KR100868523B1 - Mass propagation method through in vitro aseptic germination of Calanthe spp. - Google Patents

Abstract

In order to produce shrimp seedlings in a simple manner, the present invention is aseptically seeded from pods of shrimp eggs and aseptically germinated through in-flight cultivation to produce virus-free strains through the purification process to improve the growth of shrimp eggs. The object of the present invention is to provide a method for effectively producing high quality seedlings of shrimp eggs, the composition of which comprises: collecting shrimp pods from shrimp eggs, washing the collected shrimp pods, and then performing surface sterilization; Sowing seed of shrimp in the sterilized shrimp pod in a culture vessel containing germinated medium; Transferring the cultured seedlings sown with shrimp to a culture chamber maintained at room temperature, and culturing until the formation of protoplasts; When the protoplasts are formed, transferring them to a culture vessel containing a new medium and raising them until the seedlings are transplantable into pots; Removing the seedlings grown in the culture vessel from the culture vessel and removing the components of the agar or the medium from the roots or leaves; Wrapping the seedlings with agar or a medium component until the new roots grow; When a new root occurs in the protective member is transplanted to him in a pot and consists of covering the upper part of the pot with conception.

Shrimp eggs, aseptic germination, egg growth

Description

Mass propagation method through in vitro aseptic germination of Calanthe spp.

1 is a photograph of the formation of a protozoan from the seed of shrimp eggs

Fig. 2 is a photograph of passages grown on a seedling propagation medium, where the protoplasts develop and grow as seedlings of shrimp eggs.

Figure 3 is a photograph of the seedling growth by passage 2-3 times in seedling growth medium

The present invention relates to a method of propagation through in-flight germination of shrimp orchid plants, and more particularly, to easily sterilize pods to obtain aseptic seeds from shrimp pods and to easily inoculate the harvested seeds and sterilize germ in-flight. The present invention relates to a method for effectively propagating shrimp eggs using aseptic germination of shrimp eggs, which includes timely passage of egg seedlings and purification of cultured plants.

Shrimp eggs are very narrow compared to Japan because their habitat is limited to parts of Hallasan, the southwestern coast and southern islands of Jeju Island, and the origin of shrimp eggs is also very small compared to Japan. shrimp I (C. discolor) and gold shrimp bloom - May I (C. sieboldii) and 7-8, and I like the summer shrimp (C. reflexa) blooming in February, recently rediscovered model shrimp I (C aristulifera ) and C. izu-insulalis , an unrecorded species in Korea, have been newly discovered, and there are five species, and hybrids such as Halla shrimp and tea ceremony are found among them.

Nearly all the shrimp eggs grown in Korea have been collected and cultured in their own habitat until now, but the habitat is being destroyed enough to wonder if they can be seen even in their homes.

In particular, as the interest in shrimp eggs has increased, even shrimp eggs without horticulture have been left behind, making it difficult to see shrimp eggs on land and in the southwestern islands. The habitat is severely destroyed, and the southern bonds are also a big problem.

Therefore, it is one of the preferred methods to use germ-free germination through the breeding of shrimp eggs having excellent traits to secure shrimp eggs with horticultural characteristics along with the protection of native plants.

 The seeds of most eggplants, including shrimp eggs, are so small that they are only 0.2-1.5 mm long and only 0.3 mm wide, depending on the species, as many as 4 million in one pod. Contains seeds

Seeds of eggs consist of a single layer of epidermis and immature immature embryos and are extremely difficult to germinate because they lack the endosperm that provides the nutrients needed for germination. (共生) to receive nutrition and germination.

As in other eggs and plants, cultivation of shrimp eggs is carried out mainly by nutrition propagation, namely, abandonment division and bulb seeding. It has the advantage of being inherited, but since one nib is generated every year (one year), the proliferation rate is very low.

Other breeding methods include seed germination (種子 發芽) breeding methods, such as pollen seeding (花盆 播種) and aseptic culture (無菌 培養 法) method. Pollen seeding is a method of germinating seed of Shrimp Egg, which is planted with the help of a fungus type called Mycorrhiza or Mycorrhiza, which symbiize the root of Shrimp Egg. However, this is a simple but the germination rate is extremely low, it takes a long time from seeding to germination and flowering.

Aseptic culture method is to germinate seeds by sowing seeds in artificial medium containing various nutrients necessary for germination. This requires the equipment and tools necessary for the work and has a slightly more complicated process than pollen sowing but can increase germination rate. It has the advantage of producing flowering liquor in 3-4 years from sowing.

The present invention is to overcome the problems caused by the conventional breeding method and to produce seedlings of shrimp in a simple way aseptically seeded from the pods of shrimp eggs and aseptic germination through in-flight cultivation, followed by a virus-free bottle The purpose of the present invention is to provide a method of producing high quality seedlings of economical shrimp eggs by improving the propagation of shrimp eggs.

The present invention for achieving the above object is a step of collecting the shrimp pods from shrimp eggs and sterilization after washing the collected shrimp pods; Sowing the shrimp egg seed in the sterilized shrimp pod in the culture vessel containing the germination medium; Transferring the cultured seedlings sown with shrimp to a culture chamber maintained at room temperature, and culturing until the formation of protoplasts; When the protoplasts are formed, transferring them to a culture vessel containing a new medium and raising them until the seedlings are transplantable into pots; Removing the seedlings grown in the culture vessel from the culture vessel and removing the components of the agar or the medium from the roots or leaves; Wrapping the agar or medium components of the seedlings with a protective member until the new roots swell; When a new root occurs in the protective member is transplanted to him in a pot and consists of covering the upper part of the pot with conception.

And the method of sterilization after washing the lobster pod, sterilization after immersion in 95-97% alcohol, the medium for sowing the lobster seed, hyponex 2.5-3.5g / water L, peptone 3- A medium of gel state prepared at a rate of 5 g / L of water, 30-40 g / L of sucrose, and 7-9 g / L of agar (agar), and the new medium grown with the seedlings is placed on a medium for seeding shrimp seed. NAA 0.05mg / L water and 0.5g / L water activated carbon was added.

In addition, the method of culturing until the protoplasts are formed in the culture chamber, the culture without the light until the seeds germinate, and when germination begins, alternately maintaining a light state of 1,000 lux or more and a dark state without light alternately In addition, the method of removing the agar and the medium components of the seedlings is to wash thoroughly in uncontaminated flowing water.

The protective member to wrap until the new root of the seedlings grow is any one of a plastic bag, a plastic bag, a sealed container, the storage period is 7-10 days, the storage temperature is room temperature, when planting the seedlings in pots Root roots are conceived and planted or planted using small granules.

Hereinafter, dividing the method of propagation through aseptic germination of shrimp eggplant plants according to the present invention in detail for each step as follows.

The first step of the present invention is a step of collecting the shrimp pods from the shrimp eggs and washing the collected shrimp pods and then sterilizing, the harvesting time of the shrimp pods is artificial pollination in mid-April-early May Late September-mid October is the time to change from dark green to yellow, and the pods are wounded or opened, so it may be contaminated with mold or bacteria.

On the other hand, the reason for washing the harvested shrimp pods is to wash them out because the collected shrimp pods have various foreign substances and impurities, and the method of washing the collected shrimp pods is liquid in uncontaminated running water. Clean with a detergent (kitchen detergent) and a soft brush (or brush).

The reason for sterilizing the washed shrimp pods is to sterilize the mold or bacteria present in the shrimp pods, and the sterilization method is immersed in 95-97% alcohol for about 20-40 seconds and then the alcohol lamp or the Bunsen burner. Perform flame sterilization 2-3 times for 2 to 3 seconds on low heat. In this case, if the pod exceeds 2 to 3 seconds, the pod may burn or the air in the pod may expand and burst, so it is preferable to perform it for 2 to 3 seconds at a time (so that alcohol on the surface disappears). It is preferable that the pod is rotated so that the flame reaches the surface evenly.

The second step of the present invention is sowing the shrimp seed in the sterilized shrimp pods in a culture vessel containing a medium, which is the flame sterilized shrimp pods in a glass plate sterilized in aseptic zone Cut the pods sideways using tweezers or a knife, and the already prepared germination medium: hyponex 2.5-3.5g / L, peptone 3-5g / L and sucrose 30-40g / L, agar (agar) 7-9g Seeds are sown in a disposable plastic culture dish (φ100 × 40 mm) containing gel medium prepared at a ratio of water / L.

In the seeding method, the pods cut in the transverse direction are held with tweezers, and the seeds are dropped when the hair is dropped on the culture dish. At this time, it is appropriate to sow 1,000 ± 50 seeds per culture dish.

The third step of the present invention is a step of culturing until the protoplasts are formed after transferring the culture vessel in which the shrimp seed is sown to a culture chamber maintaining a room temperature, which is transferred to the culture chamber so that germination To make things work. Protocorm means that the size is grown to 1.5-2.5mm,

The method of culturing until the formation of the protoplasts in the culture chamber is incubated for one month with no light until germination, and when germination starts, sprinkle more than 1,000 lux lights (fluorescent) for 15-17 hours. Thereafter, the dark state for 7-9 hours is repeated 1 month-2 months and incubated.
In the fourth step of the present invention, when the protoplasts are formed, they are transferred to a culture vessel containing a new medium to form seedlings until they are 5-10 cm, which is 1.5-2.5 mm in size. When grown, they are transplanted into a new medium (passage). If this period is missed, roots and leaves are entangled with the protoplasts, causing them to become entangled (especially in the case of high sowing and germination) and depletion Because of this delay, it is preferable to transfer from the protoplast state to the culture vessel containing the fresh medium.

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The new medium for the development and growth of the protoplasts from the seedlings was the seeding medium (hyponex 2.5-3.5g / water L, peptone 3-5g / water L and sucrose 30-40g / water L, agar) The growth promoting hormone NAA (α-napthalene acetic acid) and 0.5 g / L of activated carbon were added to 7-9 g / L of water) and cultured in such a fresh medium. This is much better. Subsequent passages are then carried out two to three times at 2-3 month intervals. As described above, the protoplasts grow to form seedlings of 5-10 cm in size.
The fifth step of the present invention is to remove the seedlings grown in the culture vessel from the culture vessel to remove the components of the agar or medium in the roots or leaves, which are transplanted in a pot with the agar or the medium components in the seedlings This is to prevent fungi or bacteria because the seedlings may die. On the other hand, the method of removing the components of the agar or the medium in the seedlings may be washed clean in running water uncontaminated.

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The sixth step of the present invention is to remove the agar or medium components of the seedlings to protect them at room temperature until the new roots grow, which ensures the adaptation period before transplanting the seedlings from the culture vessel to the pollen The conditions inside the culture vessel are at a relative humidity of 90% or more and satisfactory nutrient conditions, and all growth conditions are in optimum condition. In this state, seedlings are immediately taken out of the culture vessel. When planted in pots and cultured, the relative humidity, light, nutrients, etc. changes the environment so rapidly that they can't adapt and die. Therefore, put seedlings in a protective member to protect.

The protective member is any one of a plastic bag zipper bag, a sealed container, the protection period is 7-10 days, the storage temperature is preferably room temperature. However, when the seedlings are put in a protective member and protected for 7-10 days, new roots germinate 1-2cm. On the other hand, the protective member is preferably used that the transparent light is transmitted.
In the seventh step of the present invention, when new roots germinate in a protective member, transplanting them into a pot and covering the upper part of the pot with conception, when planting the seedlings in a pot, wrap the root part with a conception (water moss) and plant it. Or planting by using a small granule or follicle is preferable, the reason for wrapping the roots of the seedlings in the conception is to protect the roots, as well as to secure moisture in the repair.

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The present invention as described above has the advantage that can increase the growth rate of the seedling plants compared with the conventional aeration and bulb seeding and pollen sowing method by cultivating the seed of shrimp eggs with a very low germination rate in the aircraft, In particular, it is possible to shorten the time and labor required for surface sterilization of shrimp pods, sowing of seeds, subculture of protozoa, and to maximize the efficiency of shrimp shrimp production by increasing the purity rate.

Claims (8)

Collecting the shrimp pods from the shrimp eggs and washing the collected shrimp pods and then sterilizing them; Sowing the shrimp seed in the sterilized shrimp pod in the culture vessel containing the medium; Transferring the cultured seedlings sown with shrimp to a culture chamber maintained at room temperature, and culturing until the formation of protoplasts; When the protoplasts are formed, transferring them to a culture vessel containing a new medium and raising them until the seedlings are transplantable into pots; Removing the seedlings grown in the culture vessel from the culture vessel and removing the components of the agar or the medium from the roots or leaves; Wrapping the agar or medium components of the seedlings with a protective member until the new roots swell; When a new root occurs in the protective member is transplanted to the pot and covering the upper part of the pot with conception; Proliferation method through aseptic germination of shrimp eggplant consisting of. The method of claim 1, wherein the method of sterilizing the washed shrimp pod after sterilization is flame sterilization after immersion in 95-97% alcohol. The medium for sowing seed of shrimp seed is hyponex 2.5-3.5g / L, peptone 3-5g / L and sucrose 30-40g / L, agar (agar) 7-9g / L Method of propagation through aseptic germination of in-flight plants of a shrimp orchid plant, characterized in that the medium is prepared in the ratio of gel. The method of claim 1 or 3, wherein the fresh medium is grown by adding 0.05 mg / L growth promoting hormone NAA and 0.5 g / L of activated carbon to the medium for seeding shrimp seed. A method of propagation through aseptic germination of shrimp orchid plants. The method of claim 1, wherein the method of culturing until the protoplasts are formed in the culture chamber is performed in the absence of light until germination, and alternately alternates the illumination light of 1,000 lux or more and the dark without illumination when germination starts. Method of propagation through aseptic germination of in-flight shrimp shrimp plants, characterized in that it is maintained and cultured. The method of claim 1, wherein the method of removing the agar or the medium component of the seedlings is a method of propagation through aseptic germination of shrimp eggplant plants, which is washed with running water that is not contaminated. The shrimp of claim 1, wherein the protective member that wraps until the new roots of the seedlings grow is any one of a plastic bag, a plastic bag, a sealed container, and a storage period is 7-10 days, and a storage temperature is room temperature. Method of propagation through aseptic germination of eggplant plants. The method of claim 1, wherein when planting seedlings in flowerpots, the root portion is covered with a conception and planted or planted using a small granule.

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