RU2479991C1 - Method of determining freeze resistance of winter barley - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to selection of plants and can be used in laboratory condition for express evaluation of freeze resistance of winter barley. To evaluate the material under study, the mellow grain is taken, crushed, and oilseed meal is obtained, which is extracted with buffer A containing 0.5 M Tris-HCl, pH 8.5, 0.05 M MgCl₂; 0.25 M sucrose, the extract is centrifuged, the obtained supernatant liquid is poured into a measuring container, and according to its volume the grain hygroscopic property is determined, and the freeze resistance is judged on it: the higher the hygroscopic property, the higher the freeze resistance.

EFFECT: invention enables to increase the speed of evaluation of freeze resistance of winter barley.

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Description

The invention relates to agriculture, in particular to plant breeding, and can be used in laboratory conditions for the rapid assessment of frost resistance, both breeding material and varieties of winter barley.

Currently, there are many methods for assessing varieties and plants on the basis of frost resistance, which are based, for example, on direct freezing of plants in refrigerators and during field trials (Shevtsov V.M., Serkin N.V., Fomenko N.P., Kostyanoy D.V. Problems of increasing winter hardiness of winter barley in the North Caucasus // Reports of the Russian Academy of Agricultural Sciences, 2007, No. 4, p.3-5).

This method is time consuming, expensive and time consuming.

A known method for assessing the frost resistance of winter wheat plants by determining the content of NH ₄ ⁺ ions in an aqueous extract from hatching seeds. An aqueous extract was prepared by immersing hatching seeds for a day in distilled water at + 2 ° С, after which NH ₄ ⁺ ions were determined in a drained solution using ion-selective electrodes per unit mass of dry matter of seeds weighed after the release of NH ₄ ⁺ ions and this indicator was evaluated frost resistance of plants in comparison with the variety (Patent No. 2025941 from 01.01.1995, the Method for assessing frost resistance of winter wheat plants. Musich VN; Cornelli B.I.). This method has been tested only on wheat, but not on barley. The disadvantage of this method is that it is more time-consuming, long-term and more expensive, requiring conditions for germination of seeds and equipment for measuring ions.

A known method for the diagnosis of the physiological state of crops, according to which the frost resistance of winter barley is determined by changing the difference in the percentage yield of poly-A ⁺⁺ -mRNA from plants grown for 8 hours at 4 ° C and plants grown at 20 ° C. The effect on the seedlings is a stress factor. First, the total RNA preparation is isolated, then the mRNA preparation is isolated in two affinity chromatography cycles and the percentage yield of poly-A ⁺⁺ -mRNA (second cycle) from poly-A ⁺ -mRNA (first cycle) is calculated and judged by the difference in the values of the experimental and control variants on the physiological state of plants (Patent No. 2084133 dated July 20, 1997. A method for diagnosing the physiological state of crops. Plotnikov V.K., Bakaldina N. B., Bibishev V. A.). This method has several disadvantages. This is a time-consuming, complex and lengthy process of technological preparation for analysis and analysis itself.

The present invention solves the problem of developing an express method for determining the degree of frost resistance of winter barley in the laboratory.

The purpose of the invention: simplification of the method, increasing efficiency, reducing the complexity.

The goal is achieved as follows. To determine the degree of frost resistance of winter barley, a number of factors affect it and evaluate the data obtained on mature grain, which is ground, the resulting meal is extracted with buffer A containing 0.5 M Tris-HCl, pH 8.5; 0.25 M sucrose and 0.05 M MgCl ₂ . The extract is centrifuged and the resulting supernatant is poured into a measuring tank. The hygroscopicity of the grain is determined by the volume of the supernatant, and frost resistance is judged by it: the higher the hygroscopicity (i.e., the lower the volume of the supernatant), the higher the frost resistance.

The technical result in the proposed method is achieved by the fact that they affect winter barley by a number of factors, evaluate the obtained data according to the proposed invention, expose the mature grain, which is crushed and extracted with buffer A containing 0.5 M Tris-HCl, pH 8.5; 0.25 M sucrose and 0.05 M MgCl ₂ , then centrifuged and the hygroscopicity of the grain is determined by the volume of the supernatant, which is used to judge frost resistance. The higher the hygroscopicity of the grain (i.e., the lower volume of the supernatant), the higher the frost resistance

A comparative analysis of the proposed technical solution with the closest analogues allows us to conclude that the claimed method meets the patentability criterion of "novelty."

Studying the state of the art in the process of conducting a patent search on all types of information publicly available in the press, we found that the method for determining the degree of frost resistance of winter barley, which includes the claimed set of features for specialists in the field of plant breeding, does not explicitly follow from the existing prior art .

Our research has established the relationship between the frost resistance of varieties of winter barley and the water-holding capacity (hygroscopicity) of ripe grain. Based on the foregoing, we can conclude that the claimed invention meets the patentability criterion of "inventive step".

The claimed technical solution meets the patentability criterion of "industrial applicability", because it can be used in agriculture, in particular in breeding, and, in addition, the description of the invention provides means and methods by which it is possible to implement a technical solution in the form in which it is described in the claims. The studies were carried out on plants of 10 varieties of winter barley of domestic and foreign selection, simultaneously freezing them in a refrigerator at critical temperatures of -12 ° and -14 ° C for 24 hours with a “closed list” when the researchers did not know the real frost resistance of the varieties during the analysis.

Table Comparison of the results of diagnostics of frost resistance of winter barley varieties by the degree of hygroscopicity of mature grain meal and by the degree of plant survival during freezing in the freezer The order of varieties as grain hygroscopicity decreases The amount of supernatant,% of the initial volume of extraction buffer The number of surviving plants with direct freezing,% of the total Pharaoh 51 84 Belogorych 51 79 NB 03435 56 76 DAI 03940 58 twenty Sarmat 58 10 Premuda 58 8 Mercedes 60 7 Zinderella 63 9 Carola 64 8 Lomerit 65 10

An example of the method

To assess hygroscopicity, the grain was crushed (milled) in a mill-grinder and the resulting meal was extracted with buffer A containing 0.5 M Tris-HCl, pH 8.5; 0.05 M MgCl ₂ ; 0.25 M sucrose in a ratio of 1: 3 (weight: volume). After centrifugation (5000 rpm, 5 minutes), the supernatant was poured into a graduated cylinder. The volume of the obtained liquid was taken as a value inversely proportional to the hygroscopicity of the grain, i.e. the larger the volume of the supernatant, the lower the hygroscopicity of the grain (ml ^-1 ). To obtain statistically significant results, hygroscopicity was measured 8-12 times.

The table shows that a comparison of the results of assessing the degree of frost resistance by hygroscopicity of meal of mature barley grain and data of direct freezing of plants in the freezer with a “closed list”, i.e. when the researchers did not know the real frost resistance of the varieties during the analysis, showed a coincidence of readings at the level of 80-90% a).

More frost-resistant varieties of winter barley were more hygroscopic. So the meal of low-frost resistant variety Lomerit gives 65% of the initial volume of the extraction buffer, while the high-frost resistant variety Pharaoh gives only 51% under the same extraction conditions.

A comparative analysis of the data showed that the claimed method is very simple to implement and makes it possible to quickly and efficiently assess the degree of frost resistance of varieties of winter barley in the laboratory.

Thus, the claimed invention ensures the achievement of the goal and the solution of the task.

Claims (1)

A method for determining the degree of frost resistance of winter barley, including exposure to a number of factors and evaluating the data obtained, characterized in that mature grain is taken as the studied material, it is ground, and meal is obtained, which is extracted with buffer A containing 0.5 M Tris-HCl, pH 8.5; 0.05 M MgCl ₂ ; 0.25 M sucrose, the extract is centrifuged, the resulting supernatant is poured into a measuring tank, grain hygroscopicity is determined by its volume and frost resistance is judged by it: the higher the hygroscopicity, the higher the frost resistance.