KR950012090B1 - Bactericide for plant tissue culture - Google Patents

Abstract

One of the berberines (I) was added at 30-100 ppm to the plant tissue culture medium as antibacterial agents. (I) esp. extracted with hot-water from Coptus Japonica Makine was stable at autoclaving, and does not have phyto-toxicity. The spectrum of (I) is to Bacillus subtilis and B. licheniformis.

Description

Additives for sterilization in plant tissue culture

Attached drawings are pictures showing organ differentiation, growth and development.

The present invention relates to an additive added to the culture medium in plant tissue culture, and more particularly, to a sterilization medium additive capable of suppressing the contamination or infection of microorganisms without affecting the growth and development of plants in the culture medium. .

Currently, tissue culture is widely used as one of asexual propagation methods for mass reproduction and aseptic reproduction of horticultural crops.

The most basic and important fact in tissue culture is that all work (preparation and cultivation) should be carried out aseptically. The causes of contamination of the plant culture are insufficient sterilization of internal and external media of the plant, contamination and work from air. Contamination due to immaturity.

Contamination from double air and from immaturity of the work can be eliminated to some extent, but the sterilization of plant tissues and aseptic medium is one of the most important tasks.

Conventional methods for sterilization of plants have been immersed in various disinfectants for a certain time in order to prevent microbial contamination of the epidermis, but in this case, the effect of disinfectant type, concentration, and soaking time on the plant varies. Almost no microorganisms present in plants can be controlled.

This is because internal infection does not occur until after plant sections have been inoculated into the medium.

In the case of the medium, nutrients and additives such as oils, inorganic substances, vitamins, and growth-active substances necessary for growth for normal growth of plants can be added to the medium, and then sterilized by autoclaving. Many of the various additives are weak to heat, and if sterilized by high-pressure steam, they are changed to other substances or their activity is low, and thus they do not show any function at all when stored for a long time.

If it is not completely sterile, microorganisms invade the medium and kill the plants.

In addition, if the plant itself is infected, even if it is sterilized under sterile conditions, it is of no use.

Such internal infection is a non-negligible problem and cannot be removed by external sterilization.

Therefore, in principle, it is best to cultivate meristems without microorganisms to solve this problem.

However, these cultures are of no use when mass breeding or other sites are needed.

On the other hand, it is possible to add antibiotics to the medium, but in this case, phytotoxicity occurs, which often prevents the plants from growing and growing normally.

The treatment of antibiotics is mostly used by filtration and sterilization, and the substances used are tetracycline, 8-Hydroxy-quinoline, achromycin, streptomycin, and rifamycin. rifamycin) and gentamycin.

However, antibiotic treatment during inflight has proved to be complicated and impractical and is currently rarely used.

Internal contamination of plants is often in the form of rod bacteria, including Bacillus licheniformis or Bacillus substilis.

Since these spores can tolerate poor environments such as heat, drying, low temperature, ultraviolet rays, and sterilizing solution, there is almost no removal method.

Accordingly, the present invention has been made to solve the above-mentioned conventional problems, by adding berberine to a substance capable of inhibiting or removing microorganisms without affecting the growth and development of the plant in the medium. It aims to provide external pollution control.

Hereinafter, the present invention will be described.

The present invention is made of a sterile medium additive characterized in that it is possible to reduce the contamination or infection of microorganisms by adding berberine to the medium (solid or liquid medium) in the culture of plant tissues.

The berberine used in the present invention is included as a main ingredient in Coptus Japonica Making and is known as a substance having an antimicrobial effect, but there is no example of using this substance as an additive material in tissue culture.

In the case of the present invention, a significant effect can be obtained even when the crude extract of berberine is used.

Here, the extraction of berberine from the sulfur sulfur is well known in the art, the berberine used in the present invention can be extracted by other known methods for extracting effective plants from various plants.

For example, monosulphurum can be extracted with an organic solvent (chloroform, alcohol, ethanol, fatty acid ester, etc.) to remove fat-soluble components and dissolved by adding hot water to the residue to extract and concentrate the active ingredient.

The present invention is not characterized in the extraction process, it is to use the berberin active ingredient in any extraction method.

The amount of berberine used in the present invention is most preferable at 30-100 ppm.

The method of using berberine is, firstly, it can be used by autoclaving after treating berberine on the medium. In this case, the emphasis is on controlling internal contamination occurring in the plant. This method may be applied to other pollution control measures rather than internal pollution from plants.

In particular, the present invention can maintain the nutrients and physiologically active substances of the medium in an optimal state by using the medium without autoclaving, so that the medium having the activity for a considerable period of time can be stored.

Thus, the berberine used in the present invention can suppress the growth of microorganisms such as bacteria and fungi invaded into the plant, and can also inhibit the growth of microorganisms in the medium from the outside or from the medium itself.

In addition, it is possible to suppress the growth of microorganisms invading the air in the container in the medium.

The following will be described by examples.

Example 1

Before the agar was added in the manufacturing process of MS base medium, 20 ppm and 50 ppm of berberine were added, respectively, and the control group was used as a control.

The pH of the medium was adjusted to 5.8 and then dispensed into each 10ml 30ml test tube.

The treatments were divided into autoclaving and non-autoclaving. Each treatment was further subdivided into aluminum foil capping and no-capping. Processed.

And after placing the medium in a tissue culture room having a temperature of 25 ℃, humidity 70% and observed at intervals of 24 hours, the results of contamination appeared as shown in (Table 1) and (Table 2).

The results showed that even if the autoclave was not sterilized, the aluminum foil was not contaminated at all when 50 ppm of berberine was added to the medium.

TABLE 1

* Number of test tubes: 30 for each berberine concentration

TABLE 2

* Number of test tubes: 30 for each berberine concentration

Example 2

This experiment was carried out to determine the external infection rate during the operation by inoculating each treatment group by injecting sterile African Violet (Saintpauliaionantha) electic callus from the aseptic culture.

As a result, as shown in (Table 3), 50 ppm of berberine treated was not contaminated at all except one container.

Therefore, the 50ppm berberine treated group was found to be usable without autoclaving.

TABLE 3

* Number of test tubes: 30 for each berberine concentration

Example 3

This experiment was conducted to investigate the effect of berberine on internal infection occurring in plants. As shown in (Table 4), the treatment of 40ppm berberine showed a complete inhibitory effect on the most contaminated microorganisms in the plant.

TABLE 4

Example 4

In order to investigate the effects of berberine on the plants, the effect on the germination of morning glory seeds was performed as follows.

After homogeneous morning glory seeds were soaked in tap water for 20 hours, uniform seeds were again selected and 30 capsules were used per treatment.

Sowing soil was mixed with Vermiculite: Pearlite: sand in a 1: 1: 1 ratio.

Treatment was performed by dipping for 2 hours in untreated (distilled water) and two concentrations of berberine, respectively, and sowing was carried out.

The concentrations of berberine used in germination experiments were 50 ppm and 200 ppm, respectively.

As a result, as shown in (Table 5), (Table 6) and (Table 7), it was shown that even at the concentration of 200ppm had no effect on the germination and growth of morning glory seeds.

TABLE 5

TABLE 6

TABLE 7

Example 5

Three months after the inoculation of the incubation in the incubation in the control medium of 200ppm and 50ppm berberine and untreated control in MS medium was observed visually three months later.

As a result, as shown in the accompanying drawings, by visual observation for 3 months, no abnormalities were observed in the organ differentiation, growth and development of the seedlings in the berberine treatment and control, and they were normally rooted and shoots developed.

There was no difference in growth rate between control and treatment.

Claims (3)

A bactericidal additive for culturing plant tissues, characterized in that 30 to 100 ppm of berberin is added to a medium composed of nutrients and physiologically active substances to reduce plant contamination. According to claim 1, Sterilization medium additive for plant tissue culture, characterized in that the sterilization treatment by autoclaving after the addition of berberine. The method of claim 1, wherein berberine is a sterilizing medium additive for culturing plant tissues, characterized in that the berberine extracted from Coptis Japonica Makine.