JPH0220281A - Method for propagating adventive root - Google Patents

Abstract

PURPOSE:To industrially propagate an adventive root in large amounts by each relatively setting motions for providing to a liquid culture material in a process for propagating the adventive root, process for dividing the adventive root and process for culturing the adventive root to low speed, high speed giving no shearing action and low speed. CONSTITUTION:In the first stage for culturing an adventive root, relatively low-speed motion suitable for propagation of the adventive root is provided to a liquid culture material with top priority to the propagation of adventive root. Then after the propagation proceeds and mass of adventive root is formed, as the second stage, relatively high-speed motion in such degree as releasing the adventive root from the base of adventive root, but hardly providing shearing action to the adventive root is provided to the liquid culture material containing the adventive root. Thereby in the third stage, relatively row-speed motion as same as the first stage is provided to a number of adventive root groups divided into one-plural pieces with top priority to the propagation of adventive root. Furthermore, as a plant capable of applying the above-mentioned propagation method, Duboisia myoporoides, Panax ginseng, Atropa belladonna, etc., is given.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for propagating adventitious roots, and more particularly to a method for propagating adventitious roots suitable for industrially propagating large amounts of adventitious roots.

[Conventional technology]

Producing useful substances by redifferentiating adventitious roots from cultured cells in a dedifferentiated state and tissue culturing them is
Research and development is being carried out on various plants such as tropane alkaloid-producing plants such as Zboisia myoporoides, periwinkle grass, and purple grass.

In order to industrially implement a method for producing useful substances through tissue culture of redifferentiated adventitious roots, it is essential to develop a method for mass propagation of good adventitious roots with high productivity of useful substances. However, a practical method for mass propagation of adventitious roots has not yet been developed. For example, for Zboisia, adventitious roots used as seeds for propagation in a liquid medium are obtained by culturing adventitious roots for about two weeks and manually cutting the adventitious root mass into pieces of 5 to 15 mm with a scalpel or cutter. The current situation is that there are many, and it is completely unsuitable for industrial mass propagation.

[Problem to be solved by the invention]

An object of the present invention is to provide a method for propagating adventitious roots that is suitable for industrially propagating adventitious roots in large quantities.

[Means to solve the problem]

The present inventors conducted research to achieve the above objective.
First, we selected Zboisia as the research material and conducted the following experiments.

The adventitious root mass of Zboisia that has been cultured for 2 weeks in a 100 ml Erlenmeyer flask is cut into 5-7 mm square pieces using a scalpel, and these are used as seeds in a liquid medium such as Murashige Skoog, which is used for normal plant tissue culture. When cultured in the medium for 2 weeks, the growth rate of adventitious roots was 5.5 times, and the total tropane alkaloid content was 0.78%.

On the other hand, as a comparative experiment, when the adventitious roots were detached from the primordium of the adventitious root mass and cultured for 2 weeks as seeds without cutting the adventitious roots, the multiplication rate of the adventitious roots was 9.6 times, and the total tropane alkaloid content was was 1.77%.

The above experimental results show that when the adventitious roots of Zboisia are cut with a scalpel or a cutter, the adventitious roots are severely damaged and are affected by culture inhibiting substances from the adventitious roots. It was found that the content of tropane-based alkaloids, which are useful substances, was significantly reduced.

Therefore, when agitating and cultivating the adventitious roots of Zboisia in a culture tank, we set certain agitation conditions (such as one using a paddle-type stirring blade) that does not cause any shearing force to the adventitious roots as follows.
An experiment was conducted in which adventitious roots were cultured while maintaining rotation at approximately 0 to 15 rpm.

2I equipped with paddle type stirring blades! , 100m in a mini jar
Adventitious roots that had been cultured for two weeks in a 1-liter flask were directly seeded and cultured with liquid aeration for two weeks while stirring at 12 rpm. The proliferation rate of the adventitious roots was 6.5 times, and the total tropane alkaloid content was 1.32%.

The above experimental results show that when the adventitious roots are simply cultured under constant agitation conditions that do not cause any shearing force to be applied to the adventitious roots, the proliferation and growth of the adventitious roots is good at the beginning of the culture; As proliferation progresses and the density of adventitious roots in the culture tank increases, a large mass of adventitious roots occurs, and not only does the center of the mass become a callus, but the rate of proliferation and growth also decreases significantly. contains almost no tropane alkaloid, which is the target product, and therefore the overall content of tropane alkaloid was found to be significantly reduced.

As a result of further research based on the various experimental results mentioned above, the present inventors found that in the first stage of adventitious root culture, the proliferation of adventitious roots was given top priority, and the proliferation of adventitious roots was placed in a liquid culture containing adventitious roots. The adventitious roots are allowed to propagate well by imparting relatively low-speed movement suitable for dividing the adventitious root mass into one or more adventitious root groups by imparting a relatively high-speed movement that is sufficient to detach the adventitious root from the adventitious root primordium and hardly imparting a shearing action to the adventitious root; In the third stage, the large number of divided adventitious root groups is given top priority to the proliferation of adventitious roots, and the liquid culture containing the adventitious roots is given a relatively low-speed movement suitable for mass proliferation of adventitious roots, so that a large number of adventitious roots can be grown in a favorable manner. It has been found that the various problems mentioned above can be satisfactorily solved by adopting a three-step process of propagation.

The present invention has been completed based on the above-mentioned new findings, and the basic feature of the adventitious root propagation method is that a liquid culture containing adventitious roots in a pre-culture tank is heated at a relatively low rate suitable for the propagation of adventitious roots. an adventitious root propagation step in which the adventitious roots are propagated to form an adventitious root mass by imparting motion; an adventitious root division step of dividing the adventitious root mass into one or more adventitious root groups by imparting a relatively high-speed movement with almost no shearing action; and a large amount of liquid culture containing the divided adventitious root groups. A method for propagating adventitious roots, comprising: a step of mass culturing adventitious roots, in which the adventitious roots are transferred into a main culture tank and cultured while imparting relatively low-speed movement suitable for mass propagation of adventitious roots;
and an adventitious root propagation step in which the liquid culture containing adventitious roots in the preculture tank is given a relatively low-speed motion to allow the adventitious roots to propagate, thereby causing the adventitious roots to propagate and form an adventitious root mass, and the liquid that has undergone the propagation step. The adventitious root mass is divided into one or more by imparting a relatively high-speed movement to the culture that is sufficient to detach the adventitious root from the adventitious root primordium of the adventitious root mass, and which hardly imparts a shearing action to the adventitious root mass. an adventitious root dividing step of dividing into adventitious root groups; a recycling step of returning a part of the liquid culture containing the divided adventitious root groups to the pre-culture tank; and a recycling step of returning part of the liquid culture to the pre-culture tank. A method for propagating adventitious roots, which comprises a step of mass culturing adventitious roots, in which the remaining parts are transferred to a main culture tank for mass culture, and cultured while imparting relatively low-speed movement.

Plant types to which the present invention can be applied include Zboisia,
Examples include Korean ginseng and belladonna.

Adventitious roots of the above plants can be obtained directly from explants consisting of stems, leaves, roots, etc. of the plants in a liquid medium containing plant hormones, or can be obtained from callus obtained by culturing the explants. Adventitious roots can also be differentiated under appropriate conditions.

For example, Zboisia can be obtained in a medium prepared by adding hormones such as indoleacetic acid to the Linsmeyer-Skoog medium used for normal plant tissue culture.

In the adventitious root propagation step, Murashige-Skoog, Linsmeyer-Skoog, White's medium, etc. are used as the medium.

Also in the process of mass propagation of adventitious roots, the medium described above is used.

The movements imparted to the liquid culture containing adventitious roots in the adventitious root propagation step, adventitious root division step, and adventitious root mass culture step consist of relatively slow, high-speed, and low-speed movements, respectively.

The form and means of movement are not particularly limited as long as they can impart the required movement in each process, but examples of the former form of movement include horizontal or vertical reciprocation, shaking, rotation, etc. Alternatively, the combined motion of these can be mentioned, and means for imparting the motion include shaking means of the culture tank, various stirring means, such as paddle type, butterfly type, flat blade turbine type, perforated plate impeller type, Examples include an air lift type, a Kablan turbine type, a draft tube type, and an air lift loop type. Among these motion imparting means, means of imparting motion by blowing air are not very preferable in terms of the dispersion effect of adventitious roots, and particularly preferable ones include paddle type,
Alternatively, a butterfly type can be mentioned.

The butterfly type stirrer that can be used in the present invention includes a type in which a disc-shaped stirring blade is attached to the lower part of a stirring rod at an angle with respect to the axis of the stirring rod, as shown in Fig. 1. Particularly preferably, the disc-shaped stirring blade has a cross-sectional shape obtained by cutting a cylinder with a perfect circle in cross section at an angle of 45 degrees to 60 degrees with respect to its central axis,
Examples include those that are attached to the stirring rod at an angle of 45 degrees to 60 degrees with respect to the axis of the stirring rod.

The degree of motion imparted in each of the above steps varies depending on the type of adventitious root used and the motion imparting means, but -
For boat fishing, in the case of horizontal or vertical reciprocating motion or shaking, the movement in each step of the adventitious root propagation step, adventitious root division step, and adventitious root mass cultivation step is 50%, respectively.
~1100rp, 200~50Qrpm, and 50~1
The range of 100 rpm is preferable, and in the case of rotation, 30 to 7 Q rpm and 100 to 300 rpm, respectively.
, and a range of 30 to 70 rpm is preferred.

The means for recycling a part of the divided liquid culture containing a large number of adventitious root groups into the preculture tank includes
．． It is possible to use means such as applying a pressure of about 5'Kg/cTIl and pumping by operating a valve.

Next, an example of the flow of the adventitious root propagation method of the present invention will be explained using the accompanying drawings.

FIG. 1 is a schematic diagram showing the flow when a paddle-type stirrer is used as a means for imparting the required movement to a liquid culture containing adventitious roots in the propagation of adventitious roots of zubo and isia.

In Fig. 1, adventitious roots obtained by shaking culture in a flask are placed in a preculture tank,
? When cultured in Orpm for 2 weeks, adventitious roots multiply about 10 times. The stirring speed was set to 10 to divide the proliferating adventitious roots.
By stirring at 0 to 300 rpm for 5 to 10 hours, divided adventitious roots are obtained. A part of the split adventitious roots is recycled to the preculture tank for propagation, and the remaining adventitious roots are pumped together with the medium to the main culture tank and cultured for 2 weeks at an agitation speed of 30 to 7 Qrpm for mass propagation.

The width of the paddle type stirrer used in the preculture tank and the main culture tank is 0.47 mm per diameter of the culture tank, and the height is 0.3 mm per 1 height of the culture tank.

[For production]

As described above, the adventitious root propagation method of the present invention gives top priority to the proliferation of adventitious roots in the adventitious root propagation step, and provides the liquid culture containing adventitious roots with a relatively low-speed movement suitable for the propagation of adventitious roots. Then, in the adventitious root division step, the adventitious roots are separated from the adventitious root primordium of the adventitious root mass into the liquid culture containing the adventitious roots at the stage where the proliferation has progressed and an adventitious root mass has been formed. The adventitious root mass is properly divided into one or more adventitious root groups by imparting relatively high-speed motion to the extent that the adventitious roots have sufficient shearing action and hardly any shearing action, thereby increasing the adventitious root mass. In addition, in the adventitious root mass culture process, the multiplication of adventitious roots is given top priority in the liquid culture containing a large number of divided adventitious root groups. By imparting a relatively low-speed movement suitable for mass propagation of adventitious roots, the adventitious roots can be propagated in large quantities.

In addition, since the movement imparted to the liquid culture containing adventitious roots in the adventitious root division step is relatively high-speed, there is an unavoidable risk of some damage to the adventitious roots. In the propagation process and adventitious root mass culture process, the liquid culture containing adventitious roots is
Since the propagation of adventitious roots is given top priority and relatively slow movement is imparted, the mass propagation of adventitious roots is carried out extremely well as a whole.

〔Example〕

Example 1 When leaves of Zboisia were placed on an agar medium (Linsmeyer-Skoog medium containing 10-5 M of naphthalene acetate and 10-6 M of benzyladenine) and cultured at 25°C for 30 days, callus formation occurred simultaneously. The adventitious roots were cut out, transferred to an Etch-Etch liquid medium supplemented with 10-5 M of indolebutyric acid, and subcultured. 50n+g (dry weight) of the adventitious roots thus obtained were placed in 100m of Etch Niche liquid medium of 20m2. When transplanted into a large Erlenmeyer flask and cultured with horizontal reciprocating shaking for 2 weeks, 50 adventitious roots grew.
5 mg was obtained. When the adventitious roots were dried and total alkaloids such as spokolamin and atropine were measured, they were found to be 0.9.
It was 6%.

Next, the adventitious roots cultured in 200 100 mfl Erlenmeyer flasks were transferred to a 6,042 volume preculture tank equipped with a paddle-type stirring blade, and after culturing at 5 Qrpm for 2 weeks, the agitation speed was set to 20 Orpm and stirred for 6 hours to form a mass of adventitious roots. is divided into small pieces, and 1710 of these pieces are recycled together with the medium into a preculture tank for use in propagation.

The remaining split adventitious roots were pumped together with the medium into a 1M main culture tank, and cultured for 2 weeks while stirring with a butterfly-type stirring blade at 7 rpm, yielding 11.25 kg (as dry roots) of adventitious roots.

The total tropane alkaloids in the dry adventitious roots were measured and found to be 1.75%.

Example 2 In Example 1, instead of using the butterfly-type stirring blade in the main culture tank, the paddle was used for two weeks of culture, and 8.33 kg (as dry roots) of adventitious roots were obtained.

The total tropane alkaloids in the dry adventitious roots were measured and found to be 0.78%.

〔Effect of the invention〕

According to the present invention, superior adventitious roots can be industrially propagated in large quantities, making a significant contribution to the field of industrial production of useful substances using adventitious roots.

[Brief explanation of the drawing]

FIG. 1 is a diagram illustrating an example of the flow of the adventitious root propagation method of the present invention. FIG. 2 is a perspective view showing an example of a butterfly type stirrer that can be used in the present invention. Applicant Representative of Research Association for New Manufacturing Technology for Chemicals Utilizing Biofunctions Patent Attorney Yusuke Hiraki Patent Attorney Sada Ishii Figure 2 Stirrer

Claims (1)

[Scope of Claims] 1. An adventitious root propagation step in which a liquid culture containing adventitious roots in a preculture tank is given relatively low-speed motion suitable for adventitious root propagation to cause adventitious roots to propagate and form an adventitious root mass. , the adventitious roots are grown by imparting relatively high-speed motion to the liquid culture that has undergone the multiplication step that is sufficient to detach the adventitious roots from the adventitious root primordium of the adventitious root mass and that hardly imparts a shearing action to the adventitious roots. Adventitious root dividing step of dividing the mass into one or more adventitious root groups, and transferring the liquid culture containing the divided large number of adventitious root groups into a main culture tank for mass culture, which is suitable for mass propagation of adventitious roots. A method for propagating adventitious roots, comprising: a step of cultivating a large amount of adventitious roots while imparting relatively low-speed motion. 2. An adventitious root propagation step in which a liquid culture containing adventitious roots in a preculture tank is given a relatively low-speed movement suitable for the propagation of adventitious roots to allow the adventitious roots to propagate and form an adventitious root mass; One or more of the adventitious root masses are created by imparting relatively high-speed motion to the liquid culture that is sufficient to detach the adventitious roots from the adventitious root primordium of the adventitious root mass and that hardly imparts a shearing action to the adventitious roots. a recycling step of returning a part of the liquid culture containing the divided adventitious root groups into the preculture tank; A method for propagating adventitious roots, comprising a step of mass-cultivating adventitious roots, in which the remaining parts other than those returned are transferred to a main culture tank for mass-culture, and cultured while applying relatively low-speed movement. 3. The adventitious root propagation method according to claim 1 or 2, wherein the motion imparted in the adventitious root propagation step, the adventitious root division step, and the adventitious root mass culture step is reciprocating motion or shaking. 4. The adventitious root propagation method according to claim 1 or 2, wherein the movement imparted in the adventitious root propagation step, the adventitious root division step, and the adventitious root mass culturing step is horizontal rotation. 5. The motion imparted to the liquid culture in the pre-culture tank in the adventitious root propagation step and the adventitious root division step is horizontal rotation at a low rotation speed and a high rotation speed, respectively. 3. The method according to claim 1 or 2, wherein the motion imparted to the liquid culture in the adventitious root mass culturing process is a composite motion of horizontal rotation at a low rotational speed and vertical reciprocating motion. Adventitious root propagation method. 6. Claim 1 or Claim characterized in that in the adventitious root propagation step, the adventitious root division step, and the adventitious root mass cultivation step, the motion imparted is a motion that appropriately combines rotation in various directions, reciprocating motion, shaking, etc. Item 2. The adventitious root propagation method according to item 2. 7. The method for propagating adventitious roots according to any one of claims 1 to 4, wherein the adventitious roots are adventitious roots of Zboisia.