JPH0585135B2 - - Google Patents

Description

[Detailed Description of the Invention] Industrial Application Field The present invention utilizes a tissue culture method to induce shoot primordia of useful plants, and uses the induced shoot primordia to multiply young seedlings in large quantities. Regarding the method.

BACKGROUND ART Seed production using tissue culture allows for the rapid supply of genetically cloned seedlings in large quantities, and has therefore been studied on many plants. The adventitious bud method and somatic embryo method have been studied for a long time as methods for producing seeds and seedlings by tissue culture, but these methods are difficult to reproduce in the process of mass propagation by tissue culture. There was a problem that many mutations occurred, making it impossible to obtain large quantities of high-quality seedlings. In recent years, in order to solve this problem, the shoot apex of annual plants such as Paplopatpus and perennial plants such as wasabi is extracted and grown into shoots by rotationally culturing them in a medium containing plant growth hormones under light irradiation. A so-called shoot primordium method for inducing primordia has been proposed (JP-A-59-132822, JP-A-59-132823, and JP-A-61-115417). This shoot primordium has high seedling ability and genetic stability.

However, in the above-mentioned shoot primordium method, the shoot apical growth point from which the shoot primordium is induced is very small, 0.2 to 1 mm, and requires skill to remove or sterilize. There was a problem that only one to a few apical growth points were obtained and the culture efficiency was very poor.

In addition, shoot primordia cannot necessarily be derived from the shoot apical growth point for all plants, and currently only about
Shoot primordia have been induced in only 50 plant species, and compared to somatic embryos, which have been induced in more than 130 plant species, this is a very small number of plants to which the shoot primordium method has been applied. was desired to expand.

Problems to be Solved by the Invention In view of the current situation, the present inventor conducted intensive research and surprisingly discovered that shoot primordia can be induced from somatic embryos other than shoot apical growth points.

The present invention has been made based on such knowledge, and it is possible to very easily induce shoot primordia from somatic embryos of plants, improve the culture efficiency thereof, and make the induced shoot primordia into seedlings to be useful. An object of the present invention is to provide a method for efficiently multiplying seedlings of plants in large quantities.

That is, the present invention provides a method that can expand the range of plants to which the shoot primordial method can be applied and supply large quantities of genetically stable seedlings of various useful plants.

Means for Solving the Problems The present invention involves inducing shoot primordia by rotationally culturing somatic embryos of useful plants under irradiation with light in a liquid medium, and statically producing the induced shoot primordia. It is characterized by propagating young seedlings by culturing them and turning them into seedlings.

The present invention is applicable to all plants in which somatic embryos can be induced, and in particular, Panax ginseng,
American ginseng, ginseng, codaceae, epimedium, wheat, barley, sugarcane, sorghum, sorghum, oat, rye, orchard grass, bamboo, soybean, alpha alpha, red clover, white clover, Japanese clover, melon, cucumber, tagashi, Chinese cabbage, rapeseed, Hanaya rhinoceros, Senkiyu, celery, tobacco, eggplant, potato, petiunia, morning glory,
Somatic embryos of plants such as sweet potato, coffee, papaya, corn poppy, fenugreek, grape, henbane, mandarin orange, orange, snapdragon, and strawberry can be used. Of course, methods for inducing shoot primordia from the shoot apical growth point have already been established, such as watermelon, pea, broad bean, etc.
For carrots, morning glories, lettuce, saladana, chisilla, crepis, daisies, cosmos, haplopoppus, rice, corn, dahlia, stevia, artemisia, porphyry, asparagus, asparagus, lily, garlic, wasabi, wasabi, eucalyptus, poplar, etc. can also be applied.

Methods for obtaining somatic embryos include inducing callus from plant explants, inducing somatic embryos after propagation, and inducing somatic embryos directly from plant explants without forming callus. However, in the present invention, somatic embryos induced by any method can be used, and somatic embryos propagated by secondary somatic embryos can also be used.

Somatic embryos are plant explants, such as roots, stems,
Leaves, flower buds, shoot apical growth points, anthers, pollen, as well as parts of plant tissues such as mericlon and multiple shoots, culture cultivation, protoplasts, etc., are treated with sodium hypochlorite and benzalkonium chloride as necessary. Or sterilize using a disinfectant such as ethanol,
After thorough washing with sterile water, the cells are adjusted to an appropriate size and cultured in a somatic embryo induction medium. The suitable medium for this somatic embryo induction medium differs depending on the type of plant, but the basic medium is Murashige, which is used for normal plant tissue culture.
Skoog medium, Linsmeyer-Skoog medium,
A white medium, Gamborg B5 medium, or a modified medium thereof, to which a plant growth hormone is added, if desired, is used. Note that the plant growth hormone used in this case is also suitable for different plants in different types, concentrations, etc., and should therefore be selected appropriately. Among the plant growth hormones, auxins include
2,4-dichlorophenoxyacetic acid, indoleacetic acid, indolebutyric acid or naphthaleneacetic acid, etc.
Examples of cytokinins include benzylaminopurine, kinetin, zeatin, isopentenyl adenine, and diphenylurea, and examples of diberlins include GA3 (GA3).
etc. can be exemplified.

Many methods for obtaining somatic embryos have already been reported for various plants (for example, "Plant Cell Culture" edited by Hiroshi Harada and Mu Komamine, p92, published by Rikogakusha, "Collection of Abstracts of the 1985 Plant Tissue Culture Symposium") ”
(See p. 161, Saga et al., etc.), so it goes without saying that these methods may also be used.

Somatic embryos induced by the above-mentioned method, particularly preferably globular embryos, heart-shaped embryos, torpedo-shaped embryos, or mature embryos, can be cultured by rotationally culturing these somatic embryos in a liquid medium under light irradiation. , to induce shoot primordia.

In this case, as a basic medium, Murashige-Skoog medium, Linsmeyer-Skoog medium, White medium, Gamborg B5 medium, or a modified medium thereof, which is used for normal plant tissue culture, can be used. Moreover, a plant growth hormone is added to this liquid medium as necessary.
In this case, examples of plant growth hormones include the above-mentioned auxins, cytokinins, and diberlins, which may be appropriately selected depending on the type of plant. The amount added in this case also differs depending on the plant, so it cannot be determined unconditionally, but it is preferably selected within the range of 0 to 100 mg/. The type and amount of plant growth hormone to be added can be determined efficiently by using the 25-square grid method.

On the other hand, the light irradiation is at the top of the rotation, from 5000 to
A setting of about 20,000 lux and 100 to 5,000 lux in the lower part is particularly preferable because induction of shoot primordia is promoted.

In addition, rotational culture is preferably performed at a rotation speed of 0.5 to 10 rpm because shoot primordia can be easily induced. Further, the culture temperature is appropriately selected within the range of 5 to 30°C.

When rotary culture of somatic embryos is performed using the method of the present invention,
A clump of shoot primordia is obtained. This color varies depending on the plant, but is generally pale green to dark green, or white to yellow. This shoot primordium initially has a smooth surface and a diameter of about 50-80 μm.
It occurs as a protuberance, and its constituent cells are uniformly small polygonal cells, and the cell division axis undergoes multiaxial division into stratified, parallel, and oblique layers. The shoot primordia in this state is the primary shoot primordium. This primary shoot primordium is
As the culture continues, the cells gradually increase in size until they reach approximately 100 μm in diameter and 60 cells, and then differentiate into two layers: epidermal and cortical. The outermost layer, the epidermal system, is a single cell layer, and the cell division axis is only parallel division; the inner cortex is a collection of many slightly larger cells, and within these cells are well-developed chloroplasts. vacuoles and storage substance granules are observed. This state is a secondary shoot primordium. When this secondary shoot primordium is further cultured, it becomes a trapezoidal bulge with a diameter of 200 μm, and then several new primary shoot primordia are generated around this. In this way, the shoot primordium continues to proliferate. When the shoot primordium grows to a size of about 15 mm, it can be divided into pieces of about 3 to 5 mm in diameter and subcultured, thereby allowing the shoot primordium to multiply in large quantities. In addition, if the culture conditions are appropriately adjusted, the cells can be spontaneously divided into aggregates of 3 to 5 mm. This subculture may be carried out by rotary culture using the same medium composition as the primary culture described above, but under irradiation with light, subculture may be carried out in an aerated agitation fermenter, an air lift fermenter, a roller bolt fermenter, or a seesaw type fermenter. It can also be carried out while forcibly stirring using a fermenter or the like.

The shoot primordium obtained as described above is transplanted to a medium for seedling formation and grown into seedlings by static culture. In this case, as a seedling culture medium, the above-mentioned basic medium may be appropriately added with the above-mentioned plant growth hormone, and the seedling formation may be carried out at a temperature of 8 to 25° C. under irradiation of light of 1000 to 4000 lux.

The present invention will be specifically described below with reference to Examples.

Example 1 (Induction of somatic embryos) Panax ginseng CAMey.
The flower buds were cut from the buds of the 5-year-old plants approximately 2 weeks after germination, and the flower stalks and flower sepals were removed. This was sterilized for 10 minutes with a 3% by weight aqueous sodium hypochlorite solution containing 0.1% by volume of Tween 20 (trade name), and then for 30 seconds with a 70% by volume ethanol solution. After washing it with sterile water, it was planted on a Murashige-Skoog agar medium supplemented with 5 ppm of 2,4-dichlorophenoxyacetic acid, and cultured stationary in the dark at a temperature of 25±1°C. Somatic embryo formation begins after 1 month of culture, and the somatic embryo formation rate after 3 months of culture is
It was 90%. This somatic embryo was transferred to a Murashige-Skoog agar medium supplemented with 1 ppm of 2,4-dichlorophenoxyacetic acid and allowed to grow.

(Induction of shoot primordium) Benzylaminopurine as a plant growth hormone
Murashige-Skoog liquid medium 10 supplemented with 1ppm
3 to 5 somatic embryos that had been induced to proliferate by the above method were placed in a 21 mmφ x 150 mm test tube and cultured in a rotary incubator. The culture temperature was 18±1°C, the light was continuously irradiated at 9000 lux at the top of the rotating incubator, and 200 lux at the bottom, and the rotation speed was 2 rpm.
Formation of shoot primordia was observed from day 113 of culture, and culture continued.
The shoot primordium formation rate in 223 days was 40%. This was subcultured and propagated under the same conditions.

(Seedling formation) A clump of approximately 5 mm in diameter of the induced propagation of the shoot primordia aggregate was placed on 10 ml of 1/2 strength Murashige-Skoog agar medium supplemented with 0.5 ppm of benzylaminopurine and 0.5 ppm of GA3 as plant growth hormones. 21 dispensed
It was placed in a mmφ x 150 mm test tube and cultured. The culture temperature was 18±1°C, and light was continuously irradiated at 200 lux. On the 46th day of culture, germination was observed from 100% of the shoot primordia, and on the 63rd day of culture, more than 10 shoots were obtained from one shoot primordium cluster with a diameter of about 5 mm.
These shoots were mixed with 1/4 strength Murashige and 1 ppm of indolebutyric acid as a plant growth hormone.
21mmφ×150mm with 10ml of Skoog agar medium dispensed
The cells were placed in test tubes and cultured. The culture temperature is 16
The temperature was ±1°C, and the light was 8000 lux for 16 hours/day. After one month of culturing, the seeds took root and were able to grow into seedlings.

Example 2 (Induction of somatic embryos) Carrot (Daucus carota Kuroda Gosun) seeds were immersed in a 3% by weight aqueous sodium hypochlorite solution to which 0.1% by volume of Tween 20 was added, the pressure was reduced with an aspirator for 5 minutes, and then the seeds were kept at room temperature. It was returned to pressure and sterilized by soaking for 20 minutes. The sterilized seeds were washed twice with sterilized water, then planted on Murashige-Skoog agar medium that does not contain plant growth hormones, irradiated with 2000 lux light for 16 hours/day at 25°C, cultured, and allowed to germinate. Ta. When the germinated sprouts are about 3 cm long,
The hypocotyl was adjusted to 1 cm in size, placed on a Murashige-Skoog agar medium supplemented with 1 ppm of 2,4 dichlorophenoxyacetic acid, and cultured in the dark at 25°C to induce callus. This callus was transplanted onto a Murashige-Skoog agar medium containing no plant growth hormones to form somatic embryos.

(Induction of shoot primordium) Benzylaminopurine as a plant growth hormone
Place 3 to 5 spherical or heart-shaped somatic embryos induced by the above method in a 21 mmφ x 150 test tube containing 1 ppm.
The cells were transplanted and cultured in rotation. The culture conditions are as in Example 1.
The same as Formation of shoot primordia was observed from day 30 of culture, and the shoots were subcultured under the same conditions to proliferate.

(Seedling formation) The shoot primordia induced and propagated as described above were placed in a 21 mmφ x 150 mm test tube into which 10 ml of 1/2 strength Murashige-Skoog agar medium containing no plant growth hormone was dispensed, and cultured. The culture conditions were the same as in Example 1 (seedling formation). Germination and rooting were observed from the shoot primordium after 30 days of culture, and the seedling formation rate was 100%.

Effects of the Invention As described above, since the present invention induces shoot primordia from somatic embryos, shoot primordia can be very easily produced even in plants where it is difficult to induce shoot primordia from the shoot apical growth point. This method not only improves culture efficiency by inducing the shoot primordia method, but also expands the range of plants to which the shoot primordium method can be applied, and has the effect of efficiently supplying genetically stable seedlings of various useful plants in large quantities. .

Claims (1)

[Scope of Claims] 1. A method for inducing shoot primordia of useful plants, which comprises rotatably culturing somatic embryos of useful plants in a liquid medium under irradiation with light. 2. A method for mass propagation of young seedlings, which comprises rotating somatic embryos of useful plants in a liquid medium under light irradiation to induce shoot primordia, which are then statically cultured to form seedlings.