MD605Z - Process for microclonal propagation of Actinidia arguta plants in vitro - Google Patents
Process for microclonal propagation of Actinidia arguta plants in vitro Download PDFInfo
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- 235000010755 mineral Nutrition 0.000 description 5
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
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Abstract
Description
Invenţia se referă la biotehnologie, în particular la un procedeu de micropropagare a plantelor de Actinidia arguta in vitro. The invention relates to biotechnology, in particular to a process for micropropagation of Actinidia arguta plants in vitro.
Actinidia arguta este o plantă liană, aparţine familiei Actinidaceae, care cuprinde mai mult de 60 specii. În natură specia se întâlneşte în sudul Siberiei, Manciuria, Nordul Chinei, Coreea, Japonia până la altitudini de 600 metri. Plantele de Actinidia arguta cresc în general în pădurile de conifere, mixte sau la marginea acestora. Actinidia arguta atinge în lungime 25 m, iar în grosime 15…18 cm. Durata vieţii acestei plante poate depăşi vârsta de 100 ani. Actinidia arguta is a liana plant, belongs to the Actinidaceae family, which includes more than 60 species. In nature, the species is found in southern Siberia, Manchuria, Northern China, Korea, Japan up to altitudes of 600 meters. Actinidia arguta plants generally grow in coniferous, mixed forests or at their edges. Actinidia arguta reaches 25 m in length, and 15…18 cm in thickness. The life span of this plant can exceed 100 years.
Actinidia arguta este o specie care se cultivă ca plantă ornamentală şi pentru obţinerea fructelor. Deoarece plantele sunt heterozigote, mărimea şi calitatea fructelor la plantele obţinute din seminţe pot varia foarte mult, de aceea este necesară multiplicarea vegetativă a genotipurilor valoroase. Dintre aceste genotipuri a fost aleasă linia Tatiana. Fructele acestei linii au o aromă similară cu cea de kiwi, care a fost descrisă că fiind o combinare a aromelor de căpşune, pepene galben, banane, smochine şi coacăză, având totodată şi particularităţi distincte. Gustul lor variază de la astringent, în timpul coacerii devreme, până la dulce la maturare deplină. Actinidia arguta is a species that is cultivated as an ornamental plant and for its fruit. Since the plants are heterozygous, the size and quality of the fruits of plants grown from seeds can vary greatly, so it is necessary to vegetatively propagate valuable genotypes. Among these genotypes, the Tatiana line was chosen. The fruits of this line have a flavor similar to that of kiwi, which has been described as a combination of strawberry, melon, banana, fig and currant flavors, but also have distinct characteristics. Their taste varies from astringent, during early ripening, to sweet at full maturity.
Raportul vitaminei C în fructul de mini-kiwi este acelaş ca la kiwi, şi anume de 37…85 mg/100 g în produsul proaspăt. În medicina tradiţională, fructele de Actinidia arguta sunt utilizate pentru tratarea bronşitei, tuberculozei, tusei convulsive, scorbutului, cariei, stomatitei etc. Actinidia arguta este numită fructul sănătăţii. Doar consumarea a 2…3 fructe poate satisface necesitatea zilnică de vitamina C. Sucul din fructele de mini-kiwi dilată vasele sangvine, reduce tensiunea arterială, stimulează activitatea inimii. The vitamin C content in mini-kiwi fruit is the same as in kiwi, namely 37…85 mg/100 g in the fresh product. In traditional medicine, Actinidia arguta fruits are used to treat bronchitis, tuberculosis, whooping cough, scurvy, caries, stomatitis, etc. Actinidia arguta is called the fruit of health. Only eating 2…3 fruits can satisfy the daily need for vitamin C. The juice from mini-kiwi fruits dilates blood vessels, reduces blood pressure, stimulates heart activity.
Înmulţirea Actinidia arguta se realizează vegetativ, cu dificultăţi mai pronunţate decât viţa de vie. Din această cauză am elaborat un procedeu de micropropagare a plantelor în condiţiile in vitro. Micropropagarea a fost realizată la linia de mini-kiwi Tatiana, dar ea este aplicabilă pentru specia de Actinidia arguta în general. Actinidia arguta propagation is carried out vegetatively, with more pronounced difficulties than the vine. For this reason, we have developed a process of micropropagation of plants under in vitro conditions. Micropropagation was carried out on the Tatiana mini-kiwi line, but it is applicable to the Actinidia arguta species in general.
Este cunoscut procedeul de micropropagare a plantelor de Actinidia arguta, ce constă în activarea mugurilor laterali. Pentru activarea şi creşterea intensivă a mugurilor laterali, explanţii primari au fost cultivaţi pe un mediu nutritiv de bază cu conţinut de săruri minerale după Murashige-Skoog (Murashige T., Skoog F. A revised medium for rapid growth and bio- assays with tabaco tissue cultures Physiology Plantarum, 1962, v.15, N 95, p.473) şi suplimentat cu 6-benzilaminopurină - 1,0 mg/l, agar - 0,6%; valoarea pH-lui a fost ajustată la 5,8 până la autoclavare [1]. The micropropagation process of Actinidia arguta plants is known, which consists in activating the lateral buds. For the activation and intensive growth of the lateral buds, the primary explants were cultivated on a basic nutrient medium containing mineral salts according to Murashige-Skoog (Murashige T., Skoog F. A revised medium for rapid growth and bio-assays with tabaco tissue cultures Physiology Plantarum, 1962, v.15, N 95, p.473) and supplemented with 6-benzylaminopurine - 1.0 mg/l, agar - 0.6%; the pH value was adjusted to 5.8 before autoclaving [1].
Însă acest procedeu are un dezavantaj, ce se manifestă prin perioada lungă de micropropagare, realizată în două etape: However, this process has a disadvantage, which is manifested by the long micropropagation period, carried out in two stages:
1) proliferarea lăstarilor din mugurii laterali; 1) proliferation of shoots from lateral buds;
2) inducerea rizogenezei. 2) induction of rhizogenesis.
Dezavantajele menţionate pot fi înlăturate cu ajutorul procedeului revendicat. The aforementioned disadvantages can be eliminated by means of the claimed process.
Problema pe care o rezolvă invenţia propusă constă în micşorarea perioadei de micropropagare, sporirea coeficientului de multiplicare a plantelor şi, respectiv, diminuarea cheltuielilor pentru a obţine o plantă nouă de Actinidia arguta. The problem solved by the proposed invention consists in reducing the micropropagation period, increasing the plant multiplication coefficient and, respectively, reducing the expenses to obtain a new Actinidia arguta plant.
Procedeul de micropropagare a plantelor de Actinidia arguta in vitro include inocularea mugurilor laterali ai minibutaşilor şi cultivarea lăstarilor obţinuţi cu inducerea rizogenezei pe un mediu nutritiv, care conţine 1⁄2 săruri minerale faţă de mediul Murashige-Skoog şi suplimentar cărbune activat 1200 mg/L, totodată cultivarea se efectuează la temperatura de 26°C, umiditatea relativă a aerului de 70%, cu o fotoperioadă de 16 ore lumină, intensitatea iluminării de 1000 lx şi pH-ul 5,8. The process of micropropagation of Actinidia arguta plants in vitro includes inoculation of the lateral buds of the mini-cuttings and cultivation of the shoots obtained by inducing rhizogenesis on a nutrient medium, which contains 1⁄2 mineral salts compared to the Murashige-Skoog medium and additionally 1200 mg/L activated carbon, at the same time the cultivation is carried out at a temperature of 26°C, relative air humidity of 70%, with a photoperiod of 16 hours of light, illumination intensity of 1000 lx and pH 5.8.
Avantajele invenţiei sunt: The advantages of the invention are:
- posibilitatea de a obţine în decurs de 10…12 săptămâni dintr-un explant 7…9 lăstari (conform invenţiei), - the possibility of obtaining 7...9 shoots from an explant within 10...12 weeks (according to the invention),
în comparaţie cu 6…7 lăstari dintr-un explant în 14…16 săptămâni (conform celei mai apropiată soluţie); compared to 6…7 shoots from an explant in 14…16 weeks (according to the closest solution);
- sporirea coeficientului de micropropagare anuală de la aproximativ 300 plante anual (cea mai apropiată soluţie) până la 8000 plante (conform invenţiei); - increasing the annual micropropagation coefficient from approximately 300 plants annually (the closest solution) to 8000 plants (according to the invention);
- în comparaţie cu cea mai apropiată soluţie, procesul de micropropagare a plantelor de Actinidia arguta prin procedeul propus se caracterizează prin reducerea timpului de menţinere in vitro din contul timpului consumat pentru inducerea rizogenezei cu aproximativ 25% şi diminuarea numărului de explanţi necesari pentru multiplicare cu 20%. - compared to the closest solution, the micropropagation process of Actinidia arguta plants through the proposed procedure is characterized by reducing the in vitro maintenance time due to the time spent inducing rhizogenesis by approximately 25% and reducing the number of explants required for multiplication by 20%.
Rezultatul invenţiei constă în sporirea coeficientului de multiplicare de la 300 plante anual (cea mai apropiată soluţie) până la 8000 (invenţia propusă) plante de Actinidia arguta, datorită modificării conţinutului mediului nutritiv (1⁄2 săruri minerale după Murashige-Skoog şi suplimentarea cu cărbune activat). Aceste modificări provoacă creşterea lăstarilor cu majorarea numărului de internoduri (ceia ce sporeşte coeficientul potenţial de multiplicare) şi paralel, inducerea rizogenezei, cu formarea unui sistem radicular bine dezvoltat. The result of the invention consists in increasing the multiplication coefficient from 300 plants per year (the closest solution) to 8000 (the proposed invention) plants of Actinidia arguta, due to the modification of the content of the nutrient medium (1⁄2 mineral salts according to Murashige-Skoog and the supplementation with activated carbon). These modifications cause the growth of shoots with an increase in the number of internodes (which increases the potential multiplication coefficient) and, in parallel, the induction of rhizogenesis, with the formation of a well-developed root system.
Exemplu de realizare a invenţiei Example of embodiment of the invention
Plantele de Actinidia arguta au fost obţinute din butaşii plantelor în vârstă de 8 ani, colectaţi din lizimetrele IGFP al AŞM. Pentru a asigura sterilizarea, ele au fost tratate cu agent de înălbire pe bază de clor de 20% pe parcursul a 15 min şi spălate de trei ori cu apă sterilă. Inocularea lăstarilor şi creşterea plantulelor a fost realizată pe mediul nutritiv Murashige-Skoog (MS), la o temperatură de 26°C, cu o fotoperioadă de 16 ore/zi şi 8 ore/noapte, iar intensitatea iluminării de 1000 lux. Actinidia arguta plants were obtained from cuttings of 8-year-old plants, collected from the IGFP lysimeters of the ASM. To ensure sterilization, they were treated with 20% chlorine bleach for 15 min and washed three times with sterile water. Shoot inoculation and seedling growth were performed on Murashige-Skoog (MS) nutrient medium, at a temperature of 26°C, with a photoperiod of 16 hours/day and 8 hours/night, and an illumination intensity of 1000 lux.
O parte din minibutaşi (varianta de control) a fost cultivată pe mediul nutritiv Murashige-Skoog, cu următorul conţinut de microelemente şi săruri minerale, mg/l: Part of the mini-cuttings (control variant) was cultivated on Murashige-Skoog nutrient medium, with the following content of microelements and mineral salts, mg/l:
NH4NO3 - 1650, KNO3 - 1900, MgSO4 · 7H2O - 370, KH2PO4 - 170, CaCl2 · 2H2O - 440, H3BO3 - 6,2, MnSO4 · 4H2O - 22,3, CoCl2 · 6H2O - 0,025, CuSO4 · 5H2O - 0,025, ZnSO4 · 7H2O - 8,6, Na2MoO4 · 2H2O - 0,25, KI - 0,83, FeSO4 · 7H2O - 27,8, Na2EDTA · 2H2O - 37,3. NH4NO3 - 1650, KNO3 - 1900, MgSO4 7H2O - 370, KH2PO4 - 170, CaCl2 2H2O - 440, H3BO3 - 6.2, MnSO4 4H2O - 22.3, CoCl2 6H2O - 0.025, CuSO4 5H2O - 0.025, ZnSO4 · 7H2O - 8.6, Na2MoO4 · 2H2O - 0.25, KI - 0.83, FeSO4 · 7H2O - 27.8, Na2EDTA · 2H2O - 37.3.
Altă parte a fost cultivată pe mediul nutritiv Murashige-Skoog supliment cu 6-benzilaminopurină, valoarea pH-lui a fost ajustată înainte de autoclavare până la 5,8 (cea mai apropiată soluţie). Another part was cultured on Murashige-Skoog nutrient medium supplemented with 6-benzylaminopurine, the pH value was adjusted before autoclaving to 5.8 (the closest solution).
A treia parte de minibutaşi a fost cultivată pe mediul nutritiv Murashige-Skoog, ce conţinea 1⁄2 săruri minerale indicate în varianta de control, suplimentat cu cărbune activat 1200 mg/l, pH-ul fiind 5,8, fără fitohormoni (conform invenţiei). În aşa fel mediile de cultivare se deosebeau după conţinutul unor componenţi. Minibutaşii din cele trei variante au fost cultivaţi la temperatura de 26°C, umiditatea relativă a aerului de 70%, cu o fotoperioadă de 16 ore/zi şi 8 ore/noapte, intensitatea iluminării de 1000 lux. The third part of the mini-cuttings was cultivated on the Murashige-Skoog nutrient medium, which contained 1⁄2 of the mineral salts indicated in the control variant, supplemented with activated carbon 1200 mg/l, the pH being 5.8, without phytohormones (according to the invention). In this way, the cultivation media differed in the content of some components. The mini-cuttings of the three variants were cultivated at a temperature of 26°C, a relative air humidity of 70%, with a photoperiod of 16 hours/day and 8 hours/night, and an illumination intensity of 1000 lux.
Creşterea şi dezvoltarea lăstarilor de la baza mugurilor laterali şi a rădăcinilor la cultivarea explantelor pe mediul nutritiv Murashige-Skoog conform invenţiei s-a observat la 7…10 zile de la momentul inoculării, iar după 30 zile lăstarii au atins în înălţime 3…5 cm. După 10…12 săptămâni de la momentul inoculării fiecare plantă a fost secţionată în minibutaşi, care ulterior au fost inoculaţi pentru următorul ciclu de dezvoltare pe acelaşi mediu nutritiv. În aşa fel, pe acelaşi mediu nutritiv a fost posibilă atât iniţierea culturii in vitro, cât şi cultivarea minibutaşilor pentru a obţine plantule, care sunt pregătite pentru cultivarea în condiţiile ex vitro. În cazul celei mai apropiate soluţii, lăstarii cultivaţi după 30 zile atingeau înălţimea de numai 1,5…2 cm. The growth and development of shoots from the base of lateral buds and roots when cultivating explants on the Murashige-Skoog nutrient medium according to the invention was observed 7…10 days after inoculation, and after 30 days the shoots reached a height of 3…5 cm. After 10…12 weeks from the moment of inoculation each plant was sectioned into mini-cuttings, which were subsequently inoculated for the next development cycle on the same nutrient medium. In this way, on the same nutrient medium it was possible to both initiate in vitro culture and cultivate mini-cuttings to obtain seedlings, which are ready for cultivation under ex vitro conditions. In the case of the closest solution, the shoots cultivated after 30 days reached a height of only 1.5…2 cm.
Spre deosebire de cea mai apropiată soluţie, care prevedea introducerea şi cultivarea iniţială a minibutaşilor pe un mediu nutritiv, iar rizogeneza pe alt mediu nutritiv (două etape de cultivare in vitro), procedeul revendicat prevede cultivarea in vitro pe un singur mediu nutritiv, într-o singură etapă. În aşa fel procedeul revendicat face posibilă micşorarea de două ori a cheltuielilor de reagenţi pentru cultivarea in vitro (după metoda cunoscută şi cea propusă), diminuarea duratei de cultivare in vitro pentru obţinerea unei noi generaţii de plantule de la 14…16 săptămâni până la 10…12 săptămâni; sporirea coeficientului de micropropagare de la 6 la 8 săptămâni. Pe parcursul a trei cicluri s-au obţinut aproximativ 700 de plante (conform invenţiei), faţă de cele 300 plante din cea mai apropiată soluţie, luând în consideraţie că în conformitate cu invenţia propusă se obţin până la patru cicluri şi jumătate pe parcursul unui an, faţă de cele trei cicluri din cea mai apropiată soluţie. Unlike the closest solution, which provided for the introduction and initial cultivation of mini-cuttings on a nutrient medium, and rhizogenesis on another nutrient medium (two stages of in vitro cultivation), the claimed process provides for in vitro cultivation on a single nutrient medium, in a single stage. Thus, the claimed process makes it possible to reduce by two times the costs of reagents for in vitro cultivation (according to the known and proposed method), to reduce the duration of in vitro cultivation for obtaining a new generation of seedlings from 14…16 weeks to 10…12 weeks; to increase the micropropagation coefficient from 6 to 8 weeks. During three cycles, approximately 700 plants (according to the invention) were obtained, compared to the 300 plants in the closest solution, taking into account that according to the proposed invention, up to four and a half cycles are obtained during a year, compared to the three cycles in the closest solution.
Prin urmare, procedeul revendicat permite de a diminua de două ori cheltuielile pentru reagenţi şi de 1,77 ori durata menţinerii in vitro pentru a obţine o nouă generaţie pregătită pentru transferul ex vitro; de a spori de 26 ori numărul de plantule multiplicate pe parcursul unui an utilizând aceeaşi suprafaţă de cultivare. Therefore, the claimed process allows to reduce by two times the expenses for reagents and by 1.77 times the duration of in vitro maintenance to obtain a new generation ready for ex vitro transfer; to increase by 26 times the number of seedlings multiplied during a year using the same cultivation area.
1. Высоцкий В.А., Бартенева Л. Б. Особенности клонального микроразмножения актинидии. Биология культивируемых клеток и биотехнология растений. Москва, Наука, 1991, с. 213...216 1. Vysotsky V.A., Barteneva L. B. Features of clonal micropropagation of actinidia. Biology of cultivated cells and biotechnology of plants. Moscow, Science, 1991, p. 213...216
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| MD1091Z (en) * | 2016-03-04 | 2017-06-30 | Институт Генетики, Физиологии И Защиты Растений Академии Наук Молдовы | Process for micropropagation of Mentha gattefossei Maire plants in vitro |
| CN111713410B (en) * | 2020-07-03 | 2022-05-31 | 四川农业大学 | Kiwi explant detoxification method |
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