Method for producing a culture strain to be subcultured

Abstract

The present invention provides a method for producing a culture strain that can be subcultured under conditions suitable for genetic recombination. (1) a culture step of culturing a plant piece to obtain a seedling; (2) a size confirmation step of confirming the size of the obtained seedlings; (3) The size of the seedling confirmed in the size confirmation step is (A) If the threshold is exceeded, the young plants are treated by removing dead leaves and roots, and the resulting culture is used for subsequent propagation. (B) If the concentration is below the threshold, the young plants are treated by leaving all healthy leaves and roots, and are used as culture lines for subsequent generations. a subculture preparation step; A method for producing a culture strain having the compound of formula (I) which is subcultured.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a culture strain that is passaged. [Background technology]

[0002] Attempts have been made to extract latex from Taraxacum kosher, a plant belonging to the Asteraceae family. For example, Patent Document 1 discloses that the amount of latex produced can be increased by applying jasmonic acid or a derivative thereof to Taraxacum kosher. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2010-142173 Summary of the Invention [Problem to be solved by the invention]

[0004] It is also envisioned that a desired gene may be introduced into Taraxacum kosher in order to increase the production of natural rubber in the plant. Various methods for introducing genes into plants are known, including the Agrobacterium method, which uses Agrobacterium.

[0005] As a result of extensive research by the present inventors, it was found that plants belonging to the Asteraceae family (particularly plants belonging to the genus Taraxacum, and especially Taraxacum koksaghyz) have high intraspecies genetic diversity, resulting in variation between individuals suitable for genetic recombination and those that are not. Therefore, in order to efficiently carry out genetic recombination, it is desirable to create clones of individuals suitable for genetic recombination and maintain them successively. As a result of extensive research by the present inventors, it was found that depending on the condition of the strain that has been maintained successively, genetic recombination may not be successful, and that there is room for improvement in terms of subculturing plants in conditions suitable for genetic recombination. The present invention aims to solve the above problems and to provide a method for producing a culture strain that can be subcultured under conditions suitable for genetic recombination. [Means for solving the problem]

[0006] The present invention includes: (1) a culture step of culturing a plant piece to obtain a seedling; (2) a size confirmation step of confirming the size of the obtained seedlings; (3) The size of the seedling confirmed in the size confirmation step is (A) If the threshold is exceeded, the young plants are treated by removing dead leaves and roots, and the resulting culture is used for subsequent propagation. (B) If the concentration is below the threshold, the young plants are treated by leaving all healthy leaves and roots, and are used as culture lines for subsequent generations. a subculture preparation step; The present invention relates to a method for producing a culture strain having the above structure. [Effects of the Invention]

[0007] The method for producing a cultured line to be subcultured of the present invention comprises: (1) a culturing step of culturing plant pieces to obtain seedlings; (2) a size confirmation step of confirming the size of the obtained seedlings; and (3) a subculture preparation step of: (A) if the size of the seedlings confirmed in the size confirmation step is equal to or greater than a threshold, removing dead leaves and roots from the seedlings to obtain a cultured line to be subcultured; or (B) if the size is less than the threshold, leaving all healthy leaves and roots from the seedlings to obtain a cultured line to be subcultured. Thus, subculture can be performed under conditions suitable for genetic recombination. DETAILED DESCRIPTION OF THE INVENTION

[0008] The method for producing a subcultured culture of the present disclosure includes: (1) a culturing step of culturing plant pieces to obtain seedlings; (2) a size confirmation step of confirming the size of the obtained seedlings; (3) The size of the seedling confirmed in the size confirmation step is (A) If the threshold is exceeded, the young plants are treated by removing dead leaves and roots, and the resulting culture is used for subsequent propagation. (B) If the concentration is below the threshold, the young plants are treated by leaving all healthy leaves and roots, and are used as culture lines for subsequent generations. a subculture preparation step; By using a subcultured culture strain produced by the method for producing a subcultured culture strain of the present disclosure, it becomes possible to perform subculture under conditions suitable for genetic recombination. Note that the production method of the present disclosure may also include other steps as long as it includes the above steps, and the above steps may be performed once or multiple times.

[0009] The reason why the above-mentioned effects are obtained is not entirely clear, but is presumed to be as follows. In the subculture preparation step, the size of the seedling confirmed in the size confirmation step is (A) If the threshold is exceeded, the young plants are treated by removing dead leaves and roots, and the resulting cultures are used for subsequent propagation; or (B) If the concentration is below the threshold, the young plants are treated by leaving all healthy leaves and roots, and are used as culture lines for subsequent generations. (A) When the density is above the threshold, the dead leaves of the seedlings are removed to maintain plant vitality, but the roots are left intact to create a culture line for subsequent propagation. Because seedlings above the threshold are in a strong plant state, removing the dead leaves and roots makes it possible to subculture them in conditions suitable for genetic modification. Furthermore, removing the roots also makes it possible to promote division. (B) If the concentration is below the threshold, all healthy leaves and roots are left in the seedlings to create a culture line for subsequent generations. Because seedlings below the threshold are small and weak as plants, leaving all healthy leaves and roots in place makes it possible to perform subculture in conditions suitable for genetic modification.

[0010] Seedlings above the threshold are particularly suitable for use in genetic modification.

[0011] As used herein, a condition suitable for genetic recombination refers to a state of a plant in which, when genetic recombination is performed using plant fragments (e.g., leaf fragments) by the Agrobacterium method, the plant fragments (e.g., leaf fragments) infected with Agrobacterium do not die until they are induced to form callus.

[0012] The plants to which the production method can be applied are not particularly limited, and can be applied to plants in general. The plants may be any of the following: seed plants, ferns, and bryophytes. The seed plants may be angiosperms or gymnosperms. The angiosperms may be monocotyledons or dicotyledons. The plants may be herbaceous plants or woody plants.

[0013] Examples of monocotyledonous plants include Orchidaceae (cypress orchid, moth orchid, vanilla, etc.), Poaceae (rice, wheat, barley, rye, corn, millet, foxtail millet, sugarcane, etc.), Cyperaceae (papyrus, etc.), Araceae (taro, etc.), Alcaligenes (arrowroot, etc.), Liliaceae (tulip, etc.), Amaryllidaceae (onion, leek, garlic, chive), Asparagaceae (asparagus, etc.), Dioscoreaceae (dioscorea, etc.), and Zingiberaceae (ginger, ginger, etc.).

[0014] Dicotyledonous plants include Asteraceae (plants of the genus Taraxacum such as dandelions, house daisies, sunflowers, lettuce, burdock, garland chrysanthemums, and butterburs), Fabaceae (soybeans, peas, adzuki beans, broad beans, and peanuts), Rubiaceae (coffee, etc.), Lamiaceae (perilla, perilla, and peppermint), Euphorbiaceae (plants of the genus Hevea such as rubber trees, poinsettias, and cassava), Malvaceae (cotton, okra, etc.), Apiaceae (carrots, parsley, and celery), and Brassicaceae (Arabidopsis, radish, rapeseed, Japanese mustard greens, Chinese cabbage, turnips, mustard greens, cauliflower, cabbage, broccoli, wasabi, and daikon radish). Examples of plant species include: Rosaceae (strawberries, apples, pears, cherry blossoms, plums, peaches, etc.), Solanaceae (eggplant, tomatoes, chili peppers, tobacco, bell peppers, potatoes, etc.), Amaranthaceae (spinach, etc.), Nymphaeaceae (water lilies, water shields, etc.), Nelumbaceae (lotus, etc.), Rutaceae (tangerines, lemons, etc.), Araliaceae (burdock, elm, etc.), Convolvulaceae (sweet potatoes, etc.), Cucurbitaceae (watermelon, melon, cucumber, bitter melon, pumpkin, loofah, etc.), Vitaceae (grapes, etc.), Pedaliaceae (sesame, etc.), Caryophyllaceae (gypsophila, carnations, etc.), Violaceae (pansies, etc.), Primulaceae (cyclamen, etc.), Ranunculaceae (Clematis, etc.), etc.

[0015] Among the above plants, dicotyledonous plants are preferred, and rubber-producing plants are more preferred.

[0016] The rubber-producing plant is not particularly limited as long as it is a plant that can produce natural rubber, and examples thereof include the genus Hevea, such as Hevea brasiliensis; the genus Sonchus, such as Sonchus oleraceus, Sonchus asper, and Sonchus brachyotus; Solidago altissima, Solidago virgaurea subsp. asiatica, Solidago virgaurea subsp. leipcarpa, Solidago virgaurea subsp. leipcarpa f. paludosa, Solidago virgaurea subsp. gigantea, and Solidago gigantea Ait. var. leiophylla. The genus Solidago, including sunflower (Helianthus annuus), white-barked sunflower (Helianthus argophyllus), Helianthus atrorubens, dwarf sunflower (Helianthus debilis), little sunflower (Helianthus decapetalus), and giant sunflower (Helianthus giganteus); the genus Helianthus, including dandelion (Taraxacum), Siberian dandelion (Taraxacum venustum H. Koidz), Japanese dandelion (Taraxacum hondoense Nakai), Kanto dandelion (Taraxacum platycarpum Dahlst), Kansai dandelion (Taraxacum japonicum), and common dandelion (Taraxacum officinale Taraxacum species such as Taraxacum weber, Taraxacum koksaghyz, and Taraxacum brevicorniculatum; fig tree (Ficus carica), rubber tree (Ficus elastica), and ficus pumila L.), Ficus erecta Thumb., Ficus ampelas Burm.f., Ficus benguetensis Merr., Ficus irisana Elm., Ficus microcarpa Lf., Ficus septica Burm.f., and Ficus benghalensis; Parthenium argentatum, Parthenium hysterophorus, and ragweed; lettuce (Lactuca serriola), and Ficus benghalensis; Among these, plants belonging to the Asteraceae family are preferred, plants belonging to the genus Taraxacum are more preferred, and Russian dandelion (Taraxacum koksaghyz) is even more preferred.

[0017] (Culture process) In the culture step, the plant explants are cultured to obtain seedlings. This culture step is not particularly limited, and may be carried out, for example, by a general method for regenerating plants from plant explants.

[0018] Plant regeneration is carried out, for example, by a production method including a callus induction step in which plant tissue fragments are cultured in a callus induction medium containing a cytokinin-based plant hormone, an auxin-based plant hormone, and a carbon source to form callus; a shoot (regeneration) induction step in which the callus obtained in the callus induction step is cultured in a regeneration induction medium containing a plant growth hormone and a carbon source to form shoots; and a rooting step in which the shoots obtained in the shoot (regeneration) induction step are cultured in a rooting medium to cause them to root.

[0019] As used herein, "callus" refers to undifferentiated plant cells or a mass of undifferentiated plant cells. Also, as used herein, "adventitious embryo" refers to embryo-like tissue induced from callus, and "adventitious bud" refers to bud-like tissue obtained from locations where buds do not normally grow, such as leaves, roots, and stem internodes. Also, as used herein, "shoot" refers to apical buds, axillary buds, adventitious buds, as well as buds differentiated from multiple buds or shoot primordia, and the elongated state of these buds. As used herein, "seedling" refers to rooted shoots.

[0020] The plant fragment is not particularly limited, and examples thereof include plant tissue fragments such as leaves, petioles, leaf blades, stems, nodes, roots, buds, axillary buds, terminal buds, petals, cotyledons, hypocotyls, anthers, seeds, etc. Of these, leaf fragments are preferred.

[0021] Furthermore, when producing a seedling transformed by the Agrobacterium method, for example, an infection step is performed in which plant-derived tissue explants are infected with Agrobacterium containing a plasmid containing a target gene or a fragment thereof and an antibiotic resistance gene, and a selective culture step is performed in which, from the tissue explants obtained in the infection step, tissue explants that have acquired the target gene are selected using an antibiotic, and the tissue explants obtained in the selective culture step may be subjected to a series of steps including the callus induction step, the shoot (regeneration) induction step, and the rooting step. The callus formed in the callus induction step in this production method is a callus transformed by the Agrobacterium method. In the above explanation, Agrobacterium is infected into tissue fragments derived from plants for transformation, but Agrobacterium may also be infected into callus.

[0022] The method of infecting plant-derived tissue fragments or callus with Agrobacterium, the method of selecting tissue fragments or callus that have acquired the target gene using antibiotics, the method of inducing callus from plant tissue fragments, the method of culturing callus to form shoots, and the method of culturing shoots to cause rooting can all be carried out according to known methods (for example, the method described in JP 2020-005538 A, etc.).

[0023] For example, by carrying out a series of steps including the callus induction step, the shoot (regeneration) induction step, and the rooting step, a seedling can be obtained from any tissue explant of any plant.

[0024] The culture in the culture step is usually carried out in a controlled environment where culture conditions such as temperature, lighting time, etc. are controlled. The culture conditions can be set appropriately.

[0025] The culture time in the culture step is preferably 1 week or more, more preferably 2 weeks or more, and even more preferably 3 weeks or more, and is preferably 6 weeks or less, more preferably 5 weeks or less, because this allows for better suppression of mutations during culture.

[0026] As described above, in the culture step, plantlets can be produced by culturing the plant pieces.

[0027] (Size confirmation process) In the size confirmation step, the size of the seedlings obtained in the culture step is confirmed. Specifically, the size of the seedlings is measured by the following method. When a plant is left in its natural, untouched state with its leaves unfolded, the diameter of the plant is measured at its longest point when viewed from directly above.

[0028] (Subculture strain preparation process) In the subculture preparation step, the seedlings are treated according to the size of the seedlings confirmed in the size confirmation step to produce cultures to be subcultured. Specifically, the subculture preparation step includes the following steps: The size of the seedling confirmed in the size confirmation step is (A) If the threshold is exceeded, the young plants are treated by removing dead leaves and roots, and the resulting culture is used for subsequent propagation. (B) If the concentration is below the threshold, the young plants are treated by leaving all healthy leaves and roots, and are used as culture lines for subsequent generations.

[0029] Preferably, the threshold is 6.5 cm.

[0030] (A) When the concentration is equal to or greater than the threshold, it is preferable to remove dead leaves and roots from young plants and leave three or more young leaves, and it is more preferable to remove dead leaves and roots from young plants and leave four to five young leaves. This tends to improve the growth of young plants.

[0031] (B) If the concentration is less than the threshold, it is preferable to leave all healthy leaves and at least 3.0 cm of roots in the young plant, and it is more preferable to leave all healthy leaves and at least 5.0 cm of roots in the young plant. Here, in this specification, the root length does not mean the length when force is applied to the root to stretch it, but the length from the boundary between the stem and the root to the tip of the root in its natural state when the plant is laid flat on a board or other flat surface.

[0032] As used herein, "dead leaves" refers to leaves that have turned brown and are inactive, while "healthy leaves" refers to leaves that are green and continue to function. Furthermore, as used herein, "young leaves" refers to healthy leaves that have expanded late. In this specification, "removing roots" means cutting the roots from the base with a scalpel or the like and physically severing them from the plant, and "leaving roots" means that the roots remain.

[0033] As described above, in the subculture preparation step, a specific treatment is performed on the seedlings depending on the size of the seedlings confirmed in the size confirmation step to produce a culture to be subcultured. By using the "culture to be subcultured" obtained in the subculture preparation step, it becomes possible to perform subculture under conditions suitable for genetic recombination.

[0034] The method for culturing (subculturing) the "cultured line to be subcultured" is not particularly limited, and may be performed by a known method for culturing young plants. Specifically, the culture may be performed, for example, in MS medium or the like. The "cultured line to be subcultured" may be transplanted directly into soil, or may be acclimatized by transferring it to artificial soil such as vermiculite before being transplanted into soil. Subculture may also be performed by carrying out the above-mentioned culturing step using a plant fragment of the "cultured line to be subcultured."

[0035] Furthermore, if the size of the seedlings confirmed in the size confirmation step is equal to or greater than the (A) threshold, the seedlings may be subcultured. However, because they are less likely to die when infected with Agrobacterium and are particularly suitable for genetic recombination, they may be used directly for genetic recombination without being subcultured. For example, for seedlings that are equal to or greater than the threshold, leaves excised from the seedlings may be used as materials for genetic recombination experiments, and the remaining seedlings may be subcultured. As described above, the genetic recombination method may involve infecting a tissue explant derived from the plant with Agrobacterium, or infecting a callus with Agrobacterium. Because long-term cultivation may result in mutations, it is preferable to use seedlings within one year of the initiation of the cultivation step or from the time of plant division for genetic recombination. This tends to result in good genetic recombination efficiency. [Example]

[0036] Below, examples (embodiments) that are considered preferable for implementation will be shown, but the scope of the present disclosure is not limited to the embodiments.

[0037] [Table 1]

[0038] In (A) and (D), materials capable of recombination with typical efficiency are obtained. In (B), materials with reduced recombination efficiency are obtained. Plant vigor increases and plant regeneration efficiency decreases. In other words, in (B), recombination is possible, but the efficiency is reduced, making it impossible to pass on the plants under suitable conditions. In (C), the plants did not grow well and were weak. Furthermore, they died after infection with Agrobacterium, making it impossible to produce recombinant plants.

[0039] From the above results, it can be seen that the method for producing a cultured line to be subcultured of the present invention comprises: (1) a culturing step of culturing plant pieces to obtain seedlings; (2) a size confirmation step of confirming the size of the obtained seedlings; and (3) a subculture preparation step of: (A) if the size of the seedlings confirmed in the size confirmation step is equal to or greater than a threshold, removing dead leaves and roots from the seedlings to produce a cultured line to be subcultured; or (B) if the size is less than the threshold, leaving all healthy leaves and roots from the seedlings to produce a cultured line to be subcultured; and therefore, it is possible to perform subculture under conditions suitable for genetic recombination.

[0040] The present invention (1) provides a method for culturing a plant explant to obtain a seedling, comprising: (2) a size confirmation step of confirming the size of the obtained seedlings; (3) The size of the seedling confirmed in the size confirmation step is (A) If the threshold is exceeded, the young plants are treated by removing dead leaves and roots, and the resulting culture is used for subsequent propagation. (B) If the concentration is below the threshold, the young plants are treated by leaving all healthy leaves and roots, and are used as culture lines for subsequent generations. a subculture preparation step; The present invention relates to a method for producing a culture strain having the formula (I) and the formula (II).

[0041] The present invention (2) is the production method according to the present invention (1), wherein the plant fragment is a leaf fragment.

[0042] The present invention (3) is the production method according to the present invention (1) or (2), wherein the plant piece is cultured for one week or more in the culture step.

[0043] The present invention (4) is the production method according to the present invention (1) or (2), wherein the plant pieces are cultured for 3 to 5 weeks in the culture step.

[0044] The present invention (5) is the production method according to any one of the present inventions (1) to (4), wherein in the subculture preparation step, the threshold value is 6.5 cm.

[0045] The present invention (6) provides a method for preparing a subculture strain, comprising: (B) If the threshold is not met, the young plants are treated by leaving all healthy leaves and at least 3.0 cm of roots. This is a production method according to any one of the present inventions (1) to (5).

[0046] The present invention (7) provides a method for preparing a subculture strain, comprising: (B) If the threshold is not met, the young plants are treated by leaving all healthy leaves and at least 5.0 cm of roots. This is a production method according to any one of the present inventions (1) to (5).

[0047] The present invention (8) provides a method for preparing a subculture strain, comprising: (A) If the threshold is exceeded, the dead leaves and roots of the young plants are removed, and three or more young leaves are left. This is a production method according to any one of the present inventions (1) to (7).

[0048] The present invention (9) provides a method for preparing a subculture strain, comprising: (A) If the threshold is exceeded, the dead leaves and roots of young plants are removed, and 4 to 5 young leaves are left. This is a production method according to any one of the present inventions (1) to (7).

[0049] The present invention (10) is the production method according to any one of the present inventions (1) to (9), wherein the plant is a plant of the Asteraceae family.

[0050] The present invention (11) is the production method according to any one of the present inventions (1) to (9), wherein the plant is a plant belonging to the genus Taraxacum.

[0051] The present invention (12) is the production method according to any one of the present inventions (1) to (9), wherein the plant is Russian dandelion (Taraxacum koksaghyz).

Claims

1. (1) a culturing step of culturing a plant piece to obtain a seedling; (2) a size confirmation step of confirming the size of the obtained seedlings; (3) The size of the seedling confirmed in the size confirmation step is (A) If the threshold is exceeded, the young plants are treated by removing dead leaves and roots, and the resulting culture is used for subsequent propagation. (B) If the concentration is less than the threshold, the young plant is treated by leaving all healthy leaves and roots, and the resulting culture is used for subsequent generations. a subculture preparation step; A method for producing a culture strain having the compound of formula (I) which is subcultured.

2. The method according to claim 1, wherein the plant fragment is a leaf fragment.

3. 3. The method according to claim 1, wherein the plant pieces are cultured for one week or more in the culture step.

4. 3. The method according to claim 1, wherein the plant pieces are cultured for 3 to 5 weeks in the culture step.

5. 3. The method according to claim 1, wherein the threshold value in the subculture preparation step is 6.5 cm.

6. In the subculture preparation step, (B) If the concentration is less than the threshold, the seedling is treated by leaving all healthy leaves and at least 3.0 cm of roots. The method according to claim 1 or 2.

7. In the subculture preparation step, (B) If the concentration is less than the threshold, the seedling is treated by leaving all healthy leaves and at least 5.0 cm of roots. The method according to claim 1 or 2.

8. In the subculture preparation step, (A) If the threshold is exceeded, the young plants are treated by removing dead leaves and roots, and leaving three or more young leaves. The method according to claim 1 or 2.

9. In the subculture preparation step, (A) If the threshold is exceeded, the young plants are treated by removing dead leaves and roots, leaving 4 to 5 young leaves. The method according to claim 1 or 2.

10. 3. The method according to claim 1, wherein the plant is a plant of the Asteraceae family.

11. 3. The method according to claim 1, wherein the plant is a plant of the genus Teraxacum.

12. 3. The method according to claim 1, wherein the plant is Taraxacum kok-saghyz.

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