RU2688464C1 - Method for non-destructive diagnostics of plant functional status ex vitro and in vitro - Google Patents

Abstract

FIELD: agriculture; forestry.SUBSTANCE: present invention relates to experimental biology, plant growing, agriculture and forestry. Method involves measuring light-scattering dynamics of photosynthesizing plant tissue in the process of illumination with monochromatic optical radiation of the blue region of the spectrum in the zone of the first absorption maximum of chlorophyll 460–480 nm. Record of chlorophyll fluorescence intensity drop of leaf area 2 mm is recordedduring first illumination with monochromatic optical radiation of blue spectrum region with power density higher than photosynthesis saturation of 600–900 W/mfor 30–180 s. Then, illumination is switched off for 30–60 s and after this dark recovery pause in the same zone of object measurements light intensity decrease again during second illumination is registered with monochromatic optical radiation of blue area of spectrum of the same power density during 30–180 s. Photosynthetic activity and resistance to photoinhibition are evaluated by the values of K= (I-I) Iand K= (I-I)/(I-I), where Iis maximum intensity of fluorescence of chlorophyll per 1–2 s of first exposure; I- steady-state level of fluorescence intensity of chlorophyll for 30–180 s of first exposure; I- maximum fluorescence intensity of chlorophyll for 1–2 s of second illumination; I- stationary level of fluorescence intensity of chlorophyll by 30–180 s of second illumination. At that, higher values of Kand K, the better the functional state of plants.EFFECT: method provides higher efficiency and reduced labour intensity of determining the functional state of plants ex vitro and in vitro.1 cl, 1 dwg, 1 tbl, 1 ex

Description

The invention relates to experimental biology, crop production, biotechnology, agriculture and forestry and can be used to assess the functional state of plants ex vitro and in vitro, including the optimization of growing conditions, storage, as well as to identify the degree of plant resistance to various adverse factors habitat.

Optical methods for assessing the functional state of plants are known, based on recording absorption spectra or reflection / transmission coefficients at certain wavelengths or fluorescence parameters of chlorophyll-containing tissues [1–4]. The functional state is judged by the differences in optical parameters obtained from measurements of test and control plants. The most accurate determination of the functional state of plants is based on comprehensive information about photosynthetic activity and its resistance to photoinhibition, but none of the known devices allows such assessments to be made in a single measuring cycle. To do this, it is necessary to carry out a time-consuming procedure for measuring optical parameters after dark adaptation of plants, then illuminate them for several hours with intense light, above the saturation threshold of photosynthesis, then also carry out a dark adaptation process for plants for several hours, and then measure optical parameters again. There is a method of assessing the functional state of plants according to the criteria of photosynthetic activity and resistance to photodestruction, based on measuring the dynamics of light scattering under the influence of blue and red radiation [5]. The implementation of this method requires precise alignment of the emitters, which is difficult to ensure constructively, the blue and red emitter illumination zones do not coincide, which leads to an incorrect assessment of the functional state of the plants. To obtain reliable information on the resistance to photodegradation of healthy plants, it is necessary to resort to rather long exposures of objects with red radiation (over 40 minutes), which makes it unacceptable to use the method for mass assessment of plants, for example, when optimizing agrotechnical growing conditions. In addition, the implementation of the analogue method involves direct contact of the sheet of the plant under study with radiators and is unacceptable for assessing the functional state of plants in an in vitro culture without compromising sterility.

The purpose of the invention is to reduce the complexity of determining the functional state of plants ex vitro and in vitro and increase its effectiveness by quantifying the photosynthetic activity of chlorophyll-containing plant tissues and their resistance to photoinhibition in a single measuring cycle.

The method is as follows. A drop in the fluorescence intensity of chlorophyll (PC) of the leaves during the first exposure to monochromatic optical radiation of the blue spectral region (in the zone of the first maximum absorption of chlorophyll 460 ... 480 nm) is recorded with a power density above the saturation of photosynthesis (600 ... 900 W / m ² ) for 30 ... 180 seconds, then the illumination is turned off for 30 ... 60 seconds and after this dark recovery pause, in the same measurement zone of the object, the decrease in the PC intensity during the second illumination is again recorded by monochromatic optical radiation of the blue region the spectrum of the same power density for 30 ... 180 seconds. The duration of the highlights is determined by the time required for the signal to go to a stationary level.

About photosynthetic activity is judged by the relative amplitude of the decrease in the intensity of PC during the first exposure by optical radiation of the blue region of the spectrum, and its resistance to photo-inhibition - by the ratio of the amplitudes of the decrease in the intensity of PC in the course of the second and first exposures. Quantitatively, photosynthetic activity and its resistance to photoinhibition are determined by indicators K of _FSA and _UFI , which are calculated by the following formulas:

- максимальная интенсивность ФХ на 1…2 секунде первой засветки;

- стационарный уровень интенсивности ФХ на 30…180 секунде первой засветки;

- максимальная интенсивность ФХ на 1…2 секунде второй засветки;

- стационарный уровень ФХ на 30…180 секунде второй засветки.Where

- the maximum intensity of PC at 1 ... 2 seconds of the first exposure;

- stationary level of PF intensity at 30 ... 180 seconds of the first exposure;

- the maximum intensity of PC on 1 ... 2 seconds of the second illumination;

- stationary level FH at 30 ... 180 seconds of the second illumination.

At the same time, photosynthetic activity and its resistance to photoinhibition are judged by the magnitude of the K _FSA and _UFI indicators, respectively. The higher the value of these indicators - the higher the photosynthetic activity and its resistance to photoinhibition.

Example. Lemon leaves with a high level of the functional state of the photosynthetic apparatus were used for measurements. For this, measurements were made of the potential quantum yield Y of photosystem 2, the magnitude of which is directly proportional to the activity of photosynthesis. For the study selected leaves with a Y value of more than 0.74 Rel. units, which were divided into 3 groups. The first group — the control (variant A), the second group (variant B) —was heat treated at + 52 ° C for 5 minutes, which led to a slight inhibition of the photosystem-2 of the photosynthesizing apparatus, and the third group (variant C) was subjected to 15 minutes heat treatment at + 56 ° C, which led to a significant inhibition of the functions of photosystem-2. Further, the leaves of all variants underwent the following measurement procedure. After a dark 30 minute adaptation, a small area of a leaf with an area of 2 mm ² was subjected to illumination by optical radiation with a wavelength of 475 nm and a power density of 800 W / m ² , during which the change in the PC intensity in the reflection mode was recorded. The duration of the illumination lasted 60 seconds, after which the source was turned off for 60 seconds. After a dark pause, in the same zone of the sheet, for 60 seconds, the dynamics of changes in the intensity of PC were recorded during the second cycle of illumination with blue radiation and then the parameters K _FSA and K _{UVI were} determined by formulas 1 and 2 (table 1). Typical qualitative curves (Fig. 1) have the following features of the response of the PSA to flare, depending on the functional state:

- in version “A” with a high functional state of the PSA of the control leaves (Fig. 1A), a rapid decrease of PC in the first seconds of exposure to blue light is observed, which is partially restored after a dark pause. At the same time, it should be noted that the relative amplitude of the intensity of the PCs (K indicator of _PSA ) is restored within 60 seconds of the dark pause by 92.72% (from 0.8106 to 0.7516 o. Units), while the absolute amplitude of the difference (the K indicator _UVI ) - only by 55.75% (from 43.49 to 24.25 conventional units);

- in variant “B” (Fig. 1B), the absolute differential of PC in the process of the first exposure is somewhat less, and after the dark pause it recovers significantly worse - only by 27.42%. At the same time, the _KSA K indicator both in the first and in the second exposure cycle remains rather high (0.7993 and 0.6215, respectively);

- in the variant “C” with a low functional state of the FSA (Fig. 1C) both the absolute and relative amplitude of the signal drop in the first cycle of illumination is significantly less than in variants “A” and “B” (Table 1). After dark adaptation, as a result of the second cycle of exposure, the already low K _FSA value decreases by almost 3 times, while the photo-inhibition resistance index (K _UVI ) changes slightly (from 27.42% to 22.98%).

These patterns are characteristic of the leaves of various plant species ex vitro and in vitro, with variations in the amplitude of the difference in light scattering curves depending on genotypic and phenotypic features.

According to the measurement results (Table 1), it is important to conclude that for a correct assessment of the functional state of plant organisms, it is necessary to evaluate both photosynthetic activity and resistance to photo inhibition at the same time.

Thus, the proposed method allows, within a single optical scheme, for a single measurement cycle, within a few minutes, the functional state of plants can be quantified by parameters reflecting the level of photosynthetic activity and resistance to photo inhibition. It takes several hours to make similar estimates using standard techniques and equipment. The method allows to evaluate the functional state of not only ex vitro plants, but also of in vitro plants without compromising sterility.

Literature

1. Veselovsky V.A., Veselova T.V. Luminescence plants. Theoretical and practical aspects. - M .: Science, 1990. - 200 p.

2. Lepedush X., Vilyevach M, Tsezar V., Lyubeshich N. Evaluation of the functional state of the photosynthetic apparatus in spruce needles with signs of chlorosis in weak and strong light by the change in chlorophyll fluorescence in vivo // Plant Physiology. - 2005. - Vol. 52, No. 2. - p. 191-197.

3. Merzlyak, M.N. Gitelson A.A., Chivkunova OB, Solovchenko A.E., Poghosyan S.I. The use of reflection spectroscopy in the analysis of pigments of higher plants // Plant Physiology. - 2003. - Vol. 50, No. 5. - p. 785-792.

4. Murata N., Takahashi S., Nishiyama Y., Allakhverdiev S.I. Photoinhibition of photosystem II under environmental stress // Biochem. Biophys. Acta - 2007. - V. 1767. - P. 414-421.

5. RF patent №2592574 for the invention "Optical method for assessing the functional state of plants" / authors Budagovskaya ON, Budagovsky AV, Goncharov S.А. - Application No. 2014148848 from 03.12.2014. - Publ. 07/27/2016 Bull. №16.

Claims (1)

An optical functional assessment of plants comprising a photosynthetic light scattering measurement dynamics of plant tissues during illumination with monochromatic optical radiation in the blue region of the spectrum zone of the first absorption maximum of chlorophyll 460-480 nm, characterized in that the recorded decline chlorophyll fluorescence intensity portion of the sheet of 2 mm ² in area the process of the first exposure to monochromatic optical radiation of the blue region of the spectrum with a power density above the saturation of photosynthesis of 600-900 V / m ² for 30-180 s and then switched off for 30-60 sec illumination and dark after reducing pauses in the same area of the object of measurement again recorded decline in light scattering intensity of the second illumination with monochromatic blue region of spectrum of the same optical radiation power density in for 30-180 s; about photosynthetic activity and resistance to photo inhibition judged by the value of indicators K _FSA = (I ¹ _max- I ¹ _cm ) / I ¹ _max and K _UVI = (I ² _max- I ² _cm ) / (I ¹ _max- I ¹ _cm ) where I ¹ _max - the maximum fluorescence intensity of chlorophyll by 1-2 from the first exposure; I ¹ _cm - stationary level of chlorophyll fluorescence intensity at 30-180 seconds; I ² _max - the maximum fluorescence intensity of chlorophyll at 1-2 from the second exposure; I ² _cm - stationary level of chlorophyll fluorescence intensity at 30-180 seconds; however, the higher the values of indicators K _FSA and K _UVI , the better the functional state of plants.

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