KR100198970B1 - A method for mass-producing Lilium sp. bu using a bioreactor - Google Patents

Abstract

The present invention relates to a method for industrially mass-producing a species of Lilium species, and more specifically, by applying a technique of tissue culture and a bioreactor culture technology, the method for producing a species of Lilium is dramatically improved. It is about a method to enable mass production of bottle-free species.

Description

A method for mass-producing Lilium sp. bu using a bioreactor}

The present invention relates to a method for industrially mass-producing a species of Lilium species, and more specifically, by applying a technique of tissue culture and a bioreactor culture technology, the method for producing a species of Lilium is dramatically improved. It is about a method to enable mass production of bottle-free species.

Lilium sp. Is a vegetative propagation of human scales. The bulb acts as a reservoir for starch and other nutrients, and contains stem growth. On the other hand, bulbs are easily infected by viral diseases caused by phytopathogens, so the quantity and quality of the bulbs can not be used continuously for many years, so growers buy new bulbs every year.

In the Netherlands, the world's major producer of lily species, the scales of virus-tested hairballs are grown in a sterile state by tissue culture, followed by prolonged purifying and benign processes in the soil, producing and selling them to the world. Although several research institutes in Korea are studying the mass production method of lily species using tissue culture method, it is dependent on tissue culture method using petri dish or bottle for bacterial culture. Most farmers rely on imports because they are difficult and difficult to mass produce for industrial production, resulting in high production costs and low growth rates. However, 68% of the cost of lily flower production costs is now accounted for, and farmers producing lily for export use mostly imported seed ball due to the quality deterioration problem. It is true.

Thus, the present inventors earnestly researched to provide a method for industrially mass-producing a species of Lilium species, and as a result, when applying the technology of tissue culture to the bioreactor culture, not only continuous culture is possible, It is found that the growth rate is much faster, the size of the reactor can be arbitrarily controlled to control the production volume, and the quality of the seed can be provided uniformly because a large amount of the seed can be incubated under the same conditions in the same incubator. The present invention has been completed.

It is therefore an object of the present invention to provide a method for industrially mass production of the species of Lilium.

In order to achieve the above object, the present inventors carried out tissue culture under various conditions in order to find a suitable culture conditions for the bioreactor culture, the species produced by the bioreactor culture under the same conditions as the tissue culture in the general soil As a result of transplantation, the degree of survival was similar to that of tissue culture.

That is, in order to achieve the above object, the method of mass production of lily lilies according to the present invention is equipped with an agitator (impeller) in the middle of the reactor, and a lot of the bottom of the reactor to inject sterile air It is characterized by using a bioreactor having an air hole,

(1) Murashige Skoog medium (hereinafter referred to as MS medium) and α-naphthalene acetic acid (hereinafter referred to as NAA) and 6-benzylaminopurine (hereinafter referred to as BAA) Incubating in a medium added with a pH of 5.6 to 5.8, a temperature of 25 ° C., and bright conditions to induce proliferation of magnetic domains; And

(2) replacing the whole medium with a hormone-free MS medium, incubating under the condition of pH 5.6 ~ 5.8, temperature 20 ~ 25 ℃, dark condition to block light to induce hypertrophy

It characterized in that it comprises a.

One-step culturing is intended to induce proliferation of magnetic domains to enable mass production of scales at the beginning of cultivation, and is cultured in a medium in which growth control hormones, oxine-type NAA and cytokinin-based BA are added. The amount of the hormone added is preferably in the range of 0.1 to 10 mg / l NAA and 0.1 to 10 mg / l BA, and preferably 0.3 mg / l NAA and 1.0 mg / l BA. In addition, oxinine 2,4-D (2,4-Dichlorophenoxy acetic acid) or cytokininine kinetin (hereinafter referred to as Ki) may be further added.

Two-stage culture is to allow a large number of multiplying cells grown in the first stage to be used as a single seed through a process of hypertrophy and maturation in a short period of time in the same incubator. Is carried out.

In addition, the bioreactor culture is carried out at a dissolved oxygen concentration of 40ppm, the rotation speed of the stirrer 100rpm can obtain the best results for the growth and enlargement of the magnetic domain, which can be confirmed in the examples described below.

Through this two-step bioreactor culture, the growth rate of the species is much faster, and a large number of species can be cultured under the same conditions in the same incubator, thereby providing a large quantity of uniform quality seeds. By shortening the training period in Esau as much as possible, the effect of promoting flowering and reducing production costs can be expected. In addition, by adjusting the size of the reactor can be adjusted arbitrarily, it can be said that it is more suitable for mass production for industrial production because it is possible to continuously culture.

Hereinafter, tissue culture under various conditions and bioreactor cultivation based on the same will be described in detail through various embodiments to find suitable culture conditions for culturing the bioreactor. However, the present invention is not limited to these examples.

Reference Example 1

Bulbs are collected from three different lilies of Oriental Casacaña, Asian Connecticut kings, and Hinamoto of longiflorums and washed overnight under running tap water. It was.

Soak the cleaned bulb in a household detergent for 2-3 minutes to remove surface dirt, wash the detergent again with tap water, put it in a beaker, add 70% ethanol and sterilize for 1 minute, and then dissolve 2% sodium hypochlorite. The bulb was again surface sterilized by shaking for 30 minutes in (Sodium hypochlorite) solution.

After sterilizing the surface, wash it 4 times or more with sterilized ionized water to remove the disinfectant solution remaining on the surface of the bulb and transfer it to sterilized Petri dish in the aseptic operation room to remove the surface area and take the growth point and healthy human tissue and cut it into 1㎠. After that, it was used as a culture material.

The basal medium was selected as MS medium and the concentrations of 4, 2,4-D and NAA of oxine type and BA and Ki of cytokinin type (0.01mg / l, 0.3mg / l, 0.5mg / l, 1 mg / l, 2 mg / l) Several combinations were made and used as experimental medium.

The incubation temperature was maintained at 25 ° C. using an air conditioner. The experiment was carried out in a culture room in which a fluorescent lamp (15 W / m 2) was turned on and 8 hours was automatically converted to a flashing state for 16 hours of the day. Weekly cultures were maintained.

From this, MS + NAA (0.3mg / l) + BA (1mg / l) medium is good for induction and proliferation of scales, and MS + NAA (0.3mg / l) medium is advantageous for induction of roots. It was concluded that using MS medium without modifiers was the optimal composition.

Subsequently, subcultures were performed at intervals of 5 to 7 weeks.

Reference Example 2

Based on the results of Reference Example 1, an optimal basic medium was selected, and as an experimental medium, MS medium, B5 medium (Gamborg's B5 medium, hereinafter referred to as B5 medium), Schenk Hildebrandt medium (hereinafter referred to as SH medium) ), A medium in which NAA (0.3 mg / l) + BA (1 mg / l) was added to Lismaier Skoog medium (hereinafter referred to as LS medium), and the basic composition of each medium is shown in Table 1 below. Is showing on.

Other culture conditions were maintained in the same manner as in Reference Example 1.

After 6 weeks of cultivation, each of the basal media was selected from five experimental groups with excellent scalp production capacity, and the number of scaffolds produced was investigated. From this, it was concluded that MS medium was the best medium.

Composition of Tissue Culture Medium (Unit: mg / l) ingredient MS badge B5 SH LS badge NH ₄ NO ₃ 1,650 - - 1,650 (NH ₄ ) ₂ SO ₄ - 134 - - KNO ₃ 1,900 2,500 2,500 1,900 H ₃ BO ₃ 6.2 3 5 6.2 KH ₂ PO ₄ 170 - - 170 NaH ₂ PO ₄ · H ₂ O - 150 300 - Na ₂ HPO ₄ - 30 - - KI 0.83 0.75 One 0.83 KCl - 300 - - Na ₂ MoO ₄ 2H ₂ O 0.25 0.25 0.1 0.25 CoCl ₂ · 6H ₂ O 0.025 0.025 0.1 0.025 CaCl ₂ · 2H ₂ O 440 150 200 440 MgSO _{4 7} H ₂ O 370 250 400 370 MnSO ₄ H ₂ O 22.3 10 10 10 ZnSO ₄ · 7H ₂ O 8.6 2 One 8.6 CuSO ₄ · 5H ₂ O 0.025 0.025 0.2 0.025 Na ₂ EDTA 37.3 18.6 20 37.26 FeSO ₄ 7H ₂ O 27.8 27.8 15 27.86 Thiamine-HCl 0.1 10 0.1 0.4 Nicotinic acid 0.5 1.0 0.5 - Pyridoxine-HCl 0.5 1.0 0.5 - Glycine 2.0 2.0 2.0 - Myo-inositol 100 100 100 100 Sucrose 30,000 20,000 20,000 30,000 Agar 6,000 6,000 6,000 6.000

Scale production capacity by basic medium Default medium Creation labor (seal) MS 20 B5 8 SH 10 LS 17

Reference Example 3

Based on the conditions selected in Reference Examples 1 and 2, an experiment was conducted to find an optimal temperature suitable for the culture. After 6 weeks of incubation in the incubator maintained at 20 ° C, 25 ° C, and 30 ° C respectively, 25 ° C was the best for the size and size of the scales and 20 ° C and 25 ° C for root differentiation and growth. There was no significant difference between.

Reference Example 4

Some of the scales induced in Reference Example 1 were cultivated in a cancerous state completely blocked by light all day, and compared after 6 weeks. As a result, the formation and hypertrophy of the bulblets were different depending on the variety. Hypertrophy was good in the cancerous state and good in the bright state.

Reference Example 5

Species cultured in sterile culture facilities were cultivated in artificial soil mixed with vermiculite (1): perlite (1), and then purified. Survival was more than% and growth was good. In addition, low temperature treatment was necessary for more than 5 weeks at 4 ℃ before transplanting to the soil.

Reference Example 6

Based on the results of Reference Examples 1 to 3, the culture was carried out using a liquid medium that had agar removed from the selected MS + NAA (0.3 mg / L) + BA (1 mg / L) medium.

As the incubator, a 250 ml triangular flask and a 500 ml roller bottle were used. As a result of comparing the magnetoforming ability according to the rotational speed (rpm), the lower the rotational speed of both incubators, the better the magnetoforming ability was. 3). The glomerular formation ability was compared after converting in scale of 1 liter by selecting only scales having a diameter of 5 mm or more after 6 weeks of culture.

Culture conditions were in accordance with Reference Example 1, but the culture was carried out in the state of blocking light referring to the results of Reference Example 4, considering the problem of oxygen supply, the amount of medium in the Erlenmeyer flask was 60 ml, In the experiment used, the amount of medium was 150 ml.

Scale production capacity according to rotation speed How Culture Rotation speed (rpm) Generation labor Erlenmeyer flask 20 510 50 272 100 62 Roller bottle 10 423 20 347 30 278

Reference Example 7

Some of the scales cultured in Reference Example 6 were cultured by changing to MS liquid medium without hormones. The other conditions of the culture were the same as in Reference Example 6 and the incubator was a Erlenmeyer flask.

As in the agar culture of Reference Example 1, a large number of generated scales grew as one species as they grew in size.

Example 1

Based on the results of Reference Examples 6 and 7, a 2.5 L batch bioreactor culture was performed as a basic step for industrial production. The type and condition of the incubator were agitated in the middle to mix the cells, inject sterilized air into the many air holes at the bottom of the reactor to adjust the dissolved oxygen concentration, and maintain the pH at 5.6 ~ 5.8 using a pH correction device. , The temperature was always maintained at 25 ℃. Experiments were conducted to determine the optimum dissolved oxygen concentration and the optimum rotational speed of the batch bioreactor. The results are shown in Table 4. The initial 6 weeks of culture was carried out in the bright state with MS + NAA (0.3mg / l) + BA (1mg / l) medium from the results of Reference Examples 1-4, and after 6 weeks, the medium was maintained with the same It was replaced with MS medium which was not added, and the whole bioreactor was covered with a black cloth and kept dark, and the culture was continued for another 4 weeks to continuously enlarge the scales. After final cultivation, the scale formation ability was compared by selecting only scales of 5 mm or more in terms of 1 L unit. The ratio of scales was expressed in terms of percentage of the total size of pieces of scales of 10 mm or more among the scales of 5 mm or more. .

Optimum Culture Conditions for Tank-type Bioreactors DO concentration (ppm) Rotation speed (rpm) Generation labor Scale ratio (%) 20 50 151 21 100 238 32 150 454 37 40 50 635 45 100 855 63 150 785 52 60 50 713 60 100 623 55 150 520 47

As a result of the experiment, it can be seen that compared to the results of Table 3, the number of flakes generated in the culture conditions of 40ppm dissolved oxygen concentration, 100rpm rotation speed of the stirrer increased up to more than 40%. Compared with Table 3, the increase in more than 40% is more significant than other culture methods, considering that the incubation lasted more than 4 weeks and the scale of 5 mm or more increased slightly.

In addition, as shown in Table 4, as a result of examining the ratio of scales, it was confirmed that favorable conditions for sclerotization, that is, a dissolved oxygen concentration of 40 ppm and a rotational speed of 100 rpm were optimal conditions for the size of scales. However, the experimental results show that when the dissolved oxygen concentration is high, the proportion of scalp is relatively high. Therefore, it can be predicted that there is a certain correlation between the growth of the scale and the dissolved oxygen concentration as well as the hormone composition of the medium.

Example 2

From the results of Example 1, a comparative experiment was conducted to confirm the effect of the replacement of the medium on the growth and enlargement of the scale in a batch bioreactor, the results are shown in Table 5. The composition of the medium was as shown in the table. Dissolved oxygen concentration and rotational speed were the same at 40 ppm and 100 rpm, respectively. The reactor was covered and incubated in the dark. Other conditions were the same as in Example 1. The calculation of the number of generated pieces and the size ratio of the pieces is the same as in Example 1.

As a result, it is not yet clear why the number of generated human populations did not increase more rapidly than the two-stage culture when cultured with MS + NAA (0.3 mg / l) + BA (1 mg / l) medium for 10 weeks. By multiplying the productivity of the total viable species, the results of the two-stage culture yielded about 10% higher results.

Medium solids comparison experiment How Culture Creation Proportion ratio (%) Comparative Example 1 Stage 1 MS badge 230 87 Tier 2 MS badge Comparative Example 2 Stage 1 MS + NAA (0.3 mg / L) + BA (1 mg / L) 1124 44 Tier 2 MS + NAA (0.3 mg / L) + BA (1 mg / L) Example 2 Stage 1 MS + NAA (0.3 mg / L) + BA (1 mg / L) 811 68 Tier 2 MS badge

Example 3

Among 100 scales produced in batch bioreactor, 100 scales with a diameter of 10 mm or more were randomly taken, and the survival rate in general soil was examined by the same process as in Reference Example 5 through the purification process in artificial soil.

Through this two-stage bioreactor cultivation according to the present invention, the growth rate of the species is much faster, and a large amount of species can be cultured under the same conditions in the same incubator, thereby providing a large quantity of uniform quality seeds. As a result, the cultivated species can be expected to substantially improve flowering and reduce production costs by minimizing the growth period in the soil. In addition, by adjusting the size of the reactor can be adjusted arbitrarily, it can be said that it is more suitable for mass production for industrial production because it is possible to continuously culture.

Claims (4)

In the mass production method of the lily bulb, In the middle of the reactor is equipped with an impeller, using a bioreactor having a lot of air holes in the bottom of the reactor to inject sterile air, (1) Murashige Skoog medium (hereinafter referred to as MS medium) and α-naphthalene acetic acid (hereinafter referred to as NAA) and 6-benzylaminopurine (hereinafter referred to as BAA) Incubating in a medium added with a pH of 5.6 to 5.8, a temperature of 25 ° C., and bright conditions to induce proliferation of magnetic domains; And (2) replacing the whole medium with a hormone-free MS medium, incubating under the condition of pH 5.6 ~ 5.8, temperature 20 ~ 25 ℃, dark condition to block light to induce hypertrophy Mass production method of the species of lily lilies, characterized in that it comprises a. The method of claim 1, wherein in the first stage culture medium, NAA is added in an amount of 0.1 to 10 mg / L, and BA is added in an amount of 0.1 to 10 mg / L. . The method for mass production of lily lilies spp. According to claim 2, wherein in the first stage culture medium, 0.3 mg / l NAA and 1.0 mg / l BA are added. The method for mass production of lily lilies species according to claim 1, wherein the first and second stage cultures are performed at a dissolved oxygen concentration of 40 ppm and a stirrer rotational speed of 100 rpm.