JPH0351377B2 - - Google Patents

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a mass propagation method for obtaining clonal propagates and disease-free seedlings in a short period of time by culturing the shoot apex of the genus Butterbur, and is applicable to agriculture. , can be used for tissue culture, etc.

[Problems to be solved by conventional technology and invention]

Butterbur (Petasites japonicus) grows wild in Japan,
It is widely cultivated. However, since the cultivated species is triploid, it is sterile and cannot produce seeds. Therefore, propagation is carried out only by division. As a result, the number of virus-infected plants has increased, resulting in poor growth and uneven growth, resulting in a decline in yield and quality, which has become a major problem. As a countermeasure, attempts are currently being made to produce disease-free seedlings using shoot tips, but a method for clonal propagation and supplying disease-free seedlings in a short period of time has not yet been found.

[Means to solve the problem]

Therefore, the present inventors made earnest efforts to solve the above problems, and as a result, they transplanted the shoot tips of the genus Butterbur to an artificial medium containing inorganic salts and plant growth hormones, and then cultured them under rotation under lighting. We have discovered a method for clonal propagation and disease-free seedlings in a short period of time by inducing, maintaining, and propagating the plants and converting them into plants on a fixed medium.

That is, the gist of the present invention is to remove the shoot apex of the genus Butterbur, transplant it to an artificial medium containing inorganic salts and plant growth hormones, and perform rotational culture under illumination to initially induce shoot primordia. Thereafter, the shoot primordium is transplanted to an artificial medium containing a different type and/or concentration of plant growth hormone from the artificial medium and subcultured, thereby allowing for continuous propagation. It consists in a production method using shoot tip culture.

The shoot primordia propagated by the method of the present invention are clumps of dividing cells that are genetically extremely stable, have a high multiplication rate, and easily convert into plants. That is, the present invention is a type of mass propagation method using vegetative reproduction, and also makes it possible to produce disease-free seedlings by using the shoot apex of the genus Butterbur.

[Effect]

The present invention will be explained in further detail.

After sterilizing the stem including the shoot apex of the genus Butterbur, the stem apex is removed and transplanted to an artificial medium containing inorganic salts, plant growth hormones, and carbon atoms. Next, rotational culture under illumination is performed to induce shoot primordia.

The lower limit of illumination intensity is 2000 for rotating culture under the above illumination.
The conditions include lux, upper limit of 10,000 lux, temperature of 18 to 28°C, and rotation speed of 0.5 to 5/min.

In static culture, shoot primordia cannot be continuously propagated even if they are induced, and it is necessary to perform initial induction of shoot primordia by rotational culture.

Examples of the above inorganic salts include Murashige-skoog, Gamborg,
A medium having a composition such as White can be used.

The above plant growth hormones include indoleacetic acid, α-naphthaleneacetic acid, etc. as auxins.
As the cytokinin, 6-benzylaminopurine, kinetin, etc. can be used, but it is preferable to use at least one type of auxin in combination with at least one type of cytokinin.

Artificial media for this initial induction include, for example, 6-benzylaminopurine 1 to 6-benzylaminopurine as cytokinin.
3ppm, α-naphthaleneacetic acid 0.2 as auxin
~2.0 ppm and 1-5% sucrose are used.

The induced shoot primordia initially has a pale red shape with many small protrusions.

Approximately one month after the initial induction, the artificial medium at the time of the initial induction is different from the type of plant growth hormone and/or
Alternatively, the initially induced shoot primordium is transplanted to a new, separate artificial medium containing different concentrations of inorganic salts and plant growth hormones, and subculture is performed for continuous propagation. It is necessary to change the type and/or concentration of plant growth hormone in the artificial medium used for this continuous propagation compared to the artificial medium used for initial induction; if this is not changed, the morphology will change and callus will form. Among the means for changing the type and/or concentration of plant growth hormones, the most effective is to use both in combination, keeping the concentration of cytokinin unchanged and reducing the concentration of only auxin. Through this continuous multiplication, the shoot primordium gradually becomes larger and becomes a pale green clump. When the shoots have grown to an appropriate size (approximately 5 mm to 10 mm), they are transplanted to the same medium containing only the plant growth hormone, and the shoot primordia are propagated as before. Thereafter, by periodically dividing and transplanting into new culture medium, it is possible to maintain and proliferate semi-permanently.

As an artificial medium for subculturing, for example, 6-benzylaminopurine 1 to 6-benzylaminopurine is used as cytokinin.
3ppm, alpha-naphthaleneacetic acid as auxin
Those containing 0.02 to 0.2 ppm and 1 to 5% sucrose are used.

The shoot primordia obtained in this manner is multiplied in large quantities and then transplanted to a fixed medium (stationary culture), whereupon a leaf primordium is formed at the tip and the shoot differentiates and grows into a plant body having stems and leaves. After that, after gradual acclimatization, it can be grown outdoors. The number of chromosomes in the obtained shoot primordia and regenerated plants is stable at 2n = 90 (X = 30).

〔Example〕

Cut the stem, including the apex of the butterbur, into approximately 1 cm pieces and sterilize them by immersing them in a 0.1% benzalkonium chloride solution for 5 minutes, and then in a 1% sodium hypochlorite solution for 5 minutes. It was extracted and used as planting material. The artificial liquid medium used was Murashige-Skoog medium, with 3% sucrose and 6% plant growth hormone.
-Benzylaminopurine and α-naphthaleneacetic acid were added to concentrations of 0, 02, 0.2, or 2 ppm, respectively.

The pH of the medium was adjusted to 5.7 to 5.8.

Add 25 ml of each medium to a test tube (27 x 200 mm)
The solution was then sterilized in an autoclave at 121°C for 15 minutes. Plant the shoot tip aseptically in the prepared medium,
24-hour illumination (lower limit of illuminance 2,000 to upper limit of 10,000 lux),
Rotary culture at a temperature of 22 (±2) °C (1 min 2
rotation). After about 1 month, 6-benzylaminopurine 2ppm, α-naphthaleneacetic acid 2ppm
In this combination, pale red shoot primordia with many small protrusions were obtained. Other combinations resulted in callus and early branching. At this point, 6-benzylaminopurine 2 ppm, α-naphthalene acetic acid
By transplanting to a medium containing a plant growth hormone concentration of 0.02 ppm, it was possible to maintain and propagate shoot primordia. It gradually grew and became a pale green shoot primordium cluster with many small protrusions. From then on, it continued to grow by dividing and transplanting regularly.

Conversion from shoot primordia to plants was performed by static culture on a fixed medium.

As a fixed medium, 1% sucrose and 0.9% agar were added to 1/2 diluted Murashige Skoog inorganic salt composition.
The pH adjusted to 5.7 to 5.8 was used. The culture conditions at this time were 16 hours of light, 1 to 2,000 lux lighting, and a temperature of 22
It was (±2)°C.

When the shoot primordium was planted in the prepared medium, it continued to proliferate for about one week, and then a green leaf primordium was formed at the tip and gradually grew into a plantlet with stems and leaves. Approximately 30-45
Rooting was observed on the second day, and after acclimatization on the rock wool, the plants were transferred to outdoor cultivation.

This plant had the same number of chromosomes as the parent plant, ie, the strain from which the shoot apex was removed, 2n = 90 (X = 30), and no variation was observed in morphology.

The growth rate of shoot primordia is approximately 7% by weight per month.
~8 times as much. Regarding the conversion of shoot primordia to plants, approximately 500 plants were obtained from 1 g of shoot primordia. In other words, more than 2,000 seedlings can be obtained from one stem tip in one year. In addition, as a result of testing using a known method, no virus was detected and the seedlings were disease-free.

〔Effect of the invention〕

By using the method of the present invention, uniform seedlings can always be stably supplied by guiding and maintaining shoot primordia and converting them into plants even in the sterile species of the genus Butterbur, for which there is no propagation method other than division. In addition, costs can be reduced by producing a large amount of plants in a short period of time.

Furthermore, without seedlings, it is possible to increase yields, make quality uniform, etc., and the effects are tremendous.

Claims (1)

[Claims] 1. Initial induction of shoot primordia, which are clonal propagators, by removing the shoot apex of the genus Butterbur, transplanting it to an artificial medium containing inorganic salts and plant growth hormones, and culturing it in rotation under illumination. After that, the shoot primordium is transplanted to an artificial medium containing a different type and/or concentration of plant growth hormone from the above-mentioned artificial medium, and subcultured to allow continuous propagation. Production method using shoot apical culture of the genus Butterbur. 2. The production method according to claim 1, characterized in that the plants are transplanted to an artificial medium of the same composition in which only the type and/or concentration of the plant growth hormone is changed, and repeated multiplication is repeated. 3. The production method according to claim 1, wherein the shoot primordium is initially induced, sustained propagation is carried out, and the shoot primordium is further transplanted to a fixed medium to differentiate and grow into a plant body. 4 Remove the stem tip of the genus Butterbur and transplant it to an artificial medium containing at least one type of auxin and at least one type of cytokinin as inorganic salts and plant growth hormones, and place it under illumination of 2,000 to 10,000 lux.
After initial induction of shoot primordia, which are clonal propagators, by rotary culture at a speed of 0.5 to 5 rotations/min, the shoot primordia are grown in the above-mentioned artificial medium with different types and/or concentrations of plant growth hormones. 1. A production method using shoot tip culture of the genus Butterbur, which is characterized by successive propagation by transplanting to different artificial media and subculturing. 5. The production method according to claim 4, characterized in that the plants are transplanted to an artificial medium having the same composition in which only the type and/or concentration of the plant growth hormone is changed, and repeated multiplication is repeated. 6. The production method according to claim 4, characterized in that the shoot primordium is initially induced, sustained propagation is carried out, and this is further transplanted to a fixed medium to differentiate and grow into a plant body.