JP2005278496A - Method for aseptically culturing eelgrasses and eelgrasses created by the same method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for aseptically culturing eelgrasses in which mass proliferation of a plant body of eelgrasses can be carried out from a partial cell of eelgrasses by utilizing aseptic culture and to provide eelgrasses obtained by the method. <P>SOLUTION: A tissue containing the growing point of sterilized eelgrasses is cultured to regenerate its plant body. The tissue is a stem tissue containing the growing point of eelgrasses. The tissue is obtained by cutting out the stem tissue from the eelgrasses and then sterilizing and cleaning the stem tissue or by sterilizing and cleaning seed of eelgrasses, sprouting and growing the seed in an aseptic state and then cutting out hycopotyl containing the growing point of eelgrass young seedlings. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for aseptic culture of eelgrass and eelgrass produced by the method.

The eel is a marine flowering plant that lives in the shallow bottom of the inner bay, which is distributed from the temperate zone to the cold zone of the northern hemisphere, and belongs to the order of the genus Pleurotus. Mallards are called “seaweeds” to distinguish them from “seaweeds” together with other underwater flowering plants because their stems, leaves, and roots are clearly differentiated and bloom to make seeds.
A place where sea lions grow in a community is called an eelgrass field. The eelgrass field has a great influence on the surrounding biological environment and water quality environment. For example, when the eels decrease in the summer, it becomes a hiding place from external enemies, and the dead leaves become organic nutrients and provide a lot of food, etc. It serves as a breeding ground for small animals such as juvenile fish, horizontal shrimp, and warekara, or a spawning ground for these. On the other hand, seaweed beds including eelgrass beds are also responsible for the purification of the marine environment by absorbing carbon dioxide, which causes global warming, or phosphorus, which is a causative agent of eutrophication. ing.

  In recent years, attempts have been made nationwide to reclaim and recover eelgrass fields in light of the decreasing tendency of marine lands due to the deterioration of the marine environment caused by land reclamation in the coastal areas and water quality deterioration of inflowing rivers. The establishment of this eelgrass field is mainly carried out by seeding seeds collected from natural eelgrass directly in the shallow water area, or by growing the collected seeds into seedlings and then seeding and transplanting the seedling seedlings in the shallow sea area. It is. In addition, since a large amount of seedlings is required to form a wide range of eelgrass fields, research on techniques for artificially breeding eelgrass seedlings has been conducted. Conventionally, as an artificial growth method for algae and the like, a sterile culture method (see Patent Document 1) in which a part of an algal body tissue is converted to protoplast (single cell) and cultured in the presence of an antibacterial substance with a concentration gradient adjusted. Is disclosed.

However, the above-mentioned creation by collecting, preserving, and sowing the natural seeds contains a contradiction that the seeds are collected from the sea while being constructed, and a large amount of natural seeds is required. There is a problem that causes it. In addition, when artificially proliferating, there is a problem that culturing under aseptic conditions is difficult because the original sea bream lived in the sea where germs spread. The above Patent Document 1 is a culture method for algae, mainly seaweeds, and does not specifically disclose a method for culturing seaweeds (sea lions) having different growth properties such as a breeding method. In addition, in the conventional research including Patent Document 1, there are some that have been successfully cultured to the extent of callus formation, but the current situation is that the regeneration of the plant body has not been achieved.
JP-A-5-68442 (paragraphs [0006] to [0007])

  The present invention has been made in order to cope with such problems, and a method for aseptically cultivating sea cucumbers that enables large-scale growth of eelgrass plants from partial cells of sea breams using aseptic culture and An object of the present invention is to provide eelgrass obtained by the method.

The aseptic culturing method of the sea cucumber of the present invention is characterized by culturing a tissue containing the sterilized sea cucumber growth point to regenerate its plant body.
The tissue is a stalk tissue including the growth point of sea cucumbers, which is obtained by cutting out the stalk tissue from sea cucumbers and then performing sterilization washing. Alternatively, the tissue is obtained by cutting out hypocotyls containing growth points of sea cucumber seedlings obtained by germinating and cultivating seeds of sea cucumbers after sterilizing and washing seeds in a sterile state. .

In the present invention, the plant of the eel family refers to an individual of the eel family whose stems, leaves, and roots are differentiated. In addition, the eel tree seedling in the present invention refers to a eel plant that has an elapsed time of 0 to 4 months after seed germination.
The tissue containing the growth point of the sterilized sea cucumber is, when using the stalk tissue of the eel, the stalk that has been sterilized by being directly sterilized and washed after being cut out from the sea cucumber and containing the growth point. In the case of using a hypocotyl of an eel seedling, it refers to the hypocotyl tissue that has been sterilized and washed at the seed stage and then germinated and grown in a sterile state to maintain the sterile state.

The culture is performed in an artificial seawater medium containing activated carbon. The culture is performed by adding an antibacterial substance.
In addition, the process from the collection of sea cucumbers to the culture is performed at a temperature of 4 ° C. to 15 ° C.

  The eels of the present invention are produced by the aseptic culturing method for the eels.

  By the aseptic culture method of the present invention, it is possible to efficiently produce aseptic cultured seedlings that are essential for the development of a mass breeding method for sea cucumbers by biotechnological techniques. In addition, clone seedlings using aseptic cultured seedlings produced according to the present invention have high uniformity and germination uniformity, and can be used as excellent varieties for eelgrass field construction.

There are eelgrass, giant eelgrass, core bream, shrimp bream, ryukyu eelgrass, ryugyuusugamo, etc. that breed in Japan.
The sterilized culture method of the sea cucumber of the present invention is a method of cultivating a tissue containing the sterilized sea cucumber growth point and regenerating the tissue to the plant body of sea cucumber. There are two processes depending on whether the stem tissue is used as the tissue containing the growth point or the hypocotyl is used, and each will be described below.
The procedure of the aseptic culture method of the present invention will be described with reference to FIG. FIG. 1 shows a process chart when a stem tissue is used as a tissue including a growth point.
Natural sea cucumbers are collected from the shallow sea bottom (S1). The peeled shellfish is peeled off and the stem tissue including the growth point is cut into a predetermined size (S2). The growth point is a part where the eel is elongated and has a nodular shape. The size to be cut out is about 5 mm to 8 mm in the major axis of the stem. By setting it to this size, the survival rate is improved while keeping the contamination rate of germs low.

The stem tissue containing the cut out growth points is immersed in a sterilization detergent or the like to be sterilized (S3). The sterilization washing is performed until the attached bacteria are sufficiently removed and the tissue can be sterilized. Alternatively, after cutting out a small piece of several centimeters from sea cucumber and sterilizing and washing it, the tissue may be further cut out from the small piece with the above size.
The sterilized stalk tissue is placed in a test tube or bottle together with an artificial medium, nutrients, osmotic pressure regulator, antibacterial substance, activated carbon, etc., and initially cultured (S4). Culturing is performed in an artificial weather apparatus at 4 ° C. to 15 ° C. under low light (200 lux to 1000 lux). The initial culture is performed until the roots, leaves, and stems are regenerated from the stem tissue to form a plant body.
After the initial culture, subculture is performed as necessary to cultivate eelgrass plants (S5). The culture conditions are the same as in the initial culture, or the medium and the like may be changed as appropriate.
It is preferable to process the general steps from (S1) to (S4) at a temperature of 4 ° C to 15 ° C. Specifically, avoiding exposure to high temperatures such as ice cooling during sampling, transportation, material procurement, operation in a clean bench, and storage of unused materials. By setting it as this temperature range, the cellular activity of the eelgrass is maintained and the survival rate is improved. In the steps (S2) to (S5), it is preferable to carry out while maintaining a sterile condition. Specifically, work is performed in a green bench, a green room, etc., and microorganisms such as bacteria are prevented from being mixed from the outside when entering a test tube or the like.

Another procedure of the aseptic culture method of the present invention will be described with reference to FIG. FIG. 2 shows a process diagram in the case where the hypocotyl of a eelgrass seedling is used as a tissue including a growth point.
Natural sea cucumber seeds are collected from the shallow sea (S6). Shells, stones and spoiled seeds mixed with the collected seeds are removed and sterilized and washed by immersing them in a sterilizing detergent (S7). The sterilization washing is performed until the bacteria attached to the outer surface of the seed have sufficiently dropped and the seed can be sterilized.
The sterilized and washed seeds are sown in a test bottle containing sea sand (autoclaved), seawater, and distilled water, and after germination, larva seedlings are grown for a predetermined period (S8). Growing seedlings are grown until the hypocotyl is large enough to be cut out, and usually takes about two months.
The hypocotyl including the growth point is cut out to a predetermined size from the base of the eel seedling (S9). The size to cut out the hypocotyl is about 1 mm to 2 mm in diameter and about 20 mm to 30 mm in length. By setting the size, the survival rate is improved.

The tissue cut out from the hypocotyl is placed in a test tube or bottle together with an artificial medium, nutrients, osmotic pressure regulator, antibacterial substance, etc., and initially cultured (S10). Culturing is performed in an artificial weather apparatus at 4 ° C. to 15 ° C. under low light (200 lux to 1000 lux). The initial culture is performed until the roots, leaves, and stems are regenerated from the tissue to become plant bodies.
After the initial culture, subculture is performed as necessary to cultivate eelgrass plants (S11). The culture conditions are the same as in the initial culture, or the medium and the like may be changed as appropriate.
Similar to the steps (S1) to (S4), it is preferable to perform the general steps from (S6) to (S10) at a temperature of 4 ° C to 15 ° C. Similarly, in the steps (S7) to (S11), it is preferable to carry out while maintaining a sterile condition.

As a disinfectant cleaning agent used for disinfecting and cleaning eelgrass tissues, any sterilizer that can sterilize bacteria that inhibit the growth of eelgrass and that does not adversely affect the physiological state of eelgrass can be used. . For example, a chlorinated detergent containing sodium hypochlorite as a sterilizing agent, and other ionic or nonionic surfactants can be used as appropriate. In order to exhibit sufficient sterilization ability and minimize adverse effects on sea cucumbers, the effective chlorine concentration of the sterilizing detergent used is preferably 0.5% to 2.5%.
In addition, ultrasonic cleaning is also preferably performed in order to remove fine deposits that cause contamination.

  As the antibacterial substance, any substance that can suppress the growth of bacteria that inhibits the growth of sea cucumbers and does not adversely affect the physiological state of sea breams can be used. For example, as cell wall biosynthesis inhibitors, carbenicillin, penicillin, methicillin, ampicillin, β-lactams such as amoxicillin, cefazolin, cefotiam, cefoperazone, cefdinir, cephemil, cephem, imipenem, panipenem, carbopenem, meropenem, etc. Monobactams such as carmonam, glycopeptides such as vancomycin, and the like, and protein synthesis inhibitors include aminoglycosides such as streptomycin, kanamycin, tobramycin and arbekacin, macrolides such as erythromycin, clarithromycin and josamycin, tetracyclines , Tetracyclines such as doxycycline and minocycline, chloramphenicol, etc. Nucleic acid synthesis inhibitors include quinolones such as nalidixic acid, nofloxacin, ofloxacin, enoxacin, rifampicin, etc., and folate synthesis inhibition as sulfa drugs such as sulfamethoxazole, sulfamine, sulfamonomethoxine, paraaminosalicylic acid , Diphenylsulfone, trimethopril and the like.

  Any osmotic pressure regulator may be used as long as it does not promote the growth of bacteria that inhibit the growth of sea cucumbers. Examples thereof include saccharides such as mannitol, glucose, sorbitol, fructose, sucrose, and salts such as sodium chloride.

  As the artificial seawater medium used for initial culture and plant culture, any medium having nutrients necessary for the growth of sea cucumbers and capable of maintaining a sterilized state can be used. For example, as a commercially available artificial medium, MS (Murashige and Skoog) medium, Daigo artificial seawater SP (manufactured by Nippon Pharmaceutical Co., Ltd.), Daigo IMK medium (manufactured by Nippon Pharmaceutical Co., Ltd.), Daigo IMK-SP medium (Nippon Pharmaceutical Co., Ltd.) Etc.).

Examples 1 to 10
Plants of eelgrass were collected at Ikenoura (Mie Prefecture: Futami Town) and Matsunase (Mie Prefecture: Matsusaka City). In order to avoid spoilage, the collected eels should be immediately peeled off the outer skin with sea soil, and the stem tissue including the growth point should be cut to a size of 5 to 6 cm. Treated with detergent (effective chlorine concentration 1%) for 10 minutes.
Place 8ml test tube with 10ml MS (Murashige and Skoog), 3wt% sucrose, 0.2M mannitol as osmotic pressure regulator, 250mg / L carbenicillin as antibacterial substance, peel off the outer skin from the stem tissue, The surrounding tissues were excised with the size shown in Table 1 and implanted one by one. A group having a stem long axis of less than 5 mm was designated as Example 1, and a group having a stem length of 5-8 mm was designated as Example 2.
Moreover, it replaced with the said mannitol, and the osmotic pressure regulator (0.2M) shown in Table 2 was added. Each group was set as Example 3-7.
A series of operations from collection to culturing was performed so that the eel was not exposed to a temperature higher than about 15 ° C. All the culture vessels were placed in an artificial weather apparatus at a constant temperature of 15 ° C. and a low light (1000 lux).
In addition, the effects of antibacterial substances and the effects of ice cooling on the stem tissue containing the sterilized growth points were examined under the conditions shown in Table 3. “No ice cooling” is performed at room temperature (25 ° C. to 30 ° C.) in a clean bench. “Ice cooling” is a culture test tube and sterilized stem tissue containing sterilized growth points, which are submerged in seawater. The container was cooled on ice. As the stem tissue, a stem having a major axis of 5 mm to 8 mm was used. Each group was set as Example 8-10.
About each Example and the comparative example, the initial growth number and the number of miscellaneous bacteria contamination were counted. The results are shown in Tables 1 to 3. The initial growth number is the number of individuals that did not die in the initial culture (excluding contamination), and the contamination number is the number of individuals in which the transparent medium became cloudy due to the occurrence of the contamination. In Example 8, after 3 weeks from the start of culture, it grew to a 1-2 cm bright green stem tissue. A gray scale photograph of the regenerated stem tissue is shown in FIG.

If the plant of the eel was sterilized only in the amount to be cultivated at one time like a crop, and the rest was put in seawater, it softened overnight and became impossible to sterilize. Therefore, it was necessary to immediately prepare all strains collected every time and disinfect them.
The growing point to be extracted had to be 5-8 mm because it died at 1-2 mm, which is the same as that of crops. Moreover, as shown in Example 1 and Example 2, even if it extracted by this magnitude | size, generation | occurrence | production of miscellaneous bacteria could be suppressed.
As shown in Examples 3 to 7, there was no great difference except that sorbitol was not suitable as the osmotic pressure regulator.
As shown in Example 9, it was found that adding 250 mg / L carbenicillin as an antibacterial substance is effective in suppressing contamination with various bacteria.

Example 11 to Example 18
Sea bream seeds were collected at Matsunase (Mie Prefecture: Matsusaka City) and Tategami (Mie Prefecture: Ago Town).
Small shells and stones mixed with seeds and spoiled seeds were removed, and surface sterilization was performed with ultrasonic cleaning for 30 minutes, 70% ethanol for 30 seconds, and 2-fold diluted kitchen detergent (effective chlorine concentration 2.5%) for 1 minute.
40 g sea sand was put into a test bottle and autoclaved, and 75 ml of filter-filtered 20% seawater (20% by weight seawater: 80% by weight distilled water) was added. 25 seeds were planted in each. Two weeks later, the number of germinated seeds and the number of germ contamination were counted. The results are shown in Table 4. In addition, the group using the seed obtained by Matsunase (collected 3 times) was set as Examples 11-13, and the group using the seed obtained by Tategami (collected twice) was set as Examples 14 and 15.
An equal amount (75 ml) of filtered 100% seawater was added to the germinated seedling seedlings. Two months later, the hypocotyl (diameter: 1 mm to 2 mm, length: 20 mm to 30 mm) containing the growth point was cut out from the base of the grown sea cucumber seedlings, Daigo artificial seawater SP (manufactured by Nippon Pharmaceutical Co., Ltd.), Daigo IMK-SP medium (manufactured by Nippon Pharmaceutical Co., Ltd.), 50% filtered seawater (50% by weight seawater: 50% by weight distilled water), 15 each of them were transplanted into 70 ml / bottle. Example 16 was transplanted into 50% filtered seawater, Example 17 was transplanted into Daigo artificial seawater SP, and Example 18 was transplanted into Daigo IMK-SP medium.
The regenerated roots were implanted in an IMK-SP solid medium (70 ml / bottle) containing 0.2 wt% activated carbon, and the foliage was immersed in an IMK-SP liquid medium (150 ml / bottle).
The culturing operation was performed so that the sea cucumber was not exposed to temperatures higher than about 15 ° C. All the culture vessels were placed in an artificial weather apparatus at a constant temperature of 15 ° C. and a low light (1000 lux).
For each example, the number of germination (or the number of stem / root regeneration) and the number of contaminated bacteria were counted. The results are shown in Table 5. Moreover, about Example 18, it grew to a 10-cm plant body three months after the culture | cultivation start. A gray scale photograph of the plant is shown in FIG. The upper left in the figure is Example 16, the upper right in the figure is Example 17, and the lower in the figure is Example 18.

By removing contaminants and spoiled seeds that cause contamination at the seed stage, the generation of germs after germination could generally be suppressed. In addition, seeds differed from region to region, and seeds collected from Matsunase produced few germs. As shown in Examples 11 to 13, the germination rate was extremely high by using 20% seawater. Amamo seeds took out leaf sheaths in about one week, and green leaf blades in about two weeks. After adding an equal amount of 10% seawater, it continued to grow for 1-2 weeks. However, when even one individual released germs from the root, the contamination spread rapidly, and the seedlings in the entire bottle often died rapidly.
Among the transplanted hypocotyls containing the growth points cut out from the seedlings that had not withered, in Example 18 transplanted to Daigo IMK-SP medium, the roots and foliage were regenerated and grown into plants up to about 10 cm. (FIG. 4). They survived for 3 months without passage. This is considered possible because the hypocotyl was completely sterilized.
When the plant body of Example 18 was subcultured to an IMK-SP solid medium and IMK-SP liquid medium containing activated carbon, the root elongation was further improved and continued to grow even after 4 months from the hypocotyl culture. It was.

  The aseptic cultured seedlings produced by the present invention have high uniformity and germination uniformity, and can be used as excellent varieties for eelgrass field construction.

It is a figure which shows the procedure of the aseptic culture method of this invention at the time of using a stem tissue. It is a figure which shows the procedure of the aseptic culture method of this invention at the time of using an hypocotyl. It is a grayscale photograph figure of the regenerated stem tissue. It is a grayscale photograph figure of the reproduced plant body.

Explanation of symbols

S1 Amamo collection process S2 Stem tissue excision process S3 Sterilization and washing process S4 Initial culture process S5 Plant body cultivation process S6 Sea cucumber seed collection process S7 Disinfection and washing process S8 Seeding and seedling raising process S9 Hypocotyl Cutting process S10 Initial culture process S11 Plant culture process

Claims (7)

  A method for cultivating eelgrass characterized by culturing a tissue containing growth points of sterilized eelgrass to regenerate the flora.   The aseptic assemblage of sea cucumbers according to claim 1, wherein the tissue is a stalk tissue including a growth point of sea cucumbers, and the stalk tissue is cut out from sea breams and then sterilized and washed. Culture method.   The tissue is one obtained by cutting out the hypocotyl containing a growth point of a eelgrass seedling obtained by germinating and growing the seeds in a sterilized state after sterilization and washing of eelgrass seeds. The aseptic culture method for sea cucumbers according to 1.   The aseptic culture method for sea cucumbers according to claim 1, 2 or 3, wherein the culturing is carried out in an artificial seawater medium containing activated carbon.   The aseptic culture method for sea cucumbers according to any one of claims 1 to 4, wherein the culturing is performed by adding an antibacterial substance.   6. The aseptic culture method for eelgrass according to any one of claims 1 to 5, wherein the steps from collection to culturing of eelgrass are performed at a temperature of 4 to 15 ° C.   A eelgrass produced by the method according to any one of claims 1 to 6.