MD46Y - Process for obtaining in vitro fruit biomass of Aronia melanocarpa (Michx.) Elliot - Google Patents

Abstract

The invention relates to biotechnology. The process according to the invention includes obtaining in vitro fruit explants from Aronia melanocarpa (Michx.) Elliot, cultivating them on the Murashige-Skoog nutrient medium, which additionally contains: naphthylacetic acid 3.0 ... 3.5 mg / L; kinetin 0.5 mg / L; Sucrose 60 g / L, having pH 5.7, at the same time the cultivation is carried out at a temperature of 18 ... 22 ° C, with day-night photoperiod respectively of 16 and 8 hours.

Description

[0001] The invention relates to biotechnology.

[0002] Aronia melanocarpa fruits characterized by a phenolic content (flavonoids, including anthocyanins, phenylpropionic acids, tannins) with antioxidant, vasoconstrictor, hypotensive, antiradiant properties are in demand as a source of medicine and healthy food. Since meeting the growing needs for chokeberry fruits by traditional in vitro methods is problematic (dependence on seasonal rotation, consequences of ecological crisis and natural disasters, application of difficult and expensive seasonal agrotechnical and protection measures), attempts have been made to obtain anthocyanin-rich biomass of chokeberry fruits under in vitro conditions as an alternative to fruit growth.

[0003] The process of obtaining the biomass of Aronia melanocarpa (Michx.) Elliot fruits of green color from explants taken from chokeberry fruits on Murashige-Skoog nutrient medium, which additionally contains, mg/L: 2,4-dichlorophenoxyacetic acid 2.0; kinetin 0.5; sucrose 60, having pH 5.7. Cultivation is carried out at a temperature of 22…24°C, with a day-night photoperiod of 16 and 8 hours respectively and aseptic conditions [1].

[0004] The shortcomings of this process consist in the fact that by inoculating chokeberry explants on aseptic nutrient media with the described hormonal formula, the assimilatory biomass of green fruits with minimal phenolic content is obtained in vitro.

[0005] The problem solved by the invention consists in obtaining anthocyanin-rich biomass of purple chokeberry fruits, rich in phenolic content, under controlled, ecologically pure, directed, continuous flow conditions as an alternative to obtaining biomass of green chokeberry fruits with minimal phenolic content.

[0006] The process, according to the invention, includes obtaining in vitro explants from Aronia melanocarpa (Michx.) Elliot fruits, cultivating them on Murashige-Skoog nutrient medium, which additionally contains: naphthylacetic acid 3.0…3.5 mg/L; kinetin 0.5 mg/L; sucrose 60 g/L, having pH 5.7, while cultivation is carried out at a temperature of 18…22°C, with a day-night photoperiod of 16 and 8 hours respectively.

[0007] The result of the invention consists in the guaranteed obtaining of aronia fruit biomass in vitro, enriched with phenolic content, in continuous flow, under controlled, ecologically pure conditions, independent of seasonal rotation, natural disasters and pedoclimatic conditions.

[0008] Embodiment of the invention

[0009] A portion of explants from Aronia melanocarpa (Michx.) Elliot fruits (control variant) was inoculated on Murashige - Skoog nutrient medium, with mineral salts content, mg/mL: NH4NO3 1650, KNO3 1900, MgSO4 · 7H2O 370, KH2PO4 170, CaCl2 · 2H2O 440, H3BO3 6.2, MnSO4 · 4H2O 223, CoCl2 · 6H2O 0.025, CuSO4 · 5H2O 0.025, ZnSO4 · 7H2O 9.6, Na2MoO4 · 2H2O 0.25, KI 0.83, FeSO4 · 7H2O 27.8, Na2EDTA 37.3 (Murashige T., Skoog F. Revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiology Plantarum, 1962, V. 15, no. 95, pp. 473-497).

[0010] The second part of explants from Aronia melanocarpa (Michx.) Elliot fruits was inoculated under aseptic conditions on Murashige-Skoog mineral salts nutrient medium supplemented with growth regulators: 2,4-dichlorophenoxyacetic acid 2.0 mg/L, kinetin 0.5 mg/L; sucrose 30 g/L, having pH 5.7, at temperatures of 22…24°C and aseptic conditions with day-night photoperiod of 16 and 8 hours respectively (the closest solution).

[0011] The third part of explants, taken from chokeberry fruits, were sterilized with chemical agent "Diacid" for 7 min, washed 3 times with sterile double-distilled water. The explants were inoculated on Murashige-Skoog nutrient medium supplemented with growth regulators: naphthylacetic acid 3.5 mg/L, kinetin 0.5 mg/L; sucrose 60 g/L, having pH 5.7, with day-night photoperiod of 16 and 8 hours respectively, at temperatures of 18…22°C and aseptic conditions.

[0012] On the control nutrient medium, only incipient callus formation of the explants was observed on the 12th...14th day from the moment of inoculation, and on the 20th day, necrotization was fixed.

[0013] The second part of explants generated a viable, green fruit biomass at the 5th...36th day after inoculation, with a minimal content of phenolic compounds (table).

[0014] Table

[0015] In vitro production of aronia fruit biomass

Composition of the nutrient medium

Light regime

Temperature, °C

result

Phenolic content

Anthocyanins, mg/100 g fruit

Flavonoids (g% rutoside)

Tannins, g%

MS + sucrose (60 g/L) + ANA (3.0…3.5 mg/L) + K (0.5 mg/L) (invention)

Day/night photoperiod - 16/8 hours

18…22

Biomass of purple fruits (anthocyanins)

680

0.483

11.2

MS + sucrose (30 g/L) + 2,4-D (2.0 mg/L) + K (0.5 mg/L) (closest solution)

Day/night photoperiod - 16/8 hours

22…24

Green fruit biomass (chlorophyll)

156

0.230

2.6

[0017] MS - Murashige-Skoog medium (1962); ANA - naphthylacetic acid; 2,4-D - dichlorophenoxyacetic acid; K - kinetin.

[0018] In the case of explants inoculated on Murashige-Skoog nutrient medium, supplemented with naphthylacetic acid 3.0…3.5 mg/L, kinetin 0.5 mg/L; sucrose 60 g/L, and the pH adjusted to 5.7 and the temperature regime 18…22°C, the initiation of callusogenesis was noted on the 5th…6th day and the maximum accumulation of viable fruit biomass, of a violet color on the 26th…30th day, characterized by an increased content of phenolic compounds: anthocyanins 680 mg/100 g of plant product; flavonoids 0.483 g%; tannins 11.2% (table).

[0019] 1. MD 3763 B1 2009.03.31

Claims (1)

1. Process for obtaining in vitro biomass of Aronia melanocarpa (Michx.) Elliot fruits, which includes cultivating explants obtained from Aronia melanocarpa fruits on Murashige-Skoog nutrient medium, which additionally contains: naphthylacetic acid 3.0…3.5 mg/L; kinetin 0.5 mg/L; sucrose 60 g/L, having pH 5.7, at the same time the cultivation is carried out at a temperature of 18…22°C, with a day-night photoperiod of 16 and 8 hours respectively