JP2840854B2 - Callus regeneration from wasabi protoplast - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to the separation of protoplasts from tissues or callus of Brassicaceae Wasabiaceae plants, and to callus induction.

(Prior art) In recent years, since the wasabi, a plant belonging to the genus Wasabi of the Brassicaceae family, has been remarkably degraded, a large-scale propagation method mainly using a plant tissue culture method such as a shoot tip culture method is intended for the stable supply of healthy wasabi seedlings. Have been proposed (JP-A-61-115417).
No. Toshio Otsuka, Agriculture and Horticulture, Vol. 63, 185, 1988).

On the other hand, regarding the application of plant tissue culture technology to wasabi, there have been reports on induction of callus from plant tissues and regeneration into plants (JP-A-59-16285; JP-A-63-1985). No. 304922).

(Problems to be Solved by the Invention) However, in the case of Wasabi genus plants, callus regeneration technology from protoplasts has not been established, and this has been a major obstacle to the introduction of new biotechnology technology into breeding of Wasabi genus plants. Was. Also, isolation and regeneration of horseradish protoplasts have been strongly desired from the practical aspects such as breeding and the fundamental scientific aspects such as elucidation of the mechanism of differentiation regulation at the gene level.

(Means for Solving the Problems) As a result of various studies, the present inventors have efficiently separated protoplasts from various tissues of the Wasabi genus plant,
In addition, the present inventors have found a method of inducing mitotic proliferation by culturing the isolated protoplasts and frequently regenerating them into calli. Further, it has been found that the present invention is also effective when the Wasabi genus plant is a regenerated plant derived from callus or protoplast. On the other hand, a method for efficiently separating protoplasts from callus itself and frequently regenerating them into calli has also been found.

In the present invention, as the Brassicaceae Wasabi genus plant, wasabi (Wasabia japonica) and lily wasabi (Wa)
sabia tenuis).

When separating protoplasts from plant tissues, roots,
Although it does not matter if the tissue of the rhizome or leaf is used,
It is preferable to use the leaf blade part of the leaf from the viewpoint of easily obtaining the material. It is also possible to sterilize the normally cultivated plants as needed to prepare the test material, but if the mother plant once sterilized by a method such as shoot apex cultivation is aseptically cloned and maintained. This is convenient because not only the subsequent sterilization treatment is not required, but also a material having a constant physiological state can be obtained, and a highly reproducible result can be obtained.

In the case of callus, it is usually maintained in a state of aseptic culture, so sterilization is not necessary, but callus maintained in static culture is transferred to a liquid shaking culture system once before use And it is easy to obtain more favorable results in terms of protoplast separation efficiency or regeneration efficiency.

The protoplast separation enzymes used in the present invention include:
Commercially available enzymes commonly used for this purpose include, for example, Cellulase Onozuka R-10, Cellulase Onozuka RS, Doriserase, Meisserase P-1, Macerozyme R-10, Pectoliase Y23, and the like. These can be used alone or in combination at an appropriate ratio. Particularly, when protoplasts are separated from leaves, a combination of cellulase Onozuka R-10 and macerozyme R-10, which have relatively mild effects, can be used. But,
Also, when separating protoplasts from callus, the combination of cellulase RS, which has relatively strong action, and pectolase Y23 seems to give better results.

After removing the enzyme solution, the separated protoplasts are removed as they are when there are few contaminants, and if there are many contaminants, the contaminants are removed by a known method, and then transferred to an appropriate medium and cultured.

As the basal medium for protoplast division and propagation used in the present invention, any of the known basal media used in ordinary protoplast culture is possible, but particularly in the case of protoplasts separated from leaves, a medium having a low ammonia content. The better result is obtained by selecting. In addition, in the case of protoplasts separated from callus, there is a relatively strong tendency to the adverse effects of ammonia, and it seems that there is no particular need to be particular about the ammonia concentration in the medium.

As the osmotic pressure adjusting agent to be added to the basic medium, mannitol, sorbitol, sucrose, glucose, etc., which are usually used, can be used without any problem. However, when culturing protoplasts obtained from leaves, it is used as a carbon source. The use of metabolized sho and the like or glucose is more preferable than non-metabolizable mannitol and sorbitol, since the osmotic pressure gradually decreases as the metabolism is increased. In any case, it is extremely important to gradually reduce the osmotic pressure in the medium at appropriate intervals during protoplast culture.

In the case of protoplasts obtained from calli, the inhibition of division by hyperosmolarity is not as pronounced as in the case of protoplasts obtained from leaves, but a proper reduction in osmotic pressure gives better results. When a non-metabolic agent such as mannitol or sorbitol is used as the osmotic pressure regulator, it is necessary to add a carbon source to the medium, but sucrose, glucose, fructose and the like usually used for this purpose are required. No problem with using

It is usually desirable to add a plant hormone to the protoplast culture medium of the present invention, and this is carried out by using generally used auxin or cytokinin alone or in an appropriate ratio. Further, although not essential for the effects of the present invention, the addition of albumin is particularly preferred for promoting the division and growth of protoplasts directly isolated from plant tissue.
This fact has already been clarified by the present inventors in Japanese Patent Publication No. 63-21473 and further supports this.

The effect of albumin addition was found to be remarkable in the early stage of protoplast culture, and albumin is considered to promote the return of protoplasts, which are particularly physiologically unstable in the early stage after separation, to the normal physiological state. Can be Similar effects are clearly observed in the case of protoplasts separated from callus, but are more pronounced in the case of protoplasts obtained from tissues. This is thought to reflect the difference in physiological state between callus cells that are already in an actively dividing state and tissue cells that are almost in a mitotic state.

The protoplast culture medium is a liquid medium without any problem, and a used medium is added (Japanese Patent Application Laid-Open No. 61-265087), or the amount of the medium is limited to such an extent that a thin film is formed on a thin layer solid medium (Japanese Patent Application Laid-Open No. No special operation such as that described in JP-A-61-265086) is required, and there is no need to add specially protected cells to the medium (Japanese Patent Laid-Open No. 232382/1987).

Incidentally, the protoplast itself is immobilized according to the purpose (for example, JP-A-62-55077; JP-A-63-301784;
Japanese Patent Application Laid-Open No. 63-313579) can be used for culturing, but the effect of the present invention is exerted completely independently of this. Wasabi protoplasts are immobilized by the method of the present invention. It should be noted that the cells grow in a liquid culture without regeneration and regenerate the callus.

By culturing horseradish protoplasts in a medium adjusted in consideration of the above points, the first division starts within a few days after the culture, and thereafter the frequency of division increases as the osmotic pressure of the medium gradually decreases, 1mm diameter within one month of culture
Are formed. The colonies thus generated can be grown on liquid or solid media and regenerated into larger calli. It is also possible to regenerate a complete plant by transferring to a special differentiation-inducing medium, but regeneration to a plant exists independently of the effects of the present invention. Is omitted. However, in order to prove that the present invention has the same effect on wasabi genus plants regenerated from rotoplasts as on natural wasabi genus plants, wasabi regenerated plants as test materials The body was also used.

(Examples) Next, the present invention will be described in more detail with reference to Examples, but these Examples are only a part of illustrations,
It does not limit the invention.

Example 1 Majutsu Wasabi (Wa) clone-proliferated by shoot tip culture
sabia japonica) was cut out and cut into small pieces with a scalpel. A 13% mannitol solution (pH 5.6) containing 0.5% cellulase Onozuka R-10, 0.25% macerozyme R-10, and 0.25% dextran sulfate potassium salt was used. ) The mixture was transferred to a 100-ml cap with a 100-ml cap containing 10 ml, and the enzyme solution was suction-permeated with a vacuum pump, and the enzyme treatment was carried out at 15 ° C overnight while standing still.

Thereafter, the protoplasts were carefully released from the softened tissue by careful pipetting, and filtered through a 50 μm nylon mesh to remove undigested tissue fragments and non-protoplastized cells. The filtrate was centrifuged at 100 g for 3 minutes to precipitate protoplasts, the enzyme solution was removed, and the protoplasts were purified by a density gradient method.

When the thus purified protoplasts were assayed by the Evans blue staining method, the viability was almost 100%.
%, And the final yield of protoplasts was about 5 × 10 ⁶ cells /
g Wet weight.

The composition of the medium used for culturing protoplasts
As shown in the table, the concentration of glucose added as an osmotic pressure regulator and a carbon source was gradually reduced from the initial 8% during the culture period to 2% in the final medium.

In addition, in the culture medium whose glucose concentration is 6% to 8%,
Bovine serum albumin to which 0.1% was added was used.
The separated and purified protoplasts were first suspended in a Petri dish containing 3 ml of an 8% glucose-supplemented medium to a concentration of 1 × 10 ⁵ cells / ml, and cultured at 25 ° C. in a dark place. On the fourth day of the culture, the cells were transferred to a fresh medium having the same composition, and on the first week, the cells were further transferred to a medium supplemented with 7% glucose. Five days later, the cells were further transferred to a medium supplemented with 6% glucose. By this time, many small colonies consisting of several to about 10 cells had already been observed, and the colony formation rate was about 40%. Thereafter, every week, when the cells were transferred to a medium supplemented with 4% glucose and a medium supplemented with 2% glucose, cell division became more active, and colonies as large as 1 mm in diameter became apparent. One month after the start of the culture, the rate of forming colonies with a diameter of 0.1 mm or more was about 10%.

Example 2 An experiment similar to that of Example 1 was conducted by selecting 10 lines from wasabi regenerated from the leaf protoplasts of 3 lines of true-married wasabi. Some of the selected regenerated wasabi strains
In addition to those whose phenotype was similar to that of the mother plant, those showing a clear displacement (for example, flower bud formation type or spotted type) were added.

In addition, as for these regenerated wasabi, as those of the mother plant, those clone-proliferated aseptically by strain separation were used. As a result, although there are differences among the strains, the final yield of protoplasts (1.3 to 8.7 × 10 ⁶ cells / g wet weight) and the colony formation rate of 0.1 mm or more after one month of culture (4.2 to 15.6%) are observed. In all strains, callus regeneration was possible. Note that these differences are apparently due to the hereditary traits of the individual lines themselves, and there is also a difference between regenerated wasabi lines derived from the same mother plant, and there is no direct correlation with the differences in phenotypes. Was.

Example 3 Clone-proliferated wasabi (Wasabia japonic)
a) Suspension culture cell line W obtained from mesophyll protoplasts
An experiment was performed using J-1. This cell line was subcultured and maintained every two weeks in Murashige-Skoog liquid medium (pH 5.6) containing 2% sucrose and 0.1 ppm 2.4-dichlorophenoxyacetic acid, and was subjected to experiments. It has been maintained for over a year at the time. 300 µm cells on day 4 of passage
To remove relatively large cell clumps,
After washing twice with a 13% mannitol solution, the cells were resuspended in a 13% mannitol solution containing 1% cellulase Onozuka RS and 0.1% pectylase Y23, and subjected to an enzyme treatment at 25 ° C. for 3 hours. Protoplasts were released by careful pipetting and filtered through a 50 μm nylon mesh to remove undigested cells. The enzyme solution was removed by centrifugation at 100 g for 3 minutes, and protoplasts were purified by a density gradient method.

The viability of the protoplasts purified in this way is
Approximately 90% by Evans blue staining, yielding a final yield of approximately 3
× 10 ⁶ / g wet weight. This protoplast was mixed with 1% sucrose, 7% mannitol, 0.1 ppm naphthalene acetic acid, and 0.1 p
The suspension was suspended at a concentration of 1 × 10 ⁵ / ml in ⁵ ml of Murashige-Skoog liquid medium (pH 5.6) to which pm-zeatin was added, and culture was started in a dark place at 25 ° C. One week later, the mixture was transferred to a medium in which the mannitol concentration was reduced to 5%.
Of medium. Up to this point, cell division had occurred at a high frequency, and a large number of several to ten colonies were observed. One week later, when the medium was transferred to a medium having a sucrose concentration of 3% except for mannitol, the proliferation was activated, and the formation rate of colonies having a diameter of 0.1 mm or more at the first month of the culture was about 15%.

Example 4 An experiment was performed in the same manner as in Example 3 using a suspension culture cell line WJP-1 obtained from regenerated wasabi mesophyll protoplasts. At the time of the experiment, the WJP-1 strain had about 8
It has been passaged every two weeks in a Murashige-Skoog liquid medium (pH 5.6) supplemented with 2% sucrose and 0.5 ppm naphthaleneacetic acid for two months.

The yield of protoplast was about 2 × 10 ⁶ / g wet weight, and the survival rate immediately after separation was about 95%. 0.1mm after one month of culture
The formation rate of the above colonies was about 10%.

(Effects of the Invention) As described in detail above, the present invention not only serves as a starting point for enabling application of techniques such as gene transfer and cell fusion in Wasabi genus plants, but also a Wasabi genus plant tissue which has been conventionally difficult. It provides a means of inducing callus formation from germ cells at a high frequency, and makes the selection or production of mutant cell lines extremely efficient, and is expected to contribute to basic science as well as practical significance.

Claims (3)

(57) [Claims] The present invention relates to a method for isolating protoplasts from plants of the genus Wasabi of the Brassicaceae family, dividing and growing them, and regenerating the callus. In this manner, the osmotic pressure in the medium is gradually increased at appropriate intervals in liquid culture without immobilizing the protoplasts. A method for regenerating calli from wasabi protoplast, which is performed while lowering. 2. The callus derived from the tissue of a plant belonging to the genus Wasabi of the Brassicaceae family is transformed into protoplasts, and is divided and propagated in liquid culture without immobilization of the protoplasts at appropriate intervals while gradually reducing the osmotic pressure in the medium. Callus regeneration method from wasabi protoplast, characterized in that callus is regenerated again. 3. The method for regenerating callus from horseradish protoplast according to (1) or (2), wherein the Wasabi genus plant of the Brassicaceae family is a plant regenerated from protoplast.