KR20030029676A - Method for seedling production of Chinese foxglove (Rehmannia glutinosa Lib.) using bioreactor - Google Patents

Abstract

PURPOSE: A method for culturing seeds of Rehmannia glutinosa using a bioreactor is provided, thereby effectively culturing high quality of Rehmannia glutinosa. CONSTITUTION: The method for culturing seeds of Rehmannia glutinosa using a bioreactor comprises the steps of: culturing stems of Rehmannia glutinosa in an MS(Murashige-Skoog) medium containing 1 mg/l of thidiazuron(TDZ) and 0.8% of agar for 4 weeks; culturing 90 of Rehmannia glutinosa shoots in 1.5 liter of an MS broth containing 1 mg/l of TDZ and 500 mg/l of MES£2-(N-morpholino)ethanesulfonic acid| in a bioreactor with aeration of 0.5 vvm for 3 weeks; culturing the shoots of Rehmannia glutinosa in a half concentration of MS medium containing 1.2% of agar and 0.3 to 0.4 mg/l of paclobutrazol in a culture bottle with 0.2 μm of polyethylene membrane filter for 4 weeks to elongate and acclimate of Rehmannia glutinosa shoots; and treating the Rehmannia glutinosa shoots with an anti-GA like growth regulator.

Description

Method for seedling production of Chinese foxglove (Rehmannia glutinosa Lib.) Using bioreactor}

The present invention relates to a method for producing a turmeric seedling, more specifically, the selection and selection of culture medium that can significantly increase the induction and growth rate of shoots from the in-plant plants of the sulfuric acid widely used as a nourishing herbal medicine for nutrition Seedling production in a flotation bioreactor.

Jihwang (地 黃) belongs to the Scrophulariaceae , the usage part is root stem, raw green sulfur, dried or dried in dried sand, dried or dried sulfur in wine and nine The boiled rice is known as Suh Ji Hwang. It contains manint, sugar, lehmannin, carotene, cytosterol, vitamin A, and catalpol.Its effects vary depending on the process of making roots, but hemostasis, antipyretic, cardiac, diuretic, detoxification It is widely used as a raw material of Chinese medicine such as diabetes, blood donor, nourishing tonic, and nervous breakdown.

Breeding of the relics is the seed or roots in the ground, but generally using the roots, seeds are used only for breeding methods for new breeds. The roots are not diseased, 6mm thick, and 6cm long, and it is better to use the middle part cut off the tip and the end. When planting the head, the roots are thin and long, but not large. On the contrary, when the roots thicker than 1㎝ in diameter are used as the roots, the amount of stalks increases.

Recently, a method for cultivating the widely used turmeric has been proposed, but there is a continuing demand for an effective culture and cultivation method for producing and obtaining better quality seedlings.

Studies on the tissue culture of Rehmannia chinensis have been conducted by Jiang and Mao (1979), who have successfully induced callus and shoot and root differentiation in seed germination, and the types and composition of tissues, media and growth regulators used in culture. Has been made. In Korea, many studies have been carried out on in vitro cultivation of turmeric, somatic embryo formation through callus suspension culture (Chae and Park, 1993; Park et al., 1995), production of artificial seeds using somatic embryos, direct somatic embryos under flask conditions Seedling production through formation (Park and Chae, 1997, 1999; Park et al., 1999) has been reported.

In-flight cultivation for disease-free mass propagation, although an excellent method, still has slow growth and high cost. Most of the research results to date have been using agar medium and small-scale culture vessels, which does not satisfy the premise of in-flight mass production of seedlings.

As one of the methods for overcoming these shortcomings, it has been reported that the large-scale cultivation method using a bioreactor has the advantage of reducing the labor required for the work and increasing the proliferation rate of the embryos (Styer, 1985).

It is therefore an object of the present invention to provide effective culture conditions and methods for producing and obtaining high quality seedlings in bioreactors.

In order to achieve the above object of the present invention, the present inventors earnestly researched, by adjusting the composition of the culture medium for turmeric shoots, by using a bioreactor to boost the air for mass production of shoots that can be used as seedlings The present invention was completed to know that the seedling of can be effectively produced and obtained.

Figure 1a shows shoots (A) induced by pre-culture in solid MS medium in accordance with a preferred embodiment of the present invention,

FIG. 1B illustrates a state (B) in which the shoots (A) obtained in FIG. 1A are cultured in a solid MS medium for 4 weeks in accordance with a preferred embodiment of the present invention and multiplication is performed.

Figure 2 is a photograph showing the root formation (A) of shoots cultured in a bioreactor, a state (B), and its root portion (C) in the bonsai pot.

According to the invention,

Cultivating turmeric stem tissue in MS (Murashige-Skoog) medium containing 1 mg / L of thidiazuron (TDZ) and 0.8% agar and culturing for 4 weeks to cultivate shoots;

Fill the bioreactor (2.5 L) with 1.5 L of MS liquid medium to which 1 mg / L of tidiazuron (TDZ) and 500 mg / L of MES [2- (N-morpholino) ethanesulfonic acid] were added. Incubating for 3 weeks while supplying sterilized air to the airlift bioreactor at 0.5 vvm;

Cultivated shoots in a bioreactor were incubated for 4 weeks in a culture medium containing 0.2 μm polyethylene membrane filter in a medium containing 1.2% agar and 0.3-0.4 mg / L of paclobutrazole in a 1/2 concentration of MS medium. To purify the shoots and seedlings of the shoots; And

Treatment with paclobutrazole as an anti-GA growth regulator to inhibit coating and improve root development when purifying shoots of turmeric grown in a bioreactor

Provided is a method for producing a sulfur-based seedling comprising a.

The concentration of paclobutrazole used in the inflation of seedlings and purifying seedlings is preferably 0.4 mg / L. In addition, it is preferable to proceed the culture under aeration conditions in order to promote root growth.

Referring specifically to the method for cultivating the rhubarb seedling of the present invention is as follows:

Order 1.

The bioreactor culture material was cultured for 4 weeks to incubate for 2 weeks in the medium of MS (Murashige-Skoog) containing 1 mg / L thidiazuron (TDZ) and 0.8% agar.

Order 2.

Fill the bioreactor (2.5 L) with 1.5 L of MS liquid medium to which 1 mg / L of Thidiazuron and 500 mg / L of MES [2- (N-morpholino) ethanesulfonic acid] were added. Incubate 90 positive shoots. The bioreactor is an air-lift bioreactor, which is incubated for 3 weeks while feeding sterilized air at 0.5vvm.

Order 3.

The shoots harvested after 3 weeks in the bioreactor were incubated for 4 weeks in a 0.2 μm polyethylene membrane filter containing medium containing 1.2% agar and 0.4 mg / L of paclobutrazol in half-strength MS medium. If you do this, seedlings are purified at the same time as root shoots.

Hereinafter, the present invention will be described in more detail by way of non-limiting examples.

Example 1:

In order to increase the production efficiency of shoots in the wormwood, two weeks of preculture in a 100ml Erlenmeyer flask containing MS liquid medium in which BA and NAA were combined with concentrations of stem sections, and then incubated for 6 weeks in 500ml Erlenmeyer flask conditions. One result is shown in Table 1.

From the table, it was found that the test cells pre-incubated in the medium treated with BA 2mg / l were more advantageous in terms of the number of shoots formed than those in the non-pre-culture. In particular, the most shoots occurred in the media treated with NAA 0.5 mg / l, and this combination was a suitable culture condition.

Petri-dish (90x20mm) has 10 stems of 0.8-1.0cm size without any nodes and incubated for 4 weeks, and shoots are formed at both ends of the sections as shown in Figure 1A. When the shoots formed here are cultured for 3 weeks in the bioreactor, the shoots are newly formed as well as the number of nodes of the cultured shoots is increased and intercalation growth occurs. Induced shoots can continue to multiply by passage (Figure 1B).

When using a bioreactor for the development of the turmeric buds, the shoots are produced in petri dishes containing solid medium and incubated in the bioreactor before the sections of the turmeric are directly inoculated into the bioreactor to generate the shoots. Incidence rate increases

Therefore, it is important to first develop shoots in petri dishes containing solid medium, and then induce shoot extension in bioreactors.

Table 1. Effect of BA and NAA combination on shoot formation after 6 week's culture of the stems got from the shoots developed in pre-culture of stem for two weeks on petri-dish in R. glutinosa

Control: without pre-culture in MS medium supplemented with BA 2 mg / l and NAA 0.1 mg / l

In order to increase shoot growth efficiency, the degree of shoot growth was compared with the culture of BA 2.0mg / L and NAA 0.5mg / L in the medium supplemented with TDZ alone at various concentrations in MS base medium. (Table 2).

It was found that the most shoots were generated in the medium treated with the concentration of 1.0 mg / L of TDZ, so that the treatment with this concentration was suitable when TDZ was used for preculture of the sulfur.

Table 2. Effect of thidiazuron (TDZ) on multiple shoots formation in Rehmannia glutinosa after 4 weeks culture.

＊; Each value represents the mean ± S.D.

†; within columns, means followed by the same letter are not significantly

different at p = 0.05 according to Duncan's multiple rage test.

Example 2:

The effect of pH was examined by comparing the case of incubating in a condition that can maintain a constant pH during the incubation period using the MES and the incubation by adjusting only before the sterilization of the medium, and at the same time, The experimental results to determine the effect of aeration by adjusting the amount to 0.5 vvm and 1.0 vvm, respectively, are shown in Table 3. Treatment with MES in the medium showed a higher shoot rate than otherwise, indicating that the pH had some effect on the shoot growth of turmeric. In addition, it was effective to adjust the amount of air injected to 0.5 vvm rather than 1.0 vvm because the amount of air generated increased a lot of bubbles in the culture bottle, which inhibited the respiration of tissues and aeration in the container. .

Table 3. Effect of pH and aeration on shoot formation in bioreactor culture of R. glutinosa after 3 weeks .

†; A: without MES and 0.5 vvm, B: without MES and 1.0 vvm

C: with MES and 0.5 vvm, D: with MES and 1.0 vvm

The shoots from 1.0mg / L-treated Thidiazuron medium were treated with bioreactor to increase shoot number and increase internode length. As a result, the most important number of nodes and shoots occurred at 1.0 mg / L thidiazuron (Table 4).

Table 4. Effect of Thidiazuron (TDZ) on shoot elongation in Rehmannia glutinosa after 3 weeks culture.

Implementation (example)

The results of investigating shoot survival and rooting rate according to MS medium and agar concentration are shown in Table 5. If the concentration of MS medium is too low and the survival rate decreases too much, use a medium of 1/2 times the medium and increase the concentration of agar, which is effective for rooting, but this also reduces the survival rate. Treatment at 1.2%, a 1.5-fold increase from 0.8% concentration, can simultaneously improve rooting and purifying the turmeric.

Table 5. Effects of agar concentration and MS medium strength on rooting from shoots of Rehmannia glutinosa harvested after 3 weeks culture in bioreactor .

*: Survival shoots / total shoots × 100 (%)

＊＊: rooted shoots / survival shoots × 100 (%)

†; within columns, means followed by the same letter are not significantly

different at p = 0.05 according to Duncan's multiple rage test.

Figure 2 shows that plants grown from air-lift type bioreactors have been grown in the greenhouse for two weeks after induction of rooting in the cabin.

In order to suppress the coating and improve root development when the shoots of sulfur incubated in a bioreactor are treated with in-flight, anti-GA-based growth regulators such as paclobutrazol, flurprimidol and chlorocholine chloride are treated at different concentrations. The results of the examination are shown in Table 6. The treatment of Paclobutrazol did not show excessive growth inhibitory effect, but it was found that the plant tissue was densified due to the increase in live weight. Especially, in the 0.3 ~ 0.4mg / L treatment, the roots were better developed than other treatments. Soil was favored in the next step.

Table 6. Effect of anti-GAs on in vitro acclimatization of Rehmannia glutinosa shoots harvested after 3 weeks culture in bioreactor.

†; within columns, means followed by the same letter are not significantly

different at p = 0.05 according to Duncan's multiple rage test.

＊; Each value represents the mean ± S.D.

To investigate the effect of aeration on the purifying and rooting of shoots harvested from the bioreactor, the filter was attached and the filter was attached under the aeration condition using Magenta GA-7 vessel (Sigma) attached with 0.2㎛ polypropylene membrane filter. Purified in a completely enclosed condition using a normal Magenta GA-7 vessel, not compared. The shoots harvested from the bioreactor were grown and grown for 4 weeks in purified medium containing paclobutrazol 0.4mg / L in MS medium diluted to 1/2. The growth of purified shoots in magenta with Membrane filter showed healthy growth compared to that in purified filter, which was superior to control in all items including grass length and root length (Table 7).

Table 7. Effect of vessel with 0.2 μm membrane filter on shoots growth of Rehmannia glutinosa harvested after 3 weeks culture in bioreactor .

＊; Each value represents the mean ± S.D.

According to the present invention as mentioned above, it can be seen that it is possible to produce and obtain by cultivating and cultivating high quality seedlings of rhubarb effectively.

Claims (3)

In the cultivation method of the foxworm seedling Cultivating turmeric stem tissue in MS (Murashige-Skoog) medium containing 1 mg / L of thidiazuron (TDZ) and 0.8% agar and culturing for 4 weeks to cultivate shoots; Fill the bioreactor (2.5 L) with 1.5 L of MS liquid medium to which 1 mg / L of tidiazuron (TDZ) and 500 mg / L of MES [2- (N-morpholino) ethanesulfonic acid] were added. Cultivating 90 lichen buds for 3 weeks while supplying sterilized air at 0.5 vvm to the air flotation bioreactor; Cultivated shoots in a bioreactor were incubated for 4 weeks in a culture medium containing 0.2 μm polyethylene membrane filter in a medium containing 1.2% agar and 0.3-0.4 mg / L of paclobutrazole in a 1/2 concentration of MS medium. To purify the shoots and seedlings of the shoots; And Treatment with paclobutrazole as an anti-GA growth regulator to inhibit coating and improve root development when purifying the shoots of turmeric grown in a bioreactor Production method of rhubarb seedling, characterized in that it comprises a. The method of claim 1, wherein producing the Rehmannia glutinosa seedling wherein wave claws boot rajol concentration used in the step of forming the purified cabin seedling and rooting of the shoots is a 0.4mg / L. The method of claim 1, wherein in order to promote root growth, the method of producing a turmeric seedling, characterized in that the culture in the bioreactor under aeration conditions.