JP5177349B2 - Raising seedlings - Google Patents

Description

  The present invention relates to a method for raising a genus genus plant, and more particularly to a method for raising a genus genus plant by micropropagation.

  Patent Document 1 is known as a method for raising a genus Vaccinium by micropropagation. Patent Document 1 targets blueberries as a genus plant of the genus Genus. In Patent Document 1, blueberry buds (buds differentiated into leaf buds) are aseptically cultured in a medium supplemented with a plant growth regulator (hereinafter referred to as “PGR”) to induce multi-buds, and dividedly transplanted into the same composition medium. Repeat aseptic growth, transplant the proliferated polyblasts to a non-added medium, induce bud elongation under aseptic conditions, insert the extended shoots into cell trays and induce rooting under non-sterile conditions, rooting A seedling raising method is disclosed in which acclimatization is continued in a culture room environment and then acclimatization in an outside air environment. On the other hand, Patent Document 2 discloses a suitable support (culture soil) for efficiently transferring from acclimatization to acclimatization in plant tissue culture and obtaining adult seedlings. It is described that a healthy plant can be grown by providing a vent hole in a container to be used and sealing the vent hole with a sterilized gas-permeable porous membrane to control ventilation. In Non-Patent Document 1, although the target plant is melon, in order to increase the yield when acclimatizing the redifferentiated individuals from the tissue cultured cells, a vent hole is provided in the redifferentiation container, and this vent hole is provided. A technique for suppressing vitrification that tends to occur in melon by sealing with a sterile gas-permeable membrane (made by Nihon Millipore, trade name “Milliseal”) and ventilating is disclosed. Non-Patent Document 2 describes that a shoot produced in a medium containing zeatin is directly transplanted into rooting-inducing culture soil.

The blueberry seedling method of Patent Document 1 is such that after the shoots are induced, the shoots are proliferated, and then the shoots are transplanted into a PGR-free medium to induce bud elongation, thereby increasing the yield of cuttings. It is a method to plan. Here, acclimatization after induction of rooting by the cell tray is performed in two stages, and in the first stage, as a preliminary acclimatization, the acclimatization is performed in a culture room environment under non-sterile conditions. Acclimatization in the open air. For this reason, there are many processes for the induction of seedlings, proliferation, extension, rooting, first acclimatization, and second acclimatization, and the entire seedling raising, which is complicated and requires a long time. Patent Document 2 and Non-Patent Document 1 disclose a technique in which a vent is provided in a container and the vent is sealed with a sterile gas vent membrane to ventilate the tissue culture for efficient acclimatization. . However, in Patent Document 2, imparting air permeability to the container only shows promotion of rooting and promotion of plant growth at the time of rooting. Non-Patent Document 1 is a technique mainly related to vitrification prevention of melon seedlings. Non-Patent Document 2 discloses a technique for transplanting shoots produced in a zeatin-containing medium to rooting-inducing culture soil. However, shoot growth after growth of multi-buds can be performed using a PGR-containing medium or a non-containing medium. It is a conventional technique to do it selectively.
JP2003-304765 JP2001-352850 Yoshiyuki Honma et al. “Overcoming Vitrification of Melon-Determined Embryos and Reduction of Seedling Period by Intrabottle Conditioning” Bull. Shizuoka Agr. Exp. Stn. [36] 75-85 (1991) Lisa J. Rowland et al. “Use of a Cytokinin Conjugate for Efficient Shoot Regeneration from Leaf Sections of Highbush Blueberry” HortScience 27 [10] 1127-1129 (1992)

  The object of the present invention is to solve the above-mentioned problems in the prior art, particularly the problem that the seedling raising process is complicated and requires a long time. That is, an object of the present invention is to provide a more efficient seedling raising method by micropropagation of a genus Genus.

  The method for raising a genus plant of the present invention includes the following steps (1) to (4).

Item 1: (1) A step of inducing a multi-bud by culturing a tissue piece of a shoot apex of a genus genus plant in a closed system in a medium containing a saccharide, a plant growth regulator, and a support,
(2) Dividing and transplanting the polyblasts induced in step (1) into a medium containing a saccharide, a plant growth regulator, and a support, and culturing under sterile or sterilized conditions while controlling ventilation; A process of multiplying multi-buds and making them healthy,
(3) A step of collecting cuttings from the shoots of the multi-buds that have become healthy seedlings in the step (2), cutting them into a culture soil, culturing them in a culture room environment, and inducing rooting; 4) A step of culturing and acclimatizing the seedling rooted in step (3) in an open air environment,
A method for raising seedlings of the genus Genus

In addition, the following specific embodiments are included in the method for raising seedlings of the genus Genus of the present invention:
Item 2. Item 2. The seedling raising method according to Item 1, wherein the genus plant is a blueberry.
Item 3. Item 3. The seedling raising method according to Item 1 or 2, wherein in step (1), tissue culture is performed in a medium containing a saccharide, a plant growth regulator, a support and an antibiotic.
Item 4. In step (1), tissue culture is performed by using sucrose as a saccharide, 1 to 10% by weight, zeatin as a plant growth regulator, 1 to 10 mg / L, agar as a support, 0.8 to 2% by weight, and antibiotics. Item 4. The seedling raising method according to Item 3, which is carried out using an MW medium containing 0.1 to 0.5% by weight.
Item 5. In step (2), culturing is carried out by using saccharides as sugars, glucose, fructose, maltose, galactose, mannitol, lactose, and sorbitol at least one selected from the group consisting of 1 to 10% by weight, and zeatin as a plant growth regulator. , A MW medium containing 0.1 to 10 mg / L of at least one selected from the group consisting of kinetin, 2-isopentenyladenine, and benzyladenine, and 0.8 to 2% by weight of agar as a support Item 5. The seedling raising method according to any one of Items 1 to 4, which is performed using
Item 6. Item 6. The seedling raising method according to any one of Items 1 to 5, wherein in the step (2), ventilation is controlled through a ventilation hole provided in the culture container.
Item 7. Item 7. The seedling raising method according to item 6, wherein the ventilation in step (2) is controlled by applying a breathable aseptic or sterilized film to a vent provided in the culture container so as to cover it.
Item 8. Item 7. The seedling raising method according to any one of Items 1 to 6, wherein in step (2), ventilation is controlled so that the ventilation frequency increases stepwise or continuously in a range of ventilation frequency of 0 to 15 times / hour.
Item 9. Item 9. The seedling raising method according to any one of Items 1 to 8, wherein in the step (3) and the step (4), the cultivation is performed using a culture soil containing shirasu.

The seedling raising method by micropropagation of the genus Genus in the present invention has the following effects or advantages.
(1) After the multi-bud induction process, the process before the rooting induction process is shortened by controlling the ventilation of the PGR-added medium container and performing growth and healthy seedling in one step.
(2) The acclimatization process is made efficient and the growth of the plant is promoted by making the multi-buds healthy seedlings before the root induction process.

Plants targeted by the seedling raising method of the present invention are Ericaceae and Vaccinium plants. Vaccinium plants are further classified into 10 sections. Of these, the fruits are used in five sections, including bilberry, cranberry, bilberry, blueberry, and the like: cyanococus section, myrtils section, oxicocus section, bitesuidia section, and baccinium section.

Blueberries, this genus Vaccinium, is an American native deciduous or evergreen shrubs or semi-tall fruit trees belonging to the Saianokokasu clause (Cyanococcus). There are many species of blueberries, roughly divided into six species, but the following three are important for fruit gardening.

(1) Highbush blueberry ( Vaccinium corymbosum L.): O'Neill, Sharp Blue, Georgia Gem, Flounder, Leveley, Spartan, Darrow, Duke, Barclay, Harrison, etc.
(2) Rabbiteye blueberry ( V. ashei Reade): Woodard, Garden Blue, Tiff Blue, Home Bell, Meyer, etc.
(3) low-bush blueberry (Lowbush blueberry, V angustifolium Aiton; V. myrtilloides Michaux.): Chigunekuto, bronze wick, Buromidon like.

  Blueberries were originally improved from wild species by the US Department of Agriculture or State University since the beginning of the 20th century. The direction of varieties improvement is easy to cultivate, and it is promoted mainly for the production of fresh fruits, which are considered to be good varieties from the viewpoint of large and sweet fruits. Today, they are cultivated in various countries around the world. The blueberry used in the present invention is not particularly limited in terms of its type, origin, origin, etc. In addition, blueberries obtained by the method of Japanese Patent Application Laid-Open No. 2007-22929 can also be used.

  Next, preferred embodiments of the present invention will be described in detail with reference to the drawings. FIG. 1 shows the flow of a seedling raising method by micropropagation according to the present invention (S1 → S2 → S3 → S4).

(1) Multiblast induction process (S1):
The said process is a process of inducing | guiding | deriving a multi-bud with respect to a genus plant. The “multi-bud” refers to those having approximately 10 or more buds.

  Specifically, this step is performed by tissue-cultivating a stem piece of a genus genus plant in a medium containing a saccharide, a plant growth regulator (PGR), and a support (shoot tip culture). Can do.

  Here, as a basic medium used for the above-mentioned shoot apex culture, Woody Plant medium (WP medium), Murashige & Skoog medium (MS medium) widely used for plant tissue culture, a combination of these media, or a composition thereof was modified. A medium can be mentioned. Preferable examples include MW medium (1 / 2MS medium + 1 / 2WP medium) in which MS medium and WP medium are combined.

  In the multi-bud induction process of the present invention, a medium in which sugars, PGR, and a support are added to such a basic medium is used. Examples of the saccharide include sugars such as sucrose, glucose, fructose, galactose, lactose, and sorbitol; and sugar alcohols such as maltose and mannitol. These may be used alone or in any combination of two or more. Sucrose is preferable.

  Examples of PGR include naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-cyclophenoxyacetic acid (2,4-DPA), indole-3-propionic acid ( Auxins such as IPA), benzofuran-3-acetic acid (BFA), and derivatives thereof; and benzyladenine (BA), kinetin, zeatin, 2-isopentenyladenine (2iP), (2-chloro-4-pyridyl) And cytokinins such as 3-phenylurea (4PU). These may be used alone or in any combination of two or more. Preferably, zeatin is used as shown in Experimental Example 1, and zeatin alone is particularly suitable.

  Here, the support is a component necessary for solidifying the medium, and a base used in a normal solid medium, for example, a gelling agent such as agar or gellan gum can be used. Preferably, agar is used practically.

  The concentration of these components in the medium varies depending on the species of the genus genus plant and cannot be specified unconditionally. For example, when an MW medium is used as the basic medium, the concentration of saccharide (preferably sucrose) is 1 to 10 weights. %, Preferably 2 to 3% by weight; PGR (preferably zeatin) concentration of 1 to 10 mg / L, preferably 2 to 5 mg / L; Support (preferably agar) concentration of 0.8 to 2% by weight Preferably, 0.8 to 1.0% by weight can be mentioned. In addition, this ratio means the total amount, when using each in combination of 2 or more types of saccharides, PGR, or a support body.

  In addition, an antibiotic such as PPM (Plant Preservative Mixture, trade name, manufactured by Nacalai Tesque Co., Ltd.) can be added to these media as other components in order to prevent the generation of mold. When the PPM is used, the total concentration in the medium is usually 0.1 to 0.5% by weight, preferably 0.1 to 0.3% by weight.

  A medium containing these components is sterilized according to a conventional method when used, and is used in a sterile state.

  The shoot tip culture is preferably performed in a closed system (airtight state) and under aseptic conditions. Specifically, the above-mentioned medium prepared in a sterilized state in a container is planted with a piece of tissue at the top of the stem of a genus genus plant, sealed with a lid, and cultured aseptically to induce multi-buds. To do.

The tissue piece at the top of the shoot is not limited, but for example, a cypress plant grown in the field, collected from, for example, an annual branch and sterilized is used. Here, the sterilization treatment is not particularly limited and can be performed according to a conventional method. For example, after rinsing with running water and ultrapure water, it is immersed in a sodium hypochlorite solution of about 5% and then sterilized and distilled. A method of washing with water can be mentioned. The shoot tip culture is preferably carried out at a temperature of 20 to 25 ° C., a relative humidity of 100%, a light period of 12 to 16 hours, and a dark period of 8 to 12 hours. In addition, the photon number of illumination used in the light period is not particularly limited, but it is usually preferable to adjust in the range of 5 to ²⁰ μmol / m ² / s. Such culture can be performed until the number of buds is about 10. Although the period varies depending on the plant species, it cannot be specified, but it can usually include about 2 to 5 months.

(2) Multiproliferation / Naturalization Step (S2):
This step is a step of growing the multi-buds obtained in the above step (S1) and then turning them into healthy seedlings. In addition, “making a healthy seedling” means bringing a plant tissue or the like into a state close to that grown in an outside air environment.

  Specifically, this step can be carried out by dividing and transplanting the multiblast obtained in the above step (S1) into a medium and culturing while controlling ventilation. Divided transplantation is carried out by dividing the polyblast into about 2 to 10 so as to be 0.5 to 0.2 g, and placing this on a medium.

  For the culture in this step, it is efficient to use the same medium as that used in the above step (S1), but it is preferable to use a medium that does not contain antibiotics. Saccharides are not necessarily required and can be used in the range of 0 (no addition) to 10% by weight, but it is preferable to add saccharides such as sucrose. As the PGR, those used in the step (S1) can be used in the same manner. Preferably, at least one selected from the group consisting of zeatin, kinetin, 2iP, and BA is used in a total amount of 0.1 to 10 mg. It is practical to use at a ratio of / L. More preferably, as shown in Experimental Example 2, it is effective to use zeatin alone, particularly at a rate of 2 to 5 mg / L. As the support, those used in the step (S1) can be similarly used, but it is practically preferable to use agar at a ratio of about 0.8 to 2% by weight.

  In this step (S2), the split transplanted strain is cultured in a closed system (airtight state) and under aseptic conditions at the beginning of transplantation, as in step (S1). It is preferable to carry out the culture while controlling the ventilation so that the air replacement increases stepwise or continuously from an airtight state to a constant open state. More specifically, by repeating the growth by split transplantation, until the stage where the shoots become a culture environment and grow at a constant rate, it is in a sterile and airtight state, and ventilation is started at the final stage of growth. Then, the cells are cultured under a ventilation condition for a certain period of time, usually about one month, to make the multi-buds into healthy seedlings.

  That is, the S2 multi-bud growth / engraftment process is a “proliferation stage” in which the proliferation by split transplantation is repeated about 2 to 3 times, and the transition from the last split transplantation to the engraftment stage becomes “proliferation → transition to healthy seedling” It can be divided into roughly three stages: "stage" and "healthy seedling stage" in which the proliferated polyblasts become healthy seedlings.

  The culture in the “growth stage” is desirably performed in an airtight state under aseptic conditions, as in the above-described multiblast induction step (S1).

  Ventilation control in the “growth → transplantation stage” and “transplantation stage” is not limited, but for convenience, a vent hole serving as an opening is provided in the main body and / or lid of the container containing the culture medium. In addition, it can be performed by controlling the air flow in and out of the vent hole. Here, the control of the air flow in and out of the vent hole is performed by, for example, sealing the vent hole with a breathable aseptic or sterilizing seal having a fine porosity, for example, Milliceal (manufactured by Nihon Millipore Corporation; trade name). It can be carried out. The number of vent holes per container and the total opening area are not particularly limited, but when the container is about 300 ml, it is desirable to provide 1 to 5 vent holes with a diameter of about 6 mmφ.

  In the “growth stage” in which the cells are cultured in a closed system (airtight state), an airtight film such as a polyester film (Diafoil Co., Ltd.) is placed on the upper surface of the air-permeable aseptic or sterilized seal attached to the ventilation hole of the culture vessel. It is preferable to perform the culture in a state where a non-breathable plastic film such as a product name “Diafoil”) is attached. Next, after the “growth stage” is completed, the airtight state is released and the next “growth → transition stage for healthy seedling” and “stage for transforming healthy seedling” are performed. Thus, a series of culture steps can be performed in the same culture vessel.

  In the “proliferation → transplantation transition stage”, it is preferable to appropriately adjust the ventilation conditions according to the growth state of the multi-buds, and the number of ventilations can be increased stepwise or continuously. Moreover, you may maintain a sterile airtight state for a fixed period. As ventilation conditions at this stage, the number of ventilations per unit time (the number of times the air in the culture vessel is replaced) is about 0 to 2.5 times / hour, preferably about 0.6 to 2.4 times / hour. Can be mentioned.

  In addition, the number of ventilations at the “healthy seedling stage” can be about 2.5 to 15 times / hour. Here, the ventilation frequency can be calculated by the following equation.

Cultivation in a series of stages of this process (“growth stage”, “growth → transition stage of healthy seedling” and “transition stage of healthy seedling”) can be performed under almost the same conditions except for ventilation conditions. Specifically, the temperature condition may be 20 to 25 ° C., and the illumination condition may be a photon number of 5 to 20 μmol / m ² / s and a range of 12 to 16 hours. Although not limited, in the “growth stage” and “growth → transition to healthy seedling transition stage”, it is necessary to proliferate the multi-buds and then to grow at a constant rate in the grown culture environment. It takes time. Although it does not restrict | limit as this period, Usually, about two months can be mentioned. In addition, a period until the plant becomes close to the one grown in the open air environment is required for the “settlement stage”. Although it does not restrict | limit as this period, Usually, about one month can be mentioned.

  As shown in FIG. 2, the healthy seedling multi-bud obtained as described above has a larger overall size and darker leaves than a multi-bud grown only in an airtight aseptic condition. For example, when taking a blueberry leaf, at this stage of making a seedling, it is possible to increase the functionality of the leaf by irradiating with ultraviolet rays and harvest the shoots that have been made into a seedling as a functional material. Hydroponics can be considered for the production of blueberry leaves in the factory. In any case, it is significantly more efficient than collecting leaves from adult trees.

(3) Rooting induction process (S3):
This step (S3) is a step of inducing rooting by cutting the cuttings prepared from the multi-buds that have been made into healthy seedlings in the above-mentioned multi-bud growth and healthy seeding step (S2) into the culture soil. is there.

  Specifically, this process is a culture soil prepared by adjusting the cuttings from the shoots of the multi-buds that have been made into healthy seedlings in the above-mentioned multi-bud propagation and healthy seeding step (S2), and filling the cell tray with this. It can be carried out by cutting and culturing in an indoor environment. The length of the cutting head to be adjusted from the chute is at least 10 mm, preferably 20 to 50 mm.

As the culture soil, a general culture soil used in the art can be used. For example, peat moss can be generally used as the culture soil, but a porous material such as zeolite, Kanuma soil, Bora soil, pumice, vermiculite or perlite may be used in appropriate combination. Furthermore, wood powder, sawdust, humus, or the fibrous composition and clay mineral shown in Patent Document 2 can be blended. As a preferable culture soil, as shown in Experimental Example 6, a mixture of peat moss and shirasu can be mentioned. The mixing ratio of the culture soil is preferably about 10 to 70% by volume of shirasu. In order to promote rooting, auxins such as indole butyric acid (IBA) or naphthalene acetic acid (NAA) may be applied to the base of the shoot. The concentration is desirably 100 to 1000 mg / L.
The culture in this step is performed in a room-controlled environment. Examples of the culture conditions in this case include a temperature condition of 20 to 25 ° C., a humidity condition of 90 to 100% RH, and an illumination condition of 20 to 50 μmol / m ² / s and a range of 12 to 16 hours. Usually, rooting starts in about 2 weeks, and the root turns in the hole of the Bragg tray in about 1 to 2 months, and a desired seedling (cell seedling) can be obtained.

  Thus, the cell seedling obtained by rooting induction is shown in FIG. 3 as an example of blueberries. The rooting state is shown in FIG.

(4) Acclimatization step (S4);
The step (S4) is a step of acclimatizing the rooted seedling obtained in the rooting induction step (S3). “Adaptation” means that the plant is brought into the outside environment.
Specifically, by transplanting the seedlings with roots obtained in the above-mentioned rooting induction step (S3) from the cell tray to, for example, a plastic pot culture soil, and culturing them in an open air environment (including a greenhouse environment) Can be implemented. Here, as the culture soil, the culture soil described in the rooting induction step can be used similarly, and a culture soil having the same composition as the culture soil used in the rooting induction step may be used. Again, a mixed soil of peat moss and shirasu is preferred. Although the acclimatization process is an outside air environment, it is preferable that the acclimatization process is performed in low light. The acclimatization step is usually performed for about 1 to 2 weeks, but it is possible to grow in this state until it is actually used (for example, planted in an alley).

  In addition, planting of blueberries for actual harvesting is usually at intervals of 2 to 3 m, but this requires a great deal of labor for harvesting leaves. As shown in FIG. 5, it is efficient to plant the blueberry seedlings for leaf harvesting at a high density and cultivate them densely, and harvest them by mechanical means such as a tea leaf reaper. This requires a large amount of seedlings. The blueberry seedling raising method by micropropagation of the present invention is suitable for constructing a consistent production system from seedling raising to planting since it can produce a large amount of substantially uniform seedlings.

  Hereinafter, the present invention will be described with reference to experimental examples and examples. However, the present invention is not limited to these experimental examples. Unless otherwise specified, “%” means “% by weight” and “parts” means “parts by weight”.

Experimental Example 1 Examination of PGR-added medium used for polyblast induction High bush blueberry Berkeley, rabbit eye blueberry home bell, and native wild natto were used as test target plants.

The shoots of each plant shoot collected in the field were washed with running water and ultrapure water, and then immersed in a 5% sodium hypochlorite aqueous solution to which Triton-X (final concentration of about 1 mL / 100 mL) was added for 15 minutes. Sterilized and then washed three times with sterile distilled water. This was used as a test sprout. The test medium is a MW medium in which WP (WoodyPlant) medium and MS (Murashige & Skoog) medium are combined in equal amounts, sucrose (final concentration 20 g / L), agar (final concentration 8 g / L), and plant growth regulator. A solid medium (pH 4.8) supplemented with (PGR) was used. As shown in Table 1, as PGR, zeatin (final concentrations: 0 mg / L, 2 mg / L, 5 mg / L) and naphthalene acetic acid (NAA) (final concentrations: 0 mg / L, 0.5 mg / L, 1 mg / L) in combination. Thus, after the preparation, on each test medium (in a culture vessel) sterilized (autoclave, 121 ° C., 15 minutes), the above-mentioned test sprouts were placed under a sterile condition and capped, and 25 ° C., The cells were cultured under continuous illumination for 24 hours at a photon number of 10 to 15 μmol / m ² / s, and the induction rate (%) of polyblasts after 2 months was examined. The polyblast induction rate (%) was obtained from the following equation.

  The results are shown in Table 1.

  As shown in Table 1, all plants have the highest polyblast induction rate in the zeatin 5 mg / L single addition group, 10.0% for Berkeley, 5.0% for homebell, and 30.0% for nuthaze. Met. From this, it is understood that it is optimal to use zeatin alone at a concentration of 5 mg / L as PGR when inducing multiblasts.

Experimental Example 2 Examination of PGR-added medium affecting multi-bud growth Each of the Barkley and Natsuhaze polyblasts (about 0.2 g including the base) obtained under the optimum conditions in Experimental Example 1 (zeatin 5 mg / L added alone) was used. Samples were collected and tested.

As shown in Table 2, each polyblast is treated with zeatin (final concentrations: 2 mg / L, 5 mg / L), benzyladenine (BA) 5 mg / L, kinetin 5 mg / L, or 2-isopentenyladenine. (2iP) Transplanted on MW medium (sterilized) or non-added sterilized MW medium (sterilized), each supplemented with 5 mg / L, at 25 ° C., with a photon number of 10-15 μmol / m ² / s 24 Cultured under aseptic conditions in a closed system under time-continuous lighting conditions, after 1 month and 2 months of fresh weight of polyblast (wet weight of the whole multi-bud) (g), and shoots (branches) of 20 mm or more The number of possible collections (number of shoots) was examined. The results are shown in Table 2.

  As can be seen from Table 2, Berkeley's multi-bud fresh weight was highest at 0.71 g in the zeatin 5 mg / L single addition group, followed by zeatin 2 mg / L single addition group (0.67 g), and 2 iP 5 mg / L. It was a single addition section (0.61 g). Natsuhaze's polyblastic fresh weight was highest at 1.18 g in the zeatin 2 mg / L single addition group, followed by zeatin 5 mg / L single addition group (1.12 g), and 2iP 5 mg / L single addition group (0.96 g). )Met. The number of shoots of 20 mm or more that can be cut is also the highest in the zeatin-added group, and the number was 10.3 in the Berkeley zeatin 5 mg / L single-added group, and 12 in the natchin zeatin 2 mg / L-added group. . From this, it can be seen that, when proliferating, the use of zeatin as PGR, in particular, by adding it to the medium at a concentration of 2 to 5 mg / L is effective for proliferating the polyblast.

Experimental Example 3 Examination of types of saccharides in the growth stage About 0.2 g of multi-buds containing the bases of Barclay and Nutzee grown under the optimum conditions of Experimental Example 2 were used. As the test medium, MW medium (sterilized) to which saccharide and zeatin as PGR were added to a final concentration of 5 mg / L was used. Here, as shown in Table 3, sucrose, glucose and fructose were used as saccharides (each addition amount: 0.058 mol / L), and a test group without addition of saccharides was set as a comparative control example. In each of the obtained test plots, the above-mentioned multi-buds were planted, cultured under aseptic conditions in a closed system under illumination conditions at 25 ° C. and a photon number of 5 to 20 μmol / m ² / s for 16 hours, and then cultured for 1 month The growth status of polyblasts was examined for the fresh weight (g) of polyblasts and the number of shoots that can be collected (shoots) of 10 mm or more. The results are shown in Table 3.

  From the results in Table 3, it can be seen that saccharides are preferably added rather than non-added, but there is no difference depending on the type of saccharide, and can be appropriately selected according to the purpose.

Experimental Example 4 Examination of Ventilation Frequency at the Stage of Making a Healthy Seed About 0.2 g of multi-buds containing the base of Barclay and Natsuhaze grown under the optimum conditions of Experimental Example 2 were used. As a test medium, as shown in Table 5, MW medium supplemented with zeatin (final concentration: 5 mg / L) as PGR and sucrose (final concentration: 20 g / L) as saccharide was used. In addition, a test group without addition of saccharide was set as a comparative control example.

  The test medium was prepared in a 60 mm × 60 mm × 100 mm polycarbonate container “Plant Box” (manufactured by IWAKI; trade name) (with lid). Culture was performed for 1 month in this state. The culture temperature was set to 25 ° C., the relative humidity in the container was set to 100%, and the relative humidity in the culture chamber (outside the container) was set to 50%.

  As a container for containing the culture medium, a container with one vent hole with a diameter of about 6 mm and a container without a vent were used. The vent hole was sealed with a breathable aseptic seal “Milliseal” (manufactured by Nihon Millipore; trade name). In order to evaluate the degree of ventilation inside and outside the culture vessel, the ventilation frequency was estimated according to the following formula based on the weight loss (mg) of the whole vessel during the culture period and the difference in relative humidity between the vessel and the culture chamber. (See Yoshiyuki Honma et al. Bull. Shizuoka Agr. Exp. Stn. [36] 75-85 (1991)). In addition, the frequency | count of ventilation here means the frequency | count of the total replacement | exchange of the air in a container in 1 hour.

  Table 4 shows the estimation results.

  From Table 4, it was estimated that the number of ventilations was 0.5 times / hour in a sealed state without vents, and 2.4 times / hour with one vent hole. Table 5 shows the results of counting the number of shoots after each month of culturing for each plant (the number of shoots with a size of 10 mm or more that can be collected) and examining the effect of the ventilation rate on healthy seedlings.

  From Table 5, it was observed that the number of shoots of 10 mm or more that can be collected tends to decrease as the number of ventilations increases. However, by increasing the number of ventilations, as shown in FIG. 2, a tendency was observed in which shoots with firm foliage were formed although the length was short. From this result, it can be seen that it is desirable to reduce the number of ventilations in the growth stage and increase the number of ventilations in the healthy seedling stage.

Experimental Example 5 Examination of the length of cuttings in the rooting induction process Cuttings were collected from the multi-buds of red pearl, Berkeley, and nuthaze grown under the optimum conditions of Experimental Example 2. The effect of the length of the cuttings on the rooting rate in the rooting induction process under non-sterile conditions was examined.

The length of the cuttings was adjusted to 10 mm, 20 mm and 30 mm for the multi-buds of each plant. This was mixed with peat moss and Bora soil at a ratio of 1: 1, and directly cut into a cell tray filled with culture soil (without PGR added) sufficiently containing water. The culture was performed under illumination conditions for 12 hours at a temperature of 25 ° C., a relative humidity of 90 to 100%, and a photon number of 20 to 50 μmol / m ² / s. The number of roots after 4 weeks from the culture was counted, and the rooting rate was calculated from the following formula. The rooting was determined to be “rooting” if 1 mm or 1 root was rooted. The results are shown in Table 6.

  As shown in Table 6, in Red Pearl, a high rooting rate of 91.7% or more was obtained when the cutting head length was 20 mm or more. On the other hand, in Berkeley, the same rooting rate was obtained when the length of the cuttings was 10 mm and 30 mm. Thus, a certain tendency was not recognized between the length of the cutting and the rooting rate, suggesting that there is a difference between species.

Experimental Example 6 Examination of culture soil in rooting induction process As shown in Table 7, the culture soil was prepared by adding Shirasu or Bora soil to peat moss to 0, 30, 50, 70, and 100% by volume, respectively. Red pearl, Berkeley or Natsuse shoots were directly cut and cultivated, and the rooting rate (%) after 4 weeks was examined in the same manner as in Experimental Example 5. The culture chamber was non-sterile and was cultured under illumination conditions at a temperature of 25 ° C., a relative humidity of 90 to 100%, and a photon number of 20 to 50 μmol / m ² / s for 12 hours.

  The results are shown in Table 7.

  As shown in Table 7, with respect to blueberries (red pearl, Berkeley), a good rooting rate was obtained with peat moss alone. In addition, even if Bora soil or Shirasu (preferably Shirasu) is added to peat moss in an appropriate ratio, the rooting rate is not adversely affected, and conversely, a good rooting rate may be promoted. I was told that there was. In general, when peat moss alone is used as the culture soil, it is difficult to work, and by adding shirasu appropriately to this, this can be improved, cutting can be easily performed, and work efficiency can be improved. The above experimental results show that peat moss can be added and mixed with components such as mullet and shirasu as culture soil.

Example 1
Axillary buds were collected from new shoots of high bush blueberry Berkeley. The collected buds are washed with running water and ultrapure water, then sterilized with 5% aqueous sodium hypochlorite solution with Triton-X added so that the final concentration is about 1 mL / 100 mL, and then with sterile distilled water. Washed 3 times. The sterilized buds sterilized above were placed on MW medium (pH 4.8) supplemented with sucrose (final concentration 20 g / L), agar (final concentration 8 g / L), and zeatin (final concentration 5 mg / L). This was cultured at 25 ° C. for 2 months under the condition of continuous illumination for 24 hours with a photon number of 10 to 15 μmol / m ² / s to induce multiblasts.

MW medium containing sucrose (final concentration 20 g / L), agar (final concentration 8 g / L), and zeatin (final concentration 5 mg / L) are placed in a sterile airtight container (60 mm x 60 mm x 100 mm). Divided and transplanted the above-mentioned multiblasts into the medium (3-6 divisions) and grown in a sterile and airtight state (culture conditions at 25 ° C., photon number 10-15 μmol / m ² / s under continuous illumination for 24 hours) . Subculture was repeated twice at 2-month intervals in the same medium and under the same conditions.

  Next, one air hole with a diameter of about 6mm is provided in the lid of this airtight container, and a sterile air-permeable milliseal (made by Nihon Millipore, Inc .; product name) is attached to this airhole, and then a confidential polyester film (diamond) is placed on the milliseal. A product name “Diafoil” manufactured by Foil Co., Ltd.) was attached.

  The above-mentioned culture medium (sucrose, agar, and zeatin-containing MW medium) newly prepared and arranged in this container is divided and transplanted after the second subculture, and the third subculture is performed for 2 months. went. During the third subculture, when the shoots grew to cover approximately half of the container, the confidential polyester film was peeled off, and the ventilation was controlled 2.4 times / hour to make healthy seedlings. It was.

The shoots of Barkley's multi-buds grown and transformed into healthy seedlings by the above method were adjusted to 20 mm under non-sterile conditions and used as cuttings. Peat moss and shirasu were mixed at a ratio of 3: 7 (volume ratio), and culture soil sufficiently containing water was prepared and filled in a cell tray. 121 cuttings were directly cut into this culture soil, and rooting treatment was performed for 4 weeks with illumination at a temperature of 25 ° C., a relative humidity of 90 to 100%, and a photon number of 20 to 50 μmol / m ² / s for 12 hours. . Then, it transplanted to the pot made from vinyl, and acclimatized under the low light outside air environment. Five weeks later, 121 seedlings having a height of about 3.5 to 6 cm were obtained. The total process time was 343 days.

  INDUSTRIAL APPLICABILITY The present invention is suitable for mass production of cypress plants, in particular, blueberry seedlings, and can be used in a seedling production business that supplies a large amount of blueberry seedlings at a low cost as local production of blueberries progresses in various places. In addition, the present invention facilitates the spread after development of a new hybrid between cypress plants. The method of the present invention can be suitably used particularly in the field of producing new cultivars and improved varieties with few cutting heads in a short period of time.

The seedling raising method of the genus Genus of this invention is shown. The multi-buds of blueberries before making them into healthy seedlings and the multi-buds after making them into healthy seedlings are shown. A blueberry cell seedling transplanted to a cell tray and induced to root is shown. The rooting state of the blueberry induced rooting in the culture soil containing Shirasu is shown. This shows the dense cultivation of blueberries for the purpose of harvesting leaves.

Claims (9)

(1) A step of inducing a multi-bud by tissue-culturing a tissue piece of a shoot apex of a genus genus plant in an airtight state in a medium containing a saccharide, a plant growth regulator, and a support,
(2) Divide and transplant the polyblast induced in step (1) into a medium containing a saccharide, a plant growth regulator, and a support, and control the ventilation of the following a to c under aseptic or sterilization. The process of culturing while performing the multiplication of the shoots and making the seedlings healthy:
a. Performing the growth stage of the multi-bud in an airtight state,
b. Transition from the growth phase to healthy seedling under the condition that the ventilation rate per unit time is 0 to 2.4 times / hour, and
c. It becomes healthy seedlings under conditions where the ventilation frequency is 2.5-15 times / hour.
(3) A step of collecting cuttings from the shoots of the multi-buds that have been turned into healthy seedlings in step (2), cutting them into culture soil, culturing them in a culture room environment, and inducing rooting; and ( 4) A step of culturing and acclimatizing the seedling rooted in step (3) in an open air environment,
A method for raising seedlings of the genus Genus The method for raising seedlings according to claim 1, wherein the genus Cypress is a blueberry. The method for raising seedlings according to claim 1 or 2, wherein in step (1), tissue culture is performed in a medium containing a saccharide, a plant growth regulator, a support and an antibiotic. In step (1), tissue culture is performed by using sucrose as a saccharide, 1 to 10% by weight, zeatin as a plant growth regulator, 1 to 10 mg / L, agar as a support, 0.8 to 2% by weight, and antibiotics. The seedling-raising method according to claim 3, which is performed using an MW medium containing 0.1 to 0.5% by weight. In step (2), culturing is carried out by using saccharides as sugars, glucose, fructose, maltose, galactose, mannitol, lactose, and sorbitol at least one selected from the group consisting of 1 to 10% by weight, and zeatin as a plant growth regulator. , A MW medium containing 0.1 to 10 mg / L of at least one selected from the group consisting of kinetin, 2-isopentenyladenine, and benzyladenine, and 0.8 to 2% by weight of agar as a support The seedling raising method in any one of Claims 1 thru | or 4 performed using. 6. The seedling raising method according to any one of claims 1 to 5, wherein in step (2), ventilation is controlled by providing a ventilation hole in the culture vessel and through the ventilation hole. The seedling raising method according to claim 6, wherein the ventilation control in the step (2) is performed by attaching a breathable aseptic or sterilized film to a vent hole provided in the culture container so as to cover the vent hole. The seedling raising method according to any one of claims 1 to 6, wherein in step (2), ventilation is controlled so that the ventilation frequency increases stepwise or continuously in a range of ventilation frequency of 0 to 15 times / hour. The seedling-raising method according to any one of claims 1 to 8, wherein in step (3) and step (4), cultivation is performed using culture soil containing shirasu.

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