JP2012196204A - Method for retaining plant for long period - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for retaining plants for a long period, in plant vegetative propagation.SOLUTION: The method for retaining plants for a long period includes performing plant vegetative propagation, using trehalose, for instance, a trehalose-containing culture medium. The method is suitably applicable to vegetative propagation of general plants which cannot retain transformation present hereditary characteristics in a progeny system by seed multiplication, due to hereditarily unfixed property or self-incompatibility of, for instance, a gene-recombination plant such as a gene-recombination ornamental plant.

Description

  The present invention relates to a long-term maintenance method of a plant, for example, a method of maintaining a plant for a long time in vegetative growth of a plant that cannot maintain the genetic characteristics of the current generation in a progeny line by seed growth, such as a genetically modified plant.

  Genetically modified plants such as genetically modified flowers are cultured, maintained, and managed by vegetative growth (cutting buds) using a medium or the like. Many genetically modified flowers use cultivated varieties that are not genetically fixed, and therefore require a great deal of time, money, and labor for maintenance during vegetative growth. For example, when cultivating and maintaining Torenia as a genetically modified flower, a conventional medium using sucrose as a carbon source can maintain the culture only for a short period (for example, up to about 30 days), and is planted in a new medium. It was necessary to change. In addition, in the production of useful flowers by gene recombination, it is necessary to maintain a large number of cultures that are several thousand lines at all times. Therefore, if the maintenance period is short, a great amount of time, cost, and labor are required. In general plant cultures such as genetically modified plants that are not genetically fixed or are unable to maintain the genetic characteristics of the current generation in progeny lines due to seed propagation due to properties such as self-incompatibility It is required to reduce the time, expense, labor, etc. when maintaining as.

  Trehalose, on the other hand, is known as a sugar responsible for so-called “dry dormancy”, which is found in plants such as squirrels and in animals such as chironomids (giving tolerance to drought stress by maintaining and protecting the structure of cells) (for example, patent documents) 1) In addition, animals and plants such as maintaining freshness of vegetables and fruits (for example, see Patent Document 2), suppressing discoloration, suppressing protein denaturation and starch aging, imparting freezing resistance (for example, refer to Patent Document 3), and suppressing moisture absorption. It is known to impart various functionalities to. Many studies on its metabolic system, transport system, and functionality have been conducted so far, but its use as a carbon source in plant culture has not been reported.

JP 2009-055832 A JP 2000-116319 A Japanese Patent Laid-Open No. 10-025209

  An object of the present invention is to provide a method capable of maintaining a plant for a long time in vegetative growth of the plant.

  The present invention is a method for long-term maintenance of a plant that performs vegetative growth of the plant using trehalose.

  Moreover, in the said long-term maintenance method of the plant, it is preferable to perform vegetative growth of a plant using the culture medium containing the said trehalose.

  In the long-term maintenance method of the plant, it is preferable that the plant is a plant that cannot maintain the genetic characteristics of the current generation in a progeny line by seed propagation.

  In the method for maintaining a plant for a long period of time, the plant is preferably a genus Torenia or Chrysanthemum.

  Further, in the long-term maintenance method of the plant, the trehalose is vegetatively grown using the trehalose in a state of being rooted by culturing with sucrose, or the trehalose in a state where leaves are removed from a node containing a plurality of axillary buds. It is preferable to perform vegetative growth of plants using

  In the present invention, by using trehalose, the plant can be maintained for a long time in the vegetative growth of the plant.

It is a figure which shows a mode that the culture of the cutting bud 70th day in Example 1-1 and Comparative Example 1-1 of this invention was observed. It is a figure which shows a mode that the culture of the cutting bud 70th day in Example 1-1 and Comparative Example 1-1 of this invention was observed from the bottom face of the container. It is a figure which shows a mode that the culture of the cutting bud 40th day in Example 1-2 of this invention and Comparative Example 1-2 was observed. It is a figure which shows a mode that the culture of the cutting bud 40th day in Example 1-5 of this invention and Comparative Example 1-5 was observed. It is a figure which shows a mode that the culture of the cutting bud 98th day in Example 3-1 and Comparative Example 3-1 of this invention was observed. It is a figure which shows a mode that the culture of the cutting bud day 157 in Example 3-1 and Comparative Example 3-1 of this invention was observed. It is a figure which shows a mode that the culture of the cutting bud 98th day in Example 3-2 and Comparative Example 3-2 of this invention was observed. It is a figure which shows a mode that the culture of the cutting bud 157th day in Example 3-2 and Comparative Example 3-2 of this invention was observed. It is a figure which shows a mode that the culture of the cutting bud 70th day in Example 4 and Comparative Example 4 of this invention was observed. It is a figure which shows a mode that the culture of the cutting bud 85th day in Example 5 of this invention and the comparative example 5 was observed. It is a figure which shows a mode that the culture of the cutting bud 144th day in Example 5 and Comparative Example 5 of this invention was observed. It is a figure which shows a mode that the culture of the cutting bud 71st day in Example 6 and Comparative Example 6 of this invention was observed. It is a figure which shows a mode that the culture of the cutting bud 130th day in Example 6 and Comparative Example 6 of this invention was observed. It is a figure which shows a mode that the culture of the cutting bud 71st day in Example 7 and Comparative Example 7 of this invention was observed. It is a figure which shows a mode that the culture of the cutting bud 130th day in Example 7 of this invention and the comparative example 7 was observed. It is a figure which shows a mode that the culture of the 2nd planting joint 25th day in Example 8 of this invention was observed.

  Embodiments of the present invention will be described below. This embodiment is an example for carrying out the present invention, and the present invention is not limited to this embodiment.

  The present inventors have found that in the vegetative growth of plants, the maintenance period of the culture can be extended, for example, by a factor of 2 or more by replacing sucrose usually used in the medium with trehalose. Thereby, the culture can be maintained for a long time, and the passage interval can be prolonged. For example, in general plants that are not genetically fixed, such as genetically modified plants, or are unable to maintain the genetic characteristics of the current generation in progeny lines due to seed propagation due to properties such as self-incompatibility, The time, expense, labor, etc. at the time of maintenance can be reduced by making the period which can be maintained by one planting as long as possible. In addition, long-term culture while maintaining a line (genetic characteristics) of a genetically modified plant or the like becomes possible. Furthermore, in the production of genetically modified plants such as useful flowers by genetic recombination, time, cost, and labor can be reduced even when maintaining a large number of cultures in the thousands.

  Trehalose used in the plant long-term maintenance method according to the present embodiment is a kind of disaccharide in which two α-glucoses are linked by 1,1-glycosides. By using trehalose, it is considered that the improvement of the rhizosphere environment in vegetative growth, the reduction of stress in nutrient absorption, etc. lead to the long-term maintenance of the culture. When trehalose is used, the growth of the plant body is rather suppressed in the rhizosphere, and as a result, accumulation of waste products and aging due to this are suppressed, leading to long-term maintenance of the culture environment.

  As the trehalose, those available as reagents and the like may be used. For example, “Trace to rice” (manufactured by H + B Life Sciences, trehalose content 90% (w / w), in dry weight) A trehalose product for food addition such as 98% (w / w)) may be used.

  Here, the vegetative growth of plants is asexual reproduction in which the next generation of plants propagates from vegetative organs such as roots, stems and leaves without going through embryos and seeds. The method of vegetative growth is not particularly limited as long as trehalose is used, and examples thereof include a method using an agar medium, a method using a liquid medium, and a method by sprouting into soil. The method using an agar medium is preferable from the viewpoint of maintaining aseptically.

  As the medium, there can be used MS (Murashige-Skoog) medium, B5 (Gamborg's B5) medium, Kano medium or the like containing trehalose.

  When a medium is used, the trehalose content is, for example, in the range of 0.1% (w / v) to 6% (w / v), usually 3% (w / v) with respect to the total weight of the medium. )

  In addition to trehalose, the medium may contain, for example, sucrose, maltose, fructose, glucose and the like.

For example, a vegetative piece of the above-ground part including nodes, axillary buds, leaves, adventitious buds, etc. of plants to be cultured and maintained is cut out and placed on a medium containing trehalose, for example, 10 ° C to 35 ° C, usually 20 ° C. in a temperature range of to 25 ° C., a photon flux density for example 0μmol / m ² / sec ~2,000μmol / m ² / sec, usually in the range of 80μmol / m ² / sec ~160μmol / m ² / sec, for example 0 What is necessary is just to culture | cultivate in a time light period / 24-hour dark period-24 hours light period / 0-hour dark period, Usually 16 hours light period / 8 hours dark period-8 hours light period / 16 hours dark period.

  The plant in which vegetative growth is performed by the plant long-term maintenance method in the present embodiment is not particularly limited, and examples thereof include plants that improve long-term maintenance by using trehalose. Such a plant may be an angiosperm or a gymnosperm. Further, the angiosperm may be a monocotyledonous plant or a dicotyledonous plant. The dicotyledonous plant may be a petal flower subclass or a joint flower subclass. Examples of the joint flower subclass include Gentian eyes, Eggplant eyes, Perilla eyes, Abagoceae, Plantains, Oleanders, Pleurotus, Rubiaceae, Tricholoma, and Chrysanthemum. In addition, examples of the petal flower subclass include bivalamata, camellia, blue mushrooms, scallops, diptera, violets, willows, scallops, azaleas, crabs, oysters, primulas, etc. It is done.

  More specific examples of such plants include, for example, rapeseed, potato, spinach, soy, cabbage, lettuce, tomato, cauliflower, soya beans, turnip, radish, broccoli, melon, orange, watermelon, leek, burdock, etc. Various edible plants, roses, chrysanthemum, hydrangea, carnations, torenia and other (flowering) garden plants, periwinkle, yew, oren, blossoms, yellowfin, ginseng, poppy, licorice, belladonna, datura, Cephaelis ipecacuanha, and other medicinal plants Examples include lilac, rowan, eucalyptus, mangrove, cedar and other trees.

  The plant long-term maintenance method according to the present embodiment is, for example, a genetically modified plant such as a genetically modified flower, or the like, which is not genetically fixed, or has a self-incompatibility, etc. It is applied to the vegetative growth of all plants that cannot maintain the genetic characteristics of the current generation. Even when maintaining large-scale cultures of such plants, the cultures can be maintained for a long time, the passage interval can be extended, and long-term maintenance of lines such as genetically modified plants is possible. Can be cultured. Examples of genetically modified plants include, for example, chrysanthemum, roses, carnations, cyclamen, gentian, eustoma, morning glory, petunia, torenia and other genetically modified flowers, rice, potato, corn, cotton, soybean, rapeseed, tobacco and other crops, Examples include trees such as poplar, eucalyptus, pine, apple, papaya, and cherry. By applying the plant long-term maintenance method according to the present embodiment, development for producing more useful genetically modified plants and the like can be performed while reducing time, cost, and labor. In addition, non-genetically modified plants can also be applied to plants that are cultured and vegetatively grown in large quantities such as cyclamen and orchids.

  Torenia is an annual plant belonging to the genus Toledia, native to Indochina. Torenia can be grown with a small plant height (for example, flowering at a minimum of about 7 cm), can be easily maintained and propagated by cuttings, has a short period of time until it blooms, and has high humidity like a plant box regardless of day length. The diploid (2n = 16 or 18) that normally blooms even under cultivation conditions is small in genome size (about 170 Mbp = almost equivalent to Arabidopsis thaliana), and has features such as established genetic recombination techniques, The plant long-term maintenance method according to this embodiment is preferably applied.

  Chrysanthemum is a plant belonging to the genus Asteraceae. Chrysanthemum has long been used as a research material for flower color, etc. because it has high commercial value and is in great demand, and a method of genetic recombination has been established for a wide variety of cultivars that can be easily maintained and propagated by cuttings. In view of the above, the plant long-term maintenance method according to the present embodiment is preferably applied.

  In the vegetative growth of the plant in the present embodiment, for example, using a medium such as sucrose medium, it is cultured for a predetermined period to obtain a young plant, and from this young plant, an above-ground nutrient tissue piece including a node is cut out. Alternatively, the culture may be performed by placing in a trehalose medium. When transferring from this sucrose medium etc. to trehalose medium, transient yellowing and wilt of leaves, which seems to be due to the transient and rapid suction of trehalose from the cut surface of the buds inserted into some buds, May be seen. In this regard, particularly in the first planting, trehalose is vegetatively grown using trehalose in a state of being rooted by culturing with sucrose, or trehalose in a state where leaves are removed from a node containing a plurality of buds. It is preferable to perform vegetative growth of plants using For example, 1) cultivate the cut buds in sucrose medium for 3 to 14 days, preferably about 1 week, and transfer to trehalose in the rooted state, or 2) insert the buds after removing all the leaves from the nodes containing multiple buds. It can be improved by using any method of budding. As a result, drastic uptake of trehalose is suppressed, and transient leaf yellowing, wilt and the like can be suppressed.

  The initial growth of the cuttings in the trehalose medium tends to be slightly inferior to that of the sucrose medium, and the difference is particularly remarkable in chrysanthemum that has been transferred from the sucrose medium to the trehalose medium in the state of the knot. Although this difference tends to be smaller in the second and subsequent planting (planting once grown in trehalose medium to trehalose medium), it tends not to be completely equivalent, so the plant species and purpose of use ( For example, it is preferable to use properly depending on whether the purpose is long-term maintenance of culture or whether it is important to nurture materials in a short period of time.

  In addition, the slow initial growth rate in trehalose medium is thought to contribute to the long-term maintenance of the culture, but the difference in the size of the plant is small for the degree of extension of the maintenance period. Considering this, it is presumed that long-term maintenance became possible as a result of some action of maintaining or improving the activity of the plant possessed by trehalose, rather than extending the time until the carbon source was depleted.

  Hereinafter, although an example and a comparative example are given and the present invention is explained more concretely in detail, the present invention is not limited to the following examples.

<Example 1-1>
25% sucrose medium (composition: 1 / 2MS medium pH 5.7, 3% sucrose, 0.38% gellan gum) containing 3% (w / v) sucrose (first grade reagent manufactured by Wako Pure Chemical Industries) Tolenia seedlings (crown violet (purple flower line)) were obtained by culturing for days. From this Torenia seedling, an above-ground vegetative tissue piece containing nodes was cut out and 1/2 MS trehalose medium (composition: 1/2 MS medium, pH 5) containing 3% (w / v) trehalose (first grade reagent manufactured by Wako Pure Chemical Industries). 7%, 3% trehalose, 0.2% gellan gum). This was cultured at 25 ° C., photon flux density 85 μmol / m ² / sec, 16 hours light period / 8 hours dark period, and the growth and wilt of the plants were observed. All Torenia plants were kept healthy up to 75 days. In addition, even in the maintenance of the plant by actual cuttings, the plant of Torenia could be maintained in a healthy manner even when the passage interval was set to 70 days, which is more than double the conventional passage.

<Comparative Example 1-1>
Culture was performed in the same manner as in Example 1-1 except that a 1 / 2MS sucrose medium containing 3% (w / v) sucrose was used instead of trehalose. Only up to 30 days, Torenia plants could only be maintained healthy. Moreover, in maintenance of the plant by actual cuttings, planting every 25 days was necessary.

  FIG. 1 shows a state in which the cultures of Example 1-1 and Comparative Example 1-1 on the 70th day after buds were observed. FIG. 2 shows the culture observed from the bottom of the container. The right side of FIGS. 1 and 2 is the Torenia plant of Example 1-1 using trehalose, and the left side is the Torenia plant of Comparative Example 1-1 using sucrose.

  As can be seen from FIG. 1, in the Torenia plant of Example 1-1 using trehalose, the healthy state was maintained even on the 70th day after cutting, and there was little unusual rooting from the above-ground part. . On the other hand, in the Torenia plant body of Comparative Example 1-1 using sucrose, the wilt of the plant was advanced and there were many different roots from the above-ground part. In addition, as can be seen from FIG. 2, in the Torenia plant of Example 1-1 using trehalose, the root density is low and a healthy state is maintained even on the 70th day after cutting. On the other hand, in the Torenia plant body of Comparative Example 1-1 using sucrose, the density of roots was high, and browning of the medium, which seems to be the accumulation of polyphenols, was confirmed. The browning of the culture medium in Comparative Example 1-1 was clearly confirmed after the 40th day after cutting. By using trehalose as in Example 1-1, it is considered that the improvement of the rhizosphere environment and the stress reduction of nutrient nutrient absorption lead to the long-term maintenance of the culture.

<Example 2>
Instead of trehalose, a 1 / 2MS medium containing 3% (w / v) “rice to treha” (manufactured by H + B Life Sciences, trehalose product for food addition, trehalose content 90% (w / w)) The culture was performed in the same manner as in Example 1-1 except that it was used. All Torenia plants were kept healthy up to 75 days. In addition, even in the maintenance of the plant by actual cuttings, the plant of Torenia could be maintained in a healthy manner even when the passage interval was set to 70 days, which is more than double the conventional passage.

  Thus, by performing vegetative growth of torenia using trehalose, the maintenance period of the culture could be extended more than twice as compared with the case of using sucrose conventionally used. As a result, the culture can be maintained for a long time, and the passage interval can be prolonged. Long-term culture was possible while maintaining strains such as genetically modified plants such as Torenia, and even when a large amount of culture was maintained, time, cost, and labor could be almost halved. The long-term maintenance method of plants that perform vegetative growth of plants using such trehalose is, for example, genetically fixed, or due to properties such as self-incompatibility, Applicable to all plants that cannot maintain genetic characteristics.

<Examples 1-2 to 1-5, Comparative Examples 1-2 to 1-5>
As a torenia, instead of “Crown Violet”, “Crown Burgundy” (Example 1-2, Comparative Example 1-2), “Crown Blue” (Example 1-3, Comparative Example 1-3), “Crown Lemon” Example 1-1, except that “Drop” (Example 1-4, Comparative Example 1-4) and inertia “Summer Wave Blue” (Example 1-5, Comparative Example 1-5) were used. Culture was performed in the same manner as Comparative Example 1-1.

  In the torenia plants of Examples 1-2 to 1-5 using trehalose, all the torenia plants could be maintained healthy up to 70 days. On the other hand, the torenia plants of Comparative Examples 1-2 to 1-5 using sucrose could only be maintained up to 40 days.

  FIG. 3 shows a state in which cultures on the 40th day after the buds of Example 1-2 and Comparative Example 1-2 were observed. The left two in FIG. 3 are the plants of Torenia “Crown Burgundy” of Example 1-2 using trehalose, and the two on the right are the plants of Torenia “Crown Burgundy” of Comparative Example 1-2 using sucrose. . Moreover, a mode that the culture was observed from the bottom face of the container is shown in the lower side of FIG. FIG. 4 shows the state of observation of the cultures on the 40th day after the buds of Example 1-5 and Comparative Example 1-5. The left two in FIG. 4 are the plants of Torenia “Summer Wave Blue” of Example 1-5 using trehalose, and the two on the right are the plants of Torenia “Summer Wave Blue” of Comparative Example 1-5 using sucrose. It is. Moreover, a mode that the culture was observed from the bottom face of the container is shown on the lower side of FIG.

  As can be seen from FIGS. 3 and 4, the torenia plants of Examples 1-2 and 1-5 using trehalose maintained a healthy state even on the 40th day after cutting. On the other hand, in the Torenia plant bodies of Comparative Examples 1-2 and 1-5 using sucrose, the wilting of the plant was slightly advanced.

<Examples 3-1 to 3-4, Comparative Examples 3-1 to 3-4>
Instead of torenia, chrysanthemum “Seimarin” (Example 3-1 and Comparative Example 3-1), “Ohira” (Example 3-2 and Comparative Example 3-2), “Shima” (Example 3-3, Comparative Example 3-3), Example 1-1 and Comparative Example 1-1, respectively, except that the diploid mushroom experiment line "Saruzawa-1" (Example 3-4, Comparative Example 3-4) was used. Culture was performed in the same manner as described above.

  In the chrysanthemum “Seimarin” plant of Example 3-1 using trehalose, all chrysanthemum plants could be maintained healthy up to 140 days. On the other hand, the chrysanthemum "Seimarin" plant of Comparative Example 3-1 using sucrose could only be maintained up to 70 days. In the chrysanthemum "Ohira" plant of Example 3-2 using trehalose, all chrysanthemum plants could be maintained healthy up to 160 days. On the other hand, the chrysanthemum "Ohira" plant of Comparative Example 3-2 using sucrose could only be maintained up to 90 days. In the chrysanthemum “Shinma” plant of Example 3-3 using trehalose, all chrysanthemum plants could be maintained healthy up to 480 days. On the other hand, the chrysanthemum “Shinma” plant of Comparative Example 3-3 using sucrose could only be maintained up to 112 days. In the chrysanthemum "Saruzawa-1" plant of Example 3-4 using trehalose, all chrysanthemum plants could be maintained healthy up to a maximum of 480 days. On the other hand, the chrysanthemum "Saruzawa-1" plant of Comparative Example 3-4 using sucrose could only be maintained up to 112 days.

  FIGS. 5 and 6 show the observation of the cultures of Example 3-1 and Comparative Example 3-1 on day 98 and day 157, respectively. The upper side of FIGS. 5 and 6 is the plant of chrysanthemum “Seimarin” of Example 3-1 using trehalose, and the lower side is the plant of chrysanthemum “Seimarin” of Comparative Example 3-1 using sucrose. FIGS. 7 and 8 show the observation of the cultures of Example 3-2 and Comparative Example 3-2 on day 98 and day 157, respectively. The upper side of FIGS. 7 and 8 is the plant of chrysanthemum “Ohira” of Example 3-2 using trehalose, and the lower side is the plant of chrysanthemum “Ohira” of Comparative Example 3-2 using sucrose.

  As can be seen from FIGS. 5 and 6, the chrysanthemum “Seimarin” plant of Example 3-1 using trehalose maintains a healthy state even on the 98th day after cutting, and is healthy even on the 157th day after cutting. It was almost maintained. On the other hand, in the chrysanthemum “Seimarin” plant of Comparative Example 3-1 using sucrose, the wilting of the plant was advanced on the 98th day after the incision. Moreover, as can be seen from FIGS. 7 and 8, the chrysanthemum “Ohira” plant of Example 3-2 using trehalose almost maintained a healthy state even on the 157th day after cutting. On the other hand, in the plant body of chrysanthemum “Ohira” of Comparative Example 3-1 using sucrose, the wilting of the plant had progressed on the 98th day after cutting.

<Example 4>
From the seedlings of chrysanthemum "Seimarin", the above-ground nutritive tissue pieces were cut out and cultured for 1 week using sucrose medium (composition: 1 / 2MS medium pH 5.7, 3% sucrose, 0.38% gellan gum). After rooting, the cells were cultured in the same manner as in Example 1-1 except that the cells were cultured using a trehalose medium.

<Comparative example 4>
From the seedlings of chrysanthemum "Seimarin", the above-ground nutritive tissue pieces were cut out and cultured for 1 week using sucrose medium (composition: 1 / 2MS medium pH 5.7, 3% sucrose, 0.38% gellan gum). After rooting, the cells were cultured in the same manner as Comparative Example 1-1 except that the cells were cultured using a sucrose medium.

  FIG. 9 shows how the cultures of Example 4 and Comparative Example 4 were observed on the 70th day of the buds. The left side of FIG. 9 is a plant body of chrysanthemum “Seimarin” of Example 4 cultured in a trehalose medium after culturing with a sucrose medium and rooted, and the right side is cultured in a sucrose medium. It is a plant body of chrysanthemum “Seimarin” of Comparative Example 4 which was cultivated using a sucrose medium after having been rooted.

  As can be seen from FIG. 9, the chrysanthemum “Seimarin” plant of Example 4 cultured using sucrose medium and rooted and then cultured using trehalose medium is healthy even on the 70th day after cutting. Was in good condition. On the other hand, after culturing using a sucrose medium to obtain a rooted state, the plant of the chrysanthemum “Seimarin” of Comparative Example 4 cultured using a sucrose medium had progressed withering of the plant.

<Example 5>
Example 1-1 except that the above-mentioned vegetative tissue pieces containing nodes were cut out from the seedlings of chrysanthemum “Seimarin” and cultured using a trehalose medium in a state where all leaves were removed from the nodes containing a plurality of buds. The culture was performed in the same manner.

<Comparative Example 5>
Comparative Example 1-1 and Except that, from the young plant of chrysanthemum “Seimarin”, the above-mentioned vegetative tissue pieces including nodes were cut out and cultured using a sucrose medium in a state where all leaves were removed from the nodes containing a plurality of buds. The culture was performed in the same manner.

  FIGS. 10 and 11 show the observation of the cultures on the 85th day and 144th day of the buds of Example 5 and Comparative Example 5, respectively. The upper side of FIGS. 10 and 11 is the chrysanthemum “Seimarin” plant of Example 5 cultured using a trehalose medium, and the lower side is the chrysanthemum “Seymarin” plant of Comparative Example 5 cultured using a sucrose medium. .

  As can be seen from FIGS. 10 and 11, the chrysanthemum “Seimarin” plant of Example 5 cultured using a trehalose medium maintained a healthy state even on the 144th day after cutting. On the other hand, in the chrysanthemum “Seimarin” plant of Comparative Example 5 cultured using a sucrose medium, the wilting of the plant progressed on the 144th day (almost 100 days after the cuttings were killed).

  Thus, in chrysanthemum "Seimarin", in particular, by using a technique of culturing using a trehalose medium in a state in which the vegetative tissue pieces including the nodes are cut out and all the leaves are removed from the nodes containing a plurality of buds, It became possible to further extend the culture maintenance period.

<Example 6, Comparative Example 6>
From the seedlings of Torenia “crown violet” obtained in the same manner as in Example 1-1, a vegetative tissue piece of the above-ground part including nodes was cut out and 1/2 MS trehalose medium containing 3% (w / v) trehalose ( Composition: 1/2 MS medium, 3% trehalose, 0.38% gellan gum) with pH values changed to 4.6, 5.0, 5.4, 5.8, 6.2 Incubation was carried out in the same manner as in 1-1 (Example 6). Moreover, it implemented using what changed pH of the 1 / 2MS sucrose culture medium containing 3% (w / v) sucrose with 4.6, 5.0, 5.4, 5.8, 6.2, respectively. Culture was performed in the same manner as in Example 1-1 (Comparative Example 6).

  FIGS. 12 and 13 show the observation of the cultures of the cuttings on day 71 and day 130 of Example 6, respectively. The upper side of FIGS. 12 and 13 is the plant body of Torenia “crown violet” of Example 6 cultured using trehalose medium, and the lower side is the plant body of Torenia “crown violet” of Comparative Example 6 cultured using sucrose medium. It is.

<Example 7, Comparative Example 7>
From the chrysanthemum “Seimarin” seedlings obtained in the same manner as in Example 3-1, the above-ground nutritive tissue pieces including knots were cut out, and the pH of a 1/2 MS trehalose medium containing 3% (w / v) trehalose was obtained. The culture was carried out in the same manner as in Example 1-1, using those obtained by changing the pH to 4.6, 5.0, 5.4, 5.8, and 6.2 (Example 7). Moreover, it implemented using what changed pH of the 1 / 2MS sucrose culture medium containing 3% (w / v) sucrose with 4.6, 5.0, 5.4, 5.8, 6.2, respectively. Culture was performed in the same manner as in Example 1-1 (Comparative Example 7).

  FIGS. 14 and 15 show the state of observation of the culture on day 71 and day 130 of the cuttings of Example 7, respectively. The upper side of FIGS. 14 and 15 is a plant of chrysanthemum “Seimarin” of Example 7 cultured using a trehalose medium, and the lower side is a plant of chrysanthemum “Seimarin” of Comparative Example 7 cultured using a sucrose medium. .

  As can be seen from FIGS. 12 to 15, the long-term maintenance effect of the culture by the trehalose medium is the same as that of the Torenia “Crown Violet” and Chrysanthemum “Seimarin”. No), it was confirmed that even when the initial pH of the medium was changed in the range of 4.6 to 6.2, it was almost stable and there was almost no difference in the effect. However, even when the sucrose medium was used, the difference due to the initial pH was hardly confirmed, and it is considered that there was almost no difference in the effect due to the initial pH due to the pH buffering ability of the plant itself.

<Example 8>
From the chrysanthemum “Seimarin” on the 72th day of the buds obtained using “trehalose medium” in the same manner as in Example 3-1, above-ground nutrient tissue pieces including nodes were cut out and cultured using trehalose medium. Further, from the chrysanthemum “Seimarin” on the 28th day of the buds obtained using the “sucrose medium” in the same manner as in Comparative Example 3-1, the above-ground nutritive tissue pieces including the knots were cut out and each was used with the sucrose medium. Cultured. In addition, the chrysanthemum “Seimarin” on the 72th day of the inserted bud and the chrysanthemum “Seimarin” on the 28th day of the inserted bud used at this time were almost the same in size of the plant body and the size of the planted ear.

  FIG. 16 shows a state where the culture on the 25th day after planting in Example 8 was observed. The right side of FIG. 16 is a chrysanthemum plant cultivated using a trehalose medium, and the left side is a chrysanthemum plant cultivated using a sucrose medium.

  As can be seen from FIG. 16, the difference in initial growth tended to be small in the second and subsequent planting.

Claims (5)

  A method for maintaining a plant for a long period of time, wherein the plant is vegetatively grown using trehalose. It is a long-term maintenance method of the plant according to claim 1,
A long-term maintenance method of a plant, comprising performing vegetative growth of the plant using a medium containing trehalose. It is a long-term maintenance method of the plant according to claim 1 or 2,
A long-term maintenance method for a plant, wherein the plant is a plant that cannot maintain the genetic characteristics of the current generation in a progeny line by seed growth. It is a long-term maintenance method of the plant according to any one of claims 1 to 3,
A long-term maintenance method for a plant, wherein the plant is a genus Torenia or Chrysanthemum.   The plant long-term maintenance method according to any one of claims 1 to 4, wherein the plant is vegetatively grown using the trehalose in a state of being cultivated using sucrose and rooted. A method for maintaining a plant for a long period of time, wherein vegetative growth of the plant is performed using the trehalose in a state where leaves are removed from a plurality of nodes.