KR20050089235A - A method for culturing isolated microspore of pepper - Google Patents

Abstract

The present invention relates to cultivation of red pepper vesicles, blending medicines taken from pepper buds and vortexing to extract vesicles, removing somatic tissue debris from the outflowed vesicles and harvesting only vesicles by centrifugation. The treated vesicles were pretreated with A medium and pretreated at 30-32 ° C. for 3 days, and the pretreated vesicles were densified with NLN medium at a density of 80,000-100,000 microspore / ml, and then cultured for 3-4 weeks in a dark state of 25-27 ° C. It is characterized by obtaining a ship.

The present invention can obtain a large amount of embryos immediately after the callus period from a semi-cellular endoplasmic reticulum in a single cell state, as well as in the field of studying embryonic development or differentiation of cells, as well as in the field of plant biotechnology such as haploid breeding, vesicle mutation, and vesicle transformation. It can be very useful for.

Description

A method for culturing isolated microspore of pepper

The present invention relates to a microspore culture method of red pepper ( Capsicum annuum L.), and more specifically to obtain a large amount of embryos (callus) immediately from the semi-aqueous vesicles in a single cell state without passing the callus period. It relates to a method for culturing the vesicles.

In general, plants are left behind through sexual reproduction in which the egg cells of the backpack and the sperm cells of the pollen are combined. In rare cases, the egg cells or sperm cells develop and form embryos, and these plants often have a normal number of chromosomes. It is called the haploid because it corresponds to half of the individual. Since these haploids can artificially double the number of chromosomes, such as drug treatment, it is possible to easily generate genetically pure strains.

In the past, mainly anther culture has been used as a method for obtaining haploids, but as it becomes possible to obtain a large amount of pears by vesicle culture in rapeseed, studies on vesicle cultivation in many plants have been made. In the culture of medicine, somatic tissue (walled tissue) and endoplasmic reticulum (follicle) vesicles are mixed, so there is a possibility that somatic cell-derived pears and vesicle pears may be mixed, and the growth of medicinal wall tissues may interfere with the generation of vesicles. But there is no such concern when culturing the vesicles. In addition, unlike the cultivation of vesicles, the starting material is a single cell population, so it is very advantageous for transduction and mutant abandonment. In the case of rapeseed, the rate of embryonic development is about 60 times higher than that of drug culture (Swanson, 1990).

However, despite these advantages, it was difficult to obtain pears or callus when only the vesicles were separated and cultured. To date, plants that can obtain large quantities of pears due to relatively easy culturing of vesicles are limited to a few plants such as wheat (Datt, 2001), barley (Datta, 2001) and tobacco, including rapeseed (Baillie et al., 1992). In most plants, even if the vesicle cultivation is not successful or successful plants, the embryo development rate is very low and cannot be used for researches for mutant induction, transformation or embryonic development. In Korea, some research institutes and seedling companies are carrying out drug culture to produce haploids.

The cultivation of red pepper sperm was made by Austrian Regner (1996) and Spain by Gonzalez-Melendi group (1995, 1996), but so far many pears have been cultivated by vesicle cultivation. No reports have been obtained, only a few multinucleic vesicles have been obtained. Recently, Barany et al. (2001) carried out vesicle cultivation according to the method of “Gonzalez-Melendi” et al. (1996) to obtain seedlings by inducing torpedo-type and cotyledonous embryos. A short report was published, but there were no pictures of developing embryos, such as torpedoes or cotyledons. A few polynuclear bodies and one or two germinated embryos appear to have been obtained.

Therefore, the present invention can obtain a large amount of pears by culturing the vesicles out of the red pepper, and can obtain plants from the embryos with cotyledons among the obtained pears, as well as the basic field of study of cell differentiation, haploid breeding To provide a vesicle cultivation method of pepper that can be utilized for development of excellent varieties by mutant breeding, vesicle transformation, and the like.

In order to achieve the above object, the method of culturing the red pepper scavenger according to the present invention, after blending the medicine taken from the red pepper buds and vortexed out the vesicles, and removing the somatic tissue debris from the outgoing vesicles by centrifugation Only the vesicles were harvested, and the harvested vesicles were pre-treated in A medium for 3 days at 30-32 ° C., and the pre-treated vesicles were harvested in NLN medium at a density of 80,000-100,000 microspore / ml, followed by cancer at 25-27 ° C. It is characterized in that the embryo is obtained by culturing for 3-4 weeks in the state.

Sucrose is suitable as a carbon source added to the NLN medium, and a preferable concentration is 6-9%.

The pretreatment medium is added to the medicine (anther) collected from the red pepper buds, and then blended at a high speed of about 17,000 to 19,000 rpm using a micro-blender, and then vortexed at a high speed. Eject the vesicles. Blending the parcels at high speed not only shortens the time out, but also increases the viability of the parcels. Other methods for extracting the vesicles include floating methods for harvesting and culturing the vesicles released into the medium as the drug is dehiscence, agitation in a liquid medium, glass rod homogenizer or There is a method of using a pestle maceration to heat buds or medicine. However, the floating method is difficult to apply because the pepper is not easy to open the medicine, the stirring method was also very low harvesting of the vesicles, the buds and the medicine was not well warmed, and the viability of the outgoing vesicles (viablility) is preferable Not.

As the pretreatment medium, A medium (Hoekstra, 1997) (medium composition see Table 1) in terms of embryogenic and cotyledonal embryogenicity. Somatic tissue debris was removed from the extracted vesicles and centrifuged to obtain only the vesicles, and then subjected to high-temperature pretreatment at 30-32 ° C. for 3 days on the A medium.

After the pretreatment, NLN medium (see Table 4 for medium composition) is preferred as the dentate medium, and sucrose is effective as a carbon source in terms of generation of torpedo-type embryos or cotyledon embryos, and its concentration (g / L) It is suitable to add 6-9%.

It is preferable that the densities of the vesicles pretreated in the dentate medium are 80,000 to 100,000 microspore / ml. If the densities are lower than the above ranges, cotyledon-time embryos do not occur, spherical embryos and torpedo-shaped embryos are lowered, and the densities are in the above-described ranges. If higher, spherical or early heart-shaped embryos are more likely, but the embryos do not grow.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited by the examples.

Example 1

(1) Growth of seedlings

Seeds of red pepper (Milyang) were sown in plastic pots with a diameter of 25 cm and transplanted one by one into a pot of 30 cm diameter when 4 weeks old. The temperature was 25 ° C., the photoperiod was 16/8 hours, and the plants were grown in a growth room maintained at 15,000 to 20,000 Lux using fluorescent lamps and metal lamps.

(2) Collection and sterilization of timely buds

About 1/4 to 3/4 of the purple buds were collected. These colored pills contain late mononuclear vesicles or early binuclear pollen. After sterilization for 7 minutes in 2% sodium hypochlorite solution and washed 4 to 5 times with sterile water.

(3) outing of the packager

In the micro-blender (Waring blender, 8575 micro container), approximately 30 buds were taken, and 4 ml of pretreatment medium (A medium, medium composition is shown in Table 1), followed by 5 seconds at a high speed of 18,000 rpm. Blended twice. After blending, the vesicles were ejected by vortexing twice at 20 seconds at high speed using a Vortex Genie 2 Mixer. Figure 1 is a photograph showing the outgoing vesicles obtained by blending and vortexing the medicine taken from the bud of the pepper. In Example 2 to investigate the effect of the pretreatment medium, two kinds of medium A and B5 medium to which 13% sucrose was added (see Table 2 for the medium composition) were used.

The micro-blender and pretreatment medium were stored in the refrigerator and used.

(4) harvesting and washing of small parcels

The extracted vesicles were filtered through 75 μm and 38 μm sieves to separate somatic tissue fragments and vesicles, and then centrifuged at about 1000 rpm for 5 minutes. It was then washed three times for 5 minutes at about 1000 rpm with pretreatment medium.

(5) pretreatment of the parcel

After adjusting the hemocytometer using a hemocytometer so that the tooth density is about 2x10 ⁵ microspore / mL in the pretreatment medium, dispense 9-12ml in a 90x20 mm petri dish, seal it with parafilm, and prevent drying. Into a 140 × 20 mm Petri dish containing filter paper soaked with sterile water, sealed with parafilm and treated at 32 ° C. for 3 days. The photo of the vesicles changed embryonically after this pretreatment is shown in FIG.

(5) washing of pretreated parcels

The pretreated vesicles were collected in a 50 ml centrifuge tube, centrifuged, and washed once with a dentate medium.

(7) the denture of the parcel

Tooth densities (NLN medium, media composition see Table 4) to adjust the densities to 80,000 to 100,000 microspore / ㎖ and dispensed 1 ml each in a 35 x 15 mm petri dish and sealed with parafilm. All media were used with a filter of 0.22 μm.

In Example 3, in which the influence of the type of medium was examined, three mediums of A2, MMS3, and AMC in addition to the NLN medium (see Tables 5 to 7 for composition) were used. In Example 4, which examined the effect of the type and concentration of carbon source in the medium, 6, 9 and 12% of sucrose and maltose were added, respectively. In Example 5 to investigate the effect of the densities in the culture was cultured by varying the densities from 4x10 ⁴ microspore / ㎖ to 16x10 ⁴ microspore / ㎖.

(8) Cultivation of vesicles

Petri dishes containing vesicles were placed in a generally used transparent plastic box, and then incubated at 27 ° C. for 3-4 weeks in the dark. Figure 3 is a photograph showing the multinucleated vesicles generated after one week of culture, Figure 4a is a photograph showing the vesicle fold magnified 20 times under a dissecting microscope after three weeks of culture.

(9) Conversion of pear seedlings

After 4 weeks of culture, cotyledonary embryos were transferred to embryonic medium (B5 solid medium containing 3% sucrose) and cultured in a low temperature incubator at 25 ° C. for 16/8 hours. At this time, the brightness was about 1,500 Lux using a fluorescent lamp.

As described above, according to the method for culturing the vesicles of the present invention, a large amount of pears were obtained and seedlings as shown in FIG.

Example 2 Analysis of Influence of Pretreatment Medium

The effect of pretreatment medium on the development of pear and callus in the vesicle culture of red pepper was investigated. Except for using B medium (Gambor et al., 1968) and A5 having the following composition as the pretreatment medium, NLN medium added with 10% sucrose was used, and the densities were approximately 100,000 microspore / ml. It carried out by the same method as Example 1. The results are shown in Table 3. As can be seen in Table 3, the incidence of pears was higher in A medium than in B5 medium, and cotyledon embryos occurred only in A medium.

Composition of A Badge ingredient Content (mg / L) CaCl ₂ 1,110 MgSO _{4 7} H ₂ O 246.47 KNO ₃ 101.10 KH ₂ PO ₄ 27.22 KI 0.17 CuSO ₄ · 5H ₂ O 0.025 Manitol 67.41 g

Composition of B5 Medium ingredient Content (mg / L) State nutrient KNO ₃ 2,500 MgSO _{4 7} H ₂ O 250 (NH ₄ ) ₂ SO ₄ 134 NaH ₂ PO ₄ · H ₂ O 150 CaCl ₂ · 2H ₂ O 150 Micronutrients   KI 0.75 H ₃ BO ₃ 3.0 MnSO ₄ 7H ₂ O 10.0 ZnSO ₄ · 7H ₂ O 2.0 Na ₂ MoO ₄ 2H ₂ O 0.25 CuSO ₄ · 5H ₂ O 0.025 CoCl ₂ · 6H ₂ O 0.025 Iron ^a FeSO ₄ 7H ₂ O 27.8 Na ₂ · EDTA 37.2 Vitamins and AA   Nicotinic Acid (B3) 1.0   PyridoxineHCl (B6) 1.0   Thiamine HCl (B1) 10.0   Myo-Inositol 100.0 Sucrose (when pretreated) 13% Sucrose (when germinated) 3% Agar (at germination) 0.8% pH 5.8 to 6.0

^a FeSO ₄ · 7H ₂ O and Na ₂ · EDTA are dissolved in distilled water by heating and stirring constantly. Mix both solutions and adjust to final volume by adding water (adjust to pH 5.5).

Effect of Pretreatment Medium on the Development of Pears and Callus in Vesicle Culture of Pepper Pretreatment badge Number of pears or callus per plate ^a Spherical boat Torpedo boat Cotyledons Abnormal times Callus A badge 23.0 ± 3.2 12.3 ± 3.0 3.8 ± 1.7 12.0 ± 3.4 7.7 ± 2.1 B5 badge 11.8 ± 2.5 2.8 ± 1.1 0.0 ± 0.0 4.7 ± 3.6 4.2 ± 1.6

^a number is mean (n = 4) ± standard deviation

Data are averaged from three independent experiments.

Example 3 Analysis of the Effect of Dental Media

The effect of alveolar medium on the development of pear and callus in the vesicle culture of red pepper was investigated. Four types of densities were used: NLN medium and MMS3 medium (Hu & Kasha, 1997), AMC medium (Kunz, 2000), and A2 medium (Touraev et al., 1996). A medium was used as the pretreatment medium, and the tooth density was about 100,000 microspore / ml. The results are shown in Table 8. As a result, the vesicle pears occurred mainly in NLN medium, and only a few spherical or heart-shaped pears were produced in other medium, but no cotyledon pears occurred.

NLN medium composition ingredient Content (mg / L) State nutrient KNO ₃ 125 MgSO _{4 7} H ₂ O 125 Ca (NO ₃ ) ₂ .4H ₂ O 500 KH ₂ PO ₄ 125 Micronutrients MnSO ₄ 4H ₂ O 22.3 H ₃ BO ₃ 6.2 ZnSO ₄ · 7H ₂ O 8.6 Na ₂ MoO ₄ 2H ₂ O 0.25 CuSO ₄ · 5H ₂ O 0.025 CoCl ₂ · 6H ₂ O 0.025   KI 0.83 Iron ^a FeSO ₄ 7H ₂ O 27.8 Na ₂ · EDTA 37.2 vitamin   Myoinositol 100   Nicotinic Acid (B3) 5   PyridoxineHCl (B6) 0.5   Thiamine HCl (B1) 0.5 Folic acid ^b 0.5   Glycine 2   Biotin 0.05 Amino acid supplements   Glutamine 800   Glutathione 30   Serine 100 Sucrose 10% pH 5.8 to 6.0

^a FeSO ₄ · 7H ₂ O and Na ₂ · EDTA are dissolved in distilled water by heating and stirring constantly. Mix both solutions and adjust to final volume by adding water (adjust to pH 5.5).

^b Folic acid and biotin are dissolved in 0.1N NaOH, respectively.

MMS3 Medium Composition ingredient Content (mg / L) State nutrient KNO ₃ 1,400 NH ₄ NO ₃ 300 KH ₂ PO ₄ 170 MgSO _{4 7} H ₂ O 370 CaCl ₂ · 2H ₂ O 440 Micronutrients   KI 0.83 MnSO ₄ 7H ₂ O 22.3 H ₃ BO ₃ 6.2 ZnSO ₄ · 7H ₂ O 8.6 Na ₂ MoO ₄ 2H ₂ O 0.25 CuSO ₄ · 5H ₂ O 0.025 CoCl ₂ · 6H ₂ O 0.025 Iron ^a FeSO ₄ 7H ₂ O 27.8 Na ₂ , EDTA, 2H ₂ O 37.3 vitamin   Myoinositol 100   Thiamine HCl (B1) 0.4   PyridoxineHCl (B6) 0.5   Nicotinic Acid (B3) 0.5 amino acid   Glutamine 500 maltose 9% pH 5.8 to 6.0

^a FeSO ₄ · 7H ₂ O and Na ₂ · EDTA are dissolved in distilled water by heating and stirring constantly. Mix both solutions and adjust to final volume by adding water (adjust to pH 5.5).

AMC Badge Composition ingredient Content (mg / L) State nutrient KNO ₃ 1,000 (NH ₄ ) ₂ SO ₄ 100 KH ₂ PO ₄ 200 MgSO _{4 7} H ₂ O 125 Ca (NO 3) ₂ 4 H ₂ O 100   KCl 35 Micronutrients   KI 0.8 MnSO ₄ 7H ₂ O 4.4 H ₃ BO ₃ 1.6 ZnSO ₄ · 7H ₂ O 1.5 Iron ^a FeCl ₃ 27.0 Na ₂ · EDTA 37.3 vitamin   Thiamine HCl (B1) 1.0 amino acid   Glutamine 1,000   Serine 100 maltose 9% pH 5.8 to 6.0

^a FeSO ₄ · 7H ₂ O and Na ₂ · EDTA are dissolved in distilled water by heating and stirring constantly. Mix both solutions and adjust to final volume by adding water (adjust to pH 5.5).

Composition of A2 Medium ingredient Content (mg / L) State nutrient KNO ₃ 1,415 (NH ₄ ) ₂ SO ₄ 231.5 KH ₂ PO ₄ 200 MgSO _{4 7} H ₂ O 92.5 CaCl ₂ · 2H ₂ O 83 Micronutrients   KI 0.375 MnSO ₄ H ₂ O 5.0 H ₃ BO ₃ 1.5 ZnSO ₄ · 7H ₂ O 1.0 NaMoO ₄ 2H ₂ O 0.125 CuSO ₄ · 5H ₂ O 0.0125 CoCl ₂ · 6H ₂ O 0.0125 Iron ^a FeSO ₄ 7H ₂ O 27.8 Na ₂ , EDTA, 2H ₂ O 37.3 vitamin   Myoinositol 50   Thiamine HCl (B1) 5.0   Nicotinic Acid (B3) 0.5   PyridoxineHCl (B6) 0.5 amino acid   Glutamine 500 MES ^b 200 maltose 9% pH 5.6 to 6.0

^a FeSO ₄ · 7H ₂ O and Na ₂ · EDTA are dissolved in distilled water by heating and stirring constantly. Mix both solutions and adjust to final volume by adding water (adjust to pH 5.5).

^b MES: [2- (N-norpolyno) ethanesulfonic acid]

Effect of Dense Medium on Pear and Callus Development in Pepper Cultivation badge Number of pears or callus per plate ^a Spherical boat Torpedo boat Cotyledons Abnormal times Callus NLN 9.6 ± 2.0 4.8 ± 2.3 3.6 ± 1.5 2.4 ± 1.1 4.8 ± 1.9 A2 2.5 ± 0.5 2.0 ± 0.6 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 MMS3 2.0 ± 0.7 1.0 ± 0.4 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 AMC 1.0 ± 0.5 2.0 ± 0.7 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

^a number is mean (n = 4) ± standard deviation

Data are averaged from three independent experiments.

Example 4 Analysis of the Effect of Types and Concentrations of Carbon Sources in the Medium

The effect of the type and concentration of carbon source in the medium on the embryo development was investigated. Except for the addition of 6, 9 and 12% maltose, which is known to be effective for embryonic development in sucrose and other plant vesicle cultivation as carbon sources, A medium was used as a pretreatment medium, and NLN medium containing 10% sucrose was used. , Tooth density was about 100,000 microspore / ㎖ was carried out in the same manner as in Example 1. The results are shown in Table 9. Only maltose was added, but only a few spherical pears occurred. Torpedo pears or cotyledon pears occurred only in the medium containing sucrose. Sucrose concentration of 9% was best for torpedo cultivars and cotyledon pears. When 12% of sucrose was added, spherical pears were generated, but torpedo-type and cotyledon pears were less likely to grow. The growth of pears was also slower, which was much smaller than that of 6% or 9% of sucrose. Also, if you add less than 6% sucrose, the pear does not grow properly. Therefore, sucrose is suitable as a carbon source in the cultivation of red pepper vesicles, and its concentration is preferably 6-9%.

Effect of Types of Carbon Sources and Their Concentrations on the Development of Pears in Cultivation of Red Pepper Party density(%) Number of pears per plate ^a rectangle Torpedo type Cotyledon abnormal Sucrose 6 8.6 ± 1.1 11.4 ± 1.1 5.2 ± 0.8 5.2 ± 0.8 9 17.2 ± 2.0 14.8 ± 1.6 5.8 ± 1.3 7.6 ± 1.6 12 18.8 ± 1.2 9.2 ± 1.7 2.2 ± 0.7 7.3 ± 1.2 maltose 6 2.0 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 9 2.5 ± 0.6 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 12 1.0 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

^a number is mean (n = 4) ± standard deviation

Data are averaged from three independent experiments.

Example 5 Analysis of Influence of Dental Density

The effect of dentate density on embryo development was investigated in the culture of red pepper vesicles. Except that the densities of the vesicles were changed in 6 steps from 4x10 ⁴ microspore / ml to 16x10 ⁴ microspore / ml, A medium was used as the pretreatment medium, and NLN medium containing 10% sucrose was used. It carried out by the method. The results are shown in Table 10. As denture density increased, spherical embryos developed more frequently. The incidence of cotyledonary embryos was highest at 10x10 ⁴ microspore / ml, followed by 8x10 ⁴ microspore / ml, and similar at 12x10 ⁴ microspore / ml and 14x10 ⁴ microspore / ml. Cotyledon embryos did not occur at 4x10 ⁴ microspore / ml, which had the lowest tooth density or 16x10 ⁴ microspore / ml, which was the highest. When the densities were more than 12x10 ⁴ microspore / ml, most of the heart-shaped embryos were torpedo-type embryos and the cotyledon or abnormal embryos were very small. Once these as can be seen in the results was the dentate density of 4x10 ⁴ microspore / ㎖ a doubling of the cotyledons period did not result in a lower low and the generation of older ships and torpedo-shaped fold, 12x10 ⁴ microspore / ㎖ or more higher in older times or earlier Cardiac embryos are more common but do not grow. Therefore, the densities suitable for the culture of red pepper vesicles are 8x10 ^{4 to} 10x10 ⁴ microspore / ml.

Effect of Dense Density on Embryonic Development in Pepper Cultivation of Red Pepper Tooth density (microspore / ml) Number of pears per plate ^a Spherical boat Torpedo boat Cotyledons abnormal ⁴ x 10 ⁴ 2.0 ± 0.5 6.0 ± 1.0 0.0 ± 0.0 0.0 ± 0.0 8 x 10 ⁴ 6.0 ± 1.0 7.3 ± 0.5 3.8 ± 0.6 1.7 ± 0.5 10 x 10 ⁴ 22.0 ± 2.5 21.0 ± 3.1 5.5 ± 1.7 5.5 ± 1.2 12 x 10 ⁴ 20.0 ± 3.4 18.3 ± 2.2 1.7 ± 0.5 4.5 ± 1.6 14 x 10 ⁴ 42.3 ± 2.0 19.0 ± 5.0 1.6 ± 0.5 16.3 ± 2.3 16 x 10 ⁴ 53.7 ± 5.5 22.0 ± 3.6 0.0 ± 0.0 21.3 ± 4.7

^a number is mean (n = 4) ± standard deviation

Data are averaged from three independent experiments.

As described above, the pepper scavenger culturing method according to the present invention can directly obtain a large amount of pears from the semi-aqueous vesicles in a single cell state without passing the callus period, and obtain plants from cotyledon-developed pears among the obtained pears. Can be.

Therefore, unlike the conventional breeding that takes a long time to grow a variety by using a haploid or double haploid produced by the vesicle culture as well as a method for culturing the red pepper vesicles according to the present invention Good varieties can be grown in a short time. In addition, callus, which has been used for mutant induction up to now, is a double-celled multicellular population, but since the vesicle culture is a semi-aquatic single-cell population, the mutant organic source can act directly on all the vesicles. By this, not only can be made into diploid homozygotes, but also useful recessive mutations can be selected and used. First of all, by using the vesicles with high embryogenicity to transform the vesicles, it is possible to produce transformants in an efficient and innovative way.

Figure 1 is a photograph showing the outgoing vesicles obtained by blending and vortexing the medicine taken from the bud of the pepper.

Figure 2 is a photograph showing the vesicles changed embryogenically after pretreatment according to the present invention.

Figure 3 is a photograph showing the multinucleated vesicles generated after one week of culture according to the present invention.

Figure 4a is a photograph showing the vesicle fold magnified 20 times under a dissecting microscope after three weeks of culture in accordance with the present invention.

Figure 4b is a photograph showing the vesicle pear of Figure 4a contained in a petri dish.

Figure 5 is a photograph showing the seedlings obtained by germinating cotyledon pear embryos among the pears obtained according to the method of culturing the red pepper vesicles of the present invention.

Claims (3)

After blending the medicine taken from the red pepper buds, vortex to extract the vesicles, remove the somatic tissue fragments from the extracted vesicles, and centrifuge to harvest only the vesicles, and harvest the collected vesicles in A medium to 30 to 32 After pretreatment at 3 ° C. for 3 days, the pretreated vesicles were densified in NLN medium at a density of 80,000 to 100,000 microspore / ml and then cultured for 3 to 4 weeks in 25 to 27 ° C. dark state to obtain embryos. Method of culturing spores. The method of claim 1, wherein sucrose is added to the NLN medium as a carbon source. The method of claim 2, wherein sucrose is added 6 to 9%.