MD31Y -   Micropropagation process of Echinacea purpurea L. Moench in vitro - Google Patents

  Micropropagation process of Echinacea purpurea L. Moench in vitro Download PDF

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MD31Y
MD31Y MDS20090037A MDS20090037A MD31Y MD 31 Y MD31 Y MD 31Y MD S20090037 A MDS20090037 A MD S20090037A MD S20090037 A MDS20090037 A MD S20090037A MD 31 Y MD31 Y MD 31Y
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Moldova
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concentration
moench
vitro
echinacea purpurea
micropropagation
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MDS20090037A
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Romanian (ro)
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Iulia SIROMEATNICOV
Anatol Jacota
Stepan Svet
Pavel Chintea
Eugenia Cotenco
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Institutul De Genetica Si Fiziologie A Plantelor Al Academiei De Stiinte A Moldovei
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Abstract

The invention refers to biotechnology, particularly to a process for micropropagation in vitro of Echinacea purpurea L. Moench plants. The process, according to the invention, includes the sterile germination of seeds, obtaining of explants, segmentation and cultivation thereof on the Murashige-Skoog nutrient medium, containing additionally steroid glycosides obtained from Solanum melongena L. egg-plant roots by extraction with boiling water, treatment of the obtained extract with butanol-1 and concentration, in a concentration of (3,0à3,5)À10-3%, at the same time cultivation is carried out at a temperature of 22à26?C, air relative humidity of 70%, with a photoperiod of 16 and 18 hours day/night and illumination intensity of 3000 lx. The result of the invention consists in increasing the somatic embryogenesis and regeneration of plants. Claims: 1

Description

Invenţia se referă la biotehnologie, în particular la un procedeu de micropropagare a plantelor de Echinacea purpurea L. Moench in vitro. The invention relates to biotechnology, in particular to a process for micropropagation of Echinacea purpurea L. Moench plants in vitro.

Echinacea purpurea L. Moench este o plantă decorativă şi medicinală preţioasă, multianuală. Acumulează şi sintetizează substanţele biologic active, este mult apreciată pentru calităţile sale terapeutice, posedă proprietăţi imunostimulente şi antivirale. În componenţa rădăcinilor se conţine substanţa anginoli. Preparatul din flori posedă proprietăţi hemostatice şi de vindecare a rănilor. Toate părţile componente ale plantei conţin glicozida steroidică echinacozidă şi ulei volatil eteric până la 1%. Echinacea purpurea L. Moench is a valuable, perennial ornamental and medicinal plant. It accumulates and synthesizes biologically active substances, is highly valued for its therapeutic qualities, possesses immunostimulating and antiviral properties. The roots contain the substance anginol. The flower preparation has hemostatic and wound-healing properties. All parts of the plant contain the steroid glycoside echinacoside and volatile essential oil up to 1%.

Este cunoscut procedeul de micropropagare a plantelor de Rhodiola rosea L., ce constă în obţinerea şi activarea meristemoizilor de la care pornesc lăstarii. Pentru activarea şi creşterea intensivă a plantulelor, explantele primare prelevate din plantule obţinute prin germinarea seminţelor au fost cultivate pe mediul nutritiv de bază ce conţine săruri minerale după Murashige-Skoog (MS) (Murashige T., Skoog F. Revised medium for rapid growth and bio-assays with tabaco tissue cultures. Physiology Plantarum, 1962, v. 15, N. 95, p. 473, suplimentat cu cărbune activat în cantitate de 1200 mg/l, valoarea pH-ului fiind ajustată la 6,5 până la autoclavare [1]. The micropropagation process of Rhodiola rosea L. plants is known, which consists in obtaining and activating the meristemoids from which the shoots start. For the activation and intensive growth of the seedlings, the primary explants taken from the seedlings obtained by seed germination were cultivated on the basic nutrient medium containing mineral salts according to Murashige-Skoog (MS) (Murashige T., Skoog F. Revised medium for rapid growth and bio-assays with tabaco tissue cultures. Physiology Plantarum, 1962, v. 15, N. 95, p. 473, supplemented with activated charcoal in a quantity of 1200 mg/l, the pH value being adjusted to 6.5 until autoclaving [1].

Dezavantajele acestui procedeu sunt: coeficientul de micropropagare scăzut, faptul că nu toate explantele asigură iniţierea creşterii meristemoizilor de la baza cărora pornesc lăstarii. The disadvantages of this process are: the low micropropagation coefficient, the fact that not all explants ensure the initiation of meristem growth from which shoots start.

Problema pe care o rezolvă invenţia constă în sporirea coeficientului de multiplicare a plantelor de Echinacea purpurea L. Moench pe calea modificării conţinutului mediului nutritiv utilizat pentru micropropagare. The problem solved by the invention consists in increasing the multiplication coefficient of Echinacea purpurea L. Moench plants by modifying the content of the nutrient medium used for micropropagation.

Esenţa invenţiei constă în aceea că procedeul include germinarea aseptică a seminţelor, obţinerea explantelor, segmentarea şi cultivarea lor pe mediul nutritiv Murashige-Skoog, ce conţine suplimentar glicozide steroidice obţinute din rădăcini de pătlăgele vinete Solanum melongena L. prin extracţie cu apă la fierbere, prelucrarea extractului obţinut cu butanol-1 şi concentrare, în concentraţie de (3,0…3,5)·10-3%, totodată cultivarea se efectuează la o temperatură de 22…26°C, umiditatea relativă a aerului de 70%, cu o fotoperioadă de 16 şi 8 ore noapte/zi şi intensitatea iluminării de 3000 lx. The essence of the invention is that the process includes aseptic germination of seeds, obtaining explants, segmenting and cultivating them on Murashige-Skoog nutrient medium, which additionally contains steroidal glycosides obtained from eggplant roots Solanum melongena L. by extraction with boiling water, processing the obtained extract with butanol-1 and concentration, in a concentration of (3.0…3.5)·10-3%, at the same time the cultivation is carried out at a temperature of 22…26°C, relative air humidity of 70%, with a photoperiod of 16 and 8 hours night/day and illumination intensity of 3000 lx.

Preparatul Somelangozidă a fost obţinut din rădăcini de pătlăgele vinete prin extracţie cu apă la fierbere, prelucrarea extractului obţinut cu butanol-1 şi conşentrare (SU 1473317 A 1988.12.15). The preparation Somelangoside was obtained from eggplant roots by extraction with boiling water, processing the obtained extract with butanol-1 and concentration (SU 1473317 A 1988.12.15).

Rezultatul invenţiei constă în sporirea embriogenezei somatice şi regenerării plantelor. The result of the invention consists in enhancing somatic embryogenesis and plant regeneration.

Exemplu de realizare a invenţiei Example of embodiment of the invention

Plantulele de Echinacea purpurea L. Moench au fost obţinute prin germinarea aseptică a seminţelor. Pentru a asigura sterilitatea, seminţele s-au tratat cu agent de înălbire pe bază de clor de 15%, timp de 20 min, spălându-se de 4...5 ori cu apă distilată sterilă. În continuare s-au inoculat pe mediul nutritiv de bază MS. Plantulele obţinute ajungând la mărimea de 7...10 cm, s-au segmentat şi cultivat în condiţii aseptice la hota cu flux de aer laminar steril. Echinacea purpurea L. Moench seedlings were obtained by aseptic seed germination. To ensure sterility, the seeds were treated with 15% chlorine bleach for 20 min, washed 4...5 times with sterile distilled water. They were then inoculated onto MS basic nutrient medium. The seedlings obtained, reaching a size of 7...10 cm, were segmented and cultivated under aseptic conditions in a sterile laminar air flow hood.

În calitate de material iniţial pentru iniţierea micropropagării de plantule au servit segmente de hipocotil şi frunze prelevate din pantulele germinate aseptic din seminţe. În vasele de sticlă de 50...100 ml s-au repartizat câte 15...20 ml de mediu nutritiv. S-au sterilizat prin autoclavare, în faza de vapori 10 min aerisire şi 15 min sterilizare în condiţiile de presiune (p=1 atm) şi temperatură (t=121°C, pH-ul fiind ajustat la 5,8...5,9). As initial material for the initiation of micropropagation of seedlings, hypocotyl segments and leaves taken from aseptically germinated seed pods served. 15...20 ml of nutrient medium were distributed in 50...100 ml glass vessels. They were sterilized by autoclaving, in the vapor phase 10 min of aeration and 15 min of sterilization under pressure (p=1 atm) and temperature (t=121°C, pH adjusted to 5.8...5.9).

Toate operaţiile au fost efectuate în condiţii strict aseptice la hota cu flux de aer laminar steril. În camera de creştere s-a menţinut un regim controlat de creştere constant, caracterizat prin temperatura de 24±2°C, umiditatea relativă a aerului de 70%, durata perioadei de iluminare 16 ore/zi şi 8 ore/noapte, intensitatea luminii 3000 lx. All operations were performed under strictly aseptic conditions in a sterile laminar flow hood. A constant controlled growth regime was maintained in the growth chamber, characterized by a temperature of 24±2°C, relative air humidity of 70%, duration of the lighting period of 16 hours/day and 8 hours/night, light intensity of 3000 lx.

O parte de explante (varianta control) s-au inoculat pe mediul de cultură de bază Murashige-Skoog, ce conţinea macroelemente (săruri minerale, mg/l): NH4NO3 - 1650, KNO3 - 1900, MgSO4 · 7H2O - 370, KH2PO4 - 170, CaCl2 · 7H2O - 440; microelemente, mg/l: H3BO3 - 6,2, MnSO4 · 4H2O - 22,3, COCl2 · 6H2O - 0,025, CuSO4 · 5H2O - 0,025, ZnSO4 · 7H2O - 8,6, Na2MoO4 · 2H2O - 0,25, KJ - 0,83, FeSO4 · 7H2O - 27,8, Na2EDTA - 37,3, Inozitol - 100, solidificat cu agar-agar 0,7%, zaharoză 3%. A part of the explants (control variant) were inoculated on the Murashige-Skoog basic culture medium, which contained macroelements (mineral salts, mg/l): NH4NO3 - 1650, KNO3 - 1900, MgSO4 · 7H2O - 370, KH2PO4 - 170, CaCl2 · 7H2O - 440; microelements, mg/l: H3BO3 - 6.2, MnSO4 · 4H2O - 22.3, COCl2 · 6H2O - 0.025, CuSO4 · 5H2O - 0.025, ZnSO4 · 7H2O - 8.6, Na2MoO4 · 2H2O - 0.25, KJ - 0.83, FeSO4 · 7H2O - 27.8, Na2EDTA - 37.3, Inositol - 100, solidified with agar-agar 0.7%, sucrose 3%.

Altă parte de explante (cea mai apropiată soluţie), s-a utilizat pe mediul de bază Murashige-Skoog, suplimentat cu cărbune activat în cantitate de 1200 mg/l, valoarea pH-ului fiind ajustată la 6,5. Another part of the explants (the closest solution) was used on Murashige-Skoog basic medium, supplemented with activated carbon in an amount of 1200 mg/l, the pH value being adjusted to 6.5.

A treia parte de explante au fost utilizate pe mediul de cultură de bază Murashige-Skoog, suplimentat cu Somelangozidă în concentraţie de 3,0...3,5·10-3. The third part of explants were used on Murashige-Skoog basic culture medium, supplemented with Somelangoside in a concentration of 3.0...3.5·10-3.

Proliferarea explantelor în varianta martor s-a remarcat foarte slab, creşterea şi dezvoltarea calusului fără zone meristematice, ca rezultat explantele inoculate pe acest mediu s-au necrotizat. The proliferation of explants in the control variant was very weak, the growth and development of callus without meristematic areas, as a result the explants inoculated on this medium became necrotic.

În al doilea caz la utilizarea mediului Murashige-Skoog cărbune activat - cea mai apropiată soluţie, proliferarea explantelor s-a realizat după 10 zile de la inoculare, inducerea, creşterea şi dezvoltarea calusului s-au observat după 18 zile. Apariţia plantulelor - după 25 zile. Creşterea şi dezvoltarea plantulelor s-au observat după o lună de zile. În acest caz micropropagarea plantulelor a constituit 3·104 plante anual. In the second case, when using the Murashige-Skoog activated carbon medium - the closest solution, the proliferation of explants was achieved after 10 days from inoculation, the induction, growth and development of callus were observed after 18 days. The emergence of seedlings - after 25 days. The growth and development of seedlings were observed after a month. In this case, the micropropagation of seedlings amounted to 3·104 plants per year.

Pe al treilea mediu, conform invenţiei, proliferarea explantelor a evoluat la 5 zile de la cultivare, la 8 zile formarea şi dezvoltarea calusului embrionar cu zone meristematice de culoare roz. Apariţia plantulelor regenerate s-a observat la 14 zile. Creşterea şi dezvoltarea plantulelor s-a observat la 20 zile de la momentul cultivării explantelor pe mediul de cultură Murashige - Skoog, suplimentat cu substanţe furostanolice Somelangozidă în concentraţie de 3,0·10-3. În această variantă raportul optimal al ingredienţilor prezenţi în mediul nutritiv de cultură artificial sporeşte considerabil coeficientul de micropropagare a plantulelor de Echinacea - cu 4·105 plante anual. Valoarea medie a masei celulare a calusului s-a caracterizat cu valori de 64,23...75,1%. Capacitatea embriogenezei somatice a constituit 77,5...83,6%, iar capacitatea de regenerare a evoluat între 74,2...76,4%. În general calusul embrionar a fost multiplicat în scopul variabilităţii genetice de micropropagare a plantulelor obţinute in vitro. On the third medium, according to the invention, the proliferation of explants evolved 5 days after cultivation, at 8 days the formation and development of embryonic callus with pink meristematic areas. The appearance of regenerated seedlings was observed at 14 days. The growth and development of seedlings was observed 20 days after the explants were cultivated on the Murashige - Skoog culture medium, supplemented with furostanol substances Somelangoside in a concentration of 3.0·10-3. In this variant, the optimal ratio of the ingredients present in the artificial culture nutrient medium considerably increases the micropropagation coefficient of Echinacea seedlings - by 4·105 plants annually. The average value of the callus cell mass was characterized by values of 64.23...75.1%. The capacity of somatic embryogenesis was 77.5...83.6%, and the regeneration capacity evolved between 74.2...76.4%. In general, the embryonic callus was multiplied for the purpose of genetic variability of micropropagation of the plantlets obtained in vitro.

1. MD 3375 G2 2007.08.31 1. MD 3375 G2 2007.08.31

Claims (1)

Procedeu de micropropagare a Echinacea purpurea L. Moench in vitro careMicropropagation process of Echinacea purpurea L. Moench in vitro which include germinarea aseptică a seminţelor, obţinerea explantelor, segmentarea şi cultivarea lor pe mediul nutritiv Murashige-Skoog, ce conţine suplimentar glicozide steroidice obţinute din rădăcini de pătlăgele vinete Solanum melongena L. prin extracţie cu apă la fierbere, prelucrarea extractului obţinut cu butanol-1 şi concentrare, în concentraţie de (3,0…3,5)·10-3%, totodată cultivarea se efectuează la o temperatură de 22…26°C, umiditatea relativă a aerului de 70%, cu o fotoperioadă de 16 şi 8 ore noapte/zi şi intensitatea iluminării de 3000 lxincludes aseptic germination of seeds, obtaining explants, segmenting and cultivating them on Murashige-Skoog nutrient medium, which additionally contains steroidal glycosides obtained from eggplant roots Solanum melongena L. by extraction with boiling water, processing of the extract obtained with butanol-1 and concentration, in a concentration of (3.0…3.5)·10-3%, at the same time cultivation is carried out at a temperature of 22…26°C, relative air humidity of 70%, with a photoperiod of 16 and 8 hours night/day and illumination intensity of 3000 lx
MDS20090037A 2009-02-23 2009-02-23 Process for micropropagation in vitro of Echinacea purpurea L. Moench MD31Z (en)

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