JP2011083260A - Chrysanthemum plant and method for creating the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a well-fragrant chrysanthemum plant; and to provide a method for creating the same. <P>SOLUTION: The fragrance-enhanced chrysanthemum plant is obtained by crossing Chrysanthemum fruticulosa or Chrysanthemum lavandulifolium as seed parent with Chrysanthemum morifolium Ramat as pollen parent each other. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a chrysanthemum plant and a production method thereof.

  Chrysanthemum is an important plant for flower gardening and has a unique aroma. Various chrysanthemum varieties have been created in the past, but inflorescence shape, number of head flowers, head flower size, head flower type, petal size, petal shape, flower color, leaf shape, leaf color, grass The aim is to improve the height, grass shape, the number of branches, etc. (Patent Document 1), and as suggested by the absence of items related to fragrance in the screening criteria for chrysanthemum varieties (Non-Patent Document 1) No improvement was made.

Japanese Patent Application Laid-Open No. 2004-321057

http://www.hinsyu.maff.go.jp/annai/sinsakijun/kijun/1480.pdf

  An object of the present invention is to provide a fragrant chrysanthemum plant and a production method thereof.

  As a result of intensive studies to solve the above problems, the present inventors have found that by crossing the cultivated species with wild species of Chrysanthemum, it is possible to create a chrysanthemum genus plant with a good fragrance and a strong fragrance, The present invention has been completed.

The present invention is summarized as follows.
[1] Chrysanthemum fruticulosa or Chrysanthemum lavandulifolium as a seed parent and Chrysanthemum morifolium Ramat. As a pollen parent, a chrysanthemum genus plant with enhanced aroma.
[2] The plant according to [1], wherein the total amount of the aromatic component is 10 times or more that of the pollen parent when the aromatic component of the plant is analyzed by ion chromatography using a headspace method.
[3] benzaldehyde, phenylacetaldehyde, linalool, methylbenzoate, limonene, 2-ethylhexanol, cis-3-hexenyl acetate, α-pinene, 1,8-cineol, myrcene, 2-phenylethyl alcohol, camphor, ethyl acetate, comprising a fragrance component selected from the group consisting of β-ferrandolene, camphene, trans-chrysanthenol, chrysantenone, trans-chrysantenyl acetate, cis-chrysantenyl acetate and cis-chrysantenol, [1] or [2] The plant according to [2].
[4] A progeny of the plant according to any one of [1] to [3].
[5] An extract obtained from the plant according to any one of [1] to [3] or the progeny of the plant according to [4].
[6] A method for producing a plant according to any one of [1] to [3], comprising crossing Chrysanthemum fruticulosa or Chrysanthemum Lavandulifolium as a seed parent and Chrysanthemum morifolium Ramat. As a pollen parent.
[7] The method according to [6], further comprising selecting a plant in which the total amount of the fragrance component is 10 times or more that of the pollen parent when the fragrance component of the plant is analyzed by gas chromatography using a headspace method. .
[8] The method according to [6] or [7], further comprising vegetative propagation.
[9] The method according to any one of [6] to [8], further comprising electric cultivation.

  The chrysanthemum plant of the present invention has a sweet scent and / or herb-like scent that is favorably felt, and has a scent that is clearly different from the unique aroma of chrysanthemum. The chrysanthemum plant of the present invention has higher aroma intensity (amount of aroma substance released) than conventional chrysanthemum plants. When it is in the state of planting, it smells like it stands up from the stock. Even in the state of cut flowers, it smells stronger than conventional chrysanthemum plants. In addition, it has a good appearance such as a large upright flower compared to the seed parent, and is excellent as an ornamental flower. It does not look dirty when it dies, and is suitable for dried flowers. The chrysanthemum genus plant of the present invention can be cultivated by electric light, and can increase the height of a plant and the size of a flower as compared with outdoor cultivation. In addition, it can contribute to the creation of electricity demand in the agricultural field, prevention of withdrawal, and activation of the flower industry. The extract extracted from the chrysanthemum plant of the present invention can be used for products such as perfumes, cosmetics, aroma oils, and fragrances. Furthermore, many wild-type chrysanthemums have functionalities that have been used for a long time, such as deodorization, air purification, and medicine, and the chrysanthemum plants of the present invention can also have similar functionalities. It can be used for purposes other than ornamental flowers.

Panel A shows a Chrysanthemum fruticulosa strain, Panel B shows a Chrysanthemum fruticulosa flower, Panel C shows a Chrysanthemum lavandulifolium strain, and Panel D shows a Chrysanthemum lavandulifolium flower. Panel A is a yellow variety of Chrysanthemum morifolium Ramat. Panel B is a yellow flower of Chrysanthemum morifolium Ramat. Panel C is a red variety of Chrysanthemum morifolium Ramat. Panel D is Chrysanthemum morifolium Ramat. Panel E is an orange variety of Chrysanthemum morifolium Ramat. Panel F is an orange variety of Chrysanthemum morifolium Ramat. Panel G is a white variety of Chrysanthemum morifolium Ramat. Panel H Shows flowers of white varieties of Chrysanthemum morifolium Ramat. Panel A shows seeds obtained from the hybrid. Panel B shows the seeding of the medium in the test tube. Panel C shows the state of germination. Panel D shows the extension. The electrophoresis image which compares the conventional CTAB method with the simple CTAB method is shown. The electrophoresis image of the hybridity test by RAPD (using primer OPA7) is shown. The electrophoresis image of the hybridity test by RAPD (using primer OPA8) is shown. The electrophoresis image of the 2nd iteration of the hybridity test by RAPD (primer OPA7 use) is shown. The electrophoresis image of the 2nd iteration of the hybridity test by PARD (using primer OPA8) is shown. Panel A shows the state of growth and growth of cultured seedlings. Panel B shows cultured seedlings at the start of acclimatization. Panel C shows plants about 4 weeks after the start of acclimatization. Panel D shows a plant about 4 weeks after planting. Panel E shows a plant about 14 weeks after planting. Panel F shows cultured seedlings in growth culture. Panel A shows a planted plant before picking. Panel B shows a planted plant before picking. Panel C shows the collected side buds. Panel D shows the state of cuttings during low temperature treatment. Panel E shows the cuttings 11 days after the start of growth. Panel F shows the cuttings 11 days after the start of growth. The survey results and the growth status in the first cultivation test of the cross in 2005 are shown. It is a continuation of FIG. It is a continuation of FIG. It is a continuation of FIG. It is a continuation of FIG. It is a continuation of FIG. It is a continuation of FIG. FIG. 17 is a continuation of FIG. It is a continuation of FIG. It is a continuation of FIG. The survey results and growth status in the second cultivation test of the cross in FY2005 are shown. It is a continuation of FIG. It is a continuation of FIG. It is a continuation of FIG. It is a continuation of FIG. It is a continuation of FIG. It is a continuation of FIG. The results of the FY2005 crossing survey are shown. It is a continuation of FIG. It is a continuation of FIG. It is a continuation of FIG. It is a continuation of FIG. It is a continuation of FIG.

1. Chrysanthemum plant of the present invention The Chrysanthemum plant of the present invention is obtained by crossing a wild species of Chrysanthemum fruticulosa or Chrysanthemum lavandulifolium as a seed parent and a cultivated species of Chrysanthemum morifolium Ramat. The aroma is enhanced more than the seed parent and the pollen parent, and there is a difference in the type and amount of the aroma component, but preferably the total amount of the aroma component is 10 times or more that of the pollen parent.

  The chrysanthemum plants of the present invention include their progenies and mutants such as branch substitutes. The chrysanthemum plant of the present invention also includes cultures of plants (including roots, stems, leaves, flowers, cuttings, shoot tips, etc.), seeds, pollen, ovules, cells, and callus. Furthermore, the chrysanthemum plant of the present invention includes not only raw ones (potted plants, cut flowers, etc.) but also processed ones (dried flowers, etc.). The Chrysanthemum plant of the present invention is more upright than the seed parent, has a higher plant height, is about 15 cm to about 150 cm, has a large flower in most hybrids, and has a head flower diameter of about 0.5 cm to about 150 cm. 5cm, there are many flowers. The flower color is, for example, yellow, but is not particularly limited. In addition, various chrysanthemum plants obtained by crossing the above combinations may be regarded as a series of plant groups (plant populations) with enhanced aroma in the present invention.

(1) Seed parent Chrysanthemum fruticulosa (panels A and B in FIG. 1) is distributed at 30 to 50 degrees north latitude and 1500 to 3000 m altitude, and Chrysanthemum lavandulifolium (panels C and D in FIG. 1) is 30 latitude north. It is a wild species distributed at an altitude of 1500 m or less, and has a thick honey-like scent. They are crowded, with thin leaves and stems, small flowers (about 0.3 cm to about 1.5 cm), and are difficult to get flowers under Japanese climatic conditions. It is not a seed. These seed parents can be obtained from the plant gene storage experiment facility attached to the Graduate School of Science, Hiroshima University (Postal Code 739-8526, 1-4-3 Kagamiyama, Higashihiroshima City, Hiroshima Prefecture).

(2) Pollen parent Chrysanthemum morifolium Ramat. Yellow varieties (hereinafter sometimes referred to as Y-4. Panels A and B in FIG. 2), Chrysanthemum morifolium Ramat. Red varieties (hereinafter referred to as R-8) 2 (panels C and D), orange varieties of Chrysanthemum morifolium Ramat. (Hereinafter sometimes referred to as Or-3, panels E and F of FIG. 2) and white varieties of Chrysanthemum morifolium Ramat. 'Sause' (Seikoen Co., Ltd.), hereinafter referred to as W-4. Panels G and H in Fig. 2 are cultivated varieties selected on the basis that the flower color is bright and dark, and the leaves are not too large. It is. Chrysanthemum morifolium Ramat. Used in the present invention is not limited to any variety. Chrysanthemum morifolium Ramat. (Y-4, R-8, Or-3 and W-4) is Chubu Electric Power Co., Ltd. (1-4 Kitakoseyama, Otakamachi, Midori-ku, Nagoya-shi, Aichi) It is available from Electric Power Co., Inc. Energy Application Laboratory.

(3) Crossing Since the seed parent is difficult to attach a flower, it is preferable to adjust the flowering of the pollen parent by electric cultivation or the like so that the pollen can be pollinated in accordance with the flowering of the seed parent. For pollination, it is preferable to use fresh pollen instead of frozen or refrigerated pollen after collection of pollen of cultivated species in order to improve fruit set. For example, according to the time when the seed parent blooms, the pollen parent pollen that bloomed on the same day is collected on the medicine wrapping paper, and after confirming that the seed parent's pistil has expanded, the pollen on the medicine wrapping paper is applied with a brush or a finger. Use it to pollinate the stigma and bag it to prevent pollination by other pollen. This is repeated during the flowering period. The seeds are collected after about 4 weeks. Of the collected seeds, immature ones can be caught and mature ones can be stored at room temperature.

(4) Breeding of hybrids In order to improve the germination rate, for example, it is preferable to sow seeds collected in a medium dispensed in a test tube or the like. As the medium, for example, MS medium can be used. Preferably, 1% to 2% sucrose and 0.1% to 0.5% gellite or agar are added to the ½ MS medium to adjust the pH to about 5.5 to 6.0. The germination conditions and the growing conditions are preferably about 3000 lux to 6000 lux, 10 hours to 16 hours, and about 20 ° C. to about 25 ° C. The hybrids grown in vitro are further cultured and propagated to increase the number of individuals. Culture growth is usually carried out by cuttings in chrysanthemum plants, but other methods such as shoot tip culture may be used. After sufficient rooting, the medium attached to the roots is washed away, planted in a pot containing horticultural soil such as vermiculite, acclimatized, and then planted. The grown chrysanthemum can be further inserted and vegetatively bred with buds, etc., and progeny can be nurtured.

  The chrysanthemum plant of the present invention can be cultivated by electric light. When heating, it is preferably about 10 ° C to about 25 ° C, more preferably about 12 ° C to about 18 ° C. Further, in the electric heating and warming cultivation, the plant height is higher and the flowers are larger than in the outdoor cultivation.

(5) Confirmation of hybrids As a method of confirming that the plant grown from the hybrid seed is a hybrid between a wild species and a cultivated species, comparison of the number of chromosomes with parents and the Genomic in situ hybridization (GISH) method However, since observation of chromosomes requires high skill acquisition and takes time, it is not efficient to target a large amount of plants. Then, based on the difference of gene fragments, it was examined whether hybrid confirmation could be performed by a simple method, and it was confirmed that it was possible by Random amplified polymorphic DNA (RAPD). If a characteristic gene fragment that can distinguish a hybrid individual or a parental wild species becomes clear, and the expression trait of the plant body matches, an individual with a promising character of the wild species can be cultivated for a long time. It can be screened at the initial stage of breeding without performing it, and it can be expected that the labor required for developing new varieties will be greatly reduced. In addition, the DNA used as a sample for the RAPD method is often extracted by the Cetyl trimethyl ammonium bromide (CTAB) method, but it takes too much time. Therefore, a simple DNA extraction method (simple CTAB method) was examined. With the DNA extracted by the simple CTAB method, RAPD data almost equivalent to the DNA obtained by the normal CTAB method can be obtained, and the time required for DNA extraction can be greatly shortened.

The simple CTAB method can be performed by the following procedure.
(A) A frozen sample of leaves (about 100 to 200 mg) is placed in a 2.0 ml tube and crushed with a micro pestle. 500 μl of 2% CTAB solution (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl) is added and mixed.
(B) Add 500 μl of CIA solution (Chloroform: Isoamylalchol = 24: 1), mix slightly for 1 minute, and centrifuge (15000 rpm, 5 minutes, room temperature).
(C) Transfer the supernatant to a new 1.5 ml tube, add 500 μl of 1% CTAB solution (1% CTAB, 20 mM EDTA, 100 mM Tris-HCl), mix, and centrifuge (15000 rpm, 5 minutes, room temperature) .
(D) After removing the supernatant, add 1 ml of 70% ethanol to the pellet and centrifuge (15000 rpm, 1 minute, room temperature). After removing the supernatant again, vacuum dry for 5 minutes. Thereafter, 100 μl of TE is added and stored at −20 ° C.

  The RAPD method is performed using the DNA obtained by the simple CTAB method as a sample. The primers used for the RAPD PCR amplification are preferably Operon's random primer series OPA-7 and OPA-8.

Base sequence of OPA-7 (SEQ ID NO: 1): GAAACGGGGTG
The base sequence of OPA-8 (SEQ ID NO: 2): GTGACGTAGG

  PCR conditions are 45 cycles, for example, initial denaturation 94 ° C., 3 minutes, denaturation 94 ° C., 1 minute, annealing 40 ° C., 2 minutes, extension 72 ° C., 2 minutes.

The obtained PCR amplification product is electrophoresed on a 1.5% agarose gel, and after staining with a nucleic acid staining solution (final ethidium bromide concentration 0.5 μg / ml), a band is detected by ultraviolet irradiation. Those having a band specific to pollen parents or a band of parents can be determined to be hybrids because they are considered not to be self-breeding.
Furthermore, a cross-species may be confirmed by preparing an STS marker.

2. Aroma component of chrysanthemum plant of the present invention The chrysanthemum plant of the present invention is preferably benzaldehyde, phenylacetaldehyde, linalool, methylbenzoate, limonene, 2-ethylhexanol, cis-3-hexenyl acetate, α-pinene, 1, 8-cineole, myrcene, 2-phenylethyl alcohol, camphor, ethyl acetate, β-ferrandrene, camphene, trans-chrysanthenol, chrysanthenone, trans-chrysanthenyl acetate, cis-chrysanthenyl acetate and cis-chrysan An aroma component selected from the group consisting of tenols is included. The characteristics of these scents are shown in Table 8 of the Examples below. The chrysanthemum plant of the present invention may further contain other components such as squalene, hexadecanoic acid, tetradecanoic acid and the like.

  In the chrysanthemum plant of the present invention, preferably, the aromatic component is analyzed by gas chromatography according to a headspace method in a state of planting, preferably at the time of flowering, and the total amount of the aromatic component is 100% (chromatography). Benzaldehyde and / or phenylacetaldehyde can be included in amounts of 1-30%, 5-25%, 10-20% in the graph data (when the total area (area) of the peak is 100%). Not. In addition, benzaldehyde and phenylacetaldehyde are respectively 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11 %, 12%, 13%, 14%, or 15% or more.

  Preferably, linalool, methylbenzoate, limonene, 2-ethylhexanol, cis-3-hexenyl acetate, α-pinene, 1,8-cineol, myrcene, 2-phenylethyl alcohol, camphor, ethyl acetate, β-ferrane Drain, camphene, trans-chrysantenol, chrysantenone, trans-chrysantenyl acetate, cis-chrysantenyl acetate and cis-chrysantenol are 0.05% or more, 0.1% or more, and 0.2%, respectively. Or more, 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more Including. However, the total amount does not exceed 100%.

  The chrysanthemum plant of the present invention has a sweet, herb-like scent and a very strong fragrance as compared with the pollen parent. Aroma is strong only with leaves and flowers. For example, when aroma components of plants such as cut flowers are analyzed by gas chromatography using the headspace method, the total amount of aroma components is preferably 10 times, 25 times, 30 times, 35 times, It is more than double, 45 times or more, or 50 times or more. If it is planted in the ground, the scent is even stronger, especially during flowering.

  Furthermore, from the group (group) of Chrysanthemum plants of the present invention, when the aromatic component of the plant is analyzed by gas chromatography using the headspace method, the total amount of the aromatic component is 10 times or more of the pollen parent, and A strain having a favorable scent (sweet scent, herb-like scent) may be screened.

  In the headspace method, the chrysanthemum plant of the present invention is covered with a glass hood while the plant is planted locally, a Teflon tube and a pump are connected to the hood, and air is circulated for 4 hours at a flow rate of 0.5 L / min. The trapping agent filled in the glass tube in the middle of the tube: The fragrance was collected with Tenax TA 50 mg, the collected fragrance was subjected to gas chromatography (GC-MS), and capillary column TC-WAX 0.25 mmφ as analysis conditions × 60 m, initial temperature 40 ° C. → final temperature 230 ° C., carrier gas: helium, inlet temperature: 250 ° C., flow rate: 1 ml / min, aroma component desorption temperature: 230 ° C.

3. The method for extracting extracts of Chrysanthemum plants of the present invention may be a conventional method, and examples thereof include a solvent extraction method. The chrysanthemum plant of the present invention used for extraction may be raw, semi-dried, dried, or lyophilized, but is preferably raw. In addition, the plant may be appropriately pulverized or cut or in the form of the plant itself, but is preferably pulverized. The plant part used as the raw material for extraction may be any of roots, stems, leaves, flowers, etc., or whole plants, but preferably contains flowers and / or leaves.

  As the solvent, water, a hydrophilic organic solvent, or a mixture thereof can be used. A hydrophilic organic solvent is preferable because an extract with higher viscosity can be obtained. Specific examples of the solvent include pure water, purified water, methanol, ethanol, butanol, propylene glycol, glycerol, 1,3-butylene glycol and other alcohols, acetone, tetrahydrofuran, acetonitrile, 1,4-dioxane, dimethyl sulfoxide, N , N-dimethylformamide, acetic acid and the like, and are not particularly limited, but methanol, butanol, propylene glycol, and 1,3-butylene glycol are preferable. Although only a hydrophilic organic solvent may be used for extraction, it is preferable to use a mixture of a hydrophilic organic solvent and water. In this case, the concentration of the hydrophilic organic solvent is preferably 10 to 90 vol%, more preferably 20 to 60 vol%, and still more preferably 25 to 35 vol%. A plurality of hydrophilic organic solvents may be mixed and used. For example, ethanol, 1,3-butylene glycol and water may be mixed. Although the addition amount of the solvent to a plant is not specifically limited, Preferably it is 100-1000 mL with respect to 100 g of plants, More preferably, it is 200-600 mL.

  Solvent is added to the plant and the extraction is preferably carried out at a temperature of 0-60 ° C, more preferably 20-30 ° C, preferably 1 hour to 7 days, more preferably 8-16 hours (overnight). After completion of the extraction, the residue may be removed by filtration, centrifugation or the like, or a concentration step or a purification step may be performed as necessary. These steps can be performed using various known methods such as vacuum concentration, freeze-drying, precipitation with a solvent such as ethanol, chromatography such as ion exchange chromatography, gel permeation chromatography, HPLC, and ultrafiltration. it can.

  The Chrysanthemum plant extract (essential oil) of the present invention preferably comprises a component selected from the group consisting of 1,8-cineol, α-pinene, p-murolen and cis-sabinol. The contents are 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more when analyzed by gas chromatography. % Or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, or 16% or more. Normally, it is said that essential oil does not contain much sabinol. The extract of the genus Chrysanthemum according to the present invention further includes benzaldehyde, phenylacetaldehyde, β-pinene, p-cymene, camphene, myrcene, limonene, linalool, methylbenzoate, 2-ethylhexanol, cis-3-hexenyl acetate. 2-phenylethyl alcohol, γ-terpinene, γ-murolene, camphor, ethyl acetate, β-ferrandolene, trans-chrysanthenol, chrysantenone, trans-chrysantenyl acetate, cis-chrysantenyl acetate, cis-chrysylene Santenol may be included.

4). Product Using Extract of Chrysanthemum Plant of the Present Invention The above extract can be used for products such as perfumes, cosmetics, aroma oils and fragrances. In addition to the extract, the above products include various components such as polyhydric alcohols such as glycerin, propylene glycol and polyethylene glycol, mucopolysaccharides such as hyaluronic acid and chondroitin sulfate and derivatives thereof, collagen, elastin, keratin, etc. Proteins and derivatives thereof, amino acids such as glycine, serine, aspartic acid, glutamic acid, arginine and derivatives thereof, saccharides such as sorbitol, trehalose, glucose, sucrose, plant extracts such as urea, ceramide, aloe extract, vitamin A , C, E and derivatives thereof, glutyrrhizic acids and derivatives thereof, niacinamide, arbutin, kojic acid, ellagic acid, linoleic acid, tranexamic acid, placental extract, superoxide dismutase, mannitol, quercetin, cation Kin, astaxanthin, coenzyme Q10, carrageenan, pectin, agar, gum arabic, guar gum, locust bean gum, xanthan gum, quince seed, gelatin, starch, and other natural products, cellulose-based polymers such as methylcellulose hydroxycellulose, carboxyvinyl Acrylic polymers such as polymers, alginate polymers such as sodium alginate, vegetable oils such as squalane, jojoba oil, castor oil, safflower oil, olive oil, paraoxybenzoates, surfactants, chelating agents, pigments , PH adjusting agents and the like may be included.

1. Crossing of Chrysanthemum Wild Species and Cultivated Species As shown in Table 1 below, the wild species Chrysanthemum fruticulosa was crossed as a seed parent, and nine cultivated species as a pollen parent. In addition, as shown in Table 2 below, a wild species Chrysanthemum lavandulifolium as a seed parent and four cultivated species as a pollen parent were crossed.

(1) Crossing method Since the flowering time of both of the above two wild species was unknown, the cultivated species were cultivated in a greenhouse capable of controlling flowering so that the pollen of the cultivated species could be pollinated during the flowering period of these wild species. . The pollen parent pollen that bloomed on the same day is collected on the medicine wrapping paper according to the time when the seed parent blooms, and after confirming that the seed parent's pistil has expanded, the pollen on the medicine wrapping paper is applied to the stigma using a brush or a finger. Pollinated and bagged to prevent pollination by other pollen. Tables 1 and 2 below show the number of pollinated flowers, the number of fruiting flowers, the number of seeds obtained, and the state of seeds.

(2) Seeding The obtained seeds were sown in a medium in a test tube in vitro.
Medium composition: 1 / 2MS, 1.5% sucrose, 0.2% gellite, hormone free, pH 5.7
Environmental conditions: Approximately 4000 lux, 12 hours long, 22 ° C

  Tables 1 and 2 below show germination numbers. 3A to 3D show seeds, seeded test tubes, germination, and elongated plants, respectively.

2. Confirmation of hybrid species The RAPD method was used as a method for confirming that a plant grown from a hybrid seed was a hybrid between a wild species and a cultivated species.

(1) Simple DNA extraction method from hybrids The simple CTAB method was performed as described above to extract hybrid DNA. FIG. 4 shows RAPD analysis data comparing the normal CTAB method and the simplified CTAB method. In the simple CTAB method, the RAPD analysis result almost equivalent to the normal CTAB method was obtained, and the time taken for DNA extraction of 24 samples could be shortened from about 6 hours to about 2 hours.

(2) Selection of Primer Using Parent Individual RAPD analysis was performed using Operon's random primer series OPA1-20 and OPB1-20 using wild seeds of seed parents and cultivated seeds of pollen parents. As a result, a band was obtained with 11 primers. As a result of analyzing these bands, it was confirmed that favorable results were obtained with the primers OPA7 (SEQ ID NO: 1), OPA8 (SEQ ID NO: 2), OPA9 (SEQ ID NO: 3), and OPA18 (SEQ ID NO: 4).

(3) Hybridity test by RAPD method using hybrid individuals. The leaves of 85 hybrids grown in good condition obtained in 1 above were collected from a culture test tube, and 2. DNA was extracted using the simple CTAB method described in 1. and RAPD analysis was performed in triplicate using primers OPA7 (SEQ ID NO: 1) and OPA8 (SEQ ID NO: 2).

  The reaction solution was prepared by mixing a final concentration of 0.5 μM primer, a final concentration of 0.2 mM dNTPs, 0.5 U of PCR enzyme (Taq polymerase) per 20 μl of reaction solution, and 1 μl of template DNA per 20 μl so that the total volume becomes 20 μl. Was added with sterile distilled water.

  In the RAPD method, PCR was carried out using the two primers described above, and the amplified product was electrophoresed.

  FIG. 5 shows an example of an electrophoresis image of an amplification product obtained by amplifying a template DNA derived from a hybrid of hybrid number 22 (C. fruticulosa × Y-4) with primer OPA7. In the figure, * indicates a pollen parent (Y-4) specific band, and Δ indicates a seed parent (C. fruticulosa) specific band. Individuals with both pollen parent-specific and seed parent-specific bands were number 22-101 (lane 2), 22-13 (lane 6), 22-17 (lane 9).

  FIG. 6 shows, as an example, an electrophoretic image of an amplification product obtained by amplifying a template DNA extracted from the hybrid No. 22 with the primer OPA8. Individuals with both pollen parent-specific and seed parent-specific bands are number 22-101 (lane 2), 22-103 (lane 3), 22-12 (lane 5), 22-13 (lane 6). 22-15 (lane 7), 22-16 (lane 8), 22-17 (lane 9), 22-18 (lane 10), 22-19 (lane 11), 22-2 (lane 12), 22 -20 (lane 13).

  The RAPD analysis using the above-mentioned primers OPA7 and OPA8 was further repeated twice, and a confirmed reproducibility was determined to be a hybrid. FIG. 7 shows an electrophoresis image of the second analysis using OPA7, and FIG. 8 shows an electrophoresis image of the second analysis using OPA8 as an example. As a result, a large number of hybrids having both pollen parent-specific bands and seed parent-specific bands were confirmed. In addition, there was a line in which a band pattern clearly different from the seed parent was confirmed (for example, lanes 3 and 4 in FIG. 8). These lines were considered to be hybrids that could not be confirmed with the primers OPA7 or OPA8, or natural hybrids.

  Tables 3 and 4 below show data of hybrids obtained by crossing each pollen parent with seed parents C. fruticulosa and C. lavanduliforium.

In addition, the STS marker was produced and the hybridity test of RAPD was also confirmed (data not shown).
3. Breeding of hybrids (1) Acclimatization and parental planting Individual individuals that were genetically confirmed to be hybrids were taken as one line and the number of individuals was secured by culture growth. Acclimatization was carried out from a line that was able to secure a sufficient number of individuals, and the parent strain was planted in an outdoor field. Since germination time, hybrid confirmation time, and growth rate differ depending on the line, acclimatization and cultivation in an outdoor field were carried out for 3 years according to the growth.

  In culture growth, the medium (1/2 MS, 1.5% sucrose, 0.2% gellite, hormone-free, pH 5.7) was planted with cuttings from fully rooted individuals, about 4000 lux, 12 hours a day. Long, it was performed under the condition of 22 ° C.

  The culture medium was washed away from the fully rooted cultured seedlings, and the cultured seedlings were planted in vermiculite for acclimatization (panels A to F in FIG. 9).

(2) Inserted buds The side buds of the parent plant that were planted and grown in the open field were folded at about 5 cm, excised leaving 3 to 4 leaves, wrapped in wet newspaper, placed in a beaker, placed in a refrigerator and kept overnight. Then, the newspaper was taken out, put in a polyethylene bag, placed in a beaker, and then stored in a refrigerator. About 2 weeks later, it was inserted into vermiculite placed in a cell tray and grown in a greenhouse for about 4 weeks (panels A to F in FIG. 10).

(3) Open field cultivation and electric warming cultivation In order to confirm the characteristics of the hybrid, outdoor cultivation and electric warming cultivation were performed. Table 5 shows the schedule for outdoor cultivation and electric heating and warming cultivation. Lighting was performed by placing a 100 W incandescent bulb at a height of about 2.0 m at a ratio of 1.5 bulbs to about 10 square meters.

4). Characteristics of hybrids (1) Growth characteristics in open field cultivation and electric warming cultivation 55 hybrids have been bred to date. In 2008, a cultivation test of 31 hybrids (crossing was carried out in 2005) was conducted to investigate the growth characteristics. Comparing the field culture and the electric heating warming cultivation, the outer petal was formed and extended even in the hybrid line without the outer petal in the outdoor field, and the whole plant was enlarged in the electric heating warming cultivation. Table 6 shows the growth characteristics survey. In the table, the number of flower arrival days in the open field is a reference value because the summer solstice is counted as the start, and the sensitivity time for short days is not clear. In the electric heating warming cultivation, the number of flower arrival days from the extinction was the largest in 54 to 60 days, and averaged 8.2 weeks. In addition, the growth failure was noticeable in 4 lines withering after planting in electric lighting and heating, but the wild species used as seed parents were cool and dry. It seemed likely to be inappropriate.

  In addition, FIGS. 11 to 26 show the seedlings, cocoons, strains and flowers of the above 31 hybrids and the survey results.

(2) Morphological features About 21 hybrids cultivated by electric heating in 2007, the uprightness of the strain, the spread of branches, the form of flowers and leaves were compared. FIG. 27 shows a comparison between a seed parent wild species (C. fruticulosa) and a hybrid (No. 22-7). In addition, FIG. 28 to FIG. 32 show the survey results of the hybrids, flowers, and leaves of the 21 strains (2005 crosses).

(3) Aromatic characteristics An aroma survey was carried out in the electric heating warming cultivation in 2007, the outdoor cultivation in 2008 and the electric heating heating cultivation.
(I) Sensory survey The fragrance continued until the flower withered immediately after flowering, and the scent and strength differed depending on the strain, but all hybrids scented more strongly than the pollen parent. As for the type of fragrance of flowers, there were some strains that included a “blue smell” or “bitter” fragrance similar to conventional chrysanthemum, but most strains felt favorably like “sweet” or “herb”. It had a strong scent (FIGS. 11 to 26). In the C.fruticulose line, a sweet honey-like sweetness was felt, and in the C.lavandulifolium line, a slight citrus sweetness was felt.

  The fragrance of the leaves was particularly strong at the time of flowering, and there were differences in the fragrance and strength depending on the line, but all the hybrids scented more strongly than the pollen parent. As for the types of scents of leaves, most of the scents felt favorably like “herbs”, and the tendency to spread to the surroundings by stimulation such as rubbing of leaves was strong.

(Ii) Aroma analysis (a) Aroma analysis of cut flowers The gas chromatographic analysis of the aromas of two hybrid lines (1-8, 22-101) and cultivated Y-4 cultivated by electric heating in 2007 using the headspace method Analyzed graphically.

  20-50 kinds of aroma components were detected. The main component was α-pinene for both hybrid and cultivated species, with the hybrid species accounting for about 53% to about 67% of the total amount of aroma components and 75% for the cultivated species (Table 7). Hereinafter, β-pinene, p-cymene, camphene, and myrcene were commonly included in both species. The characteristic of the cultivated species is that it contains a large amount of p-cymene and γ-terpinene, and this component was also found in hybrids. On the other hand, the hybrid species contained 1,8-cineole, which is not found in cultivated species, as a main component of about 10% to about 27%. Moreover, when the total amount of all the aromatic components is compared, the hybrid leaves release about 30 times the amount of the cultivated species, and the hybrid flowers release about 50 times the amount of the cultivated species. It was.

  Table 8 shows the main aroma components of the flower part of the hybrid 22-4 (C. fruticulose x Chrysanthemum morifolium Ramat.).

(B) Aroma analysis of extract An extract (essential oil) was prepared from two hybrid strains (Nos. 1-8, 22-101) by a conventional method, and the aroma component was analyzed by ion chromatography using a headspace method.

  The analysis results are shown in Table 9. The yield of the extract was about 0.04 wt% for both of the two hybrids, and the constituents of the extract contained a large amount of γ-murolen and sabinol, which are sesquiterpene hydrocarbons.

(C) Aroma analysis in ground planting The aroma of 2 hybrid lines (1-15, 22-4) in the flowering stage cultivated by electric heating in 2008 was gas in the head planting method in the state of ground planting. Analyzed by chromatography.

  To collect aroma, cover only the flower part with a glass hood while the chrysanthemum plant of the present invention is planted locally, connect a Teflon tube and a pump to the hood, and circulate air at a flow rate of 0.5 L / min for 4 hours. The aroma was collected with 50 mg of a collecting agent: Tenax TA filled in a glass tube in the middle of the tube. The collected aroma is subjected to gas chromatography (GC-MS), and the analysis conditions are capillary column TC-WAX 0.25 mmφ × 60 m, initial temperature 40 ° C. → final temperature 230 ° C., carrier gas: helium, inlet temperature: 250 ° C. The analysis was performed at a flow rate of 1 ml / min and an aroma component desorption temperature of 230 ° C.

  Α-pinene, 1,8-cineol, and myrcene, which are the main components in the analysis of cut flowers in (a) above, are contained in an amount of 1% or less, and benzaldehyde and phenylacetoaldehyde have the total amount of aroma components. It accounted for about 10 to about 20%. Other components include linalool, methyl benzoate, limonene, 2-ethylhexanol, cis-3-hexenyl acetate, 2-phenylethyl alcohol, camphor, ethyl acetate, β-ferrandrene, camphene, trans-chrysanthenol, It contained chrysanthenone, trans-chrysantenyl acetate, cis-chrysantenyl acetate, cis-chrysantenol, and the like, and was suggested to be a factor of sweet scent.

Claims (9)

  Chrysanthemum fruticulosa or Chrysanthemum lavandulifolium as a seed parent and Chrysanthemum morifolium Ramat. As a pollen parent, a chrysanthemum genus plant with enhanced aroma.   The plant according to claim 1, wherein the total amount of the fragrance component is 10 times or more of the pollen parent when the fragrance component of the plant is analyzed by ion chromatography using a headspace method.   Benzaldehyde, phenylacetaldehyde, linalool, methylbenzoate, limonene, 2-ethylhexanol, cis-3-hexenyl acetate, α-pinene, 1,8-cineol, myrcene, 2-phenylethyl alcohol, camphor, ethyl acetate, β-ferrane 3. A fragrance component selected from the group consisting of drain, camphene, trans-chrysantenol, chrysantenone, trans-chrysantenyl acetate, cis-chrysantenyl acetate and cis-chrysantenol. Plant.   A progeny of the plant according to any one of claims 1 to 3.   An extract obtained from the plant according to any one of claims 1 to 3, or a progeny of the plant according to claim 4.   The method of producing the plant of any one of Claims 1-3 including crossing Chrysanthemum fruticulosa or Chrysanthemum Lavandulifolium as a seed parent, and Chrysanthemum morifolium Ramat. As a pollen parent.   The method according to claim 6, further comprising selecting a plant having a total amount of aromatic components that is 10 times or more that of the pollen parent when the aromatic components of the plant are analyzed by gas chromatography using a headspace method.   The method according to claim 6 or 7, further comprising vegetative propagation.   The method according to any one of claims 6 to 8, further comprising electric cultivation.

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