JPH05130861A - Production of clone cell from protoplast - Google Patents

Abstract

PURPOSE:To culture the cell by entrapping and immobilizing the protoplast of a plant on calcium alginate beans, entrapping and immobilizing NAASU cells (a kind of cells) on beads larger than the above-mentioned beads, mixing both the beads with each other, separating the smaller beads from the mixture by the difference between the sizes of the beads, and subsequently culturing the small beads. CONSTITUTION:The protoplast of a plant is entrapped and immobilized on calcium alginate beads having an uniform size (particle diameter) so that the number of the dividable and multipliable protoplast is <=1per bead. NAASU cells are entrapped and immobilized on calcium alginate beads larger than the above-mentioned beads in such the extent as to be separable therefrom, and both the kinds of the beads are mixed with each other and subjected to a culturing process. Subsequently, both the kinds of beads are separated from each other by the difference between their sizes, and the smaller beads are cultured to produce many clone cells from the protoplast.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for easily producing a large number of clonal cells from plant protoplasts, which are indispensable for the creation and breeding of useful plants.

[0002]

2. Description of the Related Art When culturing only plant protoplasts in a medium containing an appropriate amount of sugars, inorganic salts, vitamins, plant hormones, etc., it is usual that the concentration of protoplasts in the medium is 10 ³ cells / ml or more, No division and proliferation of the plant, therefore colony formation is not observed (Toshiyuki Nagata, Genetic engineering of protoplasts, 1986, 1
6-17, Kodansha). Therefore, as a method for culturing protoplasts at a low cell concentration of 10 ³ cells / ml or less,
Digestion and release of callus (nurse cells) secreted or released from released single cells or small cell clusters as nutrients
Various so-called "nurse culture" to be propagated
(Edited by Komine et al., Encyclopedia of Plant Biotechnology, 1990
Year, pages 207-208, Asakura Shoten). However, all of these methods are troublesome, and although a small number of clonal cells can be obtained from protoplasts, they are not suitable for obtaining a large number of clonal cells at once.

Larkin (PJ) et al. Have succeeded in culturing plant protoplasts at a low concentration by applying calcium alginate beads to nurse culture (Plant).
Science, 1988, Vol.58, p. 203-210, E
lsevier Scientific Publishers Ireland Ltd.). Their method is to prepare protoplasts of plants (alfalfa or tobacco) entrapped and immobilized on calcium alginate beads,
Similarly, mixed culture is performed with nurse cells entrapped in calcium alginate (in this example, a high concentration of the same protoplasts is used), but this is also a low concentration, that is, calcium alginate beads 1
It has been shown that it is also possible to immobilize one protoplast per individual and grow and divide it in beads to form colonies. Larkin et al. Colored the latter with activated charcoal in order to distinguish between beads on which protoplasts of interest were immobilized and those on which nurse cells were immobilized.
The device is designed to enable the extraction and selection of beads containing the target protoplasts and their proliferative bodies using tweezers.

[0004]

However, according to the method of Larkin et al., Beads on which nurse cells are immobilized are colored with activated carbon, so that the adverse effect of activated carbon may occur depending on the type of protoplast. In addition, the target protoplasts and their proliferates must be extracted and selected with tweezers, etc., which is troublesome and inefficient. The present invention provides a method for easily producing a large number of clonal cells from protoplasts while handling a large amount of beads without worrying about the adverse effect of activated carbon on protoplasts.

[0005]

Means for Solving the Problems As a result of various studies on nurse cultures capable of efficiently handling a large amount of beads without coloring with activated carbon or the like, the present inventors have found that beads that comprehensively immobilize protoplasts are selected. It has been found that this can be achieved by making the size (particle size) of beads for entrapping and immobilizing nurse cells different so that they can be separated by a sieve or the like. Further, when the density of protoplasts used for immobilizing protoplasts on calcium alginate beads is set to 1 or less per 1 calcium alginate bead, most of the colonies formed in the beads are formed. Just 1
The present inventors have completed the present invention by finding that they are colonies grown and divided from individual protoplasts.

That is, according to the present invention, plant protoplasts are added to calcium alginate beads having a uniform size (particle size) so that the number of protoplasts capable of dividing and proliferating is 1 or less per 1 bead. Inclusive immobilization,
On the other hand, compared with the above beads, encapsulating the nurse cells in calcium alginate beads that are large enough to be separated,
The present invention relates to a method for producing a large number of clonal cells from protoplasts, which comprises culturing both of them and then culturing the smaller beads by utilizing the difference in the size of the beads to separate them.

The protoplasts used in the present invention can be prepared by a known method of treating plant tissues or cultured cells of plants with an enzyme such as cellulase or pectinase, or an improved method thereof. It may also be a cell fusion of homotopic or heterologous protoplasts prepared by these methods, or protoplasts to which genetic manipulation has been added.

In the present invention, the term "protoplasts capable of dividing / proliferating" does not mean the total number of protoplasts, but the number of effective protoplasts capable of dividing / proliferating to form colonies when mixed and cultured. The number of effective protoplasts that can divide and proliferate to form colonies divided by the total number of protoplasts is usually called the colony formation efficiency (efficiency) and is determined by the cell type, culture conditions, etc. .. When tobacco protoplasts are immobilized on calcium alginate beads, the value is about 50.
%, Which is about 15% for saffron and about 20% for Strophanthus gratas under the same conditions. Further, the smaller the density of protoplasts when immobilized on beads, the smaller the above-mentioned effective rate.

The number of protoplasts capable of dividing and proliferating (ie, the total number of protoplasts multiplied by the effective rate of colony formation) per bead is 1 or less, preferably 0.001 to 0.1. To If this number is less than 0.001, the number of beads containing clonal cells obtained will decrease, and if it exceeds 0.1, the frequency of beads containing two or more protoplast-derived cells in one bead will be obtained. Cannot be ignored.

The nurse cells of the present invention are not particularly limited as long as they are plant-derived cultured cells, but generally plant-derived cultured cells that are easy to culture are used. This may not be whole cells but protoplasts. Alternatively, inactivated cells that have been unable to grow in advance by irradiation with radiation or UV may be used.

The protoplasts and nurse cells to be proliferated / divided are usually of the same genus / same species, but may not necessarily be of the same genus / same species, and a preferable combination is appropriately selected by experiments.

The beads of uniform size in which protoplasts or nurse cells are entrapped and immobilized can be easily prepared by a known technique. That is, an appropriate concentration of protoplasts or nurse cells is suspended in a 1 to 5 wt% sodium alginate aqueous solution and dropped into a 0.05 to 0.2 M calcium chloride aqueous solution from a nozzle having an inner diameter of 0.1 mm to 3.0 mm. , Can be made.

FIG. 1 is a graph showing the relationship between the concentration of the sodium alginate aqueous solution, the inner diameter of the dropping nozzle and the diameter of the calcium alginate beads formed. In the figure, 1 indicates that the concentration of the sodium alginate aqueous solution is 3% by weight, and 2 indicates that the concentration of the sodium alginate aqueous solution is 1.5% by weight. The diameter of the alginate beads is uniform by adjusting the concentration of the sodium alginate aqueous solution in which the cells are suspended and the inner diameter of the nozzle, as shown in the relationship of FIG. 1, and the diameter is 2 mm to 6 mm. Can be of size.

The size of the two kinds of calcium alginate beads entrapping and immobilizing protoplasts or nurse cells is
The size is preferably such that the nutrients of the surrounding medium or the secretions / releases of cells can be easily transferred, and can be easily separated by a selecting means such as a sieve, and those having a size of 1 to 10 mm are usually used. Further, the particle size distribution of the beads is preferably as sharp as possible so that the beads can be selected with a sieve or the like.

The density of the nurse cells to be used is such that the density of protoplasts capable of dividing and proliferating is 1 or less per bead, but a density much higher than that is required. Therefore, nurse cells are entrapped and immobilized on large beads, and protoplasts are entrapped and immobilized on smaller beads.

The difference in size between the two types of calcium alginate beads may be such that they can be selected with a sieve or the like. If the ratio of the size (average particle size) of both beads (large beads / small beads) is in the range of approximately 1.33 to 4.0,
This selection becomes possible.

The protoplasts and nurse cells immobilized on calcium alginate beads can be cultured by liquid shaking culture using a known cell culture medium.
Examples of the cell culture medium include Murashige & Skoog medium and Rinsmeier Skoog medium.
(Linsmaier & Skoog) medium, Heller medium, White (White) medium, Nitch & Nitch medium, Cienck & Hildebrandt (Schenk & Hildebrandt)
Medium, Gamborg B5 medium, Khao and Michael
(Kao & Michayluk) 8P medium, Nagata / Takebe's medium, etc., each of which is used at the prescribed concentration depending on the protoplast and nurse cells to be cultured, or 1
Used by diluting to a concentration of / 2 to 1/10. In addition, it is usually necessary to add a plant hormone to the medium for the division / proliferation of protoplasts.

Examples of plant hormones include auxin and cytokinin, and examples of auxins include α-naphthalene acetic acid, 2,4-dichlorophenoxyacetic acid, indole acetic acid, indole propionic acid, indole butyric acid,
There are β-naphthoxyacetic acid, 2,3,6-trichlorobenzoic acid, cis-cinnamic acid, etc., and as cytokinins, kinetin, cenylaminopurine, phenylaminopurine,
There are benzylaminopurine, pentylaminopurine, naphthylaminopurine, 6-isopenteraminopurine, trans-zeatin, cis-zeatin, and the like. Among these, a protoplast that proliferates is selected and used.
Auxin is usually used at a concentration of 0.1 to 10 mg / l in the medium, and cytokinin is usually used at a concentration of 0.1% in the medium.
It is used at a concentration of 0 to 1 mg / l, and other hormones are also used at an appropriate concentration. Since the concentration of the plant hormone is optimal for growth, it is preferably determined in advance by a culture experiment.

In order to adjust the osmotic pressure of the medium, mannitol, sorbitol or the like is added at a concentration of 0.3M to 0.7M for use. Furthermore, sucrose as a carbon source,
Sugars such as glucose are added at a concentration of 5 g / l to 50 g / l and used.

As the calcium alginate beads entrapping and immobilizing the nurse cells, the beads immediately after the immobilization may be used, or the beads may be cultured for several days after the immobilization to sufficiently grow the cells in the beads and then used. ..

The beads containing the proliferate from protoplasts and the beads containing the nurse cells (or the proliferate from the nurse cells) thus cultured can be easily separated from each other by using a sieve or the like.

Depending on the type of plant cells, mannitol, sorbitol, etc., which are osmolarity regulators of the medium, may inhibit their growth. Therefore, after culturing for about 1-2 weeks, cells from protoplasts are divided to some extent. Therefore, it is advisable to transfer this to a medium containing no permeation regulator and further culture. In this way, the protoplasts immobilized within the beads will grow to a level where they can be easily observed with the naked eye. By taking these beads, transferring them to a medium suitable for growth or a medium suitable for plant regeneration, and continuing the culture, clonal cells or callus derived from a single protoplast can be obtained.

[0023]

[Function] For one bead, the average number of protoplasts capable of division / proliferation is x, and in that case, if the number of protoplasts capable of division / proliferation fixed to each bead is n, then a certain bead is n The probability of including protoplasts capable of dividing and multiplying is statistically (Poisson distribution): F (x, n) = [(x to the nth power) / n! ] Exp (-x). Therefore, the probability P that the colony formed by growing in the beads is derived from a single protoplast is P = F (x, n = 1) / [F (x, n = 1) + F (x, n ≧)] = F (x, n = 1) / [1-F (x, n = 0)] From this formula, x is 0.0001 to 1.0
The value of P at is calculated as shown in Table 1 below.

[Table 1] Table 1 Values of P when x is 0.0001 to 1.0 ──────────────────────────────── ─── x 1.0 0.5 0.3 0.25 0.1 0.05 0.01 0.001 0.0001 ────────────────────────────────── P 0.582 0.771 0.857 0.880 0.951 0.975 0.995 0.9995 0.9999 ───────────────────────────────────

From this table, when x is 1.0 to 0.25,
10% or more of the colonies formed by growing in the beads are derived from 2 or more protoplasts, and x is 0.
At 1 or less, it can be estimated that 95% or more of the formed colonies are derived from a single protoplast.

[0025]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 (1) Preparation of protoplasts Strophanthus gratus
Protoplasts were prepared from suspension-cultured cells obtained from callus. The procedure is shown below. The subculture of the suspension-cultured cells was carried out by using an MS medium containing 1 mg of 2,4-dichlorophenoxyacetic acid, 0.1 mg of kinetin and 30 g of sucrose in 1 L.
200 containing 40 ml of medium in which H was 5.7-5.8
Seed cells were suspended in a ml Erlenmeyer flask and cultured at 30 ° C. in the dark at a rotation speed of 120 rpm every two weeks for repeated culture.

On the 5th day after the start of the culture, the whole amount of the culture medium containing cells (40 ml) was taken, and this was first applied to a nylon mesh having a size of 500 μm to remove a large cell mass. Next, apply the passing liquid through a nylon mesh of 20 μm in size,
The cells remaining on this mesh were collected. Cell weight 1g
On the other hand, the enzyme reaction solution (cellulase Onozuka RS 1% by weight, pectolyase Y23 0.1% by weight, mannitol 0.5 M, pH 5.6) was suspended in 20 ml,
It was kept at 30 ° C. for 4 hours at a rotation speed of 90 rpm.

This cell suspension treated with the enzyme was filtered through a nylon mesh of 50 μm in size to remove cells or cell clumps which had not been decomposed in the cell wall, and centrifuged at 500 rpm for 5 minutes, and the precipitate was washed with a protoplast washing solution (2 0.5 M mannitol solution containing 5 mM calcium chloride, pH 5.
6) Suspended in 2 ml, this was gently poured into a 15 ml centrifuge tube containing 20% by weight and 10 ml of sucrose solution having a pH of 5.6 to form a layer, and the mixture was centrifuged at 1000 rpm for 5 minutes. Gently suck up the crude protoplast fraction in the upper layer of the sucrose solution with a Pasteur pipette, and use the above protoplast washing solution 1
Resuspended in 0 ml and centrifuged at 500 rpm for 5 minutes.
This washing operation was repeated twice, and purified protoplasts were about 2
× 10 ⁶ pieces were obtained. The obtained protoplasts were stained with Calcofluor White and then observed with a fluorescence microscope to confirm that the cell wall was removed.

(2) Immobilization of protoplasts on calcium alginate beads 3% by weight of sodium alginate [Sigma Chemical
Company No. A-21 made by SIGMACHEMICAL COMPANY
58] Prepare 100 ml of the aqueous solution, and use an autoclave to 1
High temperature sterilization was performed at 20 ° C. for 20 minutes.

1. The total number of protoplasts obtained in (1) above was 10 ⁴ (2000 protoplasts capable of dividing and multiplying when multiplied by a 20% effective colony formation rate).
The suspension was suspended in 100 ml of a 0.5 M mannitol solution (pH 6.5) containing 5 mM calcium chloride.
ml and the above 3% sodium alginate aqueous solution 100 m
l were mixed. This mixture was sterilized with 0.5 M mannitol containing 0.1 M calcium chloride aqueous solution (1000 m).
It was dripped into 1 from a syringe equipped with a needle having an inner diameter of 0.3 mm. Under the above conditions, the volume of one droplet (gel) is about 0.
It is 01 ml, and therefore, the calculation is such that protoplasts that can divide and grow at a ratio of 1 protoplast to 10 beads are fixed to the beads. After shaking this for 1 hour, the protoplast-immobilized beads in the form of spherical beads having a diameter of 2.1 to 2.3 mm were taken out and washed twice with a protoplast washing solution.

(3) Immobilization of nurse cells on calcium alginate beads 3% by weight of sodium alginate [Sigma Chemical Co.
Company No. A-21 made by SIGMACHEMICAL COMPANY
58] 200 ml of an aqueous solution was prepared and sterilized. 2 to this
20 ml of Strophanthus gratus callus cells that had been subjected to weekly liquid shaking culture were suspended in a packed cell volume. This suspension was dropped into a sterilized aqueous solution of 0.1 M calcium chloride (1000 ml) from a glass tube having an inner diameter of 0.8 mm. After shaking this for 1 hour, the plant cell-immobilized beads which became spherical beads of 3.5 to 4 mm were taken out and washed twice with sterile water.

(4) Mixed culture Mannitol was added to a final concentration of 0.5 M in an MS medium having a concentration of 1/2 each of the inorganic and organic components.
So that the sucrose is 10 g / l,
Liquid medium 20 prepared so that 2,4-dichlorophenoxyacetic acid is 1 mg / l and kinetin is 0.1 mg / l
Dispense 0 ml into a 1 L Erlenmeyer flask and store at 120 ° C, 20
It was sterilized by autoclaving for a minute. Protoplast-immobilized beads 50 prepared in (1) and (2) above
0 and 500 nurse cell-immobilized beads prepared in (3) above were placed in a flask containing the above medium, and 30
As a result of culturing with shaking at a rotation speed of 80 rpm for 2 weeks in the dark at 0 ° C., small protoplast-derived colonies could be observed in the smaller protoplast-immobilized beads. Here, 100 smaller beads were arbitrarily selected, and the number of colonies per bead was examined with a microscope. The results are shown in the following table.

[Table 2] Table 2 Distribution of the number of colonies contained in one bead ─────────────────────────────── 1 bead Number of colonies per number ────────────────────────────── 0 91 1 9 2 or more 0 ───── ──────────────────────────

(5) Proliferation and selection A culture solution containing beads on which protoplasts and nurse cells were immobilized was passed through a sterilized metal sieve having an opening of 1.75 mm, and beads of both species were collected on a sieve. Next, a medium 200 having a pH of 5.7 to 5.8 with an MS medium containing 1 mg of 2,4-dichlorophenoxyacetic acid, 0.1 mg of kinetin, and 30 g of sucrose in 1 L of these both types of beads.
Add to a 1 L Erlenmeyer flask containing 30 ml,
As a result of further culturing at 120 rpm for 2 weeks in the dark, colonies from protoplasts grew so that they could be easily observed with the naked eye.

This culture medium containing the beads is opened with a size of 3.35.
Sieved on a mm sterile metal sieve. The smaller beads, with protoplasts immobilized and grown, passed through the sieve, while the beads with immobilized and immobilized nurse cells remained on the sieve. The culture medium containing the smaller beads that passed through the sieve was then passed through a sterilized metal sieve having an opening of 1.75 mm to remove the medium, and the beads on which colonies from protoplasts were fixed were collected on the sieve. The beads were then loaded onto solid medium [1 mg 2,4-dichlorophenoxyacetic acid, 0.1 mg kinetin and 30 sucrose in 1 L].
gellite (Kelco Division of Merck (Kelco Division of Merck)
division of Merck)) to solidify by adding 0.25% by weight]
The cells were transferred to a petri dish containing cultivated and cultured statically at 30 ° C. in the dark.
After 2 weeks of culturing, a large number of callus derived from protoplasts, which had grown by breaking the beads, was obtained.

[0034]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method by which clonal cells can be easily produced from protoplasts without concern about the adverse effects of activated carbon.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the concentration of an aqueous solution of sodium alginate, the diameter of a dropping nozzle, and the diameter of calcium alginate beads formed.

[Explanation of symbols]

 1: Concentration of sodium alginate aqueous solution is 3% by weight 2: Concentration of sodium alginate aqueous solution is 1.5% by weight

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ken Sawazaki 48, Wadai, Tsukuba, Ibaraki Hitachi Chemical Co., Ltd. Tsukuba Development Laboratory

Claims (1)

[Claims] 1. A plant protoplast is entrapped and immobilized on calcium alginate beads having a uniform size (particle size) so that the number of protoplasts capable of dividing and proliferating is 1 or less per 1 bead. , On the other hand, the nurse cells are entrapped and immobilized in calcium alginate beads, which are larger than the above beads, so that they can be separated, and both are mixed and cultured.
After that, using the difference in the size of the beads to separate the two,
A method for producing a large number of clonal cells from protoplasts, which comprises culturing smaller beads.