JPH0335739A - Method for inducing seedling anlage of useful plant and method for proliferating young seedling from same induced seedling base at large amount - Google Patents

Abstract

PURPOSE:To simply and efficiently induce a seedling anlage by subjecting an adventive embryo of an useful plant to rotation culture in a liquid culture medium under irradiation of light. CONSTITUTION:An explant (e.g. root, leaf, etc.) of plant is sterilized and then subjected to rotation culture in an adventive embryo inducing culture medium to provide the adventive embryo. The adventive embryo is cultured in a plant tissue culturing medium such as Murashige and Skoog medium to which as necessary a plant growth hormone (auxins of 0-100mg/l) are added under irradiation of light (having 5000-20000lux in the upper part of rotation and 100-5000lux in the lower part) in 0.5-10rpm numbers of revolution at 5-30 deg.C to provide the aimed seedling anlage. Furthermore, the seedling anlage is grown till the size of about 15mm and the grown seedling anlage is divided into 3-5mm diameter size and subjected to subculture to proliferate the seedling anlage in large quantities and the anlage is transplanted in a culture medium for growing into seedling to which a plant growth hormone is added and then subjected to stationary culture under irradiation of light having 1000-4000lux at 8-25 deg.C.

Description

[Detailed Description of the Invention] The present invention utilizes a tissue culture method to induce shoot primordia of useful plants, and uses the induced shoot primordia to produce large numbers of young seedlings. Concerning methods for propagating.

Retire! Seed production by Li 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 the somatic embryo method have been studied for a long time as methods for producing seeds and seedlings through tissue culture, but in the process of mass propagation using U-identical culture, these methods There was a problem in that genetic mutations occurred frequently, 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 Baprobapus 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 has been proposed to induce primordia (Japanese Unexamined Patent Publication No. 132822/1982, 5
Publication No. 9-132823 and JP-A-61-115417
Publication No.). This shoot primordium has high seedling ability and genetic stability.

However, in the above-mentioned shoot primordium method, the shoot apical growth point that induces the shoot primordium is very small, 0.2 to 1 mm.
Skill is required for its removal or sterilization.
Depending on the plant, only one to a few shoots could be grown at the shoot apex, resulting in a very poor nutrient efficiency.

Furthermore, it is not necessarily possible to induce shoot primordia from the shoot apical growth point in all plants; at present, lineal primordia have been induced in only about 50 plants (but only 7, and that of somatic embryos). The number of plants induced by the shoot primordia method was very low compared to that of plants with a size of 1.130 fl or more, and there was a desire to expand the number of plants to which the shoot primordium method could be applied.

The L-engineer is also a division wing.

In view of the current situation, the present invention 4f conducted intensive research and surprisingly found that shoot primordia can be induced from somatic embryos other than shoot apical growth points.

The present invention was made based on this knowledge7.
Shoot primordia are very easily produced from somatic embryos of plants,
It is an object of the present invention to provide a method for efficiently propagating useful plant seedlings into large mice by converting the induced shoot primordia into seedlings 1- while improving the culture efficiency.

That is, the present invention provides a method for expanding the range of plants to which the 7-row primordium method can be applied, and supplying and obtaining genetically stable seedlings of various useful plants to Ogaki.

The present invention involves inducing shoot primordia by rotary culturing somatic embryos of useful plants in a liquid medium under light illumination;
2. By leaving the induced shoot primordia 4 still and turning them into seedlings. It is characterized by multiplying young.

1. The present invention is applicable to all plants from which somatic embryos can be induced, particularly Panax ginseng, American ginseng, Panax ginseng, and Aralia ginseng.

Epimedium, wheat, barley, sugar cane, juzdama, sorghum, oat, rye, millet grass,
2. Bamboo, soybean, alfalfa, red clover, white clover, cormorant, nutraceutical, melon, cucumber, tagashi, Chinese cabbage, absinna, Chinese cabbage, Chinese cabbage 5.
1, celery, tobacco, eggplant, potato, petunia,
Somatic embryos of plants such as morning glory, sweet potato, coffee, babio, poppy, fennel, grape, henbane, mandarin, orange, goldenrod, and strawberry can be used. Of course, from the shoot apical growth point, C,
A method for inducing shoot primordia has been established41, for example, watermelon, pea, fava bean, carrot, azalea,
1/Evening, Ten 1, Sarasasu, Cheshire, Clevis, Shungiku, Cosmos, Hablobugs, Rice, 1' Umo V
Saw, dahlia, stevia, a. It can also be applied to sorrel wogas, terrapokori, garlic, nebana, wasabi, yucalyptus, poplar, etc.

There are 7 methods for obtaining somatic embryos: Sewing calli from explants of plants; There are various methods for inducing somatic embryos, but in the present invention, which method 7ζ can be applied to self-expressing somatic embryos, and can also be applied to somatic embryos propagated by secondary somatic embryos. can.

Somatic embryos are plant explants, such as roots, stems, leaves, flower buds,
Shoot apical growth points, pollen, and even part of the plant tissue such as mericlon and fultips, cultured cells, protoplasts, etc., are treated with - sodium hypochlorite, etc., as necessary.
Penzalkonium chloride is sterilized using a disinfectant such as ethanol, washed thoroughly with sterilized water, prepared to an appropriate size, and cultured in a somatic embryo induction medium. Go.

Suitable somatic embryo induction media differ depending on the type of plant, but basic media include Murashige-Sukug medium, Linsmeyer-Sukug medium, which is used for normal plant tissue culture. Why I medium, Gumbo's B5 medium, or a modified medium thereof, to which a plant growth hormone is added, if desired, is used. The plant growth hormone used in this case also differs in type 5 and concentration depending on the plant, so it is best to select the plant growth hormone as appropriate. Among the auxins, 2.
4-dichlorophenoxyacetic acid, indoleacetic acid, indolebutyric acid, or naphthaleneacetic acid, etc.; cytokinins include benzylaminopurine, kinetin, zeatin, isopentenyladenine, diphenylurea, etc.; and diberlins include Jeanie three ( GA3) etc. can be exemplified.

Many methods for obtaining somatic embryos have already been reported for various plants (for example, "Plant Cell Culture" by Hiroshi Harada,
It goes without saying that these methods may also be used.

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

The medium in this case is a normal plant Mi
Murashige-Skoog medium, Linsmeyer-Skoog medium, White medium, Gamborg B5 used for culture
A medium or a modified medium thereof can be used. Moreover, a plant growth hormone is added to this liquid medium as necessary. Examples of plant growth hormones in this case include the aforementioned oxins, cytokinins, and diberlins, and may be appropriately selected depending on the type of plant. In this case, the amount added varies depending on the plant, so
-Cannot be determined generally, but O~100wg/
j! It is best to select within the range of . The 25-base method can be used efficiently to determine the type and amount of plant hormones to be added.

On the other hand, the light irradiation is at the upper part of the rotation, from 5000 to 20,0
It is particularly preferable to set the temperature to about 1,000 lux and 100 to 5,000 lux in the lower part, as this promotes the induction of shoot primordia.

Further, rotational culture is preferably performed at a rotational speed of 0.5 to 10 rp+* because shoot primordia can be easily induced.

Further, the culture temperature is appropriately selected within the range of 5 to 30°C.

When somatic embryos are rotary cultured using the method of the present invention, shoot primordia in the form of masses are obtained. This color varies depending on the plant, but
Generally, it is pale green to dark green, or white to yellow. This shoot primordium initially has a smooth surface and develops as a bulge with a diameter of about 50 to 80 μ-.The constituent cells are uniformly small polygonal cells, and the cell division axes are stratified and parallel. , the shoot primordium in this state, which undergoes multiaxial division of the oblique layer, is the primary shoot primordium. This next shoot primordium gradually increases in size by continuing the culture and has a diameter of 100 μm.
m. When the cells proliferate to about 60 cells, they differentiate into two layers: epidermal and cortical. The epidermal system of the outer layer of Jl is a single cell layer, and the cell division axis is only parallel division, and the inner cortex is a collection of many rather large cells, and within these cells are well-developed chloroplasts. , liquid bubbles, and storage substance granules are observed.

This state is a secondary shoot primordium. This secondary shoot primordium,
If the culture is continued, a trapezoidal bulge with a diameter of 200 .mu.m is formed, and several new primary shoot primordia are then generated around this bulge. In this way, the shoot primordium continues to proliferate. When this shoot primordium grows to a size of about 15+wm, it can be divided into pieces with a diameter of about 3 to 5 mm and subcultured, thereby allowing a large number of shoot primordia to grow. In addition, if the culture conditions are appropriately adjusted, it is possible to spontaneously divide into 3 to 5 agglomerates. This subculture may be carried out by rotary culture using the same medium composition as the primary culture described above, but it may be carried out in an aerated stirring fermenter, an air lift fermenter, a roller bottle fermenter, or a roller bottle fermenter under irradiation with light. It can also be carried out while forcibly stirring using a seesaw type 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, the seedling cultivation medium is prepared by adding the above-mentioned plant growth hormone to the above-mentioned basic medium, and growing it for 10 minutes at a temperature of 8 to 25°C.
It is preferable to perform this under irradiation with light of 00 to 4000 lux.

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

Dog-Example-1 (induction of somatic embryos) Panax ginseng (Pariax Binseng C,
Flower buds were cut from the buds of 5-year-old plants of A, Mey, 2 weeks after germination, and the pedicels and sepals were removed. Add this to Tmeen 20 (product name) to 0.1
10 minutes with a 3% by volume aqueous sodium hypochlorite solution, and then 30 minutes with a 70% by volume ethanol solution.
Sterilized for seconds. After washing it with sterile water, it was planted on a Murashige-Sukug agar medium supplemented with 5 pp (max) of 2,4-dichlorophenoxyacetic acid, and cultured stationary in the dark at a temperature of 2511°C. Somatic embryo formation started from the first month of culture, and the somatic embryo formation rate in three culture rows was 90%.

This somatic embryo was treated with 2,4-di-DO phenoxyacetic acid lpp.
The cells were transplanted onto a Murashige-Skoog agar medium supplemented with m and allowed to grow.

(Induction of shoot primordium) As a plant growth hormone
3 to 5 somatic embryos induced and propagated by the above method were placed in a 21++v+φX 150 mm test tube containing Murashige-Skoog liquid medium 10− supplemented with 10−, 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 vpm. Formation of shoot primordia was observed from 1130 days of culture, and the shoot primordium formation rate at 223 days of culture was 40%. This was subcultured under the same conditions and propagated (seedling formation). A mass of approximately 5 mm in diameter of the induced propagated shoot primordia aggregate was treated with hensylaminopurine 0.511 as a plant growth hormone.
11) A 4-strength Murashige-Skoog agar medium 10- supplemented with Ill and GA 3, Q, 5 ppm was dispensed into a 21 mmφ x 150 m5 test tube and cultured. The culture temperature was 18±1'c, and light was continuously irradiated at 200 lux. Germination was observed from 100% of the shoot primordia on day 46 of culture, and on day 63 of culture, more than 10 shoots were obtained from one of the acidic primordium aggregates with a diameter of about 5 openings. These shoots are treated with intlbutyric acid as a plant growth hormone.
A 1/4-strength Murashige-Sukug agar medium (taio-) supplemented with ppm was placed in a 21-diameter x 150 m5+ test tube and cultured. The culture temperature was 16±1°C, and the light was irradiated at 8000 lux for 16 hours.

It was possible to root and grow into seedlings within one month of culturing.

Dog grooming (induction of somatic embryos) Carrot (Daucus carota Kuroda Gosun) seeds are immersed in a 3% by volume sodium hypochlorite aqueous solution containing 0.1% by volume of Tween 20, and the pressure is reduced using an aspirator for 5 minutes. After that, the pressure was returned to normal, and the pressure was sterilized by soaking for 20 minutes. The sterilized seeds were washed twice with sterilized water, then planted on a Murashige-Skoog agar medium containing no 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 were about 3 cm long, the lower hypocotyl was adjusted to 1 cm, and 2,4-dichlorophenoxyacetic acid 11
) I) - was placed on a Murashige-Skoog agar medium supplemented with -, and cultured under sunny conditions at 25°C to induce callus.

This callus was transferred to a Murashige-Skoog agar medium containing no plant growth hormones to form JiiL7 somatic embryos.

(Induction of shoot primordium) Benzylanobulin lppm as a plant growth hormone
Three to five spherical or heart-shaped somatic embryos, which had been induced to proliferate by the above method, were transplanted into a 21 mm diameter x 150 old test tube, and rotary cultured.6 The culture conditions were 1. The same as in Example 1 was used. After 30 days of culture, the formation of shoot primordia was observed, and the cells were subcultured under the same conditions and multiplied.

(Seedling formation) The shoot primordia induced and multiplied above were placed on 4-strength Murashige-Skoog agar 10-
The mixture was placed in a 21 tube diameter x 150 mm test tube 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%.

As mentioned above, in the present invention, shoot primordia are induced from somatic embryos, so it is extremely easy to use even for plants in which it is difficult to induce shoot primordia from the shoot apical growth point. By inducing shoot primordia to improve culture efficiency, the shoot primordium method can be applied to a wider variety of plants, and genetically stable seedlings of various useful plants can be efficiently supplied in large quantities. be effective.

Claims (2)

[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. .