CS231241B1 - The way of directed dedifferentiation transformation of beech plants - Google Patents

Abstract

A method of directed dedifferentiation transformation of leguminous plants, characterized in that the plant organism is treated with derivatives of 2-oxo-3-benzothiazoline acetic acid of the general formula I I R /1/ where R is methoxycarbonylmethyl, ethoxycarbonylmethyl, 2-chloroethoxycarbonylmethyl, allycarbonylmethyl or hydrazinocarbonylmethyl, at a concentration of 10"* to 10"^ mol/1.

Description

(54) Method of directed dedifferentiation transformation of herbaceous plants

in.

A method of directed dedifferentiation transformation of a herbaceous plant, characterized in that the plant organism is treated with z-oxo-3-benzothiazolineacetic acid derivatives of the general formula I (1) wherein R is methoxycarbonylmethyl. ethoxycarbonylmethyl, 2-chloroethoxycarbonylmethyl, allycarbonylmethyl or hydrazinocarbonylmethyl, with a concentration of from 10 to 10 mol / l.

The invention relates to a method for directed dedifferentiation transformation of a bryophyte plant.

K induction! The most commonly used synthetic auxinoids such as beta-indolylacetic acid (IAA), alphanaphthylacetic acid (NAA), indolylbutyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), kinin derivatives , gibberellins, retardants and others / Kruše, PF, Petterson, Μ. K .: Tissue Culture, Methods and Applications, Academic Press New York and London 1973, p. 868; Novák F. J. / Edit./: Utilization of plant explanatory cultures in breeding, International Corps. symp. Czechoslovak Academy of Sciences, Prague 1977, p 650; Butenko R.G. (Edit./: Kletok rastenij culture, Kiev 1978, p. 384), which stimulate the meristemization of differentiated cells and are necessary to maintain unorganized growth of the tissue culture. This property can be used to increase biomass production by tissue culture (Venketeswaran, S., Gandhi, V .: Biomass, 2, 5-15, 1982).

In recent years, analogous effects of some other indolyl family synthetic auxinoids have also been shown. Some of these, such as benazoline (4-chloro-2-oxo-benzothiazoline-3-acetic acid) or henzotlazolileacetic acid, are equally actively involved in the inclusion and growth of calluses of many plants as 2,4-D, or even exceed them (Ingram DS, Butcher DZ Pflanzenphysiol., 66, 266 (1972).

The principle of the directed dedifferentiation transformation of the herbaceous plants according to the invention consists in impregnating the plant organism with derivatives of the general formula I

/ 1 /.

wherein R is methoxycarbonylmethyl, ethoxycarbonylmethyl, 2-chloroethoxycarbonylmethyl, allylcarbonylmethyl or hydrazinocarbonylmethyl at a concentration of 10 to 10 mol / l.

The compounds of the formula I according to the invention exert analogous morphogenetic effects with the above-mentioned phytohormonal agents, both in vivo and in vitro.

Evident transformation of plant tissues and cells occurs at a concentration of 10 µM. By de-differentiation processes, a yellow-colored callus is formed. At the stage of the primocalus, the tissue disintegrates into cells. Upon inoculation with fresh medium of an analogous composition to that of white medium during dedifferentiation, callus retains typical unorganized growth. The fresh biomass weight in the dedifferentiation process increases by 25-35%.

The degree of effectiveness of the subject compounds depends on the concentration used and the nature of the substituents bound to the core.

These biological properties, but also the economic aspect, the relatively low cost of benzothiazole synthesis, emphasize their importance as potential inducers of directed dedifferentiation transformation of the plant organism for biotechnological manipulation.

Example

In a first series of experiments under in vivo conditions to induce! dedifferentiations were used for the herbaceous plants / Pisum sativum L. var. Emerald/. After imbibition for 6 hours in distilled water. seeds germinated in a thorn in a thermostate for 72 hours. Then the germs were washed with cotton wool, washed with cotton wool, selected and for further tests, germs with a root length of 25-30 mm and 1 mm were used. The keyring sets (5 pcs) were planted in the horizontal position on a moistened filter paper in Petri dishes with an average size of 17 cm. The filter paper was saturated with the active ingredient in concentrations of 10 " ³ , 10" ⁴ , 10 "^, 10" ⁶ , 10 "\ 10" ⁸ ,

10 ^-7 mol.dm ^J. A control series of germs was incubated on filter paper which was saturated with distilled water. Induction of dedifferentiation was performed in a thermostate in a mandrel at 25 ° C - 1 ° C for 72 - 96 hours. The effect of dedifferentiation transformation was determined visually using a microscopic magnifying glass. The maximum transformation efficiency is due to 2-oxo-3-benzothiazoline acetic acid 3-ethyl ester at a concentration of 10 ^-3 mol.dm ^-3 . Indeed, derivatives were also most effective in concentra- ting IQ ^-3 mol.drn ” ³ .

Example 2

In a second series of experiments under in vitro conditions, calogenesis was induced on apical segments of the primary roots of the herbaceous plant / Viola sativa L. var. Solarka /.

The selected seeds (approximately the same size and color of the tegument) were sterilized with 5% chloramine solution for 1 hour and rinsed several times with sterile distilled water. Sterile seeds were germinated in Petri dishes on 0.8% agar medium for 48 hours in the dark at 25 ° C - 1 ° C. Sterile germs 25-30 mm and 1 mm long were used to induce calogenesis. Of these, apical segments of primary roots in the length of 10-15 mm were decapitated and hung into petri dishes in a horizontal position at 0.6% modified solid agar medium according to Murashige-Skooga. In the experimental variants, the medium contained the active substances in concentrations of 10 ^J - 10 mol.dm. In a control variant, the medium contained the aforementioned known phytohormonal agents.

After determining the fresh weight of the apical segments of the primary roots, calogenesis was induced at 15 ° C - 1 ° C in the dark for 14 days. Preparation of model material as well as cultivation were performed under aseptic conditions. The increase in fresh weight during calogenesis was determined gravimetrically. The maximum dedifferentiation efficiency was found to be 44 in 2-oxo-3-benzothiazoline acetic acid 3-ethyl ester at a concentration of 5.10 mol. dm (approx. 30% by weight. Moreover, derivatives were also most effective at concentra- tion -4-4 -3-9

5.10 mol.dm ^J. At a concentration of 10 to 10 mol / l, the increase in fresh weight was 5 to 8%.

Example 3

In a third series of experiments under in vitro conditions, calogenesis was induced on intact germs and amputated stem germs and the root of a bovine plant / Vicia faba L., var. Solarka /.

The conditions of seed sterilization were maintained as in the tests given in Example 2. Sterile seeds were sown in test tubes in variant A on a 0.6% modified solid agar medium according to Murashige-Skooga containing the active substances as in the experiments described in Example 2.

In variant B, sterile seeds were germinated in Petri dishes on 0.8% agar medium.

After germination from the seeds, the stems and roots were extirpated. The seed was replanted on medium of analogous composition as in variant A. Calogenesis was monitored for 7-14 days in the dark at 25 ° C in a thermostate. The maximal efficiency of calogenesis was found in 3-Ethyl ester of 2-oxo-3-benzothiazoline in the same way in variant A at 10 ³ mol.dm ^-3 and in variant B at 5.10 ^-4 mol.dm ^-3 . Moreover, derivatives in variants A and B. were most effective at analogous concentrations.

Compounds applied according to the invention:

1. 2-Oxo-3-benzothiazoline acetic acid 3-methyl ester

2. 2-Oxo-3-benzothiazoline acetic acid 3-ethyl ester

3. 2-Oxo-3-benzothiazoline acetic acid 3/2-chloroethyl ester

4. 2-Oxo-3-benzothiazoline acetic acid 3-allyl ester

5. 2-Oxo-3-benzothiobenzoic acid hydrazide

Claims (1)

OBJECT OF THE INVENTION A method of directed, de-differentiated transformation of bifid-like plants, characterized in that they are adapted to the plant organism with 2-oxo-benzothiazolineacetic acid derivatives of the formula I X = O (1), wherein R is methoxycarbonylmethyl, ethoxycarbonylmethyl, 2-chloroethoxycarbonylmethyl, allylcarbonyl methyl or hydrazinocarbonylmethyl, at a concentration of 10 ^µM .