JPS5950285B2 - How to grow wasabi seedlings - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for mass breeding wasabi seedlings.

Conventionally, in the wasabi culture method, it has been extremely difficult to breed wasabi seedlings in large quantities due to the low multiplication rate of wasabi.

The present inventors conducted intensive research to improve this point, and as a result, they came up with a method for increasing the number of young wasabi seedlings in large quantities by performing callus formation and redifferentiation of wasabi plant tissue.

That is, the present invention comprises a first step of inducing callus by culturing tissue of a Brassicaceae plant belonging to the genus Wasabia in a callus induction medium containing kinetin, 2.4-D and thiamine at a low temperature of 20° C. or lower; A second step in which the induced callus is grown in a growth medium to form a callus, and a third step in which the callus is cultured in a regeneration medium containing coconut milk, redifferentiated (rooted), and grown into wasabi seedlings. This is a method for increasing wasabi seedlings that consists of three steps.

Callus culture of various plant tissues is currently being carried out, but callus induction of wasabi plant tissue has not yet been achieved.

The present inventors have conducted various studies based on the idea that if callus is formed from wasabi plant tissue, it is possible to increase the number of wasabi seedlings in large quantities by redifferentiating the callus.
When plant tissues of the genus Wasabia are cultured in a callus induction medium containing kinetin, 2.4-D and thiamine at a low temperature of 20°C or lower, preferably 40 to 15°C, 2.
- It was discovered that callus was induced after 3 months.

The present invention has been made based on this discovery.

The callus induction medium is a basic medium consisting of sugar, inorganic salts, and myo-inositol, kinetin, and 2°4D.

A medium containing thiamine and yeast extract is used, but it is extremely difficult to induce callus using this basic medium alone.

The basic medium is a medium used for normal plant tissue culture, consisting of sugars such as glucose and sucrose, and inorganic salts as shown below.

Inorganic salts used: Wasabi seedling tissue is excellent as a wasabi tissue used for callus induction.

This can be done using the sterile tissue of normal wasabi seedlings, but
To obtain stable quality at will, germinate and germinate dormant seeds.
It is desirable to have cultivated one.

If this method is adopted, the first step will be as follows.

1st step: Dormant wasabi seeds were immersed in an aqueous solution containing gibberellin for several days to awaken them from dormancy, then washed with 95% ethanol, and washed with 95% ethanol.
．． Sterilize the seeds by soaking them in 6% antiformin.

After washing, the seeds are sown on a normal sugar and inorganic salt medium, cultured in a cool, dark place, and germinated. After several months, seedlings are obtained.

Next, this seedling is placed on the callus induction medium and cultured in a dark place at a low temperature of 15° C. for several months, and callus induction begins.

It is necessary to induce callus in the first step at a temperature of 20°C or lower, preferably at a temperature of 10 to 15°C, and callus will not be induced at 30°C.

The second callus propagation step is a step in which the induced callus obtained in the first step is aerobically liquid cultured or solid cultured in a callus growth medium to produce proliferated callus (cultured cells).

The callus growth medium used in this step may be the same as the callus induction medium described above, but since coconut milk promotes callus growth, it is preferable to add 10 to 20% coconut milk to it. .

The desired growth temperature for callus is 10 to 15°C. At low temperatures, callus will not die even if frozen, but will simply slow down its growth. However, if cultured at 30°C, callus will turn black and will not grow. It stops.

Therefore, the second step, like the first step, must be performed at a temperature of 20° C. or lower.

It is desirable to perform callus growth culture under aerobic conditions.
The culture method may be either solid culture method or liquid culture method, but
From an industrial standpoint, liquid culture methods are preferred.

Calli grown using this liquid culture method are called cultured cells.
It has a wasabi-like flavor and is used as a wasabi-like flavoring agent after being separated and collected from the culture solution and dried and powdered as necessary.

In order to turn the thus obtained callus into wasabi seedlings for wasabi cultivation by farmers, a third step of redifferentiating the callus is required.

It is difficult to regenerate wasabi callus using a medium that is normally used for regenerating plant callus.

Therefore, when we investigated the active ingredients of the regeneration medium, we found that it was possible to easily regenerate callus by adding coconut milk to a normal plant callus regeneration medium.

That is, the third step, the wasabi regeneration step, involves using a wasabi callus regeneration medium, which is a normal plant callus regeneration medium prepared by adding kinetin or benzyladenine, etc. to a basic medium consisting of sugars and inorganic salts, and adding 10 to 20% coconut milk to the wasabi callus regeneration medium. , second
The proliferated callus (cultured cells) obtained in the process was placed on a bed at 20°C.
The process involves culturing at the following temperature to cause budding and rooting (buds are formed approximately 6 months after being placed on the bed and roots are formed at the same time), and further culturing under the same conditions for 2 to 3 months to obtain seedlings.

The wasabi seedlings obtained by the present invention have the same pungency as the parent plant, and by actually cultivating them in the fields of farmers according to the conventional method, wasabi plants with the same pungency as the parent plant can be obtained. It is used as young seedlings for cultivation.

Example I Wasabia jap'onica Matsu
Commercially available seeds of Daruma strain, a wasabi plant of the Brassicaceae family belonging to Mura 1. Seed dormancy awakening treatment was performed by immersing Ol in 20rnl of gibberellin A3 solution (concentration 100p1N+1) in a cool, dark place at 10°C for 4 days, and the seeds were washed once with 95% ethanol and further soaked in 0.6% antiformin solution. Sterilization was performed by soaking in 50WLl and shaking gently for 30 minutes.

After sterilization, the seeds were washed three times with sterilized water, and 15 of the washed seeds were sown on the basic medium (agar plate medium) shown in Table 1 and left in a cool, dark place at 10°C for 2 months.

As a result, 10 seedlings of about 1.0 cm were obtained.

Next, add these to kinetin, 0.2772! il/11ν
2.4-Dl, 0■/! 2 Thiamin・HC/1.0～
/! 2 yeast extract (manufactured by Difco) 4.0? /II
When placed on a test tube agar medium for callus induction containing the basic medium of Fufu Table 1 and cultured for 3 months in a cool, dark place at 10°C, callus induction begins.

When these were further cultured under the same conditions for 6 months, callus pieces (referred to as primary callus) each having a diameter of about 10 mm were obtained.

These 6 pieces of callus were further subcultured on the above-mentioned callus induction agar medium, and the callus was grown to 150 cm. (wet weight)
Proliferated callus (secondary callus) was obtained.

After subdividing the secondary callus into approximately 10 callus pieces, these subdivided secondary callus were mixed with coconut milk in the basic medium shown in Table 1 at 150 y/j! ls kinetin 1.0■/! were transplanted to a regeneration medium (agar plate medium) supplemented with Fufu, and redifferentiation culture was performed in a cool, dark place at 10°C.

As a result, about 110 buds were formed after about 6 months, and rooting began at the same time.

Further, 110 seedlings were obtained by culturing for 2 months.

When these young seedlings were randomly selected and cultured in a field according to conventional methods, wasabi parent plants with almost the same quality as wasabi plants obtained by ordinary farmers were obtained.

Example 2 Approximately 2.4F (wet weight) of proliferated callus (secondary callus) obtained in the same manner as in Example 1 was shaken in a flask with a liquid medium for callus growth (composition is the same as the medium for callus induction). Culture was performed.

In this flask shaking culture, the flask filling volume is 20ml.
/ 500yrtl flask, shaking width; 7Cffl, 1
The test was carried out for one month in a cool, dark place at 10°C under the conditions of 0.050 s c i 1 Fm.

When the cultured cells were separated and collected from the obtained culture solution, wasabi cultured cells with a wet weight of 18.07 cm were obtained.

This had a wasabi-like flavor.

The thus obtained wasabi cultured cells 101 (wet weight) were placed on the above-mentioned callus regeneration agar medium (however, benzyladenine was used at 1.0 μl/l in place of kinetin),
As a result of redifferentiation using the same method as in Example 1, nine seedlings were obtained.

Claims (1)

[Claims] 1 Cultivate plant tissue of the Brassicaceae family belonging to the genus Wasabia at a low temperature of 20°C or lower using a callus induction medium containing kinetin, 2.4-D and thiamine to induce and proliferate callus, and grow the proliferated callus. A method for increasing wasabi seedlings, which comprises regenerating them into seedlings using a regeneration medium containing coconut milk and kinetin.