JP5792933B2 - Clone seedling production method - Google Patents

Description

  The present invention relates to a method for producing cloned seedlings, and more particularly, to a method for producing cloned seedlings that can improve the rooting rate from plant shoots, and in particular, it is difficult to form adventitious roots. The present invention relates to a method for producing a clone seedling suitable for a genus plant or a sandalwood genus plant.

  Seedlings from seeds are called seedlings. Seed breeding crops grow this, but in vegetative breeding crops such as fruit trees and strawberries, seedlings are miscellaneous and are not used for purposes other than breeding by seedling selection methods. In most of the main fruit trees, some flower trees, garden trees, fruit vegetables, etc., cut off the grafts, that is, part of the plant body (joint ears) for the purpose of propagation, and bond them to other plant bodies (rootstocks). The seedlings are produced by training independent individuals. In addition, a plant body that serves as a platform on which a breeding head is brought into contact is called a rootstock. The rootstocks and ears in the grafts retain the genetic characteristics of the grafts that interact with each other, but exhibit nutritional variation.

  On the other hand, roots generated from a part of one's own nutrition are called self-roots. Seedlings obtained from cuttings and cuttings are called self-rooted seedlings. Usually, the root system when seedlings or cuttings are propagated is distinguished from the case of grafted seedlings. Trees that are primarily nutrient-absorbed by their roots are called self-rooted trees.

  When cutting, cuttings are extremely vulnerable to drying because there is no water replenishment by strong root pressure. In addition, the cut surface is not covered with the epidermis and the internal tissue is exposed, so that pathogens can easily enter and the resistance is remarkably low. In addition, there is no supplement of new nutrients (photosynthesis, etc. in addition to fertilizer components), and at the same time as cuttings, consumption for maintaining survival such as rooting and budding begins, but some of the nutrients contained in the cuttings Greatly affects the rooting rate of cuttings. In addition, the insertion floor is an important element in terms of moisture management and disease management of the cut.

  The moisture state of the cutting floor is closely related to the water absorption capacity of the cutting head. Since the water absorption ability of the cuttings depends on the water absorption from the cut end, a certain amount of humidification is required. The most suitable soil moisture content is considered to be a moisture state of about 50 to 60% with respect to the liquid phase of the inserted bed immediately after irrigation. This state is a suitable condition not only for supplying water but also for supplying oxygen to the cuttings in the rooting process. As an insertion bed, a material that has a high ratio between the liquid phase and the gas phase in the three-phase distribution (for example, the sum of the ratio between the liquid phase and the gas phase is 80% or more) and is aseptic is suitable. . Thus, in general cuttings, it is necessary to increase the water content of the root bed of cuttings.

  In the case of cuttings, there is no nutritional variation like rootstocks and ears in grafts. However, it is difficult to cut a plant such as a pear genus plant or a sandalwood genus plant. Therefore, these plants are propagated by means other than cuttings. For example, pear plants are propagated by grafting, and sandalwood plants are propagated by seeds. However, since the grafted seedlings are affected by rootstocks as described above, there is a problem that they are difficult to become uniform seedlings due to nutritional variation. Therefore, it is required to improve the rooting rate of cuttings in plants that are difficult to root. In addition, grafted seedlings have a problem in that the growth and fruiting properties are changed afterwards because nutrient absorption from roots and the like are greatly changed as compared to cuttings. Furthermore, in the case of propagation by seeds, there has been a problem that the traits are segregated as described above. Therefore, it is highly possible to make self-root seedlings by cutting, which greatly contributes to agricultural development.

  Examples of methods for improving the rooting rate of cuttings of plants that are difficult to root include, for example, a method of sprouting cuttings in the dark when obtaining cuttings (yellowing treatment) (The science reports of the Hyogo University of Agricultural.Series agile chemical chemistry 4 (1) pp. 60-64, 1959 (Non-patent Document 1), a method of treating cuttings with various plant hormones (auxin etc.) Bioresources, Mie University 12, pp. 44-47, 2001 (non-patent document 2)), polyvinylpyrrolidone is mixed with the medium, and the cutting end of the cutting head is mixed. To remove the tannin component released from the plant (Agricultural and Food Industry Research Organization, 1999 Kanto Tokai Agricultural Research Result Information “Rooting Promotion Technology in Tissue Culture of Kakiwai-rootstock” (Non-patent Document 3) )) Has been reported so far. However, even with these improvements, there was a plant whose rooting rate was not sufficiently improved.

The science reports of the Hyoguro University of Agricultural. Series of aggressive chemistry 4 (1) pp. 60-64, 1959 The Bulletin of the Experimental Farm, Facility of Bioresources, Mie University 12, pp. 44-47, 2001 National Institute of Agricultural Sciences, Food and Industrial Technology, 1999 Kanto Tokai Agricultural Research Result Information “Rooting Promotion Technology in Tissue Culture of Kakiwagi Rootstock” http: // web08. affrc. go. jp / top / seika / 1999 / kan-tou / narc99K320. html

  The present invention promotes the formation of adventitious roots from plant shoots and improves the rooting rate, thereby improving the productivity of cloned seedlings by cutting methods and the like, particularly clone seedlings belonging to plant species with low rooting ability. It aims to improve. Furthermore, the purpose is to enable mass production and rapid production of cloned seedlings and open the way to its industrial use.

  As a result of intensive studies, the present inventors have found that the rooting rate from the shoots can be improved by paying attention to the relationship between the liquid retention rate of the support and the formation of adventitious roots from the plant shoots. Specifically, the support used for cutting is usually one that can absorb and hold water and liquid medium well, but on the other hand, it can support only a certain amount of water and liquid medium. Found that the body is suitable. In other words, adventitious root formation and root elongation are promoted and shoot death is suppressed by culturing shoots using a support having a liquid retention represented by a specific formula of 80% or less. As a result, the present invention has been completed.

The present invention provides the following [1] to [1].
[1] A rooting bed is prepared by holding water and / or a liquid medium on a support having a liquid retention of 80% or less represented by the following formula 1, and plant shoots are inserted into the rooting bed. A method for producing a clonal seedling, wherein the seedling is cultured and adventitious roots are formed from the shoot.
(Formula 1) Liquid retention rate (%) = {saturated mass of liquid that can be retained on the support / (saturated mass of liquid that can be retained on the support + dry mass of the support)} × 100
[2] The method for producing a clonal seedling according to [1] above, wherein the solid phase ratio in the three-phase distribution of the support is 40% or more.
[3] The method for producing a cloned seedling according to [1] or [2], wherein the support has a liquid retention represented by the following formula 2 of 90% or less.
(Formula 2) Liquid retention (%) = (mass of liquid retained on the support 6 days after the start of culture / mass of liquid retained on the support at the start of culture) × 100
[4] The method for producing a clonal seedling according to any one of [1] to [3], wherein the culturing is performed in a sealed culture vessel.
[5] The method for producing a cloned seedling according to any one of [1] to [4] above, wherein the plant is a pear genus plant or a sandalwood genus plant.
[6] A method for preparing a root bed for plant shoot insertion, wherein a support having a liquid retention of 80% or less represented by the following formula 1 is held with water and / or a liquid medium.
(Formula 1) Liquid retention rate (%) = {saturated mass of liquid that can be retained on the support / (saturated mass of liquid that can be retained on the support + dry mass of the support)} × 100
[7] The method for preparing a root bed according to the above [6], wherein the ratio of the solid phase in the three-phase distribution of the support is 40% or more.
[8] The method for preparing a root bed according to the above [6] or [7], wherein the support has a liquid retention represented by the following formula 2 of 90% or less.
(Formula 2) Liquid retention (%) = (mass of liquid retained on the support 6 days after the start of culture / mass of liquid retained on the support at the start of culture) × 100

  According to the present invention, adventitious root formation from shoots can be promoted, and withering of shoots, especially insertion sites can be suppressed, and rooting rate can be improved, so that the productivity of cloned seedlings can be improved. it can. Such an effect of the present invention is remarkable in plant species having low rooting ability, in particular, pear genus plants and sandalwood genus plants. Therefore, the present invention contributes to the large-scale and rapid production of cloned seedlings of a wide variety of plant species, and in particular, even to plant species with low rooting ability, the cloned seedlings are produced in large quantities and rapidly. And open the way to its industrial use.

FIG. 1 is a graph showing the transition of the liquid retention rate of Examples 1 and 2 and Comparative Example 1. FIG. 2 is a graph showing the transition of the liquid retention rate of Example 3 and Comparative Example 2.

(Target plant)
The present invention can be applied to any plant. In particular, it is preferably applied to a woody plant, and more preferably applied to a woody plant having a lower rooting ability than a herbaceous plant because the effects of the present invention can be remarkably exhibited. Examples of woody plants include Eucalyptus plants, Pinus plants, Prunus plants (Prunus spp., Prunus mume, Prunus tomentosa, etc.), avocados, etc. Genus (Avocado) plant, Mangofera plant (Mangofera indica etc.), Acacia plant (Acacia) plant, myrtle genus (Myrica) plant, Quercus genus plant (Quercusactis plant, etc.) Plants of the genus Grapes (Vitis), plants of the genus Apple (Malus), plants of the genus Rose (Rosa), plants of the genus Camellia, plants of the genus Jacaranda (Jaka) Randa (Jacaranda mimosifolia, etc.), Alligator (Persea) plants (Avocado (Persea americana), etc.), Pears (Pyrus serotina Rehder, Pyrus pyrifolis, Pyrus pyrifolis) (Sandalwood; etc.). Among these, when applied to eucalyptus, pine, cherry, mango, avocado, acacia, bayberry, cucumber, grapes, apples, roses, camellia, ume, yusu raume, jakaranta, pear, sandalwood, etc., the effect of the present invention is more exhibited. Yes. Among them, Eucalyptus plants, Pinus plants, Mango plants, Pear plants, and Sandalwood plants are preferable.In particular, if the present invention is applied to Pear plants and Sandalwood plants that are known to be difficult to root, great effects can be obtained. It is done.

(shoot)
Shoots refer to all tissues that have rooting ability. Examples of the tissue include branches, stems, apical buds, buds, adventitious buds, leaves, cotyledons, hypocotyls, adventitious embryos, shoot primordia, and the like. The origin of the shoot is not particularly limited, and it may be obtained from a plant individual growing in a greenhouse or outdoors, may be a cultured tissue obtained by a tissue culture method, or may be a natural plant body. It may be a departmental organization. A cloned seedling refers to a seedling obtained by rooting these tissues.

  The origin of the shoot is not particularly limited, and it may be obtained from a plant individual growing in a greenhouse or outdoors, may be a cultured tissue obtained by a tissue culture method, or may be a natural plant body. It may be a departmental organization. The shoot can be efficiently obtained from the main plant of the cutting head and the multi-bud. Among them, cuttings (cutting ears obtained from the mother plant), polyblasts obtained by aseptically culturing organs collected from the mother plant, or foliage obtained by aseptically growing the organs Preferably there is. When cuttings are obtained from a plant individual, the branches may be cut and used as cuttings. In the case of woody plants, green branches (current year branches) and mature branches (branches extending before the previous year) are usually used, and in the case of herbaceous plants, buds and leaves are usually used. When branches are used as cuttings, it is also effective to excise part of the leaves in order to suppress the transpiration of the leaves attached to the branches and promote the formation of adventitious roots.

  When cuttings are obtained by tissue culture, multi-buds are induced, adventitious buds extending from these tissues are cut off from the vicinity of the roots, and this can be used as cuttings. The multiblast can be induced using a known method in each plant. For example, in order to obtain the cuttings to be used in the present invention by forming multi-buds from the above-mentioned woody plants, it is carried out as follows.

  First, tissues such as apical buds and axillary buds are collected from a plant as a material, and the collected tissues are subjected to sodium hypochlorite aqueous solution having an effective chlorine content of 0.5 to 4% or peroxidation having an effective chlorine content of 5 to 15%. Surface sterilization is performed by immersing in an aqueous hydrogen solution for 10 to 20 minutes. Next, this is washed with sterilized water, inserted into a solid medium to open the buds, and the elongated shoots are subcultured in a medium having the same composition to form multi-buds. In the case of pear, the solid medium contains 1 to 5% by mass of sucrose, 0.02 to 1 mg / l of benzyladenine (hereinafter abbreviated as BA) as a plant hormone, 0.2 to 0.3% by mass of gellan gum or Murashige-Skoog containing 0.5 to 1% by mass of agar (hereinafter abbreviated as MS). Since the adventitious buds are actively differentiated from the multi-buds thus formed and extend, in the present invention, the adventitious buds that have been extended may be cut out and used as cuttings. The multiblasts themselves can be maintained and proliferated by appropriately dividing them and culturing them in a medium having the same composition as the medium used for the formation of the multibuds.

(Support / Root bed)
In the present invention, the support is a support for supporting a plant shoot and is also called an insertion floor. The support is preferably one that can be held with the shoots inserted during the cultivation period. The material of the support is not particularly limited, but a material that can hold water and / or a liquid medium is preferable, and a material that can hold water and / or a liquid medium at a specific ratio for a long period of time (such as not flowing out or evaporating) is preferable. Thereby, water and / or a liquid culture medium can be hold | maintained for a long period of time, and the liquid supply amount to the shoot of a plant can be supplied efficiently.

In the method of the present invention, first, a root bed is prepared by holding water and / or a liquid medium on a support. The liquid retention rate of the support is expressed by the following formula 1, which needs to be 80% or less.
(Formula 1) Liquid retention rate (%) = {saturated mass of liquid that can be retained on the support / (saturated mass of liquid that can be retained on the support + dry mass of the support)} × 100

  The saturated mass of the liquid that can be held on the support means the mass of the liquid remaining in the support after irrigating the support with a sufficient amount of water and / or liquid medium and allowing excess water and / or liquid to flow out. Say. Specifically, water and / or liquid medium (for example, 4-fold diluted B5 liquid medium) is irrigated on the support, and the excess water and / or liquid medium is removed for a certain period of time, for example, 10 to 30 minutes. Left under conditions of high temperature (eg, constant temperature in the range of 20-30 ° C.), humidity (eg, constant humidity in the range of 50-90%), carbon dioxide concentration (eg, constant concentration in the range of 300-2000 ppm) It can be calculated by measuring the total mass of the subsequent root bed and subtracting the dry mass of the support from the total mass of the root bed.

  The lower limit of the liquid retention rate of the support is not particularly limited, but is preferably 50% or more, more preferably 70% or more, and particularly preferably 75% or more.

  In the present invention, the ratio of the solid phase in the three-phase distribution of the support is preferably 40% or more, more preferably 45% or more, and even more preferably 50% or more. The support having a liquid retention of 80% or less and a solid phase ratio of 40% or more promotes adventitious root formation from shoots during the culture period, suppresses shoot death, and increases rooting rate. Can be improved.

  The method for measuring the ratio of the solid phase in the three-phase distribution can be measured by a conventionally known method for a support containing water and / or a liquid medium, for example, a soil three-phase meter (manufactured by Daikai Rika Kogyo Co., Ltd.). , Trade name DIK-1120).

  In the three-phase distribution, the gas phase ratio and the liquid phase ratio are not particularly limited, but the gas phase ratio is preferably 5% or more and 30% or less, and preferably 5% or more and 15% or less. It is more preferable. The ratio of the liquid phase is preferably 30% or more and 55% or less, and more preferably 40% or more and 50% or less. In addition, it cannot be overemphasized that the ratio of each phase of three phase distribution is a ratio in case the sum total of each ratio is 100%.

  The material of the support is preferably one that can hold water and / or a liquid medium in a relatively low content for a long period of time (such as not letting out or evaporating). The support is preferably one that can be infiltrated with water and / or a liquid medium, and among them, one that can be substantially uniformly wetted with water and / or a liquid medium is preferable.

Further, in the present invention, it is preferable that the support can hold water and / or liquid medium at a constant low rate for a long time. Preferably, the upper limit of the liquid retention of the root bed of the following formula 2 is not particularly limited, but is preferably 90% or less, more preferably 80% or less, and further preferably 75% or less. More preferred.
(Formula 2) Liquid retention (%) = (mass of liquid retained on the support 6 days after the start of culture / mass of liquid retained on the support at the start of culture) × 100

  The “mass of the liquid retained on the support at the start of culture” in Formula 2 (hereinafter referred to as “mass A”) refers to the water and / or contained in the support when the root bed is prepared at the start of the culture in the present invention. It means the mass of the liquid medium, and can be rephrased as “the mass of water and / or liquid medium contained in the rooting bed at the start of culture”.

  “The mass of the liquid retained on the support 6 days after the start of the culture” indicates that the culture is started with the support having the mass A of water or the liquid medium retained thereon, and then water and liquid medium are not added. It means the mass of the liquid remaining on the support after culturing for a day, and can be rephrased as “the mass of liquid contained in (or remains in) the root bed 6 days after the start of culture”.

  The measurement of “the mass of the liquid retained on the support 6 days after the start of the culture” can actually be performed as follows.

  By using a support having a liquid retention of 90% or less, the amount of water retained by the root bed can be maintained at a lower level during the cultivation period, and adventitious root formation from shoots during the cultivation period is promoted. In addition, shoot death can be suppressed, and the rooting rate can be improved. In addition, the lower limit of the liquid retention property of the root bed of Formula 2 is not particularly limited, but is preferably 50% or more, and more preferably 70% or more.

  Examples of the support material include natural soils such as sand (river sand, mountain sand), akadama soil and kanuma soil; One of these can be selected and used. Two or more types can be selected and mixed. Among them, red jade earth, vermiculite, perlite, and peat moss are preferable, and a combination of red jade earth (especially fine red jade earth) and peat moss, and a combination of vermiculite, perlite, and peat moss are preferable.

  Sand (river sand, mountain sand) and pearlite can be used as a material for the support in order to adjust the liquid retention rate low. The mixing ratio of sand and / or pearlite is preferably 50% by mass or more and more preferably 60% by mass or more with respect to the total mass of the support.

  In natural soil and artificial soil, the particle size of the fine particles constituting the soil is preferably about 0.5 to 7 mm, and more preferably about 0.7 to 5.5 mm. In the case of fine grained red crust, it is preferably 0.5 to 4.5 mm, more preferably 1 to 3 mm. In the case of vermiculite and pearlite, the thickness is preferably about 1 to 5.5 mm, and more preferably 1.5 to 5 mm.

  When using a culture vessel, insert the chute directly into the support or in a state where it is filled in a tray (plug tray, cell tray, etc.) as necessary, and then put it in the culture vessel to add water and / or liquid. The root bed can be prepared by retaining (eg, swelling, wetting) the medium. It is also possible to hold water and / or liquid medium on a support (or a support filled in a tray as described above), insert a chute, and then install it in a culture vessel to serve as a root bed. Good.

  The pH of the support during the culture period is preferably adjusted to 4-8. Especially, since growth of miscellaneous bacteria etc. can be suppressed by adjusting to the acidity of about pH 4 (for example, pH 4-6), it is more preferable.

  In forming adventitious roots from shoots, culturing is performed by inserting shoots into a rooting bed prepared by holding water and / or liquid medium on the support. That is, these cuttings cut from the tissue are inserted into the root bed and cultured.

  In the present invention, shoots are cultured with their bases inserted into the root bed. Plants naturally recognize the top and base, and adventitious roots are usually formed from the base, so by inserting shoots in this way, the formed adventitious roots remain as they are and contain the necessary nutrients. This is because it can extend into the root bed. In the preparation of the root bed, at least one of water and liquid medium may be held on the support, and both water and liquid medium may be held on the support. The amount of water and / or liquid medium retained on the support (ie, the amount of water and / or liquid medium contained in the rooting bed) is preferably near the saturation mass of the support, and the mass of the rooting bed More preferably, the amount is 200 to 350%.

  The liquid medium is not particularly limited as long as it is suitable for rooting. For example, Murashige-Skoog (MS) medium, Rinsmeier-Skoog medium, Gamborg B5 medium, white medium, niche niche medium, etc. Is diluted. Among these, a B5 medium or a diluted one thereof is preferable, a B5 medium diluted 2 to 6 times is more preferable, and a D5 medium diluted 3 to 5 times is even more preferable. If necessary, one or two or more types selected from components such as plant hormones, carbon sources, antioxidants, inorganic components, vitamins, and amino acids may be added to these basic media.

  Plant hormones include auxins (eg, naphthalene acetic acid (NAA), indole acetic acid (IAA), p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4D), indole butyric acid (IBA) and their derivatives. Etc.) are exemplified by cytokinins (for example, benzyladenine (BA), kinetin, zeatin, and derivatives thereof). Of these, auxins are preferable. Among them, indole butyric acid (IBA), naphthalene acetic acid (NAA) and the like are preferable because they are easily available and easy to use, and IBA is even more preferable. The addition ratio of the plant hormone to the basic medium is preferably 1 to 50 mg / l, more preferably 2 to 10 mg / l.

  A sucrose is illustrated as a carbon source. The addition ratio of sucrose to the basic medium is not particularly limited, but is usually 5 to 30 g / l. In addition, when using the liquid culture medium which added the carbon source, it is preferable to perform cutting head culture | cultivation in aseptic environment. Thereby, propagation of microorganisms can be suppressed. Further, instead of using a carbon source, carbon dioxide gas can be added to the culture environment. The preferable concentration of carbon dioxide in the culture environment is 300 ppm to 2000 ppm, preferably 300 to 1500 ppm, more preferably about 800 ppm to 1500 ppm. This is preferable because it is not necessary to culture the cuttings in a sterile environment.

  Examples of the antioxidant include ascorbic acid and sulfite. Examples of the inorganic substance include elements such as nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, iron, manganese, zinc, boron, molybdenum, chlorine, iodine, and cobalt, and inorganic salts containing these. Further, examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B4), pyridoxal, pyridoxamine, calcium pantothenate, inositol, nicotinic acid, nicotinamide and / or riboflavin (vitamin B2). Examples of amino acids include glycine, alanine, glutamic acid, cysteine, phenylalanine, and lysine.

  As a liquid culture medium, commercially available liquid fertilizer etc. other than said specific example may be sufficient. Hyponex (registered trademark) is exemplified as the liquid fertilizer.

  In addition, it is preferable to have a cut surface or a cut portion in a portion that comes into contact with the root bed when the chute is inserted into the root bed, because formation of adventitious root from the chute is promoted. This is thought to be due to the fact that the cells at the site were damaged by the cut or cut of the shoot, which became a physiological stimulus and promoted adventitious root formation from the nearby site. For this reason, one or more small scratches may be artificially made with a cutter or the like in the portion of the shoot that is inserted into the root bed prior to culture, but the shoot is usually removed from the original individual or tissue. Since it is obtained by cutting out a branch and has a cut surface at its base, this effect can usually be obtained simply by inserting the base of the shoot into a medium or the like. At this time, if the shoot is cut obliquely, stimulation due to cell damage increases and the contact area between the cut surface and the culture soil also increases, so that the formation of adventitious roots is further promoted.

  Furthermore, adventitious root formation from shoots can be promoted by preliminarily treating the shoots before culture with auxin. The auxin treatment may be performed by immersing the shoot in an auxin solution having a concentration of 5 to 100 ppm, or applying a powder mixed with 1 to 20 mg of auxin per gram of talc to the cut surface of the shoot.

(Culture container)
In culturing, a culture vessel can be used. The culture vessel is not particularly limited. For example, it may be a closed type (a humidity that can be maintained at 90% or higher) or an open type, but a closed type is preferred. By using a closed culture vessel, it becomes easy to place the chute under high humidity, so that the transpiration of the leaves on the branches is suppressed, and the conventional partial excision of the leaves can be omitted. In addition, the humidity can be easily maintained.

  The culture container is more preferably a container capable of supplying carbon dioxide gas into the container. As such a culture container, a container having an opening covered with a carbon dioxide permeable membrane is exemplified. The shape of the opening is not particularly limited. The material for the carbon dioxide permeable membrane is not particularly limited, and examples thereof include polytetrafluoroethylene. Further, the pore diameter of the membrane is not particularly limited, and examples thereof include those having a thickness of about 0.1 μm to about 1 μm.

(Culture conditions)
In the present invention, if the shoot is inserted into the root bed prepared by holding water and / or liquid medium on the support and cultured, other culture conditions (temperature, humidity, culture period, light intensity, etc.) There is no particular limitation. For example, the temperature can usually be set to 23 to 28 ° C., and the average humidity can be usually set to 40% or more, preferably 50% or more, more preferably 90% or more. Usually, adventitious roots are formed from the base of shoots in 4 to 8 weeks from the start of culture. During the culture period, water or liquid medium may be added to the root bed (support), but at an appropriate time (usually after the second half of the culture period, for example, when the culture period is 8 weeks, the 5th to 6th weeks) It is preferable to suppress the addition of a minimum amount (for example, to the extent that the root bed is moistened).

Light intensity is expressed as a photosynthetic photon flux density, with preferably about 10μmol / m ² / s~ about 1000 micro mol / m ² / s, about 50μmol / m ² / s~ about 500μmol / m ² / s More preferably. In either case, rooting from the shoot is usually observed in about 2 weeks to about 5 weeks.

  In addition, the culture is preferably performed under irradiation with light containing a wavelength component of 650 nm to 670 nm and a wavelength component of 450 nm to 470 nm in a ratio of 9: 1 to 7: 3. It is more preferable to carry out under irradiation of light containing at a ratio of 8: 2. By cultivating by irradiating light containing such a wavelength component, rooting from the shoot can be further promoted.

  Shoots with adventitious roots formed are used as seedlings by continuing cultivation for a certain period of time to enrich the roots and then transplanting them to rooting seedling containers or seedling fields as rooting seedlings. be able to. What is necessary is just to set suitably the conditions at the time of growing a seedling, conditions, such as temperature and light intensity, so that it may be suitable for the plant. In the case where shoots are adventitious buds derived from multi-buds or cultured tissues, it is usually necessary to undergo an acclimatization process before transplanting to a seedling container or the like.

[Action]
In the present invention, adventitious roots can be formed from the shoots by culturing plant shoots in a rooting bed in which water and / or liquid are held on a support having a liquid retention of 80% or less. The reason is guessed as follows.

  When plant shoots are inserted into the root bed (ie, prepared by holding the support with water and / or liquid medium) and cultured, the shoots are in contact with the root bed (support). From the portion, water and components of the liquid medium contained in the support are absorbed by the shoot, and adventitious roots are formed. At this time, it is considered that by using a support having a liquid retention rate of 80% or less, the insertion site becomes callus and adventitious root formation is promoted. In particular, in plants with a long period of rooting, such as pear plants and sandalwood plants, most of the shoots inserted until adventitious roots are formed often die, By culturing in an environment with a low ratio, shoot death is suppressed, and adventitious root formation is further promoted.

  Hereinafter, the present invention will be described in more detail with reference to examples.

[Example 1]
The current year branches of Pyrus serotina Rehder were used as cuttings. In addition, the cutting leaves were cut in half and suppressed transpiration, and when the cuttings were densely planted, they were inserted after the cuttings of adjacent cuttings were not overlapped. .

  On the other hand, a support (mixed medium-1 (vermiculite (particle size 2 mm or less): perlite)) added with IBA 2 mg / l as a plant hormone and wetted with a 4-fold diluted B5 liquid medium with a saturated mass that can be retained by the support. (Particle diameter 1.5-5 mm): peat moss (diameter 10 mm or less) = 1: 2: 1)), commercially available plug tray (upper diameter 5 cm × width 5.2 cm, lower diameter 2 cm × width 2. 4 cm and a depth of 7.8 cm) to prepare a root bed. 67 Hosui and 45 Kosui were inserted into the root bed. This plug tray was placed in a rectangular-shaped polycarbonate culture vessel (maximum size: 48 cm long × 68 cm wide × 20.8 cm high). The temperature in the container was 28 ° C. and the humidity was 50% or more. In a culture chamber adjusted to a carbon dioxide concentration of 1000 ppm, a temperature of 25 ° C., and a humidity of 60%, under light irradiation containing a wavelength component of 650 to 670 nm and a wavelength component of 450 to 470 nm at a ratio of 8: 2 (40 μmol photons m− 2 s-1).

Table 1 shows the results of measuring the three-phase distribution of soil of mixed soil-1 as a support with a soil three-phase meter (trade name: DIK-1120, manufactured by Daiki Rika Kogyo Co., Ltd.). In addition, Table 2 shows the dry mass and post-hydrous mass of mixed soil-1, the saturated mass of liquid that can be held on the support, the liquid retention rate, and the liquid retention property 6 days after the start of culture. The culture was continued under the above conditions for 24 days, and the transition of the amount of liquid (liquid maintenance rate) maintained in the support during the culture period was calculated by the following formula 3 and shown in FIG.
(Expression 3) Liquid retention rate (%) = (mass of liquid retained on support during measurement / mass of liquid retained on support at the start of culture) × 100

  The liquid retention was calculated according to Equation 1, and each mass of Equation 1 was measured according to the following procedure. First of all, each of the predetermined dry mass (see Table 2) is placed on a tray having a caliber of φ1.2 cm at the bottom of the market and having a hole of that size (upper caliber is 3 cm long × 3 cm wide, 4.5 cm deep). The support was filled. A tray filled with a support was placed in a polycarbonate container (maximum dimension: 11.5 cm long x 11.5 cm wide x 6.5 cm high) with a tubular shape with a slightly protruding body. 150 ml of water was injected and the bottom irrigated for about 2 hours. The tray filled with the support was taken out from the container and allowed to stand for about 10 minutes, and the mass of the support holding the liquid with a saturated mass (mass after water content in Table 1) was measured. By subtracting the dry mass of the support from the mass, the saturated mass of the liquid that can be held on the support (the mass of the liquid in Table 1) was calculated. These numerical values were substituted into Equation 1 to calculate the liquid retention rate.

  Further, the liquid retention was calculated according to Equation 2, and each mass of Equation 2 was measured according to the following procedure. The support holding the liquid was cultured for 6 days under the above conditions. The mass of the support holding the liquid 6 days after the start of the culture was measured, and the mass of the liquid held on the support 6 days after the start of the culture was calculated by subtracting the dry mass of the support from the mass. This value and the mass of the liquid retained on the support at the start of the culture (the mass of the liquid in Table 1) were substituted into Equation 2 to calculate the liquid retention.

  Furthermore, although the liquid maintenance factor was computed according to Formula 3, the measurement of each mass of Formula 3 was performed according to the following procedure. The support holding the liquid was cultured under the above conditions for 24 days. Every 3 days from the start of culture, the mass of the support holding the liquid at the time of measurement is measured, and the mass of the liquid held on the support at the time of measurement is calculated by subtracting the dry mass of the support from the mass. did. The numerical value and the mass of the liquid retained on the support at the start of the culture (the mass of the liquid in Table 1) were substituted into Equation 3 to calculate the liquid retention rate.

  After the start of culture, the rooting situation was observed, and after 8 weeks from the insertion, the number of roots and the rooting rate were shown in Table 3 (Hosui) and Table 4 (Kosui). The rooting rate was 60% in Hosui and 62% in Kosui.

[Example 2]
As in Example 1, pear (Hosui and Kosui) current year branches were used as cuttings. Substrate (mixed soil-2 (fine grained red crust (particle size 1 mm to 3 mm): wetted with a 4-fold diluted B5 liquid medium with a saturated mass that can be retained by the support) to which IBA 2 mg / l was added as a plant hormone Peat moss = 1: 1)) is packed into a commercially available plug tray (upper diameter is 5 cm x width 5.2 cm, lower diameter is 2 cm x width 2.4 cm, depth 7.8 cm) Prepared. On this root bed, 45 cuttings of Hosui and 90 Kosui were inserted. This plug tray was placed in a rectangular culture container made of polycarbonate (maximum dimension: 48 cm long × 68 cm wide × 20.8 cm high).

  In a culture chamber adjusted to a carbon dioxide concentration of 1000 ppm, a temperature of 25 ° C., and a humidity of 60%, under light irradiation containing a wavelength component of 650 to 670 nm and a wavelength component of 450 to 470 nm at a ratio of 8: 2 (40 μmol photons m− 2s-1). Table 1 shows the results of measuring the three-phase soil distribution of the mixing soil-2 as a support in the same manner as in Example 1. Moreover, the dry mass and the post-moisture mass measured for the mixing soil-2 in the same manner as in Example 1, the saturated mass of the liquid that can be held on the support at the start of the culture, the liquid retention rate, and the liquid retention after 6 days from the start of the culture Are shown in Table 2, respectively.

  The cells were cultured for 24 days under the above conditions. The transition of the amount of liquid maintained in the support during the culture period (liquid retention rate) was calculated in the same manner as in Example 1 and is shown in FIG.

  The rooting situation after the start of the culture was observed, and after 8 weeks from the insertion, the number of rooted cuttings and the rooting rate were shown in Table 3 (Hosui) and Table 4 (Kosui). The rooting rate was 42% in Hosui and 19% in Kosui.

[Comparative Example 1]
As in Example 1, pear (Hosui and Kosui) current year branches were used as cuttings. A support wetted with a 4-fold diluted B5 liquid medium supplemented with 2 mg / l of IBA as a plant hormone (porous support made of foamed phenol resin, “Oasis (registered trademark)” manufactured by Smither Oasis (length 2 cm × width 2 cm × 3 cm deep)) was placed on a styrene foam tray, and 54 cutting water and 54 cutting water were inserted. It was placed in a rectangular culture container made of polycarbonate (maximum dimension: 48 cm long x 68 cm wide x 20.8 cm high). The temperature in the container was 28 ° C. and the humidity was 50% or more.

  In a culture chamber adjusted to a carbon dioxide concentration of 1000 ppm, a temperature of 25 ° C., and a humidity of 60%, under light irradiation containing a wavelength component of 650 to 670 nm and a wavelength component of 450 to 470 nm at a ratio of 8: 2 (40 μmol photons m− 2s-1). Table 2 shows the dry mass and post-moisture mass of the support oasis, the saturated mass of the liquid that can be held on the support, the liquid retention rate, and the liquid retention after 6 days from the start of culture. The liquid retention rate during 24 days of culture under the above conditions is shown in FIG.

  The rooting situation after the start of the culture was observed, and after 8 weeks from the insertion, the number of rooted cuttings and the rooting rate were shown in Table 3 (Hosui) and Table 4 (Kosui). The rooting rate was 0% in Hosui and 0% in Kosui.

[Example 3 and Comparative Example 2]
A closed rectangular-shaped polycarbonate culture vessel (maximum dimensions: 11.5 cm in length × 11.5 cm in width × height) that uses the current branch of Hosui as the cutting head and the plug tray can maintain the humidity at 90% or higher. A 6.5cm cube with a slightly overhanging body and a circular opening with a polytetrafluoroethylene film with a pore diameter of 0.45μm (Millipore, trade name: Milliceal) on the top surface The test was carried out in the same manner as in Example 2 and Comparative Example 1 (Example 3 and Comparative Example 2 respectively) except that one was installed. The temperature in the container was 28 ° C. and the humidity was 90% or more. The results are shown in Table 5 and FIG.

  From the results of Examples 1 to 3 and Comparative Examples 1 to 2, the method of the present invention promotes adventitious root formation from shoots of the genus Pear, suppresses shoot death, and from shoots. It became clear that the rooting rate improved.

[Example 4, Example 5 and Comparative Example 3]
The test was conducted in the same manner as in Example 1, Example 2 and Comparative Example 1 (Example 4, Example 5 and Comparative Example 3, respectively) except that the current year branch of sandalwood was used as the cutting head. In addition, the liquid retainability of Example 4, Example 5, and Comparative Example 3 was 74.4%, 77.0%, and 96.3%, respectively. The results are shown in Table 6.

  From the results of Example 4, Example 5 and Comparative Example 3, the method of the present invention promotes adventitious root formation from shoots of sandalwood plants, suppresses shoot death, It was revealed that the root rate was improved.

  From the above results, it has been clarified that adventitious root formation from plant shoots is promoted, shoot death is suppressed, and rooting rate from shoots is improved by the method of the present invention.

Claims (4)

A support having a liquid retention of 80% or less represented by the following formula 1 is prepared with a root bed by holding water and / or a liquid medium, and the root bed contains a pear plant or a sandalwood plant. A method for producing a cloned seedling, in which a shoot is inserted and cultured, and adventitious roots are formed from the shoot.
(Formula 1) Liquid retention rate (%) = {saturated mass of liquid that can be retained on the support / (saturated mass of liquid that can be retained on the support + dry mass of the support)} × 100   The method for producing clonal seedlings according to claim 1, wherein the ratio of the solid phase in the three-phase distribution of the support is 40% or more. The clonal seedling production method according to claim 1 or 2, wherein the support has a liquid retention represented by the following formula 2 of 90% or less.
(Formula 2) Liquid retention (%) = (mass of liquid retained on the support 6 days after the start of culture / mass of liquid retained on the support at the start of culture) × 100   The method for producing a clonal seedling according to any one of claims 1 to 3, wherein the culturing is performed in a closed culture vessel.

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