KR20150103879A - Producing method of orchid seedlings - Google Patents

Abstract

The present invention relates to a method for producing orchid seedlings and, more specifically, to a method for producing orchid seedlings through regeneration after forming protocorm from orchid seeds. According to the method for producing orchid seedlings according to the present invention, a higher germination rate is shown by culturing orchid seeds having a very low germination rate in a natural state, aseptically in vitro; and orchid seedlings can be produced in volume for a short period of time. In addition, seedlings produced by the method of the present invention show significantly better growth compared to seedlings produced by solid culturing, and thus good seedlings can be produced for a short period of time.

Description

I. PRODUCING METHOD OF ORCHID SEEDLINGS

The present invention relates to a method for producing egg seedlings, and more particularly, to a method for producing seedlings through regeneration after forming protocorms from seeds.

Orchidaceae (Orchidaceae) is distributed in about 700-800 species and 20,000-25,000 species worldwide. It is known that Orchidaceae belongs to the most species among the flowering plants. Most orchidaceous seeds are very fine, 0.05-6.0 mm in length, 0.01-0.9 mm in width, and 0.31-24 μg in weight, and some species have 20-50 and more than 4,000,000 seeds in one pod . Most plants and plants do not contain endosperm and cotyledons in most paper seeds, but are composed of seeds that surround the abdomen and abdomen.

Eggs and seeds, which can not be supplied with nutrients for germination from the endosperm, are no longer developed without the infection of the proper fungus after absorbing and expanding the water. In the natural state, seeds form protocom by infiltration of mycorrhiza, and rhizoid develops and shoots and roots are generated from the divisional tissue. Germination through these mycoplasma is one of the key features of eggs and plants and is called symbiotic germination (Dearnaley et al., 2007).

In the past, growers and collectors have collected orchids from the wild, but with the development of asymbiotic germination methods of seeds, mass production of eggs and plants has become possible. The development of non-symbiotic germination techniques enabled seed propagation in most eggs and plants, but in some cases, it is difficult to germinate in the cabin, which is caused by the presence of substances that inhibit the seedling's opacity and germination Lee et al., 2007). In order to overcome this, studies are under way to improve the germination rate through ultrasonic treatment, low-temperature pre-chilling, and the like.

On the other hand, Bletilla striata ( Bletilla striata ) is an egg and plant which grows in Korea, Japan and China. In Korea, it lives in Mokpo, Jindo, Growth has thin leaves compared to other eggs and plants, but it also has pores on the front side of the leaves, so it can grow well in strong sunlight. It is also used as a flower material because of its high ornamental value of leaves and beautiful harmony with flowers. However, the larvae are classified as vulnerable (VU) vulnerable species in the list of rare plants in Korea established by the Korean Forest Service and they are facing extinction due to harvesting and local development around their native areas. .

Dearnaley et al., Further advances in orchid mycorrhizal research, Mycorrhiza 17: 475-486, 2007 Lee et al., Developmental changes in endogenous abscisic acid concentrations and asymbiotic seed germination of a terrestrial orchid, Calanthe tricarinata Lindl. J. Amer. Soc. Hort. Sci. 132: 246-252, 2007

Therefore, it is an object of the present invention to provide a production method of egg seedlings and a culture medium for egg planting regrowth for effective mass production of egg including grown eggs.

In order to achieve the above object, the present invention provides a method for producing seedlings by forming protocom from egg seeds and regenerating the protocom.

In one embodiment, the seeds seeded in the medium are cultivated in a rotifer or in a bioreactor culture to form protocom; And transplanting the protocom into a regeneration medium and culturing the seedlot.

Another embodiment may be that the seeds seeded are sterile and sterile.

In another embodiment, the rotation speed of the culture bottle in the rotary culture may be 2 to 10 rpm.

Another embodiment may be that the amount of air injected in the bioreactor culture is 0.1 to 3 vvm.

In another embodiment, the carbon source content in the regeneration medium may be 20 to 30 g per 1 L of the medium.

Another embodiment may be that the medium for regeneration comprises the content of components listed in Table 1 below for 1 L of medium.

ingredient content Ammonium nitrate (NH ₄ NO ₃₎ 412 to 2475 mg Calcium chloride (CaCl ₂ ) 83 to 499 mg Magnesium sulfate (MgSO ₄ ) 45 to 272 mg Potassium nitrate (KNO ₃₎ 475 to 2850 mg Potassium phosphate (KH ₂ PO ₄ ) 42 to 255 mg Boric acid (H ₃ BO ₃ ) 1 to 10 mg Cobalt chloride (CoCl ₂ .6H ₂ O) 0.006 to 0.04 mg Copper sulfate (CuSO ₄ · 5H ₂ O) 0.006 to 0.04 mg Ferric sodium EDTA (C ₁₀ H ₁₂ FeN ₂ NaO ₈ .3H ₂ O) 10 to 64 mg Manganese sulfate (MnSO ₄ .H ₂ O) 4 to 26 mg Molybdic acid (Na ₂ MoO ₄ .2H ₂ O) 0.06 to 0.4 mg Potassium iodine (KI) 0.2 to 2 mg Zinc sulfate (ZnSO ₄ .7H ₂ O) 2 to 13 mg Glycine (C ₂ H ₅ NO ₂ ) 0.5 to 3 mg Myoinositol (C ₆ H ₁₂ O ₆ ) 25 to 150 mg Nicotinic acid (C ₆ H ₅ NO ₂ ) 0.1 to 1 mg Pyridoxine (C ₈ H ₁₁ NO ₃ .HCl) 0.1 to 1 mg Thiamine (C ₁₂ H ₁₇ CIN ₄ OS · HCl) 0.25 to 1.5 mg

In another embodiment, the regeneration medium comprises HP (Hyponex) inorganic salts, wherein the weight ratio of HP inorganic salts and carbon sources in the regeneration medium is 1:20 to 9:20, N): potassium (K) may be 6 to 25: 15 to 30.

In another embodiment, the regeneration medium may contain 1 mg or less of naphthalene acetic acid per 1 L of the medium.

In another embodiment, the egg is grown ( Bletilla < RTI ID = 0.0 > striata , Dendrobium , Bovine Cypripedium , Oncidium or Cymbidium .

The present invention also provides a medium for egg planting regeneration comprising 20 to 30 g of carbon source per liter of culture medium.

One embodiment may be one containing the content of the ingredients listed in Table 1 for 1 L of medium.

Another embodiment includes HP (Hyponex) inorganic salts wherein the weight ratio of HP inorganic salts and carbon sources in the medium is 1:20 to 9:20 and the ratio of nitrogen (N): potassium (K) May be from 6 to 25: 15 to 30.

Another embodiment may be one containing less than 1 mg of naphthalene acetic acid per liter of medium.

The production method of the present invention is advantageous in that it can cultivate aseptically an egg seed having an extremely low germination rate in a natural state and mass-produce seedlings for a short period of time at a high germination rate. In addition, the seedlings according to the production method of the present invention exhibit remarkably good growth as compared with the seedlings grown through the solid culture, so that healthy seedlings can be produced in a short period of time.

Fig. 1 is a photograph of seed pods grown sufficiently after about 180 days.
2 is a photograph showing an example of a bioreactor.
Fig. 3 is a result of Example 1, and the left photograph shows the solid culture, and the right photo shows the bioreactor culture.
FIG. 4 is a photograph of protocorm seedlings and solid culture seedlings from a bioreactor culture after culturing for 12 weeks in a regeneration medium. In the photographs on the left side, solid culture medium seedlings are shown, and on the right side, protocom will be
Figure 5 shows the number of leaves and no. Of roots after the 12-week incubation of protocorm seedlings and solid culture seedlings from the bioreactor culture in regeneration medium, As the average number per plant.
Figure 6 shows the fresh weight of the protocorm seedlings and solid culture seedlings from the bioreactor culture after 12 weeks of cultivation in the regeneration medium for the average weight per plant ).
Fig. 7 shows the leaf length, root length (pseudobulb) and the number of pods after the incubation of the protocorm seedlings and the solid culture seedlings from the bioreactor culture for 12 weeks in the regeneration medium. width) as the average length per plant.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for cultivating seedlings seeded on a culture medium, comprising the steps of: culturing a rotifer or culturing a bioreactor to form protocom; And transplanting the protocom into a regeneration medium and culturing the seedlot.

In the present invention, eggs contain all plants belonging to the Orchidaceae. For example, in the present invention I is grown (Bletilla striata , Dendrobium , bovine Cypripedium , Oncidium or Cymbidium , and preferably Bletilla, striata .

The seeds can be harvested from seed pods. For example, it is possible to harvest the seeds grown from the paddy field, and the day after pollination is mature enough to be about 180 days. The pods collected in the seeds just before the opening and the pale brown Yellow-green seeds are preferred (Fig. 1).

The seeds may be sterilized and sterile. The sterilization of seeds can be accomplished by extracting the seed pod from the egg, opening the seed pod and disinfecting the seed inside the pod with a disinfectant.

In the rotavirus culture or the bioreactor culture, a liquid medium is used. For the supply of air to the liquid medium, the rotation of the incubation vessel causes the exchange of the gas in the reactor through the injection of air using the air pump in the bioreactor culture. At this time, the egg seeds in the liquid medium are rotated and cultivated and the polarity is destroyed, so that protocomb is formed without seedling generation and rooting. The medium used for the rotavirus culture or the bioreactor culture may include MS inorganic salts, sucrose as a carbon source, and the like. The rotavirus culture or the bioreactor culture can be cultured for 4 to 6 weeks, and the culture conditions such as pH, temperature and the like can be appropriately adjusted according to the purpose of the invention.

In the present invention, the culture of the rotary bottle is carried out by attaching seeds to the inside of a cylindrical culture bottle, rotating the bottle with a rotary incubator, and rotating the culture bottle in the rotary bottle culture, And can be suitably adjusted to form. For example, the rotation speed of the culture bottle may be 2 to 10 rpm, preferably 4 to 6 rpm.

The bioreactor culture in the present invention is cultured using a bioreactor. The amount of air to be injected in the bioreactor culture can be appropriately adjusted to form a protocome from the seed. For example, the amount of air injected in the bioreactor culture may be 0.1 to 3 vvm, preferably 0.1 to 0.7 vvm. It is preferable to connect the supplementary flask containing the medium to the bioreactor to supplement the medium which is evaporated due to the injection of air in the bioreactor culture (see FIG. 2).

The formed protocom can be transplanted into a regeneration medium and cultured to produce a large amount of healthy egg seedlings.

The regeneration medium may include a carbon source. Specifically, the carbon source may include monosaccharides such as glucose and disaccharides such as sucrose. The content of the carbon source in the regeneration medium is preferably 20 to 30 g per 1 L of the medium. If the content of carbon source is less than 20 g per 1 L of medium, the rootstock is somewhat poor. If it exceeds 30 g, protocom can be damaged or become transparent.

The regeneration medium may include MS inorganic salts or HP (Hyponex) inorganic salts. The MS inorganic salt or HP inorganic salt may be included in the medium in a proper amount to induce regrowth of protocom.

With respect to MS mineral salts, the medium for regeneration may comprise the contents of the components listed in Table 1 above for 1 L of medium.

Regarding HP inorganic salts, HP inorganic salts include nitrogen (N) and potassium (K), and HP inorganic salts have a weight ratio of nitrogen (N): potassium (K) of 6 to 25:15 to 30 . The HP inorganic salt may optionally contain phosphorus (P), and the weight ratio of nitrogen (N): phosphorus (P): potassium (K) in the HP inorganic salt may range from 6 to 25: 0 to 30:15 to 30 .

In the regeneration medium, the weight ratio of HP inorganic salts and carbon source may be 1:20 to 9:20.

The regeneration medium may contain an appropriate amount of a growth regulator such as naphthalene acetic acid (NAA) or benzylaminopurine (BA). However, when 1 L of the medium contains more than 1 mg of naphthalene acetic acid in the culture medium, dedifferentiation is induced in the root portion contacting with the culture medium to form a PLB (protocormlike body), and the regemination medium is prepared by dissolving naphthalene acetic acid Is preferably 1 mg or less.

The regeneration medium may include an agar or the like to form a solid medium, and the culture conditions such as pH and temperature may be appropriately adjusted according to the purpose of the invention.

The time period during which the protocolums are transplanted into the regeneration medium and cultured can be appropriately adjusted according to the purpose of the invention, and it is preferable to cultivate them for 8 to 12 weeks in general.

The present invention also provides a medium for protocom regeneration of eggs.

It is preferable that the regeneration medium contains 20 to 30 g of carbon source per 1 L of the medium.

The medium for regeneration may include MS inorganic salts or HP inorganic salts, and the specific contents thereof are as described above.

In addition, the regeneration medium may contain an appropriate amount of a growth regulator such as naphthalene acetic acid (NAA), benzylaminopurine (BA), etc., as described above.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are provided to illustrate the present invention, and the present invention is not limited by the following examples, but may be variously modified and changed.

Example  1. Bred Protocom ( protocorm ) Judo

In the Garden of Korea Flower Garden (Seoul, Seongbuk-gu, Anam-dong 5-ga 1), seeds grown in natural moisture were opened and the seeds in the pods were placed in a conical tube and a disinfectant with 1-2 drops of Tween- After disinfection by hand for 1 minute in 99.9% ethanol: 5% sodium hypochlorite solution: distilled water = 5: 5: 90), disinfectant was removed by sterilized water three times or more thoroughly in sterilized water. Sterilized seeds were sterilized using sterilized tweezers and inoculation loops and seeded on solid medium, rotary culture, and bioreactor culture ( Bletilla striata seed germination patterns were compared. The composition and content of inorganic salts of MS medium used in each culture are shown in Table 2 below, and the conditions in each culture are as follows.

<Solid culture>

The medium for solid culture was prepared by adding 30 g · L ^-1 sucrose to the MS medium, adjusting the pH to 5.8, adding 8 g · L ^-1 agar, sterilizing for 15 minutes, and cultivating petri- Each dish was used in a 30 mL aliquot.

<Rotary bottle culture>

The culture medium for the rotavirus culture was prepared by adding 30 g · L ^-1 sucrose to the MS medium, adjusting the pH to 5.8, and dispensing 100 mL in a 1 L culture bottle. For the exchange of gas inside and outside the culture bottle, a plug with a PTFE (polytetrafluoroethylene) membrane filter with a pore size of 0.2 μm was used. The rotation speed of the culture bottle was changed by a rotation culture apparatus (R ₂ P ^® roller culture apparatus, WHEATON ^® ) And adjusted to 6 rpm.

<Bioreactor Culture>

The medium for the bioreactor culture was prepared by adding 30 g · L ^-1 sucrose to the MS medium, adjusting the pH to 5.8, adding 500 mL of the medium to a 700 mL volume of a balloon type bubble bioreactor Followed by high pressure sterilization. Air using the air pump (ZP-40A, SHIN HWA HITECH) and was injected, the amount of air injected ^{air flow meter (RATE-MASTER ®} RMA-14-SSV, DWYER ®) used was adjusted to 0.2 vvm. The injected air and the air exiting the bioreactor were filtered through a venting filter (Midisart ^® 2000, SATORIUS ^® ) with a pore size of 0.2 μm. A supplementary flask containing 500 mL of medium was connected to the bioreactor to supplement the medium which evaporated due to the injection of air.

Furtherance Content (per liter) Ammonium nitrate (NH ₄ NO ₃₎ 1650 mg Calcium chloride (CaCl ₂ ) 332.2 mg Magnesium sulfate (MgSO ₄ ) 180.7 mg Potassium nitrate (KNO ₃₎ 1900 mg Potassium phosphate (KH ₂ PO ₄ ) 170 mg Boric acid (H ₃ BO ₃ ) 6.2 mg Cobalt chloride (CoCl ₂ .6H ₂ O) 0.025 mg Copper sulfate (CuSO ₄ · 5H ₂ O) 0.025 mg Ferric sodium EDTA (C ₁₀ H ₁₂ FeN ₂ NaO ₈ .3H ₂ O) 42.11 mg Manganese sulfate (MnSO ₄ .H ₂ O) 16.9 mg Molybdic acid (Na ₂ MoO ₄ .2H ₂ O) 0.25 mg Potassium iodine (KI) 0.83 mg Zinc sulfate (ZnSO ₄ .7H ₂ O) 8.6 mg Glycine (C ₂ H ₅ NO ₂ ) 2 mg Myoinositol (C ₆ H ₁₂ O ₆ ) 100 mg Nicotinic acid (C ₆ H ₅ NO ₂ ) 0.5 mg Pyridoxine (C ₈ H ₁₁ NO ₃ .HCl) 0.5 mg Thiamine (C ₁₂ H ₁₇ CIN ₄ OS · HCl) 1 mg

Growth seeds sown in each culture were cultured for 4 weeks, and the results are shown in Table 3 and FIG.

Table 3 shows the effect of solid culture, rotary culture, and bioreactor culture on the formation of protocom, which shows the protocom formation rate, caliber diameter and fresh weight in each culture. In relation to caliber diameter, The diameters of the pseudobulbs were measured, and the diameter of the protocom was measured by the protocom in the rotavirus culture and the bioreactor culture. In Table 3, the protocomb formation rate represents the proportion of protocom formed in the whole grown seeds, which means 0% for '-', 40 ~ 60% for ++ and 60 ~ 80% for '+++'. In Table 3, a, b, and c indicate statistically significant differences. No statistically significant differences are found in the same alphabet, and statistically significant differences are found in the other alphabets Averages in different alphabets in one column indicate significant differences at the P &lt; 0.05 level by Duncan's multiple test.

Protocom formation rate Diameter (mm) Fresh weight (mg / 100ea) Solid culture - 1.1 b 82 c Incubation ++ 2.15 a 478 b Bioreactor Culture +++ 2.16 a 619 a

The left photograph of FIG. 3 shows the solid culture and the right photograph shows the bioreactor culture.

As shown in FIGS. 2 and 3, the germination rate (98.5% in solid culture, 98.0% in rotavirus culture, 98.5% in bioreactor culture) Were different depending on the culture method. On the other hand, in the solid culture, the leaves and roots were normally grown from the seeds. In the rotifer culture and the bioreactor culture, however, shoot formation and rooting did not occur from the majority of the seeds, and the seeds became large after the protocomb formation. In the bioreactor culture, the protocome formation rate was higher than that in the rotifer culture, because the air injected into the bioreactor caused most of the grown seeds to continuously circulate in the culture medium to form protocombs, Seems to be due to its attachment to the culture bottle, rotation along the bottle surface, and development into seedlings. Also, in rotavirus culture and bioreactor culture, live weight was much heavier than live weight in solid culture. The results showed that the liquid medium increased the rate of nutrient uptake of the seeds and promoted the enlargement of the sphere. Direct injection of air into the bioreactor culture seemed to make the supply of oxygen and the exchange of air inside the vessel more smooth than in the rotary culture.

Example  2. Grow From Protocom Seedling  subdivision

2.1 Type and concentration of medium

In order to determine the medium composition of the optimization for regeneration of the seedlings grown in the bioreactor culture of Example 1, seeds were cultivated at 25 ± 1 ° C and 65 μmol / m ^-2 s ^-1 for 16 hours under a growth condition. The grown protocom from the bioreactor culture of Example 1 was cultured for 8 weeks.

Table 4 shows the types and concentrations of regeneration medium for protoplasts grown in protoplasts. MS medium was the composition shown in Table 2, and HP (Hyponex) (Hyponex Japan Corp., Ltd.) medium was N ), P (phosphorus): K (potassium) = 7: 6: 19, and MS inorganic salts, HP inorganic salts, sucrose and agar. 1/4, 1/2, 1, and 2 of the medium indicate the concentrations of the inorganic salts, 1 MS indicates that the content of the inorganic salts listed in Table 2 is added per 1 L of the medium, Lt; / RTI &gt; of the inorganic salts listed in Table 2 above per liter, and 1/4 MS shows the addition of 1/4 of the inorganic salts listed in Table 2 per liter of medium, Shows the addition of the two-fold content of the inorganic salts listed in Table 2 per liter of the medium, and the HP medium was also applied to HP inorganic salts as the same.

badge MS inorganic salts HP Inorganic Salt Sucrose Baby 1/4 MS 1.11 g 0 g 30 g 8 g 1/2 MS 2.21 g 0 g 30 g 8 g 1 MS 4.41 g 0 g 30 g 8 g 2 MS 8.82 g 0 g 30 g 8 g 1/4 HP 0 g 0.75 g 30 g 8 g 1/2 HP 0 g 1.5 g 30 g 8 g 1 HP 0 g 3 g 30 g 8 g 2 HP 0 g 6 g 30 g 8 g

The results of the cultivation are shown in Table 5, and Table 5 shows the survival rate in each medium.

badge Survival rate 1/4 MS 53.3% 1/2 MS 61.7% 1 MS 66.7% 2 MS 3.3% 1/4 HP 58.3% 1/2 HP 56.7% 1 HP 66.7% 2 HP 18.3%

As shown in Table 5, both the MS medium and the HP medium were able to induce regrowth to protoplasts as seedlings. The survival rate was the highest at 66.7% in both 1MS and 1HP medium. The survival rate in 2MS medium and 2HP medium was very poor, 3.3% and 18.3%, respectively. Therefore, the type and concentration of the medium for regeneration is 1MS medium or 1HP medium.

2.2 Carbonic  density

In order to determine the concentration of the carbon source in the medium for regeneration into the seedlings of the grown protocom, the concentration and the concentration of the carbon source as shown in the following Table 6 were measured in the same manner as in Example 2.1 (1MS medium) Were cultured.

Table 6 below shows the content of each component contained in 1 L of regeneration medium for seedlings of protocom grown.

MS inorganic salts sucrose agar 4.41 g 10 g 8 g 4.41 g 20 g 8 g 4.41 g 30 g 8 g 4.41 g 40 g 8 g 4.41 g 50 g 8 g

The culture results are shown in Table 7 below. Table 7 shows the survival rate, leaf number, root length, rooting ratio, logarithm, root length, and fresh weight according to the sucrose concentration. In Table 7, a, b, c, and ab indicate statistically significant differences. There is no statistically significant difference in the same alphabet and statistically significant difference in the other alphabets (The mean, indicated by different alphabets in one column, indicates a significant difference at the level of P <0.05 by the Tukey's honest significant difference test).

The sucrose concentration (g · L ^-1 ) Survival rate (%) Number of leaves (ea) Length (mm) Rooting rate (%) Logarithm (ea) Root (mm) Fresh weight (mg) 10 98.8 a 4.8 a 11.4 ab 31.3 b 1.3 b 6.7 b 328.2 a 20 90.0 ab 4.3 ab 12.6 a 67.5 a 1.5 ab 9.0 ab 375.2 a 30 91.3 ab 3.8 b 11.7 a 73.8 a 1.7 ab 10.0 ab 348.4 a 40 78.8 b 3.8 b 11.5 a 68.8 a 2.0 a 11.7 a 394.0 a 50 76.3 b 3.9 b 09.0 b 63.8 a 1.7 ab 8.7 ab 329.3 a

As shown in Table 7, over 90% of the individuals survived in 10 to 30 g · L ^-1 sucrose, but the survival rates in the sucrose at 40 and 50 g · L ^-1 were respectively 78.6% and 76.2%, respectively. Rooting rate of adventitious roots was the highest at 30 g ㆍ L ^-1 sucrose, and was the lowest at 31.8% at 10 g ㆍ L ^-1 sucrose. 10 to 30 g ㆍ L ^-1 sucrose showed no significant difference in fresh weight and fresh weight, but the root length was slightly inferior to 10 g ㆍ L ^-1 sucrose 6.7 mm. Therefore, the optimum sucrose concentration in the medium for regeneration is preferably 20 to 30 g · L ^-1 .

2.3 Concentration of Growth Regulators

In order to investigate the effect of the concentration of the growth regulator in the medium on the regeneration of protocom grown in the seedlings of the seedlings grown in the same manner as in Example 2.1 (1MS medium) described in Table 8 below, Growth protocom from the bioreactor culture was cultured.

Table 8 shows the content of each component contained in 1 L of the medium for regeneration into the seedlings of the grown protocom, NAA means naphthalene acetic acid, and BA means benzylaminopurine, respectively.

MS inorganic salts sucrose agar NAA BA 4.41 g 30 g 8 g 0 mg 0 mg 4.41 g 30 g 8 g 0.5 mg 0 mg 4.41 g 30 g 8 g 1 mg 0 mg 4.41 g 30 g 8 g 0 mg 0.5 mg 4.41 g 30 g 8 g 0 mg 1 mg 4.41 g 30 g 8 g 0.5 mg 0.5 mg 4.41 g 30 g 8 g 0.5 mg 1 mg 4.41 g 30 g 8 g 1 mg 0.5 mg 4.41 g 30 g 8 g 1 mg 1 mg

The results of the cultivation are shown in Table 9 below. Table 9 shows the survival rate, leaf number, leaf area, rooting ratio, logarithm, root length, and fresh weight according to the concentration of growth regulators. In Table 9, a, b, c, and ab indicate statistically significant differences. There is no statistically significant difference in the same alphabet and statistically significant difference in the other alphabets (The mean, indicated by different alphabets in one column, indicates a significant difference at the level of P <0.05 by the Tukey's honest significant difference test).

Growth Regulators (mg · L ^-1 ) Survival rate (%) Number of leaves (ea) Leaf length (mm) Rooting rate (%) Logarithm (ea) Root (mm) Fresh weight (mg) NAA BA 0 0 92.2 ab 3.9 a 16.2 a 62.7 a 1.4 a 8.6 a 34.0 b 0.5 0 97.5 a 3.4 a 13.6 a 43.6 a 1.1 a 5.6 a 40.9 ab One 0 82.5 b 4.1 a 15.3 a 65.2 a 1.5 a 7.5 a 55.0 a 0 0.5 96.3 a 3.9 a 18.0 a 61.0 a 1.4 a 6.0 ab 30.2 ab 0 One 95.0 a 3.7 a 13.2 a 34.2 a 1.3 a 4.8 b 24.8 b 0.5 0.5 80.0 a 4.0 a 14.1 a 68.8 a 1.6 a 6.1a 44.6 ab 0.5 One 85.0 a 4.4 a 14.4 a 61.8 a 1.4 a 6.2 a 49.4 ab One 0.5 79.7 a 4.5 a 15.0 a 80.4 a 2.1 a 6.4 a 60.6 a One One 82.8 a 4.7 a 13.4 a 90.6 a 1.8 a 6.4 a 59.4 a

As shown in Table 9, the grown protocom from the bioreactor culture was able to regenerate seedlings without treatment of growth regulators. There was no significant difference in survival rate, leaf number, length, and logarithmic values after 8 weeks of incubation. However, treatment with NAA alone or NAA and BA mixed treatment promoted root enlargement, resulting in a significant increase in fresh weight. However, in the medium supplemented with 1.0 mg ㆍ L ^-1 NAA, PLB (Protocorm like body) was formed by induction of dedifferentiation at the root portion abutting the medium. Therefore, low-concentration NAA treatment is preferable for seedling growth.

Example  3. Growing up Solid medium seedlings Protocom  Growth comparison

The solid medium seedlings cultured for 4 weeks on seeds grown in accordance with the above Example 1 and the grown protocom obtained by seeding the seeds in a bioreactor for 4 weeks were cultured for 30 days in 30 ml of MS medium supplemented with the composition of Table 2 g · L ^-1 sucrose and 8 g · L ^-1 agar were added and 120 mL was added to each 80 × 130 (mm) culture bottle). Twenty individuals were transplanted into the medium at a temperature of 25 ± 1 ° C. and a light intensity of 65 μmol · m ^-2 ㆍ s ^-1 , Growth of 16 hr single-day condition The leaf number, the length of the root, the root length, the root diameter and the fresh weight were measured after culturing for 12 weeks in an incubator. The results are shown in Table 10 and Figs. .

division Number of leaves (ea) Length (mm) Logarithm (ea) Root (mm) Top Diameter (mm) Fresh weight (mg) Solid badge grave 3.6 19.7 3.6 16.7 3.0 43.6 Protocom 4.2 34.8 4.1 22.4 4.3 123.0

FIG. 4 is a photograph of protocorm seedlings and solid culture seedlings from a bioreactor culture after culturing for 12 weeks in a regeneration medium. In the photographs on the left side, solid culture medium seedlings are shown, and on the right side, protocom will be

Figure 5 shows the number of leaves and no. Of roots after the 12-week incubation of protocorm seedlings and solid culture seedlings from the bioreactor culture in the regeneration medium, As the average number per plant.

Figure 6 shows the fresh weight of the protocorm seedlings and solid culture seedlings from the bioreactor culture after 12 weeks of culture in the regeneration medium for the average weight per plant ).

Fig. 7 shows the leaf length, root length (pseudobulb) and the number of pods after the incubation of the protocorm seedlings and the solid culture seedlings from the bioreactor culture for 12 weeks in the regeneration medium width) as the average length per plant.

5 to NS, ** as described in Figure 7. *** If the difference is shown in that if there is a statistically significant difference, P <0.05 level, *, P <0.01 ** If there is a difference in level, P < 0.001, and NS (nonsignificant) if there is no statistically significant difference.

As shown in FIG. 4 to FIG. 7, the protocom from the bioreactor culture showed better growth than the solid culture medium, 1.8 times in the plant height, 1.5 times in the diameter of the upper diameter, and 2.8 times in the living body.

Claims (13)

Culturing the seeds seeded in the medium in a rotator culture or in a bioreactor culture to form a protocorm; And
And transplanting the protocom into a regeneration medium and culturing the seedlot. The method according to claim 1, wherein the seeded seed is sterilized and sterile. The method according to claim 1, wherein the rotation speed of the culture bottle in the rotary bottle culture is 2 to 10 rpm. The method of claim 1, wherein the amount of air injected in the bioreactor culture is 0.1 to 3 vvm. The method according to claim 1, wherein the carbon source content in the regeneration medium is 20 to 30 g per 1 L of the medium. The method according to claim 1, wherein the regeneration medium contains the components listed in Table 1 for 1 L of the medium.
[Table 1]

6. The regeneration medium according to claim 5, wherein the regeneration medium comprises HP (Hyponex) inorganic salts,
The weight ratio of HP inorganic salts and carbon sources in the regeneration medium is 1:20 to 9:20,
Wherein the weight ratio of nitrogen (N): potassium (K) in the HP inorganic salt is 6 to 25: 15 to 30. The method according to claim 1, wherein the regeneration medium contains naphthalene acetic acid in an amount of 1 mg or less based on 1 L of the medium. The method of claim 1, wherein the eggs are grown ( Bletilla striata , Dendrobium , Cypripedium , Oncidium , or Cymbidium . The method of producing the seedlings is described below. A medium for protocorm regeneration of eggs containing 20 to 30 g of carbon source per liter of medium. 11. The medium for protocom regrowth of eggs according to claim 10, wherein the regeneration medium comprises the content of the components listed in Table 1 for 1 L of the medium. 11. The method according to claim 10, wherein the regeneration medium comprises HP (Hyponex) inorganic salts,
The weight ratio of HP inorganic salts and carbon sources in the regeneration medium is 1:20 to 9:20,
Wherein the HP inorganic salt has a weight ratio of nitrogen (N): potassium (K) of 6 to 25: 15 to 30. [Claim 11] The culture medium for regrowth treatment according to claim 10, wherein the regeneration medium contains naphthalene acetic acid in an amount of 1 mg or less based on 1 L of the medium.

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