JP2019154284A - Clone plant seedling production method using bamboo charcoal, clone plant production method, method for imparting bacteria resistance and/or insect resistance, additive for tissue culture, and culture medium composition - Google Patents

Abstract

To provide a novel tissue culture technique for obtaining a clone plant that has excellent insect resistance and bacteria resistance and can be cultivated without agrochemicals.SOLUTION: The present invention provides a method for producing a clone seedling by tissue culture of plant tissue, including the step of performing the tissue culture in the presence of bamboo charcoal.SELECTED DRAWING: Figure 5

Description

  The present invention relates to plant tissue culture and cultivation techniques.

A method of collecting plant tissue and cells from a plant body and culturing them is known. Examples of such tissue culture methods include shoot tip culture for culturing shoot apex, cocoon culture for culturing cocoons, and protoplast culture for culturing protoplasts.
In these tissue culture methods, the collected tissue is cultured under certain conditions to induce a cell mass called callus. Callus has different properties from the cell from which it originates, such as abnormal cell division, and has multipotency to produce various tissues and sometimes totipotency to produce somatic embryos.
By adjusting the conditions for culturing the callus, the plant can be re-differentiated to obtain a new plant individual. This plant individual is a clone having the same genetic information as the plant used for tissue culture.
In addition, organ culture for culturing organs such as leaves, embryo culture for culturing immature embryos, and the like are known. Cloned plants can also be obtained by these methods.

  Various studies have been conducted on the medium composition during tissue culture. In general, in addition to inorganic salts, vitamin B group, saccharides, growth regulators such as auxin and kinetin, amino acids, coconut milk, yeast extract and the like are used as necessary (for example, non-patented) Reference 1).

  It is known to use activated carbon as an additive to a culture medium for tissue culture (Patent Documents 1 to 9, etc.). Since activated carbon has an adsorbing ability, it is added to the medium for the purpose of removing growth-inhibiting substances released into the medium and for the purpose of gradual release of active ingredients.

JP 2017-0555670 A JP 2016-140318 A Japanese Patent Laid-Open No. 2005-27896 JP 2003-310070 A JP 2001-025330 A JP-A-11-308934 Japanese Patent Laid-Open No. 06-237658 JP 05-236833 A JP-T-2001-511652

“Methods of Plant Tissue Culture” Chemistry and Biology, Vol. 6, No. 3, 178-184.

  In tissue culture, it is necessary to maintain sterility, and it is common practice to add antibiotics to the medium as part of it. However, the clonal plant obtained by such a method is affected by being exposed to antibiotics in the tissue culture process, and may be inferior in insect resistance or fungus resistance. For this reason, in the process of cultivating clone seedlings in soil, the use of agricultural chemicals is essential, and there is a problem that it is very difficult to carry out agricultural chemical-free cultivation.

  In order to solve such problems, an object of the present invention is to provide a novel tissue culture technique for obtaining a clonal plant that is excellent in insect resistance and bacteria resistance and can be cultivated without agricultural chemicals.

The present invention for solving the above problems is a method for producing a cloned seedling by tissue culture of a plant tissue,
A clonal seedling production method comprising a step of tissue culture in the presence of bamboo charcoal.
ADVANTAGE OF THE INVENTION According to this invention, the fault of the conventional tissue culture technique called insect resistance and a bacteria-resistant deterioration can be solved, and the clone plant applicable also to agrochemical-free cultivation can be obtained.

In a preferred embodiment of the present invention, tissue culture is performed on a solid medium supplemented with bamboo charcoal.
In a liquid medium, it is necessary to stir in order to disperse oxygen in the medium and to disperse bamboo charcoal in the medium. However, in the case of a solid medium, tissue culture is simply performed without adding operations such as stirring. be able to.

  Tissue culture is performed in a medium in which a composition containing bamboo charcoal is laminated on a solid medium. Even with such a simple culture medium configuration, the present invention exhibits the effect effectively.

  In a preferred form of the invention, the tissue culture is a callus culture. A cloned plant obtained by applying the present invention and redifferentiating callus is excellent in resistance such as insect resistance and fungus resistance.

  In a preferred form of the invention, tissue culture is performed in the absence of antibiotic. According to the present invention, the risk of contamination is low without adding antibiotics. Moreover, the clonal plant which was excellent in resistance can be obtained by setting it as the form which does not use antibiotics.

The present invention also relates to a clonal plant production method comprising a clonal seedling production step for producing a clonal seedling by the above-described clonal seedling production method and a cultivation step for cultivating the clonal seedling in soil.
The cloned plant produced by the present invention is excellent in resistance.

In the preferable form of this invention, the agrochemical chosen from an insecticide and a disinfectant is not used in the said cultivation process.
Since the clone seedling produced as described above is excellent in resistance, pesticide-free cultivation is possible in the cultivation of the seedling.

  The present invention also relates to a method for imparting bacterial resistance and / or insect resistance to a plant, comprising a step of tissue culture in the presence of bamboo charcoal.

  The present invention also relates to an additive for tissue culture made of bamboo charcoal, which is used for imparting bacterial resistance and / or insect resistance to plants.

  The present invention further relates to a medium composition for tissue culture of plants, characterized in that it contains bamboo charcoal.

  According to the present invention, it is possible to produce a clonal plant having excellent resistance such as insect resistance and bacteria resistance.

Drawing substitute photograph showing child stock cut out from stock of banana parent stock. Drawing substitute photograph showing growth tissue cut out from center of banana seedling. A drawing-substituting photograph showing a state of cutting out germinating tissue generated as a result of primary culture. The drawing substitute photograph showing a mode that the cut germinated tissue was placed on the agar medium. Drawing substitute photograph showing bamboo charcoal laminated solid medium. A drawing-substituting photo showing a tissue culture on a bamboo charcoal solid medium. A drawing-substituting photo showing a tissue culture on a bamboo charcoal solid medium. A drawing-substituting photo showing a tissue culture on a bamboo charcoal solid medium. Drawing substitute photograph showing the appearance of transplanting cloned seedlings into aseptically treated soil. Drawing substitute photograph showing a state of soil cultivation while fertilizing and managing clone seedlings. Drawing substitute photo showing the appearance of cloned seedlings being cultivated in nursery pots.

  The present invention relates to a method for producing a cloned seedling by culturing a plant tissue and a clonal plant obtained by cultivating the cloned seedling.

  The kind of plant which can apply this invention is not limited. For example, Papayaceae (Caricaceae), Pineappleaceae (Bromeliaceae), Musaceae (Musaceae), Cucurbitaceae, Myrtaceae Myrtaceae, Oxalidaceae, Moraceae, Moraceae Rubiaceae, Laureaceae, Passifloraceae, Spindeaceae, Closiaceae, Ebenaceae, Rubiaceae, Rutaceae , Cactaceae, Rosaceae It can be exemplified application to plants.

  More specifically, the genus Papaya (Carica), the genus Ananas, the genus Musa, the genus Lacanca Siraitia, the genus Vanjiro (Psidium), the genus Averrhoa, the genus Ficus, and the genus Theobroma. ), Coffea, Cinnamumum, Passiflora, Ganoderma (Litchi), Garcinia, Diospyros, Casimiroa, Vanilla Examples include plants belonging to the genus Phenix, Hylocereus, and Cerasus.

The tissue culture to which the present invention can be applied is not limited. The shoot tip culture for culturing the shoot tip, the culm culture for cultivating the cocoon, the protoplast culture for culturing the protoplast, the organ culture for culturing organs such as leaves, and the immature embryo are cultured. Examples include embryo culture.
In particular, it is preferable to apply the present invention to so-called callus culture in which a clonal seedling is produced by inducing callus of a collected plant tissue such as shoot apex culture, anther culture, or protoplast culture and redifferentiating the callus.

The present invention is characterized by including a step of performing tissue culture in the presence of bamboo charcoal. The form of bamboo charcoal used in the present invention is not limited, but is preferably a powder.
Bamboo charcoal includes true bamboo, Miso bamboo, black bamboo, Shino bamboo, Chishima bamboo, Kenmon bamboo, Yatake, okame bamboo, Chinese bamboo, bamboo bamboo, pale bamboo, cloth bag bamboo, turtle shell bamboo, four-sided bamboo, arabic bamboo, bamboo shoot, metropolitan, female bamboo Any bamboo-derived material may be used, but bamboo bamboo charcoal derived from Miso bamboo is preferred.

  The method for producing bamboo charcoal is not limited, and the carbonization temperature is preferably 200 ° C. or higher, more preferably 500 ° C. or higher, more preferably 800 ° C. or higher, further preferably 1000 ° C. or higher, more preferably 1100 ° C. or higher, and still more preferably 1100 ° C. ~ 1300 ° C.

  In general, there are multiple processes for producing cloned seedlings by tissue culture, including a callus induction process for inducing callus from tissue, a callus culture process for culturing callus, a shoot formation process for forming shoots, and a rooting process for generating roots. These steps are included. In the present invention, any process may be performed in the presence of bamboo charcoal.

The medium for tissue culture may be a liquid medium or a solid medium. It can be appropriately selected depending on the plant species to be produced, the purpose of each step in tissue culture, and the like.
The composition of the medium is not particularly limited, and is a basic medium generally used for plant tissue culture containing inorganic components, vitamins, and carbon sources, and if necessary, amino acids, plant hormones, coconut milk, yeast extract. Etc. can be used.

  Examples of the inorganic component include elements such as nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, iron, manganese, zinc, boron, molybdenum, chlorine, iodine and cobalt, and inorganic salts containing these. Examples of inorganic salts include potassium nitrate, ammonium nitrate, ammonium chloride, sodium nitrate, potassium monohydrogen phosphate, sodium dihydrogen phosphate, potassium chloride, magnesium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, Examples thereof include copper sulfate, sodium sulfate, calcium chloride, magnesium chloride, boric acid, molybdenum trioxide, sodium molybdate, potassium iodide, cobalt chloride, and hydrates thereof. As an inorganic component, 1 type (s) or 2 or more types can be selected and used from the said specific example.

  As the carbon source, compounds such as carbohydrates such as sucrose and derivatives thereof; organic acids such as fatty acids; primary alcohols such as ethanol can be used. As a carbon source, 1 type (s) or 2 or more types can be selected and used from the said specific example.

  As vitamins, for example, biotin, thiamine (vitamin B1), pyridoxine (vitamin B4), pyridoxal, pyridoxamine, calcium pantothenate, inositol, nicotinic acid, nicotinamide and / or riboflavin (vitamin B2) are used. Can do. As vitamins, 1 type (s) or 2 or more types can be selected and used from the above specific examples.

  As amino acids, for example, glycine, alanine, glutamic acid, cysteine, phenylalanine and / or lysine can be used. As the amino acids, one or more of the above specific examples can be selected and used.

As plant hormones, for example, auxins and cytokinins can be used.
Examples of auxins include naphthalene acetic acid (NAA), indole acetic acid (IAA), p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4D), indolebutyric acid (IBA), and derivatives thereof. One or more selected from these or a combination of two or more may be used.
Examples of the cytokinins include benzyladenine (BA), kinetin, zeatin, and derivatives thereof, and one or more selected from these can be used in combination.
As plant hormones, only auxins, only cytokinins, or a combination of both auxins and cytokinins can be used.

  Examples of the basic medium include MS medium, Rinsmeier Skoog medium, white medium, Gamborg B-5 medium, and niche niche medium.

  The medium composition in each step constituting tissue culture, such as a callus induction step, a callus culture step, a shoot formation step, and a rooting step, can be appropriately designed by adjusting the concentration of plant hormones by a conventional method.

  When tissue culture is performed using a liquid medium in the presence of bamboo charcoal, it is preferable to perform the culture using a bamboo charcoal-dispersed liquid medium in which bamboo charcoal is dispersed. When using bamboo charcoal-dispersed liquid medium, it is necessary to supply oxygen in the liquid, and from the viewpoint of uniformly dispersing bamboo charcoal in the medium, cultivate with a rotary incubator, shaker, etc. Is preferred.

On the other hand, a solid medium is preferable because an operation such as stirring is not required unlike a liquid medium. As the solid medium used in the present invention, a solid medium solidified by a gelling agent such as agar, agarose, carrageenan, gellan gum can be preferably exemplified.
When tissue culture is performed with a solid medium in the presence of bamboo charcoal, a solid medium containing bamboo charcoal may be prepared and cultured on the bamboo charcoal-containing solid medium.

In the present invention, tissue culture may be performed in a bamboo charcoal laminated solid medium in which a composition containing bamboo charcoal is laminated on a solid medium.
Examples of the “composition containing bamboo charcoal” include a dispersion composition such as water or an aqueous solution in which bamboo charcoal is dispersed, and a powder composition composed of bamboo charcoal or powder other than bamboo charcoal and bamboo charcoal.

  In the present specification, the culture may be carried out using any of the above-described medium compositions of bamboo charcoal dispersion liquid medium, bamboo charcoal-containing solid medium, and bamboo charcoal laminated solid medium, but bamboo charcoal laminated solid medium can be particularly preferably used. .

  The bamboo charcoal content in the entire medium composition is preferably 1 to 10% by weight, more preferably 2 to 5% by weight, and even more preferably 3 to 4% by weight, but is not limited to these concentration ranges.

  In general, various antibiotics such as carbenicillin, vancomycin and cefotaxime are used in tissue culture. In the present invention, it is preferable not to use antibiotics in tissue culture. According to the method of the present invention, a clonal seedling or a clonal plant having excellent resistance can be obtained without using an antibiotic.

  The clonal seedling obtained by tissue culture by the above-described method and redifferentiating the plant can be used for soil cultivation. The specific method of the cultivation process which grows a clone seedling can be suitably selected by a conventional method according to the kind of plant.

  In the cultivation process, agricultural chemicals are often used in general. However, since the clonal seedlings or clonal plants produced by the method of the present invention are imparted with resistance, they can be subjected to pesticide-free cultivation without using pesticides such as insecticides and fungicides.

  The child stock generated from the stock of the banana parent strain was dug out and cut out (FIG. 1). The cut out stock was cut into round pieces, the central growth tissue was cut out (FIG. 2), and this was sterilized with alcohol. The growing tissue was first cultured and germinated (FIG. 3), and the generated buds were cut and placed on an agar medium spread in a bioflask (FIG. 4). Water containing bamboo charcoal powder dispersed on an agar medium on which germinating cells were placed was added to obtain a bamboo charcoal laminated solid medium (FIG. 5). The bamboo charcoal powder used here is obtained by carbonizing Somune bamboo at a carbonization temperature of 1200 ° C. Bamboo charcoal powder was added to 3 to 4% by weight of the whole medium. Antibiotics were not added to the bamboo charcoal laminated solid medium.

  Tissue culture of banana germinating cells was continued on bamboo charcoal laminated solid medium. 6 to 8 show photographs taken over time of the growth of bananas during the tissue culture process. As the banana clone seedlings grew, the amount of bamboo charcoal powder in the bioflask decreased. This is thought to be because banana clone seedlings took bamboo charcoal powder into the cells.

  Mold did not occur in the bioflask during the tissue culture process in this test example. On the other hand, as a comparative example, tissue culture was similarly performed on a plurality of samples using an agar medium (with no bamboo charcoal powder and no antibiotics) having the same composition as in the above example, but a sample in which mold was generated in the bioflask Has occurred.

  This result shows that contamination problems are usually manifested in tissue culture unless antibiotics are used, but by conducting tissue culture in the presence of bamboo charcoal, contamination problems can be solved without using antibiotics. Is shown.

The clonal seedlings of Examples obtained in this way were transferred to aseptic culture soil subjected to steam treatment at 160 ° C., and soil cultivation was performed (FIG. 9). On the 100th day after planting, the humidity was adjusted to the same humidity as the culture conditions in the bioflask, and the cultivation was carried out under fertilization management for the purpose of adjusting the ultraviolet rays (FIG. 10). After 100 days from the fixed planting, the plants were replanted into seedling pots and cultivated in an environment directly exposed to sunlight (FIG. 11). After that, banana clone plants could be cultivated without problems until fruit set and harvest. In this cultivation process, no pesticide was used.
On the other hand, the comparative clone plant grown on the soil under the same conditions died due to the effects of pests and bacteria.

  This result shows that the clonal plant obtained by tissue culture in the presence of bamboo charcoal is excellent in resistance such as insect resistance and bacteria resistance and can be used for agricultural chemical-free cultivation.

  The present invention can be applied to plant tissue culture and cultivation methods.

Claims (10)

A method for producing a cloned seedling by culturing a plant tissue,
A method for producing clonal seedlings, comprising a step of performing tissue culture in the presence of bamboo charcoal.   The clonal seedling production method according to claim 1, wherein tissue culture is performed on a solid medium to which bamboo charcoal is added.   The clonal seedling production method according to claim 2, wherein tissue culture is performed in a medium in which a composition containing bamboo charcoal is laminated on a solid medium.   The clonal seedling production method according to any one of claims 1 to 3, wherein the tissue culture is callus culture.   The method for producing a clonal seedling according to any one of claims 1 to 4, wherein tissue culture is performed in the absence of an antibiotic.   A clone comprising: a clone seedling production step for producing a clone seedling by the clone seedling production method according to any one of claims 1 to 5; and a cultivation step for growing the clone seedling in soil. Plant production method.   The method for producing a clonal plant according to claim 6, wherein an agricultural chemical selected from insecticides and fungicides is not used in the cultivation step.   A method for imparting bacterial resistance and / or insect resistance to a plant, comprising a step of tissue culture in the presence of bamboo charcoal.   An additive for tissue culture made of bamboo charcoal, which is used to impart plant resistance and / or insect resistance to plants. A medium composition for plant tissue culture, comprising bamboo charcoal.