JPH05236950A - Culture of protoplast - Google Patents

Abstract

PURPOSE:To easily produce numerous clonal cells from a large quantity of protoplasts while handling them. CONSTITUTION:Plant cells protoplasts are comprehensively immobilized onto stringy calcium alginate gel and cultured together with nurse cells; in this case, the distance between adjacent two protoplasts is set at 1-20mm on average.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing protoplasts, and more particularly to a method for culturing plant-derived protoplasts effective for creating and breeding 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 of culturing protoplasts at a low cell concentration of 10 ³ cells / ml or less, the secreted or released product of callus (nurse cells) in contact with released single cells or small cell clusters is divided as a nutrient.・ Various kinds of so-called "nurse culture" (Komamine et al., Ed., Plant Biotechnology Encyclopedia, 1990, 207)
~ 208 pages, 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.

[0005] Larkin et al. Distinguish the beads in which the desired protoplasts are entrapped and immobilized from the beads in which the nurse cells are entrapped and immobilized. The latter is colored with activated charcoal, and the beads containing the desired protoplasts and their proliferative bodies are treated with tweezers or the like. The device is designed to enable extraction and selection.

[0006]

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. It is an object of the present invention to provide a method for easily producing a large number of clonal cells from a large number of protoplasts without worrying about the adverse effects of activated carbon.

[0007]

Means for Solving the Problems As a result of various studies on nurse cultures capable of efficiently handling a large number of protoplasts without coloring them with activated carbon or the like, the present inventors have found that protoplasts of plant cells are cord-like alginate. When the cells are entrapped and immobilized on calcium gel and cultured together with the nurse cells, the protoplasts entrapped and immobilized on the string-like calcium alginate gel are spaced at an average value of 1 to 20 mm between two protoplasts that are close to each other The inventors have found that the object can be achieved and completed the present invention.

That is, according to the present invention, when protoplasts of plant cells are entrapped and immobilized on a string-like calcium alginate gel and cultured together with a nurse cell, the above-mentioned protoplasts entrapped and immobilized on the string-like calcium alginate gel are closely spaced. The present invention relates to a method for culturing protoplasts, wherein the average value between individual protoplasts is 1 to 20 mm.

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.

[0010] The entrapping immobilization of protoplasts on a string of calcium alginate gel is carried out by immobilizing protoplasts in an amount of 1 to 5
After suspending in a wt% sodium alginate aqueous solution,
From the nozzle with an inner diameter of 0.1 mm to 1.0 mm,
It is carried out by extrusion into a 0.2 M calcium chloride aqueous 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 wt% sodium alginate aqueous solution, and a 0.1 M calcium chloride aqueous solution is supplied from a nozzle having an inner diameter of 0.3 mm. When extruded inside, the thickness is about 0.9
A mm-like string gel is obtained, and using a 0.8 mm nozzle, a string-like gel having a thickness of about 1.5 mm is obtained.

The thickness of the string-like gel for entrapping immobilization of protoplasts is preferably as thin as possible for formation of colonies and separation of colonies, but if it is too thin, mechanical strength is lowered and it tends to break during handling. On the other hand, if it 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 gel is preferably in the range of 0.5 mm to 2.0 mm.

The concentration (density) of protoplasts entrapped and immobilized in a string-like calcium alginate gel is such that the average distance between two protoplasts is 1 to 20 m.
m, preferably 2 to 10 mm. If the distance between two adjacent protoplasts is less than 1 mm, it will be difficult to cut off the part after forming a colony. If the distance exceeds 20 mm, a string-like gel will be formed when a large number of protoplasts are processed. Must be unusually long.

The distance (interval) between the protoplasts entrapped and immobilized on the string-like calcium alginate gel can be set by adjusting the concentration of the protoplasts entrapped and immobilized. For example, a protoplast with a diameter of 1.5 mm
If immobilized on a string-like gel at a concentration of 100 / ml,
The average spacing of protoplasts is about 5 mm, and the average spacing of protoplasts is about 1.6 mm when immobilized on a string gel having a diameter of 0.9 mm at a concentration of 1000 cells / ml.

As the nurse cells used in the present invention, preferable cells are appropriately selected in relation to the protoplasts to be proliferated / divided. Usually, a protoplast to be propagated / divided is often of the same genus and species, but is not necessarily limited to the same genus and species. Further, the nurse cells may be whole cells or protoplasts. Inactivated cells that have been unable to grow in advance by irradiation with radiation or UV may be used.

As the nurse cells, cells not entrapped and immobilized may be used as they are, but in order to obtain operability during medium exchange and string-like calcium alginate gel entrapped and immobilized protoplasts in which no nurse cells are attached. It is preferable that the nurse cells are entrapped and immobilized on a suitable carrier, for example, beads of calcium alginate gel.

The entrapping immobilization of the nurse cells on the calcium alginate beads can be easily carried out by a known technique. That is, the nurse cells are suspended in a 1 to 5 wt% sodium alginate aqueous solution, and the inner diameter is 0.1 mm to 3.0 mm.
Can be added dropwise to a 0.05 to 0.2 M aqueous solution of calcium chloride from the nozzle to prepare the calcium alginate beads with immobilized nurse cells.

When the nurse cells are entrapped and immobilized on calcium alginate gel beads, the beads immediately after the immobilization may be used, or the cells may be cultured in the beads for several days after the immobilization. It can also be used after sufficient growth.

The protoplast and nurse cells immobilized on a string of calcium alginate gel and 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.
Skoog medium, Linsmaier & Sk
oog medium, Heller medium, White medium, Nitch & Nitch medium, Cienck
Schenk & Hildebrandt medium, Gamborg B5 medium, Kao & Michayl
uk) 8P medium, Nagata / Tatebe's medium, etc., and each component is used at the concentration as prescribed according to the protoplasts and nurse cells to be cultured, or 1/2 to 1/10
Use after diluting to the concentration of. Further, addition of plant hormones to the medium is usually necessary for the division / proliferation of protoplasts.

Examples of plant hormones include auxin and cytokinin, and examples of auxins include α-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, indoleacetic acid, indolepropionic acid, indolebutyric 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, ciszeatin and the like, and among these, those that protoplasts are selected and used.

Auxin is usually 0.1 to 10 m in the medium.
Cytokinin is usually used at a concentration of 0.01 to 1 mg / l in the medium, and other hormones are also used at an appropriate concentration. Since the concentration of plant hormones is optimal for growth,
Preferably, the concentration is determined beforehand 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 to 50 g / l for use.

Depending on the type of plant cell, mannitol, sorbitol, etc. added to adjust the osmotic pressure of the medium may inhibit the growth of protoplasts. In such a case, after culturing for 1 to 2 weeks, it is advisable to transfer to a medium containing no osmotic pressure adjusting agent such as mannitol or sorbitol and continue the culturing.

The string-like calcium alginate gel containing proliferatives from protoplasts thus cultured is washed while being pulled up with tweezers or the like, or is passed through a sieve having an appropriate mesh so that nurse cells (or nurse cells) (Prolifers from cells) or these can be easily separated from entrapped and immobilized beads.

A string-like calcium alginate gel containing protoplast-derived proliferates as it is, or a gel portion containing protoplast-derived proliferates is cut off with scissors or a scalpel while visually or visually confirming. Can be transplanted to an appropriate solid medium for growth and cultured to obtain a single protoplast-derived callus or regenerated plant body.

[0025]

EXAMPLES The present invention will be specifically described below with reference to examples. (1) Preparation of protoplasts Strophanthus gratus
Protoplasts were prepared from suspension-cultured cells obtained by forming callus from leaves. The procedure is shown below.

The subculture of suspension-cultured cells was 2,4 in 1 L.
-Dichlorophenoxyacetic acid 1 mg, kinetin 0.1 m
pH of 5.7-5.
The seed cells were suspended in a 200 ml Erlenmeyer flask containing 40 ml of the medium No. 8 and the culture was repeated every 2 weeks at 30 ° C. and 120 rpm in the dark.

On the 5th day after the start of the culture, the whole amount of the culture solution containing cells (40 ml) was taken, and this was first applied to a nylon mesh of 500 μm in size 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 liquid (cellulase Onozuka RS 1% by weight, pectolyase Y23 0.1% by weight, mannitol 0.5M, pH 5.6) was suspended in 20 ml, and
Incubation was carried out at 90 ° C. in a dark place at 90 rpm for 4 hours.

The 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 ml of a sucrose solution having a pH of 5.6 and 10 ml to be overlaid, 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 10 m of the above protoplast washing solution.
resuspended in 1 and centrifuged at 500 rpm for 5 minutes. This washing operation is repeated twice, and the purified protoplasts are added to about 2 × 1.
I got 0 ⁶ . A part of this was sampled, stained with Calcofluor White, and then observed with a fluorescence microscope to confirm that the cell wall was removed.

(2) Preparation of string-like calcium alginate gel in which protoplasts are entrapped and immobilized 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.

5 × 10 protoplasts obtained in (1) above
^{Four of} them were suspended in 100 ml of a 0.5 M mannitol solution (pH 6.5) containing 2.5 mM calcium chloride, and 100 ml of this suspension was mixed with 100 ml of the 3% aqueous sodium alginate solution. This mixed solution was extruded into 1000 ml of 0.1 M calcium chloride aqueous solution containing sterilized 0.5 M mannitol from a syringe equipped with an injection needle having an inner diameter of 0.3 mm to form a string-like gel having a thickness of about 0.9 mm. .. After shaking this for 1 hour, the string-like gel was taken out and washed twice with a protoplast washing solution.

(3) Preparation of nurse cells entrapped and immobilized in calcium alginate beads 3% by weight of sodium alginate [Sigma Chemical Co.
Company (SIGMACHEMICAL COMPANY) No.A-2158]
200 ml of an aqueous solution was prepared and sterilized. Strophantus gratas (Stro
20 ml (packed cell volume) of callus cells of Phanthus gratus) was suspended. This suspension was added to 1000 ml of a sterilized 0.1 M calcium chloride aqueous solution with an inner diameter of 0.
It was dripped from an 8 mm glass tube. 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 an MS medium having a concentration of 1/2 of the predetermined concentrations of the inorganic component and the organic component to a final concentration of 0.5M.
Dissolve so that sucrose 10 g / l, 2,
20 ml of a liquid medium prepared so that 1 mg / l of 4-dichlorophenoxyacetic acid and 0.1 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-shaped 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 kept at 80 ° C. in the dark at 30 ° C.
As a result of shaking culture for 2 weeks at a rotation speed of rpm, many small colonies derived from protoplasts were observed in the string-like gel.

(5) Proliferation and selection A culture solution containing a string-like gel on which protoplasts are immobilized and beads on which nurse cells are immobilized is passed through a sterilized metal sieve having an opening of 1.75 mm to remove the medium, and then the sieve is placed. The string gel and beads were collected. This string-like gel and beads were mixed with 1 m of 2,4-dichlorophenoxyacetic acid in 1 L.
g, kinetin 0.1 mg and sucrose 30 g
100 containing 20 ml of medium (pH 5.7 to 5.8)
After being placed in a ml Erlenmeyer flask and continued to be cultured for 2 weeks, colonies grown from protoplasts could be easily confirmed with the naked eye. A string-like gel containing this colony was taken out with tweezers, and the gel was aseptically cut with scissors so as to contain one colony each. 1L of this fragment
MS solid medium (pH 5.7 to 5.8) containing 2,4-dichlorophenoxyacetic acid (1 mg), kinetin (0.1 mg), sucrose (30 g), and medium solidifying agent, Gelrite (trade name of Kelco Division of Merck). One by one, and static culture was performed in the dark at 30 ° C. Two weeks after transplanting to the above-mentioned solid medium, callus that broke the string-like gel and proliferated could be obtained from any of them.

[0034]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily produce clonal cells from individual protoplasts while handling a large amount of protoplasts without fear of adverse effects of activated carbon, and to obtain callus or regenerated plant therefrom. A simple protoplast culture method could be provided.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Riko Iwata 48, Wadai, Tsukuba City, Ibaraki Prefecture Hitachi Chemical Co., Ltd. Tsukuba Development Laboratory

Claims (1)

[Claims] 1. Two protoplasts in which protoplasts of plant cells are entrapped and immobilized in a string-like calcium alginate gel and, when cultured together with a nurse cell, are entrapped and immobilized in a string-like calcium alginate gel in close proximity to each other. A method for culturing protoplasts, characterized in that the mean value between the two is 1 to 20 mm.