KR101539009B1 - Method for plantlet formation of Cypripedium macranthum Sw. using bioreactor - Google Patents

Abstract

The present invention relates to a method of seedling formation of Cypripedium macranthum Sw. Using a bioreactor, and more particularly, to establish an optimum condition for culturing a bag in a balloon type bioreactor, , And it can be usefully used for mass proliferation of bovine pancreas, because it is excellent in protocorm, new bud, and seedling planting rate.

Description

[0001] The present invention relates to a method for forming a seedling of a bovine egg using a bioreactor. using bioreactor}

The present invention relates to a method of seedling formation of a bacterium ( Cypripedium macranthum Sw.) Using a bioreactor.

Cypripedium macranthum Sw. ( Cypripedium macranthum Sw.) Is a stable perennial plant belonging to the orchidaceae family. The Cypripedium genus that is native to our country is called Cypripedium 4 varieties such as macranthum Sw., Cyp. guttatum var koreanum , Cyp. calceolus , Cyp. japonicum , Cyp. Guttatum for albiflorum ). Bamboo grass grows in the shade at the foot of the mountain, but grows on the grass in the bamboo grass, climbs up to the side, grows to the side, root is lowered from the hill, 25-40 cm in length and has multicellular hairs. The leaves are 3-5, and the flower of light purplish red color is bloomed at the end of the main stem in May-July, and the characteristic lip is transformed into a peculiar shape like pouch, which is one of the wild flowers with high ornamental value.

In the ornamental value is very outstanding already abroad jungyimyeo one of the most popular jasaengran in the bags field is in Japan, the United States has been commercialized distribution, particularly in the commercial, I sold the company in Vermont Lady's Slipper of the US company Cypripedium in such bags is It sells the plant Gwangneung dragon flower for $ 70 per person, which is one of the most valuable commercial plants. Despite its high potential for industrialization, the native bamboo field has been limited to use as a horticultural plant because it is impossible to breed seeds in the natural state, and it tends to be degraded due to insufficient cultivation.

In 2005, the Wildlife Conservation Act was enacted, related laws were enacted, and conservation restoration projects for rare rare plants were actively pursued mainly by the Ministry of Environment in accordance with the Forest Biodiversity Conservation Master Plan. Currently, there are 610 species of rare plants designated by the Korean Forest Service. Among them, Gwangneung Yogang flower is designated as 65 and Bokbun column is designated as No. 66 among the five species of bamboo grass. However, Decrease in the number of habitats is a serious situation.

Therefore, it was necessary to develop an efficient breeding method for flower growth and industrialization in accordance with the restoration and propagation of the bifurcation line designated as an endangered species and the development of promising native plants. However, the known seedling breeding method has a low germination rate, a long incubation period including the germination period, and a tendency to be degenerated or dead, resulting in limitations on mass cultivation and cultivation. Therefore, it is necessary to improve the seed germination rate of bamboo seedlings and to establish a mass culture system that optimizes seedling formation from protocorm (germinal mass) after germination, Development was essential.

Since conventional culture methods using conventional solid media are performed in an intensive culture form using a small scale culture container, it takes a lot of time and labor, and there is a disadvantage that the volume utilization of the culture container is low and automation is impossible. To overcome these disadvantages, various types and sizes of bioreactors have been applied to plant cell and organ culture, and a culture system for increasing mass proliferation efficiency through process automation and optimization has been put into practical use. Examples of commercialization development include taxol cancer drugs (attention), biologics and diabetic drugs (wild ginseng). The culture type using the bioreactor can provide an appropriate level of air to the inside of the vessel, which makes it possible to utilize more than 90% of the reactor volume and to control the amount of air, acidity, temperature, and liquid medium supply.

Accordingly, the present inventors have established an optimum condition for culturing a bovine bag in a balloon type bioreactor in order to find a method for mass proliferation for preservation, restoration and horticulture of endangered species species bovine egg As a result, it was confirmed that the rate of formation of the raw gut, new bud, and seedling plant of the bumblebug was remarkably excellent, so that the seedling formation method using the bioreactor was useful for mass proliferation of the bumblebug column The present invention has been accomplished on the basis of these findings.

SUMMARY OF THE INVENTION

1) Cypripedium macranthum Sw.) in flight ( in vitro ) seeding medium to form a protocorm; And

2) Inoculating the raw mass of step 1) into a bioreactor and culturing the same in a bioreactor culture medium to form a seedling.

In order to achieve the object of the present invention,

1) Cypripedium macranthum Sw.) in flight ( in vitro ) seeding medium to form a protocorm; And

2) Inoculating the raw mass of step 1) into a bioreactor and culturing in a bioreactor culture medium to form a seedling.

The present invention relates to a method of forming a seedling of Cypripedium macranthum Sw. Using a bioreactor, and more specifically, it has been found that an optimal condition for culturing a bovine bag was established in a balloon type bioreactor, Since the formation rate of protocorm, new bud, and seedling plant of bovine rice bran is significantly superior, the seedling formation method using the bioreactor is useful for mass proliferation of bovine rice bran. .

Brief Description of the Drawings Fig. 1 is a cross- macranthum Sw.) seed seed sterilization method.
FIG. 2 is the seed bags on board (in vitro) is a diagram showing a source goeche (protocorm) germination after sowing seeds.
FIG. 3 is a graph showing the initial formation of a new bud and a seedling according to the seeding density of the raw goat.
FIG. 4 is a graph showing the initial formation of a new baby and seedling according to the amount of air supplied into the bioreactor.
FIG. 5 is a graph showing the initial formation of newborn seedlings and embryo seedlings according to the content of sucrose in the culture medium.
Fig. 6 is a diagram showing the initial formation of newborn seedlings and embryo seedlings according to the culture medium supply method and cultivation method.
Fig. 7 is a graph showing the content of sucrose remaining in the medium by incubation period.
Fig. 8 is a view showing the seedlings and seedling plants of the bovine rice field one year after the glasshouse planting.

Hereinafter, the present invention will be described in detail.

The present invention

1) What is a bag (Cypripedium macranthum Sw.) Seeds on board (in vitro) Seeding medium to form a protocorm; And

2) Inoculating the raw mass of step 1) into a bioreactor and culturing in a bioreactor culture medium to form a seedling.

The same seedling formation method as that of the present invention can be used for the bushbone field in step 1).

The seeds of the step 1) are preferably those obtained by cross-pollination of the seeds of the bovine cells and artificially crossing the seeds of the bovine seeds, and then collecting the ovaries after 60 to 70 days.

The seeds of the step 1) are preferably separated from the ovary and the seed surface is immersed in 70% ethanol for 20 to 40 seconds, followed by immersion in 2% sodium hypochlorite (NaOCl) solution for 10 to 15 minutes , It is more preferable to immerse in 70% ethanol for 30 seconds.

Preferably, the sterilized seed is washed with sterilized water for 2 to 4 times for 10 to 15 minutes, but is not limited thereto.

Flight planting medium of step 1) is 1/2 Phytamax Orchid Maintenance Medium (w / o Charcoal, NH 4 NO 3 412.50 mg / ℓ, MgSO 4 45.175 mg / ℓ, KNO 3 475.00 mg / ℓ, KH 2 PO 4 42.50 _{mg / ℓ, CaCl 2 83.0 mg} / ℓ, CoCl 2 · 6H 2 O 0.00625 mg / ℓ, ZnSO 4 · 7H 2 O 2.65 mg / ℓ, Na 2 MoO 4 · 2H 2 O 0.0625 mg / ℓ, MnSO 4 · H ₂ O 4.225 mg / l, H ₃ BO ₃ 1.55 mg / ℓ, KI 0.2075 mg / ℓ, Na 2 EDTA 18.62 mg / ℓ, FeSO 4 · 7H 2 O 13.93 mg / ℓ, CuSO 4 · 5H 2 O 0.00625 mg / ℓ and sucrose (sucrose) 10.00 g / ℓ) Inositol 20 to 30 mg / l, Thiamine HCl 0.2 to 0.3 mg / l, Nicotinic acid 0.12 to 0.13 mg / l, Pyridoxine HCl 0.12 to 0.13 mg / / L and 3 to 8% of coconut water. Preferably, it is a medium prepared by adding 25 mg / L of myoinositol, 0.25 mg / L of thiamine HCl, 0.125 mg / L of nicotinic acid, (Pyridoxine HCl) 0.125 mg / L, and coconut water 5%.

The bioreactor culture medium of step 2) is 1/2 Phytamax Orchid Maintenance Medium (w / o Charcoal, NH ₄ NO ₃ 412.50 mg / ℓ, MgSO ₄ 45.175 mg / ℓ, KNO ₃ 475.00 mg / l, KH ₂ PO ₄ 42.50 mg / l, CaCl ₂ 83.00 mg / l of CoCl ₂ .6H ₂ O, 2.65 mg / l of ZnSO ₄ .7H ₂ O, 2.65 mg / l of Na ₂ MoO ₄ .2H ₂ O, 4.225 mg / l of MnSO ₄ .H ₂ O , H ₃ BO ₃ 1.55 mg / ℓ, KI 0.2075 mg / ℓ, Na 2 EDTA 18.62 mg / ℓ, FeSO 4 · 7H 2 O 13.93 mg / ℓ, CuSO 4 · 5H 2 O in sucrose 10 0.00625 mg / ℓ and sucrose 10.00 g / ℓ) To about 20 mg / l, myoinositol 90-110 mg / l, thiamine HCl 0.5-1.5 mg / l, nicotinic acid 0.3-0.8 mg / l, pyridoxine HCl 0.3-0.8 mg / l, coconut water 7-13% Plant Preservative Mixture), 0.7-1.3 ml, and preferably 20 g / l sucrose, 100 mg / l myoinositol, 1 mg / l thiamine HCl, 0.5 mg / l nicotinic acid, 0.5 mg / l pyridoxine HCl, l, coconut water 10% and 1 ml of PPM.

The bioreactor culture medium of step 2) was diluted with 1/4 Murashige & Skoog Medium (Included Modified Vitamins, NH ₄ NO ₃ 412.50 mg / l, MgSO ₄ 45.135 mg / l, KNO ₃ 475.00 mg / l, KH ₂ PO ₄ 42.50 mg / ℓ, CaCl ₂ MnSO ₄揃 H ₂ O 4.225 mg / ℓ, MnSO ₄揃 H ₂ O, 0.025 mg / ℓ, CoCl ₂揃 6H ₂ O 0.00625 mg / ℓ, ZnSO ₄揃 7H ₂ O 2.15 mg / ℓ, Na ₂ MoO ₄揃 2H ₂ O 0.0625 mg / , H ₃ BO ₃ 1.55 mg / ℓ, KI 0.2075 mg / ℓ, FeNaEDTA 9.175 mg / ℓ, CuSO 4 · 5H 2 O 0.00625 mg / ℓ and Glycine (Glycine) in the 0.50 mg / ℓ) of sucrose of 20 to 30 g / ℓ, myoinositol 70 to (Pyridoxine HCl) 0.3 to 0.4 mg / l, coconut water 7 to 13% and PPM 0.7 to 1.3 mg / l, And the medium prepared by adding sucrose 30 g / l, myoinositol 75 mg / l, thiamine HCl 0.75 mg / l, nicotinic acid 0.375 mg / l, pyridoxine HCl 0.375 mg / / l, 10% coconut water and 1 ml of PPM.

The cultivation in step 2) is preferably carried out under a dark condition by inoculating 2 to 4 g of a raw mass of 3 to 7 mm per liter of the medium, and it is more preferable to culture the raw mass of 5 mm per liter of the medium .

The bioreactor culture medium of step 2) is preferably supplied in an immersion culture system without netting.

Preferably, the bioreactor culture medium of step 2) is replaced with fresh medium every 4 to 5 weeks after the culture.

The bioreactor in step 2) is preferably injected with 0.1 to 0.2 vvm air, and more preferably injected with 0.2 vvm air.

In a specific embodiment of the present invention, in order to reduce the seed germination period of bamboo seedlings and increase the germination rate of seeds, the present inventors cross-pollinated the bamboo seedlings bloomed from the end of April to the middle of May, After seeding, seeds inside the ovary (pod containing seeds) of about 60-70 days were collected, washed and disinfected, and washed with 1/2 Phytamax Orchid Maintenance Medium (w / o Charcoal, NH ₄ NO ₃ 412.50 mg / _{, MgSO 4 45.175 mg / ℓ,} KNO 3 475.00 mg / ℓ, KH 2 PO 4 42.50 mg / ℓ, CaCl 2 83.0 mg / ℓ, CoCl 2 · 6H 2 O 0.00625 mg / ℓ, ZnSO 4 · 7H 2 O 2.65 mg / L, Na ₂ MoO ₄ .2H ₂ O 0.0625 mg / L, MnSO ₄ .H ₂ O 4.225 mg / L, H ₃ BO ₃ 1.55 mg / ℓ, KI 0.2075 mg / ℓ, Na 2 EDTA 18.62 mg / ℓ, FeSO 4 · 7H 2 O 13.93 mg / ℓ, CuSO 4 · 5H 2 O 0.00625 mg / ℓ and sucrose (sucrose) 10.00 g / ℓ) 25 mg / l of myo-inositol, 0.25 mg / l of thiamine HCl, 0.125 mg / l of nicotinic acid, 0.125 mg / l of pyridoxine HCl and 5% of coconut water As a result of culturing liquid (shaking) in a dark state in which light was blocked by putting it in a culture bottle (outer diameter: 81 mm, height: 132 mm) containing 100 ml of the prepared medium, the bovine bag improved seed germination rate and contamination rate And seeds were germinated 3-4 months later to form a lozenge (see FIGS. 1 and 2, and Tables 1 and 2).

In order to confirm the optimal culture condition of the bacterium used for mass culture of the bacterium, the present inventors used the bacterium obtained by the seed disinfection and cultivation method as a 1/2 bacterium of Phytamax Orchid Maintenance Medium (w / o Charcoal, NH₄NO₃412.50 mg / l, MgSO4₄ 45.175 mg / l, KNO₃ 475.00 mg / l, KH₂PO₄ 42.50 mg / l, CaCl₂83.00 mg / l, CoCl₂· 6H₂O 0.00625 mg / l, ZnSO₄· 7H₂O 2.65 mg / l, Na₂MoO₄2H₂O 2.65 mg / l, MnSO₄· H₂O 4.225 mg / l, H₃BO₃ 1.55 mg / l, KI 0.2075 mg / l, Na₂EDTA 18.62 mg / l, FeSO₄· 7H₂O 13.93 mg / l, CuSO₄· 5H₂Inocosol 100 mg / l, Thiamine HCl 1 mg / l, Nicotinic acid 0.5 mg / l, sucrose 20 g / l, , 1/4 Murashige & Skoog Medium (Including Modified Vitamins, NH), 0.5 mg / l of Pyridoxine HCl, 10% of coconut water and 1 ml of Plant Preservative Mixture (PPM)₄NO₃ 412.50 mg / l, MgSO4₄ 45.135 mg / l, KNO₃ 475.00 mg / l, KH₂PO₄ 42.50 mg / l, CaCl₂  83.01 mg / l, CoCl₂· 6H₂O 0.00625 mg / l, ZnSO₄· 7H₂O 2.15 mg / l, Na₂MoO₄2H₂O 0.0625 mg / l, MnSO₄· H₂O 4.225 mg / l, H₃BO₃  1.55 mg / l, KI 0.2075 mg / l, FeNaEDTA 9.175 mg / l, CuSO₄· 5H₂O), 30 g / L of sucrose, 75 mg / l of Myo-Inositol, 0.75 mg / l of thiamine HCl, 1 g of nicotinic acid, , 0.375 mg / l of pyridoxine HCl, 0.375 mg / l of pyridoxine HCl, 10% of coconut water and 1 ml of PPM (Plant Preservative Mixture, Plant Cell Technology Co.) As a result of confirming the degree of starch growth and the initial shape of new bud and seedlings, the growth rate in the bioreactor was excellent when the inoculum density of 2 g of the bovine lozenge was high, and the optimal inoculum density was 2 To 4 g (see Fig. 3 and Tables 3 to 6).

The present inventors have also found that, in order to determine optimal culture conditions for a bioreactor-use bag for mass culture, the present inventors found that a bovine cell obtained by the above-described seed sterilization and culture method was cultured in an optimal bioreactor culture medium As a result of examining the degree of growth of bovine bag according to the amount of air supplied in the bioreactor, and the initial shape of the newborn and seedling seedlings, the growth rate of the bovine bran was excellent in the bioreactor in which the air was adjusted by 0.2 vvm air supply, It was confirmed that the optimal air supply amount for the culture of the bag was 0.2 vvm (see Fig. 4 and Table 7 and Table 8).

The present inventors have also found that, in order to determine optimal culture conditions for a bioreactor-use bag for mass culture, the present inventors found that a bovine cell obtained by the above-described seed sterilization and culture method was cultured in an optimal bioreactor culture medium As a result of examining the degree of growth of the bovine bag according to the sucrose content in the bioreactor and the initial shape of the newborn and seedling seedlings, it was found that the bovine seedlings cultured with the culture medium containing 3% sucrose had a good growth, Of sucrose was found to be 3% (see Fig. 5 and Tables 9 and 10).

The present inventors have also found that, in order to determine optimal culture conditions for a bioreactor-use bag for mass culture, the present inventors have found that the bacterium obtained by the above-mentioned seed disinfection and culturing method is a bioreactor in which a raw material is cultured, As a result of examining the degree of growth and the initial shape of the newborn and seedling according to the culture method, it was found that the rye culture system, the immersion culture system without netting and the temporary immersion method using Ebb & Flood (Ebb & Flood culture system), the growth of the bovine cells cultured by the continuous immersion method without netting was excellent, and it was confirmed that the bioreactor-used bovine bag was the optimum culture medium for culturing and culture method See Table 12 and Table 13).

In addition, the bovine bag obtained by the seed disinfection and culture method described above was cultured in a sucrose-containing medium containing 3% sucrose, which had been confirmed to have an optimal sucrose content in the medium, to sustain the growth of the original plant, As a result of analysis of the residual content of sucrose in the medium when cultured for 6 weeks according to the culture medium feeding method and culturing method to confirm the timing of replacing with new medium during the incubation period to prevent depletion of sucrose, The sucrose consumption rate was the highest in the immersion culture system which had the best growth rate, the highest growth rate of the newborn and seedling formation, and the lowest sucrose content in the culture medium. In the parking lot, the sucrose residue content in the medium was 0.7%, and in the fifth week of culture, It was confirmed that the residual sucrose content was abruptly decreased to about 0.2%. Thus, it was confirmed that the content of sucrose in the medium was reduced to 1% or less, It was confirmed that it was appropriate to replace the medium with the medium and to sustain the growth of the raw mass (see FIG. 7).

In order to confirm optimal culture conditions for a bioreactor used for mass culture, the present inventors used a 3 ℓ or 5 ℓ balloon bioreactor and a bovine granule about 5 mm in size, The feed amount of the air injected into the bioreactor is 0.2 vvm and the sucrose content in the medium is 3% (30 g / l). The cycle of replacing with fresh medium is 4 to 5 As a result of feeding the culture medium and culturing the raw masses by a continuous immersion method in which the webs were not spaced apart from each other at regular intervals, six to twelve weeks later, seedlings were formed from the raw masses of bovine bags, And after 1 year of cultivation, the seedlings differentiated from the newborn were identified (see FIG. 8).

Therefore, since the bovine seeds cultured and established in the optimum condition in the balloon type bioreactor of the present invention are excellent in the formation of raw masses and the rate of formation of newborn and seedling plants, The method of seedling formation can be useful for the mass proliferation of bovine eggs.

Hereinafter, the present invention will be described in detail with reference to examples.

It should be noted, however, that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

< Example  1> Bags  Artificial seeding of seeds

In the conventional seedling seeding method, the entire ovary collected is cleaned and disinfected, the surface of the ovary is completely sterilized, and then the incision is made longitudinally using a tissue culture scalpel (an artificial knife, mes). Then, seeds Is shaken on a solid medium for in-flight seeding and cultured. However, in the conventional seed sowing method, the seed germination period only takes at least one year. Therefore, in order to reduce the seed germination period and increase the seed germination rate, seeds were artificially sown as described below.

Specifically, a bovine flower bloomed from the end of April to mid May (for testing in the greenhouse of the Chungcheongbuk-do Agricultural Research and Extension Service) was cross-pollinated and artificially mated. After 60-70 days of seeding, Pods contained). The collected ovaries were cut longitudinally with a dissection knife for tissue culture, and seeds contained in the ovary were collected and washed and disinfected. The seed surface was sterilized in 70% ethanol for 30 seconds in a clean bench, and the seed surface was disinfected. The seed surface was disinfected and immersed in 2% sodium hypochlorite (NaOCl) solution for 10-15 minutes and once sterilized. And washed. Thereafter, the washed and disinfected seeds were seeded with 25 mg / l Myo-Inositol in a 1/2 Phytamax Orchid Maintenance Medium (w / o Charcoal, P0931, SIGMA) containing the ingredients shown in Table 1 below, A culture bottle (100 ml) prepared by adding 0.25 mg / l of thiamine HCl, 0.125 mg / l of nicotinic acid, 0.125 mg / l of pyridoxine HCl and 5% of coconut water 81 mm in outer diameter, 132 mm in height) and cultured in a dark state (light shade) in which light was blocked.

1/2 Phytamax Orchid Maintenance Medium (w / o Charcoal) ingredient content NH ₄ NO ₃ 412.50 mg / l MgSO ₄ 45.175 mg / l KNO ₃ 475.00 mg / l KH ₂ PO ₄ 42.50 mg / l CaCl ₂ 83.0 mg / l CoCl ₂ .6H ₂ O 0.00625 mg / l Na ₂ MoO ₄ .2H ₂ O 0.0625 mg / l MnSO ₄ .H ₂ O 4.225 mg / l H ₃ BO ₃ 1.55 mg / l KI 0.2075 mg / l Na ₂ EDTA 18.62 mg / l FeSO ₄ .7H ₂ O 13.93 mg / l CuSO ₄ · 5H ₂ O 0.00625 mg / l Sucrose 10.00 g / l The pH of the medium 5.8

As a result, as shown in FIG. 1 and FIG. 2, it was confirmed that the directly disinfected seeds germinated 3-4 months later to form protocorm (protocorm) having a stranded or branched structure (Figures 1 and 2)

Furthermore, as shown in Table 2, when the seeds were directly disinfected and the seeds were directly disinfected and cultured in liquid (shaking), the germination ratio was 6.5 times higher than the conventional method in which the seeds were opened and the seeds were immediately cultured in a solid medium after sterilization And the contamination rate is reduced by 81%. Thus, the method of seed disinfection and the culture form of the bag have a significant effect on the germination rate. As a result, the sodium hypochlorite used in the disinfection process, It was confirmed that the germination rate of the seed was improved by improving the impermeability by the wax layer of the seed coat due to dissociation of the seed coat (seed surface) and the movement of moisture and nutrients through the sucking and pores (Table 2).

< Example  2> Raw stratum  Bioreactor use by inoculation density Bags  Identify optimal culture conditions

A bioreactor is a device for cultivating microorganisms, animals, or plant cells using a biological reaction or for producing a specific substance. An aerobic bioreactor is a device in which air is injected from the outside and a culture medium to be. This type of bioreactor has low initial installation cost, low contamination rate, and a bubble column type with a sparge attached to the bottom of the reactor is used so that the oxygen transmission rate is high and the hydrodynamic stress of the culture can be minimized It is widely used in mass culture. Therefore, in order to confirm the optimal culture condition of the bacterium using the bioreactor, the bacterium obtained by the seed disinfection and cultivation method of the above Example 1 was cultured in a bioreactor cultured on a raw mass, , And the initial shape of newborn and seedlings were measured.

Specifically, in a 3 L or 5 L ballon type bioreactor (Samsung Scientific), a raw mass 3 to 4 months after in-flight sowing of Example 1 was inoculated, and when culturing, 1 liter of culture medium After 2, 4, 6 and 8 g inoculum of about 5 mm in sugar size and incubated under the dark condition with blocking of light, growth characteristics and growth characteristics of each gonad New buds, and seedlings were compared. 20 g / l of sucrose, 100 mg / l of Myo-Inositol, 1 ml of thiamine HCl (manufactured by Sigma), 1/2 Phytamax Orchid Maintenance Medium (w / o Charcoal, P0931, Sigma) ) Was prepared by adding 1 mg / L of 1% nitric acid, 0.5 mg / L of nicotinic acid, 0.5 mg / L of pyridoxine HCl, 10% of coconut water and 1 mL of Plant Preservative Mixture (PPM) 1 or Murashige & Skoog Medium (Including Modified Vitamins, M0245, Duchefa) containing the components shown below in Table 4, sucrose 30 g / l, myo-inositol 75 mg / l, thiamine HCl Medium 2 prepared by adding 0.75 mg / L of thiamine HCl, 0.375 mg / L of nicotinic acid, 0.375 mg / L of pyridoxine HCl, 10% of coconut water and 1 mL of PPM was added to the 3 L balloon Type bioreactor, 0.8-1.0 L was added to the 5-L balloon-type bioreactor, and 1.3-1.7 L was added to the 5 L balloon bioreactor. Investigated after, growth characteristics, and were compared to Shin and seedling initial shape.

1/2 Phytamax Orchid Maintenance Medium (w / o Charcoal) ingredient content NH ₄ NO ₃ 412.50 mg / l MgSO ₄ 45.175 mg / l KNO ₃ 475.00 mg / l KH ₂ PO ₄ 42.50 mg / l CaCl ₂ 83.00 mg / l CoCl ₂ .6H ₂ O 0.00625 mg / l ZnSO ₄ .7H ₂ O 2.65 mg / l Na ₂ MoO ₄ .2H ₂ O 0.0625 mg / l MnSO ₄ .H ₂ O 4.225 mg / l H ₃ BO ₃ 1.55 mg / l KI 0.2075 mg / l Na ₂ EDTA 18.62 mg / l FeSO ₄ .7H ₂ O 13.93 mg / l CuSO ₄ · 5H ₂ O 0.00625 mg / l Sucrose 10.00 g / l The pH of the medium 5.8

1/4 Murashige & Skoog Medium (Including Modified Vitamins) ingredient content NH ₄ NO ₃ 412.50 mg / l MgSO ₄ 45.135 mg / l KNO ₃ 475.00 mg / l KH ₂ PO ₄ 42.50 mg / l CaCl ₂ 83.01 mg / l CoCl ₂ .6H ₂ O 0.00625 mg / l ZnSO ₄ .7H ₂ O 2.15 mg / l Na ₂ MoO ₄ .2H ₂ O 0.0625 mg / l MnSO ₄ .H ₂ O 4.225 mg / l H ₃ BO ₃ 1.55 mg / l KI 0.2075 mg / l FeNaEDTA 9.175 mg / l CuSO ₄ · 5H ₂ O 0.00625 mg / l Glycine 0.50 mg / l The pH of the medium 5.8

As a result, as shown in Fig. 3 and Tables 5 and 6, in the case of inoculation of 2 g and 4 g in the inoculum density of the ingested masses of both medium 1 and medium 2 compared to the inoculum density of 6 and 8 g, It was confirmed that the rate of plant growth developed from newborns was high because of good growth, number of rooting, low rooting rate, high rate of newborn formation and excellent growth of newborn. In particular, when the initial density of the culture inoculated into the bioreactor is high, the doubling time required for cell division is relatively short, so that the development stage of the cultured organism progresses rapidly, And 8 g, it was confirmed that the mortality rate of roots was 22% or more and 2 to 2.6 times higher than that of 2 g and 4 g inoculations. Thus, it was confirmed that the optimal inoculum density was 2 to 4 g (Fig. 3, and Table 5 and Table 6).

< Example  3> Bioreactor usage by air supply Bags  Identify optimal culture conditions

In order to confirm the optimum culture condition of the bifurcation using the bioreactor as in the above <Example 2>, the bifurcation bag obtained by the seed disinfection and culture method of <Example 1> The growth rate of the bumblebee seedlings and the initial shape of newborns and seedlings were measured.

Specifically, as in Example 2, a 3 L or 5 L balloon-shaped bioreactor was inoculated with about 2 to 4 g per 1 L of the culture medium of about 5 mm in size, and the supply amount of air into the bioreactor was set at 0.05, 0.1 , 0.2 and 0.4 vvm for 60 days, respectively. The growth characteristics were investigated, and the initial morphological characteristics of newborn and seedlings were compared.

As a result, as shown in FIG. 4, and Table 7 and Table 8, in the 0.05 vvm treatment which supplied the least amount of air and the 0.4 vvm treatment which supplied the greatest amount of air, the root kill rate was 25% It was confirmed that the growth of the raw mass was not performed normally compared with 0.1 to 0.2 vvm. The amount of air supplied to the inside of the bioreactor influences the concentration of dissolved oxygen in the culture medium and the water absorption rate of the culture medium. When the amount of air supplied is small, the culture medium and the culture medium are not rotated smoothly. The required amount of moisture absorption and the oxygen supply required for respiration are relatively low. Conversely, when a large amount of air is supplied, since excessive air is supplied into the bioreactor from the initial stage of culture, the rotation rate of the culture medium and the culture medium is excessively high, so that strong hydrodynamic stress is applied to the culture medium. Therefore, by confirming that the maximum shoot length, weight, rooting number and newborn formation rate were shown when air was supplied at 0.2 vvm, it was confirmed that in the bioreactor in which the air was supplied at a flow rate of 0.2 vvm, (Fig. 4, and Table 7 and Table 8). It was confirmed that the optimal air supply amount for culturing the bioreactor-used bag was 0.2 vvm.

< Example  4> Use of bioreactor according to sucrose content Bags  Identify optimal culture conditions

In order to confirm the optimum culture condition of the bacterium using the bioreactor as in the above <Example 2>, the bacterium obtained by the seed disinfection and cultivation method of <Example 1> The growth of bamboo shoots and the initial shape of newborns and seedlings were measured.

Specifically, when a 3 L or 5 L balloon-shaped bioreactor is inoculated with about 2 to 4 g per 1 L of a raw material having a size of about 5 mm, the content of sucrose in the medium is set to 1 , 3, 5, 7, and 9% for 60 days. Growth characteristics of the seedlings were investigated. At this time, the supply amount of air injected into the bioreactor was adjusted to 0.2 vvm.

As a result, as shown in Fig. 5, and Tables 9 and 10, as the sucrose content was lower, the root mortality rate of the raw mass was decreased, the rate of newborn formation and the rate of plant formation were increased, and especially, 3% sucrose And it was confirmed that the number of rooting and the length of the newborn baby were the maximum. As the sugar concentration in the medium increases, the osmotic pressure and the viscosity of the medium increase due to the suppression of the absorption of water, inorganic elements and carbon sources necessary for cell division and expansion, thereby reducing the growth of the cultured medium. , Proline content, and DDPH activity. When sucrose was treated more than 5%, the hydrogen peroxide, proline content and DDPH activity were greatly increased, and it was confirmed that the defense mechanism by moisture stress was strongly expressed. Therefore, through the above results, it was found that the bacterium cultured in the culture medium containing 3% sucrose in the bioreactor was excellent in the rate of newborn and seedling formation, and the optimum content of sucrose in the bioreactor used bacterium was 3% 30 g / L) (FIG. 5 and Tables 9 and 10).

< Example  5> Badge supply  And use of bioreactor according to cultivation method of culture Bags  Identify optimal culture conditions

In order to confirm the optimal culture condition of the bifurcation using the bioreactor as in the above <Example 2>, the bifurcation bag obtained by the seed disinfection and culture method of <Example 1> Growth of bamboo shoots and initial shape of newborns and seedlings were measured by feeding and culturing methods.

Specifically, as in Example 2, when a 3 L or 5 L balloon-shaped bioreactor was inoculated with about 2 to 4 g per 1 L of the raw gristle of about 5 mm in size, (RBC) culture system, Immersion cultures system, and temporary immersion using Ebb & Flood. The culture medium of the present invention can be used as a culture medium, After culturing for 60 days, the growth characteristics were investigated and the initial morphology of the newborn and seedlings was compared. At this time, the supply amount of air injected into the bioreactor was adjusted to 0.2 vvm.

As a result, as shown in FIG. 6, and Table 12 and Table 13, the bovine bag cultured in the continuous immersion system without netting was a plant having a root mortality rate of 10% or less, a rate of newborn formation, Were 78.6% and 80.7%, respectively, and it was confirmed that the growth rate of raw giardia and newborn was good. In addition, since an appropriate amount of air of 0.2 vvm is injected into the incubator, an immersion culture system in which the culture is immersed in the medium throughout the incubation period, And the growth rate of the newborn baby is maximally increased. As a result, it was found that the bovine cells cultured by the continuous immersion method without the mesh was superior in the rate of newborn and seedling formation, (Fig. 6, and Table 12 and Table 13). The culture medium for culturing pouch was the optimal culture medium and the culturing method was a non-net immersion culture system.

< Example  6> Using bioreactor Bags  Confirmation period of medium change for optimal culture

The bovine bag obtained by the seed disinfection and culturing method of Example 1 described above was cultured in the medium containing 3% sucrose which had been confirmed to have the optimum sucrose content in the medium, During the incubation period to prevent the depletion of sucrose, which is the source of carbon source energy, the residual content of sucrose in the medium was analyzed in order to confirm the timing of replacing with new medium for 6 weeks according to the culture medium feeding method and culturing method.

Specifically, when a 3 L or 5 L balloon type bioreactor is inoculated with about 2 to 4 g per 1 L of the raw gross mass of about 5 mm in size, as in the above <Example 2> Cultures were immersed in a culture medium using a culture method, a continuous culture method, a continuous culture method, a continuous culture method, a continuous immersion culture method, an immersion culture system and a temporary immersion method using Ebb & Flood, (5 to 10 ㎖) were collected at the interval of one week after the initiation of the incubation for 6 weeks in the bioreactor culture medium.

As a result, as shown in Fig. 7, in the immersion culture system in which the growth rate of the raw gut and newborn was the best and the growth rate of the newborn and the seedling formation was the highest, the sucrose consumption rate The content of sucrose in the medium was the lowest, and the content of sucrose in the medium was 0.7% in the 4th week of culture. In the 5th week of culture, the amount of sucrose remained in the medium was decreased to 0.2%. In addition, the cabin (in (in the present invention, the term &quot; raw material &quot;) cultivated in a limited environment, such as in vitro , requires the absorption of water, inorganic elements, and carbon source necessary for growth and development from the medium to synthesize an organic material. Even if a medium having the same chemical composition is used As the cultivation period elapsed, the degree of growth of the cultivar was different depending on the cultivation method, so that the absorption rate of the carbon source absorbed from the culture medium was also different. Generally, when the amount of carbon source remaining in the medium is lowered to 1% or less due to a high growth rate of the culture, a new medium containing a carbon source may be added or a new one may be added to sustain the growth of the culture. Therefore, through the above results, the growth of the raw material was continued by replacing with a new medium containing 3% sucrose at 4 to 5 weeks after the culture, in which the content of sucrose, which is a carbon source in the medium, . On the other hand, when the replacement period of the new medium is too short, it is important to select a suitable culture period because the growth and aging of the culture medium grows quickly and the labor required for replacing or adding the medium is consumed. In the present invention, (See FIG. 7).

< Example  7> Using bioreactor Bags  Identification of seedling and seedling plant in bovine egg under optimal culture conditions

<7-1> Use of bioreactor Bags  Confirmation of embryo formation of bovine lamb under optimal culture condition

From the above <Examples 1 to 6>, optimum culture conditions were established by using bioreactor bags, and seedling formation of the bovine cells cultured under optimal culture conditions was confirmed.

Specifically, a 3 L or 5 L balloon bioreactor was inoculated with about 2 to 4 g per 1 L of a raw mass of about 5 mm in size, and the supply amount of air injected into the bioreactor was 0.2 vvm, The content was 3% (30 g / l), and the time of replacing with fresh medium was 4 to 5 weeks after the incubation, and the medium was supplied and the raw gristle was cultured by the continuous immersion method without netting.

As a result, after 6 to 12 weeks, it was confirmed that a faithful new baby was developed from the raw bovine body and the seedling was formed.

<7-2> Using a bioreactor Bags  Under optimal culture conditions Bags Plants  Confirm formation

The growth of seedlings and the formation of seedlings were confirmed in the bags obtained by the method described in Example <7-2> above.

Specifically, the seedlings of the cultured bovine longeum obtained by the method described in the above Example <7-2> were transferred to a glasshouse, and then the soil of rotten leaves such as leaf mold, mulch, grass and trees was decayed. It is cultivated in a mixed soil with a ratio of 8: 2 mixed with Masato (磨沙 土, which is good for drainage and water and nutrients) and Masato Was cultivated for 1 year in a greenhouse adjusted to about 2,000 Lux. The stem and leaf emerged from the seedling of the seedlings during the cultivation period and the young plants (seedlings) were differentiated. In order to enhance the growth of the upper part of the young plant and the development of the newborn baby, 1,000 to 2,000 times of 4 kinds of compound fertilizer (N: P: K = 20-20-20) is sprayed 3 to 6 times at 4 to 6 weeks Respectively.

As a result, as shown in Fig. 8, the cultured bovine seedlings were planted in a glasshouse and grown for about one year, and the degree of growth was confirmed. The seedlings were seeded in a glasshouse and cultivated for about one year. Were identified.

Claims (10)

1) Cypripedium macranthum Sw. Was cross-pollinated and cross-pollinated. The seeds of Cypripedium macranthum Sw. Harvested 60-70 days after ovation were washed with 70% ethanol , Immersing in a 2% sodium hypochlorite (NaOCl) solution for 10 to 15 minutes, and disinfection and culturing in a seeding medium in vitro to form a protocorm ; And
2) Inoculating a bioreactor in which 0.1 to 0.2 vvm of air is supplied with 2 to 4 g of raw masses of 3 to 7 mm per 1 liter of the medium of step 1) and immersion culture (immersion culture system, and culturing under a dark condition in a bioreactor culture medium containing 3% sucrose, and then replacing the bioreactor culture medium every 4 to 5 weeks to form seedlings (seedlings) A method for forming a seedling of a bovine egg yolk using the method.
2. The method according to claim 1, wherein the bioreactor in step 2) is a balloon type.
delete Flight planting medium in said step 1) according to claim 1, wherein the 1/2 Phytamax Orchid Maintenance Medium (w / o Charcoal, NH 4 NO 3 412.50 mg / ℓ, MgSO 4 45.175 mg / ℓ, KNO 3 475.00 mg / ℓ , KH ₂ PO ₄ 42.50 mg / l, CaCl ₂ 83.0 mg / l, CoCl ₂揃 6H ₂ O 0.00625 mg / l, ZnSO ₄揃 7H ₂ O 2.65 mg / ℓ, Na ₂ MoO ₄揃 2H ₂ O 0.0625 mg / l, MnSO ₄ .H ₂ O 4.225 mg / l, H ₃ BO ₃ 1.55 mg / ℓ, KI 0.2075 mg / ℓ, Na 2 EDTA 18.62 mg / ℓ, FeSO 4 · 7H 2 O 13.93 mg / ℓ, CuSO 4 · 5H 2 O 0.00625 mg / ℓ and sucrose (sucrose) 10.00 g / ℓ) Inositol 20 to 30 mg / l, Thiamine HCl 0.2 to 0.3 mg / l, Nicotinic acid 0.12 to 0.13 mg / l, Pyridoxine HCl 0.12 to 0.13 mg / / L, and 3 to 8% of coconut water. The method for forming seedlings using the bioreactor according to claim 1,
The method according to claim 1, wherein the bioreactor culture medium of step 2) is 1/2 Phytamax Orchid Maintenance Medium (w / o Charcoal, NH ₄ NO ₃ 412.50 mg / ℓ, MgSO ₄ 45.175 mg / ℓ, KNO _{3 475.00 mg / ℓ, KH 2 PO} 4 42.50 mg / L, CaCl ₂ 83.00 mg / l of CoCl ₂ .6H ₂ O, 2.65 mg / l of ZnSO ₄ .7H ₂ O, 2.65 mg / l of Na ₂ MoO ₄ .2H ₂ O, 4.225 mg / l of MnSO ₄ .H ₂ O _{, H 3 BO 3 1.55 mg /} ℓ, KI 0.2075 mg / ℓ, Na 2 EDTA 18.62 mg / ℓ, FeSO 4 · 7H 2 O 13.93 mg / ℓ, CuSO 4 · 5H 2 O 0.00625 mg / ℓ and sucrose 10.00 g / l), sucrose 10 to 20 g / l, myoinositol 90 to 110 mg / l, thiamine HCl 0.5 to 1.5 mg / l, nicotinic acid 0.3 to 0.8 mg / l, pyridoxine HCl 0.3 to 0.8 mg / 13% and 0.7 to 1.3 ml of a PPM (Plant Preservative Mixture).
The method of claim 1, wherein the bioreactor culture medium of step 2) is 1/4 Murashige & Skoog Medium (Including Modified Vitamins, NH 4 NO 3 412.50 mg / ℓ, MgSO 4 45.135 mg / ℓ, KNO 3 475.00 mg / ℓ , KH ₂ PO ₄ 42.50 mg / L, CaCl ₂ MnSO ₄揃 H ₂ O 4.225 mg / ℓ, MnSO ₄揃 H ₂ O, 0.025 mg / ℓ, CoCl ₂揃 6H ₂ O 0.00625 mg / ℓ, ZnSO ₄揃 7H ₂ O 2.15 mg / ℓ, Na ₂ MoO ₄揃 2H ₂ O 0.0625 mg / , H ₃ BO ₃ 1.55 mg / ℓ, KI 0.2075 mg / ℓ, FeNaEDTA 9.175 mg / ℓ, CuSO 4 · 5H 2 O 0.00625 mg / ℓ and Glycine (Glycine) in the 0.50 mg / ℓ) of sucrose of 20 to 30 g / ℓ, myoinositol 70 to (Pyridoxine HCl) 0.3 to 0.4 mg / l, coconut water 7 to 13% and PPM 0.7 to 1.3 mg / l, Wherein the culture medium is a culture medium prepared by adding the culture medium to the culture medium.
delete delete delete [2] The method according to claim 1, wherein the bioreactor in step 2) is injected with 0.2 vvm air.

Images