CS231242B1 - Method of Guided Dedlferencial Transformation Bean Plant - Google Patents
Method of Guided Dedlferencial Transformation Bean Plant Download PDFInfo
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Abstract
Vynález sa týká spósobu usmernenej dediferenciačnej transformácie bóbovitých rastlín. Podstata spósobu podlá vynálezu spočívá v tom, že na rastlinný organizmus sa pósobí derlvátmi kyseliny 2-oxó-3-ben- zotiazolín octovej všeobecného vzorca I (I) kde R je kyslík R1 je nietylester kyseliny octovej, etylester kyseliny octovej, 2-chlóretyl- ester kyseliny octovej, alylester kyseliny octovej, hydrazid kyseliny octovej o koncentrácii 10-4 až 10"3 mol.dm"3. Látky všeobecného vzorca I aplikované podlá vynalezu spósobujú analogické, morfo- génne procesy a morfologické efekty ako fytohormonálne látky, a to v podmienkach in vivo aj in vitro. Čerstvá hmotnost biomasy v procese dediferenciácie sa zvyšuje o 25 až 35 %.The invention relates to a method of directed dedifferentiation transformation of leguminous plants. The essence of the method according to the invention consists in that the plant organism is treated with derivatives of 2-oxo-3-benzothiazoline acetic acid of the general formula I (I) where R is oxygen R1 is methyl acetate, ethyl acetate, 2-chloroethyl acetate, allyl acetate, acetic acid hydrazide at a concentration of 10-4 to 10"3 mol.dm"3. The substances of the general formula I applied according to the invention cause analogous, morphogenic processes and morphological effects as phytohormonal substances, both in vivo and in vitro conditions. The fresh weight of biomass in the dedifferentiation process increases by 25 to 35%.
Description
231242 2
Vynález sa týká spósobu usmernenej dediferenciačnej transformáeie bĎbovitých rastlín. K indukcii dediferenciačnej transformáeie buniek a pletiv rastlín sú najčastéjšie použí-vané syntetické auxinoidy, ako je kyselina beta-indolyloctová (IAAJ, kyselina alfa-naftyl-octová (NAA), kyselina indolylmaslová (IBA1, kyselina 2,4-dichlórfenoxyoctová (2,4-D);, deri-váty kinínu, gibereliny, retardanry a iné (Kruše, P. F., Petterson, Μ. K.: Tissue culture,Methoda and Aplications, Academie Press New York and London 1973, p. 868; Novák, F. J. /Edit./:Využití kultur rostlinných explantátu ve šlechtění, Sbor. Mezinár. Symp. ČSAV, Praha 1977,p. 650; Butenko, R. G. /Edit./: Kultura kletok rastenij, Kyjev 1978, p. 384), ktoré stimulujúmeristenizáciu diferencovaných buniek a sú nevyhnutné pre udržanie·neorganizovaného rastupletivovej kultúry. Táto ich vlastnost móže byť využitá pri zvyšovaní produkcie biomasypomocou pletivových kultúr (Venketeswaran, S., Gandhi, V.: Biomass, 2, 5-15, 1982). V posledných rokoch však boli dokázané analogické účinky aj niektorých iných syntetickýchauxinoidov indolylového radu. Niektoré z nich, například benazolin (4-chlčr-2-oxo-benzotiazo-lín-3-octová kyselina) alebo benzotiazoliloctová kyselina sú rovnako aktivně v iniciáciia raste kalusov mnohých rastlín ako 2,4-D, alebo ju dokonca prevyšujú (Ingram, D. S., Butcher,D. N.: Z. Pflanzenphysiol., 66.206, 1972).
Podstata spósobu usmernenej dediferenciačnej transformáeie bĎbovitých rastlín spočíváv tom, že na rastlinný organizmus sa pĎsobi derivátmi 2-R-3-R^-substituovaných benzotiazóli-ových solí všeobecného vzorca
kde R je vodík R1 je alyl, metoxykarbonylmetyl) ethoxykarbonylmetyl, propoxykarbonylmetyl, alyl- oxykarbonylmetyl, pentoxykarbonylmetyl, izopropoxykarbonylmetyl, butyl, propargyl,propyl X- je bróm, chlór, jód, -4 -3-3 o koncentrácii 10 až 5.10 mol.dm
Preukazná transformácia rastlinných pletiv a buniek nastáva pri koncentráciách 10 až-3 —3 * 5.10 mol.dm . Dediferenciačnými procesmi dochádza k vytvoreniu kalusu krémovo-žltej farby. V štádiu primokalusu pletivo sa rozpadává na buňky. Po preočkovaní na čerstvé médium analo-gického zloženia, ako bolo médium počas dediferenciácie, kalus zachovává typický neorganizo-vaný rast. Čerstvá hmotnost biomasy v procese dediferenciácie sa zvyšuje o 28-30 %. Pri -7 -9 -3 koncentrácii 10 až 10 mol.dm je zvýšenie 7 až 10 % hmot.
Zlúčeniny aplikované podlá vynálezu spĎsobujú analogické morfogenné procesy a morfologic-ké efekty ako horeuvedené fytohormóny, a·to v podmienkach in vivo aj in vitro.
Stupeň účinnosti předmětných látok závisí od použitéj koncentrácie, od charakteru sub-stituentov viazaných na jádro benzotiazolu a od aniónu.
Uvedené biologické vlastnosti, ale aj ekonomický aspekt, relativné nízké náklady nasyntézu benzotiazolov zvýrazňujú ich význam ako potencionálnych induktorov usmernenej dedi-ferenciačnej transformáeie rastlinného organizmu na účelom biotechnologických manipulácii. Přikladl V prvej sérii pokusov v podmienkach in vivo k indukcii dediferenciácie boli použitéklíčence bóbovitej rastliny (Pisum sativum L., var. Smaragd). Po imbibícii (6 h) v destilova- 3 231242 nej vodě semená klíčili v tme v termostate 72 hodin. Potom boli klíčence po omytí od perlituosušené papierovou vatou, selektované a na áalšie testy boli použité klíčence s koreňomdlhým 25-30 mm - 1 mm. Súbory klíčencov (5 ks) boli vysadené v horizontálněj polohe na navlh-čený filtračný papier do Petriho misiek priemernej velkosti 17 cm. Filtračný papier bolnasýtený účinnou látkou v koncentráciách 5.10 3, 10 3, 10 10 10 10 , 10-3, 10 ® mol. •dm 3. Kontrolná séria klíčencov bola inkubovaná na filtračnom papieri, ktorý bol nasýtenýdestilovanou vodou. Indukcia dediferenciácie sa uskutočnila v termostate v tme pri 25 °C - 1 °Cpočas 72 až 96 hodin. Efekt dediferenciačnej transformácie bol stanovený vizuálně pomocou mikroskopickej lupy. Maximálnu transformačnú účinnost spósobuje 3-Etoxykarbonylmetylbenzoti-- -3-3 zoliumbromid pri koncentrálcii 5.10 mol.dm . Ostatně deriváty boli najúčinnejšie taktiež -3 -3 pri koncentrácii 5.10 mol.dm Přiklad 2 V druhej sérii pokusov v podmienkach in vitro kalogenáza bola indukovaná na apikálnychsegmentoch primárných koreňov bóbovitej rastliny (Vicia·sativa L. var. Solařka) . Selektované··semená (přibližné rovnakej velkosti a farmy osemenia) boli sterilizované roztokom 5% chlor-aminu 1 hodinu a niekolkokrát opláchnuté sterilnou destilovanou vodou. Sterilně semená klíčiliv Petriho miskách na 0,8% agarovom médiu 48 hodin v tme pri 25° - 1 °C. K indukcii kalogenézyboli použité sterilně klíčence 25-30 - 1 mm dlhé. Z nich boli dekapitované apikálne segmentyprimárných koreňov v dlžke 10-15 mm, ktoré boli vysadené do Petriho misiek v horizontálněj polohe na 0,6 % modifikované pevné agarovo médium podlá Murashige-Skooga. V pokusných varian--3 -9 -3 toch médium obsahovalo účinné látky v koncentráciách 5.10 -10 mol.dm . V kontrolnej variante médium obsahovalo vyššie uvedené známe fytohormonálne látky.
Po stanovení čerstvej hmotnosti apikálnych segmentov primárných koreňov kalogenézabola indukovaná pri teplote 25 °C v tme počas 14 dní. Příprava modelového materiálu, akoaj kultivácia, boli robené v aseptických podmienkach. Nárast čestvej hmotnosti počas kalogenézybol stanovený gravimetricky. Maximálna dediferenciačná účinnost bola zistená u 3-Etoxykarbonyl- metylbenzotiazóliumbromidu pri koncentrácii 10 3 mol.dm 3. Ostatně deriváty boli najúčinnej--3 -3 šie taktiež pri koncentrácii 10 mol.dm Příklad 3 V třetej sérii pokusov v podmienkach in vitro kalogenéza bola indukovaná na intaktnýchklíčencoch a klíčencoch s amputovanou stonkou a koreňom bóbovitej rastlinyi(Viacia sativaL., var. Solarka). Podmienky sterilizácie semien boli zachované ako v testoch uvedených v příklade 2. Sterilně semená boli vysadené do skúmaviek v A. variante na 0,6% modifikovanépevné agarovo médium podlá Murashige-Skooga s obsahom účinných látok ako v pokusoch uvede-ných v příklade 2. V B. variante sterilně semená klíčili v Petriho miskách na 0,8% agarovom médiu. Povyklíčení zo semien boli extirpované stonky a kořene. Semeno bolo znova vysadené na médiumanalogického zloženia ako v A. variante. Kalogenéza bola sledovaná počas 7-14 dní v tme pri teplote 25 °C-^ 1 °C v termostate. Maximálna účinnost kalogenézy bola zistená u 3-Etoxy--3 -3 karbonylmetylbenzotiazoliumbromidu v A. variante v koncentrácii 5.10 mol.dm a v B. vari-—3 -3 ante v koncentrácii 10 mol.dm . Ostatně deriváty v A. a B. variante testov boli najúčin- nejšie pri analogických koncentráciách.
Zlúčeniny aplikované podlá vynálezu: • 1. 3-Alylbenzotiazóliumbromid 2. 3-Metoxykarbonylmetylbenzotiazóliumbromid 3. 3-Etoxykarbonylmetylbenzotiazóliumbromid 4. 3-Propoxykarbonylmetylbenzotiazóliumbromid 5. 3-Alyloxykarbonylmetylbenzotiazóliumbromid
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The present invention relates to a method for the directed dedifferentiation transformation of marbled plants. For the induction of dedifferentiation transformation of cells and plant tissues, synthetic auxinoids such as beta-indolylacetic acid (IAAJ, alpha-naphthyl-acetic acid (NAA), indolylbutyric acid (IBA1, 2,4-dichlorophenoxyacetic acid (2,4 Kinin derivatives, gibberellins, retardants and others (Kruse, PF, Petterson, K. K .: Tissue culture, Method and Aplications, Academic Press New York and London 1973, p. 868; Novak, FJ / Edit./:Use of Plant Explant Cultures in Breeding, Choir International Symp., CSAV, Prague 1977, p. 650; Butenko, RG / Edit./: Kultura kletok rastenij, Kyiv 1978, p. 384), which stimulate the differentiation of differentiated cells and are indispensable for the maintenance of unorganized growth media culture, which can be used to increase the production of biomass by tissue cultures (Venketeswaran, S., Gandhi, V .: Biomass, 2, 5-15, 1982). analogical key also some other synthetic auxinoids of the indolyl group. Some of them, for example, benazoline (4-chloro-2-oxo-benzothiazolino-3-acetic acid) or benzothiazolileacetic acid, are equally active in initiating the growth of calluses of many plants such as 2,4-D, or even exceed them (Ingram, DS, Butcher, DN: Z. Pflanzenphysiol., 66, 206, 1972).
The principle of the directed dedifferentiation transformation of the fungal plants is that the plant organism is treated with derivatives of the 2-R-3-R 6 -substituted benzothiazolium salts of the formula
wherein R is hydrogen R 1 is allyl, methoxycarbonylmethyl) ethoxycarbonylmethyl, propoxycarbonylmethyl, allyloxycarbonylmethyl, pentoxycarbonylmethyl, isopropoxycarbonylmethyl, butyl, propargyl, propyl X- is bromo, chloro, iodo, -4-3-3 of 10-5.10 mol.dm
The evident transformation of plant tissues and cells occurs at concentrations of 10 to -3-3 * 5.10 mol.dm. Differentiation processes result in the formation of a callus of creamy-yellow color. In the primocalous stage, the tissue is disintegrating into cells. After inoculation on fresh medium analogous to the medium during dedifferentiation, the callus retains the typical unorganized growth. Fresh weight of biomass in the process of dedifferentiation increases by 28-30%. At -7-9 -3 concentrations of 10 to 10 mol.dm, the increase is 7 to 10% by weight.
The compounds of the present invention produce analogous morphogenic processes and morphological effects such as the above phytohormones, both in vivo and in vitro.
The degree of efficacy of the subject matter depends on the concentration used, the nature of the substituents bound to the benzothiazole core and the anion.
The stated biological properties, as well as the economic aspect, the relative low cost of benzothiazole synthesis, emphasize their importance as potential inducers of the directed dedifferentiation transformation of the plant organism for biotechnological manipulations. Example 1 In a first series of experiments under in vivo conditions to induce dedifferentiation, germs of a buoy plant (Pisum sativum L., var. Emerald) were used. After imbibition (6 h) in distilled 231242 water, the seeds were germinated in the dark in a thermostate for 72 hours. Thereafter, after washing from perlite-dried paper wool, the germs were selected and other germs with a length of 25-30 mm-1 mm were used for further testing. The germination sets (5 pcs) were planted in a horizontal position on a moistened filter paper in a diameter of 17 cm Petri dishes. Filter paper saturated with the active ingredient in concentrations of 5.10 3, 10 3, 10 10 10 10, 10 -3, 10 ® mol. • dm 3. A control series of germs was incubated on filter paper saturated with distilled water. Induction of dedifferentiation was performed in a dark thermostate at 25 ° C - 1 ° C for 72-96 hours. The effect of dedifferentiation transformation was determined visually by microscopic magnification. The maximum transformation efficiency is caused by 3-ethoxycarbonylmethylbenzothiazol-3-ol bromide at a concentration of 5.10 mol.dm. Moreover, the derivatives were also most effective at -3.10 at a concentration of 5.10 mol.dm. Example 2 In a second series of experiments under in vitro conditions, calogenase was induced on the apical segments of the primary roots of the buoy plant (Vicia sativa L. var. Solařka). The selected seeds (approximately the same size and seed size) were sterilized with 5% chloro amine for 1 hour and rinsed several times with sterile distilled water. Sterile seeds of germinant petri dishes on 0.8% agar medium for 48 hours in the dark at 25 ° -1 ° C. Sterile germs 25-30 - 1 mm long were used to induce calogenyzyne. Of these, apical segmented primary roots were decapitated in 10-15 mm length, planted in Petri dishes at a horizontal position of 0.6% modified agar medium according to Murashige-Skooga. In experimental variations 3 -9-3, the medium contained active substances in concentrations of 5.10 -10 mol.dm. In the control variant, the medium contained the above known phytohormone agents.
After determination of the fresh weight of the apical segments of the primary roots of the calogens, it was induced at 25 ° C in the dark for 14 days. The preparation of the model material, such as cultivation, was performed under aseptic conditions. An increase in the weight of the body during the calcogenyzybol determined gravimetrically. The maximum dedifferentiation efficiency was found for 3-ethoxycarbonylmethylbenzothiazolium bromide at a concentration of 10 3 mol.dm 3. Moreover, the derivatives were the most effective - 3 -3, also at a concentration of 10 mol.dm Example 3 In a third series of experiments under in vitro conditions, on intact germs and germs with an amputated stem and root of a buoy plant (Viacia sativaL., var. Solarka). Seed sterilization conditions were maintained as in the tests given in Example 2. Sterile seeds were seeded into tubes in A. variant for 0.6% modified solid agar medium according to Murashige-Skooga containing the active ingredients as in Examples 2. V B. variant sterile seeds germinated in petri dishes on 0.8% agar medium. Seed germination was extirpated with stems and roots. The seed was replanted on a medium analogous composition as in the A variant. Calogenesis was monitored for 7-14 days in the dark at 25 ° C → 1 ° C in the thermostate. The maximum efficacy of the calcogenesis was found for 3-Ethoxy-3-3 carbonylmethylbenzothiazolium bromide in the A variant at a concentration of 5.10 mol.dm and in B. vari-3 -3 ante at a concentration of 10 mol.dm. Moreover, the derivatives in A. and B. test variants were most effective at analogous concentrations.
Compounds of the Invention: 1. 3-Allylbenzothiazolium bromide 2. 3-Methoxycarbonylmethylbenzothiazolium bromide 3. 3-Ethoxycarbonylmethylbenzothiazolium bromide 5. 3-Propoxycarbonylmethylbenzothiazolium bromide 5. 3-Allyloxycarbonylmethylbenzothiazolium bromide
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| Application Number | Priority Date | Filing Date | Title |
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| CS8210069A CS231242B1 (en) | 1982-12-31 | 1982-12-31 | Method of Guided Dedlferencial Transformation Bean Plant |
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| Application Number | Priority Date | Filing Date | Title |
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| CS8210069A CS231242B1 (en) | 1982-12-31 | 1982-12-31 | Method of Guided Dedlferencial Transformation Bean Plant |
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| CS1006982A1 CS1006982A1 (en) | 1984-03-20 |
| CS231242B1 true CS231242B1 (en) | 1984-10-15 |
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