RU2652390C1 - Method for determination of potential winter hardiness of soft winter wheat - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to agriculture. Method includes seed selection for analysis, seedling production, and evaluation of winter hardiness characteristics in seedlings of seedling tissues. In this case, the selected seeds are germinated by keeping them at a temperature of 2–5 °C for 2–4 weeks, then they receive cuttings from the tillering node 20–40 mcm thick and the fluorescence intensity of the seedling tissues is measured at 365 nm, 470 nm and 546 nm wavelengths. Potentially winter hardy is considered sprouts with increased fluorescence intensity. To measure the fluorescence, the AxioImager Z1 fluorescence microscope is used.

EFFECT: method makes it possible to shorten the duration and simplify the process of obtaining a selection material with increased potential winter hardiness in the early stages of selection work.

1 cl, 2 dwg, 2 tbl, 1 ex

Description

The invention relates to agriculture, namely, to assess the potential winter hardiness of winter soft wheat plants, and can be used in the selection of winter-hardy varieties of winter grain crops.

Winter cereal crops are the most productive component of agrocenoses, but in the Russian Federation, large losses of winter grain crops occur due to damage and death of plants under adverse wintering conditions, the main of which is the effect of low temperatures in the late autumn, winter and early spring. The task of determining the winter hardiness of winter crops in a simple and quick way is relevant.

A known method for determining the frost resistance of winter wheat plants by freezing whole plants at a temperature gradually decreasing with an interval of one day (-7 ° C, -10 ° C, -13 ° C, -16 ° C). Then they are transferred to thaw in a chamber with a temperature of 2 ° C. After a day, these plants are planted in the soil, and after three weeks, the number of overgrown plants is taken into account. Frost resistance is expressed as a percentage of the number of those frozen at a certain temperature (Aleksidze G.Ya., Korolev N.P., Semenov I.L., Vyskrebentseva E.I. // Plant Physiology. 1983. T. 30. V. 6. C . 1069).

The disadvantage of this method is the duration and complexity.

A known method for determining the frost resistance of crops by freezing leaf fragments, followed by staining with resorcinol, which stains cold-killed leaf fragments in dark blue, while freezing time is used as an estimate of frost resistance, in which leaf fragments are irreversibly damaged by cold (RU patent 2370942 C1, op. October 27, 2009).

The disadvantage of this method is the need for growing adult plants.

Closest to the claimed method, the prototype, is a method for selecting winter-hardy winter wheat plants, including sowing, growing, sampling, their evaluation according to breeding and economic characteristics, where sampling is carried out in the phase of wax ripeness of seeds, and medium-sized plants are selected as winter-hardy - 81-110 cm with a reddish color of the stems, and the assessment of the characteristics is carried out according to the content of flavonoids, and plants with a flavonoid content of more than 8 mg / g are selected as winter-hardy. Selected plants can be used in winter wheat breeding to create winter-hardy varieties of this crop (patent RU 2575100 C1, October 10, 2016).

The disadvantage of this method is the need for growing samples in the field, which significantly increases the time to assess the sign of severity of winter hardiness of winter wheat samples.

The objective of the invention is to develop a simple and less durable method that allows in laboratory conditions to determine the potential winter hardiness of winter soft wheat plants.

Technical result: reducing the duration and simplifying the process of obtaining breeding material with increased potential winter hardiness in the early stages of breeding.

The technical result is achieved by the proposed method, which consists in the following.

Varieties of winter soft wheat are selected for analysis, soaked in water and allowed to bend. Stuck grains are rolled up in filter paper and placed in a refrigerator at a temperature of 2-5 ° C. After two to four weeks, the seedlings are used for analysis.

For this, on a freezing microtome (Cryostat NM 550), sections of a tillering site of a seedling (i.e., the base of a seedling) 20–40 μm thick are obtained and then the fluorescence intensity of the seedling tissue (without any color) is determined using a fluorescence microscope when fluorescence is excited by radiation with lengths waves 365, 470 and 546 nm. Comparing the results, genotypes with increased fluorescence intensity, with increased winter hardiness, are selected.

The defining differences of the proposed method, in comparison with the prototype, are:

1) for analysis in the laboratory using two to four week old seedlings, which allows to accelerate the selection process (in the prototype, sampling is carried out at later phases of plant development);

2) sections of tillering nodes are prepared from seedlings and potential winter hardiness is assessed by the fluorescence intensity of seedling tissues measured using a fluorescence microscope with wavelengths of 365, 470 and 546 nm, while samples with increased fluorescence are selected as winter hardy, which can significantly simplify and speed up the process selection by reducing the time to obtain estimates and a significant reduction in the complexity of analyzes.

The intensity of fluorescence of seedling tissues, directly related to the amount of aromatic glycosides in the seedlings, which contribute to the gelification of cell juice and freezing of water in an amorphous state, without the formation of ice crystals, which are the main damaging factor in the winter (Cheyniera., Et al Plant phenolics: Recent advances on their biosynthesis, genetics, and ecophysiology. Plant Physiology and Biochemistry. 2013. V. 72. P. 1-20).

All flavonoids are optically active, able to fluoresce in UV light, have characteristic UV spectra characterized by the presence of two absorption maxima, and IR spectra.

The proposed method is illustrated by the following example of a specific implementation.

Example

FF and OO genotypes of winter common wheat at the locus of aromatic alcohol dehydrogenase, which differ in winter hardiness, were used as samples for analysis. Table 1 presents the data on overwintering of the FF and OO genotypes for three seasons (2013/2014, 2014/2015 and 2015/2016), where winter hardiness was determined as the number of overwintered plants in relation to the number of seeds sown (in brackets - as a percentage of the number of seeds sown). The nonparametric Pearson criterion χ ² allows us to evaluate the significance of differences between the genotypic groups of FF and OO.

Stuck caryopsis in the amount of 10 pieces of each genotype was rolled up in filter paper and placed in a refrigerator at a temperature of 2-5 ° C. After two to four weeks, the seedlings in the tillering phase were used for analysis. Five seedlings, selected according to the best phenotypic characteristics, received tillering sections on a 30 micron thick freezing microtome and then determined the fluorescence intensity of the seedling tissue (without any color), on an Axiolmager Z1 fluorescence microscope with AroTote attachment and AxioVision program (Zeiss company) fluorescence radiation with wavelengths of 365, 470 and 546 nm. On sections, fluorescence was measured visually and by recording the numerical values of the fluorescence intensity (Table 2). Comparing the results, genotypes with increased fluorescence and, accordingly, with a high content of aromatic glycosides were selected.

From table 2 it can be seen that the more winter-hardy FF genotype of winter common wheat has increased fluorescence in the blue and green range and slightly larger in the red range. These results allow the use of fluorescence intensity to select potentially more winter-hardy genotypes.

In FIG. 1 shows an autofluorescence recording of a slice of the OO genotype, and FIG. 2 - FF genotype. In FIG. 1, 2 it is seen that the FF genotype has a pronounced increased fluorescence in the blue spectrum and for it there are jumps in the blue spectrum from 0 to 450 nm, and the OO genotype has a smaller amplitude - from 0 to 65 nm. There are also differences in the red and green spectrum.

Using the proposed method will allow:

1) to simplify the assessment of winter hardiness of genotypes (samples) of winter forms due to the exclusion of manual labor (prototype), because in the present method, sections of seedlings are made using a cryotome, and the fluorescence intensity of the seedling tissue is measured using a fluorescence microscope;

2) speed up the analysis process, because the sampling is carried out in the phase of two to three week old seedlings, and in the prototype in the later phase of the wax ripeness of seeds;

3) reduce the cost of analysis, since the inventive method is intended for use in laboratory conditions that do not require the cost of sowing in the field, for sowing work (including the use of pesticides and fungicides).

A method for determining the potential winter hardiness of winter soft wheat plants

Claims (2)

1. A method for determining the potential winter hardiness of winter soft wheat plants, including selecting seeds for analysis, obtaining seedlings and evaluating winter hardiness signs in the tissue sections of seedlings, characterized in that the selected seeds are germinated by holding them at a temperature of 2-5 ° C for 2-4 weeks, then sections of tillering nodes with a thickness of 20-40 microns are obtained from them and the fluorescence intensity of seedling tissues at wavelengths of 365 nm, 470 nm and 546 nm is measured, and seedlings with increased intensity are considered potentially winter-hardy fluorescence. 2. The method according to p. 1, characterized in that the AxioImager Z1 fluorescence microscope is used to measure fluorescence.

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