JPH04187083A - Method for culturing protoplast - Google Patents

Abstract

PURPOSE:To efficiently culture a plant cell protoplast effective on the production and breeding of an useful plant by including and immobilizing the plant cell protoplast in string-like calcium alginate gel and culturing the immobilized protoplast with a nurse cell. CONSTITUTION:For example, a plant cell protoplast is suspended in 1-5wt.% aqueous solution of sodium alginate and then pushed out from a nozzle having 0.1-1.0mm inner diameter into 0.05-0.2M aqueous solution of calcium chloride to include and immobilize the protoplast in string-like calcium alginate gel, and the protoplast is cultured together with a nurse cell included and immobilized in beads of the calcium alginate gel to carry out culture of the plant cell protoplast effective on the production and breeding of an useful plant.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for culturing protoplasts, and more particularly to a method for culturing plant-derived protoplasts that is effective for the creation and breeding of useful plants.

[Conventional technology]

When culturing plant protoplasts in a medium containing appropriate amounts of sugars, inorganic salts, vitamins, plant hormones, etc., the concentration of protoplasts in the medium must be 103 cells/mL or higher for cell division and proliferation to occur. , therefore no colony formation is observed (Toshiyuki Nagata, Genetic Engineering of Protoplasts, 1986, pp. 16-17, Kodansha).

Therefore, as a method for culturing protoplasts at a low concentration, the so-called "nurse culture" is a method in which released single cells or small cell clusters are brought into contact with callus (nurse cells) that divide and proliferate using the secretions or extrusions as nutrients. ed., Plant Biotechnology Encyclopedia, 1990, 20
7-208, Asakura Shoten).

Larkin (La) cultivates plant protoplasts at low concentrations.
rkin, P. J.) et al. (Plant 5cie
nce, 1988, Vol. 58.203-210,
Elsevier Scientific Publicis
Hers Ireland Ltd.) has applied calcium alginate beads to nurse culture. This method involves mixing plant (alfalfa or tobacco) protoplasts entrapping in calcium alginate beads with nurse cells (in this example, a high concentration of the same protoplasts) also entrapping in calcium alginate. This allows protoplasts to grow and divide even at a low concentration, ie, 50 cells/mL. In addition, in order to distinguish between beads on which the target protoplasts were comprehensively immobilized and beads on which nurse cells were comprehensively immobilized, Larkin et al. colored the latter with activated charcoal and used tweezers to select beads containing the target protoplasts and their proliferators. are doing.

[Problem to be solved by the invention]

However, in Larkin et al.'s method, beads on which nurse cells are immobilized are colored with activated carbon, so there is concern that activated carbon may have an adverse effect on some types of protoplasts. In addition, when handling a large amount of beads, sorting is time consuming and inefficient.

An object of the present invention is to provide a method for culturing protoplasts that does not have the influence of activated carbon on protoplasts and can efficiently handle a large number of protoplasts.

[Means to solve the problem]

As a result of various studies on nurse culture that can efficiently handle large numbers of protoplasts without coloring them with activated carbon, etc., the present inventors have comprehensively immobilized protoplasts in a string-shaped calcium alginate gel, and have developed a system that allows both nurse cells and nurse cells to be immobilized together. They have discovered that the above objectives can be achieved by mixed culturing of the following, and have completed the present invention.

That is, the present invention relates to the following (1) to (3).

(1) A method for culturing protoplasts, which comprises entrapping and immobilizing plant cell protoplasts in a string-like calcium alginate gel and culturing them together with nurse cells.

(2) The method for culturing protoplasts according to (1) above, wherein the string-like calcium alginate gel has a diameter of 0.5 to 2.0 mm.

(3) The method for culturing protoplasts according to (1) or (2) above, wherein the nurse cells are entrappingly immobilized on calcium alginate gel beads.

The protoplasts used in the present invention can be prepared by a known method of treating plant tissues or cultured plant cells with enzymes such as cellulase and pectinase, or improved methods thereof.

For entrapping immobilization of protoplasts in a string-like calcium alginate gel, the protoplasts are suspended in a 1 to 5% by weight sodium alginate aqueous solution, and then the protoplasts are
．． 0.05~0.2 from 1mm~1.0mm nozzle
This can be done by extruding M into an aqueous calcium chloride solution. The thickness of the string-like calcium alginate gel can be adjusted by the inner diameter of the nozzle used. For example, protoplasts are suspended in a 1.5% by weight sodium alginate aqueous solution, and a 0.1M calcium chloride aqueous solution is injected through a nozzle with an inner diameter of 0.3 mm. When extruded inside, the thickness is approximately 0.9mm
A string-like gel of approximately 1.5 mm in thickness is obtained when a 0.8 mm nozzle is used.

The thickness of the string-like gel that comprehensively immobilizes protoplasts is
A thinner material is preferable for colony formation and colony separation, but if it is too thin, mechanical strength decreases and it is likely to break during handling. Furthermore, if the gel is too thick, it becomes difficult to supply oxygen and nutrients to the protoplasts immobilized in the gel, and the colony formation rate decreases. Therefore, the thickness of the string-like gel is 0.5 mm to 2.5 mm. A range of Omm is preferred.

The nurse cells used in the present invention are not particularly limited as long as they are plant-derived cultured cells, but for boat fishing, plant-derived cultured cells that are easy to culture are used. Even if this is a complete cell,
It does not matter if it is a protoplast. Alternatively, inactivated cells that have been rendered incapable of proliferating by irradiation with radiation or UV may also be used.

As nurse cells, cells that have not been entrapping-immobilized may be used as they are, but in order to improve operability during medium exchange and to obtain string-like calcium alginate gel-entrapping protoplasts to which nurse cells are not attached, it is necessary to use nurse cells. It is preferable to entrapping and immobilize the cells on a suitable carrier, such as beads of calcium alginate gel.

Entrapping immobilization of nurse cells onto calcium alginate beads can be easily performed using known techniques. That is, nurse cells are suspended in a 1 to 5% by weight aqueous sodium alginate solution, and the inner diameter is 0.1 mm to 3.0 mm. Calcium alginate beads on which nurse cells are immobilized can be produced by dropping the beads into a 0.05 to 0.2 M calcium chloride aqueous solution from an Omm nozzle.

If the nurse cells are comprehensively immobilized on calcium alginate gel beads, the beads may be used immediately after immobilization, or they may be cultured for several days after immobilization to allow the cells to sufficiently proliferate within the beads. It can also be used after

The types of protoplasts and nurse cells to be proliferated and divided are usually of the same genus and species, but they do not necessarily have to be of the same genus and species, and a preferable combination is selected as appropriate based on experiments.

Protoplasts and nurse cells immobilized on string-shaped calcium alginate gels and calcium alginate beads can be cultured by liquid shaking culture or the like using a known cell culture medium.

As a cell culture medium, for example, Murashige Skoog (
Murashige & Skoog medium, Linsmaier & Skoog medium
g) Medium, Heller medium, white (W
hite) medium, niche/niche (Nitch & N
1tch) medium, Schenk-Hildebrand (Sche
NK & Hildebrandt medium, Gamborg B5 medium, Kao and Michael (
Kao & Michaeluk) 8P medium, oblong/oblong medium, etc. are available, and depending on the protoplasts and nurse cells to be cultured, each component can be used at the prescribed concentration, or at a concentration of 1/2 to 1/10. Use diluted. Furthermore, the division and proliferation of protoplasts usually requires the addition of plant hormones to the medium.

Examples of plant hormones include auxin and cytokinin, and auxins include α-naphthalene acetic acid and
Examples of cytokinins include kinetin, senylaminopurine, There are phenylaminopurine, benzylaminopurine, pentylaminopurine, naphthylaminopurine, 6-bentelaminopurine, trans-zeatin, cis-zeatin, etc., and those that allow protoplast proliferation are selected from these.

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 to 10 mg/L in the medium.
It is used at a concentration of 0.01 to 1 mg/L, and other hormones are also used at appropriate concentrations. Since there is an optimal concentration of plant hormones for growth, the concentration is preferably determined in advance through culture experiments.

Further, in order to adjust the osmotic pressure of the medium, mannitol, sorbitol, etc. are added at a concentration of 0.3M to 0.7M. Furthermore, sugars such as saccharose and glucose are added and used as a carbon source at a concentration of 5 g/L to 20 g/L.

The string-shaped calcium alginate gel containing the protoplasts cultured in this way is removed from the nurse cells (or from the nurse cells) by being pulled up with tweezers and washed, or passed through an appropriate mesh sieve. (proliferating cells) or these can be easily separated from entrapping immobilized beads.

〔Example〕

Hereinafter, the present invention will be specifically explained with reference to Examples.

(1) Preparation of protoplasts
Protoplasts were prepared from suspension cultured cells obtained by callus formation from the leaves of P. usgratus. The procedure is shown below.

For subculture of suspension cultured cells, 1 mg of 2,4-dichlorophenoxyacetic acid, 0.1 mg of kinetin, and 3 mg of sucrose were added in IL.
Seed cells were suspended in a 200 mL Erlenmeyer flask containing 40 mL of MS medium containing 0 g and adjusted to pH 5.7-5.8, and cultured every 2 weeks at 30 °C in the dark at a rotation speed of 12 Orpm. This was done by repeating.

On the 5th day after the start of culture, the entire volume of the culture solution containing cells (40 mL
) was first passed through a 500 μm nylon mesh to remove large cell clusters. Next, the passed liquid was passed through a nylon mesh having a size of 20 μm, and the cells remaining on the mesh were collected. Enzyme reaction solution (Cellulase Onozuka R81% by weight, Pectolyase Y230, 1% by weight, Mannitol 0.5M, p
Suspend in 20 mL of H5,6), 30°C, dark, 90 rpm.
The temperature was kept at a rotation speed of m for 4 hours.

The enzyme-treated cell suspension was filtered through a 50 μm nylon mesh to remove undegraded cells or cell aggregates, centrifuged at 500 rpm for 5 minutes, and the precipitate was washed with protoplast washing solution (2.5 mM 0.5M mannitol solution containing calcium chloride, pH 5, 6) 2
mL of the suspension, and this was gently poured into a 15 mL centrifuge tube containing 10 mL of a 20% by weight sucrose solution at pH 5, 6 to form an overlay, and centrifuged at 1000 rpm for 5 minutes.

The crude protoplast fraction in the upper layer of the sucrose solution was gently sucked up with a Pasteur pipette, resuspended in 10 mL of the above protoplast washing solution, and centrifuged at 500 rpm for 5 minutes. This washing operation was repeated twice to obtain approximately 2×10 6 purified protoplasts.

The obtained protoplasts were stained with calcofluor white and observed under a fluorescence microscope to confirm that the cell wall had been removed.

(2) Preparation of string-shaped calcium alginate gel entrapping and immobilizing protoplasts 3% by weight sodium alginate [Sigma Chemical Co., Ltd.
Company (SIGMA CHEMICAL COMP)
100 mL of A-2158) aqueous solution manufactured by ANY)
It was prepared and sterilized at high temperature in an autoclave at 120°C for 20 minutes.

2.5 5×10' protoplasts obtained in (1) above
0.5M mannitol solution containing mM calcium chloride (
pH 6,5) and 100 mL of this suspension.
L and 100 mL of the above 3% sodium alginate aqueous solution
were mixed. 0, containing sterile 0,5M mannitol
This mixed solution was extruded into a 1M calcium chloride aqueous solution (10,100O) through a syringe equipped with a needle with an inner diameter of 0.3 mm to form a string-like gel with a thickness of about 0.9 mm. After shaking this for 1 hour, the string-like gel was taken out and washed twice with protoplast washing solution.

(3) Preparation of nurse cells entrapping immobilized on calcium alginate beads 3% by weight sodium alginate [Sigma Chemical Co., Ltd.
Company (SIGMA CHEMICAL COMP)
200 mL of an aqueous solution (No. A-2158) manufactured by ANY) was prepared and sterilized. This was then cultured with liquid shaking for two weeks.
gratus) callus cells were suspended in a packed cell volume of 20 mL. This suspension was poured into a sterilized 0.1M calcium chloride aqueous solution (10100O) into a tube with an inner diameter of 0.8 m.
It was dropped from a glass tube of m. After shaking this for 1 hour, 3
．． The plant cell-immobilized beads, which had become spherical beads of 5 to 4 mm, were taken out and washed twice with sterile water.

(4) Mixed culture inorganic components and organic components are each 1/2 of the specified concentration (
7) Concentration (7) Mannitol was dissolved in MS medium to a final concentration of 0.5M, and sucrose was further dissolved at 10M.
g/L, 1 mg of 2.4-dichlorophenoxyacetic acid
20 mL of a liquid medium containing 1 mg/L of kinetin was dispensed into a 100 mL Erlenmeyer flask and sterilized by autoclaving at 120° C. for 20 minutes. 2 mL of the protoplast-immobilized string gel prepared in (2) above and 50 nurse cell-immobilized beads prepared in (3) above were placed in a flask containing the above medium, and the mixture was heated at 8 Orpm in the dark at 30°C. As a result of two weeks of shaking culture at a rotating speed, many small colonies derived from protoplasts were observed in the string-like gel on which protoplasts were immobilized.

(5) Proliferation and selection A culture medium containing a string-like gel immobilized with protoplasts and beads immobilized with nurse cells was prepared with a 1.75 mm opening.
The medium was removed through a sterilized metal sieve, and the string-like gel and beads were collected on the sieve. The above procedure (4) was repeated, and the culture was continued for another two weeks to sufficiently proliferate the cells in the gel.

Next, the culture solution containing these string-like gels and beads was passed through a sterilized metal sieve with an opening of 4.75 mm, and the beads on which the nurse cells had been immobilized and grown were passed through the sieve, and the protoplast cells were immobilized thereon. Only the grown gel strings were collected on the sieve.

〔Effect of the invention〕

The present invention has provided a method for culturing protoplasts that is free from the influence of colorants such as activated carbon and can efficiently handle a large number of protoplasts.

Claims (1)

[Scope of Claims] 1. A method for culturing protoplasts, which comprises entrapping and immobilizing plant cell protoplasts in a string-like calcium alginate gel and culturing them together with nurse cells. 2. The thickness of the string-shaped calcium alginate gel is 0 in diameter.
．． The method for culturing protoplasts according to claim 1, wherein the protoplasts have a diameter of 5 to 2.0 mm. 3. Claim 1 or Claim 2, wherein the nurse cells are comprehensively immobilized on beads of calcium alginate gel.
The described method for culturing protoplasts.