RU2711577C1 - Method for increasing potato plant productivity in optimum and stress growing conditions - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology and can be used in agriculture to increase yield of potato minitubers in hydroponic conditions or on peat. Method involves treating plants with a solution of biologically active substances. During adaptation to liquid nutrient medium area of assimilating surface of plants, content of photosynthetic pigments in them and efficiency of crawfish formation by single treatment of root system of plants with solution of 24-epicastasterone in concentration of 0.01–1 nM for 4–5 hours. Also, activity of antioxidant enzymes is increased, and toxic inorganic ions are supplied by treatment of plant root system with solution of 24-epibrassinolide in concentration of 0.01–1 nM for 4–5 hours before salting.EFFECT: technical result is increase in potato plant productivity both under normal growing conditions and under action of chloride salinity.1 cl, 4 dwg, 3 tbl

Description

FIELD OF TECHNOLOGY

The invention relates to biotechnology and can be used in agriculture to increase the yield of healthy (virus-free) minitubers in hydroponic conditions or on peat; in the chemical protection of plants from the action of abiotic stressors during subsequent cultivation on open ground or in protected ground structures.

BACKGROUND

From the scientific literature it is known that at present, the production of 90% of all world agronomic products is achieved through the cultivation of thirty types of crops, the vast majority of which are glycophytes. Potatoes are the fourth most important food crop in the world after rice, wheat and corn. Wild potato plants are relatively resistant to salinization, while modern varieties, which are the product of long-term selection, are significantly less salt tolerant. Potatoes are irrigated crops, so they often face the adverse effects of salinization.

Currently existing agronomic and engineering technologies to minimize soil salinization are not able to solve this problem at the global level due to their low efficiency, high labor costs and negative impact on the environment.

The increase in plant resistance to stress is largely determined by hormonal factors. Among phytohormones as effective and environmentally friendly stress-protective regulators, steroid hormones of plant brassinosteroids are of the greatest interest.

A known method of increasing the resistance of rapeseed plants to the damaging effect of intense chloride salinization by the steroid hormone of plants - 24-epibrassinolide (EBL). Rapeseed plants were cultivated on a liquid nutrient medium for three weeks, the next two weeks the plants were subjected to chloride salinity of 125 mM with a single addition to the solution simultaneously with the onset of salinization of EBL at a concentration of 0.01 μM. It has been shown that the protective effect of the hormone is expressed in reducing the negative effect of salinization on the photosynthetic apparatus of plants by stabilizing the transcription of chloroplast genes (Pat. RU 2514641).

The main disadvantage of this method is the need to use a high concentration of the active substance - 0.01 μm, leading to increased consumption of phytohormones.

There is a method of increasing the yield of potatoes and tomatoes by three times during the growing season foliar feeding of plants with a solution of the phytohormone 6-benzylaminopurine with a concentration of 100 μM at a flow rate of 300-400 l / ha (Pat. RU 2243658).

The main disadvantage of this method is the multiple treatment of the aerial parts of plants with a high concentration of the hormone, which is accompanied by an increase in time and financial costs.

A known method of regulating tuberization and productivity of potato plants when processing potato plants with brassinolide, 24-epibrassinolide and 28-homobrassinolide. The method increases the accumulation of photosynthetic pigments, accelerates tuberization and increases plant productivity (Pat. RU 2660918, nice for the prototype).

The main disadvantage of the prototype method is its relatively low efficiency, for example, homobrassinolide and epibrassinolide increased the number of minitubers only by 6 or 23%, respectively. Another disadvantage of the prototype method can be considered an indirect assessment of the effect of hormones. There is no experimental data on the functioning of the assimilation apparatus, which is important for the final productivity of plants. In addition, in the known method, the effectiveness of hormonal effects is considered only under optimal growing conditions, the ability of phytohormones to increase the productivity and yield of potato plants under the action of technogenic stress factors is not proved.

OBJECT OF THE INVENTION

The aim of the invention is to develop an economical way to increase the productivity of potato plants, both under normal growing conditions and under the action of chloride salinity using environmentally friendly phytohormones.

The problem is solved by short-term treatment of the root system of potato plants with steroid phytohormones: 24-epicastasterone and 24-epibrassinolide.

SUMMARY OF THE INVENTION

To solve the problem in a method of increasing the productivity of potato plants, including short-term treatment of the root system of potato plants with a solution of a biologically active substance, in the process of adaptation of plants to a liquid nutrient medium, the area of the assimilating surface, the content of photosynthetic pigments in them and the efficiency of stolonogenesis are increased by processing the root system of plants once a solution of 24-epicastasterone in a concentration of 0.01-1 nM for 4-5 hours, and also increase activity l antioxidant enzymes, increase the efficiency of photosynthesis and reduce the excess intake of toxic inorganic ions by treating the plant root system with a solution of 24-epibrassinolide at a concentration of 0.01-1 nM for 4-5 hours before salt exposure.

It is known that plant productivity is determined not only by its mass, but also by its photosynthetic activity of leaves, which is directly determined by the area of the assimilating surface and the content of photosynthetic pigments [Zivcak et al., 2017]. Under the conditions of chloride salinization, it is important to maintain not only the optimal photosynthetic activity, but also to limit the flow of toxic inorganic ions or neutralize their excess by stimulating the enzymatic and non-enzymatic protective systems. During the experimental selection of the most effective growth hormones, their concentration from 0.01 nM to 1 nM and duration of exposure, special attention was paid to these characteristics. Hormone treatment of the root system ranged from 1 to 24 hours, the optimal period was at least 4 hours, an increase in the soaking time of more than 5 hours up to one day does not produce a noticeable effect. Among the studied phytohormones, the most significant results were shown by ketone-containing (24-epicastasterone) and lactone-containing (24-epibrassinolide) steroid hormones.

A significant increase in plant productivity in the absence of stressors and the protective effect of steroid hormones of various chemical structures during chloride salinization in relation to potato plants is illustrated in tables and figures. The stimulating and stress-protective effects of brassinosteroids have been proven with hormone concentrations of 0.01 - 1 nM (10 ^-9 - 10 ^-11 M), mainly 0.1 nM, with a duration of exposure of 4-5 hours for EPA and from 4 to 5 hours for EBL (as examples in the tables and figures, data are presented only for these plant treatment options).

EXPLANATORY TABLES AND GRAPHICS

Table 1 presents the growth indicators of potato plants. The letters indicate significant differences (p˂0.05) between the options.

Table 2 shows the photochemical activity of the photosynthetic apparatus of plants. Letters indicate significant differences (p˂0.05) between options

Y (II) is the effective quantum yield of PS II;

ETR is the electron transfer rate;

qP and qL are the photochemical quenching coefficients of fluorescence, which estimate the proportion of open reaction centers of PS II;

qN is the coefficient of non-photochemical quenching of fluorescence (reflects the degree of dissipation of absorbed energy into heat);

NPQ - non-photochemical quenching of fluorescence: quantitative assessment of non-photochemical quenching, alternative to qN calculations;

Y (NO) - quantum outputs of unregulated non-photochemical dissipation of light energy in PS II;

Y (NPQ) - quantum outputs of controlled non-photochemical dissipation of light energy in PS II;

Fo 'is the minimum level of fluorescence excited by measuring light of low intensity, so that under its action all the reaction centers of PSII remain open;

Fm 'is the maximum fluorescence yield at the moment of supply of a strong light pulse with closed reaction centers of PS II.

FV / FM - maximum quantum output of photosystem II (FS II).

Table 3 shows the dynamics of inorganic ions in different parts of the plant.

In FIG. 1, 2, 3, 4 shows histograms reflecting the physiological state of potato plants using the following indicators as examples: photosynthetic pigments, osmotic potential of cell contents, proline, malondialdehyde, antioxidant enzymes.

The effects of hormones were tested both under optimal conditions and with subsequent salt stress factor (NaCl at a concentration of 125 mM).

MODES FOR CARRYING OUT THE INVENTION

The examples illustrate the effect of steroid hormones on the plant with different chemical structure of the molecule (ketone-containing 24-epicastasterone or lactone-containing 24-epibrassinolide). The experiments were conducted on plants of Solanum tuberosum L. of the mid-ripening variety Lugovskaya (identifier 8301891). Under optimal conditions, this type of potato yields a consistently high yield; its tubers are characterized by high shelf life and resistance to a number of diseases, including late blight. Improved potato regenerant plants in vitro were obtained from the apical meristem and cultivated for 30 days on an agarized nutrient medium Murashige and Skoog with half the medium composition (½ MS). The roots of the plants were washed from the agar medium and the microclones were weekly adapted to the liquid half medium Murashige and Skoog (½ MS) and air conditions under L36W / 77 Fluora fluorescent lamps (Osram, Germany) at a photon flux density of 100–150 μmol ∙ m-2 ∙ s-1 in a phytotron with a 16-hour photoperiod and a temperature of 20 ± 3 ° C.

After two weeks of growth in a hydroponic setup in MS medium, the plants were transferred for a short time to the same medium in the absence (control version) or with the addition of 24-epicastasterone (EPA) to various concentrations in the range of 0.01-1 nM. We also studied the effect of salt stress factor on control and hormone-treated specimens: plants were placed on a nutrient medium MS in the absence (to assess the effectiveness of hormonal effects under optimal growth conditions), and stress conditions were also simulated (presence of NaCl at a concentration of 125 mM).

The obtained fresh biomass of plant material was evaluated by the gravimetric method. To assess the level of photosynthetic pigments, potato leaves were ground in 96% ethanol, the homogenate was centrifuged (10 min at 8 thousand rpm, MiniSpin "Eppendorf" centrifuge, Germany). The optical density of the sample (supernatant) was evaluated on a Genesys 10 ThermoElectron spectrophotometer (USA). The concentration of pigments in the alcohol extract was calculated according to the method proposed by H.K. Lichtenthaler, 1987.

Fluorescence indices reflecting the photochemical activity of the photosynthetic apparatus of plants were measured using a PAM fluorimeter (Junior-PAM, Heinz-Walz, Germany). Fluorescence parameters were recorded after a 20-minute dark adaptation.

Extraction and determination of free proline was carried out according to the method proposed by L.S. Bates, 1973.

The osmotic potential of cell exudate was determined on a Osmomat 030 cryoscopic osmometer (Germany) in accordance with the manufacturer's instructions. Cellular juice was squeezed from thawed plant leaf samples.

Determination of the activity of antioxidant enzymes (superoxide dismutase (EC 1.15.1.1) and peroxidase (EC 1.11.1.7) in plant leaves was evaluated according to the methods described by Ch. Beauchamp, 2002. The protein concentration in the obtained enzyme preparations was evaluated according to the method proposed by A.A. Esen, 1978.

The content of inorganic ions (sodium, potassium, chlorine, aluminum, phosphorus, magnesium, calcium, sulfur, and iron) was determined in leaves, stems, and roots by energy dispersive analysis using an EDAX Quanta 200 3D electron-scanning scanning electron microscope (Netherlands). Data are presented in atomic percent (At%) of the total number of elements at a given point in the sample (100 At%) minus the fraction of carbon and oxygen. Scanning each option was performed at least six times.

As key indicators of the response of plants to hormonal and salt effects, integral indicators were used: the dimensions of the axial organs, the total area of the assimilating surface, the number of tiers and stolons.

The results of the experiments showed an increase in plant productivity under optimal growing conditions and a decrease in the effect of the negative impact of chloride salinity on the growth and physiological parameters of potatoes.

Under optimal plant growing conditions, exogenous 24-epibrassinolide has virtually no effect on the growth performance of potato plants, while its predecessor, 24-epicastastron, increases the number of stolons, tiers and the total leaf surface area by 46, 27 and 18%, respectively (tab. 1). The content of green pigments (chlorophyll a and b) increased with 4 or more hours of exposure to EPA to a greater extent for chlorophyll b (an average of 33%).

It is known that NaCl in high concentrations not only has a direct toxic effect on cellular metabolism and causes osmotic stress, but also stimulates the generation of ROS and the development of oxidative stress. The main cause of oxidative stress in this case is associated with stomata closure, reduced availability of CO ₂ and increased electron excitation energy, which is accompanied by intense ROS generation.

To identify the protective effect of steroid hormones under the action of an abiotic stressor, we evaluated their effectiveness against the background of chloride salinity (see Table 1). The 125 mM NaCl concentration used in the experiment suppresses stolonation by 4.4 times and reduces the total leaf surface area by 2.0 times. The length of the axial organs decreased by 25%, the total mass of plants in response to the action of the stressor decreased by 1.5 times. Treatment of plants with hormones reduces the inhibitory effect of salinity of 125 mM NaCl on the root length, the area of the assimilating apparatus, and the number of tiers, leaves, and stolons. The negative effect of NaCl is manifested not only on growth characteristics, but also on the content of photosynthetic pigments; the level of green pigments in the control decreased by 15-20% (Fig. 1).

The effectiveness of the protective action in relation to the area of the leaf surface and stolonation depends on the chemical structure of the substance. So, the protective effect of EPA is noted at the level of stolon formation, while the specificity of the action of EBL is manifested in stimulation of the development of the assimilating apparatus (Table 1). The negative effect of chloride salinization on the accumulation of photosynthetic pigments is completely removed by preliminary processing of EPA; however, the positive effect of exogenous EBL was most pronounced - the content of chlorophylls and carotenoids exceeded the control values by 20% (Fig. 1). Additional experiments with 28-homobrassinolide prior to the onset of chloride salinization (NaCl 100 mM) also confirmed that lactone-containing steroid hormones are more effective in controlling stress due to the accumulation of proline and carotenoids with pronounced antioxidant and stress-protective properties.

 Given the key role of the photosynthetic apparatus in plant productivity and their survival under adverse growing conditions, the evaluation of indicators reflecting the functioning of the plant assimilation apparatus is of great importance.

The maximum quantum efficiency of photosystem II (F _v / F _m ) of the control plants was 0.83, chloride salinity reduced this indicator to 0.78, which indicates a malfunction of the photosynthetic apparatus (Table 2). In accordance with this, the effective quantum yield of PS II (Y (II)) decreased by 20%. The coefficients of photochemical quenching of fluorescence qP and qL in response to the action of the stressor also decreased. In addition, a decrease in the efficiency of PS II is evidenced by a decrease in the electron transfer rate ETR and an increase in the quantum yield of controlled utilization Y (NPQ).

Short-term treatment of plants with hormones reduces the subsequent inhibitory effect of salt in relation to the maximum and effective quantum yields of photosystem II, as well as the photochemical quenching coefficients of fluorescence qP to control values. In response to hormonal effects, the electron transfer rate and the photochemical quenching coefficient qL were partially restored. Regarding the quantum yield of regulated utilization, the specificity of action was revealed for ketone and lactone-containing steroid compounds - EPA completely restored this indicator, while EBL kept this indicator at the salt control level (Table 2).

The osmotic potential of the cellular contents of the leaves during the experiment in the control plants was (- 0.75 MPa). Chloride salinization caused a decrease in the chemical potential of cell exudate to (- 1.4 MPa), which was 1.8 times lower than the corresponding values of the control variant (Fig. 2). Hormonal priming with subsequent salt stress did not have a significant effect on the flow of water into plants compared with the action of a stressor (Fig. 2); keeping the osmotic potential at a low level allows the plant to restore the direction of the gradient of the water potential between the nutrient medium and plant cells and absorb water from an environment with a low water potential.

Inorganic ions play a decisive role in the formation of the osmotic potential of cellular contents. We analyzed the content of sodium, chlorine, potassium, calcium, magnesium, aluminum, phosphorus, sulfur and iron ions in the leaves, stems and roots of potato plants. As follows from the data obtained (Table 3), in the absence of a stressor, the atomic concentration (At%) of sodium in the underground part of plants was approximately three times higher than its content in leaves and stems, and At% of potassium ions, on the contrary, was higher in the above-ground part . Among the predominant ions, potassium ions can be noted. In response to the effect of NaCl on the root system, At% of inorganic ions changed fundamentally. The bulk of the nine analyzed ions were sodium, chlorine and potassium ions. The maximum increase in the percentage of sodium and chlorine ions is shown for the aerial part — about 9 and 6 times, respectively, and the minimum was observed in the roots — 4 times (Table 3). At% K ⁺ decreased in all parts of the plant in response to the action of the stressor by about half. The atomic concentration of calcium, sulfur and iron decreased by about half in the aerial part and by 15% in the roots of plants; the proportion of magnesium ions in all parts of the plant decreased by 16-33%.

Treatment of potato plants with a lactone-containing 24-epibrassinolide followed by chloride salinization led to a decrease in the atomic fraction of sodium chloride ions in all parts of the plant and a slight increase in At% potassium ions, which indicates a decrease in the negative effect of the stressor (Table 3).

The authors suggest that a decrease in the concentration of inorganic ions when exposed to phytohormones occurs due to an increase in the efficiency of barrier mechanisms that delay the flow of toxic ions into plants. The protective effect of short-term treatment with ketone-containing 24-epicastasterone was less pronounced and was manifested only in a decrease in the atomic fraction of sodium ions; Despite the decrease in At% of chlorine ions in the stems of plants, an increase was observed in the roots, which cannot guarantee the positive effect of this hormone on the accumulation of chlorine ions in the plant.

When plants adapt to a violation of their water status and to the toxic effect of an excess of inorganic ions, primarily sodium, an important role is played by compatible osmolytes with the properties of chemical chaperones. We have revealed organ-specificity in the accumulation of universal compatible osmolyte proline in potato plants in response to acting factors. The proline content in all parts of plants in the absence of a stressor was 5.4–6.5 μM / g wet weight (Fig. 3). Chloride salinization activated the accumulation of proline in the leaves and stem of plants by 19 and 10 times, respectively (Fig. 3). The accumulation of proline under stress contributes to the preservation of cellular homeostasis. This is evidenced by the fact that proline under stress plays the role of not only an osmoregulator, although this is extremely important during salinization, but it also implements a number of other stress-protective functions, such as the functions of a chemical chaperone, antioxidant, a regulator of expression of stress-regulated genes, a source carbon, nitrogen, and reducing equivalents, intracellular pH-stat regulator, etc.) (Mansour MMF, Ali EF, 2017). The content of proline during short-term hormonal exposure followed by chloride salinity depends on the chemical structure of the hormone. Thus, root treatment of a plant with lactone-containing EBL to salt stress (NaCl 125 mM) contributed to a more effective decrease in proline levels compared to its content in plants exposed to EPA, especially for stems and leaves (Fig. 3).

With the development of oxidative stress in response to the action of chloride salinity, the plant responds with the formation of a cellular antioxidant system, which includes, in addition to low molecular weight organic compounds, antioxidant enzymes superoxide dismutase (SOD), peroxidase, etc. Chlorine salinity contributed to a slight activation of enzymatic systems in the leaves of potato plants; the activity of SOD and guaiacol-dependent peroxidase increased by 32 and 12%, respectively (Fig. 4). Short-term treatment of plants with EPA and EBL before stress exposure caused an increase in superoxide dismutase activity by 54-76% (Fig. 4A), peroxidase by 3 times and practically does not depend on the chemical structure of the hormones used (Fig. 4B).

Thus, it has been experimentally shown that short-term root pretreatment of potato plants with 24-epicastasterone increases the number of stolons, tiers, the total leaf surface area and the content of chlorophylls in the absence of a stressor. The root pretreatment of potato plants with 24-epibrassinolide reduces the negative effect of subsequent chloride salinity on growth indicators, the content of photosynthetic pigments, the maximum and effective quantum yields of the second photosystem, and the photochemical quenching coefficients of fluorescence qP. The protective effect of hormones is achieved by reducing the atomic fraction of chlorine and sodium ions and increasing the activity of antioxidant enzymes, while the proline content decreases. A more pronounced protective effect under the action of chloride salinity showed a lactone-containing EBL.

Based on these results, a method is proposed for increasing the productivity of potato plants under optimal growing conditions and its agronomic stability under conditions of chloride salinity according to the claimed claims.

Used sources

1. Pat. RU 2514641. Published: 04/27/2014.

2. Pat. RU 2243658. Publisher: 10.01.2005.

3. Pat. RU 2660918. Publisher: 07/11/2018.

4. Mansour M.M.F., Ali E.F. Evaluation of proline functions in saline conditions // Phytochemistry. 2017. Vol. 140. P. 52–68.

5 Zivcak M., Olsovska K., Brestic M. Photosynthetic Responses Under Harmful and Changing Environment: Practical Aspects in Crop Research // Photosynthesis: Structures, Mechanisms, and Applications. 2017. P. 203-248.

Claims (1)

A method of increasing the productivity of potato plants under optimal and stressful growing conditions, including short-term treatment of plants with a solution of a biologically active substance, characterized in that, in the process of adaptation to a liquid nutrient medium, the area of the assimilating surface of plants is increased, the content of photosynthetic pigments in them and the efficiency of stolon formation are processed once by root the plant system with a solution of 24-epicastasterone in a concentration of 0.01-1 nM for 4-5 hours, and also increase the active antioxidant enzymes and reduce the excess intake of toxic inorganic ions by treating the plant root system with a solution of 24-epibrassinolide at a concentration of 0.01-1 nM for 4-5 hours before salt exposure.

Images