MD894Z - Process for in vitro production of Rhodiola rosea L. callus biomass - Google Patents

Process for in vitro production of Rhodiola rosea L. callus biomass Download PDF

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Publication number
MD894Z
MD894Z MDS20140133A MDS20140133A MD894Z MD 894 Z MD894 Z MD 894Z MD S20140133 A MDS20140133 A MD S20140133A MD S20140133 A MDS20140133 A MD S20140133A MD 894 Z MD894 Z MD 894Z
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Moldova
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callus
rhodiola rosea
biomass
nutrient medium
vitro production
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MDS20140133A
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Romanian (ro)
Russian (ru)
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Татьяна КЭЛУГЭРУ-СПЭТАРУ
Мария КАУШ
Александру ДАСКАЛЮК
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Институт Генетики, Физиологии И Защиты Растений Академии Наук Молдовы
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Priority to MDS20140133A priority Critical patent/MD894Z/en
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Abstract

The invention relates to biotechnology, namely to a process for in vitro production of Rhodiola rosea L. callus biomass.The process, according to the invention, comprises cultivation of callus from leaves of sterile shoots of Rhodiola rosea L. on the agarized Murashige-Skoog nutrient medium supplemented with 1.5 mg/l of 6-benzylaminopurine, 0.5 mg/l of α-naphthylacetic acid and 0.1% sterile aqueous solution of 5% of dry substances obtained from the biomassofgreen alga Spirogira sp. by extraction with ethanol.

Description

Invenţia se referă la biotehnologie, şi anume la un procedeu de obţinere a biomasei calusului de Rhodiola rosea L. in vitro. The invention relates to biotechnology, namely to a process for obtaining Rhodiola rosea L. callus biomass in vitro.

Rhodiola rosea L. reprezintă o plantă valoroasă care, datorită metaboliţilor secundari ce se conţin în rădăcini şi rizomi, este folosită pe larg în medicină şi farmaceutică. Tocmai de aceea Rhodiola rosea L. este colectată intensiv din masivele naturale şi în ultimii ani ea a dispărut de pe mai multe creste montane, unde planta creştea spontan. Cultivarea artificială a speciei necesită condiţii specifice, din aceasta cauză întâmpină mai multe dificultăţi. O cale de soluţionare a problemei este obţinerea principiilor active caracteristice pentru Rhodiola rosea L. din biomasa celulelor calusului obţinută pe cale biotehnologică. Rhodiola rosea L. is a valuable plant that, due to the secondary metabolites contained in its roots and rhizomes, is widely used in medicine and pharmacy. That is why Rhodiola rosea L. is intensively collected from natural massifs and in recent years it has disappeared from several mountain ridges, where the plant grew spontaneously. Artificial cultivation of the species requires specific conditions, which is why it encounters several difficulties. One way to solve the problem is to obtain the active principles characteristic of Rhodiola rosea L. from the biomass of callus cells obtained by biotechnology.

Este cunoscută metoda de cultivare a celulelor calusului în vitro [1]. Pentru proliferarea celulelor calusului a fost utilizat mediul nutritiv Murashige-Skoog (MS) (1962), suplinit cu 6-benzilaminopurină (BA) 1,5 mg/l, acid α-naftilacetic (ANA) 0,5 mg/l; valoarea pH-ului fiind ajustată la 5,8 înainte de autoclavare [1]. Din punct de vedere economic acest procedeu necesită îmbunătăţire, deoarece cultivarea calusului pe parcursul unei perioade optimale nu asigură acumularea semnificativă a biomasei. Acest neajuns poate fi înlăturat cu ajutorul procedeului propus. The method of culturing callus cells in vitro is known [1]. For the proliferation of callus cells, the Murashige-Skoog (MS) (1962) nutrient medium was used, supplemented with 6-benzylaminopurine (BA) 1.5 mg/l, α-naphthylacetic acid (ANA) 0.5 mg/l; the pH value being adjusted to 5.8 before autoclaving [1]. From an economic point of view, this process requires improvement, since the cultivation of callus during an optimal period does not ensure significant accumulation of biomass. This shortcoming can be eliminated with the help of the proposed process.

Problema pe care o rezolvă invenţia constă în sporirea proliferării celulare şi a acumulării biomasei calusului de Rhodiola rosea L. The problem solved by the invention consists in increasing cell proliferation and biomass accumulation of Rhodiola rosea L. callus.

Procedeul, conform invenţiei, include inocularea frunzelor prelevate de la plantulele sterile de Rhodiola rosea L. pe un mediu nutritiv, obţinut prin adăugarea la mediul nutritiv agarizat Murashige-Skoog a 1,5 mg/l 6-benzilaminopurină şi 0,5 mg/l acid α-naftilacetic, ajustarea pH-ului până la 5,8, autoclavare şi adăugarea unei soluţii apoase sterilizate de 5% de substanţe uscate obţinute din biomasa algei verzi Spirogira sp. prin extragere cu etanol în cantitate de 0,1%, după care se efectuează cultivarea calusului în decurs de 40 zile la temperatura de 26°C şi umiditatea relativă a aerului de 70% cu o fotoperioadă de 16 ore lumină şi 8 ore întuneric. The process, according to the invention, includes inoculating leaves taken from sterile Rhodiola rosea L. seedlings on a nutrient medium, obtained by adding 1.5 mg/l 6-benzylaminopurine and 0.5 mg/l α-naphthylacetic acid to the Murashige-Skoog agarized nutrient medium, adjusting the pH to 5.8, autoclaving and adding a sterilized aqueous solution of 5% dry substances obtained from the biomass of the green alga Spirogira sp. by extraction with ethanol in an amount of 0.1%, after which the callus is cultivated for 40 days at a temperature of 26°C and a relative air humidity of 70% with a photoperiod of 16 hours of light and 8 hours of darkness.

Soluţia apoasă obţinută din biomasa algei verzi Spirogira sp. (în continuare preparatul Reglalg, Autorizaţie AA nr. 0448 eliberată din 21.02.2003 de Centrul de Stat pentru Atestarea Produselor Chimice şi Biologice de Protecţie şi Stimulare a Plantelor. Grupa a 4-a de toxicitate) prezintă un complex de substanţe biologic active, obţinute din biomasa algei filamentoase verzi Spirogira sp. crescută în mediul organic timp de 10…12 zile, uscată până la umiditatea de 5…7% şi fărâmiţată până la starea de praf, totodată înainte de extragere biomasa se tratează cu cloroform, apoi cu apă, iar extragerea finală se realizează cu alcool etilic în raport de 1:(4…6) cu diluţia ulterioară cu apă. Soluţia finală conţine 5% de substanţă uscată. The aqueous solution obtained from the biomass of the green algae Spirogira sp. (hereinafter the Reglagl preparation, AA Authorization No. 0448 issued on 21.02.2003 by the State Center for Certification of Chemical and Biological Products for Plant Protection and Stimulation. 4th toxicity group) presents a complex of biologically active substances, obtained from the biomass of the green filamentous algae Spirogira sp. grown in organic medium for 10…12 days, dried to a humidity of 5…7% and crushed to a state of dust, at the same time before extraction the biomass is treated with chloroform, then with water, and the final extraction is carried out with ethyl alcohol in a ratio of 1: (4…6) with subsequent dilution with water. The final solution contains 5% of dry matter.

Avantajele procedeului propus constau în simplicitatea lui, sporirea substanţială a biomasei acumulate, conţinutului pigmenţilor asimilatori şi costul redus al preparatului. The advantages of the proposed process consist in its simplicity, the substantial increase in accumulated biomass, the content of assimilatory pigments and the reduced cost of the preparation.

Rezultatul constă în sporirea proliferării celulare şi a acumulării biomasei calusului de Rhodiola rosea L. cu 38%, a concentraţiei clorofilei a, b şi a carotenoidelor în celulele calusului cu 92%, 49% şi, respectiv, 53% faţă de procedeul cunoscut. The result consists in increasing cell proliferation and biomass accumulation of Rhodiola rosea L. callus by 38%, the concentration of chlorophyll a, b and carotenoids in callus cells by 92%, 49% and, respectively, 53% compared to the known process.

Exemplu de realizare a invenţiei Example of embodiment of the invention

Pentru inducerea calusului au fost prelevate frunze de la plantulele sterile de Rhodiola rosea L., obţinute din seminţe prealabil sterilizate. Segmente foliare cu dimensiunea de 0,3…0,5 cm au fost inoculate pe mediul MS suplimentat cu 1,5 mg/l BA şi 0,5 mg/l ANA, agar 0,6% [1]. Valoarea pH-lui a fost ajustată până la 5,8, înainte de autoclavare. Pentru menţinerea calusului, la fiecare 40 de zile de cultivare, fragmente de calus cu masa de aproximativ 2 g erau transferate pe mediul MS proaspăt preparat [1]. For callus induction, leaves were taken from sterile Rhodiola rosea L. seedlings, obtained from previously sterilized seeds. Leaf segments with a size of 0.3…0.5 cm were inoculated onto MS medium supplemented with 1.5 mg/l BA and 0.5 mg/l ANA, 0.6% agar [1]. The pH value was adjusted to 5.8, before autoclaving. To maintain callus, every 40 days of cultivation, callus fragments with a mass of approximately 2 g were transferred onto freshly prepared MS medium [1].

O parte din explanţi a fost cultivată pe mediul nutritiv MS [1] (cea mai apropiată soluţie), alta - pe acelaşi mediu suplimentat după autoclavare cu soluţie apoasă sterilizată de Reglalg în concentraţie finală de 0,001% (invenţia propusă). În aşa fel, mediile de cultivare se deosebeau numai prin prezenţa Reglalgului. Explanţii celor două variante au fost cultivaţi la temperatura de 26°C, cu fotoperioada de 16 ore lumină (2000 lux) şi 8 ore întuneric. Umiditatea relativă a aerului în camera de cultivare a fost egală cu 70%. Part of the explants was cultivated on the MS nutrient medium [1] (the closest solution), another - on the same medium supplemented after autoclaving with sterilized aqueous solution of Regalg in a final concentration of 0.001% (the proposed invention). Thus, the cultivation media differed only in the presence of Regalg. The explants of the two variants were cultivated at a temperature of 26°C, with a photoperiod of 16 hours of light (2000 lux) and 8 hours of darkness. The relative humidity of the air in the cultivation room was equal to 70%.

Pentru analiză de la fiecare variantă au fost luate câte 3 vase, au fost prelevate celulele calusului, masa proaspătă a lor fiind determinată prin cântărire. Ulterior din masa calusului au fost extraşi componenţii clorofilieni, conţinutul clorofilei a, b şi al carotenoidelor fiind determinat prin metoda spectrofotometrică. For analysis, 3 pots were taken from each variant, callus cells were sampled, their fresh mass was determined by weighing. Subsequently, chlorophyll components were extracted from the callus mass, the chlorophyll a, b and carotenoid content was determined by spectrophotometric method.

Conform datelor prezentate în tabel, calusurile cultivate conform celei mai apropiate soluţii şi conform invenţiei se deosebeau prin sporirea biomasei cu 38% şi a conţinutului clorofilei a, b şi al carotenoidelor cu 92%, 49% şi, respectiv, 53%. Prin urmare, suplimentarea mediului de cultivare cu un volum de soluţie apoasă sterilizată de Reglalg a asigurat sporirea acumulării biomasei şi a conţinutului clorofilei a, b şi al carotenoidelor în celulele calusului de Rhodiola rosea L. (vezi tabelul). According to the data presented in the table, the calli cultivated according to the closest solution and according to the invention differed in the increase in biomass by 38% and in the content of chlorophyll a, b and carotenoids by 92%, 49% and, respectively, 53%. Therefore, the supplementation of the cultivation medium with a volume of sterilized aqueous solution of Reglag ensured the increase in the accumulation of biomass and the content of chlorophyll a, b and carotenoids in the callus cells of Rhodiola rosea L. (see table).

Tabel Table

Procedeu de cultivare Biomasa calusului, g Clorofila a, mg/g Clorofila b, mg/g Carotenoide, mg/g Conform celei mai apropiate soluţii 12 ± 0,16 1,27 ± 0,01 0,75 ± 0,04 1,14 ± 0,01 Conform invenţiei 16,6 ± 0,15 2,44 ± 0,03 1,12 ± 0,01 1,74 ± 0,03Cultivation process Callus biomass, g Chlorophyll a, mg/g Chlorophyll b, mg/g Carotenoids, mg/g According to the closest solution 12 ± 0.16 1.27 ± 0.01 0.75 ± 0.04 1.14 ± 0.01 According to the invention 16.6 ± 0.15 2.44 ± 0.03 1.12 ± 0.01 1.74 ± 0.03

1. Călugăru-Spataru T. Optimizarea mediului de cultivare a calusului de Rhodiola Rosea L. şi marcarea proteică a etapei de dezvoltare a lui. Materialele simpozionului ştiinţific internaţional consacrat aniversării a 60-a de la fondarea Grădinii Botanice (Institut) al AŞM, Chişinău, 7-9 noiembrie 2010, p. 50-55 1. Călugăru-Spataru T. Optimization of the Rhodiola Rosea L. callus cultivation environment and protein marking of its development stage. Materials of the international scientific symposium dedicated to the 60th anniversary of the founding of the Botanical Garden (Institute) of the Academy of Medical Sciences, Chisinau, November 7-9, 2010, p. 50-55

Claims (1)

Procedeu de obţinere a biomasei calusului de Rhodiola rosea L. in vitro, care include inocularea frunzelor prelevate de la plantulele sterile de Rhodiola rosea L. pe un mediu nutritiv, obţinut prin adăugarea la mediul nutritiv agarizat Murashige-Skoog a 1,5 mg/l 6-benzilaminopurină şi 0,5 mg/l acid α-naftilacetic, ajustarea pH-ului până la 5,8, autoclavare şi adăugarea unei soluţii apoase sterilizate de 5% de substanţe uscate obţinute din biomasa algei verzi Spirogira sp. prin extragere cu etanol în cantitate de 0,1%, după care se efectuează cultivarea calusului în decurs de 40 zile la temperatura de 26°C şi umiditatea relativă a aerului de 70% cu o fotoperioadă de 16 ore lumină şi 8 ore întuneric.Process for obtaining Rhodiola rosea L. callus biomass in vitro, which includes inoculating leaves taken from sterile Rhodiola rosea L. seedlings on a nutrient medium, obtained by adding 1.5 mg/l 6-benzylaminopurine and 0.5 mg/l α-naphthylacetic acid to the Murashige-Skoog agarized nutrient medium, adjusting the pH to 5.8, autoclaving and adding a sterilized aqueous solution of 5% dry substances obtained from the biomass of the green alga Spirogira sp. by extraction with ethanol in an amount of 0.1%, after which the callus is cultivated for 40 days at a temperature of 26°C and a relative air humidity of 70% with a photoperiod of 16 hours of light and 8 hours of darkness.
MDS20140133A 2014-10-20 2014-10-20 Process for in vitro production of Rhodiola rosea L. callus biomass MD894Z (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109452174A (en) * 2018-12-21 2019-03-12 西藏诺迪康药业股份有限公司 The fast propagating culture medium and propagation method of rhodiola
RU2818083C1 (en) * 2023-11-17 2024-04-24 Федеральное государственное автономное образовательное учреждение высшего образования "Национальный исследовательский Томский государственный университет" Method of increasing raw productivity of rhodiola rosea linnaeus

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MD634G2 (en) * 1996-04-26 1997-07-31 Институт Физиологии Растений Академии Наук Республики Молдова Method of receive of the biological-active substance
MD1659G2 (en) * 1999-08-05 2001-11-30 Институт Физиологии Растений Академии Наук Республики Молдова Process for presawing treatment of winter wheat seeds
MD3375G2 (en) * 2007-03-23 2008-03-31 Институт Генетики, Физиологии И Защиты Растений Академии Наук Молдовы Process for micropropagation in vitro of Rhodiola rosea L. plants
MD497Z (en) * 2011-11-14 2012-11-30 Институт Генетики, Физиологии И Защиты Растений Академии Наук Молдовы Process for cultivation of tomatoes
  • 2014

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MD634G2 (en) * 1996-04-26 1997-07-31 Институт Физиологии Растений Академии Наук Республики Молдова Method of receive of the biological-active substance
MD1659G2 (en) * 1999-08-05 2001-11-30 Институт Физиологии Растений Академии Наук Республики Молдова Process for presawing treatment of winter wheat seeds
MD3375G2 (en) * 2007-03-23 2008-03-31 Институт Генетики, Физиологии И Защиты Растений Академии Наук Молдовы Process for micropropagation in vitro of Rhodiola rosea L. plants
MD497Z (en) * 2011-11-14 2012-11-30 Институт Генетики, Физиологии И Защиты Растений Академии Наук Молдовы Process for cultivation of tomatoes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Călugăru-Spataru T. Optimizarea mediului de cultivare a calusului de Rhodiola Rosea L. şi marcarea proteică a etapei de dezvoltare a lui. Materialele simpozionului ştiinţific internaţional consacrat aniversării a 60-a de la fondarea Grădinii Botanice (Institut) al AŞM, Chişinău, 7-9 noiembrie 2010, p. 50-55 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109452174A (en) * 2018-12-21 2019-03-12 西藏诺迪康药业股份有限公司 The fast propagating culture medium and propagation method of rhodiola
RU2818083C1 (en) * 2023-11-17 2024-04-24 Федеральное государственное автономное образовательное учреждение высшего образования "Национальный исследовательский Томский государственный университет" Method of increasing raw productivity of rhodiola rosea linnaeus

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