KR101289655B1 - Method for producing polyploid of Miscanthus sinensis - Google Patents

Abstract

The present invention relates to a method for modifying the genome composition of wild type pampas antelope for transformation, and the pampas antelope produced by the method so that the wild pamper can be used as an industrial, environmental improvement or feed plant biomass. Using wild-type diploids, we induced an artificial diploid and developed a diploid with a hybrid diploid genome in which the pampas genome and the cells of diploid and tetraploid coexist. As a result of assaying the ploidy and genome composition of the induced haploid pampas grass, it was confirmed that the ploidy.
The new drainage pampas grasses with drainage genome genome composition according to the method of the present invention are grown on non-cultivated land for biomass, and are a resource of biomass for supplying valuable carbon materials for economical and precious carbon materials that can produce alternative energy for global warming and carbon emission. Can be used as

Description

Method for producing polyploid of Miscanthus sinensis

The present invention provides a method for producing a diploid pampas antelope having a diploid genomic composition of at least a diploid or tetraploid (4n) having a highly productive trait for use in native wild type pampas grasses for agricultural farming purposes. It relates to a drainage pampas grass plant, seeds of the plant, pollen and pear, biomass produced from the plant and a composition for producing the plant.

The development and use of renewable energy is essential to reduce the greenhouse gases generated by the excessive consumption of fossil fuel oil and coal. Bioenergy crops are an alternative to transport fuels and chemicals that currently make up the bulk of fossil fuels. For such chemical materials, the mass production of biomass should be carried out first. Biomass refers to bioorganisms including microorganisms, plants, and microorganisms that are propagated by photosynthesis to fix carbon dioxide by receiving solar energy and animals that live on them. The biomass is used to make solids (wood), liquids (fuel diesel, ethanol), gases (biogas), and the like. Such biomass raw materials are industrialized as biofuels or fine chemicals through a process of biorefinery. Biorefinery is a field for producing bio-based chemical products (organic acids, amino acids, polyols, biopolymers) or biofuels (bioethanol, biodiesel, biobutanol) using biotechnology (biocatalyst). Chemical material production, bioplastics / bioenergy, and functional biomaterials / bioprocessing technology. Biofuel is a fuel obtained from biomass. It uses not only living organisms but also by-products from metabolic activities such as animal excretion as fuels, or bioethanol, biodiesel, Biogas and the like.

Among the cellulosic biomass, herbaceous plants have emerged as important biomass crops because they have the advantage of ensuring high harvestability with low cultivation cost even in the unsuitable area, i.e., the environment unsuitable for crop cultivation. It is silver grass. Pampas grass is not a cultivated plant and is classified as a wild weed and is a strong herb that colonizes the soil conditions of the low mountains and fields of the northern hemisphere. Pampas grass is the most environmentally friendly biomaterial and it is desirable to develop it as a versatile crop. It is native to Northeast Asia including Korea, and about 17 species have been reported worldwide. Pampas grasses are mainly double genus Miscanthus sinensis and M. sacchariflorus . M. x giganteus ( M. sinensis x M. sacchariflorus ) , a natural hybrid triploid selected for biomass research, has been studied for biomass production. Pampas vulgaris is a low-temperature C4 plant with 43-70% cellulose content, a potential maximum yield of 35 tons per ha, and wheat cultivation, so it is attracting much attention as a plant resource for biomass production. Since the speed and dry weight of silver grass do not meet the mass production and economic feasibility of biomass, the development of new silver grass with improved dry weight, yield and growth rate is required.

In the case of drainage plants, the expression of agricultural economic dominance is predominant, and the useful traits such as seedlessness due to increased yield or infertility are much higher than those of haploid or diploid, which are mainly used for breeding and industrializing new varieties. Therefore, it is necessary to develop wild grass that exists in nature that has suitable characteristics as a plant for biomass, and to develop it as an industrial plant for the purpose of producing biomass by doubling the numerical composition of the genome.

The drainage breeding technique used in the present invention was developed into a technique suitable for mass production of biomass by administering a lot of effort and time. Induced tetrapolyploid pterygium with a tetraploid (4n) genome composition using the wild weed, Miscanthus sinensis 2n = 2x = 38, to meet the demand of large quantities of biomass for carbon materials for industrial use. Developed. Miscanthus to date There is no public disclosure of artificial drainage in sinensis ).

The present invention has been made in accordance with the requirements as described above, in the present invention is a drainage plant with improved traits to increase the dry weight or cultivation to be used for industrial or environmental improvement or biomass from the existing wild pampas grass An effective chromosomal doubling system has been developed for differentiation.

In order to solve the above problems, the present invention is a Miscanthus sinensis , to provide a drainage of 2n = 2X).

The present invention also provides a drainage pampas grass plant produced by the above method.

The present invention also provides a seed of the above-mentioned drainage pampas grass plant.

In addition, the present invention provides a pollen of the drainage pampas grass plant.

In addition, the present invention provides an pear of the drainage Pampas grass plant.

In addition, the present invention provides a biomass produced from the drainage Pampas grass plant.

In addition, the present invention provides a composition for producing the Drainage Pampas grass.

Drainage pampas grass plants of the present invention is characterized by a higher development of rhizome, higher number of buns, longer leaf length, and larger leaf area than the wild type, resulting in high biomass productivity. High value as a bioenergy crop.

1 shows the production of sterile plants in the process of callus formation and regeneration into plants (A: callus induction, B: shoot induction from callus, C: growth of shoots, D: of individual plants) growth). EH in FIG. 1 shows the process of inducing regeneration of chromosomal doubling plants from colchicine treated callus (E: colchicine treated callus, F: induction of shoots from treated callus, G: shoot growth, H: chromosome doubling plant Growth).
Figure 2 is a cell analyzer to determine whether the chromosome doubled by measuring the amount or chromosome amount of the genome in the nucleus by extracting a single cell from the young leaves in order to determine whether the chromosome doubled from the plant derived from colchicine-treated seed-derived callus ( A graph of a graph analyzed by a flow cytometer. When the diploid is determined based on the maximum absorption point of the spectrophotometer of about 50, the quadruple shows an absorption point of 100 or more.
3 is a diagram of a graph of chromosome multiplication of single cells in young leaves by flow cytometer to confirm whether or not chromosome doubled from newly generated plants from colchicine treated seeds, showing plants of 3n and 4n.
4 is a graph of chromosome multiplication of single cells in young leaves from a colchicine-treated callus by flow cytometer, showing the plant of 4n, 5n and 6n. Indicates.
FIG. 5 is a graph of chromosomal amplification of single cells in young leaves by flow cytometer in order to confirm whether chromosome doubled from newly-developed plants from colchicine-treated callus-derived differentiators. 4n, 5n and 6n Represents a plant.
Figure 6 shows the process of inducing the regeneration of seed-derived callus used in the present invention and the chromosomal doubling plants after colchicine treatment. A: Treated redifferentiated by colchicine concentration, B: Culture of treated redifferentiated, C: Induced dialect from treated redifferentiated, D: Generated new bullet.
FIG. 7 is a diagram of a graph of a chromosome analysis of single cells in young leaves with a flow cytometer in order to confirm whether chromosomal multiplication of newly generated plants from colchicine treated seedlings is shown.
8 is a growth comparison picture of 2n, 4n coma and 4n chromosome doubling plants among colchicine-treated parent plants 2n and colchicine-treated ploidy doubled plants. You can see that the chromosomal doubling plants are larger and have more bulking than the parent plants.

In order to achieve the object of the present invention, the present invention is a Miscanthus sinensis ) provides a method for producing drainage.

The way to produce the pampas drainage

(1) removing the seed of the pampas grass seed;

(2) treating columine (colchicine) on the seed to prevent somatic cell division;

(3) washing the colchicine treated seeds with sterile water;

(4) inducing germination by sowing the washed seeds onto the soil; And

(5) selecting and transferring the germinated seedlings to pots and growing them in a greenhouse.

After step (5), the flow cytometry can determine the ploidy of the plant.

Another way to produce the pampas drainage

(1) removing the seed of the pampas grass seed;

(2) sterilizing the seed;

(3) inducing germination by sowing the seeds in a 1 / 2MS base medium;

(4) selecting the germinated seedlings;

(5) treating somatic cell division by treating colchicine with the selected seedlings;

(6) washing the colchicine-treated seedlings with sterile water and then transferring to a 1 / 2MS base medium to induce new powder; And

(7) transferring the derived plants to pots and growing them in a greenhouse.

After step (7), flow cytometry can confirm the ploidy of the plant.

Another way to produce the pampas drainage

(1) removing the seed of the pampas grass seed;

(2) sterilizing the seed;

(3) culturing the seed in a callus induction medium;

(4) selecting the callus by culturing the induced callus in a callus holding medium;

(5) treating somatic cell division by treating colchicine with the selected callus;

(6) washing the colchicine-treated callus with sterile water and then transferring to regeneration medium to differentiate shoots;

(7) transferring the differentiated shoots to a 1 / 2MS base medium to induce rooting; And

(8) transferring the rooted plant to a pot and growing in a greenhouse.

After step (8), flow cytometry can confirm the ploidy of the plant.

Another way to produce the pampas drainage

(1) removing the seed of the pampas grass seed;

(2) sterilizing the seed;

(3) culturing the seed in a callus induction medium;

(4) selecting the callus by culturing the induced callus in a callus holding medium;

(5) transferring the selected callus to regeneration medium to induce shoots and rooting;

(6) treating colchicine with the re-differentiated seedlings to prevent somatic cell division;

(7) washing the colchicine treated seedlings with sterile water, and then transferring to a 1 / 2MS base medium to induce new powder; And

(8) transferring the plant to a pot and growing in a greenhouse.

After step (8), flow cytometry can confirm the ploidy of the plant.

Specifically, the callus induction medium may be MS medium containing 1 to 3 mg / L of 2,4-D and 30 g / L of sucrose, but is not limited thereto. Preferably, the callus induction medium may be MS medium containing 2 to 3 mg / L of 2,4-D and 30 g / L of sucrose, and most preferably, the callus induction medium of 3 mg / L MS medium comprising 2,4-D and 30 g / L of sucrose.

Specifically, the colchicine treatment may be one to three days of 100 ~ 800 μM colchicine, but is not limited thereto. Preferably, the colchicine treatment may be one or two days treatment of 300 or 600 μM of colchicine, respectively.

Specifically, the callus regeneration medium may be MS medium containing 1 to 3 mg / L of BA and 30 g / L of sucrose, but is not limited thereto. Preferably, the callus regeneration medium may be MS medium containing 2 mg / L BA and 30 g / L Sucrose.

The colchicine is a cell division inhibitor treated to plant tissues for the purpose of inducing chromosomal doubling by inhibiting cell division, and may be replaced with any cell division inhibitor having the same effect. The cell division inhibitors are oralzaline, dinitroaniline and etafluralin (N-methyl-N-2-methyl-2-propenyl) -2,6-dinitro-4- (trifluoromethyl) benzaninyl May be, but is not limited thereto.

"Plant tissue" is all tissues of a plant at the time of cultivation or culture. The term may include all plants, plant cells, plant organs, protoplasts, cell cultures, or groups of plant cells composed of structural and functional units.

Cell division inhibitors may be treated on the seed prior to sowing. Seeds are soaked in clear water for 5-6 hours, and then the seeds are soaked in a colchicine solution or dinitiroaniline solution for 24 hours. The seeds are washed with water before sowing. Dry seeds can be directly immersed in the chemical solution without pre-immersion in water.

In addition, cell division inhibitors can be treated to new seedlings. Soil temperature is maintained at 29 ~ 31 ℃ for germination after sowing. A drop of colchicine or dinitroaniline solution is added to the shoot apex between the cotyledon as soon as the seedlings emerge from the soil. The colchicine solution is applied to the growing point in the morning or evening for three consecutive days.

In addition, after treating a cell division inhibitor to the apical tip of the germinated seed, the germinated seed can be planted in the soil. Seeds are germinated in an incubator at 30 ° C., the tops of the germinal seeds of the germinated seeds are inverted and soaked in a 30 ° C. incubator for 10-15 hours in a colchicine or dinitroaniline solution.

Preferably, after removing the seed of the pampas grass seed, the seed may be treated with 100 ~ 400μM colchicine with stirring for 2 days. The colchicine treated seeds may be washed with sterile water, and the washed seeds may be sown in the soil to induce germination.

Preferably, after removing the seed of the pampas grass seed, it can be surface sterilized with stirring for 15 minutes in 70% ethanol and washed three times with sterile water. Then, 0.8% NaOCl solution was added and surface sterilized with stirring for 30 minutes, washed 5 times with sterile water, transferred to sterile filter paper to remove water, and then seeded on 1 / 2MS base medium to induce germination. have. The seedlings may be passaged to select seedlings, and 100 to 800 μM colchicine may be treated for 24 hours. The colchicine treated seedlings were washed with sterile water and then transferred to 1 / 2MS rooting induction medium to which 0.01-0.1 mg / L IAA and 15 g / L sucrose were added to induce new powder.

Preferably, after removing the seed of the pampas grass seed, it can be surface sterilized with stirring for 15 minutes in 70% ethanol and washed three times with sterile water. Then, 0.8% NaOCl solution was added and surface sterilized with stirring for 30 minutes, washed 5 times with sterile water and transferred to sterilized filter paper to remove water, followed by 1-3 mg / L 2,4-D and 30 g / L Sudose. Callus can be induced by densification on the added MS medium. The induced callus may be passaged in a callus growth medium to select callus, and the selected callus may be placed on a callus growth medium of liquid containing 300 to 600 μM of colchicine for 1 to 2 days. The colchicine-treated callus was washed with sterile water and then transferred to MS medium containing 1 to 3 mg / L of BA and 30 g / L of sucrose to differentiate shoots, and differentiated shoots were 0.01 to 0.1. Roots of shoots can be induced by transferring to 1/2 MS rooting induction medium with mg / L IAA and 15 g / L Sucrose added.

Preferably, after removing the seed of the pampas grass seed, it can be surface sterilized with stirring for 15 minutes in 70% ethanol and washed three times with sterile water. Then, 0.8% NaOCl solution was added and surface sterilized with stirring for 30 minutes, washed 5 times with sterile water and transferred to sterilized filter paper to remove water, followed by 1-3 mg / L 2,4-D and 30 g / L Sudose. Callus can be induced by densification on the added MS medium. The induced callus was passaged in a callus growth medium to select callus, transferred to MS medium containing 1 to 3 mg / L of BA and 30 g / L of sucrose to induce shoots, and induced shoots Roots of shoots can be induced by transferring to 1/2 MS rooting induction medium with 0.01-0.1 mg / L IAA and 15 g / L sucrose. The re-differentiated seedlings may be placed on 1/2 MS basal medium of liquid containing 200-800 μM colchicine for 1-3 days. The colchicine treated seedlings were washed with sterile water, and then transferred to 1/2 MS rooting induction medium to which 0.01-0.1 mg / L IAA and 15 g / L sucrose were added to induce new powder.

The present invention also provides a drainage pampas grass plant produced by the above method. Compared with the wild type, the drainage pampas fern plant has more features of rhizome, a large number of buns, a long leaf length, and a larger leaf area.

In addition, the present invention provides a seed of the produced Drainage Pampas grass plant.

In addition, the present invention provides a pollen of the produced drainage pampas grass plant.

In addition, the present invention provides the circumference of the produced drainage pampas grass plants.

In addition, the present invention provides a biomass produced from the produced Drainage Pampas grass plant.

In addition, the present invention provides a composition for the production of drainage pampas grass, containing colchicine. The drainage pampas quince plant production composition may contain 100 to 800 μM of colchicine, preferably 300 or 600 μM of colchicine, respectively.

Hereinafter, the present invention will be described in more detail with reference to Examples. These implementations are only intended to illustrate the present invention, and the content of the present invention is not limited to the following examples.

Selection of materials

Pampas grass used in the present invention is a wild grass that grows on the Korean peninsula ( Miscanthus sinensis ) and its seeds (Table 1).

Pampas Grass ( Miscanthus) sinensis ) 's List of Genes number line Collection date (year, month, day) Collection Collection 014 Prairie 2010.06.10 strain Gimhae 1, Gyeongsangnam-do South Korea 027 Prairie 2010.11.27 Seed, rhizome Miryang 1, Gyeongsangnam-do South Korea

Example  1: Establishment of efficient tissue culture system using mature seeds of pampas grass

1. Plant Materials and Seed Sterilization

As plant material for tissue culture of Miscanthus sinensis , mature seeds of Korean native species were used (Table 1). Sterilization of the seed for callus induction was surface sterilized with stirring for 15 minutes in 70% ethanol after removing the seed, and washed three times with sterile water. Then, 0.8% NaOCl solution was added and the surface was sterilized for 30 minutes with stirring. After washing 5 times with sterile water, it was transferred to sterilized filter paper to remove water, and then healed in callus induction medium.

2. Embryonic development Callus  Judo

The basic callus induction medium to induce callus from mature seeds of P. falciparus was MS (Murashige and Skoog, 1962) medium added with 30 g / L sucrose and 0.8% agar. In addition, 2,4-D (2,4-dichlorophenoxyacetic acid) is widely used to induce embryogenic callus in monocots (Tishu et al. 1987 Plant Cell, Tissue and Organ Culture 9: 245-252, Oldach. Et al. 2001 Plant Cell Rep. 20: 416-421). In this experiment, 2,4-D of auxin (auxin), a plant growth regulator, was treated in different concentrations in the medium to investigate the efficiency of induction of embryogenic callus. Sterilized seeds in the medium were wound and then cultured in a 24 ± 2 ℃ incubator.

In order to investigate the optimum cultivation effect according to the concentration of 2,4-D added to callus induction medium, Cultured in callus-induced medium, callus generation efficiency was different according to 2,4-D concentration according to the type of Pampas grass (Table 2).

In case of Pampas oleracea, callus development efficiency was the highest at 3mg / L at 61.7% at 2,4-D concentration, and 60.3% at 2mg / L and 55.6% at 1mg / L. The higher the callus tends to increase. Thus, 2,4-D 3mg / L was the most appropriate concentration of callus induction from pampas grass mature seeds.

2,4-D Concentration Conditions for Callus Induction from Pampas Seeds line 2,4-D (mg / L) Number of bad seeds () Number of callus induced () Callus induction rate (%) Prairie One 54 30 55.6 2 58 35 60.3 3 60 37 61.7

3. Plant regeneration

In order to regenerate the plant from the callus derived from the blue-grown seeds, 0.8% of MS (Murashige and Skoog, 1962) medium with 30 g / L sucrose and agar were used. Four-week-old embryogenic callus was used to investigate plant regeneration efficiency. In addition, as a study of callus induction and regeneration using various tissues of silver grass, auxin 2,4-D and cytokinin BA were treated alone or in combination to investigate the type and concentration of proper plant growth regulator. Callus was placed on the medium and incubated for 4 weeks at 24 ± 2 ° C., 16 hours of spot / 8 hours of dark conditions. After incubation, shoots formed in each treatment group were examined by redifferentiated individuals.

In order to investigate the efficiency of plant regeneration from the callus derived from mature seeds, the regeneration efficiency according to the types of plant growth regulators was determined by treating alone or in combination with auxin 2,4-D and cytokinin BA. It appeared differently (Table 3, FIG. 1).

In case of Pampas oleracea, the re-differentiation efficiency was the highest at 50.0% when BA 2mg / L was treated, and when mixed with BA 2mg / L and 2,4-D 1mg / L, it was 20.0% at BA and 2,4-D. It was confirmed that treatment of BA alone was more effective. As such, regeneration of the pampas grass from mature seed-derived callus showed that BA 2 mg / L was the most appropriate concentration.

Condition of Regeneration of Callus Induced from Pampas Seed line Plant growth regulator
(mg / L) Dirty
Number of callus () Number of callus-derived shoots () Plant regeneration rate (%) Prairie BA 2 10 5 50.0 BA 2; 2,4-D 1 10 2 20.0

Example  2: Induction of homogenous drainage pampas grasses using colchicine

Induction of Chromosome Double Plants Using Pampas Seed and Colchicine

Pampas Grass ( Miscanthus) In order to induce the chromosomal embryos of sinensis ), seeds were treated with colchicine (Sigma Co., USA) and germinated in the green house, and germination began 4 days after sowing, and there was no significant difference between the control and the control. After 4 weeks of sowing, the ground part grew more than 40 ~ 50cm, and 10 individuals were selected and planted based on the superior individuals in each experimental section. Plants of the colchicine treatment was longer and broader than the control. In addition, two weeks after the gestation of chromosomes, flow cytometric analysis was performed by collecting leaves of three controls, 27 treatments, and a total of 30 leaves. The graph analyzed by the flow cytometer of FIG. 3 is a diagram for determining whether the chromosome is doubled by extracting single cells from young leaves and measuring the amount or chromosome amount of the genome in the nucleus. In the case of tetraploid, when the maximum absorption point of the spectrophotometer is set to about 50, the absorption point of the tetraploid shows 100 and higher (FIG. 2). Flow cytometry confirmed exactly that the chromosome of the pampas grass was 2n, and one in 100uM treatment, one in 400uM treatment (Table 4, Figure 3).

Induction of Chromosomal Doping Plants after Colchicine Treatment with Pampas Grass Seeds line Colchicine treatment concentration
(μM) Number of seeds treated
(dog) Drainage Number of plants tested Number of chromosomal doubling plants Prairie 0 100 3 0 100 200 9 One 200 200 9 0 400 200 9 One

2. Origin of Pampas Seeds Callus and  Induction of Chromosomal Dope Plants Using Colchicine

Pampas Grass ( Miscanthus) Induction of callus from mature seeds of sinensis ), we investigated the number of shoots generated in callus of each treatment group after concentration treatment, 3 ~ 4 weeks, and differentiated shoots into complete plants. Flow cytometry was performed to determine whether the chromosome doubled (Table 5, Figure 1).

For 1 day treatment of colchicine, the callus-derived efficiency of callus-derived non-colchicine-treated control was the lowest at 3.0%, and the highest incidence was 18.2% at 300uM colchicine. On the contrary, the callus-derived efficiency of callus-derived shoots was highest at 15.2%, and the lowest at 3.0uM of 300uM colchicine. The 600uM colchicine treatment was the same as 12.1% in both the 1 day treatment and 2 days treatment. In addition, the shoots induced rooting and induced differentiation into a complete plant to obtain a total of 31 individuals, and as a result of confirming whether the chromosomal doubled through flow cytometry, four in 300uM treatment group and 9 in 600uM treatment group, total Thirteen chromosomal embryos obtained Pampas ventus (FIG. 4).

As such, in the case of the daily treatment of the control group not treated with colchicine, the callus-derived efficiency was lower than that of the colchicine treatment. On the contrary, in the two-day treatment, the incidence of shoot growth was higher than that of the colchicine treatment. It is thought that in the daily treatment control group not treated with colchicine, callus was more severe than the treatment treated with colchicine to inhibit shoot induction. In addition, the treatment group treated with colchicine by concentration showed the highest shoot rate and chromosome doubling rate in the daily treatment of 300uM colchicine. As a result, it was confirmed that the treatment of 300 μM colchicine for 1 day was the most favorable condition for inducing chromosome doubling using callus and colchicine.

Induction of Chromosomal Doping Plants after Colchicine Treatment with Pampas Callus line Colchicine Treatment Concentration ( u M) Colchicine Processing Period (Days) Number of callus processed () Number of shoots in callus Number of plants tested for ploidy Chromosome doubling
Vegetative
several) Pampas Grass




0 One

33 1 (3.0%) 2 0 300 33 6 (18.2%) 13 4 600 33 4 (12.1%) 13 9 0 2

33 5 (15.2%) 2 0 300 33 1 (3.0%) - - 600 33 4 (12.1%) One 0

3. Pampas Grass Callus  origin With subdivided bodies  Induction of Chromosomal Dope Plants Using Colchicine

Pampas Grass ( Miscanthus) Induction of callus regeneration from mature seeds of sinensis ), 4 weeks after colchicine treatment, concentrations of newly generated water in each treatment were investigated. Analysis was performed to determine whether the chromosome doubled (Table 6, Figure 5).

In the control group not treated with colchicine, the powder generation efficiency was 20%, and the colchicine treatment group showed 10% powder generation efficiency only at 200 uM. In addition, the generated powder was obtained by inducing rooting and differentiating into a complete plant to obtain a total of four individuals, and as a result of confirming whether the chromosome was doubled through flow cytometry, all four were chromosomal doubled plants (FIG. 6). However, no powdering occurred at the concentrations of colchicine 400 and 800 uM, which is thought to be because the treatment period was too long after three days of colchicine treatment. Therefore, it was confirmed that colchicine treatment with silver seedlings is more effective for long-term treatment at low concentration or for short-term treatment at higher concentration than for long-term treatment at high concentration.

Induction of Chromosome Doping Plants after Colchicine Treatment Using Callus Derivatives Colchicine treatment
Concentration ( uM ) Number of plants treated Number of messages generated Number of plants tested for ploidy Chromosome doubling
Vegetative
several) 0 10 2 4 0 200 10 One 4 4 400 10 0 - - 800 10 0 - -

4. Pampas Grass With seedlings  Induction of Chromosomal Dope Plants Using Colchicine

By inducing seedlings from mature seeds of Miscanthus sinensis , the colchicine was treated by concentration, and after 4 weeks, the number of newly produced cereals was examined in each treatment group, and the generated powders were differentiated into complete plants. Flow cytometry was performed to determine whether chromosome doubled. As a result, one individual was obtained from the 400 uM treatment group and one individual from the 800 uM treatment group to secure a total of two chromosomal doubling plants (Table 7, FIG. 7).

Induction of Chromosomal Doping Plants after Colchicine Treatment Using Pampas Seedling Colchicine concentration (u M) Number of plants treated Number of messages generated Number of plants tested for ploidy Chromosome doubling
Vegetative
several) 0 60 22 2 0 200 60 15 9 0 400 60 10 9 One 800 60 12 9 One

Example  3: Confirmation of Drainage Genome Composition of Drainage Pampas Grass

To investigate the doubling of chromosomes of colchicine-treated Pampas oleracea for ploidy induction, chromosome doubling was assayed using flow cytometric analysis (Part cytometry, Partec GmbH, Munster, Germany). The chromosomal doubling was performed by flow cytometry analysis of leaves of control and treatment individuals for each method of induction of pampas grass (Table 8).

Flow cytometry was able to accurately identify the chromosomes of Pampas grass. When the colchicine was treated with seed, the result of ploidy was very small, but the efficiency of chromosome doubling was high when the shoot was induced after treatment with the seed-derived callus. In addition, the induction rate of the drainage was high even when the callus was treated in the regeneration body. All of the untreated controls showed 2n, but the ploidy was obtained from chromosomal doubled plants with 2n and 4n or 4n composition (Table 8). In addition, compared to the control chromosomal doubling plants showed a longer elongation and the number of minutes was also found to increase significantly (Fig. 8).

Pampas chromosome doubling results series
number Colchicine Treatment Concentration (uM) Chromosome Number and Genome Composition ^a Chromosome doubling ^b Chromosome doubling method M1 0 2n - Treatment of colchicine using callus M3 300 4n o M5 300 4n o M7 300 4n o M12 300 4n o M18 600 4n o M24 600 4n o M27 600 4n o M30 600 6n o M31 600 4n o M32 600 4n o M34 600 4n o M35 600 4n o M36 600 4n, 5n o M40 0 2n - Colchicine Treatment Using Callus-derived Regeneration System M44 200 2n, 4n o M45 200 4n o M46 200 2n, 4n o M47 200 4n o MC01 0 2n - Treatment of colchicine with seeds MC06 100 3n o MC25 400 4n o MC31 0 2n - Colchicine treatment using inflight seedlings MC45 400 4n o MC58 800 4n o Drainage analysis with ^a flow cytometer
^b Drainage (o), no change (-)

Claims (13)

delete delete In the method of producing a drainage of Miscanthus sinensis ,
(1) removing the seed of the pampas grass seed;
(2) sterilizing the seed;
(3) culturing the seed in a callus induction medium;
(4) selecting the callus by culturing the induced callus in a callus holding medium;
(5) inhibiting somatic cell division by treating the selected callus with 300 to 600 μM of colchicine for 1 to 3 days;
(6) washing the colchicine treated callus with sterile water and transferring the callus regeneration medium to differentiate shoots;
(7) transferring the differentiated shoots to a 1 / 2MS base medium to induce rooting; And
(8) a method for producing a pampas drainage, comprising transferring the rooted plant to a pot and growing in a greenhouse. delete The method of claim 3, wherein the callus induction medium is MS medium comprising 1 to 3 mg / L of 2,4-D and 30 g / L of sucrose. delete The method of claim 3, wherein the callus regeneration medium is characterized in that the MS medium containing 1-3 mg / L BA and 30 g / L Sucrose. Drainage Pampas grass plant produced by the method according to claim 3. Seed of a Drainage Pampas Grass Plant according to claim 8. Pollen of drainage pampas grass plants according to claim 8. Distribution of drainage pampas grass plants according to claim 8. Biomass produced from the Drainage Pampas grass plant according to claim 8. delete

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