KR20160046999A - Callus induction medium, shoot induction regeneration medium for Chrysanthemum anther culture, and preparing method of Chrysanthemum haploid plantlet by anther culture using the same - Google Patents

Abstract

The present invention relates to a medium for callus induction for culturing a Dendranthema grandiflorum anther, a differentiation medium for shoot induction, and a method for preparing a Dendranthema grandiflorum haploid plant by anther culture using the same. As a haploid plant can be converted into a diploid, to be efficiently used for a variety breeding material by combination of various pure lines, a medium for callus induction capable of culturing Dendranthema grandiflorum anther, a differentiation medium for shoot induction, and a method for preparing a Dendranthema grandiflorum haploid plant by anther culture using the same are provided. According to the present invention, before culturing Dendranthema grandiflorum anther, as Dendranthema grandiflorum anther is pre-treated at low temperatures and cultured in a medium having a specific composition, while a callus differentiation rate and a shoot induction rate of Dendranthema grandiflorum anther are improved, a Dendranthema grandiflorum haploid plant can be prepared.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a callus induction medium, a shoot induction regeneration medium, and a preparing method of Chrysanthemum haploid plantlet anther culture using the same}

The present invention relates to a callus induction medium for cultivation of chrysanthemum, a differentiation medium for shoot induction, and a method for producing chrysanthemum haploid plant by drug culture using the same, and more particularly, The present invention relates to a callus induction medium used for the production of haploid plants, a differentiation medium for shoot induction, and a method for producing chrysanthemum haploid plants by drug culture using the same.

Drainage refers to the phenomenon that the number of chromosomes of any organism is a multiple of that of an ordinary individual, and is commonly found in plants. For example, in the case of mulberry, the basic number of chromosomes is 14, which is referred to as monoploid (semi-hydrophobic), biplot or triplet depending on the number of chromosomes overlapping.

Among them, the number of chromosomes in the normal population of half the plant is called haploid (haploid). This haploid can artificially double the number of chromosomes by medicinal treatment, so that genetically pure lines can easily be created. On the other hand, since the haploid plant does not exist in pairs, there is no relationship between dominance and hunger, and all traits are present.

More specifically, the haploid body can be obtained by culturing a male gametophyte or a magnetic gametophyte in a cabin. Haploid production can generally be accomplished in a shorter period of time compared to the method of breeding a tearing crop, and it can be applied to crop breeding to develop a child line for F1 seed production or express a feisty trait Haploid material is useful for geneticists and breeders by showing the possibility of interbreeding with different mobility. It is advantageous to obtain a haploid plant and perform systematic and scientific breeding using the haploid plant.

An anther culture has been used to acquire haploids. Anchor cultivation means culturing a microspore contained in anther wall, which is somatic tissue.

For example, Patent Document 1 is directed to a method for producing transgenic rice in a short period of time using an agar culture and a transgenic technique. More particularly, the present invention relates to a method for transforming a haploid callus derived from a rice plant, The present invention discloses a method for producing stable transgenic rice in a short period of time by eliminating the cell mutagen. According to this, transformed rice can be produced by transforming and regenerating haploid callus derived from a drug, and thus it can be usefully used for the development of a new variety of rice.

As another example, Non-patent reference 1 relates to a haploid plant produced through the cultivation of a flowerpot of a peony, and the effect of the low temperature pretreatment (5 ° C / 10 days) on the change of the cultured flower buds and the callus and embryo development And the survival rate of pollen is disclosed.

On the other hand, chrysanthemum is a very popular crop that is cultivated mainly as cut flower, differentiation, and perennial flower plant in the world, and its market is rapidly increasing because of its commercial and ornamental value. Many studies have been conducted on the transformation of chrysanthemum using the regeneration ability of plants to improve the quality, quality and genetic characteristics. However, chrysanthemum is a hybrid plant, and genotypes are widely distributed throughout the genome. Therefore, systematic cultivation of varieties by a combination of various pure line systems is insufficient. Therefore, it is required to study the efficient mass production of the pure system which can be used as a breeding model in chrysanthemum.

Korean Patent No. 10-1212058 (Published on March 15, 2011)

Callus and Embryogenesis by Pollen Cultivation of Peony Pellets, Young Sook Kim, Byung Ki Lee, Ji-Won Lee, 22 (1) 13-17 1598-6365

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems, and an object of the present invention is to provide a callus inducing medium for cultivation of chrysanthemum, a method of producing a chrysanthemum haploid plant .

To this end, the present invention relates to a process for preparing a pharmaceutical composition comprising 0.002 to 0.02 part by weight of 6-benzylaminopurine (6-BA) and 2,4-dicholorophenoxyacetic acid per 100 parts by weight of sucrose. acid in an amount of 0.001 to 0.01 part by weight based on the total weight of the culture medium for inducing callus induction.

According to a preferred embodiment of the present invention, the callus inducing medium may further comprise 0.25 to 2.5 parts by weight of casein hydrolyzate based on 100 parts by weight of sucrose.

Another embodiment of the present invention is a method for producing a microcapsule comprising 0.004 to 0.02 parts by weight of 6-benzylaminopurine (6-BA) and 0.0002 to 0.001 part by weight of naphthaleneacetic acid (NAA) per 100 parts by weight of sucrose , ≪ / RTI > wherein the culture medium contains MS medium.

Another aspect of the present invention is to isolate the chrysanthemum anther from the bud of chrysanthemum bud, inoculate the chrysanthemum drug into a callus induction medium, and then incubate at 20-25 ° C for 10-20 hours A step of irradiating fluorescence and culturing for 35 to 60 days to induce callus; The callus induced in step 1 is cultured in a differentiation medium at 25 to 27 DEG C for 10 to 50 days to induce shoot;

Culturing the shoots in a rooting medium at 25 to 27 ° C for 10 to 50 days to produce roots; And a fourth step of screening the haploid plant from the horsetail. The present invention also provides a method for producing a mums haploid plant by drug cultivation.

According to a preferred embodiment of the present invention, the chrysanthemum anther may include a microparticle, and the microparticle may be a uninucleate stage to 2 nucleus.

According to a preferred embodiment of the present invention, the chrysanthemum flower bud is washed with water for 15 to 20 minutes, immersed in an aqueous ethanol solution for 10 to 50 seconds, immersed in sodium hypochlorite for 15 to 30 minutes, washed with water for 3 to 10 minutes And the surface is sterilized by washing.

According to a preferred embodiment of the present invention, the chrysanthemum drug inoculated on callus induction medium in step 1 may be incubated after being kept in a dark room for 24 to 48 hours at 3 to 5 캜 in a sealed state.

According to a preferred embodiment of the present invention, the callus induction medium may be pH 5.6 to pH 6.0.

According to a preferred embodiment of the present invention, the first step may be performed for 30 to 40 μmol / m ² / s for 10 to 15 hours and then for 35 to 45 days.

According to a preferred embodiment of the present invention, the callus induction medium is prepared by adding 0.002 to 0.02 part by weight of 6-benzylaminopurine (6-BA) to 2,4-dihydropyrimidine group per 100 parts by weight of sucrose, , And 0.001 to 0.01 part by weight of dichlorophenoxyacetic acid (2,4-dicholorophenoxyacetic acid).

According to a preferred embodiment of the present invention, the callus inducing medium may further comprise 0.25 to 2.5 parts by weight of casein hydrolyzate based on 100 parts by weight of sucrose.

According to a preferred embodiment of the present invention, the calli induction of the first stage may be performed at 60 to 95%.

According to a preferred embodiment of the present invention, the differentiation medium is prepared by adding 0.004 to 0.002 part by weight of 6-benzylaminopurine (6-BA) to 100 parts by weight of sucrose and adding 0.004 to 0.002 part by weight of naphtaleneacetic acid , NAA) in an amount of 0.0002 to 0.001 part by weight.

According to a preferred embodiment of the present invention, the shoot-induction of the two stages may be performed at 60 to 85%.

According to a preferred embodiment of the present invention, the rooting medium may include MS medium containing 0.0002 to 0.001 part by weight of naphthaleneacetic acid (NAA) relative to 100 parts by weight of sucrose.

According to a preferred embodiment of the present invention, the Chrysanthemum morifolium Ramat. Varieties may include Yes morning.

According to the present invention, there is provided a method for preparing a haploid plant capable of improving the callus differentiation rate and shoot induction rate of a chrysanthemum drug by pretreating it at a low temperature before culturing the chrysanthemum drug and culturing it in a medium having a specific composition.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an image showing a method for producing a chrysanthemum haploid plant according to an embodiment of the present invention; FIG.
Fig. 2 is an image showing the chromosomes observed in the root end cells of the yeast Morning variety chrysanthemum.
FIG. 3 is an image showing a chromosome observed in root tip cells of a haploid plant according to the present invention. FIG.

The terms used in the context of the invention, "chrysanthemum (Chrysanthemum)" is generally the bottom of the stem mokjilhwa about 1 m height and the leaf is displaced has a divergent form with gitkkol. Chrysanthemum is the oldest flower of oriental plant cultivated in the Orient, widely cultivated for ornamental purposes and has many horticultural varieties. Chrysanthemums are not only the largest part of the domestic flower industry except roses, but also used as ornamental, edible and insecticides. Chrysanthemum is a hybrid plant, and the degree of heterogeneity of genotypes throughout the genome is very high. Therefore, there is a problem that systematic variety breeding by combination of various pure line systems is insufficient.

The term "anther" as used in the specification of the present invention is one of the most important factors for the formation of seed and the deletion of fruit. The reproductive process of the higher plants consists of two firearms, ie, surgery and pistil. The surgery consists of medicine and surgery, including pollen, and the medicine is differentiated from the cool tissue of the firearms. Surgical tissues of the spleen produce tissues that form the drug from each of the three glands, and each tissue develops into reproductive function (microspore, pollen formation) and non-reproductive function (supporting tissue, tissue organization). The pollen developed from the microspores divides into nutritional nuclear cells and reproductive cells, and is released into the air through the process of opening the pollen sachet. In the plant, the male organ is a drug containing the operating table and pollen, and the development of various tissues and cells in the drug eventually plays an important role in the production and scattering of the pollen. When the medicine in the anther is cultured, the drug can divide and form a complete plant through morphological changes such as embryogenesis. In this case, the chromosome of this plant is only half of the somatic cell like flowerpot, If it is a haploid plant.

As used herein, the term "polyploid" means the number of chromosome assemblies of an organism cell. Drainage is monoploid (semi-permeable), diploid, and triple. The number of chromosomes in chrysanthemums is 9 and the number of chromosomes in somatic cells (2n) is 18, 36, 54, 72, and 90. Generally, the cultivated countries generally have diploids based on hexaploid (2n = 54), and they have an average of 56 chromosomes

The term "haploid " as used in the specification of the present invention means a cell or an individual having the number of chromosomes (n) of the diploid body produced by meiosis. There is a pair of basic chromosomes consisting of half a number, and its nuclear image is called half of it. Half the number of chromosomes as a result of meiosis in nuclear alternation becomes half generation. The half-generation creature returns to the renaissance with crystal. In half-life creatures, athletes are only zygotes, and generational shifts do not appear.

As used herein, the term "callus" is an undifferentiated amorphous cell mass, which is a typical tumor tissue formed by a cleavage tissue around a wound when a plant is injured. Plants are composed of dividing tissues that are largely dividing cells and permanent tissues that are not. When cells of the first dividing tissue are put in nutrient medium, callus is formed. Thereafter, the callus is formed into a somatic embryo, from which it can be differentiated into plants. Callus is therefore often referred to as "plant stem cells. &Quot;

The term " regeneration "as used in the specification of the present invention means a process of regenerating a plant having a full function from the organ or cell of a plant. In the technology of culturing a plant cell, There is no ability to grow to have. However, by changing the cultivation conditions, the roots of irregular buds or roots are formed, which is the starting point for regeneration of the plant.

Hereinafter, the callus inducing medium for cultivating chrysanthemum according to the present invention, the differentiation medium for shoot induction, and the method for producing chrysanthemum haploid plant by drug culturing using the same will be described in detail.

The present invention relates to a process for the preparation of a pharmaceutical composition comprising 0.002 to 0.02 part by weight of 6-benzylaminopurine (6-BA) and 2,4-dicholorophenoxyacetic acid per 100 parts by weight of sucrose. And 0.001 to 0.01 part by weight of a culture medium for inducing callus induction for cultivating chrysanthemums.

The callus induction medium is to provide a nutrient composition for callus differentiation from a chrysanthemum drug, and the MS medium is a basic medium for propagating callus, which generally has a content of NO ₃ -N, NH ₄ -N and K Which is advantageous for growing plant cells. The 2,4-dicholorophenoxyacetic acid plays a role in elongation and division of cells. The 6-benzylaminopurine (6-BA) promotes cell division and delays cell aging. Sucrose also serves to supply carbohydrates to the cultured tissue. The callus inducing medium is preferably prepared by adding 0.002 to 0.005 parts by weight of 6-benzylaminopurine (6-BA) and 2,4-dichlorophenoxyacetic acid (2-benzylaminopurine) to 100 parts by weight of sucrose. , 4-dicholorophenoxyacetic acid) in an amount of 0.001 to 0.003 part by weight. When the callus inducing medium is out of the above-mentioned composition range, callus differentiation and shoot induction rate are lowered, and it is difficult to obtain a haploid plant through drug cultivation of chrysanthemum.

The callus inducing medium may further comprise 0.25 to 2.5 parts by weight of casein hydrolyzate based on 100 parts by weight of sucrose, more preferably 0.25 to 2.5 parts by weight of casein hydrolyzate based on 100 parts by weight of sucrose, 0.6 part by weight. The casein hydrolyzate serves as a pH buffering substance, and the callus inducing medium containing the casein hydrolyzate has an effect of improving callus differentiation rate of chrysanthemum.

At this time, the MS medium may be an MS medium generally used for preparing the medium, and is not particularly limited. In addition, the MS medium can control the total amount of the medium to be produced, and is preferably 1 L to 10 L, but is not particularly limited thereto.

In addition, the present invention relates to a process for producing a microcapsule containing 0.004 to 0.02 part by weight of 6-benzylaminopurine (6-BA) and 0.0002 to 0.001 part by weight of naphthaleneacetic acid (NAA) per 100 parts by weight of sucrose. Wherein the microorganism is cultured in the presence of a culture medium. More preferably, the differentiation medium for cultivating the chrysanthemum is 0.005 to 0.01 part by weight of 6-benzylaminopurine (6-BA) and 100 parts by weight of naphthaleneacetic acid (6-BA) per 100 parts by weight of sucrose. NAA) in an amount of 0.00025 to 0.0005 part by weight. If it is outside the above range, the shoot induction rate is deteriorated.

The present invention relates to a method for producing chrysanthemum haploid plants by drug culturing, which method comprises separating the chrysanthemum anther from a bud of a chrysanthemum and then spraying the chrysanthemum drug to callus induction medium After inoculation, the cells are incubated at 20 to 25 ° C for 10 to 20 hours with fluorescent light for 35 to 60 days to induce callus.

The callus induced in step 1 is cultured in a differentiation medium at 25 to 27 DEG C for 10 to 50 days to induce shoot; Culturing the shoots in a rooting medium at 25 to 27 ° C for 10 to 50 days to produce roots; And a fourth step of screening the haploid plant from the postal matter. Hereinafter, the present invention will be described in more detail by step.

In the method for producing a mums haploid plant according to the present invention, in step 1, an anther is separated from a bud of a chrysanthemum, and the chrysanthemum is administered to callus induction medium After inoculation, the callus can be induced by incubating at 20 to 25 ° C for 10 to 20 hours with fluorescence for 35 to 60 days.

At this time, the chrysanthemum anther may include a micropore, and the micropore may be an uninucleate stage to 2 nuclei, preferably 1 nuclei to 2 nuclei. When the development stage of the chrysanthemum is 1 nucleus, the diameter of the bud is about 0.5 to 1 cm, preferably 0.5 to 0.8 cm, and the calyx at the center is closed, and the petals of the outside are light yellow. When the microparticle contained in the anthera is not a single nucleus group, the incidence of microspheres is low due to the development stage of microspheres, so that callus differentiation is not easily caused, so that it is difficult to achieve microbial cultivation. This means that in the plant tissue culture, the outer part or callus from the plant tissue, the indeterminate from the cultured cell, or a structure similar to the embryo is differentiated and formed.

The chrysanthemum flower bud is washed with water for 15 to 20 minutes, immersed in an aqueous ethanol solution for 10 to 50 seconds, and then added to sodium hypochlorite for 15 to 30 minutes It may be immersed and washed with water for 3 to 10 minutes to sterilize the surface. More preferably, the chrysanthemum buds are washed with water for 18 to 20 minutes, immersed in 50 to 80% ethanol aqueous solution for 20 to 50 seconds, then immersed in sodium hypochlorite for 20 to 30 minutes, washed with water for 5 to 10 minutes To sterilize the surface. As described above, the surface of the chrysanthemum is washed and sterilized several times, so that the subsequent culturing process can be performed in a sterilized state.

In the first step, the chrysanthemum drug may be cultured according to callus induction conditions known in the art. Preferably, the chrysanthemum drug is cultured in callus induction medium it is preferable to induce callus by incubating at 20 to 25 ° C for 10 to 20 hours with fluorescence for 35 to 60 days and more preferably to induce callus induction in a constant temperature chamber at 23 to 25 ° C. It is advisable to incubate for 35 to 45 days with fluorescence at 30 to 40 μmol / m ² / s for ~ 15 hours. When the culture conditions for inducing the callus are out of the above range, the incidence of indomethacin is low due to the stage of development of the microspheres, and callus differentiation is not easily caused, so that it is difficult to achieve microspheres culture.

At this time, in step 1, the chrysanthemum is sealed and stored in a dark room at 3-5 ° C for 24-48 hours, and then cultured in a callus induction medium. More preferably, the chrysanthemum is incubated at 3-4 ° C for 30-48 hours It is better to mature at low temperature in the dark room. When the pretreatment as described above is performed, callus induction and shoot initiation rate are more excellent than those in the case of not pretreating in the dark room at low temperature.

The callus inducing medium may be pH 5.6 to pH 6.0, and preferably pH 5.7 to pH 5.9. When the pH of the culture medium is out of the above-mentioned range, the callus induction to be performed later becomes poor.

In the method for producing a mums haploid plant by drug culture according to the present invention, the callus inducing medium is added to 100 parts by weight of sucrose in an amount of 6-benzylaminopurine (6-BA) of 0.002 to 0.02 And an MS medium containing 0.001 to 0.01 part by weight of 2,4-dicholorophenoxyacetic acid. More preferably, 0.002 to 0.005 parts by weight of 6-benzylaminopurine (6-BA) and 2,4-dichlorophenoxyacetic acid (2,4-dichlorophenoxyacetic acid) are added to 100 parts by weight of sucrose. ) In an amount of 0.001 to 0.003 part by weight.

At this time, the MS medium may be an MS medium generally used for preparing the medium, and is not particularly limited. In addition, the MS medium can control the total amount of the medium to be produced, and is preferably 1 L to 10 L, but is not particularly limited thereto.

The callus induction medium is intended to provide a nutrient composition for differentiation of callus from a chrysanthemum drug. The MS medium is a basic medium for propagating callus, and generally has NO ₃ -N, NH ₄ -N and K It has a high content and is easy to grow plant cells. The 2,4-dicholorophenoxyacetic acid plays a role in elongation and division of cells. The 6-benzylaminopurine (6-BA) promotes cell division and delays cell senescence. Sucrose also serves to supply carbohydrates to the cultured tissue.

When the callus inducing medium is out of the above-mentioned composition range, callus differentiation and shoot induction rate are lowered, and it is difficult to obtain a haploid plant through drug cultivation of chrysanthemum.

In the method for producing chrysanthemum haploid plants according to the present invention, it is preferable that the callus inducing medium further contains 0.25 to 2.5 parts by weight of casein hydrolyzate based on 100 parts by weight of sucrose, more preferably 0.25 to 2.5 parts by weight It is preferable to further include 0.25 to 0.6 part by weight of casein hydrolyzate relative to 100 parts by weight of sucrose. When the callus inducing medium further comprises casein hydrolyzate, the callus inducing and shoot inducing rate are more excellent than those not including the casein hydrolyzate.

In the method for producing chrysanthemum haploid plants by drug cultivation according to the present invention, the callus induction of the first step is carried out by adding 6-benzylaminopurine, 6-benzylaminopurine, or the like to 100 parts by weight of sucrose, BA) and 0.001 to 0.003 parts by weight of 2,4-dicholorophenoxyacetic acid, and then sealed and incubated at 3-5 占 폚 It is preferable to incubate in a dark room for 24 to 48 hours, preferably for 36 to 48 hours. The callus differentiation and shoot induction rate may be superior to those cultured in the above range.

At this time, the callus induction of the first step may be performed at 60% or more, preferably 60 to 95%, more preferably 64 to 92%. The callus induction rate was calculated as a ratio of the number of chrysanthemum drugs that succeeded in inducing callus to the number of introduced chrysanthemum drugs. The callus induction rate can be shown by culturing the chrysanthemum drug in the callus induction medium according to the present invention.

In the method for producing chrysanthemum haploid plants according to the present invention, in the step 2, the callus induced in the above step 1 is cultured in a differentiation medium at 25 to 27 ° C for 10 to 50 days, ), More preferably at 25 to 27 DEG C for 20 to 40 days. The shoot is generated after a culture period of about one month, and it is preferably carried out until shoots grow to a length of 2 to 3 cm. When the above condition is exceeded, there is a problem that the shoot induction rate is lowered.

At this time, 0.004 to 0.02 part by weight of 6-benzylaminopurine (6-BA) and 0.0002 to 0.001 part by weight of naphthaleneacetic acid (NAA) are added to 100 parts by weight of sucrose, , ≪ / RTI > wherein the culture medium contains MS medium. More preferably, the differentiation medium for cultivating the chrysanthemum is 0.005 to 0.01 part by weight of 6-benzylaminopurine (6-BA) and 100 parts by weight of naphthaleneacetic acid (6-BA) per 100 parts by weight of sucrose. NAA) in an amount of 0.00025 to 0.0005 part by weight. If it is outside the above range, the shoot induction rate is deteriorated.

At this time, the MS medium may be an MS medium generally used for preparing the medium, and is not particularly limited. In addition, the MS medium can control the total amount of the medium to be produced, and is preferably 1 L to 10 L, but is not particularly limited thereto.

At this time, the shoot induction in the second step may be performed at 60% or more, preferably 60 to 85%, and more preferably 60 to 80%. According to the present invention, an excellent shoot induction ratio as described above can be exhibited by culturing in the seedling-inducing differentiation medium according to the present invention.

In the method for producing chrysanthemum haploid plants according to the present invention, in step 3, the shoots are cultivated in a rooting medium at 25 to 27 DEG C for 10 to 50 days, more preferably at 25 to 27 DEG C For 20 to 40 days, to prepare a roots-bearing micro-organism. If the above condition is exceeded, there is a problem that the growth of roots from shoots is not easy.

In the method for producing a chrysanthemum haploid plant according to the present invention, the rooting medium is an MS medium containing 0.0002 to 0.001 part by weight of naphthaleneacetic acid (NAA) per 100 parts by weight of sucrose, . At this time, the sucrose is preferably 20 to 40 g / L. The rooting medium contains naphthaleneacetic acid (NAA), which promotes root growth.

In the method for producing a mums haploid plant by drug culture according to the present invention, the haploid plant may be screened from the microorganism using the flow cytometer in step 4 above. More specifically, the root end cells of the plants were collected and pretreated in colchicine for 1 to 3 hours. The root tip cells were fixed in a mixed solution of ethanol and acetic acid for about 1 to 2 days, After decomposition, the chromosome number can be measured by staining with a foilgen solution.

In this case, the chrysanthemum (Chrysanthemum morifolium Ramat . ) Varieties preferably include Yes morning. The Yes morning (Yes morning) is a D. grandiflorum variety developed at Chungnam Chungnam Chrysanthemum Experiment Station . In the cultivation for producing the chrysanthemum haploid plants according to the present invention, the above varieties can be used as donor plants (plants giving donors in experiments to prove the presence of substances that maintain the flowering of plants) . When the above-mentioned Yes Morning is used as the chrysanthemum varieties, the chrysanthemum haploid plant can be produced through the production method according to the present invention.

[ Example ]

Example  One. Yes of Morning  Drug cultivation

The micropore contained in the chrysanthemum anther is thoroughly rinsed for 15 to 20 minutes in tap water flowing through a flower embryo (flower bud) of the chrysanthemum, the uninucleate stage. After 30 seconds in 70% ethanol and then 20 minutes in sodium hypochlorite (1.5% active chlorate), the surface was sterilized by washing three times (5 minutes each) in sterilized distilled water.

About 200 chrysanthemums were separated using a sterile surgical scalpel. 200 chrysanthemum extracts separated from each other by using tweezers were blended with 1.0 mg of 2,4-dichlorophenoxyacetic acid, 2.0 mg Solid callus induction medium was prepared to include 6-benzylaminopurine of 90 g of sucrose, 250 mg of casein hydrolyzate and 2.75 g of Gel-rite, and the callus induction medium And transferred to a 100 mm plastic Petri dish containing 20 ml of the medium. At this time, the induction medium was adjusted to pH 5.8. The Petri dish was sealed and stored in a dark room at 4 ° C for 24 hours to perform pretreatment.

The chrysanthemum treated with the pretreatment was cultured at 20 째 C for 16 hours at 20 째 C with 20 袖 mol / m ² / s of fluorescence. The chrysanthemum drug was subcultured weekly and cultured for a total of 60 days at a temperature of about 25 to 27 DEG C to conduct haploid callus induction and then transferred to differentiation medium.

The differentiation medium was prepared so as to contain 2.0 mg of 6-benzylaminopurine, 0.1 mg of naphthaleneacetic acid (NAA), 30 g of sucrose and 2.75 g of gelite in 1 L of MS medium, shoots were induced by culturing in differentiation medium at a temperature of about 25 to 27 DEG C for 1 month.

The shoots were grown to a length of 2 to 3 cm, removed aseptically, separated into vials containing a rooting medium containing 30 g of 1 L of MS medium sucrose, 0.1 g of NAA and 3 g of gelite, . The microorganisms grown to a root length of about 6 to 10 cm were taken out from the vials, and the medium adhered to the roots was washed away by running tap water to prevent microbial infection. Thereafter, the plantlets were transferred to pots containing soil, and the pots were transferred to the growth phase and stored under a controlled environment of about 25 to 27 ° C and (70% or more humidity) for about 7 to 15 days (see FIG. 1).

Example  2-12. Yes of Morning  Drug cultivation

Except that the pretreatment was carried out in the same manner as in Example 1 but under the same conditions as in Table 1 below.

Chrysanthemum variety Callus induction medium Preprocessing time
(time) Chrysanthemum Medicine Number Example 1 Yes Morning A1 24 200 Example 2 Yes Morning A1 48 200 Example 3 Yes Morning A1 - 200 Example 4 Yes Morning A2 24 200 Example 5 Yes Morning A2 48 200 Example 6 Yes Morning A2 - 200 Example 7 Yes Morning A3 24 200 Example 8 Yes Morning A3 48 200 Example 9 Yes Morning A3 - 200 Example 10 Yes Morning A4 24 200 Example 11 Yes Morning A4 48 200 Example 12 Yes Morning A4 - 200

The composition of the callus induction medium is shown in Table 2 below.

A1 A2 A3 A4 Basic medium (1L) MS MS MS MS 6-BA (mg / L) 2 2 2 2 2,4-D (mg / L) One One One One casein
Hydrolyzate
(mg / L) - 250 - 250 Sucrose (g / L) 90 90 45 45 Gel-rite (g / L) 2.75 2.75 2.75 2.75

Example  13-24. Yes of Morning  Drug cultivation

The microcapsules were prepared in the same manner as in the Example of Table 3, except that the pretreated chrysanthemum drug was cultured for 45 days while being irradiated with fluorescence of 35 μmol / m ² / s for 14 hours every day.

Example Medium and culture conditions For callus induction
Incubation period
(Work) Fluorescence Photon Quantity Example 13 Example 1 45 35 μmol / m ² / s Example 14 Example 2 45 35 μmol / m ² / s Example 15 Example 3 45 35 μmol / m ² / s Example 16 Example 4 45 35 μmol / m ² / s Example 17 Example 5 45 35 μmol / m ² / s Example 18 Example 6 45 35 μmol / m ² / s Example 19 Example 7 45 35 μmol / m ² / s Example 20 Example 8 45 35 μmol / m ² / s Example 21 Example 9 45 35 μmol / m ² / s Example 22 Example 10 45 35 μmol / m ² / s Example 23 Example 11 45 35 μmol / m ² / s Example 24 Example 12 45 35 μmol / m ² / s

Example  25-36. Yes of Morning  Drug cultivation

The microcapsules were prepared in the same manner as in the Example of Table 3, except that the pretreated chrysanthemum drug was cultured for 45 days while being irradiated with fluorescence of 50 μmol / m ² / s for 14 hours every day.

Example Medium and culture conditions For callus induction
Cultivation period (days) Fluorescence Photon Quantity Example 25 Example 1 45 50 μmol / m ² / s Example 26 Example 2 45 50 μmol / m ² / s Example 27 Example 3 45 50 μmol / m ² / s Example 28 Example 4 45 50 μmol / m ² / s Example 29 Example 5 45 50 μmol / m ² / s Example 30 Example 6 45 50 μmol / m ² / s Example 31 Example 7 45 50 μmol / m ² / s Example 32 Example 8 45 50 μmol / m ² / s Example 33 Example 9 45 50 μmol / m ² / s Example 34 Example 10 45 50 μmol / m ² / s Example 35 Example 11 45 50 μmol / m ² / s Example 36 Example 12 45 50 μmol / m ² / s

Comparative Example  1-24. Chrysanthemum culture

The same procedure as in Example 1 was carried out except that the material was prepared in the same manner as in Example 1, except that the conditions were as shown in Table 5 below.

Breed name Callus
Induction medium Pretreatment time (hours) Comparative Example 1 Hi Maya A1 24 Comparative Example 2 Hi Maya A1 48 Comparative Example 3 Hi Maya A1 - Comparative Example 4 Hi Maya A2 24 Comparative Example 5 Hi Maya A2 48 Comparative Example 6 Hi Maya A2 - Comparative Example 7 Hi Maya A3 24 Comparative Example 8 Hi Maya A3 48 Comparative Example 9 Hi Maya A3 - Comparative Example 10 Hi Maya A4 24 Comparative Example 11 Hi Maya A4 48 Comparative Example 12 Hi Maya A4 - Comparative Example 13 Piece pink A1 24 Comparative Example 14 Piece pink A1 48 Comparative Example 15 Piece pink A1 - Comparative Example 16 Piece pink A2 24 Comparative Example 17 Piece pink A2 48 Comparative Example 18 Piece pink A2 - Comparative Example 19 Piece pink A3 24 Comparative Example 20 Piece pink A3 48 Comparative Example 21 Piece pink A3 - Comparative Example 22 Piece pink A4 24 Comparative Example 23 Piece pink A4 48 Comparative Example 24 Piece pink A4 -

Experimental Example  Chrysanthemum Haploid  Manufacture of plants

Haploid plants were separated by measuring the drainage properties of the microorganisms prepared in Examples 1 to 12 and Comparative Examples 1 to 24 according to the present invention. To accomplish this, 35 replicates were randomly selected from the grower, and the drainage was measured using a flow cytometer (Partec, GmbH) of Chungnam Agricultural Research and Extension Service (Korea, budget).

The analysis for the drainage measurement was performed by Naing et al ., (2013). Specifically, root end cells pretreated with 0.3% colchicine for 2 h were fixed in a 3: 1 solution of ethanol: acetic acid for 1 day, then hydrolyzed in 1 N HCl at 60 ° C for 5 min, Lt; / RTI > Chromosomes were observed through the crushed specimen to observe the chromosomes in the acetic acid carmine, and the flowability of the plants was determined by flow cytometry (Partec GmbH). The results are shown in Table 6 and Fig. 2 to Fig. 3.

Chrysanthemum
kind Callus
Induction medium Pretreatment
time
(time) Chrysanthemum Medicine Number Fluorescence Photon Quantity
(μmol / m ² / s) For callus induction
Incubation period
(Work) Callus
Induction rate (%) Shinshu
Induction rate (%) Example 1 Yes Morning A1 24 200 20 60 74 61 Example 2 Yes Morning A1 48 200 20 60 79 64 Example 3 Yes Morning A1 - 200 20 60 64 56 Example 4 Yes Morning A2 24 200 20 60 79 66 Example 5 Yes Morning A2 48 200 20 60 85 69 Example 6 Yes Morning A2 - 200 20 60 68 60 Example 7 Yes Morning A3 24 200 20 60 86 69 Example 8 Yes Morning A3 48 200 20 60 89 73 Example 9 Yes Morning A3 - 200 20 60 72 67 Example 10 Yes Morning A4 24 200 20 60 89 76 Example 11 Yes Morning A4 48 200 20 60 92 79 Example 12 Yes Morning A4 - 200 20 60 78 73 Example 13 Yes Morning A1 24 200 35 45 81 68 Example 14 Yes Morning A1 48 200 35 45 84 72 Example 15 Yes Morning A1 - 200 35 45 75 60 Example 16 Yes Morning A2 24 200 35 45 84 69 Example 17 Yes Morning A2 48 200 35 45 89 75 Example 18 Yes Morning A2 - 200 35 45 80 64 Example 19 Yes Morning A3 24 200 35 45 88 72 Example 20 Yes Morning A3 48 200 35 45 90 77 Example 21 Yes Morning A3 - 200 35 45 79 65 Example 22 Yes Morning A4 24 200 35 45 90 84 Example 23 Yes Morning A4 48 200 35 45 95 89 Example 24 Yes Morning A4 - 200 35 45 81 76 Example 25 Yes Morning A1 24 200 50 45 68 55 Example 26 Yes Morning A1 48 200 50 45 72 59 Example 27 Yes Morning A1 - 200 50 45 60 49 Example 28 Yes Morning A2 24 200 50 45 74 63 Example 29 Yes Morning A2 48 200 50 45 77 68 Example 30 Yes Morning A2 - 200 50 45 64 54 Example 31 Yes Morning A3 24 200 50 45 78 69 Example 32 Yes Morning A3 48 200 50 45 82 75 Example 33 Yes Morning A3 - 200 50 45 69 66 Example 34 Yes Morning A4 24 200 50 45 80 70 Example 35 Yes Morning A4 48 200 50 45 83 78 Example 36 Yes Morning A4 - 200 50 45 75 63 Comparative Example 1 Hi Maya A1 24 200 20 60 66 44 Comparative Example 2 Hi Maya A1 48 200 20 60 72 52 Comparative Example 3 Hi Maya A1 - 200 20 60 60 40 Comparative Example 4 Hi Maya A2 24 200 20 60 75 55 Comparative Example 5 Hi Maya A2 48 200 20 60 78 58 Comparative Example 6 Hi Maya A2 - 200 20 60 64 41 Comparative Example 7 Hi Maya A3 24 200 20 60 84 69 Comparative Example 8 Hi Maya A3 48 200 20 60 85 72 Comparative Example 9 Hi Maya A3 - 200 20 60 77 60 Comparative Example 10 Hi Maya A4 24 200 20 60 89 85 Comparative Example 11 Hi Maya A4 48 200 20 60 95 90 Comparative Example 12 Hi Maya A4 - 200 20 60 80 76 Comparative Example 13 Piece pink A1 24 200 20 60 72 46 Comparative Example 14 Piece pink A1 48 200 20 60 75 54 Comparative Example 15 Piece pink A1 - 200 20 60 62 42 Comparative Example 16 Piece pink A2 24 200 20 60 75 57 Comparative Example 17 Piece pink A2 48 200 20 60 78 60 Comparative Example 18 Piece pink A2 - 200 20 60 68 43 Comparative Example 19 Piece pink A3 24 200 20 60 84 71 Comparative Example 20 Piece pink A3 48 200 20 60 89 74 Comparative Example 21 Piece pink A3 - 200 20 60 72 62 Comparative Example 22 Piece pink A4 24 200 20 60 89 87 Comparative Example 23 Piece pink A4 48 200 20 60 91 89 Comparative Example 24 Piece pink A4 - 200 20 60 79 73

According to the above Table 3, it can be confirmed that when the pretreatment was carried out in both of Examples 1 to 36 and Comparative Examples 1 to 24, the calli induction rate and the shoot induction rate were better, and that 48 hours Lt; RTI ID = 0.0 > callus induction < / RTI > and shoot induction.

In addition, when the A2 and A4 medium supplemented with 250 mg / L of casein hydrolyzate were used for the pretreatment and callus induction medium, the callus induction and shoot inducing effects were somewhat increased. And, when the content of sucrose contained in the callus induction medium was low, it was confirmed that callus induction and shoot inducing effect were better.

More specifically, it was confirmed that when cultured in the medium of A4, the best callus induction rate and shoot induction rate were shown, and it was confirmed that most favorable effect was obtained when cultured in the medium of A4 for 48 hours .

Further, in Examples 13 to 24, in which the cultivation for callus induction was carried out by irradiating fluorescence with a photon quantity of 30 to 40 μmol / m ² / s for 35 to 45 days, the calli induction rate and the shoot induction rate .

In addition, the callus induction rate and shoot induction rate are different for each cultivar.

Through these results, it can be seen that the variety of chrysanthemum, pre - treatment temperature, culture condition and composition ratio of induction medium act as a main factor for callus induction and shoot induction.

On the other hand, in the case of three randomly selected microbial cells prepared in Examples 1 to 36, the number of chromosome of haploid was 2n = 27 as a result of flow cytometry analysis. As a donor plant, 'Yes Morning' has 2n = 54 chromosome numbers, it can be seen that horsetail plants can be produced by cultivating chrysanthemum drug by the method according to the present invention.

Claims (16)

0.002 to 0.02 parts by weight of 6-benzylaminopurine (6-BA) and 0.001 to 0.01 part by weight of 2,4-dicholorophenoxyacetic acid are added to 100 parts by weight of sucrose, By weight of MS medium.
2. The callus induction medium for chrysanthemum phloem culturing according to claim 1, wherein the callus inducing medium further comprises 0.25 to 2.5 parts by weight of casein hydrolyzate based on 100 parts by weight of sucrose.
0.004 to 0.02 parts by weight of 6-benzylaminopurine (6-BA) and 0.0002 to 0.001 part by weight of naphthaleneacetic acid (NAA) relative to 100 parts by weight of sucrose. Wherein the culture medium for chrysanthemum is cultivated.
The chrysanthemum drug is inoculated into a callus induction medium and then irradiated with fluorescence at 20 to 25 ° C for 10 to 20 hours, Lt; / RTI > for 1 day to induce callus;
The callus induced in step 1 is cultured in a differentiation medium at 25 to 27 DEG C for 10 to 50 days to induce shoot;
Culturing the shoots in a rooting medium at 25 to 27 ° C for 10 to 50 days to produce roots; And
And screening the haploid plant from the microorganism. ≪ Desc / Clms Page number 19 >
5. The method according to claim 4, wherein the chrysanthemum anther comprises a microparticle, and the microparticle is a uninucleate stage to a nucleus.
The method according to claim 4, wherein the chrysanthemum flower bud is washed with water for 15 to 20 minutes, immersed in an aqueous ethanol solution for 10 to 50 seconds, immersed in sodium hypochlorite for 15 to 30 minutes, washed with water for 3 to 10 minutes, Wherein the plant is sterilized.
[5] The method according to claim 4, wherein the chrysanthemum inoculated on callus induction medium in step 1 is sealed and stored in a dark room at 3-5 [deg.] C for 24-48 hours and then cultured. Gt;
5. The method according to claim 4, wherein the callus inducing medium has a pH of 5.6 to pH 6.0.
[6] The method according to claim 4, wherein the step 1) is performed for 30 to 40 탆 ol / m ² / s for 10 to 15 hours and incubated for 35 to 45 days. .
5. The method according to claim 4, wherein the callus induction medium is prepared by adding 0.002 to 0.02 part by weight of 6-benzylaminopurine (6-BA) to 2,4-dichlorophenoxyacetic acid And 0.001 to 0.01 part by weight of 2,4-dicholorophenoxyacetic acid.
11. The method according to claim 10, wherein the callus inducing medium further comprises 0.25 to 2.5 parts by weight of casein hydrolyzate per 100 parts by weight of sucrose.
5. The method according to claim 4, wherein the callus induction of step 1 is performed at 60 to 95%.
5. The method according to claim 4, wherein the differentiation medium comprises 0.004 to 0.002 parts by weight of 6-benzylaminopurine (6-BA) and 0.0002 to 0.002 parts by weight of naphthaleneacetic acid (NAA) relative to 100 parts by weight of sucrose. ≪ / RTI > to about 0.001 part by weight of the culture medium.
[5] The method according to claim 4, wherein the shoot induction at the second step is performed at 60 to 85%.
5. The method according to claim 4, wherein the rooting medium comprises MS medium containing MS medium containing 0.0002 to 0.001 part by weight of naphthaleneacetic acid (NAA) relative to 100 parts by weight of sucrose (Method for producing chrysanthemum haploid plants by drug cultivation).
16. The method according to any one of claims 4 to 15, wherein the Chrysanthemum morifolium Ramat . Varieties include Yes morning.

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