KR101627182B1 - Method for germinating turion separated from Spirodela polyrhiza - Google Patents

Abstract

 The present invention provides a germinating germination process separated from frogs which comprises heat-shocking (frosting) the frog isolated from frog to 60-70 DEG C, and cultivating the heat-treated frog. The present invention can be applied to the biological and industrial use of frog rice as a biowag since frog can be easily germinated at a high rate irrespective of the storage period regardless of the storage period. It is expected to be a cornerstone in raising the castle.

Description

(Method for germinating turion separated from Spirodela polyrhiza)

The present invention relates to a method of germinating a submerged poultry separated from frog, and the present invention is characterized by improving the germination rate of a submerged poultry through a predetermined heat shock to a slipped poultry isolated from frog.

Hydrophytes have the flexibility to become morphologically diverse by the transformation of the various organs of the plant to the habitat environment. Some aquatic plants undergo structural deformation into very shrunken plants that do not form roots, stems, leaves, and other organs to survive and adapt to the particular underwater environment they are in.

Extremely reduced plant populations ( Spirodela polyhriza ) develops into a very small individual consisting of three parts: lobes, connective roots, and roots (Lee, 1996). They are infertile aquatic plants of 5 to 8 mm in size, reproductive reproducting with frond, and multiply the plants at very high speeds, with the plants doubling in a few days (Landolt, 1986; Appenroth, 2003). When an aquatic plant finishes its life cycle, it forms a seed as in land plants, or it forms a special structure that can keep the growth stopped, and enters a dormancy in the winter season. A perennial herb plant, frog, is a dormant structure that forms a turion, a kind of winter snow, and maintains its dormancy in winter. During the growing period, the thalli increase rapidly by rapid division and multiply the individual to occupy most of the plant volume. These lobsters stop nutrient breeding and form turion primordium inside the leaf tissue when the temperature starts to fall (Kwak & Kim, 2008). Afterwards, the sleeping is separated from the mother frond and sinks to the bottom of the water. When the temperature starts to rise in the next year, it floats on the surface of the water and repeats the life cycle that develops as a new plant. In their sleep, vegetative sheath and frond primordium, which were enclosed in the lyophilized tissues, germinate due to the rise in temperature after the dormant period, resulting in the thallus. This characteristic of frogbill sleeping to maintain dormancy at the time of temperature drop has been proposed as a representative model system species in the study of the dormant structure formation and development process of aquatic plants (Smart, 1996). Extremely reduced frog rice plants consisting of only thallus, connective roots and roots have the structural features of resuming the sleeping and thyroid gland in the thighs and lining tissues, allowing them to resume their growth rapidly after the dormant period or directly into the dormant state (Kwak & Kim, 2008), which has a significant role in the circulation of life in reduced plants (Newton et al., 1978; Sibasaki & Oda, 1979; Kim & Kim, 2000;

On the other hand, the foliage of frogs has a high potential value as an animal feed, because the protein content per dry weight is up to 45% (Chang et al., 1977; Faskin, 1999). In addition, it is used as a medicinal material of cardiovascular system as well as diaphoresis by drying the outpost of frog rice in one room. In addition, mass production of medical proteins useful for the human body has been carried out using rapid multiplication of frog rice and simple mass culture system (Yamamoto et al., 2001). In addition, the International Agency for Recognition of Toxicity to Ecotoxicology (ISO), an internationally recognized authority, uses frog rice as a biomarker. Thus, frog rice has a very high value for pharmacological and industrial applications and also occupies an important position in biological diagnosis such as evaluation of water quality toxicity. In general, the use of frog rice is carried out through adult or fronds. In the natural state, when the cold winter or the environment deteriorates, the frog falls from the frog's adult body and sinks. When the temperature rises, the frog falls to the surface and forms a frond As a result, seed breeding rarely occurs, and individuals are kept in active breeding by sleeping. Therefore, it is necessary to study how to improve the germination rate of ducklings in dormant state to improve biological and industrial utilization of frog.

SUMMARY OF THE INVENTION The present invention has been made under these circumstances, and an object of the present invention is to provide a method capable of effectively germinating a sleeping state separated from frog.

The inventors of the present invention confirmed that it is possible to effectively germinate sleeping regardless of the storage time of the frog separated from the frog by heat-shocking the frog separated from the frog to a predetermined condition, and completed the present invention .

In order to solve the above-mentioned object, the present invention provides a method for producing frog rice, comprising the steps of: (a) heat-treating a frog separated from frog rice at 60 to 70 ° C; and (b) The frog separated from the frog in step (a) is preferably in a dormant state, and the frog separated from the frog in the step (a) Is stored in the dark condition before the heat treatment and in the temperature condition of 0 to 25 DEG C for less than 2 months and more preferably in the cold condition of 0 to 5 DEG C. In addition, And the treatment time is preferably 30 seconds to 10 minutes. The medium for culturing the slides of step (b) is preferably Steinberg's medium or Hoagland's medium. The culture temperature during the culture period of step (b) is 15 ~ 35 ℃, the light irradiation amount is 50 ~ 150 μ㏖ photon / ㎡ · s, the incubation time is preferably 2-4 days.

When the method according to the present invention is used, it is possible to easily germinate the frog separated from the frog, irrespective of the storage period, so that the frog can be used immediately when the frog is needed as the bio-wear. Therefore, the present invention is expected to be a cornerstone for enhancing biological and industrial utilization of frog rice.

FIG. 1 is a photograph showing the germination state of a latte which is stored, heat-treated, and cultured on a Steinberg medium. 1 (a), (b), (c) and (d) show the results of Test Example 1, Test Example 2, Test Example 3 and Test Example 4, respectively.
FIG. 2 is a photograph showing the germination state of the slug that has been stored, heat-treated, and cultured based on the calligraphy medium. Figures 2 (a), 2 (b), 2 (c) and 2 (d) show the results of Test Example 4, Test Example 5, Test Example 6 and Test Example 7, respectively.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for effectively germinating a sleeping state separated from a frog, wherein the germination method of the frog separated from the frog is characterized in that (a) the frog separated from the frog is heat-treated at 60 to 70 ° C the method comprising the steps of: (a) heat shocking the frozen rice, and (b) cultivating the heat-treated frozen rice. The germination method of frozen rice separated from frog rice according to the present invention will be described step by step.

Frog To sleep  Step of heat treatment

In the present invention, the step of heat treating the frog separated from the frog is performed by heating the frog separated from the thallus of the frog or the mature frog separated from the thallus of the adult frog and then stored under predetermined conditions at a predetermined temperature condition . At this time, the slug separated from the frozen rice used in the present invention is in a dormant state, and preferably it is stored in a dark condition before the heat treatment and at a temperature condition of 0 to 25 ° C for less than 2 months, for example, 10 days to 50 days Preferably from 15 days to 45 days. Also, considering the germination rate of the submerged hull by the heat treatment, it is preferable that the submerged frog separated from the frog is stored under the dark condition before the heat treatment and the cold condition at 0 to 5 캜. In addition, it is preferable that the slug separated from the frog is stored in a culture medium for the slug, and the type of the medium for slug culture is not particularly limited, and for example, Steinberg's medium or Hoogland medium (Hoagland's medium), among which Steinberg's medium is more preferable. It is also preferable that the heat treatment temperature of the immersed frog separated from the frog is 60 to 70 ° C. When the heat treatment temperature of the immobilized frog separated from the frog is lower than 60 캜, even when the slipped after the heat treatment is immersed on the water surface, the growth of the thallus is not smooth and the germination rate may not be improved. In addition, when the heat treatment temperature of the frog separated from the frog is more than 70 캜, the frog is denatured by heat, so that it does not smoothly float on the water surface when the frog is incubated after the heat treatment, There is a fear that it will not be achieved. In addition, the heat treatment time of the immature frog separated from the frog is not particularly limited, but it is preferably from 30 seconds to 10 minutes, and from the viewpoint of improving the germination rate of the immature child by heat treatment and minimizing the denaturation of the immature child by heat treatment, To 5 minutes, and most preferably 45 seconds to 2 minutes. In view of the fact that the heat treatment of the frog separated from the frog is immediately terminated when the frozen food is dried, it is preferable that the heat treatment is carried out in a liquid medium heated to a predetermined temperature. The liquid medium includes Steinberg's medium or Hoagland's medium.

Heat-treated To sleep  Culturing step

In the present invention, the step of incubating the heat-treated submerged pellets is performed by placing the heat-treated submerged pellets in a predetermined culture medium and culturing the pellets for a predetermined time at a predetermined temperature and light irradiation condition. At this time, the medium for cultivating the heat-treated frozen dough can be selected from various known media used for incubation of frogs separated from frog, for example, Steinberg's medium or Hoagland's medium, And a Steinberg's medium is preferable considering the germination rate of the submerged horses. The incubation time of the heat-treated submerged poultry is not particularly limited, but is preferably at least 2 days, more preferably from 2 to 4 days, most preferably from 3 to 4 days, from the viewpoint of ensuring reliability against the germination rate of submerged poultry . It is preferable that the light irradiation amount is 50 to 150 mu mol photon / m 2 · s, more preferably 75 to 150 μmol photon / m 2 · s, and 90 to 120 μmol photon / M < 2 > s. In addition, the culture temperature at the time of culturing the heat-treated frozen dough is preferably 15 to 35 ° C, more preferably 20 to 30 ° C.

When the method of the present invention is used, it is possible to effectively germinate slices separated from the thallus of adult frogbish irrespective of the storage period. Generally, it is known that the germination rate of the subspecies is very low even when the subspecies isolated from the thrips of the fowl populations are cultured under optimal culture conditions for not more than 2 months in the refrigerated condition. However, in the thallus of the adult populations using the method of the present invention It may be possible to germinate at least 90% of the slug stored immediately after it has been separated or for less than 2 months, for example 1 month, after it has been separated from the thallus of mature frog.

Hereinafter, the present invention will be described more specifically by way of examples. However, the following embodiments are intended to clearly illustrate the technical features of the present invention, and do not limit the scope of protection of the present invention.

One. Steinberg  badge( Steinberg medium ) And Ho Grand  badge( Hoagland's medium )

Table 1 below shows the components and concentrations contained in the stain bug medium used as the artificial medium used in the examples of the present invention. As shown in Table 1, the Stainberg's culture medium contains a total of 5 stock solutions, each of which consists of a solution containing an inorganic component or an organic component necessary for culturing the frog. The pH of the Stainberg medium is 7 ± 0.2.

Stock solution The kind of constituent constituting the stock solution The concentration of constituents in the stock solution (g / l) Volume of stock solution per liter of artificial medium (ml / l) Ⅰ KNO ₃ 17.5 20 K ₂ HPO ₄ 4.5 KH ₂ PO ₄ 0.63 Ⅱ MgSO ₄ 7H ₂ O 5 20 Ⅲ _{Ca (NO 3) 2 4H 2} O 14.75 20 IV H ₃ BO ₃ 0.12 One ZnSO ₄ 7H ₂ O 0.18 Na ₂ MoO ₄ 2H ₂ O 0.044 MnCl ₂ 4H ₂ O 0.18 V FeCl ₃ 6H ₂ O 0.76 One Na ₂ -EDTA ₂ H ₂ O 1.5

Table 2 below shows the components and concentrations contained in the callus medium used as the artificial medium used in the examples of the present invention. As shown in Table 2, the calligraphy medium contains a total of six stock solutions, each of which consists of a solution containing an inorganic component or an organic component necessary for culturing the frog. The pH of the callus medium is 7 ± 0.2.

Stock solution The kind of constituent constituting the stock solution The concentration of constituents in the stock solution (g / l) The stock solution per liter of artificial medium
Volume (ml / l) Ⅰ MgSO ₄ 7H ₂ O 246 1.0 Ⅱ K ₂ HPO ₄ 236 2.3 Ⅲ _{Ca (NO 3) 2 4H 2} O 136 0.5 IV KNO ₃ 101 2.5 V H ₃ BO ₃ 2.86 0.5 MnCl ₂ 4H ₂ O 1.82 ZnSO ₄ 7H ₂ O 0.22 _{Cu (SO 4) 2 6H 2} O 0.09 Na ₂ MoO ₄ 2H ₂ O 0.09 VI FeCl ₃ 6H ₂ O 0.484 20 Na ₂ -EDTA ₂ H ₂ O 1.5

2. From frog rice Sleepy  Detach and store

The lice isolated from the thallus of mature domestic frog populations were collected and placed in a plastic tube filled with a Steinberg or Hoagland medium and stored for a certain period of time in a light condition, that is, in a dark condition.

3. Collected Sleepy  Germination test according to heat treatment conditions

The germination rate of the submerged hatchlings was investigated by heat treatment without any separate storage. Specific experimental methods are as follows. The stain bug medium was heated to a predetermined temperature, and then the licked sample was placed thereon and heat-treated at a predetermined temperature for a predetermined period of time. Ten heat-treated licks were immediately transferred to a petridish filled with about 15 ml of Stainberg's medium and irradiated at a dose of 90 ~ 100 mu mol photon / m < 2 >Lt; RTI ID = 0.0 > 25 C < / RTI > for about 72 hours. The germination rate was calculated by observing the number of germinated germinated after completion of incubation of the heat - treated gibbous. The germination rate was calculated by observing the number of germinated germs after completion of incubation. The experiment under the same conditions was repeated 10 times in total.

Table 3 shows the germination rate of the slugs according to the heat treatment temperature and time.

Heat treatment conditions of harvested lambs Germination rate of silkworm (%)
Temperature (℃) Time (min) 35 One 0 45 One 0 55 One 0 60 One 68 65 One 72 70 One 70 75 One 0 85 One 0 95 One 0 35 5 0 45 5 0 55 5 0 60 5 69 65 5 73 70 5 71 75 5 31 85 5 0 95 5 0 No heat treatment 0

4. Refrigerated for 30 days after collection Sleepy  Sleep according to heat treatment condition Rate of injury  And germination rate test

The lice isolated from the thallus of domestic frogs were collected and placed in a plastic tube filled with stainbug medium and stored for 30 days under light-blocked condition, that is, in a dark condition at about 4 ° C under dark conditions. Thereafter, the stain bug medium was heated to a predetermined temperature, and then a frozen slurry kept for 30 days was added thereto and heat-treated at a predetermined temperature for a predetermined period of time. Ten heat-treated licks were immediately transferred to a petridish filled with about 15 ml of Stainberg's medium and irradiated at a dose of 90 ~ 100 mu mol photon / m < 2 >Lt; RTI ID = 0.0 > 25 C. < / RTI > The rate of surface flotation was calculated by observing the number of sleeping babies on the surface when 48 hours had elapsed after the initiation of heat-treated incubation, and the germination rate was calculated by observing the number of germinated germs when 72 hours had elapsed after the initiation of incubation Respectively. In addition, the frozen slides stored for 30 days in the control were immediately incubated under the same conditions without heat treatment, and the number of sluggers rising on the surface of the water when 48 hours had passed from the start of the culture was measured to calculate the water surface flotation rate. The germination rate was calculated by observing the number of germinated germs when 72 hours had elapsed. The experiment under the same conditions was repeated 10 times in total.

Table 4 below shows the water flotation rate and germination rate according to the heat treatment temperature and time of the frozen slip kept for 30 days after harvesting.

Heat treatment conditions of frozen lambs refrigerated for 30 days Sleep injury rate of sleeping (%)
Germination rate of silkworm (%) Temperature (℃) Time (min) 35 One 25 10 45 One 50 10 55 One 80 13 60 One 96 93 65 One 100 100 70 One 98 95 75 One 55 35 85 One 0 0 95 One 0 0 35 5 50 11 45 5 55 12 55 5 90 15 60 5 98 91 65 5 100 92 70 5 98 89 75 5 60 30 85 5 0 0 95 5 0 0 No heat treatment 22 20

As shown in Table 4, when the lice stored for 30 days were heat-treated at a temperature of less than 60 ° C. and then cultured, a part of the louse was injured, but most of the louse was not germinated. In addition, when 30 days of frozen storage was heat treated at a temperature of 75 ° C or higher, the color of the frog began to change from dark green to yellowish brown. When heat treated at a temperature of 85 ° C or higher, appear.

5. Storage for 30 days after collection Sleepy  Germination rate test according to storage conditions, heat treatment conditions and medium for culture

The frog isolated from the thallus of the domestic frog populations was collected and placed in a plastic tube filled with a Steinberg or Hoagland medium. The frozen sample was frozen at 4 ° C in dark condition or at room temperature in 25 ° C And stored for 30 days. Thereafter, the stainbug medium or the horseradish medium was heated to a temperature of 65 ° C, and then stored for 30 days in a refrigerator or a room temperature, and heat treated at 65 ° C for 1 minute or 5 minutes. Ten heat-treated licks were immediately transferred to a Petri dish filled with about 15 ml of Stainbug's medium or horseradish medium, and irradiated with light of 90 to 100 mu mol photon / m < 2 > Light condition) and 25 < 0 > C. The germination rate was calculated by observing the number of germinated germs when 72 hours had passed since the start of the culture. The control group was also incubated for 30 days in the stainbug medium or the hogrland medium for 30 days in the same condition without heat treatment and at room temperature. The number of germinated germinated at 72 hours after the initiation of culture was observed The germination rate was calculated. The experiment under the same conditions was repeated 10 times in total.

Table 5 shows the germination rate according to whether or not heat-treated slips were stored under predetermined conditions for 30 days after collection.

Test Example Storage conditions (storage solution, storage temperature, storage time) Heat treatment conditions (heat treatment medium, heat treatment temperature, heat treatment time) Culture medium for culture Sleepy
Germination rate (%) Test Example 1 Steinberg badge, 4 ℃, 30 days Stainbug badge, 65 ° C, 1 minute Steinberg badge 100 Test Example 2 Steinberg badge, 4 ℃, 30 days Steinberg badge, 65 ° C, 5 minutes Steinberg badge 92 Comparative Test Example 1 Steinberg badge, 4 ℃, 30 days No heat treatment Steinberg badge 20 Test Example 3 Steinberg badge, 25 ℃, 30 days Stainbug badge, 65 ° C, 1 minute Steinberg badge 84 Test Example 4 Steinberg badge, 25 ℃, 30 days Steinberg badge, 65 ° C, 5 minutes Steinberg badge 100 Comparative Test Example 2 Steinberg badge, 25 ℃, 30 days No heat treatment Steinberg badge 10 Test Example 5 Hoagland badge, 4 ℃, 30 days Hoagland medium, 65 캜, 1 min Ho Grand Badge 84 Test Example 6 Hoagland badge, 4 ℃, 30 days HoGrand medium, 65 ° C, 5 min Ho Grand Badge 100 Comparative Test Example 3 Hoagland badge, 4 ℃, 30 days No heat treatment Ho Grand Badge 17 Test Example 7 Hoagland medium, 25 ℃, 30 days Hoagland medium, 65 캜, 1 min Ho Grand Badge 67 Test Example 8 Hoagland medium, 25 ℃, 30 days HoGrand medium, 65 ° C, 5 min Ho Grand Badge 67 Comparative Test Example 4 Hoagland medium, 25 ℃, 30 days No heat treatment Ho Grand Badge 8

FIG. 1 is a photograph showing the germination state of a latte which is stored, heat-treated and cultured on a Steinberg medium. 1 (a), (b), (c) and (d) show the results of Test Example 1, Test Example 2, Test Example 3 and Test Example 4, respectively. FIG. 2 is a photograph showing the germination state of the slug that is stored, heat-treated, and cultured on the basis of a calligraphy medium. Figures 2 (a), 2 (b), 2 (c) and 2 (d) show the results of Test Example 4, Test Example 5, Test Example 6 and Test Example 7, respectively.

As shown in Table 5, FIG. 1 and FIG. 2, when the predetermined heat treatment conditions were satisfied, the germination rate of the submerged germinated was high regardless of the storage temperature and culture medium. In addition, the germination rate of the submerged flounder was more favorable when stored in a refrigerated condition or when a stain bug medium was used as a culture medium.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the scope of protection of the present invention is not limited to the specific embodiments but should be construed as including all embodiments belonging to the claims attached hereto.

Claims (7)

(a) heat-treating the frog separated from the frog to 60 to 70 ° C, and
(b) culturing the heat-treated embryo for 2 to 4 days at a temperature condition of 15 to 35 占 폚 and a light irradiation dose of 50 to 150 占 퐉 photon / m2 占 퐏; Way.
The germinating germ of frogs separated from frogs according to claim 1, wherein the slice separated from the frog in step (a) is in a dormant state.
The method according to claim 1, wherein the frozen slices isolated from the frogs of step (a) are stored for a period of less than 2 months under a dark condition and a temperature condition of 0 to 25 캜 before heat treatment. Germination method.
[4] The germinating germ of frost separated from frogs according to claim 3, wherein the frozen slices separated from the frogs are stored at 0 to 5 [deg.] C before the heat treatment.
delete The method of germinating germs separated from frogs according to claim 1, wherein the medium for culturing the slug of step (b) is Steinberg's medium or Hoagland's medium.
delete

Images