DD271712A1 - METHOD FOR DETERMINING THE BIOLOGICAL ACTIVITY OF PHASEOLOTOXIN, DERIVATIVES OF PHASEOLOTOXINE OR PHASEOLOTOXINE-RELATED COMPOUNDS - Google Patents

Abstract

A method is described for determining the biological activity of phaseolotoxin, derivatives of the phaseolotoxin and phaseolotoxin-like compounds. According to the new method, the biological activity is determined by means of cell cultures of Lycopersicon peruvianum, Chenopodium album and / or Phaseolus coccineus cv. "Red Kidney" by the determination of selected physiological parameters, such as the growth, the content of free amino acids, the protein content, various enzymes of the metabolism, determined. The cell cultures can be cultured both on solidified media and submersed in liquid media at 20-30C. The determination of inhibitory sites is also possible. The samples to be tested are added immediately after vaccination or at the latest after 24 hours and after 1-5 days, the result can be determined.

Description

Field of application of the invention

A number of micro-bleachable as well as accessible by chemical synthesis substances have phytotoxic or phyto-effective properties. For the development of environmentally friendly active ingredients for agriculture and horticulture, it is necessary to possess test systems that can be used to quickly find effective substances. Biotests are best suited for such investigations because they allow immediate conclusions about the biological activity of the respective products. Plant cell cultures are particularly suitable for this, since their application allows the consideration of a variety of specific metabolic properties of plant cells. One such metabolically active substance is the paseolotoxin. It is highly phytotoxic, for example to the bean. It induces, inter alia, chlorosis and symptoms of so-called fatty spot disease. Resistance breeding makes it possible to obtain resistant plants against this phytotoxin. The bioassay according to the invention with plant cell cultures serves for the faster finding of effective components.

Characteristic of the known technical solutions

For the rapid identification of phaseolotoxin and phaseolotoxinderivaten some biosynthetic or enzyme tests have been used. For example, Staskawicz and Panapoulos found that the phaseolotoxin inhibits the growth of some microorganisms, such as E. coli (Phytopathol.69 (1979) 663) .Other method works with greenhouse grown plants (e.g., beans, chard ) on whose leaves the toxin is applied

defined time, the degree of chlorosis is determined (Plant Physiology 60 (1977) 723, Phytopathol.Z.96 (1979) 215). Due to the specific inhibition of the enzyme ornithincarbamyltransferesis, it is possible to detect this toxin very sensitively (Z. Alleg. Mikroblol., 23 (1983) 485) .These methods have various drawbacks Once, the bacterial system with E. coli only allows general conclusions about cytotoxic On the other hand, the tests with whole plants require cultivation under well-defined conditions in the greenhouse, in addition they require a longer time until the results are available.

Target animal invention

It is the object of the invention to develop cell culture systems, with the aid of which it is possible to determine the biological activity of phaseolotoxin, derivatives of phaseolotoxin or phaseolotoxin-like compounds quickly, quantifiable, reproducible and in an easily manageable manner without expensive pre-purification, and thus selection method or method Make screening programs more effective.

Explanation of the essence of the invention The invention has for its object to develop a bioassay system, the determination of the biological activity of Phaseolotoxin, derivatives of the Phaseolotoxins and phaseolotoxinfiähnlicher connections allows fast information By influencing the biological activity of plant cells gives and with its help besides the finding more effective Principles a quantification of the effects is possible. According to the invention, the object is achieved in that in plant cell cultures growth and / or selected Metabolic parameters and their influence on the examined by the microwave culture filtrates, solutions and / or

enriched or pure substances whose biological activity is to be determined, are determined and that by use of low concentrations of the substances to be examined, a selection of resident plant cells takes place.

The focus is on cell cultures of Lycopersicon peruvianum, Chenoposium album, a weed, and / or Phaseolus

coccineus cv. "Red Kidney", one of the host plants of the phaeolotoxin-producing strain of Pioudomonas spp., May be used both as an emmeric booster on solidified media and as a submerged cow in liquid media. or aggregates of

Protoplasts possible. The cultivation takes place on or in known media, the C-waste, mineral salts, Trace elements, small amounts of other organic substances and / or addition of phyto-effective substances

such as Medash according to Murashige and Skoog, Linsmeyer and Skoog, Gamborg or media modified according to the cell line used or the material to be examined. The use of different cell cultures makes it possible to draw conclusions about specific effects of the substances or mixtures of substances to be investigated.

The samples to be tested may contain both phaseolotoxin, derivatives of phaseolotoxin produced by partial synthetic Variations, feeding experiments and / or re-isolations from microwaves or other culture solutions as well Culture filtrates or partially purified or enriched culture filtrates. The partial cleaning or Enrichment can be carried out in a manner known per se with, for example, activated carbon, ion exchangers or extraction steps

respectively. These samples are added immediately or up to 24 hours after vaccination. Sterilization can be done by sterile filtration or autoclaving, depending on the nature of the samples.

The determination of the biological activity can take place in various ways. Once the culture can be on solid media

are plated and as in the search nt ch antibiotics, a Hemmhoftest is possible. On the other hand, an analysis of the physiological state of the cells is possible, wherein one or more parameters can be characterized, such as the growth, the content of soluble amino acids, the protein content, the content of nucleic acids, the

Redox status, the reducing or oxidizing potential, the content of inorganic and / or organic phosphates

or activities of typical enzymes of the metabolism. These measurements are possible with intact, submerged shake cultures or protoplasts. For the characterization of the influence one determines generally one or more characteristic parameters for the culture and the physiological state, whereby usually several parameters should be determined side by side. The methods and methods to be used for the determination correspond to the generally known procedures adapted to the particular cell line and samples to be examined.

It has proven to be advantageous in some cases if the media and / or samples used are arginine-poor or -free. The invention is characterized in that the statements about the degree of influence Jer used cell culture after

1-5 days, generally after 24-72 hours can be taken. Surprisingly, all used

Cell cultures a reaction that is reproducible and quantifiable at any time. To test the Zeükulturen are used with a preculture age of 4-12 days, preferably 5-8 days, and the Cultivation at 20-30 ⁰ C, preferably 25-29 ⁰ C, carried out, wherein the suspension cultures and protoplasts in the liquid Media should be shaken for better oxygen and nutrient supply.

-3- 271712 Execution Bibles ·

The following examples are intended to illustrate the invention. The culture media used for the cell cultures are in accordance with the usual regulations and are among others in the book: EFGeorge, DJMPuttock, HJ George "Plant Culture Media", Volume 1: "Formulations and Uses", Exegetics Ltd., England, 1987, too Find. Provisions on biochemical methods can be found in the relevant instructions.

example 1

A culture of Lycopersicon peruvianum was transferred 1:10 ratio to arginine-free MS medium and Phaseolotoxin added to the culture medium in concentrations of 2-8μΜ. There is a growth inhibition between 10 and 15% observed after '* * 4 days.

Claims (12)

1. A method for determining the biological activity of Phaseolotoxin, derivatives of Phaseolotoxins or phaseolotoxinähnlichen compounds, characterized in that in plant cell cultures, the growth and / or selected metabolic parameters and their influence by the examined microwave culture filtrates, solutions and / or enriched or pure Substances whose biological activity is to be determined are determined and that by using low concentrations of the substances to be investigated, a selection of resistant plant cells takes place. 2. The method according to claim 1, characterized in that cell cultures according to Lycopersicon peruvianum, Chenopodium album and / or Phaseolus coccine'us cv. "Red Kidney" can be used. 3. Process according to claims 1 and 2, characterized in that the cell cultures are cultured in media with C sources, N sources, mineral salts, trace elements, small amounts of other organic substances and with additions of phyto-effective substances, the cultivation both emers as callus culture, submersed ate suspension culture as well as protoplast culture is possible. 4. The method according to claim 3, characterized in that the media or test samples used are argininarm or free. 5. Process according to claims 1 to 4, characterized in that the influencing of the cell cultures by determining the growth, the content of proteins, nucleic acids, amino acids, reducing, oxidizing substances or other metabolites, by the determination of regulatory important or other enzyme activities and / or carried out by a Hemmhoftest. 6. The method according to claim 5, characterized in that this influence on the cell cultures is determined after 1 to 5 days. 7. The method according to claim 6, characterized in that this determination is carried out 24 to 72 hours after addition of the sample to be examined. 8. V experienced according to claims 1 to 7, characterized in that the addition of the samples to be tested is 0-24 hours after the inoculation and that the samples are sterile filtered or autoclaved prior to addition. 9. The method according to claims 1 to 7, characterized in that the cultivation of the cell cultures at temperatures of 20-3O ⁰ C and in the suspension or protoplast cultures with shaking. 10. The method according to claim 9, characterized in that the cultivation is carried out at 25-29 ⁰ C. 11. The method according to claims 1 to 10, characterized in that are used as precultures for testing 4-12 days old customs cultures. 12. The method according to claim 11, characterized in that 5 to 8 days old cell cultures are used as precultures for testing.