Method for growing plants with high content of carotenoids and anthocyanins

Abstract

FIELD: closed-type crop production.SUBSTANCE: illumination is increased from time t1 to time t2, from time t2 the set illumination is maintained until time t3, from time t3 the illumination is reduced until time t4. During the light phase, plants are additionally affected by temperature changes and wind loads created by a fan. In the light phase, several periods of complex impact on plants are distinguished, lasting from the moment of time t0 to the moment of time t16. From the moment of time t4 the set illumination is maintained until the moment of time t5. From the moment of time t5, the illumination is increased until the moment of time t6, from the moment of time t6, the set illumination is maintained until the moment of time t7. From the moment of time t7, the illumination is reduced until the moment of time t8, and from the moment of time t8, the set illumination is maintained until the moment of time t9. From the moment of time t9, the illumination is increased until the moment of time t10, from the moment of time t10, the set illumination is maintained until the moment of time t11, from the moment of time t11, the illumination is reduced until the moment of time t12, from the moment of time t12, the set illumination is maintained until the moment of time t13. From the moment of time t13, the illumination is increased until the moment of time t14, and from the moment of time t14, the set illumination is maintained until the moment of time t15. From the moment of time t15 the illumination is reduced until the moment of time t16. The set temperature is maintained from time t0 to time t5. From the moment of time t5 the temperature is increased until the moment of time t6, from the moment of time t6 the set temperature is maintained until the moment of time t7. From the moment of time t7 the temperature is reduced until the moment of time t8, from the moment of time t8 the set temperature is maintained until the moment of time t9. From the moment of time t9 the temperature is increased until the moment of time t10, from the moment of time t10 the set temperature is maintained until the moment of time t11. From the moment of time t11 the temperature is reduced until the moment of time t12, from the moment of time t12 the set temperature is maintained until the moment of time t13, from the moment of time t13 the temperature is reduced until the moment of time t14. From the moment of time t14, the set temperature is maintained until the moment of time t15, from the moment of time t15, the temperature is increased until the moment of time t16. The fan is turned on at time t9. From the moment of time t9 the wind force is increased until the moment of time t10, from the moment of time t10 the set wind force is maintained until the moment of time t11. From the moment of time t11 the wind force is reduced until the moment of time t12, at which time the fan is turned off. The fan is turned on at time t13. From the moment of time t13 the wind force increases until the moment of time t14. From the moment of time t14, the set wind force is maintained until the moment of time t15, from the moment of time t15, the wind force is reduced until the moment of time t16, at which time the fan is turned off, the values of illumination and temperature at the moments of time t0, t4, t8, t12 and t16 are optimal for plants. The moment of time t16 of the current period of complex impact on plants coincides with the moment of time t0 of the next period of complex impact on plants.EFFECT: increase in the efficiency of formation of carotenoids and anthocyanins in plants.1 cl, 3 dwg

Description

[0001] The invention relates to indoor crop production and can be used to grow vegetables, greens, and medicinal herbs in greenhouses, phytotrons, growboxes, etc. The invention makes it possible to grow plants with increased levels of carotenoids and anthocyanins—important natural compounds with antioxidant properties that can be used to prevent cancer—in both industrial and domestic conditions.

[0002] A method for growing plants with increased carotenoid content is known [1 - Russian Federation Patent No. 2649338, IPC: A23K 10 / 30 Method for obtaining plant material with increased carotenoid content], which includes sowing herbaceous plants of the Poaceae family in late periods no earlier than mid-July, preliminary summer mowing, and final harvesting of plant material after reaching low positive air temperatures.

[0003] The disadvantages of the known method [1] lie in its narrow scope of application. This narrow scope of application is due to the fact that the known method can only increase carotenoid content in herbaceous plants adapted to cold climates (such as Yakutia) and cannot be applied to other plant species and in other climatic zones. Furthermore, the known method [1] cannot be used in individual greenhouses and phytotrons.

[0004] A method for growing plants with an increased content of carotenoids is also known [2 - Russian Federation Patent No. 2661086, IPC: C12N 1 / 12; C12P 23 / 00; C12R 1 / 89 Method for cultivating the microalgae COELASTRELLA RUBESCENS for obtaining carotenoids and lipids], which includes cultivating by a two-stage enrichment culture method on a nutrient medium, maintaining a photoperiod of 15 hours of light and 9 hours of darkness at stage I, and transferring the resulting culture to stage II with a 24-hour lighting regime.

[0005] The disadvantages of the known method [2] lie in its narrow scope of application. This narrow scope of application is due to the fact that the known method can only increase carotenoid content in the microalgae COELASTRELLA RUBESCENS and cannot be applied to other plant species. Furthermore, the known method [2] cannot be used in individual greenhouses and phytotrons.

[0006] The closest in technical essence to the claimed method is the method for growing plants selected as a prototype [3 - Russian Federation Patent No. 2811128, IPC: A01G 9 / 14 Method for lighting greenhouses], in which each day is divided into a dawn phase, a day phase and a sunset phase, and information on the current state of illumination comes from light sensors to the control device, which compares it with the illumination value that is optimal for a given period of time, the crop being grown and the phase of its development, determines the adjustments necessary to bring the value of the current illumination into line with the optimal value for a given period of time, and sends the corresponding commands on adjustments to the actuators, the illumination is set in accordance with the dawn phase, the day phase and the sunset phase using light-emitting devices, the luminous flux of which in total corresponds to the illumination that is optimal for a given period of time,the crop being grown and the phase of its development, whereby the illumination corresponding to the dawn phase is obtained by increasing the illumination to 100% of the required illumination for the crop being grown in the greenhouse by increasing the power of the light-emitting devices, whereby the total power of the light-emitting devices ensures the optimal illumination value for the crop being grown.

[0007] When implementing the prototype method [3], all photobiologically active compounds, including carotenoids and anthocyanins, are formed in plants; however, this formation is insufficiently effective. This is due to the fact that carotenoids and anthocyanins are particularly actively formed in plants under various stress conditions, including complex stresses that activate temperature and mechanical stressors. However, in the prototype method [3], it is impossible to implement the effect of dosed complex stresses (including temperature and mechanical stress) on plants due to the lack of appropriate technical means. This determines the low efficiency of the known method [3].

[0008] The technical problem that the proposed invention is aimed at solving is the possibility of growing plants with an increased content of carotenoids and anthocyanins in both industrial greenhouses and home conditions (phytotrons and growboxes).

[0009] To solve the technical problem, a method is proposed for growing plants with an increased content of carotenoids and anthocyanins, in which plants are exposed to light radiation in the light phase, while the illumination is increased from time t1 to time t2, from time t2 the set illumination is maintained until time t3, and from time t3 the illumination is reduced until time t4.

[0010] According to the invention, plants in the light phase are additionally affected by a change in temperature and a wind load created by a fan, while in the light phase, several periods of complex impact on plants are distinguished, lasting from the moment of time t0 to the moment of time t 16 , from the moment of time t4, the set illumination is maintained until the moment of time t5, from the moment of time t5, the illumination is increased until the moment of time t6, from the moment of time t6, the set illumination is maintained until the moment of time t7, from the moment of time t7, the illumination is reduced until the moment of time t8, from the moment of time t8, the set illumination is maintained until the moment of time t9, from the moment of time t9, the illumination is increased until the moment of time t 10 , from time t 10 maintain the set illumination until time t 11 , from time t 11 reduce illumination until time t 12 , from time t12 maintain the set illumination until time t 13 , from time t 13 increase the illumination until time t 14 , from time t 14 maintain the set illumination until time t 15 , from time t 15 reduce illumination until time t 16 , maintain the set temperature from time t0 to time t5, from time t5 increase the temperature until time t6, from time t6 maintain the set temperature until time t7, from time t7 decrease the temperature until time t8, from time t8 maintain the set temperature until time t9, from time t9 increase the temperature until time t 10 , from time t 10 maintain the set temperature until time t 11 , from time t 11reduce the temperature to time t 12 , from time t 12 maintain the set temperature until time t 13 , from time t 13 lower the temperature to time t 14 , from time t 14 maintain the set temperature until time t 15 , from time t 15 increase the temperature to time t 16 , turn on the fan at time t9, from time t9 increase the wind force until time t 10 , from time t 10 maintain the set wind force until time t 11 , from time t 11 reduce the wind force until time t 12 , at which the fan is turned off, the fan is turned on at time t 13 , from time t 13 increase the wind force until time t 14 , from time t 14maintain the set wind force until time t 15 , from time t 15 reduce the wind force until time t 16 , at which the fan is turned off, the values ​​of illumination and temperature at times t0, t 4, t8, t 12 and t 16 are optimal for plants at time t 16 the current period of complex impact on plants coincides with the time t0 of the next period of complex impact on plants.

[0011] The combination of distinctive features and properties of the proposed method are not known from the literature, therefore it meets the criteria of novelty and inventive step.

[0012] The technical result of the invention is to increase the efficiency of carotenoid and anthocyanin formation in plants by repeatedly creating periods of dosed complex stress (light, temperature, and mechanical) during the light phase. This stimulates enhanced biosynthesis of these photobiologically active compounds. A specially selected activation and deactivation cycle for each period of the three stressors (light, temperature, and mechanical) allows for high levels of synthesized compounds to be maintained in the plant even after all stress factors have ceased to act.

[0013] The method for growing plants with increased content of carotenoids and anthocyanins is illustrated in Fig. 1, Fig. 2 and Fig. 3.

[0014] Fig. 1 shows a diagram explaining the allocation of several periods of complex effects on plants in the light (day) phase.

[0015] Figure 2 shows a generalized time diagram of a plant’s response to stress. This diagram corresponds to Figure 1 of the work [4 - Lichtenthaler H.K. The Stress Concept in Plants: An Introduction / / Annals New York Academy of Sciences, 1998, Volume 851, pp. 187-198].

[0016] Figure 3 shows a time diagram (cyclogram) illustrating the application of light, temperature, and wind (mechanical) stress to a plant during one period of complex treatment in accordance with the proposed method, as well as the level of antioxidant compounds in the grown plant. In the time diagram shown in Figure 3, the horizontal axis represents time. The vertical axis represents the values ​​of illumination, temperature, wind strength, and antioxidant compound content in the plant within the greenhouse.

[0017] The theoretical basis for the claimed method is as follows. During their life, plants experience various types of abiotic (factors of the inanimate environment) and biotic (factors of the living environment) stress [4], [5 - Plant and stress. Lecture course / Ekaterinburg: Ural State University, 2008, 267 p.], [6 - Zagoskina N.V., Nazarenko L.V. Reactive oxygen species and the antioxidant system of plants / / Bulletin of Moscow State Pedagogical Univ. Series: Natural Sciences, 2016, No. 2, pp. 9-23].

[0018] Abiotic stressors are divided into physical and chemical. Physical stressors include: high and low light; high and low temperatures; mechanical stress; moisture deficiency or excess; and elevated radiation levels. Among chemical factors that can cause stress, the most common are: salts and xenobiotics (gases, pesticides, industrial waste, heavy metals).

[0019] Biotic stressors are typically represented by pathogens: fungi, bacteria, viruses, etc.

[0020] In the work [5] it is noted that regardless of the cause of stress, during the onset of stress (primary inductive stress response) in plants, in the form of a non-specific reaction of cells and the organism as a whole to extreme influences, the following changes occur.

[0021] Membranes' permeability increases due to changes in the molecular composition of their components. This leads to a reversible release of potassium ions from the cell and the entry of calcium ions from the cell wall, vacuole, ER, and mitochondria. Membrane depolarization occurs. Increased membrane permeability and inhibition of H+-ATPase lead to cytoplasmic acidification. A decrease in cytoplasmic pH promotes the activation of hydrolases, most of which have an optimum pH in an acidic environment. As a result, polymer breakdown processes are intensified. Protein synthesis is inhibited, and the conformation of protein molecules changes. Polysomes disintegrate, and mRNAs of "pre-stress" proteins are hydrolyzed or interact with specific proteins, forming "stress granules" in the cytoplasm. Transcription and replication processes are inhibited. At the same time, expression of repressed genes and synthesis of a number of stress proteins occur at this stage. The assembly of cytoskeletal elements is activated, which leads to an increase in the viscosity of the cytoplasm.The rate of photosynthesis is inhibited due to changes in the structure of proteins and lipids in the thylakoid membranes. Respiration is initially activated, but then inhibited, as is photosynthesis, and ATP levels decrease [5].

[0022] It should be especially noted that regardless of the type of stressor, the most life-threatening processes associated with reactive oxygen species (ROS) are activated in the plant [5], [6], [7 - Poleskaya O.G. Plant cell and reactive oxygen species, Moscow: KDU, 2007, 139 p.].

[0023] The term ROS refers to oxygen species with extremely high reactivity that can oxidize virtually all classes of biological molecules - proteins, membrane lipids, DNA molecules, etc. ROS include singlet oxygen l O2, hydroxyl radical HO • , superoxide anion radical O2 •- , hydroperoxide radical HO2 •, hydrogen peroxide H2O2. In normally functioning cells, the ROS content is maintained at a low level, since special enzymatic systems are busy eliminating them [5], [6], [7]. Under any stressful conditions, the ROS content in cells begins to increase rapidly, developing into oxidative stress.

[0024] Singlet oxygen molecules are believed to l O2 can trigger apoptosis processes, i.e. programmed cell death [8 - Vargas F. Et al. Photoinduced Apoptosis by Photosensitizer Drugs - in: Frontiers in Cell Apoptosis Research / edited by Erlich SR, New York, Nova Biomedical Books, 2007, 228 p.], [9 - Martusevich A.A. Molecular and cellular mechanisms of action of singlet oxygen on biosystems / / Modern technologies in medicine, 2012, No. 2, pp. 128-134].

[0025] The hydroxyl radical HO is considered a very strong oxidizing agent. •This radical is incapable of intracellular migration, as it immediately reacts with biological molecules. It not only initiates membrane destruction and protein degradation by interacting with the residues of many amino acids, but also disrupts carbohydrate bridges between nucleotides, breaking DNA and RNA chains. It is believed that such processes may be factors in natural mutagenesis [5].

[0026] The damaging effect of active oxygen species in plants is counteracted by the antioxidant defense system. In the work [10 - Pradedova E.V. Classification of the antioxidant defense system as a basis for rational organization of experimental research of oxidative stress in plants / / Plant Physiology, 2011, Vol. 58, No. 2, pp. 177-185] various options for classifying antioxidant compounds in plants are considered: by their catalytic activity (enzymatic and non-enzymatic); by molecular weights (low molecular weight and high molecular weight); by localization (intracellular, cell-membrane and extracellular), etc. From the point of view of the authors of the claimed method, the most successful classification is the one related to the chemical nature of the antioxidant compound, which is manifested by the presence in the structure of the molecule of certain functional groups associated with the manifestation of antioxidant properties.Based on this, all antioxidant compounds can be divided into those with indirect (mediated) action and those with direct (directed) action

[10] . Indirect-action antioxidant compounds are capable of reducing the effects of free radical oxidation at the plant level as a whole, but are ineffective at the cellular level

[10] . Naturally, direct-action antioxidant compounds appear to be more effective, among which are polyenes (substances with several unsaturated bonds that are easily oxidized, binding ROS and thereby protecting other biomolecules from oxidation) and proton donors (i.e., substances with a mobile hydrogen atom, capable of intercepting free radicals)

[10] .

[0027] Polyenes include carotenoids and retinoids.

[0028] Proton donors include phenols (flavonoids, tocopherols, phenol and naphthol derivatives, catechins, etc.), nitrogen-containing heterocyclic substances (including melatonin), and thiols.

[0029] The classes of these compounds are extremely extensive (for example, more than 6,500 varieties of flavonoids alone are currently known). Therefore, when describing the claimed method, carotenoids (as substances with several unsaturated bonds) and anthocyanins (as proton donors) will be mentioned as typical compounds. However, the essence of the claimed method and its scope of application are not limited to carotenoids and anthocyanins, but include any antioxidant compounds of plants. Therefore, in the description of the present invention, along with mentioning carotenoids and anthocyanins (as the most important antioxidant compounds used for the prevention of cancer), the general term antioxidant compounds (AOCs) will be used.

[0030] The antioxidant properties of carotenoids and anthocyanins in plants are described in many sources, for example [11 - Maslova TG et al. Functions of carotenoids in the leaves of higher plants (Review) / / Journal of General Biology, 2020, Vol. 81, No. 4, pp. 297-310], [12 - Demmig-Adams B. et al. Linking the xanthophyll cycle with thermal energy dissipation / / Photosynthesis Research, 2003, No. 76, pp. 73-80], [13 - Demmig-Adams B. et al. In vivo functions of carotenoid in higer plants / / The FASEB Journal, 1996, Volume 10, Issue 4, pp. 403-412], [14 - Cappellini F. et al. Anthocyanins: From Mechanisms of Regulation in Plants to Health Benefits in Foods / / Frontiers in Plant Science, 2021, Volume 12, October], [15 - Sarma AD et al. Anthocyanin-DNA copigmentation complex: mutual protection against oxidative damage / / Phytochemistry, 1999, No. 52, pp. 1313-1318], [16 - Lorenc-Kukula K. et al.Ectopic Expression of Anthocyanin 5-O-Glucosyltransferase in Potato Tuber Causes Increased Resistance to Bacteria / / Journal of Agricultural and Food Chemistry, 2004, December, Volume 53, Issue 2, pp. 272-281], [17 - Gould KS, Lister C. Flavonoid functions in plants - in: Flavonoids: chemistry, biochemistry and applications, Boca Raton, CRC Press LLC, 2006, pp. 397-441], [18 - Makarevich AM et al. Functions and properties of anthocyanins in plant materials / / Proceedings of BSU, 2010, Volume 4, Issue 2, pp. 1-11], [19 - Jaakola L. et al. Activation of flavonoid biosynthesis by solar radiation in bilberry (Vaccinium myrtillus L.) leaves / / Planta, 2004, Volume 218, pp. 721-728], [20 - Neill SO et al. Antioxidant activities of red versus green leaves in Elatostema rugosum / / Plant, Cell and Environment, 2002, Vol. 25, No. 4, pp. 539-547], etc.

[0031] At the same time, from the point of view of carotenoids and anthocyanins as substances found in the human diet, the following can be noted.

[0032] It is known that the taste of tomatoes, for example, is determined by the concentration of substances such as carotenoids, carbohydrates, fatty and organic acids, polyphenols, and amino acids in their juice and pulp. A rich, bright, and distinct taste is observed in fruits with a significant concentration of carotenoids [21 - What determines the taste of tomatoes / / https: / / news.myseldon.com / ru / news / index / 232142354]. Carotenoids are precursors of volatile substances associated with the sensory qualities of tomatoes and flavor-forming compounds such as apocarotenoids-VOCs (apocarotenoid-volatile organic compounds).

[0033] Carotenoids are natural organic pigments colored red, orange, or yellow. Based on their chemical composition, carotenoids can be divided into two large groups: carotenes (including beta-carotene and lycopene) and xanthophylls (including lutein and zeaxanthin). Carotenoids function as antioxidants in the human body. Carotenoids are provitamins A, metabolic precursors of vitamin A; beta-carotene is the most important of them. Both vitamin A and beta-carotene, being powerful antioxidants, are used in the prevention and treatment of cancer, in particular, by preventing tumor recurrence after surgery. Both vitamin A and beta-carotene protect brain cell membranes from the destructive effects of free radicals, with beta-carotene neutralizing the most dangerous types of free radicals: oxygen radicals and polyunsaturated acid radicals.The antioxidant action of beta-carotene plays an important role in the prevention of heart and arterial diseases, it has a protective effect in patients with angina, and also increases the level of “good” cholesterol (HDL) in the blood.

[0034] Another carotenoid, lycopene, protects against atherosclerosis by preventing oxidation and accumulation of low-density cholesterol on artery walls. It is also the most potent carotenoid in terms of cancer protection, particularly against breast, endometrial, and prostate cancer [22 - Massaretto IL Recovering Tomato Landraces to Simultaneously Improve Fruit Yield and Nutritional Quality Against Salt Stress / / Frontiers in Plant Science, 2018, November, vol. 9]. Lycopene also helps strengthen bones, protect against atherosclerosis and cardiovascular disease, kidney disease, and helps maintain skin elasticity and strong hair.

[0035] Lutein and zeaxanthin are the main carotenoids that protect the eyes: they help prevent cataracts and reduce the risk of macular degeneration (the most important organ of vision), which is the cause of blindness in one in three cases.

[0036] Carotenoids themselves are non-toxic, and their formation into vitamin A is enzymatically limited. Therefore, when consuming foods containing carotenoids, an overdose of vitamin A does not occur, and therefore the upper tolerable intake level has not been established [23 - Nilova L.P., Potoroko I.Yu. Carotenoids in plant food systems / / Bulletin of the South Ural State University, series "Food and Biotechnology", 2021, Vol. 9, No. 4, pp. 54-69].

[0037] In ripe red tomatoes, lycopene is the predominant carotenoid in the range from 0.9 to 7.7 mg / 100 g; while the amount of beta-carotene is from 0.4 to 3.1 mg / 100 g; lutein - from 0.1 to 0.6 mg / 100 g

[23] , [24 - Kondratieva I.Yu., Golubkina N.A. Lycopene and beta-carotene in tomato / / Vegetables of Russia, 2016, No. 4, pp. 80-83]. New varieties of tomatoes are constantly being created using genetic engineering methods. For example, the tomato variety "Xantomato" is enriched with zeaxanthin, the amount of which is 3.9 mg / 100 g, which reaches 50% of the total amount of carotenoids in the fruits [25 - Hermanns AS et al. Carotenoid Pigment Accumulation in Horticultural Plants / / Horticultural Plant Journal, 2020, No. 6, pp. 343-360].

[0038] Anthocyanins are plant polyphenolic compounds important for humans, which are abundant in peppers, raspberries, eggplants, and red cabbage. The following types of biological activity have currently been proven for anthocyanins: anticarcinogenic properties; protection against cardiovascular diseases; reduction of capillary fragility and permeability; anti-inflammatory effect; antimicrobial activity; improved visual acuity and restoration of rhodopsin [26 - Pisarev D.I. et al. Biological activity of plant polyphenols. Prospects for the use of anthocyanins in medical practice. Scientific News. Series "Medicine. Pharmacy", 2012, No. 10, Issue 18 / 2, pp. 17-24].

[0039] The work [27 - Koldayev V.M., Kropotov A.V. Anthocyanins in Practical Medicine / / Pacific Medical Journal, 2021, No. 3, pp. 24-28] notes that the medical effects of anthocyanins are mainly associated with the inactivation of free radicals due to their antioxidant properties, as well as their ability to modulate intracellular mitochondrial antioxidant systems. In addition, anthocyanins activate tumor suppressor genes, induce apoptosis of cancer cells, restore genomic DNA and increase its stability

[27] .

[0040] Anthocyanins also help reduce inflammatory reactions in the intestines during the consumption of excess fats and carbohydrates. Blueberries, which contain large amounts of anthocyanins, are used for the treatment and prevention of diabetes [28 - Chehri A. Phytochemical and pharmacological anti-diabetic properties of bilberries (Vaccinium myrtillus), recommendations for future studies / / Primary Care Diabetes, 2022, vol. 16, iss. 1, pp. 27-33].

[0041] Thus, from a medical point of view, the common properties of carotenoids and anthocyanins are their antioxidant properties, which can be used to prevent cancer. The prospects of using carotenoids to create drugs with antioxidant, antimutagenic, and anticarcinogenic properties are discussed in the work of employees of the Research Institute of Experimental Diagnostics and Therapy of Tumors [29 - Shashkina M.Ya. et al. Carotenoids as a basis for creating therapeutic and prophylactic agents / / Russian Biotherapeutic Journal, 2009, Vol. 8, No. 4, pp. 91-97]. Carotenoids and anthocyanins are not formed in the human body, so all the needs for these compounds are met by a person through plant foods

[29] . At the same time, a low level of carotenoids in food and human blood plasma is an indicator of cancer risk [30 - Jeong NH et al. Plasma carotenoids, retinal and tocopherol levels and risk of ovarian cancer / / Acta Obsest Ginecol. Scand., 2009, Volume 88, pp. 457-462]. Some aspects of the use of anthocyanins in medicinal plants are discussed in the work [31 - Maslennikov P.V. et al. Content of anthocyanin and carotenoid pigments in medicinal plants / / Electronic journal "Vestnik MGOU", www.evestnik-mgou.ru, 2013, No. 1, pp. 1-14].

[0042] So, as shown above, carotenoids and anthocyanins have antioxidant properties for both the plant and the human body.

[0043] Enhanced and targeted production of carotenoids and anthocyanins in plants can be induced by exposure to a stressor. The problem is that after the stressor ends, the concentration of carotenoids and anthocyanins in the plant begins to decrease.

[0044] Thus, the essence of the claimed method consists in the justified use of three successive types of complex stressors with a certain dependence of intensity on time for each of them, with the aim of increasing the concentration of carotenoids and anthocyanins in the plant while simultaneously maintaining this concentration (or slightly decreasing it) after the end of the action of the last of the stressors.

[0045] Due to the long activation time and the practically unpredictable deactivation time, for the purposes of this method, it is not advisable to use abiotic stresses associated with water (including excess water or lack of water), chemicals, etc., as well as any types of biotic stresses (fungi, bacteria, insects, weeds, etc.).

[0046] Of all the stressors, those that can be quickly activated and deactivated were selected for use in the claimed method, namely: light stress, temperature stress and mechanical stress.

[0047] Under all types of the above stresses or their combinations (light stress, temperature stress and mechanical stress), protective compounds in the form of carotenoids and anthocyanins are formed in the plant. It should be especially emphasized that the molecular and metabolic response of plants to a combination of several stress factors cannot be directly extrapolated from the response of plants to each of these factors when they act separately [32 - Mittler R. Abiotic stress, the field environment and stress combination / / TRENDS in Plant Science, 2006, Vol. 11, No. 1, pp. 15-19]. This makes it possible to use the plant response to the combined action of various stressors (complex stress) in such a way as not only to increase the level of protective compounds in the plant, but also to fix it at a high level.

[0048] The implementation of the claimed method occurs as follows.

[0049] Plants are grown in a controlled environment, such as protected or enclosed ground in a greenhouse, phytotron, or growbox. The following parameters are adjustable in the greenhouse, phytotron, or growbox: light level, temperature, and wind strength. Light level is determined, for example, by LED phytolights or other artificial lighting devices (possibly as a supplement to natural sunlight). Temperature is regulated by appropriate air heating and cooling devices. Wind can be generated by a fan-type device or a specialized air blower. During this process, light level, temperature, and wind strength can reach values ​​that are stressful for the plants being grown.

[0050] During the light (daytime) phase (see Fig. 1), several periods of complex influence on plants are distinguished, in each of which the plants are subjected to a dosed effect of stressors (excessive illumination, increased temperature, wind load, decreased temperature) according to the cyclogram shown in Fig. 3.

[0051] The general plant response to stress is described in [4] and can be illustrated by the diagram in Fig. 1. After the active abiotic factor (illumination, temperature, wind force) leaves the optimal zone and reaches the stress value, the plant enters a state of so-called "alarm." If the stress factor does not lead to the plant reaching its minimum resistance (and, accordingly, does not lead to acute damage), the plant enters a state of resistance, in which so-called "hardening" of the plant occurs, including through the production and accumulation of AOS. If the stress factor continues to act for a sufficiently long time, this can lead to the depletion of the plant's reserves and its death.With a relatively short-term exposure to a stressor, the plant enters a regenerative state, characterized, among other things, by "adapting" to new living conditions and accepting new optimal values ​​for the factors involved. The principles of the general plant response to stress described above (Fig. 2) are utilized in the proposed method.

[0052] From time t0 (see Fig. 3) to time t1, the parameters of three variable abiotic factors (illumination, temperature, and wind strength) are maintained at optimal levels, while the fan (or other wind-generating device in the greenhouse) is turned off. Thus, from time t0 to time t1, normal plant development occurs at optimal temperature and illumination values ​​and in the absence of wind. During this time, AOS biosynthesis occurs in the plant at normal rates.

[0053] From time t1 to time t2, the illumination is increased to a level that causes stress in the plant but does not lead to its death. The specific value of this increased illumination that causes stress in the plant depends on the type of crop being grown, its stage of development, and other factors. As an example, for a tomato crop, the optimal illumination level (photon irradiance) can be between 200 μmol / (s⋅m 2 ) up to 300 µmol / (s⋅m 2 ) depending on the variety and other conditions [33 - Ryabin Ya. What kind of light do tomatoes like? Creating a light menu / https: / / growergood.ru / blog / kak-osveschat-tomaty]. In accordance with this, the illumination value (photon irradiance), at which a tomato experiences light stress, can be taken at a level exceeding the illumination on a bright sunny day under direct sunlight in mid-latitudes, that is, more than 1500 μmol / (s⋅m 2 ).

[0054] Then, from time t2 to time t3, maintain the illumination at the selected stress illumination level (for example, for a tomato crop, it is 1500 μmol / (s⋅m 2 )). At this time, the plant is in a state of resistance and increased AOS biosynthesis occurs in it.

[0055] Интенсивный биосинтез АОС в растениях во время действия светового стресса рассмотрен во многих работах, например в [34 - Stewart J.J. et al. Growth and Essential Carotenoid Micronutrients in Lemna gibba as a Function of Growth Light Intensity / / Frontiers in Plant Science, 2020, May, vol. 11, Article 480], [35 - Logan B.A. et al. Acclimation of leaf carotenoid composition and ascorbate levels to gradients in the light environment within an Australian rainforest / / Plant, Cell & Environment, 1996, Vol. 19, Issue 9, pp. 1083-1090], [36 - Logan B.A. et al. Antioxidants and xanthophyll cycle-dependent energy dissipation in Cucurbita pepo L. and Vinca major L. upon a sudden increase in growth PPFD in the field / / Journal of Experimental Botany, Vol. 49, №328, pp. 1881-1888], [37 - Izuhara T. et al. Elevated Levels of Specific Carotenoids During Acclimation to Strong Light Protect the Repair of Photosystem II in Synechocystis sp. PCC 6803 / / Frontiers in Plant Science, 2020, July, vol.11, Article 1030] and others.

[0056] To prevent the plant from entering a state of exhaustion (see Fig. 2), from time t3 to time t4 (see Fig. 3), the illumination level is reduced to the optimal value, i.e., the illumination is returned to the optimal level. If, after time t4, the plant were no longer exposed to any stress factors, a slightly elevated AOC level would be observed in the plant, which would slowly decrease over time (in Fig. 3, this process is shown by the dashed line).

[0057] To maintain high AOS formation in the plant and simultaneously preserve its viability, according to the proposed method, after the plant returns to optimal conditions at time t4, the initial stressor is not intensified (which could be achieved in our case by further increasing illumination), but the following actions are performed. From time t4 to time t5, illumination is maintained at an optimal level, allowing the plant to regenerate. Then, at time t5, the stress factor of increased illumination is activated (according to the algorithm described previously), as well as an additional stress factor, namely, increased temperature. Furthermore, from time t5 to time t6, the temperature is increased to a level that causes stress in the plant but does not lead to its death.The specific value of elevated temperature that causes stress in a plant depends on the type of crop being grown, its stage of development and other factors. When determining this value of stress elevated temperature, it should be taken into account that for heat-loving plants the optimal temperature is from 25 to 30 degrees. These include zucchini, tomatoes, cucumbers, peppers, etc. For cold-resistant plants the optimal temperature is from 20 to 25 degrees. These include dill, spinach, turnips, lettuce, etc. [38 - msk.teplicy.ru]. At the same time, short-term exposure to very high temperatures (about 45 degrees) can be detrimental to plants [39 - Klimentova E.G. Adaptation and resistance of plants. - Ulyanovsk, Ulyanovsk State University, 2006, 53 p.]. Taking into account the fact that stress reactions of plants occur even with temperature fluctuations from 6 to 8 degrees above the optimal value [40 - Physiology of resistance of agricultural plants. A short course of lectures.- Saratov: Saratov State Agrarian University, 2014], it is recommended to increase the temperature by 6 degrees relative to the optimal value starting from time t5. This elevated stress temperature is maintained from time t6 to time t7. From time t7 to time t8, the temperature is reduced to the optimal value. Thus, starting from time t5, simultaneously with the temperature increase, illumination is increased according to the algorithm described above.

[0058] The plant response to the stressor in the form of elevated temperature is described, for example, in [41 - Saidi et al. Heat perception and signaling in plants: a tortuous pathway to thermotolerance / / New Phytologist, 2011, Volume 190, Issue 3, pp. 556-565], [42 - Bita CE Plant tolerance to high temperature on a changing environment: scientific fundamentals and production of heat stress-tolerant crops / / Frontiers in Plant Science, 2013, July], [43 - Allakhverdiev SI Heat stress: an overview of molecular responses in photosynthesis / / Photosynth. Res., 98, pp. 541-550]. It is important that under the action of a stressor in the form of elevated temperature, increased AOS biosynthesis occurs.

[0059] Thus, when implementing the proposed method, beginning at time t5, the plant is artificially induced to specifically and intensively form AOS using the combined action of two different stressors. It should be noted that, according to the proposed method, the various stressors are not activated simultaneously, but rather staggered over time (see Fig. 3). Thus, from time t1 to time t4, the plant is exposed to the stressor of increased illumination, and from time t5 to time t8, the plant is exposed to a complex stressor of increased illumination and elevated temperature. This is done to prevent the plant from adapting to environmental conditions by constantly changing environmental factors, thereby expending accumulated AOS.

[0060] After the activation of an additional stressor in the form of elevated temperature (in addition to increased illumination), this elevated temperature is maintained constant from time t6 to time t7. During this time, the plant experiences increased AOS biosynthesis and, simultaneously, adaptation to the new living conditions [4]. At time t8, illumination and temperature return to optimal values ​​and are maintained at this optimal level until time t9.

[0061] Similarly to what was described earlier, to maintain a high intensity of AOS formation in the plant, and at the same time, to preserve its viability, at time t9, the action of previously acting stressors is not intensified (which could be achieved in our case by further increasing illumination and temperature), but at time t9, additional activation of a new stress factor, namely mechanical (wind) load, is performed. In this case, from time t9 to time t 12The plant is exposed to a complex stressor, including increased light, elevated temperature, and mechanical stress (wind load). The wind load corresponding to the stress level is determined experimentally, depending on the type of crop being grown, its stage of development, and other factors. It is recommended to adjust the fan speed to select a wind speed that causes the plants to bend significantly but does not cause critical damage (break or bruise).

[0062] Intensive AOS biosynthesis in plants under mechanical stress is discussed in [41 - Li X. et al. Mechano-stimulated modifications in the chloroplast antioxidant system and proteome changes are associated with cold response in wheat / / BMC Plant Biology, 2015, 15:219], [42 - Slesak I. et al. Antioxidant response system in the short-term post-wounding effect in Mesembryanthemum crystallinum leaves / / Journal of Plant Physiology, 2008, Volume 165, Issue 2, pp. 127-137], [43 - Chandru HK et al. Kinetics of wound-induced activation of antioxidant enzymes in Oryza sativa: differential activation at different growth stages / / Plant Science, 2003, Volume 164, Issue 6, pp. 935-941] and others.

[0063] At time t 12 the values ​​of illumination, temperature and wind load are returned to optimal values, which are maintained at the optimal level until time t 13 .

[0064] Similarly to what was described earlier, in order to maintain a high intensity of AOS formation in the plant, and at the same time, to maintain its viability, at time t 13 they do not increase the effect of previously acting stressors (which could be achieved in our case by further increasing illumination, temperature and wind load), but at time t 13 perform additional activation of a new stress factor, namely the action of a low temperature. In this case, from the moment of time t 13 until time t 16 the plant is exposed to a complex stressor in the form of increased illumination, low temperature and mechanical stress (wind load).

[0065] Intensive AOS biosynthesis in plants under low temperature stress has been discussed in many studies, for example, in [44 - Jahed KR et al. Coping with the cold: unveiling cryoprotectants, molecular signaling pathways, and strategies for cold stress resilience / / Frontiers in Plant Science, 2023, August, Volume 14], [45 - Orvar et al. Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane fluidity / / Plant Journal, 2000, Volume 23, pp. 785-794], [46 - Bhattacharjee S. Reactive Oxygen Species in Plant Biology, Springer, 2019, pp. 107-125], [47 - Prasad TK. Acclimation, Hydrogen Peroxide, and Abscisic Acid Protect Mitochondria against Irreversible Chilling Injury in Maize Seedlings / / Plant Physiol, 1994, Volume 105, pp. 619-627].

[0066] It should be emphasized that, at the same level of increased illumination, a lower temperature is perceived by the plant as a stronger stress than an increased temperature, since the lower the temperature, the lower the activity of the photosynthetic apparatus of the plant, and the higher the level of AOS produced in the plant to protect against light stress at the same level of illumination [48 - Titova M.S. Comparative analysis of the accumulation of carotenoids in needles / / Pacific Medical Journal, 2014, No. 2, pp. 48-50].

[0067] At time t 16 the values ​​of illumination, temperature and wind load are returned to optimal values.

[0068] Thus, in accordance with the proposed method, from time t1 to time t 16The plant is sequentially exposed to increasing complex stressors: from time t1 to time t4, the plant is exposed to a stress factor in the form of increased illumination; from time t5 to time t8, the plant is exposed to a complex stress factor in the form of increased illumination and increased temperature; from time t9 to time t 12 act as a complex stress factor in the form of increased illumination, increased temperature and mechanical stress; from the moment of time t 13 until time t 16act as a complex stress factor in the form of increased illumination, decreased temperature, and mechanical stress. For each of the complex stressors, a certain transient activation stage is provided, short enough for the activated stressor to be an unexpected phenomenon for the plant, and nevertheless, does not lead to irreversible damage. Such transient processes take place from time t1 to time t2; from time t5 to time t6; from time t9 to time t 10 ; from time t 13 until time t 14 .

[0069] It should be noted that the stress factors described above are applied to the plant with increasing intensity: each subsequent stressor is more powerful than the previous one. This is done so that, under the action of the next stressor, the intensity of AOS production in the plant is greater than under the action of the previous stressor. In this case, the stressors are separated from each other in time by short time intervals, during which the plant is in a state of regeneration (see Fig. 3). The states of regeneration, in which the plant accepts the achieved content of AOS in the tissues (accumulated after the action of stress) as a new standard, occupy the time from time t4 to time t5; from time t8 to time t9; from time t 12 until time t 13(see Fig. 3). This logic of organizing complex stress effects on a plant that increase over time, simultaneously with their separation from each other by the states of plant regeneration (after “hardening” of plants), makes it possible to achieve a stable increase in the content of AOS (carotenoids and anthocyanins) in the plant while maintaining the value of this content in plant tissues for a long time (see the lower graph in Fig. 3). This is fully consistent with the modern scientific view on the “memory” of environmental stress in plants, according to which plants have developed the ability to remember stress, which helps them adapt to new stress factors [49 - Song ZT Environmental Stress Memory in Plants / / Frontiers in Plant Science, 2024, Volume 15, April], [50 - Hilker M. Stress priming, memory, and signaling in plants / / Plant, Cell & Environment, 2019, Volume 42, pp. 753-761], [51 - Liu H. et al.Priming crops for the future: rewiring stress memory / / Trends in Plant Science, 2022, Volume 27, No. 7, pp. 699-716], [52 - Crisp PA et al. Reconsidering plant memory: Intersections between stress recovery, RNA turnover, and epigenetics / / Science Advances, 2016, Volume 2]. Sometimes in the scientific literature, the term “priming” and similar terms are used instead of the term “memory” of plants, and the term “stress preparation” and similar terms are used instead of the term “hardening”, but the essence of the phenomenon does not change: the effect of a stress factor on a plant allows the plant to adapt to such conditions of existence through various mechanisms, including the accumulation and maintenance of a high concentration of antioxidant compounds, such as carotenoids and anthocyanins, in its organs.

[0070] Of particular importance is the application in each of the periods of complex impact on the plant (from the moment of time t1 to the moment of time t 16) as the final stressor, specifically low temperature. The fact is that the reduction in accumulated AOS levels (after the end of stressors) is an energy-consuming process for the plant. Accordingly, if the end of stressors is preceded by a temporary period of low temperature, the reduction in accumulated AOS levels will be slower, allowing the plant to maintain high AOS levels for a longer period. Importantly, the rate of AOS biosynthesis in the plant is many times greater than the rate of decline in accumulated AOS levels.

[0071] In Fig. 3, the distances between adjacent moments of time t are shown conditionally, that is, without respect to scale. The specific values ​​of each of the moments of time (from t0 to t 16) should be selected empirically based on achieving a compromise between normal plant development (from time t0 to time t1) and enhanced biosynthesis of AOS in the plant under the influence of dosed stressors (from time t1 to time t 16 ).

[0072] The proposed method may be particularly attractive for use in home greenhouses, phytotrons, and growboxes, since it allows for the cultivation of plants with increased carotenoid and anthocyanin content using accessible and technologically advanced procedures. This means that, in essence, natural medicinal compounds for the prevention of cancer can be obtained from growing familiar crops such as tomatoes or peppers.

[0073] Thus, in comparison with the prototype [3], the claimed method for growing plants with an increased content of carotenoids and anthocyanins has increased efficiency, due to the use of a controlled process of intensification of AOS biosynthesis, which is realized by the sequential activation over time of dosed complex abiotic nature-like stressors, such as excessive illumination, high and low temperatures and wind load.

Claims

A method for growing plants with an increased content of carotenoids and anthocyanins, in which plants are exposed to light radiation in the light phase, with illumination increasing from time t1 to time t2, with the set illumination maintained from time t2 to time t3, with illumination decreasing from time t3 to time t4, characterized in that the plants in the light phase are additionally exposed to a change in temperature and wind load created by a fan, with several periods of complex exposure to plants being distinguished in the light phase, lasting from time t0 to time t 16, from the moment of time t4, the set illumination is maintained until the moment of time t5, from the moment of time t5, the illumination is increased until the moment of time t6, from the moment of time t6, the set illumination is maintained until the moment of time t7, from the moment of time t7, the illumination is reduced until the moment of time t8, from the moment of time t8, the set illumination is maintained until the moment of time t9, from the moment of time t9, the illumination is increased until the moment of time t 10 , from time t 10 maintain the set illumination until time t 11 , from time t 11 reduce illumination until time t 12 , from time t 12 maintain the set illumination until time t 13 , from time t 13 increase the illumination until time t 14 , from time t 14 maintain the set illumination until time t 15 , from time t 15reduce illumination until time t 16 , maintain the set temperature from time t0 to time t5, from time t5 increase the temperature until time t6, from time t6 maintain the set temperature until time t7, from time t7 decrease the temperature until time t8, from time t8 maintain the set temperature until time t9, from time t9 increase the temperature until time t 10 , from time t 10 maintain the set temperature until time t 11 , from time t 11 reduce the temperature to time t 12 , from time t 12 maintain the set temperature until time t 13 , from time t 13 lower the temperature to time t 14 , from time t 14 maintain the set temperature until time t15 , from time t 15 increase the temperature to time t 16 , turn on the fan at time t9, from time t9 increase the wind force until time t 10 , from time t 10 maintain the set wind force until time t 11 , from time t 11 reduce the wind force until time t 12 , at which the fan is turned off, the fan is turned on at time t 13 , from time t 13 increase the wind force until time t 14 , from time t 14 maintain the set wind force until time t 15 , from time t 15 reduce the wind force until time t 16 , at which the fan is turned off, the values ​​of illumination and temperature at times t0, t4, t8, t 12 and t 16 are optimal for plants at time t 16the current period of complex impact on plants coincides with the time t0 of the next period of complex impact on plants.

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