JP4886213B2 - Eustoma plant cultivation method - Google Patents

Description

  The present invention relates to a method for cultivating Eustoma plants. Specifically, the present invention relates to a method for flowering a plant belonging to the genus Eustoma in a small container, and a plant belonging to the genus Eustoma obtained by the method.

Eustoma (Eustoma grandiflorum) is a high-quality plant with a plant height of 80 cm or more, and is mainly cultivated in facilities such as open spaces or vinyl houses for cut flowers. In the natural state, it blooms from early summer to summer, so cut flower production is also prosperous in the high temperature period from spring to summer, and flowering in winter is difficult. Eustoma was originally a high-quality plant, but individuals that flowered at low plant heights were discovered, and selection and improvement were added to develop fertile varieties for potting.
On the other hand, for the purpose of providing a new ornamental method, the development of plants that can be admired as they are by cultivating them in a sealed container and blossoming has been promoted for various flowers (for example, Patent Documents 1 to 6). reference). Plants cultivated and flowered in sealed containers (1) Can be enjoyed easily without the need for watering, etc. (2) Small and can be moved appropriately without choosing a place (3) Various advantages such as being able to watch over a relatively long period of time are expected.

Japanese Patent Laid-Open No. 05-207828 JP 05-207829 A Japanese Patent Laid-Open No. 06-078641 JP 07-327534 A Japanese Patent Application Laid-Open No. 08-056514 JP 2001-000060 A

  As a result of the breeding improvement, the current dwarf Eustoma has been improved to the point where it usually blooms at a plant height of about 20 cm. If the plant height at the time of flowering can be lowered to about 10cm, it can be made to bloom in a small container, and its commercial value is high. In view of the above background, an object of the present invention is to provide a cultivation method for flowering an eastoma in a small container.

In view of the above-mentioned object, the present inventors have repeatedly studied to find a cultivation condition that allows the Eustoma genus plant to bloom while suppressing elongation, that is, a cultivation condition that allows the Eustoma genus plant to bloom well with a low plant height. As a result, it became clear that switching to aeration culture at the time when a predetermined period passed after sowing was an effective cultivation condition. It has also been found that increasing the sucrose concentration in the medium in the late stage of culture is an effective cultivation condition. Furthermore, it was also found that the content ratio of ammonia nitrogen and nitrate nitrogen in the nitrogen source in the medium used in the late stage of culture affects the flowering of the eastoma. In addition, it was found that the light irradiation conditions at the time of cultivation were also important for the plant height and flowering rate of the eastoma. Cultivation of dwarf Eustoma plants under the cultivation conditions found as a result of the study allows them to flower in a good condition at a plant height of around 10 cm, and therefore the flowering state (or cocoon state) in a small sealed container ) Eustoma plants can be obtained.
The present invention provides the following configuration based on the above results. That is, the present invention provides a first culturing step for obtaining a plant body by aseptically cultivating a seed of a fertile genus of Eustoma genus for 4 to 12 weeks under non-aerated conditions, and the plant body under aerated conditions. And a second culturing step for carrying out aseptic culture.
In one embodiment of the present invention, 10 to 60 g / l of sucrose as a carbon source prepared in a sealed container having a vent hole 4 to 8 weeks after the start of the second culture step, nitrogen Plants are transplanted to a solid medium containing a source, a phosphate source and a potassium source, and aseptic culture is continued under aerated conditions.
One embodiment of the present invention is characterized in that an MS solid medium having a sucrose content of 10 g / l to 30 g / l is used in the culture before transplantation in the second culture step.
One embodiment of the present invention is characterized in that the sucrose content of a solid medium used for culturing after transplantation in the second culturing step is 20 g / l to 60 g / l.
One embodiment of the present invention is characterized in that the solid medium used for culturing after transplantation in the second culturing step contains ammonia nitrogen and nitrate nitrogen in a molar ratio of 1: 3 to 1:10.
One aspect of the present invention is characterized in that the solid medium used for culturing after transplantation in the second culturing step contains calcium chloride and magnesium sulfate.
One embodiment of the present invention is characterized in that the illuminance during light irradiation in the second culturing step is 4,000 lux to 10,000 lux .
Another embodiment of the present invention is characterized in that the illuminance during light irradiation is 4,000 lux to 10,000 lux throughout the entire culture period.
As another aspect, the present invention provides a plant belonging to the genus Eustoma, which is produced by any of the above cultivation methods and has a plant height of 8 cm to 10 cm with buds or flowers in a sealed container.

  The present invention relates to a method for cultivating a plant belonging to the genus Eustoma, and each of the following culturing steps, that is, a step of obtaining seedlings by aseptically culturing under aerated conditions for 4 to 12 weeks after sowing seeds of fertile genus (First culturing step), characterized in that the seedling body is subjected to a step of aseptic culturing under aerated conditions (second culturing step). Hereinafter, the structure, conditions, etc. are demonstrated in detail for every culture | cultivation process. In addition, about the conditions which are not mentioned in a specification, what is necessary is just to employ | adopt the normal conditions in cultivation of a Eustoma genus plant.

1. First culture step In this culture step, first, seeds of dwarf varieties of the genus Eustoma are sown in an appropriate medium. As the dwarf varieties of the genus Eustoma, for example, the following can be preferably used.
Various varieties of the Tom Sam series (Fukuhanaen Tanae Co., Ltd.) (white thumb, pink thumb, pink picot thumb, etc.)
As shown below, white thumb and pink thumb are registered as varieties.
Name: Product registration number: Product registration date White thumb: No. 6570: July 14, 1998 Pink thumb: No. 6572: July 14, 1998

  The seeds are sown in a medium prepared in advance in a container. For example, a necessary amount of a medium prepared by adding predetermined components is placed in a culture vessel, and then subjected to autoclaving to obtain a sterilized medium. Seeds are sown in the medium thus prepared. As the medium here, for example, an MS solid medium (Murashige-Skoog solid medium) or an equivalent thereof can be used. For the composition of the MS solid medium, see, for example, Introduction to Tissue Culture (Seibundo Shinko). For the solidification of the medium, a known gelling agent such as gellite (Sigma Aldrich (USA)), gellan gum, agar or the like can be used.

  The sucrose concentration in the medium is preferably 20 g / l to 30 g / l. This is because if the sucrose concentration is too low or too high, growth will be poor.

The container used for the culture is not particularly limited as long as it is light transmissive. For example, various containers such as glass, plastic, and polycarbonate can be used. In order to increase the cultivation efficiency, it is preferable to employ a container having a size capable of sowing a plurality of seeds together.
In order to culture in a sterilized state, seeds are usually sown after sterilization. Sterilization can be carried out in a conventional manner. As an example of the sterilization method, a treatment of immersing in an aqueous solution of sodium hypochlorite having an effective chlorine concentration of about 0.5% (usually containing about 0.02% of a surfactant such as Tween 20) for about 10 to 20 minutes can be mentioned. The sterilized seed is sown after being washed several times (for example, three times) with sterilized distilled water.
The temperature at the time of culture shall be suitable for germination and growth of Eustoma plants. Specifically, the light irradiation (day temperature) is, for example, 20 ° C. to 30 ° C., preferably 22 ° C. to 28 ° C., more preferably about 25 ° C., and the light non-irradiation (night temperature) is, for example, 20 ° C. to 30 ° C. The temperature is preferably 22 ° C to 28 ° C, more preferably 22 ° C to 25 ° C.

The light / dark cycle is, for example, a light condition (light irradiation) of 16 hours to 20 hours, and a dark condition (light non-irradiation, dark room) of 4 hours to 8 hours. Instead of continuously irradiating light under bright conditions, light may be irradiated intermittently or intermittently.
The illuminance during light irradiation is, for example, 1,000 lux to 6,000 lux, preferably 2,000 lux to 6,000 lux, and more preferably 4,000 lux to 6,000 lux. In addition, it is good also as the illumination intensity at the time of light irradiation being the same (for example, about 5,000 lux) through the whole culture | cultivation period (1st culture process-2nd culture process). In this way, it is not necessary to adjust the illuminance for each culture process, and the operation is simplified.

The first culture step is performed under non-aerated conditions and aseptic conditions. Therefore, normally, after seeding, the culture vessel is cultured in a sealed state. The culture method of the present invention is characterized by culturing for a predetermined period after sowing under non-aerated conditions (first culture process) and then cultivating by switching to aerated conditions (second culture process). Switching to aeration culture is performed for the purpose of preventing vitrification and suppressing the growth of plant height. As shown in the Examples described later, as a result of the study by the present inventors, it became clear that the aeration culture start time (in other words, the non-aeration culture period) affects the flowering rate, plant height, number of flowers, and flower color. It was. The aeration culture start time in the present invention is set based on the knowledge. Preferably, aeration culture is performed after 4 weeks to 12 weeks after seeding, more preferably after 4 weeks to 10 weeks after seeding, even more preferably after 4 weeks to 8 weeks after seeding, and most preferably after 4 weeks to 6 weeks after seeding. Start.
As a result of the first culturing step, plants having a diameter of 1 cm to 2 cm and a plant height of about 0.5 cm to 1.5 cm are typically obtained.

2. Second culture process In the second culture process, the plant obtained in the first culture process is aseptically cultured under aerated conditions. For example, the plant body obtained in the first culturing step is switched to aeration culture by transplanting the plant body to another container that can be cultured under aeration conditions. As a container suitable for culture | cultivation on aeration conditions, the airtight container which has a vent can be illustrated. Specifically, a sealed container in which a through-hole is provided in a part of the main body or the lid and a gas permeable membrane is coated thereon can be used. You may implement | achieve an aeration state using a cotton plug. If the first culture step is performed in a container that can be switched from non-aeration culture to aeration culture, transplantation is not required when shifting from the first culture step to the second culture step. The material of the container is not particularly limited as long as it is light transmissive. For example, various containers such as glass, plastic, and polycarbonate can be used.

  As the medium here, for example, an MS solid medium or an equivalent thereof can be used. The sucrose concentration in the medium is preferably 10 g / l to 40 g / l. This is because if the sucrose concentration is too low, the flower does not bloom, and if it is too high, the growth is poor.

  In a preferred embodiment of the present invention, the culture medium is switched 4 to 8 weeks after the start of the second culturing step, and aseptic culture under aerated conditions is continued. Thus, one embodiment of the present invention is characterized in that the medium is switched during aeration culture. The medium is switched mainly for the purpose of growth suppression (preventing the plant from becoming too large). For example, the medium is switched by transplanting a plant obtained by culturing for a predetermined period from the start of aeration culture to a predetermined medium prepared in another container. As a container here, the airtight container which has a vent hole can be employ | adopted. The transplantation time is preferably 5 to 7 weeks after the start of the second culture step, and more preferably about 6 weeks after the start of the second culture step. By transplanting at this time, it becomes easy to obtain a plant that blooms at a desired plant height.

As a medium before the medium switching operation (hereinafter referred to as “first medium”), for example, an MS solid medium having a sucrose concentration of 10 g / l to 40 g / l, preferably a sucrose concentration of 20 g / l to 30 g / l, or its Use the equivalent. On the other hand, as a medium after the medium switching (hereinafter referred to as “second medium”), for example, Hyponex medium (powder Hyponex (manufactured by Hyponex Japan Co., Ltd., nitrogen content 6.5%, internal ammonia nitrogen 1%, nitrate nitrogen 5.5%, Sucrose (eg, 10 g / l to 60 g / l) dissolved in an appropriate concentration (eg, 10 g / l to 15 g / l, preferably about 13 g / l) of water-soluble phosphoric acid 6% and water-soluble potassium 19% ), Calcium chloride (for example, 440 mg / l) and magnesium sulfate (for example, 370 mg / l) and solidified) or the like can be used.
For the solidification of each medium, known gelling agents such as gellite (Sigma Aldrich (USA)), gellan gum, agar and the like can be used.

  As a result of the study by the present inventors, the content ratio of ammonia nitrogen and nitrate nitrogen in the nitrogen source in the medium used in the later stage of the aeration culture (that is, the culture after the medium switching operation) is the plant height, the number of flowers, and the flower color. It became clear that it affected. In particular, it has been found that a higher ratio of nitrate nitrogen tends to be preferable in order to obtain a good flower color. Based on these findings, the content ratio of ammonia nitrogen and nitrate nitrogen (ammonia nitrogen: nitrate nitrogen) in the medium (in the second medium) after the medium switching operation is preferably 1: 3 to 1:10. Yes, more preferably 1: 4 to 1: 7, and even more preferably 1: 6. In addition, the content ratio of ammonia nitrogen and nitrate nitrogen in the Hyponex medium is 1: 6.

For the purpose of sufficiently solidifying the medium, it is preferable to use a medium supplemented with calcium chloride and magnesium sulfate as the second medium.
On the other hand, the sucrose content in the medium used in the later stage of the aeration culture (that is, the culture after the medium switching operation) affects the flowering rate, plant height, number of flowers and flower color, and there is an appropriate range of sucrose content. It became clear. Based on this finding, the sucrose concentration in the medium (in the second medium) after the medium switching operation is preferably 10 g / l to 60 g / l, more preferably 20 g / l to 60 g / l, still more preferably 30 g. / l to 60 g / l, even more preferably 40 g / l to 60 g / l, most preferably about 40 g / l.

About the culture | cultivation temperature and light-dark cycle in a 2nd culture process, the conditions similar to a 1st culture process are employable. However, the illuminance during light irradiation is preferably set to be relatively high. This is because when the illuminance is low, the plant height elongation is not sufficiently suppressed. For example, the illuminance is 4,000 lux or more, preferably 4,500 lux or more, more preferably 5,000 lux or more. The upper limit here is not particularly limited, but is, for example, 10,000 lux. Specific examples of illuminance are about 4,000 lux, about 4,500 lux, about 5,000 lux, and about 5,500 lux.
When the medium is switched during the second culture step as described above, the culture temperature and / or the light / dark cycle may be the same or different before and after the medium switching operation. From the viewpoint of simplifying the operation, it is preferable to unify the culture temperature and the light / dark cycle conditions during the second culture step.

Typically, by continuing the culture before the medium switching operation for 4 to 8 weeks, a plant body having a diameter of about 2.0 cm to 4.0 cm and a plant height of about 1.5 cm to 3.0 cm is obtained. After culturing for 4 to 10 weeks, a plant body having a diameter of 5 cm to 7 cm and a plant height of about 8 cm to 10 cm can be obtained. In order to moderately suppress the elongation, the culture period before the medium switching operation is preferably about 5 weeks to about 7 weeks.
After the medium switching operation, water is supplied to the medium as necessary. For example, an amount of water corresponding to the amount of water decrease in the medium is replenished every 2 to 4 weeks. Water may be replenished to the medium even before the medium switching operation.

  In one aspect of the present invention, as described above, transplantation is performed twice in total, that is, transplantation associated with switching from the first culture step to the second culture step and transplantation associated with medium switching during the second culture step. However, transplantation is performed by omitting transplantation between the first culture step and the second culture step, for example, or by performing transplantation twice or more during the second culture step, for example. The number of times may be three or more. However, in terms of cultivation efficiency, it can be said that a total of 2 transplants is appropriate.

  As another aspect, the present invention provides a plant of the genus Eustoma having a cocoon or a flower in a sealed container produced by any one of the above cultivation methods. A plant of the genus Eustoma provided by the present invention is typically characterized in that it has a plant height of about 8 cm to 10 cm and has a bud or flowering state.

<Examination of cultivation conditions for Eustoma>
The following experiments were conducted in order to find out the conditions for flowering the eusumae under a lower plant height than before.
1. The effect of the start of aeration culture on flowering of osteoma.The optimal time for disclosure of aeration culture is predicted by the following method based on the prediction that the timing of switching from non-aeration culture to aeration culture after sowing is an important factor for flowering of osteoma. investigated.
(1) Materials and methods Variety: Pink Picotisam (Tom Sam Series, Fukuhanaen Tanae Co., Ltd.)
Sowing: Seed is immersed in an aqueous solution of sodium hypochlorite (containing 0.02% Tween20) with an effective chlorine concentration of 0.5% for 15 minutes, washed three times with sterile distilled water, and then the MS solid in a glass container (mayonnaise bottle) It was seed | inoculated to the culture medium (sucrose content 30g / l).
Transplantation: Switching from non-aeration culture to aeration culture at the time of the first transplantation, 8 test zones (transplantation from 2nd to 16th week after seeding) will be provided according to the difference of the first transplantation time (starting time of aeration culture) It was. A second transplant was performed 10 weeks after sowing. Aeration culture was continued after the second transplantation. In the control group, aeration culture was performed throughout the entire culture period.
Medium: MS solid medium (sucrose content 30 g / l) was used for seeding and for the first transplantation. Hyponex medium (powder Hyponex (Hyponex Japan, Inc., nitrogen content 6.5%, internal nitrogen 1%, nitrate nitrogen 5.5%, water-soluble phosphate 6%, water-soluble potassium 19%) 13 g / l added with calcium chloride 440 mg / l, magnesium sulfate 370 mg / l, sucrose 40 g / l, gellite 3 g / l). Each medium was sterilized (autoclaved) after adjusting the pH to about 5.8 before use.
Culture temperature: 25 ° C. during illumination and 25 ° C. during non-illumination (dark conditions) throughout the entire culture period.
Illumination conditions: 16 hours (4 o'clock to 20 o'clock) illumination (continuous light irradiation) throughout the entire culture period. Illuminance during lighting was about 5,000 lux.
Evaluation items: Flowering rate, number of days required for flowering, plant height, number of flowers, and flower color were evaluated. In addition, 20 individuals were prepared for each test group, and the average value thereof was used for each evaluation. As for the flower color, the evaluation point is 0 for white as a whole, 1mm for the tip of the petal is dark pink and the background color is white (pico tea bloom), 1 for the background color, and 3mm for the tip of the petal is dark pink and the ground color is white ( A typical pico tea bloom) was rated 2, the petal tip 1/2 was rated light pink, and the whole petal was rated pink 4.
(2) Results Relationship between aeration culture start time and flowering rate (Fig. 1), relationship between aeration culture start time and number of days required for flowering (Fig. 2), relationship between aeration culture start time and plant height (Fig. 3), aeration culture start time And the relationship between the number of flowers (FIG. 4) and the relationship between the start time of aeration culture and the flower color (FIG. 5). The following matters are derived from the graphs of FIGS.
The group that started aeration culture 4 weeks after sowing (4 weeks) had the highest flowering rate (FIG. 1). Next, the flowering rate in the 6-12 week group was high, and the flowering rate in the 2 week group was slightly low (FIG. 1). In the 14-week group, the 16-week group, and the non-ventilated group, the flowering rate was extremely low or did not flower (FIG. 1).
When the number of days from the second transplant to flowering was examined for the flowered individuals, it was about 70 days within the scope of this experiment, regardless of the timing of starting aeration culture (FIG. 2).
The plant height was shortened to about 8 cm in 2, 4, and 6 weeks (FIG. 3). On the other hand, when aeration was started after 8 weeks after sowing, it became longer than 8 cm (FIG. 3).
The number of flowers that arrived per individual (number of flowers) was 3 or more in 4 to 10 weeks (FIG. 4). In 2 weeks and 12 weeks, the number of flowers slightly decreased (FIG. 4).
The flower color was the original pico tea blooming until the 10th week (the ground color is white and the tip of the petal is colored), but in the 12th week and the 16th week the pink colored part becomes larger and the pico tea does not bloom. A trend was observed (Figure 5).
From the above results, the timing of starting aeration culture is preferably 4 to 12 weeks later, more preferably 4 to 10 weeks later, more preferably 4 to 8 weeks later, and most preferably 4 weeks after sowing. You can conclude that it is later.

2. Effect of medium composition on flowering of eustasis Optimum medium composition based on the prediction that the composition of the medium used in the late stage of culture (the medium used for the second transplantation in this experiment) is an important factor for flowering of eustoma The following method was used to examine.
(1) Materials and methods Variety: Pink Picotisam (Tom Sam Series, Fukuhanaen Tanae Co., Ltd.)
Sowing: Seed is immersed in an aqueous solution of sodium hypochlorite (containing 0.02% Tween20) with an effective chlorine concentration of 0.5% for 15 minutes, washed three times with sterile distilled water, and then the MS solid in a glass container (mayonnaise bottle) It was seed | inoculated to the culture medium (sucrose content 30g / l).
Transplantation: The first transplantation was performed 6 weeks after seeding, and aeration culture was performed after transplantation. A second transplant was performed 10 weeks after sowing. Aeration culture was continued after the second transplantation.
Medium: MS solid medium (sucrose content 30 g / l) was used for seeding and for the first transplantation. Depending on the medium used for the second transplantation, there are 3 test groups, namely MS (1) group (MS medium, ammonia nitrogen: nitrate nitrogen = 1: 2), MS (2) group (based on MS medium) , Ammonia nitrogen: nitrate nitrogen = 1: 6) and Hypo section (Hyponex medium, ammonia nitrogen: nitrate nitrogen = 1: 6). Each medium was sterilized (autoclaved) after adjusting the pH to about 5.8 before use.
Culture temperature: 25 ° C. during illumination and 25 ° C. during non-illumination (dark conditions) throughout the entire culture period.
Illumination conditions: Illumination (continuous light irradiation) was performed for 16 hours (4 o'clock to 20 o'clock) throughout the entire culture period. Illuminance during lighting was about 5,000 lux.
Evaluation items: Plant height, number of flowers, and flower color were evaluated. In addition, 13 individuals were prepared for each test group, and the average value thereof was used for each evaluation. As for the flower color, the evaluation point is 0 for white as a whole, 1mm for the tip of the petal is dark pink and the background color is white (pico tea bloom), 1 for the background color, and 3mm for the tip of the petal is dark pink and the ground color is white ( A typical pico tea bloom) was rated 2, the petal tip 1/2 was rated light pink, and the whole petal was rated pink 4.
(2) Results The relationship between the medium composition and plant height (FIG. 6), the relationship between the medium composition and the number of flowering (FIG. 7), and the relationship between the medium composition and flower color (FIG. 8) are shown. The following matters are derived from the graphs of FIGS.
The plant height was the shortest in the Hypo group, followed by the MS (2) group (ammonia nitrogen: nitrate nitrogen = 1: 6) (FIG. 6).
The number of flowering was high in the Hypo ward, and decreased in both the MS (1) and MS (2) wards (FIG. 7).
In the Hypo and MS (2) wards, the flower color was originally bloomed in the pico tea, but in the MS (1) ward, the whole petal became light pink and did not bloom (Fig. 8). This result is estimated as follows when considered together with the experimental results of sugar concentration described below. Ammonia nitrogen: nitrate nitrogen = 1: 2 MS (1) medium (general MS medium), because the amount of plant growth is large, sugar contained in the medium is absorbed early, and flower buds develop During the flowering period, the sugar concentration in the medium decreased, and as a result, it was thought that the picotee did not bloom.
From the above results, it is preferable to use a medium having a high nitrate nitrogen ratio in the nitrogen component as the medium used for the second transplantation. Specifically, it can be concluded that a hyponex medium is appropriate.

3. The effect of sucrose concentration in the medium on the flowering of the eustema Predicted that the sucrose concentration in the medium used in the late stage of the culture (the medium used for the second transplantation in this experiment) is an important factor for the flowering of the euthomas Under the above, the optimum sucrose concentration was examined by the following method.
(1) Materials and methods Variety: Pink Picotisam (Tom Sam Series, Fukuhanaen Tanae Co., Ltd.)
Sowing: Seed is immersed in an aqueous solution of sodium hypochlorite (containing 0.02% Tween20) with an effective chlorine concentration of 0.5% for 15 minutes, washed three times with sterile distilled water, and then the MS solid in a glass container (mayonnaise bottle) It was seed | inoculated to the culture medium (sucrose content 30g / l). Aeration was started 4 weeks after sowing.
Transplantation: The first transplantation was performed 6 weeks after seeding, and aeration culture was continued. A second transplant was performed 10 weeks after sowing. Aeration culture was continued after the second transplantation.
Medium: MS solid medium (sucrose content 30 g / l) was used for seeding and for the first transplantation. The medium used for the second transplantation is a Hyponex medium containing a predetermined concentration of sucrose (powder Hyponex (manufactured by Hyponex Japan, nitrogen content 6.5%, internal ammonia nitrogen 1%, nitrate nitrogen 5.5%, water-soluble) Phosphoric acid 6%, water-soluble potassium 19%) 13g / l with calcium chloride 440mg / l, magnesium sulfate 370mg / l, sucrose with the prescribed concentration and gellite 3g / l)) 7 test zones (sucrose concentration 0-60 g / l) were prepared. Each medium was sterilized (autoclaved) after adjusting the pH to about 5.8 before use.
Culture temperature: 25 ° C. during illumination and 25 ° C. during non-illumination (dark conditions) throughout the entire culture period.
Illumination conditions: Illumination (continuous light irradiation) was performed for 16 hours (4 o'clock to 20 o'clock) throughout the entire culture period. Illuminance during lighting was about 5,000 lux.
Evaluation items: Flowering rate, number of days required for flowering, plant height, number of flowers, and flower color were evaluated. In addition, 13 individuals were prepared for each test group, and the average value thereof was used for each evaluation. As for the flower color, the evaluation point is 0 for white as a whole, 1mm for the tip of the petal is dark pink and the background color is white (pico tea bloom), 1 for the background color, and 3mm for the tip of the petal is dark pink and the ground color is white ( A typical pico tea bloom) was rated 2, the petal tip 1/2 was rated light pink, and the whole petal was rated pink 4.
(2) Results Relationship between sucrose concentration and flowering rate (Fig. 9), relationship between sucrose concentration and number of days required for flowering (Fig. 10), relationship between sucrose concentration and plant height (Fig. 11), sucrose concentration and number of flowers (FIG. 12) and the relationship between sucrose concentration and flower color (FIG. 13). The following matters are derived from the graphs of FIGS.
When the sugar concentration in the medium was 5 g / l or less, it hardly flowered (FIG. 9). On the other hand, at 10 g / l or more, the flowering rate was 50% or more. In this range, the flowering rate is high under the conditions of sugar concentrations of 10 g / l, 20 g / l, and 40 g / l (FIG. 9).
When the number of days from the second transplant to flowering was examined for the flowered individuals, it was about 70 days regardless of the sugar concentration (FIG. 10).
The plant height became shorter as the sugar concentration in the medium increased, and was about 8 cm under the conditions of 40 g / l and 60 g / l (FIG. 11).
The number of flowers increased as the sugar concentration in the medium increased (FIG. 12). The number was 3 or more under the conditions of 40 g / l and 60 g / l (FIG. 12).
The flower color bloomed with a sugar concentration of 20 g / l or more, but when it was 10 g / l or less, the whole was colored pale pink and did not bloom (Fig. 13). In order to obtain a good flower color, a sugar concentration of 15 g / l or more is considered necessary.
From the above results, the concentration of sucrose added to the hyponex medium used for the second transplantation is preferably 10 g / l to 60 g / l, more preferably 20 g / l to 60 g / l, still more preferably It can be concluded that it is 30 g / l to 60 g / l, even more preferably 40 g / l to 60 g / l, most preferably about 40 g / l.

4). The effect of illuminance on the flowering of the eustoma The optimal illuminance was examined by the following method, assuming that the illuminance during lighting was an important factor for the flowering of the eustoma.
(1) Materials and methods Variety: Pink Picotisam (Tom Sam Series, Fukuhanaen Tanae Co., Ltd.)
Sowing: Seed is immersed in an aqueous solution of sodium hypochlorite (containing 0.02% Tween20) with an effective chlorine concentration of 0.5% for 15 minutes, washed three times with sterile distilled water, and then the MS solid in a glass container (mayonnaise bottle) It was seed | inoculated to the culture medium (sucrose content 30g / l). Aeration was started 4 weeks after sowing.
Transplantation: The first transplantation was performed 6 weeks after seeding, and aeration culture was continued. A second transplant was performed 10 weeks after sowing. Aeration culture was continued after the second transplantation.
Medium: MS solid medium (sucrose content 30 g / l) was used for seeding and for the first transplantation. Hyponex medium (powder Hyponex (Hyponex Japan, Inc., nitrogen content 6.5%, internal nitrogen 1%, nitrate nitrogen 5.5%, water-soluble phosphate 6%, water-soluble potassium 19%) 13 g / l added with calcium chloride 440 mg / l, magnesium sulfate 370 mg / l, sucrose 40 g / l, gellite 3 g / l). Each medium was sterilized (autoclaved) after adjusting the pH to about 5.8 before use.
Culture temperature: 25 ° C. during illumination and 25 ° C. during non-illumination (dark conditions) throughout the entire culture period.
Illumination conditions: Illumination (continuous light irradiation) was performed for 16 hours (4 o'clock to 20 o'clock) throughout the entire culture period. Four test zones (fluorescent lamp number 1: about 2,000 lux, fluorescent lamp number 2: about 3,000 lux, fluorescent lamp number 3: about 5,000 lux, fluorescent lamp number 4: about 6,500 lux) were established depending on the difference in illuminance during lighting. .
Evaluation items: Flowering rate, plant height, and number of flowers were evaluated. In addition, 10 individuals were prepared for each test group, and the average value thereof was used for each evaluation.
(2) Results The relationship between illuminance and flowering rate (FIG. 14), the relationship between illuminance and plant height (FIG. 15), and the relationship between illuminance and number of flowers (FIG. 16) are shown. The following matters are derived from the graphs of FIGS.
The number of fluorescent lamps installed on the culture shelf was 3 lights (illuminance of about 5,000 lux) or more, the flowering rate was high, and the plant height was shortened (FIGS. 14 and 15). The highest flowering rate was obtained when three fluorescent lamps were used. On the other hand, although there was no significant difference in the number of flowers due to the difference in the number of fluorescent lamps, the maximum number of flowers was shown when three fluorescent lamps were used (FIG. 16).
From the above results, it is preferable that the illuminance at the time of illumination is high, and specifically 5,000 lux or more is considered appropriate. Considering the flowering rate and the number of flowers, the optimal number of fluorescent lamps installed on the culture shelf is 3 (about 5,000 lux).

<Preparation of an flower that blooms in a small container>
Eustoma was cultivated under the conditions considered to be optimal from the above experimental results. The specific cultivation procedure was as follows.
(1) The seeds of Pink Picotisam (tomsum series, Fukuhanazono Tanae Co., Ltd.), a dwarf cultivar, are seeded in an aqueous solution of sodium hypochlorite (containing 0.02% Tween20) with an effective chlorine concentration of 15% After soaking and washing three times with sterilized distilled water, it was seeded on a sterilized MS solid medium (sucrose content 30 g / l, pH before sterilization, about 5.8) in a polycarbonate plant box (360 ml). In addition, 36 seeds were sown together at intervals. After sowing, the plant box is transferred to a culture shelf equipped with a fluorescent lamp and placed in a light-dark cycle environment with 16 hours of illumination (illuminance of about 5,000 lux, temperature of about 25 ° C) and 8 hours of non-lighting (temperature of about 25 ° C). It was. This light-dark cycle was adopted throughout the entire culture period.
(2) Switching to aeration culture 4 weeks after sowing. The first transplantation was performed 6 weeks after sowing. In other words, 5 seedlings grown in a strain with a diameter (leaf spread) of about 1.5 cm and a plant height of about 1 cm were prepared in a sterilized MS solid medium (sucrose content 30 g / l, Transplanted to a pH of about 5.8)) before sterilization. The ventilation state was ensured by attaching two 1 cm diameter ventilation membranes (for example, Millisea manufactured by Millipore) to the lid of the mayonnaise bottle.
(3) The second transplantation was performed 4 weeks after the first transplantation (10 weeks after sowing). In other words, one seedling that grows to about 3 cm in diameter (leaf spread) and about 2 cm in height is the same container as the mayonnaise bottle used for the first transplant (sealed container with a breathable membrane on the lid). Sterilized Hyponex medium (powdered Hyponex (manufactured by Hyponex Japan, nitrogen content 6.5%, internal nitrogen 1%, nitrate nitrogen 5.5%, water-soluble phosphate 6%, water-soluble potassium 19%) It was transplanted to 13 g / l with calcium chloride 440 mg / l, magnesium sulfate 370 mg / l, sucrose 40 g / l, gellite 3 g / l, pH before sterilization about 5.8)). Aeration conditions were the same as those after the first transplantation.
(4) After the second transplantation, 10 ml of sterilized water was replenished to the medium in the container at intervals of 3 to 4 weeks.

  By the above cultivation procedure, about 16 weeks after sowing, the plant grew to a plant with a plant height of about 8 cm (average value) and a flowering number of about 3.5 (average value). It was. In this way, we succeeded in flowering the flowers in good condition with a plant height of 10 cm or less in a closed container.

  According to the cultivation method of the present invention, it is possible to blossom in a state where the plant height is low. Therefore, it is possible to obtain a flowering state (or cocoon state) elastomer in a small container. The cultivation method of the present invention, that is, the method of blooming the eusta in an airtight container is a kind of plant tissue culture, and can be flowered all year round regardless of the season by cultivating under artificial lighting in a temperature-controlled room. . In carrying out the method of the present invention, everything other than the container can be made of a flammable material. If such a form is adopted, disposal after viewing is easy. As described above, the elastomer obtained by the method of the present invention has a new form different from cut flowers and potted plants, and has a very high commercial value.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The graph shows the relationship between the start of aeration culture and the flowering rate. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the start time of aeration culture and the number of days required for flowering is shown in a graph. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The graph shows the relationship between the aeration culture start time and the plant height. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The graph shows the relationship between the aeration culture start time and the number of flowers. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the aeration culture start time and the flower color is shown in a graph. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the medium composition and plant height is shown in a graph. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the medium composition and the number of flowers is shown in a graph. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the medium composition and flower color is shown in a graph. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the sugar concentration in the medium and the flowering rate is shown in a graph. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the sugar concentration in the medium and the number of days required for flowering is shown in a graph. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the sugar concentration in the medium and the plant height is shown in a graph. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the sugar concentration in the medium and the flowering rate is shown in a graph. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the sugar concentration in the medium and the flower color is shown in a graph. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the illuminance during illumination and the flowering rate is shown in a graph. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the illumination intensity and the plant height is shown in a graph. It is the result of the experiment implemented in order to determine the cultivation conditions of an eustoma. The relationship between the illuminance during illumination and the number of flowers is shown in a graph.

Claims (9)

A first culturing step for obtaining a plant body by aseptic culture under non-aerated conditions for 4 to 12 weeks after sowing seeds of a fertile genus of Eustoma;
A second culturing step for aseptically culturing the plant body under aeration conditions;
A method for cultivating Eustoma plants, comprising:   Four to eight weeks after the start of the second culturing step, 10 g / l to 60 g / l of sucrose prepared as a carbon source in a sealed container having a vent, a nitrogen source, a phosphate source, and The cultivation method according to claim 1, wherein the plant body is transplanted to a solid medium containing a potassium source, and aseptic culture is continued under aerated conditions.   The cultivation method according to claim 2, wherein an MS solid medium having a sucrose content of 10 g / l to 30 g / l is used in the culture before transplantation in the second culturing step.   The cultivation method according to claim 2 or 3, wherein a sucrose content of the solid medium used for culturing after transplantation in the second culturing step is 20 g / l to 60 g / l.   5. The solid medium used for culturing after transplantation in the second culturing step contains ammonia nitrogen and nitrate nitrogen in a molar ratio of 1: 3 to 1:10. The cultivation method in any one of.   The cultivation method according to any one of claims 2 to 5, wherein the solid medium used for culturing after transplantation in the second culturing step contains calcium chloride and magnesium sulfate. The cultivation method according to any one of claims 1 to 6, wherein the illuminance at the time of light irradiation in the second culturing step is 4,000 lux to 10,000 lux . The cultivation method according to any one of claims 1 to 6, wherein the illuminance at the time of light irradiation is 4,000 lux to 10,000 lux throughout the entire culture period. A plant of the genus Eustoma having a plant height of 8 cm to 10 cm in a state of having a bud or a flower in a sealed container, produced by the cultivation method according to claim 1.

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