WO2017101691A1 - The method for cultivation of plants using metal nanoparticles and the nutrient medium for its implementation - Google Patents

The method for cultivation of plants using metal nanoparticles and the nutrient medium for its implementation Download PDF

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WO2017101691A1
WO2017101691A1 PCT/CN2016/108424 CN2016108424W WO2017101691A1 WO 2017101691 A1 WO2017101691 A1 WO 2017101691A1 CN 2016108424 W CN2016108424 W CN 2016108424W WO 2017101691 A1 WO2017101691 A1 WO 2017101691A1
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nanoparticles
nutrient medium
zinc
iron
copper
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PCT/CN2016/108424
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French (fr)
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Hui Zhao
Min Liu
Yu Chen
Jinying LU
Huasheng LI
Qiao SUN
Nechitaylo Galina SEMENOVNA
Zhigach Alexey NIKOLAEVICH
Leypunsky Ilya OVSEEVICH
Rakhmetova Alla ALEKSANDROVNA
Bogoslovskay Olga ALEKSANDROVNA
Glushchenko Natalia NILOLAEVNA
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Shenzhou Space Biotechnology Group
Emanuel Institute Of Biochemical Physics Of Russian Academy Of Sciences (Ibcp Ras)
V.L. Talrose Institute For Energy Problems Of Chemical Physics Of Russian Academy Of Science (Inepcp Ras)
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Priority to CN201680068482.1A priority Critical patent/CN108471713B/en
Publication of WO2017101691A1 publication Critical patent/WO2017101691A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G31/00—Soilless cultivation, e.g. hydroponics
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00—Cultivation of specific crops or plants not otherwise provided for
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00—Cultivation of specific crops or plants not otherwise provided for
    • A01G22/05—Fruit crops, e.g. strawberries, tomatoes or cucumbers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
    • A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/16—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/16—Heavy metals; Compounds thereof
    • A01N59/20—Copper
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the proposed invention relates to the field of bio-and nanotechnology in plant cultivation and can be used for production of high quality planting material of agricultural plants, to conduct biotechnology research, to improve the quality of agricultural products.
  • the invention can be used in aeroponic and hydroponic technologies, and also for the creation of life-support systems forastronauts under conditions of long space flight.
  • Nanotechnological preparations such as NanoGro, GreenLift, AgBion, etc. are already known, but research in the field of developing new preparations and the ways of using nanotechnology to improve the efficiency of agricultural production are continued. Recent advances in this area are summarized in particular in reviews [Azamal, HusenKhwaja Salahuddin Siddiqi. "Phytosynthesis of nanoparticles: concept, controversy and application” //Nanoscale Res. Lett., 2014, V. 9, No1, p. 229; L.R. Khot et al. "Applications of nanomaterials in agricultural production and crop protection: A review” //Crop Protection, V. 35, May 2012, p. 64] .
  • at least one component of the wide range of substances including carbon compounds, phosphorus, nitrogen, boron and other elements necessary for the nutrition and development of plants and also salts, chelates and metal oxides or other compounds, can be used as a coating.
  • nanoparticles of metals such as noble metals, iron, copper, etc. are used as "means of delivery" of micro fertilizers into plant tissues and cells, but their own independent role as nutrient components cannot be estimated.
  • the known nano-preparations are intended to stimulate plant growth in the open and protected ground.
  • modern development of biotechnology is inextricably linked to the plants and tissue cultures growing using artificial media with a balanced composition of nutrients necessary for proper growth and development of plants.
  • titan dioxide nanoparticles nanoanatase
  • MS medium Murashing-Skoog medium
  • TiO 2 nanoparticles at a concentration of 30 mg/l demonstrated the best effects: the enhancement of seed germination indexand viability potential, the increase in the length of roots and sprouts, the gain in green mass and other parameters.
  • the invention [EP 2499107 A1, Pub. 19.09.2012] is taken as a prototype for our declared method of cultivation of plants using nanoparticlesas active factors. It was shown, that introduction of multilayer carbon nanotubes into MS medium at the effective concentrations of 0.01 –0.2 pg/ml accelerates the germination of tomato seeds and increases the percentage of germination.
  • the tomato plants which were grown on a nutrient medium with carbon nanotubes hadthe larger volume of biomass than in the control and at the same time did not differ from the control by the root length. The authors associate this effectwith the increase in water absorption processes in seeds with carbon nanotubes assisting.
  • the nutrient media such as Murashing-Skoog, Gamborg, Heller etc. are known, which contain the balanced nutritional complex of obligatory components necessary for the development of plants, including organic substances –vitamins, carbohydrates, amino acids and/or protein hydrolysates–and also inorganic salts containing nitrogen, phosphorus, boron, potassium, calcium, magnesium, sulfur, iron, and trace elements such as manganese, zinc, copper, molybdenum, etc.
  • the prototype of a nutrient medium for implementation of the declared method is a widely applied Murashing-Skoog nutrient medium (MS medium) [Murashing T., Skoog F. "A received medium for rapid growth and bio-assays with tobacco tissue culture” . Physiol. Plant. 1962. V. 15, pp. 473-497] . Table 1 shows the known composition of the agar nutrient medium of the prototype.
  • MS medium containsthese metals in ionic form as salts.
  • the object of the invention is to provide a method for cultivation of plants on a nutrient medium containing nanoparticles of essential elements that provides the improvement of seed germination, and also the improvement of morphometric and/or physiological parameters of plants with the aim to obtain improved high quality planting material.
  • the task is solved by the proposed method for cultivation of plants comprising the germination of seeds and subsequent cultivation of plants both under aseptic conditions on agar nutrient medium containing nanoparticles, wherein said nanoparticles are nanoparticles of iron, or nanoparticles of zinc, or nanoparticles of copper, or a combination thereof.
  • Another object of the invention is to propose a composition of nutrient medium for implementationof the claimed method.
  • Thistask is solvedbythe proposed alternatives of agar nutrient medium which contain the components included in the composition of the Murashing-Skoog medium, namely, vitamins: ⁇ , ⁇ 6 and ⁇ 1 , Glycine, Sucrose, Mesoinositol, helating agent Na 2 EDTA ⁇ 2H 2 O, inorganic salts: NH 4 NO 3 , KNO 3 , CaCl 2 ⁇ 2H 2 O, MgSO 4 ⁇ 7H 2 O, KH 2 PO 4 , KI, H 3 BO 3 , MnSO 4 ⁇ 7H 2 O, NaMoO 4 ⁇ 2H 2 O and CoCl 2 ⁇ 6H 2 O, as well as iron, zinc and copper, wherein iron or zinc or copper or combination thereof is included into nutrient medium in the form of nanoparticles of iron or nanoparticles of zinc or nanoparticles of copper or combination thereof respectively.
  • the nutrient medium can include chitosan.
  • the concentration of nanoparticles ofelectro neutraliron in the nutrient medium is 10.0 –0.06 mg/l.
  • the concentration of nanoparticles of electro neutralzinc in the nutrient medium is 3.0 –0.016 mg/l.
  • the concentration of nanoparticles of electro neutral copper in the nutrient medium is 0.04 –0.00016 mg/l.
  • the technical result of the invention isthe stimulation of seed germination and the improvement of morphometric and/or physiological parameters of the grown plants.
  • the essence of the invention is in the fact that cultivation of plants on the traditional nutrient media, but modified in such a way that the salts of essential metals, particularly iron sulfate, or zinc sulfate, or copper sulfate or ironsulfate and zinc sulfate and copper sulfate simultaneously are replaced by nanoparticles of these metals in electro neutral state while other components of the nutrient medium remain unchanged, leads to better seed germination and to improvedmorphometric and/or physiological parameters of plants grown on these media compared with the plants grown on unmodifiednutrient media.
  • the invention is based on the results of the authors’research regarding the influence of metal nanoparticles on the structure and function of various biosystems.
  • the electro neutral metal nanoparticles are characterized by polyfunctional and prolonged action, low toxicity, which is 7 –50 times lower than that of the corresponding metals in ionic forms, and are capableto distribute actively through organs and tissues of plants and to stimulate the vital processesat biotic doses, i.e., at the doses, which are 10 –50 times lower than the maximum tolerated doses.
  • Chitosan which is known as absorbent for metabolic products of the growing plants and microflora, and also as plant growth stimulating factor, may be incorporated in the claimed nutrient medium. Chitosan is known to possess high sorption activity against bacteria, exhibits growth promoting effect, increases adaptive potential of plants, protects plants from infections, promotes higher crop productivity [Nyanikova G.G., Mametnabiev T.E., Kalinkin I.P., Gepetskaya M.V., Komissarchik S.M., Eldinova E.Y. "Applications of chitosan" http: //science. spb. ru/] .
  • Fig. 1 ⁇ –1C show the electronic microscopy images of iron, zinc, copper nanoparticles, and histograms of the size distributions for metal particles: 1 ⁇ –iron nanoparticles; 1B –copper nanoparticles; 1C –zinc nanoparticles.
  • Fig. 2A –2D show the effect of metal nanoparticles at various concentrations and chitosan (100mg/l) on germination ofVenice tomato seeds: 2A –the nutrient mediawith iron nanoparticles; 2B –the nutrient mediawith zinc nanoparticles; 2C –the nutrient mediawith copper nanoparticles; 2D –the nutrient mediawith combinations of iron, zinc and copper nanoparticles.
  • Fig. 3A –3D showthe effect of metal nanoparticles at various concentrations and chitosan (100mg/l) onthe LJ-king pepper root length: 3A –the nutrient media with iron nanoparticles; 3B –the nutrient media with zinc nanoparticles; 3C –the nutrient media with copper nanoparticles; 3D –the nutrient media with combinations of iron, zinc and copper nanoparticles.
  • Fig. 4 showsthe photo of theLJ-king pepper plants grown on a nutrient media containing zinc nanoparticles and chitosan: Zn-b 0 –the control, Zn-b3 –with zinc nanoparticles at a concentration of 0.016 mg/l in a nutrient medium; Zn-b2 –with zinc nanoparticles at a concentration of 0.08 mg/l in a nutrient medium; Zn-b4 –with zinc nanoparticles at a concentration of 0.4 mg/l in a nutrient medium.
  • Fig. 5 ⁇ –5D show the effect of metal nanoparticles at various concentrations and chitosan (100 mg/l) on theHY-2 tomato root length: 5A –the nutrient media with iron nanoparticles; 5B –the nutrient media with zinc nanoparticles; 5C –the nutrient media with copper nanoparticles; 5D –the nutrient media with combinations of iron, zinc and copper nanoparticles.
  • Fig. 6 ⁇ –6D show the effect of metal nanoparticles at various concentrations and chitosan (100 mg/l) on theroot activity ofLJ-king pepper, HY-2 tomato and Venic Canal tomato: 6A –the nutrient media with iron nanoparticles; 6B –the nutrient media with zinc nanoparticles; 6C –the nutrient media with copper nanoparticles; 6D –the nutrient media with combinations of iron, zinc and copper nanoparticles.
  • Fig. 7 ⁇ –7C show the effect of metal nanoparticles at various concentrations and chitosan (100 mg/l) on the chlorophyll content in theleaves of plants: 7A –with iron nanoparticles in a nutrient medium for LJ-king pepper; 7B –with zinc nanoparticles in a nutrient medium for HY-2 tomato; 7C –with copper nanoparticles in a nutrient medium for LJ-king pepper.
  • the nanoparticles of electro neutral iron, zinc and copper are manufactured as described in [AS SSSRNo 814432 //Bull. inventions. 1981, No 11, ⁇ . 25] , using the apparatus Migen-3, described in [Jigatch A.N., Leipunskii I.O., Kuskov M.L., Stoenko N.I., Storozhev V.B. "An apparatus for the production and study of metal nanoparticles" , Instrum. Exp. Tech. 43 (2000) 839-845] .
  • the estimation of the shape and size of nanoparticles was performed by the method of Transmission Electron Microscopy (TEM) using LEO 912 AB OMEGA device.
  • TEM Transmission Electron Microscopy
  • phase composition of nanoparticles was performed by the method of X-ray Diffraction analysis (XRD) using the ADP-1 Diffractometer ( Russia) .
  • the TEM images were processed by means of the computer Micran 25 program based on measurement of the diameter of at least thousand of particles. On the basis of the data obtained a distribution curve of metal nanoparticles according to their size were plotted and the average diameter of particles was counted.
  • the TEM images and curves of size distributions of metal nanoparticles are presented in Fig. 1A –1C.
  • the curve of the size distribution of iron nanoparticles lies in the area of 5 –80 nm.
  • An average diameter of iron particles is 27.0 ⁇ 0.51 nm.
  • the curve of the size distribution of zinc nanoparticles lies in the area of 10 –200 nm.
  • An average diameter of zinc particles is 54.0 ⁇ 2.8 nm.
  • the curve of the size distribution of copper nanoparticles lies in the area of 5 –225 nm.
  • An average diameter of copper particles is 79.0 ⁇ 1.24 nm.
  • iron, zinc and copper nanoparticles are single-crystal structures with a round regular shape covered with a semitransparent oxide film.
  • the results of XRD-analysis demonstrate that the crystal phases content of iron nanoparticles are follows: the iron metal phase is equal to 53.6%, the iron oxide phase (Fe 3 O 4 ) is equal to 46.4%, and the thickness of oxide film is 3.5 nm.
  • Zinc and copper nanoparticles consist of the crystal metal phase with oxide film thickness of 1.0 –0.5 nm.
  • the formulation of any known balanced nutrient mediums containing all vital for plants organic and inorganic macro-and micronutrients can be used as a basis.
  • electroneutraliron nanoparticles, or electro neutral zinc nanoparticles, or electro neutralcopper nanoparticles, or a combination thereof are inserted into themedium instead of the salt of iron, or salt of zinc, or salt of copper, or combination of these salts respectively, while other components of the nutrient medium used as a basis, remain without changes.
  • the ranges of concentrations of iron, zinc and copper nanoparticles in the embodiments of the invention are chosen taking into account the content of these metals in the ionic form in the medium taken as a basis. So, in terms of metal, the concentration of ionic iron in the base MSmedium is 5.6 mg/l, the concentration of electroneutral iron nanoparticles in the nutrient media in embodiments of the invention is 10.0 –0.06 mg/l; the concentration of ionic zinc in terms of metal in the base MSmedium is 1.96 mg/l, the concentration of electroneutral zinc nanoparticles in the nutrient media in embodiments of the invention is 3.0 –0.016 mg/l; the concentration of ionic copper in terms of metal in the base MSmedium is 0.0064 mg/l, the concentration of electroneutral copper nanoparticles in the nutrient media in embodiments of the invention is 0.04 –0.00016 mg/l.
  • the MS nutrient medium is prepared in accordance with the formulation given in Table 1.
  • the nutrient medium is sterilized at a temperature of 120°C under a pressure of 0.1 MPa for 20 min in an autoclave "Hirayama, HVE-50" (Japan) .
  • the Murashing-Skoog nutrient medium prepared as described above is used as a control.
  • a weighed portion of 37.3 mg of Na 2 EDTA ⁇ 2H 2 O is dissolvedin 1 l of water while stirring on a magnetic stirrer for 5 minutes.
  • the stock iron nanoparticle suspension is diluted by sterile distilled water up to required concentrations with the subsequent dispersing and cooling stages performed as described above.
  • a stockaqueous suspension of zinc nanoparticles 600 mg of powder, containing the nanoparticles of electro neutral zinc, is put into a flask and 200 ml of distilled sterile water is added. Then the sample is dispersed for 30 sec. with a power of 99%, and the suspension is cooled in an ice bath for 30 sec. to a temperature 10 –15°C. The dispersing process is repeated three times.
  • the stock zinc nanoparticle suspension is diluted by sterile distilled water up to required concentrations with the subsequent dispersing and coolingstages performed as described above.
  • the stock aqueous suspension of copper nanoparticles is diluted by sterile distilled water up to necessary concentrations with the subsequent dispersing and cooling stages performed as described above.
  • aqueous suspensions with iron zinc and copper nanoparticlesin different ratios
  • 2 ml of each aqueous suspension in a desired concentrations, prepared as described above (see 3.2, 3.4 and 3.6) are mixed, and the sample is adjusted with sterile distilled water to 10 ml and then dispersed and cooled as described above for nanoparticles of iron (see 3.2. ) .
  • 50 ml of the nutrient media prepared as described above are immediately dispensed into 200 ml sterile bottles, sealed with polyethylene film or polypropylene caps and leaved in a sterile room to cool.
  • the nutrient media prepared according to the described procedures were further used for seed germination and plant cultivation.
  • the samples of the nutrient media containing metal nanoparticles with the addition of Tyanshi chitosan at a concentration of 100 mg/l are prepared similarly as the Murashing-Skoog nutrient medium with the addition of chitosan as described in paragraph 2.
  • the invention is realized as follows:
  • the seeds of plants are placed into sterile culture vessels, 3 seeds in each vessel, on the surface of the agar nutrient medium containing, in accordance with the invention, nanoparticles of iron or nanoparticles of zinc, or nanoparticles of copper, or combinations of these nanoparticles in various proportions.
  • the vessels are put on shelves in a climatic room at 22 –25°C and 36%humidity and are illuminated with 3500 –3000 Lux 12/12 hoursa day. Separate sets of experiments were carried out usingthe standard MS medium (control) and usingthe nutrient media containing Tyanshi chitosan and/or metal nanoparticles (test) .
  • the seed germination capacity is estimated on the 15th day and morphometric and physiological parameters of the grown plants are measured on the 40th day after the start of plant cultivation.
  • Example 1 Effect of metal nanoparticles and chitosan in the composition of nutrient medium on tomato seeds germination.
  • Seed germination is characterized by the value of germination index, defined as the percentage of the number of germinated seeds in relation to the total number of seeds taken. The index of seed germination is determined on the 15th day after plant cultivation starts.
  • Fig. 2A –2D show the effect of replacement of metal salts by nanoparticles of metals in the composition of the nutrient medium and the effect of chitosan supplement (100 mg/l) on the Venice tomato seed germination.
  • Fig. 2A demonstrates an increase of thegermination index of tomato seedsby 13 –27% (as compared to the control) on the nutrient medium containing the iron nanoparticles at concentrations of 0.6 –10.0 mg/l.
  • the addition of chitosan to the nutritious medium containing iron nanoparticles demonstrates an additional increase in germination index up to 23 –33%.
  • zinc nanoparticlessupplemented to the nutrient medium at concentrations of 0.2 –3.0 mg/l entails a 2 –3 fold increase in seed germination index compared with the control.
  • the addition of chitosan into the nutrient medium containing 1.0 mg/l of zinc nanoparticles induces further increase of germination index up to 13%.
  • blend of iron, zinc and copper nanoparticles introduced into the nutrient medium instead of salts of these metals also provides the improvement of tomato seed germination, moreover the higher the concentration of nanoparticles in the medium, the higher the tomato seed germinationindex.
  • iron nanoparticles (10.0 mg/l) + zinc nanoparticles (3.0 mg/l) + copper nanoparticles (0.04 mg/l) enhances tomato seed germination up to 2.7 fold compared with the control.
  • Example 2 Effect of metal nanoparticles in the composition of nutrient medium on morphometric indices of pepper and tomato plants.
  • Fig. 3A –3D showthe effect of metal nanoparticles in the composition of nutrient medium on the LJ-king pepper root length.
  • Fig. 3B shows that pepper root length increases by 71, 80 and 62%respectively compared with the controlif pepper is cultivated on the nutrient medium containing zinc nanoparticles ata concentrationsof 0.4, 0.008 and 0.016 mg/l instead of zinc ions (1.96 mg/l in terms of metal) .
  • the optimal concentration of zinc nanoparticles appears to be 5 –122 timeslower than the concentration of ionic zinc in MS nutrient medium.
  • Fig. 3C illustrates an increase ofthe pepper root length by 34, 57 and 54%compared with the control when pepper grows on a nutrient medium containing copper nanoparticles at concentrations of 0.00016, 0.0008 and 0.004 mg/l, respectively, instead of copper ions (0.0064mg/l in terms of metal) .
  • concentration of copper nanoparticles is 1.6 –40timeslower than the concentration of copper ions in the MS nutrient medium.
  • Fig. 3D demonstrates that pepper root length increases by 7 –58%compared with the control when pepper is cultivated on a nutrient medium containing the combinations of Fe, Zn and Cu nanoparticles in different proportions instead of iron, zinc and copper ions.
  • the exception is the experiment, in which the nanoparticles are in the minimal concentrations of the tested (iron –0.06 mg/l, zinc –0.016 mg/land copper –0.00016 mg/l) .
  • Fig. 3D shows a significant synergistic effect of the combined action of low concentrations of metal nanoparticles and chitosan, resulting the increase of pepper root length by 40%compared with the control.
  • Fig. 4 presents a photo of thesprout ofLJ-king pepper grown on a nutrient medium in which zinc salt is replaced by zinc nanoparticles in combination with chitosan. It is obvious that the presence of zinc nanoparticles in a nutrient medium at concentrations of 0.016 mg/l –0.4 mg/l and chitosan contributes to a more active development of the pepper root system compared with the control: root length increases 1.6 –1.8 times. The supplement of chitosan into the nutrient medium containing zinc nanoparticles at a concentration of 0.4 mg/l, gives an additional increase of root length by 30%.
  • Fig. 5A –5D show the effect of metal nanoparticles and chitosan in the composition of the nutrient medium on the HY-2 tomato root length.
  • the root length is increased by 1, 17 and 38%respectively when tomatoes are grown on a nutrient medium containing zinc nanoparticles at concentrations 0.016, 0.08 and 0.4 mg/l respectively instead of zinc in the ionic form. This result increases by 17, 2 and 15%respectively with chitosan supplement (see Fig. 5B) .
  • tomato root length increases by 7%and 10%respectively in comparison with control.
  • addition of chitosan promotes a further increase in the tomato root length.
  • a combined use of iron nanoparticles (3.0 mg/l) , zinc nanoparticles (0.4 mg/l) , copper nanoparticles (0.004 mg/l) and chitosan in a nutrient medium gives again in a tomato root length by 70%in comparison with control.
  • the inclusion in the culture medium of metal nanoparticles has a positive effecton sprout lengthand green mass of pepper and tomato plants.
  • the introduction of zinc nanoparticles into a nutrient medium ata concentration of 0.08 mg/l promotes an increase in theHY-2 tomato sprout length by 1.2 times.
  • Chitosan supplement to a nutrient medium further increases this index by 17%.
  • the green mass ofLJ-king pepper growing on a nutrient medium containing zinc nanoparticles (0.4 mg/l) and chitosan is 42%higher than in the control.
  • the use of a nutrient medium with zinc nanoparticles ata concentration of 0.08 mg/l for growing of Venice tomato increases the green mass by 80%in comparison with control.
  • Example 3 Effect of metal nanoparticles in the composition of nutrient media on physiological indices of pepper and tomato plants.
  • test values areobtained on the nutrient medium with metal nanoparticles, the control values –on the Murashing-Skoog nutrient medium.
  • the root activity is determined by the reduction of chloride 3-phenyltetrazolium as described in [Adebusoye O. Onanuga, Ping’a n Jiang, Sina Adl. "Effect of phytohormones, phosphorus and potassium on cotton varieties (Gossypium hirsutum) root growth and root activity grown in hydroponic nutrient solution”//Journal of Agricultural Science. 2012, Vol. 4, N 3, pp. 93-110] .
  • Fig. 6A –6D show the effect of metal nanoparticles, introduced into the nutrient medium instead of corresponding metal salts, onLJ-king pepper and Venice tomato root activity.
  • the cultivationof Venice tomato plants on the media with iron nanoparticles at concentrationsof 0.6, 3.0 and 10.0 mg/l leads toanincrease in the root activity by 112, 125 and 76%respectively compared with the control.
  • Fig. 6C demonstrates the increase in the root activity of pepper and tomatoes plants grown on the nutrient media with copper nanoparticles.
  • Cultivation ofVenice tomato on the nutrient medium, containing copper nanoparticles at concentrations 0.0008, 0.004 and 0.04 mg/l increasestheroot activity by 65, 10 and 12%, respectively, as compared to control.
  • Cultivation of pepper on the nutrient medium containingthe combinations of nanoparticles: (0.06 mg/l of iron +0.016 mg/l of zinc + 0.00016 mg/l of copper) , and (0.3 mg/l of iron + 0.08 mg/l of zinc + 0.0008 mg/l of copper) , and (3.0 mg/l of iron + 0.4 mg/l of zinc + 0.004 mg/l of copper) instead of these metals in ionic form results in the increase of the pepper root activity by 98, 51 and 91%respectively.
  • Fig. 7A demonstrates the effects of iron nanoparticles added into the nutrient medium instead of ferrous sulfate on the chlorophyll content in the leaves of LJ-king pepper plants. It is shown that iron nanoparticles at concentrations 0.3 mg/l and 3.0 mg/l in the nutrient media enlarge the chlorophyll content by 5%and 27%respectively as compared to control. Addition of chitosan into the nutrient medium together with iron nanoparticles at concentrations 0.06 mg/l and 0.3 mg/l leads to a further increase of chlorophyll content in pepper leaves by 6%and 10%respectively.
  • Fig. 7B shows the effect ofaddition of zinc nanoparticles into the nutrient medium instead of zinc sulfate on the chlorophyll content in HY-2 tomato leaves.
  • Zinc nanoparticles at concentrations 0.08 mg/l and 0.4 mg/l increase the chlorophyll content in HY-2 tomato leaves by 56%and 108%, respectively.
  • Additional introduction of chitosan into the nutrient media increases this index by 48%and 20%if the concentrations of zinc nanoparticles are 0.016 mg/l and 0.08 mg/l respectively.
  • Fig. 7C shows the effect of copper nanoparticle supplement to the nutrient medium instead of copper sulfate on chlorophyll content in leaves ofLJ-king pepper.
  • Copper nanoparticles added into the nutrient medium increase thechlorophyll content in LJ-king pepper leaves. The maximum effect is observed at the lowest tested copper concentration of 0.00016 mg/l, wherein the chlorophyll content is 59%higher than in the control.
  • chlorophyll content in leaves of pepper has increased by 75%.
  • the method of growing plants based on the use of the nutrient medium, in which the salts of iron, zinc and copper are partially or completely replaced by electroneutralnanoparticles of these metals is claimed.
  • the method allows producinghealthy plants with compact stems with a developed and active root system, which can be used as high-quality planting material.
  • the proposed nutrient medium can be used inbiotechnological researches, for improving of the quality of agricultural products, for aeroponic and hydroponic technologies.
  • the proposed method of growing plants on a nutrient media containing iron, copper and zinc nanoparticles can be used to create life-support systems for astronauts in conditions of prolonged space flights.

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Abstract

A method for cultivation of plants is used in the field of bio- and nanotechnology in plant cultivation and can be used for production of high quality planting material of agricultural plants, for implementation of biotechnology research, to improve the quality of agricultural products. The method can be used in aeroponic and hydroponic technologies and also for the creation of life-support systems for astronauts under conditions of long space flight. The method for cultivation of plants comprises the seed germination and subsequent cultivation of plants on the aseptic agar nutrient medium containing a set of essential for plant growth organic and inorganic components, including iron, zinc and copper, wherein iron or zinc, or copper or a combination thereof is in the form of electro neutral metal nanoparticles. Additionally, the nutrient medium can include chitosan. The method allows to improve the seed germination and to enhance the physiological and morphological indices of plants, such as the root length and root activity, the content of chlorophyll in the leaves, the sprout length, and the green mass yield.

Description

THE METHOD FORCULTIVATION OF PLANTS USING METAL NANOPARTICLES AND THE NUTRIENT MEDIUM FOR ITS IMPLEMENTATION
The proposed invention relates to the field of bio-and nanotechnology in plant cultivation and can be used for production of high quality planting material of agricultural plants, to conduct biotechnology research, to improve the quality of agricultural products. The invention can be used in aeroponic and hydroponic technologies, and also for the creation of life-support systems forastronauts under conditions of long space flight.
Application of nanomaterials as plant protection agents and fertilizer contributes to improving plant resistance to adverse weather conditions and diseases, improving yield and quality of agricultural crops. Nanotechnological preparations such as NanoGro, GreenLift, AgBion, etc. are already known, but research in the field of developing new preparations and the ways of using nanotechnology to improve the efficiency of agricultural production are continued. Recent advances in this area are summarized in particular in reviews [Azamal, HusenKhwaja Salahuddin Siddiqi. "Phytosynthesis of nanoparticles: concept, controversy and application" //Nanoscale Res. Lett., 2014, V. 9, №1, p. 229； L.R. Khot et al. "Applications of nanomaterials in agricultural production and crop protection: A review" //Crop Protection, V. 35, May 2012, p. 64] .
Nanofertilizersin the form of metal nanoparticles (NP) coated by the substances with nutritious micro additives or their precursorsare described in the application [WO2013121244 A1, published 22.08.2013] . According to this invention, at least one component of the wide range of substances, including carbon compounds, phosphorus, nitrogen, boron and other elements necessary for the nutrition and development of plants and also salts, chelates and metal oxides or other compounds, can be used as a coating. In this technical solution, nanoparticles of metals such as noble metals, iron, copper, etc. are used as "means of delivery" of micro fertilizers into plant tissues and cells, but their own independent role as nutrient components cannot be estimated.
The methods of seed treatment [UA 33863, published 10.07.2008] and the ways of cultivation of grain and vegetable crops [UA 92875, published 10.12.2010 and UA 92876, published 10.12.2010] , including the use of colloidal solutions of nanoparticle blends of biogenic trace elements –zinc, manganese, iron, copper, molybdenum, cobalt, in combination with chemicals, known for their biological role, were described. As in the previous analogue, in these technical solutions an independent role of nanoparticleas nutrient components can not be detected. Moreover theintroductioninto compositions of organic productsmay cause environmental stress and adversely affect the quality of the product.
The known nano-preparations, as a rule, are intended to stimulate plant growth in the open and protected ground. At the same time, modern development of biotechnology is inextricably linked to the plants and tissue cultures growing using artificial media with a balanced composition of nutrients necessary for proper growth and development of plants.
The widespread use of biotechnologies associated with creation and maintenance of collections of valuable species, with the need for the rapid multiplication of the clone of the plant and production in large quantities the vegetative progeny of hard propagated under common conditions forms of plants, as well as with modern requirements to the quality of planting material, the need for its improvement and testing.
Also it is of great importance to use the artificial nutrient media in the development of autonomous life-support systems, for example, for long-term spaceflights.
The solution of all these tasks supposes the use of advanced techniques, the improvement of technology of cultivation of plants in order to obtain planting material free from viral, fungal and bacterial diseases, mites and nematodes.
In this respect the use of nanostructures in different techniques for improvement and cultivation of planting material and possibly for the clonal reproduction of plants, is a promising course. This is even more so important becausecultivation factors and conditions are significant for plants growing. In addition to light and temperature the composition of nutrient mediumisa very important factor for plant cultivation； so, the changesof media due to the introduction of nanostructure forms ought to influence the quality parameters of growing plants.
The addition of titan dioxide nanoparticles (nanoanatase) to the Murashing-Skoog medium (hereinafter MS medium) at a concentration of 10 –40 mg/l is known to be usedtoincrease seed germination of parsley and improve the quality ofseedling [Dehkourdi E H. Mosavi M. Biol Trace Elem Res. 2013, Nov., 155 (2) , p. 283] . TiO2 nanoparticles at a concentration of 30 mg/l demonstrated the best effects: the enhancement of seed germination indexand viability potential, the increase in the length of roots and sprouts, the gain in green mass and other parameters. However, it isnot possible yet to consider nanoparticles of titan dioxide as acceptable stimulator of plant growth in crop production because there remain doubts about their safety for living organisms. For example, it has been shown that inhalation of titanium dioxide powder increases the probability of cancer progress in rats, beinga potentially carcinogenicfactor for human [http: //www. neboleem. net/dioksid-titana. php] .
According to the set of essential features, the invention [EP 2499107 A1, Pub. 19.09.2012] is taken as a prototype for our declared method of cultivation of plants using nanoparticlesas active factors. It was shown, that introduction of multilayer carbon nanotubes into MS medium at the effective concentrations of 0.01 –0.2 pg/ml accelerates the germination of tomato seeds and increases the percentage of germination. The tomato plants which were grown on a nutrient medium with carbon nanotubes hadthe larger volume of biomass than in the control and at the same time did not differ from the control by the root length. The authors associate this effectwith the increase in water absorption processes in seeds with carbon nanotubes assisting. However, it is known that carbon nanotubes penetrating the human body through the skin, by inhalation or orally, can destroy cells like asbestos. The ability of nanotubes to get deeply into tissues without leaving a chance to immune system to destroy them can cause cancerogenic effect [Shvedova A.A., Kisin E.R., Yanamala N., Tkach A.V. et. all. "MDSC and TGFβ are required for facilitation of tumor growth in the lungs of mice exposed to carbon nanotubes" //Cancer Research, 2015, V. 75 (8) , pp. 1-9] . Therefore, some special research controlling distribution, accumulation and removal of  carbon tubes from plantsisrequiredto apply them into practice of plant cultivation. This makes it unlikely the use of carbon nanotubes in agriculture in the near future.
The nutrient media, such as Murashing-Skoog, Gamborg, Heller etc. are known, which contain the balanced nutritional complex of obligatory components necessary for the development of plants, including organic substances –vitamins, carbohydrates, amino acids and/or protein hydrolysates–and also inorganic salts containing nitrogen, phosphorus, boron, potassium, calcium, magnesium, sulfur, iron, and trace elements such as manganese, zinc, copper, molybdenum, etc. The prototype of a nutrient medium for implementation of the declared method is a widely applied Murashing-Skoog nutrient medium (MS medium) [Murashing T., Skoog F. "A received medium for rapid growth and bio-assays with tobacco tissue culture" . Physiol. Plant. 1962. V. 15, pp. 473-497] . Table 1 shows the known composition of the agar nutrient medium of the prototype.
Table1. Murashing-Skoog medium composition
Figure PCTCN2016108424-appb-000001
Such metals as iron, copper and zinc are essential elements participating in regulatory and oxidation-reduction processes in plants as a part of coenzymes. MS medium containsthese metals in ionic form as salts.
The object of the invention is to provide a method for cultivation of plants on a nutrient medium containing nanoparticles of essential elements that provides the improvement of seed germination, and also the improvement of morphometric and/or physiological parameters of plants with the aim to obtain improved high quality planting material.
The task is solved by the proposed method for cultivation of plants comprising the germination of seeds and subsequent cultivation of plants both under aseptic conditions on agar nutrient medium containing nanoparticles, wherein said nanoparticles are nanoparticles of iron, or nanoparticles of zinc, or nanoparticles of copper, or a combination thereof.
Another object of the invention is to propose a composition of nutrient medium for implementationof the claimed method.
Thistask is solvedbythe proposed alternatives of agar nutrient medium which contain the components included in the composition of the Murashing-Skoog medium, namely, vitamins: РР, В6 andВ1, Glycine, Sucrose, Mesoinositol, helating agent Na2EDTA×2H2O, inorganic salts: NH4NO3, KNO3, CaCl2×2H2O, MgSO4×7H2O, KH2PO4, KI, H3BO3, MnSO4×7H2O, NaMoO4×2H2O and CoCl2×6H2O, as well as iron, zinc and copper, wherein iron or zinc or copper or combination thereof is included into nutrient medium in the form of nanoparticles of iron or nanoparticles of zinc or nanoparticles of copper or combination thereof respectively.
In addition the nutrient medium can include chitosan.
The concentration of nanoparticles ofelectro neutraliron in the nutrient medium is 10.0 –0.06 mg/l.
The concentration of nanoparticles of electro neutralzinc in the nutrient medium is 3.0 –0.016 mg/l.
The concentration of nanoparticles of electro neutral copper in the nutrient medium is 0.04 –0.00016 mg/l.
The technical result of the invention isthe stimulation of seed germination and the improvement of morphometric and/or physiological parameters of the grown plants.
The essence of the invention is in the fact that cultivation of plants on the traditional nutrient media, but modified in such a way that the salts of essential metals, particularly iron sulfate, or zinc sulfate, or copper sulfate or ironsulfate and zinc sulfate and copper sulfate simultaneously are replaced by nanoparticles of these metals in electro neutral state while other components of the nutrient medium remain unchanged, leads to better seed germination and to improvedmorphometric and/or physiological parameters of plants grown on these media compared with the plants grown on unmodifiednutrient media.
The invention is based on the results of the authors’research regarding the influence of metal nanoparticles on the structure and function of various biosystems. [Glushchenko N.N., Bogoslovskayа O.A., Olhovskayа I.P. "Physical and chemical regularities of biological effect of high-disperse powders of metals" //Chemical physics. 2002, V. 21, №4, P. 79-85； Publications on the website: http: //nanobiology. narod. ru] .
It has been shown that the electro neutral metal nanoparticles are characterized by polyfunctional and prolonged action, low toxicity, which is 7 –50 times lower than that of the corresponding metals in ionic forms, and are capableto distribute actively through organs and tissues of plants and to stimulate the vital processesat biotic doses, i.e., at the doses, which are 10 –50 times lower than the maximum tolerated doses.
Further chitosan which is known as absorbent for metabolic products of the growing plants and microflora, and also as plant growth stimulating factor, may be incorporated in the claimed nutrient medium. Chitosan is known to possess high sorption activity against bacteria, exhibits growth promoting effect, increases adaptive potential of plants, protects plants from infections, promotes higher crop productivity [Nyanikova G.G., Mametnabiev T.E., Kalinkin I.P., Gepetskaya M.V., Komissarchik S.M., Eldinova E.Y. "Applications of chitosan" http: //science. spb. ru/] . We have used сhitosan Taynshi (China) , widely used inChinese medicine as detoxicator. As soon as chitosan was supplemented into the nutrient medium, any bacterial flora, fungi or mold contamination were not occur. In some cases as it will be shown below, the synergism of combined action of chitosan and nanoparticles of metals in the nutrient medium was registered, resulting in further improvement of seed germination and of physiological and morphologic parameters of plants cultivated in these conditions.
The list of drawings explaining the essence of the invention and brief description thereof is placed below.
Fig. 1А –1Cshow the electronic microscopy images of iron, zinc, copper nanoparticles, and histograms of the size distributions for metal particles: 1А –iron nanoparticles； 1B –copper nanoparticles； 1C –zinc nanoparticles.
Fig. 2A –2D show the effect of metal nanoparticles at various concentrations and chitosan (100mg/l) on germination ofVenice tomato seeds: 2A –the nutrient mediawith iron nanoparticles； 2B –the nutrient mediawith zinc nanoparticles； 2C –the nutrient mediawith copper nanoparticles； 2D –the nutrient mediawith combinations of iron, zinc and copper nanoparticles.
Fig. 3A –3D showthe effect of metal nanoparticles at various concentrations and chitosan (100mg/l) onthe LJ-king pepper root length: 3A –the nutrient media with iron nanoparticles； 3B –the nutrient media with zinc nanoparticles； 3C –the nutrient media with copper nanoparticles； 3D –the nutrient media with combinations of iron, zinc and copper nanoparticles.
Fig. 4 showsthe photo of theLJ-king pepper plants grown on a nutrient media containing zinc nanoparticles and chitosan: Zn-b0 –the control, Zn-b3 –with zinc nanoparticles at a concentration of 0.016 mg/l in a nutrient medium； Zn-b2 –with zinc nanoparticles at a concentration of 0.08 mg/l in a nutrient medium； Zn-b4 –with zinc nanoparticles at a concentration of 0.4 mg/l in a nutrient medium.
Fig. 5А –5D show the effect of metal nanoparticles at various concentrations and chitosan (100 mg/l) on theHY-2 tomato root length: 5A –the nutrient media with iron nanoparticles； 5B –the nutrient media with zinc nanoparticles； 5C –the nutrient media with copper nanoparticles； 5D –the nutrient media with combinations of iron, zinc and copper nanoparticles.
Fig. 6А –6D show the effect of metal nanoparticles at various concentrations and chitosan (100 mg/l) on theroot activity ofLJ-king pepper, HY-2 tomato and Venicе tomato: 6A –the nutrient media with iron nanoparticles； 6B –the nutrient media with zinc nanoparticles； 6C –the nutrient media with copper nanoparticles； 6D –the nutrient media with combinations of iron, zinc and copper nanoparticles.
Fig. 7А –7C show the effect of metal nanoparticles at various concentrations and chitosan (100 mg/l) on the chlorophyll content in theleaves of plants: 7A –with iron nanoparticles in a nutrient medium for LJ-king pepper； 7B –with zinc nanoparticles in a nutrient medium for HY-2 tomato； 7C –with copper nanoparticles in a nutrient medium for LJ-king pepper.
The nanoparticles of electro neutral iron, zinc and copper are manufactured as described in [AS SSSR№ 814432 //Bull. inventions. 1981, № 11, С. 25] , using the apparatus Migen-3, described in [Jigatch A.N., Leipunskii I.O., Kuskov M.L., Stoenko N.I., Storozhev V.B. "An apparatus for the production and study of metal nanoparticles" , Instrum. Exp. Tech. 43 (2000) 839-845] .
The estimation of the shape and size of nanoparticles was performed by the method of Transmission Electron Microscopy (TEM) using LEO 912 AB OMEGA device.
The analysis of phase composition of nanoparticles was performed by the method of X-ray Diffraction analysis (XRD) using the ADP-1 Diffractometer (Russia) .
In order to determinethe average diameter of nanoparticles the TEM images were processed by means of the computer Micran 25 program based on measurement of the diameter of at least thousand of particles. On the basis of the data obtained a distribution curve of metal nanoparticles according to their size were plotted and the average diameter of particles was counted.
The TEM images and curves of size distributions of metal nanoparticles are presented in Fig. 1A –1C. The curve of the size distribution of iron nanoparticles lies in the area of 5 –80 nm. An average diameter of iron particles is 27.0 ± 0.51 nm. The curve of the size distribution of zinc nanoparticles lies in the area of 10 –200 nm. An average diameter of zinc particles is 54.0 ± 2.8 nm. The curve of the size distribution of copper nanoparticles lies in the area of 5 –225 nm. An average diameter of copper particles is 79.0 ± 1.24 nm.
According to Fig. 1A –1C, iron, zinc and copper nanoparticles are single-crystal structures with a round regular shape covered with a semitransparent oxide film.
The results of XRD-analysis demonstrate that the crystal phases content of iron nanoparticles are follows: the iron metal phase is equal to 53.6％, the iron oxide phase (Fe3O4) is equal to 46.4％, and the thickness of oxide film  is 3.5 nm. Zinc and copper nanoparticles consist of the crystal metal phase with oxide film thickness of 1.0 –0.5 nm.
For preparation of the nutrient medium according to the invention the formulation of any known balanced nutrient mediums containing all vital for plants organic and inorganic macro-and micronutrients can be used as a basis. Herewith, to prepare the nutrient medium in accordance with the invention, electroneutraliron nanoparticles, or electro neutral zinc nanoparticles, or electro neutralcopper nanoparticles, or a combination thereof are inserted into themedium instead of the salt of iron, or salt of zinc, or salt of copper, or combination of these salts respectively, while other components of the nutrient medium used as a basis, remain without changes.
To demonstrate the possibility of implementation of the invention we used an agar Murashing-Skoog nutrient medium formulation (see Tab. 1) as a basis for preparation of the embodiments of the nutrient medium composition according to the invention.
The ranges of concentrations of iron, zinc and copper nanoparticles in the embodiments of the invention are chosen taking into account the content of these metals in the ionic form in the medium taken as a basis. So, in terms of metal, the concentration of ionic iron in the base MSmedium is 5.6 mg/l, the concentration of electroneutral iron nanoparticles in the nutrient media in embodiments of the invention is 10.0 –0.06 mg/l； the concentration of ionic zinc in terms of metal in the base MSmedium is 1.96 mg/l, the concentration of electroneutral zinc nanoparticles in the nutrient media in embodiments of the invention is 3.0 –0.016 mg/l； the concentration of ionic copper in terms of metal in the base MSmedium is 0.0064 mg/l, the concentration of electroneutral copper nanoparticles in the nutrient media in embodiments of the invention is 0.04 –0.00016 mg/l.
Preparation of a base stock MSnutrient medium (control) and of embodiments ofthe nutrient media according to the invention is described below.
1. Preparation of the MS nutrient medium (control) .
The MS nutrient medium is prepared in accordance with the formulation given in Table 1.
1.1. Preparation of stock solution of major salts (macronutrients) .
The weighed portions (in mg) of: NH4NO3 –33000, KNO3 –38000, CaCl2×2H2O –8800, MgSO4×7H2O –7400, KH2PO4 –3400 are dissolved in 1 liter of water by stirring on a magnetic stirrer "IKA RH basic 2" (Germany) for 5 minutes.
1.2. Preparation of stock solution of minor salt (micronutrients) .
The weighed portions (in mg) of: KJ –166, H3BO3 –1240, MnSO4×7H2O –4460, ZnSO4×7H2O –1720, Na2MoO4×2H2O –50, CuSO4×5H2О –5, CoCl2×6H2O –5are dissolved in 1 liter of water by stirring on a magnetic stirrer for 5 minutes.
1.3. Preparation of stock solution of iron chelate.
The weighed portions (in mg) ofNa2EDTA×2H2O –37.3and FeSO4×7H2O –27.8 are dissolved in 1 liter of water by stirring on a magnetic stirrer for 5 minutes.
1.4. Preparation of stock solution of vitamins and organics.
The weighed portions (in mg) of: Mesoinositol –100000, Nicotinic acid (PP) –500, Pyridoxine-HCl (B6) –500, Thiamine-HCl (B1) –500, Glycine –2000, are dissolved in 1 liter of water by stirring on a magnetic stirrer for 5 minutes.
Prepare weighed portions (in mg) of sucrose in a powder form –30000, agar-agar –7000
1.5. Preparation of MS nutrient medium.
To prepare 1 l of the medium, 50 ml of the stock solution of macronutrients and 5 ml of the stock solution of micronutrients and5 ml of the stock solution of chelated iron and 1 ml of the stock solution of vitamins and organics are dissolved in 800 ml water by stirring on a magnetic stirrer for 5 minutes. Then the weighed portions of sucrose and agar-agar areadded and the volume is adjusted to 1000 ml.
The nutrient medium is sterilized at a temperature of 120℃ under a pressure of 0.1 MPa for 20 min in an autoclave "Hirayama, HVE-50" (Japan) .
The Murashing-Skoog nutrient medium prepared as described above is used as a control.
2. Preparation of the nutrient medium with the addition of chitosan.
100 mg ofchitosanTyanshi (China) is added to 1 l of the sterile cooled to 45℃ MS nutrient medium prepared as described above, and mixed bystirring on a magnetic stirrer for 2 minutes.
3. Preparation of thenutrient media with metal nanoparticles.
3.1. Preparation of the solution of chelating agent.
A weighed portion of 37.3 mg of Na2EDTA×2H2O is dissolvedin 1 l of water while stirring on a magnetic stirrer for 5 minutes.
3.2. Preparation of aqueous suspension of iron nanoparticles.
To preparea stockaqueous suspension of iron nanoparticles, 2000 mg of powder, containing thenanoparticles of electro neutral iron, is put into a flask and 200 ml of distilled sterile water is added. Then the sample is dispersed by "Scientz JY 92-IIN" dispergator (China) for 30 sec. with a power of 99％and thesuspension is cooled in an ice bath for 30 sec. to a temperature of 10 –15℃. The dispersing process is repeated three times.
9.0 ml of sterile distilled water is added to 1.0 ml of the prepared suspension and dispersing and cooling processes are carried out as described above.
To prepare thesuspensions with smaller concentrations of iron nanoparticles, the stock iron nanoparticle suspension is diluted by sterile distilled water up to required concentrations with the subsequent dispersing and cooling stages performed as described above.
3.3. Preparation of nutrient medium with iron nanoparticles.
10 ml of the stock aqueous suspension of iron nanoparticles or itsappropriate dilution (see3.2. ) is added to 1 l of sterile, cooled to 45℃ nutrient medium prepared as described in paragraph 1, but withoutadding of the stock solution of iron chelate (see 1.3. ) , then 5 ml of chelating agent (see 3.1. ) is added to the solution instead of stock solution of iron chelate. The mixture is stirred on a magnetic stirrer for 2 minutes.
3.4. Preparation of aqueous suspension of zinc nanoparticles.
To prepare a stockaqueous suspension of zinc nanoparticles, 600 mg of powder, containing the nanoparticles of electro neutral zinc, is put into a flask and 200 ml of distilled sterile water is added. Then the sample is dispersed for 30 sec. with a power of 99％, and the suspension is cooled in an ice bath for 30 sec. to a temperature 10 –15℃. The dispersing process is repeated three times.
9.0 ml of sterile distilled water is added to 1.0 ml of the prepared suspension and dispersing and cooling processes are carried out as described for iron powder (see 3.2. ) .
To prepare suspensions with smaller concentrations of zinc nanoparticles, the stock zinc nanoparticle suspension is diluted by sterile distilled water up to required concentrations with the subsequent dispersing and coolingstages performed as described above.
3.5. Preparation of nutrient medium with zinc nanoparticles.
10 ml of the stock aqueous suspension of zinc nanoparticles or its appropriate dilution (see3.4) is added to 1 l of sterile, cooled to 45℃ nutrient medium, prepared according to paragraph 1but without adding of zinc sulfate. The mixture is stirred on a magnetic stirrer for 2 minutes.
3.6. Preparation of aqueous suspension of copper nanoparticles.
To prepare a stock aqueous suspension of copper nanoparticles, 8 mg of powder, containing the nanoparticles of electro neutral copper is put into a flask and 200 ml of distilled sterile water is added. Then the sample is dispersed for 30 sec. with a power of 99％, and suspension is cooled in an ice bath for 30 sec. to a temperature of 10 –15℃. The dispersing process is repeated three times.
9.0 ml of distilled sterile water is added to 1.0 ml of prepared suspension and dispersing and cooling process is carried out as described for iron powder (see 3.2. ) .
To preparesuspensions with smaller concentrations of copper nanoparticles, the stock aqueous suspension of copper nanoparticles is diluted by sterile distilled water up to necessary concentrations with the subsequent dispersing and cooling stages performed as described above.
3.7. Preparation of nutrient medium with copper nanoparticles.
10 ml of the stock aqueous suspension of copper nanoparticles or of its appropriate dilution (see. 3.6) is added to 1 l of sterile, cooled to 45℃ nutrient medium, prepared as described in paragraph 1 but without adding of copper sulfate. The mixture is stirred on a magnetic stirrer for 2 minutes.
3.8. Preparation of aqueous suspension with iron and zincand copper nanoparticles.
To prepare aqueous suspensions with iron, zinc and copper nanoparticlesin different ratios, 2 ml of each aqueous suspension in a desired concentrations, prepared as described above (see 3.2, 3.4 and 3.6) , are mixed, and the sample is adjusted with sterile distilled water to 10 ml and then dispersed and cooled as described above for nanoparticles of iron (see 3.2. ) .
3.9. Preparation of nutrient medium with iron and zinc and copper nanoparticles.
10 ml of metal nanoparticle suspension prepared as described above (see. 3.8. ) is added to 1 l of sterile, cooled to 45℃ nutrient medium prepared as described in paragraph 1, but without adding of the stock solution of iron chelate (see 1.3. ) , of zinc sulphate (ZnSO4×7H2O) and copper sulfate (CuSO4×5H2O) . Then 5 ml of the solution of chelating agent (see3.1. ) is added. The mixture is stirred on a magnetic stirrer for 2 minutes.
50 ml of the nutrient media prepared as described above (see 3.3., 3.5., 3.7. and 3.9) are immediately dispensed into 200 ml sterile bottles, sealed with polyethylene film or polypropylene caps and leaved in a sterile room to cool.
The nutrient media prepared according to the described procedures were further used for seed germination and plant cultivation.
The samples of the nutrient media containing metal nanoparticles with the addition of Tyanshi chitosan at a concentration of 100 mg/l are prepared similarly as the Murashing-Skoog nutrient medium with the addition of chitosan as described in paragraph 2.
The invention is realized as follows:
The seeds of plants are placed into sterile culture vessels, 3 seeds in each vessel, on the surface of the agar nutrient medium containing, in accordance with the invention, nanoparticles of iron or nanoparticles of zinc, or nanoparticles of copper, or combinations of these nanoparticles in various proportions.
The vessels are put on shelves in a climatic room at 22 –25℃ and 36％humidity and are illuminated with 3500 –3000 Lux 12/12 hoursa day. Separate sets of experiments were carried out usingthe standard MS medium (control) and usingthe nutrient media containing Tyanshi chitosan and/or metal nanoparticles (test) .
The seed germination capacity is estimated on the 15th day and morphometric and physiological parameters of the grown plants are measured on the 40th day after the start of plant cultivation.
Experimental results obtained from 7–10 replications are statistically processed by the Microsoft Excel Statistica 6.0computer program. The reliability (p) of the results is evaluated using the U-criterion by Mann-Whitney [Mann H.B., Whitney D.R. Ann. Math. Statist. 1947, V. 18, №1, pp. 50-60] . The differences between two samples are statistically significant at 0.001≤ p≤ 0.1.
The following are the examples ofgermination and growing of pepper and tomato plants on the nutrient media according to the embodiments of the invention with various concentrations of iron or zinc, or copper nanoparticles, or combinations thereof. These examples only illustrate the possibility of implementation of the invention, but do not limit all possible variants of its realization.
Example 1. Effect of metal nanoparticles and chitosan in the composition of nutrient medium on tomato seeds  germination.
Seed germination is characterized by the value of germination index, defined as the percentage of the number of germinated seeds in relation to the total number of seeds taken. The index of seed germination is determined on the 15th day after plant cultivation starts.
Fig. 2A –2D show the effect of replacement of metal salts by nanoparticles of metals in the composition of the nutrient medium and the effect of chitosan supplement (100 mg/l) on the Venice tomato seed germination.
Fig. 2A demonstrates an increase of thegermination index of tomato seedsby 13 –27％ (as compared to the control) on the nutrient medium containing the iron nanoparticles at concentrations of 0.6 –10.0 mg/l. The addition of chitosan to the nutritious medium containing iron nanoparticles, demonstrates an additional increase in germination index up to 23 –33％.
As seen from Fig. 2B, zinc nanoparticlessupplemented to the nutrient medium at concentrations of 0.2 –3.0 mg/l entails a 2 –3 fold increase in seed germination index compared with the control. The addition of chitosan into the nutrient medium containing 1.0 mg/l of zinc nanoparticles induces further increase of germination index up to 13％.
As seen from Fig. 2С, copper nanoparticles at concentrations of 0.0008 –0.04 mg/l in the nutrient medium improves tomato seeds germination in 2.0 –2.8 times compared with the control. Chitosan addition into a nutrient medium containing copper nanoparticles at concentrations of 0.0008 and 0.04 mg/l induces extra augmentation of seed germination up to 20％.
As seen from the Fig. 2D, blend of iron, zinc and copper nanoparticles introduced into the nutrient medium instead of salts of these metals also provides the improvement of tomato seed germination, moreover the higher the concentration of nanoparticles in the medium, the higher the tomato seed germinationindex. For example, the combination: iron nanoparticles (10.0 mg/l) + zinc nanoparticles (3.0 mg/l) + copper nanoparticles (0.04 mg/l) enhances tomato seed germination up to 2.7 fold compared with the control. Chitosan addition to the nutrient medium containing a combination: iron nanoparticles (3.0 mg/l) + zinc nanoparticles (1.0 mg/l) + copper nanoparticles (0.004 mg/l) generates a further increase in tomato seed germinationindex by 13％.
Example 2. Effect of metal nanoparticles in the composition of nutrient medium on morphometric indices of  pepper and tomato plants.
Morphometric indices of plant –sprout length, root length, and green mass yield –were determined on the 40th day from the start of cultivation of plants on the nutrient media containing metal nanoparticles in the conditions described above. The results are presented as the magnitudes of percentage ratio between the indices obtained in the experiment (test) and in control.
Fig. 3A –3D showthe effect of metal nanoparticles in the composition of nutrient medium on the LJ-king pepper root length.
As can be seen from Fig. 3A, cultivation of pepper on the nutrient medium containing iron nanoparticles at concentrationsof 0.06, 0.3 and 3.0 mg/l instead of iron in the ionic form (5.6 mg/l in terms of metal) , causes the increase of root length by 54, 118 and 102％respectively compared with the control. This positive effect is observed when the concentration of iron nanoparticles in the medium is almost two orders lower than that of iron in the ionic form.
Fig. 3B shows that pepper root length increases by 71, 80 and 62％respectively compared with the controlif pepper is cultivated on the nutrient medium containing zinc nanoparticles ata concentrationsof 0.4, 0.008 and 0.016 mg/l instead of zinc ions (1.96 mg/l in terms of metal) . Thus the optimal concentration of zinc nanoparticles appears to be 5 –122 timeslower than the concentration of ionic zinc in MS nutrient medium.
The supplement of chitosan into a nutrient medium containing0.4 mg/l zinc nanoparticles causes an additional increase of root length by 30％.
Fig. 3C illustrates an increase ofthe pepper root length by 34, 57 and 54％compared with the control when pepper grows on a nutrient medium containing copper nanoparticles at concentrations of 0.00016, 0.0008 and 0.004 mg/l, respectively, instead of copper ions (0.0064mg/l in terms of metal) . Thus, a positive effect is observed when the concentration of copper nanoparticles is 1.6 –40timeslower than the concentration of copper ions in the MS nutrient medium.
Fig. 3D demonstrates that pepper root length increases by 7 –58％compared with the control when pepper is cultivated on a nutrient medium containing the combinations of Fe, Zn and Cu nanoparticles in different  proportions instead of iron, zinc and copper ions. The exception is the experiment, in which the nanoparticles are in the minimal concentrations of the tested (iron –0.06 mg/l, zinc –0.016 mg/land copper –0.00016 mg/l) .
However Fig. 3D shows a significant synergistic effect of the combined action of low concentrations of metal nanoparticles and chitosan, resulting the increase of pepper root length by 40％compared with the control.
A similar synergetic effect was also obtained in the case of the nutrient medium containing chitosan and a combination: iron nanoparticles (0.3 mg/l) + zinc nanoparticles (0.08 mg/l) + copper nanoparticles (0.0008 mg/l) .
As an illustration, Fig. 4 presents a photo of thesprout ofLJ-king pepper grown on a nutrient medium in which zinc salt is replaced by zinc nanoparticles in combination with chitosan. It is obvious that the presence of zinc nanoparticles in a nutrient medium at concentrations of 0.016 mg/l –0.4 mg/l and chitosan contributes to a more active development of the pepper root system compared with the control: root length increases 1.6 –1.8 times. The supplement of chitosan into the nutrient medium containing zinc nanoparticles at a concentration of 0.4 mg/l, gives an additional increase of root length by 30％.
Fig. 5A –5D show the effect of metal nanoparticles and chitosan in the composition of the nutrient medium on the HY-2 tomato root length. Tomatoes grown on a nutrient medium containing iron nanoparticles at a concentration of 0.06, 0.3 and 3.0 mg/l instead of iron in the ionic form, exhibitthe increase in root length by 58, 31 and 3％respectively (see Fig. 5A) .
The root length is increased by 1, 17 and 38％respectively when tomatoes are grown on a nutrient medium containing zinc nanoparticles at concentrations 0.016, 0.08 and 0.4 mg/l respectively instead of zinc in the ionic form. This result increases by 17, 2 and 15％respectively with chitosan supplement (see Fig. 5B) .
The replacement of copper ions at a concentration of 0.0064 mg/l by copper nanoparticles at a concentration of 0.0008 mg/l leads to an increase in the tomato root length by 24％ (See Fig. 5C) .
When tomato is grown on a nutrient medium containing combinations of metal nanoparticles (0.3 mg/l iron + 0.08 mg/l zinc + 0.0008 mg/l of copper) and (3.0 mg/l iron + 0.4 mg/l zinc + 0.004 mg/l copper) rather than metals inionic form, tomato root length increases by 7％and 10％respectively in comparison with control. In some experiments the addition of chitosan promotes a further increase in the tomato root length. For example, a combined use of iron nanoparticles (3.0 mg/l) , zinc nanoparticles (0.4 mg/l) , copper nanoparticles (0.004 mg/l) and chitosan in a nutrient medium gives again in a tomato root length by 70％in comparison with control.
The inclusion in the culture medium of metal nanoparticles has a positive effecton sprout lengthand green mass of pepper and tomato plants. Thus, the introduction of zinc nanoparticles into a nutrient medium ata concentration of 0.08 mg/l promotes an increase in theHY-2 tomato sprout length by 1.2 times. Chitosan supplement to a nutrient medium further increases this index by 17％.
Introduction of iron nanoparticles into a nutrient medium at a concentration of 3.0 mg/l promotes an increase in the LJ-king pepper sprout length by 4.7％. Chitosan supplement to a nutrient medium further increases this index by 8.9％.
Introduction of zinc nanoparticles into a nutrient medium at a concentration of 0.2 mg/l promotes an increase in the Venice tomato sprout length by 15.5％.
The green mass ofLJ-king pepper growing on a nutrient medium containing zinc nanoparticles (0.4 mg/l) and chitosan is 42％higher than in the control. The use of a nutrient medium with zinc nanoparticles ata concentration of 0.08 mg/l for growing of Venice tomato increases the green mass by 80％in comparison with control. The nutrient medium containing a combination of nanoparticles: (3.0 mg/l of iron + 0.4 mg/l of zinc + 0.004 mg/l of copper) gives a gain of the Venice tomato green mass yield 1.3 times higher than control.
Example 3. Effect of metal nanoparticles in the composition of nutrient media on physiological indices of pepper  and tomato plants.
The physiological indices of growing plants –root activity and chlorophyll content in leaves –are registered on the 40th day from the start of cultivation of plants in the conditions described above.
The results are presented as the percent ratio of the test indexvalues to control one. The test values areobtained on the nutrient medium with metal nanoparticles, the control values –on the Murashing-Skoog nutrient medium.
The root activity is determined by the reduction of chloride 3-phenyltetrazolium as described in [Adebusoye O. Onanuga, Ping’a n Jiang, Sina Adl. "Effect of phytohormones, phosphorus and potassium on cotton varieties (Gossypium hirsutum) root growth and root activity grown in hydroponic nutrient solution"//Journal of Agricultural Science. 2012, Vol. 4, N 3, pp. 93-110] .
Fig. 6A –6D show the effect of metal nanoparticles, introduced into the nutrient medium instead of corresponding metal salts, onLJ-king pepper and Venice tomato root activity.
It is obvious from Fig. 6A that theroot activity of LJ-king pepper is increased by 59％and 58％whenpepper is cultivated on the nutrient medium containing iron nanoparticles ata concentration of 0.06 and 0.3 mg/l respectively. The root activityof HY-2 tomato plants, cultivatedon the nutrient media, containing iron nanoparticles atconcentrationsof 0.06, 0.3 and 3.0 mg/l, is increased by 37, 34 and 48％respectively in comparison with the control.
The cultivationof Venice tomato plants on the media with iron nanoparticles at concentrationsof 0.6, 3.0 and 10.0 mg/l leads toanincrease in the root activity by 112, 125 and 76％respectively compared with the control.
A similar effect is observed when zinc ions in the nutrient medium are replaced by zinc nanoparticles (see Fig. 6B) . The root activity of LJ-king pepper is increased by 31, 56 and 38％compared with the control when the plants are grown on a nutrient mediacontaining zinc nanoparticles at the concentrations 0.016, 0.08 and 0.4 mg/l  respectively. In these conditions theHY-2 tomato root activity isincreased by 86, 8 and 29％respectively compared to control.
When Venice tomato plants are grown on a nutrient medium containing zinc nanoparticles at concentrations 0.2, 1.0 and 3.0 mg/l, the root activity increases by 25, 15 and 42％respectively compared with the control.
Fig. 6C demonstrates the increase in the root activity of pepper and tomatoes plants grown on the nutrient media with copper nanoparticles.
Cultivation of pepper on the nutrient mediacontaining copper nanoparticles at concentrations 0.00016, 0.0008 and 0.004 mg/l instead of ionic copper results in the increasing of LJ-king pepper root activity by 18, 61 and 21％respectively. The root activity of a HY-2 tomato increases by 43, 151 and 149％respectively in comparison with control.
Cultivation ofVenice tomato on the nutrient medium, containing copper nanoparticles at concentrations 0.0008, 0.004 and 0.04 mg/l, increasestheroot activity by 65, 10 and 12％, respectively, as compared to control.
The majority of experiments shows thatcombined replacement of ionic iron, ionic zinc and ionic copper by nanoparticles of these metals in the nutrient medium promotes positive effects on the root activity (see Fig. 6D) .
Cultivation of pepper on the nutrient medium, containingthe combinations of nanoparticles: (0.06 mg/l of iron +0.016 mg/l of zinc + 0.00016 mg/l of copper) , and (0.3 mg/l of iron + 0.08 mg/l of zinc + 0.0008 mg/l of copper) , and (3.0 mg/l of iron + 0.4 mg/l of zinc + 0.004 mg/l of copper) instead of these metals in ionic form results in the increase of the pepper root activity by 98, 51 and 91％respectively.
Cultivation of HY-2 tomato on the nutrient medium, containing the combinations of nanoparticles: (0.06 mg/l of iron + 0.016 mg/l of zinc + 0.00016 mg/l of copper) and (0.3 mg/l of iron + 0.08 mg/l of zinc + 0.0008 mg/l of copper) instead of these metals in ionic form increases the HY-2 tomato root activity by 33％, and 46％respectively in comparison with control.
Cultivation of Venice tomato in the nutrient medium containing the combinations of nanoparticles: (0.6 mg/l of iron + 0.2 mg/l of zinc + 0.0008 mg/l of copper) and (3.0 mg/l of iron + 1.0 mg/l of zinc + 0.004 mg/l of copper) instead of these metals in ionic form increases root activity by 43％and 48％respectively in comparison with control.
The supplement of chitosan to a nutrient medium containing metal nanoparticles, in most cases promotesan additional gain of the root activity of plants.
The chlorophyll content in leaves is evaluated as described in ["Measurement and Characterization by UV-VIS Spectroscopy UNIT F4.3" //Current Protocols in Food Analytical Chemistry, 2001, F4.3.1-F4.3.8] . The results received are presented in Fig. 7A –7C.
Fig. 7A demonstrates the effects of iron nanoparticles added into the nutrient medium instead of ferrous sulfate on the chlorophyll content in the leaves of LJ-king pepper plants. It is shown that iron nanoparticles at concentrations 0.3 mg/l and 3.0 mg/l in the nutrient media enlarge the chlorophyll content by 5％and 27％respectively as compared to control. Addition of chitosan into the nutrient medium together with iron nanoparticles at concentrations 0.06 mg/l and 0.3 mg/l leads to a further increase of chlorophyll content in pepper leaves by 6％and 10％respectively.
Fig. 7B shows the effect ofaddition of zinc nanoparticles into the nutrient medium instead of zinc sulfate on the chlorophyll content in HY-2 tomato leaves. Zinc nanoparticles at concentrations 0.08 mg/l and 0.4 mg/l increase the chlorophyll content in HY-2 tomato leaves by 56％and 108％, respectively. Additional introduction of chitosan into the nutrient media increases this index by 48％and 20％if the concentrations of zinc nanoparticles are 0.016 mg/l and 0.08 mg/l respectively.
Fig. 7C shows the effect of copper nanoparticle supplement to the nutrient medium instead of copper sulfate on chlorophyll content in leaves ofLJ-king pepper. Copper nanoparticles added into the nutrient medium increase thechlorophyll content in LJ-king pepper leaves. The maximum effect is observed at the lowest tested copper concentration of 0.00016 mg/l, wherein the chlorophyll content is 59％higher than in the control.
With the introduction of chitosan in the nutrient medium containing 0.0008 mg/l of nanoparticles of copper, chlorophyll content in leaves of pepper has increased by 75％.
It is important to note that the replacement of ionic forms of iron, zinc and copper in the nutrient medium by electro neutral metal nanoparticles of these metals does not cause any disturbances in the development of plants: the plants remain upright, havingwell-developed leaf plates and keeping the alternation of leaves and other species-specific characteristic features. In additionthe plants are resistant to bacterial, mold and fungal infection.
Thus, the method of growing plants based on the use of the nutrient medium, in which the salts of iron, zinc and copper are partially or completely replaced by electroneutralnanoparticles of these metals is claimed. The method allows producinghealthy plants with compact stems with a developed and active root system, which can be used as high-quality planting material.
The proposed nutrient medium can be used inbiotechnological researches, for improving of the quality of agricultural products, for aeroponic and hydroponic technologies. The proposed method of growing plants on a nutrient media containing iron, copper and zinc nanoparticles can be used to create life-support systems for astronauts in conditions of prolonged space flights.

Claims (10)

  1. A method for cultivation of plants comprising the germination of seeds and subsequent cultivation of plants under aseptic conditions on agar nutrient medium containing nanoparticles, wherein said nanoparticles are nanoparticles of electro neutral iron, or nanoparticles of electro neutral zinc, or nanoparticles of electro neutral copper, or a combination thereof.
  2. The method of claim1, wherein the said nutrient medium additionally contains chitosan.
  3. The method of claims 1 and 2, wherein said nutrient medium contains nanoparticles of electro neutral iron at a concentration of 10.0 –0.06 mg/l.
  4. The method of claims 1 and 2, wherein said nutrient medium contains nanoparticles of electro neutral zinc at a concentration of 3.0 –0.016 mg/l.
  5. A method of claim 1 and 2, wherein said nutrient medium contains nanoparticles of electro neutral copper at a concentration of 0.04 –0.00016 mg/l.
  6. The nutrient medium for the implementation of method in accordance with claim 1, which contain the components included in the composition of the Murashing-Skoog medium, namely, vitamins: РР, В6 and В1, Glycine, Sucrose, Mesoinositol, Agar-agar, helating agent Na2EDTA×2H2O, inorganic salts: NH4NO3, KNO3, CaCl2×2H2O, MgSO4×7H2O, KH2PO4, KI, H3BO3, MnSO4×7H2O, NaMoO4×2H2O and CoCl2×6H2O, as well as iron, zinc and copper, wherein iron or zinc or copper or combination thereof is included into nutrient medium in the form of nanoparticles of iron or nanoparticles of zinc or nanoparticles of copper or combination thereof respectively.
  7. The nutrient medium of claim6wherein saidnutrient medium additionally contains chitosan.
  8. The nutrient medium of claim 6 and 7 wherein said nutrient medium contains nanoparticles of electro neutral iron at concentration of 10.0 –0.06 mg/l.
  9. The nutrient medium of claim 6 and 7 wherein nutrient medium contains nanoparticles of electro neutral zinc at concentration of 3.0 –0.016 mg/l.
  10. The nutrient medium of claim 6 and 7 wherein said nutrient medium contains nanoparticles of electro neutral copper at concentration of 0.04 –0.00016 mg/l.
PCT/CN2016/108424 2015-12-17 2016-12-02 The method for cultivation of plants using metal nanoparticles and the nutrient medium for its implementation WO2017101691A1 (en)

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