Composition of nutrient medium for culturing isolated soybean cells in vitro

Abstract

FIELD: biotechnology.SUBSTANCE: composition of a nutrient medium for culturing isolated soybean cells in vitro, including: Gamborg B5 medium, ascorbic acid, mesoinositol, nicotinic acid, pyridoxine-HCI, thiamine-HCI, sucrose and phytohormones 2,4-D and 6-BAP. The volume of the finished product is calculated for 1 liter: Gamborg B5 medium, including: (NH4)2SO4 – 134.0 mg / l, KNO3 – 2500.0 mg / l, CaCl2×2H2O – 150.0 mg / l, MgSO4×7H2O – 250.0 mg / l, KI – 0.75 mg / l, Na2 EDTA – 37.3 mg / l, FeSO4×7H2O – 28.0 mg / l, H3BO3 – 3.0 mg / l, MnSO4×7H2O – 250.0 mg / l, ZnSO4×7H2O – 2.0 mg / l, Na2MoO4×2H2O – 0.25 mg / l, CuSO4×5H2O – 0.025 mg / l, CoCl2×6H2O – 0.025 mg / l, agar-agar – 8000.0 mg / l; additionally: ascorbic acid – 2.0 mg / l; mesoinositol – 100.0 mg / l; nicotinic acid – 1.0 mg / l; pyridoxine HCl – 1.0 mg / l; thiamine HCl – 10.0 mg / l; sucrose – 25000.0 mg / l; phytohormone 2,4-D – 2.5 mg / l; phytohormone 6-BAP – 1.5 mg / l; distilled water – the rest.EFFECT: invention can be used for culturing isolated soybean cells in vitro.1 cl, 3 tbl, 1 ex

Description

[0001] The invention relates to the field of agriculture, namely to agricultural biotechnology and can be used for cultivating isolated soybean cells in vitro.

[0002] To obtain regenerated plants with a complex of new traits and properties from various plant elements, it largely depends on the composition of the nutrient medium and the concentration of phytohormones and vitamins. (Bespalova, E.S. Soybean regeneration in vitro culture (review) / E.S. Bespalova, K.M. Ershova, Yu.V. Ukhatova / / Proceedings on applied botany, genetics and breeding. - 2021. - Vol. 182, No. 4. - Pp. 148-155. - DOI 10.30901 / 2227-8834-2021-4-148-155. - EDN UDXSNN; Stroeva, N.S. Obtaining Medicago varia regenerant plants by inducing callus formation of leaf explants in vitro culture / N.S. Stroeva, V.G. Darkhanova / / Science and education. - 2017. - No. 1 (85). - Pp. 110-113. - EDN YHLWJL).

[0003] To assess the current patent situation in the area under study, a search of protection documents was conducted for the period from 2000 to 2025.

[0004] In the scientific and available patent documentation reviewed, the authors did not find a nutrient medium composition identical to the proposed nutrient medium composition for cultivating isolated soybean cells in vitro.

[0005] RU Patent No. 2729461 C1 (published August 6, 2020) describes a method for the in vitro surface sterilization of soybeans. The invention relates to the field of biotechnology. It is a method for the in vitro surface sterilization of soybeans, comprising obtaining the beans, sterilizing them with an agent, washing, isolating explants, culturing them in a nutrient medium, and recording them. A distinctive feature of the method is that the beans are sterilized in a solution of the "AKhD-2000 Express" agent for 1.5 minutes, followed by two rinses with sterile distilled water. The agent solution can be reused up to seven times. The proposed invention makes it possible to increase the yield of sterile and viable soybean explants, accelerate the selection process in breeding, and minimize the harmful effects of the sterilizing agent, which will simplify the method of surface sterilization of soybeans in vitro.

[0006] The disadvantage of this method is that the quality of the original beans and seeds used in the experiments is not taken into account: seeds with high germination rates, without signs of damage from diseases and pests, are expected to have high efficiency of introduction into in vitro culture.

[0007] From the article by Rozhanskaya O.A., Gorshkova E.M. / In vitro culture as a source of biodiversity for soybean breeding / / Siberian Bulletin of Agricultural Science. 2019. Vol. 49. No. 4. Pp. 24-31. DOI: 10.26898 / 03708799-2019-4-3 It is known that the tissues of the apexes and cotyledon nodes of the somaclonal line R21 differed from the original variety SibNIIK 315 in increased activity of callus formation, regeneration and shoot growth. Considering that the tissues of the regenerated R21 plants repeatedly went through the regeneration cycle according to the recurrent regeneration protocol, we believe that the high morphogenic activity of the tissues of the R21 line is the result of in vitro autoselection.

[0008] From the article by Bespalov, E.S. Soybean regeneration in vitro culture (review) / E.S. Bespalova, K.M. Ershova, Yu.V. Ukhatova / / Works on applied botany, genetics and breeding. - 2021. - Vol. 182, No. 4. - Pp. 148-155. - DOI 10.30901 / 2227-8834-2021-4-148-155. - EDN UDXSNN it is known that the efficiency of soybean regeneration in vitro culture depends on the content of growth regulators (commonly found - BAP, IAA, kinetin) and vitamins (B5), carbohydrate sources (maltose, sucrose, glucose, fructose) in the nutrient medium, as well as on the genotype and type of explant. In the future development of work on genomic editing of soybean samples, it is most expedient to use cotyledon nodes, cotyledons and stem segments of seedlings obtained from seeds as explants.

[0009] The technical objective of the proposed invention is to expand the range of nutrient medium compositions for cultivating isolated soybean cells in vitro due to the developed nutrient medium composition.

[0010] The technical result is achieved by creating a nutrient medium for culturing isolated soybean cells in vitro by modifying the nutrient medium with components that promote the formation of embryo-like structures and regenerated plants. The advantages of the proposed nutrient medium composition for culturing isolated soybean cells in vitro include the most effective formation of regenerated plants.

[0011] The novelty lies in the fact that the proposed composition of a nutrient medium for culturing isolated soybean cells in vitro and the influence of endogenous factors on these processes are unknown in the prior art. The inventive step lies in the possibility of forming a full-fledged regenerated plant by culturing isolated soybean cells in vitro in the proposed nutrient medium composition.

[0012] The implementation of the proposed invention is achieved by the proposed composition of the nutrient medium for cultivating isolated soybean cells in vitro (Table 1).

[0013] Table 1

[0014] Components of the environment Concentration, mg / l (NH4)2SO4 134,0 KNO3 2500,0 CaCl2×2H2O 150,0 MgSO4×7H2O 250,0 KI 0,75 Na2 EDTA 37,3 FeSO4×7H2O 28,0 H3BO3 3,0 MnSO4×7H2O 250,0 ZnSO4×7H2O 2,0 Na2MoO4×2H2O 0,25 CuSO4×5H2O 0,025 CoCl2×6H2O 0,025 Ascorbic acid 2,0 Mesoinositol 100,0 Nicotinic acid 1,0 Pyridoxine HCl 1,0 Thiamine HCl 10,0 Sucrose 25000,0 Phytohormone 2,4-D 2,5 Phytohormone 6-BAP 1,5 Agar-agar 8000,0

[0015] To obtain 1 liter of nutrient medium for culturing isolated soybean cells in vitro, the remaining volume is made up with distilled water, i.e. the rest is distilled water.

[0016] When implementing the proposed invention, the basic Gamborg medium (Gamborg medium B5) is taken as a basis (Table 2).

[0017] Table 2

[0018] Components of the environment Concentration, mg / l (NH4)2SO4 134,0 KNO3 2500,0 CaCl2×2H2O 150,0 MgSO4×7H2O 250,0 KI 0,75 Na2 EDTA 37,3 FeSO4×7H2O 28,0 H3BO3 3,0 MnSO4×7H2O 250,0 ZnSO4×7H2O 2,0 Na2MoO4×2H2O 0,25 CuSO4×5H2O 0,025 CoCl2×6H2O 0,025 Agar-agar 8000,0

[0019] The distinctive feature of the proposed nutrient medium is the introduction of the following into the composition: ascorbic acid - 2.0 mg / l, mesoinositol - 100.0 mg / l, nicotinic acid - 1.0 mg / l, pyridoxine-HCI - 1.0 mg / l, thiamine-HCI - 10.0 mg / l, sucrose - 25000.0 mg / l and phytohormones: 2,4-D - 2.5 mg / l and 6-BAP - 1.5 mg / l (Table 1).

[0020] This composition and concentrations contribute to the most effective formation of regenerated plants by 3.83-7.50% compared to other nutrient media and concentrations, which is confirmed by the data given in Example 1 of the invention (Table 3).

[0021] Examples of the implementation of the invention of the composition of the nutrient medium for culturing isolated soybean cells in vitro were carried out at the Center for Genomic Selection and Biotechnology of the Belgorod State Agrarian University named after V.Ya. Gorin and confirmed by examples:

[0022] Example 1

[0023] The nutrient medium for cultivating isolated soybean cells is prepared on the basis of the basic nutrient medium of Gamborg (B5), using the appropriate amount of stock solutions of salts and vitamin solutions for its preparation. Before preparing the basic Gamborg nutrient medium (B5), 25,000.0 mg / l of sucrose are placed in a 1-liter beaker, distilled water is added to make up to 0.3 l, and after it has dissolved, the required amounts of stock solutions of salts and vitamins are added according to the prescription for B5 - the basic Gamborg nutrient medium, except for agar-agar, then the following are added to the composition: ascorbic acid - 2.0 mg / l, mesoinositol - 100.0 mg / l, nicotinic acid - 1.0 mg / l, pyridoxine-HCL - 1.0 mg / l, thiamine-HCL - 10.0 mg / l, and phytohormones: 2,4-D - 2.5 mg / l and 6-BAP - 1.5 mg / l. Check the pH of the solution, which should not exceed 5.8, and adjust if necessary with a 0.1% solution of KOH (potassium hydroxide).In another beaker, heat agar-agar, previously soaked at 8000.0 mg / L in 0.2 L of distilled water to swell. Mix the agar-agar with the prepared solution and bring the total volume to 1 L with distilled water. Pour the prepared nutrient medium into glass test tubes one-third full, seal with cotton stoppers, and sterilize in an autoclave for 45 minutes at 121°C.

[0024] Under sterile conditions in a laminar flow hood, isolated soybean cells are placed into test tubes on a nutrient medium using sterile instruments, spreading them evenly across the entire surface of the medium. Callus formation and the emergence of normally developed and functional plants using a modified nutrient medium and observing all conditions are 3.83-7.50% higher than comparable methods (Table 3).

[0025] Table 3

[0026] Composition of the nutrient medium and growth regulators Isolated soybean cells planted, pcs. New growths received pcs. % Murashige-Skoog base medium without hormones + ascorbic acid 2.0 mg / l 600 2,0 0,33 Basic Murashige-Skoog medium, ascorbic acid 2.0 mg / l + mesoinositol - 100.0 mg / l, nicotinic acid - 1.0 mg / l + pyridoxine HCl - 1.0 mg / l + thiamine HCl - 10.0 mg / l + sucrose - 25,000 mg / l + 2.4 D - 1.5 mg / l + 6-BAP - 0.5 mg / l 600 13,0 2,17 Murashige-Skoog base medium, ascorbic acid 2.0 mg / l + mesoinositol - 100.0 mg / l, nicotinic acid - 1.0 mg / l + pyridoxine HCl - 1.0 mg / l + thiamine HCl - 10.0 mg / l + sucrose - 25,000 mg / l + 2.4 D - 2.5 mg / l + 6-BAP - 1.5 mg / l 600 22,0 3,67 Basic Gamborg medium (B5) without hormones + ascorbic acid 2.0 mg / l 600 8,0 1,33 Gamborg's basic medium (B5), ascorbic acid 2.0 mg / l + mesoinositol - 100.0 mg / l, nicotinic acid - 1.0 mg / l + pyridoxine HCl - 1.0 mg / l + thiamine HCl - 10.0 mg / l + sucrose - 25,000 mg / l + 2.4 D - 1.5 mg / l + 6-BAP - 0.5 mg / l 600 22,0 3,67 The proposed nutrient medium for culturing isolated soybean cells in vitro: basic Gamborg medium (B5), ascorbic acid 2.0 mg / l, mesoinositol - 100.0 mg / l, nicotinic acid - 1.0 mg / l, pyridoxine-HCl - 1.0 mg / l, thiamine-HCl - 10.0 mg / l, sucrose - 25000.0 mg / l, phytohormones 2,4 D - 2.5 mg / l, and 6-BAP - 1.5 mg / l 600 45,0 7,5

[0027] Thus, the obtained composition of the nutrient medium for cultivating isolated soybean cells in vitro solves the technical problem, which consists in expanding the range of nutrient media for cultivating isolated soybean cells, in the most effective formation of regenerated plants.