KR20050000279A - Method for production of seed Dioscorea opposita Thunb. by use of tissue culture technology - Google Patents

Abstract

PURPOSE: A method for production of virus-free seed of Dioscorea opposita Thunb. using tissue culture technology is provided, thereby inhibiting the tissue degradation of Dioscorea opposita Thunb. by virus infection during vegetative propagation, and mass-producing virus-free seed of Dioscorea opposita Thunb. CONSTITUTION: The method for production of seed Dioscorea opposita Thunb. using tissue culture technology comprises the steps of: obtaining excellent stem cuttings containing 1 or 2 gnarls by growing point cultivation and virus test; planting the excellent stem cuttings through cutting technique to acclimate the stem cuttings; supplying the stem cuttings with nutrient solution to grow a ground part; and producing the virus-free seed of Dioscorea opposita Thunb.

Description

Method for production of seed Dioscorea opposita Thunb. by use of tissue culture technology}

The present invention relates to a seed production method of round yam to fundamentally solve the virus infection problem that causes a decrease in production yield and quality deterioration, and more specifically, the tissue culture plant using tissue culture technology in the production of seed yam The present invention relates to a mass production method of disease-free cattle that inserts and purifies and swells at the same time to mass-produce sterile plants and then grow the sterile plants to produce seedlings.

Hemp is also known as "acid powder" and belongs to the genus (Dioscoreaceae). It is a crop that mainly breeds tuber by the tuber. It is a crop that shows degeneration due to viral infection during the process of vegetative breeding.

There are about 600 species of horses around the world, and about 10 species are used for food. Cultivated areas can be divided into Asia, Central Africa and Central America, of which 96% of the world's total production comes from Africa. Hemp is the most cultivated next to potato, cassava and sweet potatoes, and the highest daily yield is after potatoes.

Horses can be divided into rainy, short and round horses in the form of tuber roots. The horses produced in Korea are mainly used for medicinal purposes and are used as some health foods, but some introduced horses have a high content of irradiated or internally As it is purple, it is necessary to review demand development for various uses, such as the use of health food additives or food coloring such as bread and sweets.

As with most trophic crops, the damage caused by the virus is severe in the "horse", which leads to a decrease in crop yield and quality deterioration. Since such viral diseases cannot be solved by pesticides, it is true that no significant measures have been proposed until now.

A method of producing seedlings using plant tissue culture technology, which has been developed so far, can be classified into four categories:

1) By rainy season (Dioscorea opposita Thunb. Cv. Nagaimo):

(Hajime Araki et al. 1992. Japan.Soc. Hort. Sci. 60 (4): 851-857)

Shoot induction: modified MS medium (nitrogen level decreased 1/10, NAA 0 ~ 0.1mg / l, BA 0.1mg / l)

Callus induction: NAA 1.0 mg / l Callus is a form similar to shoot primordium

Shoot regeneration: MS medium, BA 0-0.1 mg / l, NAA 0.01-0.1 mg / l

2) Dioscorea composita Hemsl:

(S. Alizadeh et al. 1998. Plant Cell, Tissue and Organ Culture 53: 107-112)

In-flight sterile plants were treated with 2, 8 and 10% sucrose concentrations and 2.5 uM of kinetin to produce in-flight globules.

3) Purification of four hemp plants in the genus Dioscorea :

(H.N. Asemota et al. 1997. Top.Agri. (Trinidad) 74 (3): 243-247)

99% survival when grown in mixed soil and sand soil at 1: 1 ratio after 4 weeks of incubation in rooting medium at 75 ± 2 ℃

4) Round horse ( Dioscorea opposita Thunb.):

(T. Okamoto et al. 2001. J. of Japan.Crop Science 70 (2): 179-185)

Cultivation material: Fertilization by extracting the tip of round horse grown in greenhouse

Superculture: Cultured by adding NAA 0.01mg / l and BAP 0.1mg / l to 1/2 LS medium

Subculture: When the leaves are re-differentiated 60 days after the start of culture, and 4 to 5 nodes grow, the nodes of the in-flight plant including liquid solution are cultured. The cultured seedlings were purified for 2 weeks, and then cultivated in a box filled with 30 liters of culture soil.

In general, the production of horseshoe-free cattle begins with cultivation of the growth point of the hemp plant, and forms stems, and the stems are subcultured in the culture vessel, and the stems are produced by cutting, or the cultured stems are cultured. Different conditions may be used to produce microtubers. However, these methods are small scale research in a laboratory such as a Erlenmeyer flask or a test tube, and mass production by these methods is difficult, and in particular, the development of tissue culture technology using round hemp has been attempted, but the results have not been achieved.

In the case of applying the tissue culture technology developed so far to the round hemp, the stems that have undergone the culture culture propagation stage after the growth point area with little viral infection density in the plant are collected in order to prevent the yield reduction caused by the virus infection. In general, it is possible to breed as a young woman running on the soil or nutrient propagules or above ground, but in the case of round horses, the young woman is rarely produced, so breeds only by the method of nutrition propagation. In the case of round horses, degeneration is caused by viral infection during the breeding process, which reduces the yield.

Accordingly, a first object of the present invention is to provide a tissue culture system showing a high proliferation rate capable of producing disease-free cattle in large quantities using tissue culture technology with round hemp material.

A second object of the present invention is to provide a method for producing disease-free cattle by inserting sterile plants derived from the tissue culture system of the present invention.

A third object of the present invention is to find a method for producing microtubers directly in the cabin.

The ultimate object of the present invention is to fundamentally solve the virus infection problem that leads to a decrease in production yield and quality, thereby suppressing and / or preventing tissue degeneration caused by viral infection in the propagation process, thereby lowering the growth rate in the package As a breed of round horses, it provides a way to mass-produce disease-free cattle.

In order to prevent such a decrease in yield, the present inventors collected a growth point having almost no virus infection density among round horse plants, and then produced disease-free seed through a tissue culture propagation step to mass-produce disease-free cattle as a breed of round horses. The present invention has been completed.

Figure 1a and Figure 1b is a process diagram schematically showing the process of producing a yam of round hemp using tissue culture according to a preferred embodiment of the present invention,

Figure 2a and Figure 2b is a photograph showing the harvesting time and post-harvest view of the seed of the round yam produced by the interpolation method in accordance with a preferred embodiment of the present invention, respectively,

Figure 3a and Figure 3b is a photograph showing the appearance of the in-flight and in-flight after the harvest of round horses produced in accordance with a preferred embodiment of the present invention, respectively.

According to the present invention, a method for producing round horse disease-free seedling is first obtained by culturing growth points and virus assay, and obtaining fine seedling seedlings that have been mass-produced. Producing a step, feeding the nutrient solution to a formal seedling seedlings to grow the ground portion, and the step of producing a sima (cow, 우). The present invention provides a method for mass-producing disease-free cattle as propagules of round hemp with low growth rate in the package.

When explaining the round horse disease-free seed production method according to the present invention in detail:

Step 1: obtaining basic stem

The juvenile growth point of round hemp is collected and cultured to obtain basic stem. At this time, the seedlings were harvested from the seedlings generated from the upper to sixth to seventh nodes when the stems were planted with 9 to 10 stems, and the culture was carried out using MS medium which is generally used for plant tissue culture. It is carried out using a medium in which fluprimidol is added to a medium in which the concentration of the nitrogen source in the composition is reduced to 1/2, and a base stem is obtained from about 6 weeks after the induced stem elongation. Incubation is also performed under conditions of an illumination cycle with a temperature of 25 ° C. ± 1 ° C., illuminance of 3,000 lux or more and a ratio of light and dark to 16: 8 (hours).

Step 2: propagation of the basic stem

The basic stem obtained in the previous section is cut by 1 to 2 nodes, and this is incubated in stem kidney medium for 5 weeks to extend the stem. These elongated round-bottom plants were then subcultured every six weeks. Culture conditions are the same as the culture conditions of the first step.

Step 3: Production of Bovine Cattle by Trapation of Tissue Cultured Plants

Plant stems mass-proliferated in Petri dishes are cut so as to include at least one node, secure a seedling seedling, planting it on the top soil and cultivating for 3 to 5 months to harvest disease-free cattle.

Step 3-1: Production of Inflight Tools:

Mass-proliferated disease-free plants are cultured in in-flight induction medium and inflight production medium to produce round-bottom incubation. At this time, it is preferable to incubate for 4 to 6 weeks in the incubation induction medium, 8 to 10 weeks in the incubation medium. Round globule induction medium contains 6-benzyl aminopurine (BA), indole acetic acid (IAA), and paclobutrazol (paclobutrazol). The incubation medium does not contain plant growth regulators.

Cultivation is carried out under conditions of an illumination cycle with a temperature of 20 to 28 ° C. and a ratio of light and dark to 12:12 (hours).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention according to the gist of the present invention is not limited by the following examples.

Example 1 Induction and Proliferation of Basic Plants

After extracting round hemp slices into pots, the liquid growth point was collected from the elongated stems of the plants, and cultured medium (825 mg / L ammonium nitrate, 950 mg / L potassium nitrate, 370 mg / L magnesium sulfate, 170 mg / L dihydrophosphate). Potassium salt, 0.83 mg / L potassium iodide, 6.2 mg / L boric acid, 15.1 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 0.25 mg / L sodium molybdate dihydrate, 0.025 mg / L copper sulfate pentahydrate pentahydrate), 0.025 mg / L cobalt chloride hexahydrate, 3.671 mg / L EDTA, 100 mg / L myo-inositol, 0.5 mg / L nicotinic acid, 0.5 mg / L pyridoxine Incubated with pyridoxin) -HCl, 0.5mg / L thiamine-HCl, 2mg / l glycine, 0.5-2.0mg / L flupyprimidol, pH 5.7-5.8 After enzymatic immunoassay, individuals not infected with the virus were selected.

Selected individuals were incubated for 5 weeks in a medium to which 1.0 to 2.0 mg / L BA was added instead of fluprimidol included in the electric culture medium, followed by extension, and growth medium (825 mg / L ammonium nitrate, 950 mg / L potassium). nitrate, 185mg / L magnesium sulfate, 85mg / L potassium dihydro phosphate, 0.415mg / L potassium iodide, 3.1mg / L boric acid, 7.52mg / L manganese sulfate, 4.3mg / L zinc sulfate, 0.125mg / L sodium molybdate dihydrate , 0.0125mg / L cupric sulfate pentahydrate, 0.0125mg / L cobalt chloride hexahydrate, 1.835mg / L EDTA, 100mg / L myo-inositol, Nitsch vitamin, pH 5.7∼5.8) Was 6 to 7 times.

Example 2 Production of Disease-Free Cows by Spine

Production of round hemp disease-free cattle of the present invention may be carried out by preparing a seedling seedling so that 1 to 2 nodes of the plants grown in the cabin are included and planting them on top soil.

Example 2-1: Sliding Rate According to Treatment of Rooting Seedling Root of Tissue Cultured Plants

As a result of immersing and inserting a commercial rooting promoter (trade name; oxyberine) into the distal end of the prepared seedling seedlings, the lubrication rate increased more than about 13% compared to the control.

TABLE 1

Enhancement of Sliding Rate by Insertion Treatment of Tissue Cultured Plants

repeat Active rate (normal growth number / total population) IBA processing No treatment One 88.4% (107/121) 70.5% (62/88) 2 87.0% (100/115) 89.7% (113/126) 3 86.7% (130/150) 62.1% (100/161) Average 87.4 ± 0.9 (337/386) 74.1 ± 14.1 (275/375)

Example 2-2 Growth Comparison of Carrot Seedlings According to Hormones and Treatment Methods

As a result of comparing hormones on seedling seedlings by treatment type and treatment method, immersion in IBA (trade name: Oxyberine) showed the best results with the height of 14.9cm and the number of branches 2.9.

TABLE 2

Comparison of Growth according to Hormone Types and Treatments in Tissue Cultured Plants

process Extra long (cm) Number of branches () T 1 6.48 ± 1.91 1.1 ± 0.3 T 2 3.43 ± 1.55 0.8 ± 0.8 T 3 9.02 ± 2.42 1.9 ± 0.6 T 4 11.12 ± 4.48 2.5 ± 0.7 T 5 14.88 ± 8.08 2.9 ± 1.3

T1: no treatment,

T2: 1/8 MS medium + IBA 5 mg / l + Kinetin 2 mg / l + IAA1 mg / l + NAA1 mg / L after inoculation into the soil

T3: IBA (trade name Oxyberine) 2mg / ℓ dilution after irrigation in the soil after cutting

T4: 2 hours soaked in 10 mg / l of IAA

T5: 2 hours immersion in 5mg / l of IBA (brand name Oxyberine)

Example 3: Production of In-flight Tool

In-flight induction medium (1650 mg / L ammonium nitrate, 1900 mg / L potassium nitrate, 370 mg / L magnesium sulfate, 170 mg / L potassium dihydro phosphate, 0.83 mg / L potassium iodide, 6.2 mg) / L boric acid, 15.1mg / L manganese sulfate, 8.6mg / L zinc sulfate, 0.25mg / L sodium molybdate dihydrate, 0.025mg / L cupric sulfate pentahydrate, 0.025mg / L cobalt chloride hexahydrate, 3.671mg / L EDTA, 100mg / L myo-inositol, 0.5mg / L nicotinic acid, 0.5mg / L pyridoxin-HCl, 0.5mg / L thiamin-HCl, 2mg / l glycine, 0.5-1.0mg / L benzyl aminopurine, 0.5-1.0mg / L indole After incubation for 5 weeks in acetic acid, 0.1 ~ 0.5mg / L paclobutrazol, pH 5.7 ~ 5.8), incubation medium (1650mg / L ammonium nitrate, 1900mg / L potassium nitrate, 370mg / L magnesium sulfate, 170mg / L potassium) dihydro phosphate, 0.83mg / L potassium iodide, 6.2mg / L boric acid, 15.1mg / L manganese sulfate, 8.6mg / L zinc sulfate, 0.25mg / L sodium molybdate dihydrate, 0.025mg / L cupric sulfate pentahydrate, 0.025mg / Incubated in L cobalt chloride hexahydrate, 3.671mg / L EDTA, 100mg / L myo-inositol, Nitsch vitamins, pH 5.7∼5.8) for more than 8 weeks.

At this time, the in-flight produced in the cabin 3 to 5 per stem of the plant body was formed to produce 15 to 25 inflight per one petri dish.

According to the present invention, by providing a method of producing disease-free cattle as a breeding body of round horses with low growth rate in the package, the production efficiency of the round horses can be dramatically increased and the possibility of mass production that can meet the increasing demand of round horses It is expected to be realized.

Claims (4)

In the round horse disease-free seed production method, Obtaining high-growth fine seedlings grown through growth point culture and virus assay, Formulating and purifying the fine seedling by the cutting method; Feeding the nutrient solution to the formal seedling seedlings to grow the ground portion; And Round horse disease-free seed production method comprising the step of producing a cow. The method of claim 1, wherein the cutting method comprises the steps of preparing a seedling seedlings to include 1 to 2 nodes of the plants grown in the cabin; And Round horse disease-free seed production method comprising the step of immersing and inserting the rooting promoter in the prepared end of the seedling seedlings. The method of claim 1 or 2, wherein the rooting promoter is oxyberine, immersed in oxyberine 2 to 10 mg / l diluent for 2 hours, and then irrigated on topsoil. In the production method of round hemp diseased seedling, after collecting round hemp slices in a pot, liquid growth points are collected from the elongated stems of plants that appear, and then subcultured in culture medium, and then grown in large quantities through growth point culture and virus assay. Obtaining an insert; Producing globules in Petri dishes using plants grown on board; Growing the globules; And Round horse disease-free seed production method comprising the step of producing a cow.