (Chlorella vulgaris)
vulgaris) A T CC11468, Chlorella saturophila var. (Chlorella 5a
ccharophila var, 5accharop
hila) AT CC30408, Chlorella ellipsoide
ea) Prepared using ATCC11466. After culturing the microalgae under ATCC-specified culture conditions, the culture solution was centrifugally filtered to obtain a filtrate, which was concentrated 100 times using an evaporator. This concentrated solution is processed using a mosaic charged membrane desalter (Desarton).
DS-103: Desalted with Tosoh Corporation) and 0.45μ
The resulting filtrate was used as a microalgae culture filtrate. Microalgae extract is obtained by collecting the bacterial cells, freeze-drying them, suspending the bacterial cells to a concentration of 3% in water, extracting with hot water at lOO'C for 60 minutes, and centrifuging to obtain the supernatant liquid. was obtained by filtration using a 0.45 μm membrane filter.

(III) Cultivation of cultured carrot cells Using the cultured carrot cells prepared in (I), add 1% of the Chlorella vulgaris cell extract and culture filtrate obtained in (II) to the medium. Table 1 shows the results of culturing for 12 days in the culture medium and the medium without additives.

Table-1
It was measured as follows according to 38, Published 3 years ago, Tennen, Junior and Senior High School, Furuya, Breakfast Shoten), p. 38.

Cellulase Onozuka R-102%, Marcelozyme R
-10i%, driselase 2%, calcium chloride (Ca
C1,・2H,O) 0.5%, mannitol 0.7
Using M, 30℃, 60 minutes, amplitude 7 countries, number of shaking 9 times
The cells were shaken at 0 times/min, then 50 times/min for 90 minutes, and the number of liberated protoplasts was counted using a hemocytometer at a depth of 0.1 mm.

Example 2 It is known that the cultured carrot cells produced in Example 1 (1) undergo morphological differentiation and form somatic embryos, but the microalgae extract and microalgae culture filtrate at this time were The effect of addition was investigated.

The medium was the same as that used in Example 1 (1).

A basic medium that does not contain the plant hormone 2.4-D was used. The extract and culture filtrate of Chlorella satsurophylla cells prepared in (II) of Example 1 were added to the medium at a rate of 1.
Table 2 shows the results of microscopic observation on the 10th, 20th, and 30th days of somatic embryos formed by culturing in a medium with and without additives at a temperature of 25°C for 30 days in the dark.
Shown below.

Table-2 ×1. Somatic embryo formation rate: The value obtained by dividing the number of somatic embryos by the number of total cells or cell clusters.

*2. O: Changes in morphology such as germination and rooting from somatic embryos occurred quickly, and growth was rapid.

×: Changes in the morphology of germination and rooting from somatic embryos did not occur promptly.

Example 3 In a growing point culture of Cadrea, an orchidaceous plant, 0.5% each of the microalgae extract and the microalgae culture filtrate prepared in (II) of Example 1 were added to the medium to form protocorm-shaped spheres ( The effect on the rPLBJ (hereinafter referred to as rPLBJ) was investigated.

The material is Leriocattleya (Lael), which belongs to the Cadrea family.
This is a PLB derived from the meristem near the growing point of the lateral bud of A. iocattleya). The PLB culture medium used was Hyponex (7-6-19) containing 7% potato juice and 2% sucrose as a carbon source.

The initially induced PLBs were expanded to 15, one PLB was divided into four, and the divided 60 PLBs were adjusted.

Add 0.5 of the Chlorella ellipsoidea bacterial cell extract and culture filtrate prepared in (■) of Example 1 to the PLB culture medium.
Table 3 shows the results of culturing for one month in a medium with % addition and a medium without addition.

Table-3 *(1) The number of PLBs did not increase.

(2) The number of PLBs has increased.

(3) Differentiated into buds and roots.

Example 4 The effects of adding microalgae extract and microalgae culture filtrate on regeneration of plants from tobacco callus were investigated.

The material is tobacco (Nicotiana tabacum).
L, cv.

This is a callus derived from the pith tissue of the stem of Bright Yellow. For callus culture, use Murashige
&S koog medium was used, and indole acetic acid (1 mg/Q) and kinetin (0 mg/Q) were added as plant hormones.
, 1■/Q) was subcultured on an agar medium supplemented with.

As a basic medium for inducing differentiation of buds, Murashige
& Indole acid, a plant hormone, in Skoog medium! ! (0.1mg/Q) and kinetin (1mg
/Q) was used.

Add 1.5% of the Chlorella ellipsoidea extract and culture filtrate prepared in (II) of Example 1 to the above basic medium.
A medium with the addition and a medium without the addition were prepared. For each medium, cut the subcultured callus into 5 mm square pieces using a razor blade, and place the callus sections into test tubes.
Table 4 shows the results of 14-day culture using 25 transplanted plants, each transplanted at a ratio of one per plant.

Table-4 Example 5 For cultured carrot cells producing β-carotene.

The effects of adding microalgae extract and microalgae culture filtrate were investigated.

The medium used was Murashige & Skoog medium, to which 3% sucrose and 2.4-D 1/Q were added, the pH was adjusted to 5.5 to 5.7, and 1% agar was added to the agar solid medium. was used as the basal medium.

The Chlorella saturophila extract prepared in (It) of Example 1 and the culture filtrate were cultured in a medium to which 1% of the basic medium was added and in a medium without any additives at a temperature of 25°C for 50 days in the dark. The amount of β-carotene accumulated within the cells was measured.

The amount of β-carotene was determined by measuring the fresh weight of cultured cells, crushing the cells in a mortar, and adding a small amount of acetone to extract β-carotene.

Add 3m1) of petroleum ether and add β to the petroleum ether layer.
-Carotene was transferred, and the absorbance of the petroleum ether layer at 453 nm was measured using a spectrophotometer, and the amount of β-carotene accumulated in the cultured cells was calculated.

The results are shown in Table-5.

Table 5 [Effects of the invention] According to the present invention, in plant tissue culture, by adding microalgae extract and/or microalgae culture filtrate,
This method can promote cell proliferation and differentiation, allow efficient culture, and can serve as a treatment method for minimizing the adverse effects of synthetic plant hormones. Therefore, by culturing plants under certain conditions, it is possible to induce the tissues and cells of their organs into a form suitable for the purpose, and it is possible to improve the production ability of cells that produce useful substances. Alternatively, since it also has the effect of promoting cell growth (proliferation), it is possible to easily produce a large amount of useful substance-producing cells.

Claims (1)

[Claims] 1. A method for culturing plant tissue, which comprises culturing plant tissue using a medium containing a microalgae extract and/or a microalgae culture filtrate.