JPS63245668A - Tissue culture of plant - Google Patents

Abstract

PURPOSE:To sprout a callus cell to form a seedling row and to effect the growth of callus cell in high efficiency, by culturing a callus cell of a plant in a proper medium in the presence of a plant hormone containing gibberellin. CONSTITUTION:Callus cell of a plant (e.g. broad bean or radish) is cultured in a proper medium in the presence of plant hormone containing gibberellin. The callus cell is sprouted to form a seedling row by this process. The process is effective in producing a large amount of high-quality seedling of a plant. Combination of the process with the technique of cell fusion enables even the production of a large amount of seedling of various novel plants imparted with nitrogen capability of root nodule bacteria of leguminous plant. The concentration of gibberellin in the medium is preferably 0.1-20mg/l. Auxins and/or cytokinins may be used in combination with gibberellin as necessary.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to plant tissue culture, and particularly to a method for germinating callus cells to form shoots.

[Conventional technology]

Plant tissue culture has recently attracted attention as a field of biotechnology. Among these, callus culture is already applied to the production of plant secondary metabolites such as andocyanin and shikonin because it can be cultured in large quantities in tanks. Furthermore, the cultured callus cells can be used for cell fusion as protoplast cells by dissolving the cell wall.

Therefore, it is also useful as a means of breeding useful new plant varieties. Furthermore, due to totipotency (totipotency), which is a characteristic of plants, if callus cells are allowed to germinate and grow, a complete plant with exactly the same genetic characteristics as the original plant from which the callus was derived, i.e. Clone plants can be obtained. Therefore, callus culture is an extremely effective means for producing large quantities of excellent seedlings.

Since this is carried out for the above-mentioned purposes, promoting germination and rooting and increasing the growth efficiency of callus cells are important issues in callus culture.

Among these problems, as a method for promoting germination and rooting, a method of adding auxins and cytokinins to the culture medium has been conventionally used.

On the other hand, no particular method has been proposed so far for the purpose of increasing the growth efficiency of callus cells.

[Problem that the invention seeks to solve]

Conventionally used methods for promoting callus cell germination and rooting are effective against callus cells derived from a range of plants and have been successful in that range. However, it is not effective against callus derived from any plant. Examples of plants for which conventional methods are not effective include legumes such as soybeans and edamame, and cruciferous plants such as radish and rapeseed. In cultured callus cells derived from these plants, rooting can be promoted by adding auxins and cytokinins, but germination is not promoted. Furthermore, there has been no suitable means for germinating callus cells derived from these plants. Therefore, shoot bodies have not yet been obtained from callus cells derived from the above plants.

Furthermore, since there is no suitable method to improve the growth efficiency of callus cells, when applying callus culture to the production of plant metabolic products, the only way to increase production efficiency is to increase the proliferation of callus cells. There were certain limits to its effectiveness.

The present invention has been made in view of the above circumstances, and provides a method for culturing plant tissues that can germinate the callus cells that have not yet been successfully germinated, and can also improve the growth rate of callus cells. It is something to do.

[Means and effects for solving the problem] The plant tissue culture method according to the present invention is characterized in that callus cells of a plant are cultured in an appropriate medium in the presence of a plant hormone containing gibberellin to cause germination. That is.

The gist of the invention is the use of gibberellin in plant callus culture. Gibberellins, like auxins and cytokinins, are plant growth regulators. Gibberellin has been widely used in the production of seedless grapes and watermelons, but there are few examples of its use in plant tissue culture, and no known examples of its use in callus culture.

The present invention has succeeded in making it possible to germinate callus cells, which could not be germinated conventionally, or to enlarge callus cells and increase their growth efficiency by using this gibberellin in callus culture.

Therefore, the present invention is particularly meaningful when applied to callus cells that cannot be germinated by these conventional methods. However, the scope of application of the present invention is not limited to these plant-derived callus cells, and even when applied to callus cells that germinate using conventional methods, effects such as shortening the culture period required for germination can be obtained. It will be done.

Gibberellin is a general term for growth-promoting substances of higher plants produced by the rice bacanae disease fungus, and the four types A1 to A4 (hereinafter referred to as GA to GA4) are the most well-known. Although any of these may be used in the present invention, GA
3 is the most active.

As for its usage, it may be added to a medium for callus culture, or it may be added to a medium for inducing callus and introduced into callus cells. Furthermore, when culturing a plant that will serve as a callus-inducing mother, it may be introduced into the plant by adding it to the medium or the like. The amount of gibberellin to be used varies depending on the form of use, type of callus, and other specific conditions, but for example, when added to the culture medium of callus cells, 0. I IItg~20mg/J! degree is desirable. This is because if it is too small, it will be ineffective, and if it is too large, the callus will rapidly deteriorate during long-term culture.

In the present invention, other plant hormones other than gibberellin are used in combination as appropriate. Plant hormones used in combination include those conventionally used in callus culture, such as auxins and cytokinins shown below. In some cases, gibberellin can be used alone to cause germination, but depending on the type of plant, germination may occur only when a combination of these is used.

Auxins> Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (
2,4-D), 1-naphthalene acetic acid (NAA), cytokinins> kinetin, 6-benzyladenine (BA), zeatin As a medium for culturing callus in the present invention, conventionally used ones can be used as they are. Can be used. For example, MS medium (Urashlgc-koog medium), White medium, Gautley medium, Gamborg medium,
Examples include LS medium (Insmaler-Koog medium) and modified media based on these.

As mentioned above, the present invention is particularly useful when applied to callus cells that cannot be germinated by conventional methods. Callus that can germinate for the first time by applying the present invention,
For example, it is derived from the following plants.

Leguminae plants of the genus Broad Bean (broad beans, etc.), soybean genus (soybeans, etc.), genus Peas (peas, etc.), cowpea genus (cowpeas, etc.), genus Azuki (adzuki bean, etc.) Cruciferous plants of the genus radish (radish, radish, etc.), rapeseed Genus (
(Brassica rape, Chinese cabbage, cabbage, Komatsuna, Mizuna, etc.) Note that callus can be induced from the above-mentioned plants by any conventional method. For example, seeds, leaves, stems, roots, and other tissues that have been thoroughly washed with ion-exchanged water are cut into tissue pieces of appropriate size, and then sterilized with an appropriate disinfectant such as sodium hypochlorite or ethanol. do. After further washing with sterile water, callus can be induced by culturing in an appropriate medium. Alternatively, after sterilizing plant seeds in a conventional manner, aseptically inoculating them into a sterile germination medium (usually a 0.8% agar medium containing 3% sucrose) and leaving them in a dark or light place to produce sterile seedlings. The hypocotyl is about 5 long
Callus may be induced by cutting into pieces or by cutting leaves into approximately 3 x 3 mrtr to prepare 4 to 5 samples and culturing them in an appropriate medium.

Note that the callus cells to which the present invention is applied may be, for example, callus obtained from a haploid plant obtained by pollen culture. It may also be callus obtained from polyploid plants such as diploid, triploid, and tetraploid plants obtained by treatment with colchicine.

〔Effect of the invention〕

According to the present invention, even callus cells that could not be germinated conventionally can be germinated to form shoots. Therefore, it is not only an effective means for producing large quantities of excellent seedlings of these plants, but also significantly increases the possibility of culturing callus. For example, by combining this technology with cell fusion technology, it becomes possible to mass produce seedlings of various excellent new plant species that have been endowed with the ability to fix atmospheric nitrogen through rhizobia of leguminous plants.

Furthermore, the enlargement of callus cells themselves by application of the present invention is expected to be a powerful means of increasing production efficiency when producing plant metabolic products by culturing callus.

〔Example〕

The present invention will be explained in detail below. The media, culture conditions, and growth regulators used in the following examples are as follows.

Medium> MS medium containing 3% saccharose and 0.8% agar.Culture conditions>Culture temperature 25℃, 16 hours of light at 2000 lux.Growth regulators> ■NAA (1-naphthalene acetic acid)/BA (6-benzyl adenine)/gibberellin (GA3) combination.

Added to culture medium.

■ Indole-3-butyric acid (IBA)/Kinetin (K
)/Gibberellin (G3) combination. Added to culture medium.

Example 1 Approximately 5 vt from sterile germinated seedlings of commercially available early edamame
A stem piece of X. This callus was then incubated under the above culture conditions for 6
Cultured for a week. In this example, culture was carried out using the combination (2) above as a growth regulator.

A total of 64 culture experiments were conducted by varying the concentration of each growth regulating substance within the following ranges.

NAA: 4 levels in the range of O to 5B/no BA: o, 5 to 4M! 4 steps GA3 in i/no range
: As a result of the above callus culture in four stages in the range of 0 to 519/I, no germination occurred at all in the culture example with GA3 concentration of 0, and no shoot formation was observed.

On the other hand, in the culture examples to which GA3 was added, germination occurred and shoot formation was observed in the following cases.

NAA concentration: orllj/no BA 6 degrees: 0.51151/I GA3 concentration: 1. Oj2!7/I! Next, the fresh weight of the callus after culturing and the dry weight after drying at 90° C. for 24 hours were examined, and the following results were obtained. First, in a culture example using NAA and GA3 in combination,
An increase in the fresh weight of callus was observed.

In addition, the ratio of dry weight/fresh weight is approximately 0.0 in the culture example without the addition of GA3. l, whereas GA3. 0 in the case of supplemented culture
．． It decreased to 0.07-0.08. This result indicates that the presence of GA3 promoted hypertrophy of callus cells.

Furthermore, when we examined the condition of callus cells after culturing, we found that GA
In the culture example without the addition of 3, the callus easily collapsed and was crumbly. However, in the case of culturing in the presence of GA3, as the concentration increased, soft callus with elasticity was obtained.

Example 2 Early edamame, 26/I! Aseptic germination was performed using MS medium supplemented with GA3. Furthermore, callus was induced from the hypocotyl by a conventional method, and then the callus was cultured in the same manner as in Example 1.

As a result, callus germinated and shoot formation was observed when NAA, BA, and GA3 were at the following concentrations.

NAA concentration: 0η/noBA concentration: 1.0 @/noGA3 concentration = 0.1 or 5.0 And it was small.

Example 3 Approximately 5 stem pieces were prepared from sterile germinated seedlings of commercially available pill Komatsuna, and callus was induced using the stem pieces by a conventional method. Next, this callus was cultured for 6 weeks under the above-mentioned predetermined culture conditions. In this example, both culture using the combination (2) and culture using the combination (2) as growth regulators were carried out.

In addition, the concentration of each growth regulator was varied within the following range, and a total of 96 culture experiments and a total of 144 culture experiments were conducted for each combination.

Combination ■〉 NAA: 4 levels BA in the range of 0 to 516/): o
, 6 steps in the range of i to 4η/no GA3: 0 to 5 B
Combination in 4 stages in the range of /〉 IBA: 4 stages in the range of 0 to 5tj /〇 K: o
, 6 steps in the range of t~4η/l G A3: 0~1
As a result of the above callus culture in 6 stages in the range of 0.6/no, GA
In the case of culture at 0 concentration of 3, no germination occurred at all, and no shoot formation was observed.

On the other hand, in the culture examples to which GA3 was added, germination occurred and shoot formation was observed in the following cases.

Combination ■〉 Two sides of I to ■ III NAA concentration (El/)) 2 5 5 BA concentration CKI/, e) 4 1 2GA3
Concentration (double/, i?) 0. L O, 10, 1 combination■> IBA concentration: 5/K la degree: 0.5 all! GA3 concentration: 0.1 #/1 Next, in the same manner as in Example 1, the fresh weight and dry weight of the callus after culture were examined. As a result, the fresh weight is GA3
An increase was seen with the addition of . In the combination of ■, the weight increase in the low concentration range of GA3 is large, and in the combination of
The increase was large when the concentration was 1.0 cases/no or more.

Furthermore, as in Example 1, the dry weight/fresh weight ratio is 0.08 to 0.12 in culture examples without GA3 addition, while it is 0.05 to 0 in culture examples with GA3 addition. ．．
It decreased to 08. This result indicates that the presence of GA3 promoted hypertrophy of callus cells.

Furthermore, as a result of examining the condition of callus cells after culture, GA3
Calli cultured without additives were green and quite compact, whereas calli cultured in the presence of GA3 were pale, bright, swollen, and soft.

Example 4 Approx.
A stem piece of rnm was prepared, and callus was induced using this as a material by a conventional method. Next, this callus was cultured for 6 weeks under the above-mentioned predetermined culture conditions. A total of 32 culture experiments were carried out using the combination (2) above as growth regulators and varying the concentration of each growth regulator within the following range.

NAA: 4 levels in the range of 0.2 to 2η/no BA: 0.
4 levels GA3: 0 or 21 in the range of 2 to 2 IIg/no
As a result of the above two-stage callus culture at 19/no, no germination occurred at all in the culture example with GA3 concentration of 0, and no shoot formation was observed.

In contrast to this, in the culture examples in which GA3 was added, germination occurred and shoot formation was observed in the following cases.

NAA concentration: 2.0 M/, l? BA concentration: 2.01G/J! G A 3 'ta degree: 2.01517 no In addition, when the fresh weight of callus after culture was examined, an increase in the fresh weight of callus and a decrease in the dry weight/fresh weight ratio were observed due to the addition of GA3. The effect of promoting hypertrophy of callus cells was also revealed. In addition, calli cultured in the presence of GA3 were in a softer and swollen state compared to calli cultured in a GA3-free medium.

Applicant's representative Patent attorney Takehiko Suzue Procedural amendment March 31, 1988 Director General of the Patent Office Kunio Ogawa 1, Indication of case Patent Application No. 1983-76232 2, Title of invention Method for cultivating tissue of plants 3, Relationship with the case of the person making the amendment Patent applicant Seno Sanbe 4, agent UBE Building 6, 3-7-2 Kasumigaseki, Chiyoda-ku, Tokyo;
Subject of the amendment The statement "It was in a state" on page 17, line 10 of the specification of description 7, contents of the amendment has been corrected as follows.

Record It was a state.

Example 5 To examine the effect of gibberellin administration on azuki beans,
The following experiment was conducted. The variety of azuki beans is Dainagon (
A native species) was used.

■ Effect of GAE 1 degree during culture on shoot formation,
Interaction between GAE and auxin: 0.2 during sterile germination
The obtained stem pieces were cut into lengths of approximately 5 mm and cultured using MS medium supplemented with tny/Q GAE and MS medium without the addition. The culture medium is basically MS medium, containing BA (0.4 mg/Q), GAE (0~
7.0 my/Q>, auxin NAA, PC
L, 2.4-D (add a combination of 0, 0, 21n9/(!, >), add 3% sucrose and 0.8% agar, +
) Adjusted to H5.8. The culture conditions were a temperature of 25°C and a temperature of 16°C.
It was set as time illumination (35001ux).

As a result, shoot formation was GAE 0.1■, / , &,
, was confirmed most often in auxin-free. In particular, those treated with GA3 during aseptic germination were good.

Addition of auxin promoted callus formation and rooting, and shoot formation was not effective. Furthermore, when GAE was added, both the number of shoots and shoot elongation were better than when using the conventional method.

■ Effects of different parts of stem pieces on shoot formation and relationship with callus formation: During cultivation, shoot formation was examined by placing the stems on a bed so that the parts of the stem pieces were clearly defined, and the weight of callus was also measured.

As a result, we found that shoot formation due to differences in stem segment location was
In samples treated with G A 3 during sterile germination and cultured without auxin, the area from the center of the hypocotyl to the root was good, but under other conditions, the attachment of cotyledons was good at the root. In areas with good shoot formation, a somatic embryo-like shape was observed, whereas in areas with poor shoot formation, a swollen callus shape was observed.

-■ Shadow of GAa addition concentration and light duration during aseptic germination in shoot formation 9: GA3 addition concentration during aseptic germination was adjusted from 0 to 1.0IR9/(1
The effect on shoot formation was investigated by culturing for 50 days in two cases in which the light duration was varied within the range of 16 hours and 24 hours.

As a result, the concentration of GA3 added during aseptic germination was 0.1 to 0.
51 ng/R was good. Regarding the influence of irradiation time, 24-hour irradiation was particularly better than 16-hour irradiation.

■ Difference between 2mm and 5mm stem hole size in shoot formation: Culture medium is BA (0,4m9/Q,), GAE
(0,1■/Q), and culture was performed using the material with stem hole diameters of 2 mm and 5 mm.

As a result, in shoot formation, the diameter of the stem hole is 2 m when the diameter is 5.
It was better than m.

When the shoots obtained in the above experiments were transferred to a rooting medium (IB8, 1 ig/g), rooted and raised, 41 plants regenerated and set fruit. It was done.

Claims (7)

[Claims] (1) A method for germinating callus cells, which comprises germinating plant callus cells by culturing them in an appropriate medium in the presence of a plant hormone containing gibberellin. (2) The method for germinating callus cells according to claim (1), wherein the callus cells are derived from a leguminous plant or a cruciferous plant. (3) The leguminous plant is a plant of the soybean genus, broad bean genus, or pea genus (
2) The callus cell germination method described in section 2). (4) The method for germinating callus cells according to claim (2), wherein the plant of the family Cruciferae is a plant of the genus Brassica or radish. (5) The plant hormone containing gibberellin contains auxins and/or cytokinins in addition to gibberellin, according to any one of claims (1) to (4). How to germinate callus cells. (6) The plant hormone containing gibberellin is added to the appropriate medium.
The method for sprouting callus cells according to any one of items 1) to (5). (7) The concentration of gibberellin added is 0.1 mg to 20 mg.
mg/l.
) The callus cell germination method described in section 2.