Modified composition of nutrient medium for inducing formation of soybean regenerated plants in vitro

Abstract

FIELD: biotechnology.SUBSTANCE: modified composition of a nutrient medium for inducing the formation of soybean regenerated plants in vitro, including ammonium nitrate (NH4NO3) – 1650 mg / l; potassium nitrate (KNO3) – 1900 mg / l; calcium chloride dihydrate (CaCl2×2H2O) – 440 mg / l; magnesium sulfate (MgSO4×7H2O) – 370 mg / l; potassium phosphate (KH2PO4) – 170 mg / l; disodium salt of ethylenediaminetetraacetic acid (Na2 EDTA) – 37.3 mg / l; ferrous sulfate (FeSO4×7H2O) – 27.95 mg / l; boric acid (H3VO3) – 6.2 mg / l; manganese sulfate (MnSO4×4H2O) – 22.3 mg / l; zinc sulfate (ZnSO4×H2O) – 8.6 mg / l; sodium molybdate (Na2MoO4×2H2O) – 0.25 mg / l; copper sulfate (CuSO4×5H2O) – 0.025 mg / l; thionyl chloride (CoCl2×H2O) – 0.025 mg / l; agar-agar – 7000 mg / l, according to the invention the following components are additionally introduced: glycine – 1.0 mg / l; mesoinositol – 120 mg / l; ascorbic acid – 3.0 mg / l; nicotinic acid – 1.0 mg / l; pyridoxine-HCl – 1.5 mg / l; thiamine-HCl – 1.0 mg / l; biotin – 0.03 mg / l; sucrose – 25000 mg / l; 6-BAP – 1.0 mg / l; indole-butyric acid (IBA) – 1.5 mg / l; starch – 9000 mg / l; activated carbon – 3.0 mg / l; water is the rest.EFFECT: expanding the range of nutrient medium compositions for inducing the formation of soybean regenerated plants in vitro.1 cl, 3 tbl

Description

[0001] The invention relates to the field of agriculture, namely to agricultural biotechnology.

[0002] To assess the current patent situation, a search of patent protection documents was conducted for the period from 1992 to 2021. The documents were analyzed in the following areas: agricultural biotechnology, soybean breeding and seed production, cytology, and genetics.

[0003] RU patent No. 17466951 A1 (published 16.07.1992) is known, which describes a method for isolating soybean anthers, which consists in separating the anthers from the somatic tissue by preliminary filtration through a sieve followed by flotation. The essence of the invention is that buds are selected, sterilized in a 0.1% solution of HgCl2 for 10 minutes, placed in a container with a liquid whose osmotic pressure is equal to or close to the osmotic pressure inside the microspores, for example, a physiological solution (8.5 g / l NaCl), crushed using a mixer, primary cleaning of the anthers from somatic tissue is carried out on a sieve and the crushed tissue of the buds is separated from the anthers by electroflotation, then the anthers are transferred to a sieve, where they are washed with a fresh physiological solution (8.5% g / l NaCl).This improves the quality of anther purification and reduces anther loss during the extraction process, as the size of the gas bubbles during electroflotation corresponds to the size of the soybean anthers. In this case, the gas bubbles do not merge with each other, as each carries a charge of the same sign on its surface.

[0004] The disadvantage of this method is that a 0.1% HgCl2 solution is used as a sterilizing agent, which is toxic both for the operator performing the sterilization process and for the introduced explant, which leads to tissue necrosis and death of the introduced parts.

[0005] From the article by Vishnyakov, M. A. Requirements for the source material for soybean breeding in the context of modern biotechnologies / M. A. Vishnyakova, I. V. Seferova, M. G. Samsonova / / Agricultural biology. - 2017. - Vol. 52, No. 5. - P. 905-916. - DOI 10.15389 / agrobiology.2017.5.905rus. - EDN ZRXNYH it is known that the creation of early maturing varieties for all soybean-growing regions of the country as one of the urgent needs and the search for the necessary source material have long been in the center of attention of the curators of the VIR soybean collection (decoding). For all the traits considered in the review, modern data on their genetic determinism, the degree of study of the genomic organization of the corresponding genes, information on certain QTL and their mapping are provided.It was concluded that the main requirement for the source material for modern soybean breeding is that the range of crop use areas should be based on a diversity of specialized varieties with specified parameters for specific application purposes and with different adaptive capabilities.

[0006] A technical solution is known from the article Evaluation of the efficiency of introducing soybean samples (Glycine max (L.) Merr.) from the VIR collection into in vitro culture / E.S. Korshikova, K.M. Ershova, Yu.A. Moksheninova, Yu.V. Ukhatova / / Works on applied botany, genetics and breeding. - 2021. - Vol. 182, No. 4. - P. 137-142. - DOI 10.30901 / 2227-8834-2021-4-137-142. - EDN FWTFEJ. During the work, the influence of various seed sterilization techniques was assessed (single-stage, using a commercial bleach, and two-stage, combining the effects of a chlorine-containing preparation and hydrogen peroxide), the influence of the type of explant (epicotyls, hypocotyls, cotyledon nodes, segments of cotyledon leaves) and the phytohormonal composition of the nutrient medium were assessed: (1) MS + 1.13 mg / l BAP + 0.5 mg / l GA and (2) MS + 1 mg / l BAP + 0.1 mg / l IAA).The conducted research revealed that the two-stage seed sterilization method is most effective during the initiation stage of in vitro soybean culture; hypocotyls, epicotyls, and cotyledon nodes exhibit the highest callus formation capacity in both nutrient media types. A disadvantage of this method is that the nutrient medium composition for isolated anthers is not indicated, only for vegetative elements.

[0007] The technical objective of the proposed invention is to expand the range of nutrient medium compositions for inducing the formation of soybean regenerated plants in vitro.

[0008] The technical result is achieved by modifying the basic nutrient medium by introducing new elements into the composition that have a beneficial effect on the formation of soybean regenerated plants in vitro.

[0009] Implementation of the proposed invention.

[0010] To create a modified composition of the nutrient medium for inducing the formation of soybean regenerated plants in vitro, the basic Murashige-Skoog nutrient medium is taken as a basis (Table 1), to which the following components are additionally included: glycine - 1.0 mg / l; mesoinositol - 120 mg / l; ascorbic acid - 3.0 mg / l; nicotinic acid - 1.0 mg / l; pyridoxine HCl - 1.5 mg / l; thiamine HCl - 1.0 mg / l; biotin - 0.03 mg / l; sucrose - 25,000 mg / l; 6 - BAP-1.0 mg / l; indole butyric acid (IBA) - 1.5 mg / l; starch - 9000 mg / l; activated carbon - 3.0 mg / l; water - the rest (Table 2). Growing regenerated plants is carried out in cultivation vessels

[0011] The development of regenerated soybean plants in a modified nutrient medium is 58% higher than the development of plants grown in the basic Murashige-Skoog nutrient medium (Table 3)

[0012] Table 1 - Classic composition of Murashige-Skoog nutrient medium

[0013]

[0014] Table 2 - Modified nutrient medium for inducing the formation of soybean regenerated plants in vitro, pH 5.8

[0015]

[0016] Development of a modified nutrient medium composition for inducing the formation of soybean regenerated plants in vitro was carried out at the Center for Genomic Selection and Biotechnology of the V.Ya. Gorin Belgorod State Agrarian University.

[0017] Example 1.

[0018] The work is carried out in a laminar flow cabinet. To obtain regenerated plants, soybean embryoid structures are placed on a nutrient medium prepared in a 1-liter container with the following composition: ammonium nitrate (NH4NO3) - 1650 mg / l; potassium nitrate (KNO3) - 1900 mg / l; calcium chloride dihydrate (CaCl2×2H2O) - 440 mg / l; magnesium sulfate (MgSO4×7H2O) - 370 mg / l; potassium phosphate (KH2PO4) - 170 mg / l; disodium salt of ethylenediaminetetraacetic acid (Na2EDTA) - 37.3 mg / l; iron sulfate (FeSO4×7H2O) - 27.95 mg / l; boric acid (H3BO3) - 6.2 mg / l; manganese sulfate (MnSO4×4H2O) - 22.3 mg / l; zinc sulfate (ZnSO4×H2O) - 8.6 mg / l; sodium molybdate (Na2MoO4×2H2O) - 0.25 mg / l; copper sulfate (CuSO4×5H2O) - 0.025 mg / l; thionyl chloride (CoCl2×H2O) - 0.025 mg / l; agar-agar - 7000 mg / l; glycine - 1.0 mg / l; mesoinositol - 120 mg / l; ascorbic acid - 3.0 mg / l; nicotinic acid - 1.0 mg / l; pyridoxine HCl - 1.5 mg / l; thiamine-HCl - 1.0 mg / l; biotin - 0.03 mg / l; sucrose - 25000 mg / l; 6-BAP-1.0 mg / l;indolebutyric acid (IBA) - 1.5 mg / l; starch - 9000 mg / l; activated carbon - 3.0 mg / l; the missing volume to 1 liter is filled with water.

[0019] The survival rate of regenerated plants on the modified nutrient medium was 3-6% higher than on the basic Murashige-Skoog medium.

[0020] Table 3 - Yield of soybean regenerated plants under in vitro conditions

[0021]

[0022] The resulting modified composition of the nutrient medium solves the technical problem, which consists in expanding the range of nutrient medium compositions for inducing the formation of soybean regenerated plants in vitro.

Claims

Modified composition of nutrient medium for inducing formation of soybean regenerated plants in vitro, including ammonium nitrate (NH4NO3) - 1650 mg / l; potassium nitrate (KNO3) - 1900 mg / l; calcium chloride dihydrate (CaCl2×2H2O) - 440 mg / l; magnesium sulfate (MgSO4×7H2O) - 370 mg / l; potassium phosphate (KH2PO4) - 170 mg / l; disodium salt of ethylenediaminetetraacetic acid (Na2EDTA) - 37.3 mg / l; iron sulfate (FeSO4×7H2O) - 27.95 mg / l; boric acid (H3BO3) - 6.2 mg / l; manganese sulfate (MnSO4×4H2O) - 22.3 mg / l; zinc sulfate (ZnSO4×H2O) - 8.6 mg / l; sodium molybdate (Na2MoO4×2H2O) - 0.25 mg / l; copper sulfate (CuSO4×5H2O) - 0.025 mg / l; thionyl chloride (CoCl2×H2O) - 0.025 mg / l; agar-agar - 7000 mg / l, characterized in that the following components are additionally added: glycine - 1.0 mg / l; mesoinositol - 120 mg / l; ascorbic acid - 3.0 mg / l; nicotinic acid - 1.0 mg / l; pyridoxine HCl - 1.5 mg / l; thiamine HCl - 1.0 mg / l; biotin - 0.03 mg / l; sucrose - 25000 mg / l; 6-BAP - 1.0 mg / l;Indolebutyric acid (IBA) - 1.5 mg / l; starch - 9000 mg / l; activated carbon - 3.0 mg / l; water - the rest.;

Images