Preparation and use of sand substrate in agrochemical research

Preparation of sand substrate for precise experiments

Soil, due to its heterogeneity and high adsorption capacity, does not allow for the creation of strictly controlled conditions for plant nutrition. To circumvent this limitation, the sand culture method is used. It allows for the accurate assessment of the influence of individual elements on plant metabolism and development, the study of ion synergism and antagonism, as well as the determination of precise nutrient uptake coefficients. Unlike soil, in a sand substrate, elements are distributed evenly, do not bind physico-chemically, and are in direct contact with the roots.

• Sand particle diameter — 0.2–0.4 mm
• Iron oxide content — no more than 1%
• Aluminum oxide content — 1–5%
• Uniform sand moisture — in the 20–25 cm layer

Fine quartz or white river sand, completely cleaned of organic matter and silt impurities, is used as a substrate. In terms of its physical properties, it must be similar to soil so as not to interfere with normal root growth. The chemical composition of sand depends on its origin, but silicon oxide predominates in any type.

To remove extraneous chemical impurities and prepare a high-quality substrate, the sand must undergo mandatory treatment before setting up the experiment.

1. Wash the sand with tap water to rinse away organic residues and the finest particles.
2. Drain the water, pour hydrochloric acid over the sand, cover the vessels with glass, and leave them in the vegetation house.
3. Stir the sand daily with wooden sticks for one week.
4. Wash the acid out of the sand, first with tap water and then with distilled water, until the reaction to the chloride ion disappears (checked with silver nitrate, AgNO₃).
5. Spread the sand in a thin layer on carts and air-dry it.
6. Sieve the dry material twice: first through a 1 mm mesh screen, then through a 0.25 mm screen.

Selection of vegetation vessels and nutrient mixtures

For sand cultures, shorter vessels are chosen compared to soil cultures. Capillary rise of water in sand is weak, so stable moisture is maintained only in a layer 20–25 cm high. If deeper vessels are used, the top layer of sand will dry out quickly. To increase the water-holding capacity of the substrate, finely crushed glass or powdered barium sulfate is added to it.

To ensure normal plant development, nutrient mixtures are added to the vessels. They are subject to strict requirements: they must contain all necessary elements in an easily accessible form and maintain an optimal medium pH throughout the entire growing season. Nitrogen salts affect acidity the most, as nitrogen is consumed in the largest quantity. To stabilize the solution reaction, nitrogen and phosphorus are introduced in the form of balanced conjugate pairs.

How different types of nutrient mixtures work

When conducting vegetation experiments, the choice of nutrient mixture determines not only the availability of nutrients but also the chemical stability of the root zone. All formulations used in agrochemistry are divided into three main types depending on their effect on substrate acidity. Understanding these mechanisms helps to more precisely manage plant nutrition under conditions of protected ground and sand cultures.

The first type of mixture is based on readily soluble salts, where the chemically acidic salt KH2PO4 is combined with the physiologically alkaline Ca(NO3)2. When growing plants in such a medium, the pH value inevitably shifts to the alkaline side. Most classical formulations used in agrochemistry and plant physiology, including Hellriegel's, Knop's, and Hoagland's media, belong to this type.

The second type of nutrient mixture uses KNO3 as a weakly physiologically alkaline nitrogen source and the poorly soluble salt Fe3(PO4)2 as a phosphorus source. During the hydrolysis of iron phosphate, a weak base and a strong orthophosphoric acid are formed, which neutralizes the alkalinization from nitrates. The presence of poorly soluble salts (including Ca3(PO4)2 and CaSO4) maintains the ion concentration in the solution at a low but constant level due to the gradual dissolution of the precipitate. Such a mechanism effectively mimics the natural conditions of plant nutrition in the soil solution.

The third type of mixture, developed by domestic agrochemists, combines readily soluble and poorly soluble compounds. The basis here is Helriegel's medium, in which nitrogen is replaced by ammonium nitrate NH4NO3, and phosphorus is added in the form of calcium phosphate CaHPO4·2H2O. The stability of the solution is ensured by the buffering capacity of calcium phosphate, the hydrolytic acidity of iron sulfate Fe2(SO4)3, and the balance between the physiological acidity of ammonium nitrogen and the alkalinity of nitrate nitrogen.

Formulations of universal and specialized media

To conduct accurate experiments, it is necessary to strictly observe the dosages of components when preparing solutions or packing containers. Below are the verified compositions of universal media, as well as specialized formulations for individual crops.

• Nitrogen dose in Knop’s medium — 1.00 g/l Ca(NO3)2
• Nitrogen dose in Chirikov’s medium — 1.000 g/kg KNO3
• Phosphorus in Belousov’s medium — 0.36 g/kg KH2PO4
• CaCO3 supplement for buckwheat — 55.5 mg/kg on the 20th day

Knop's medium in Treyman's modification (wheat): 2.04 g/L (g/kg of sand 3)2·4H2O; 0.431 – KNO3; 0.25 – KH2PO4; 0.12 – KCl; 0.51 – MgSO4·7H2O; 19.36 mg/L (mg/kg of sand) FeCl3·6H2O; 26.3 – MnSO4·5H2O; 13.2 – ZnSO4·7H2O; 1.96 – CuSO4·5H2O; 8.58 – H3BO3; 0.126 – Na2MoO4·2H2O; 0.238 mg/L (mg/kg of sand) CoSO4·7H2O.

Hoagland's medium in Treyman's modification (wheat): 1.38 g/L (g/kg of sand 3)2·4H2O; 0.78 – KNO3; 0.23 – NH4(H2PO4); 0.51 g/L (g/kg of sand) MgSO4·7H2O; 19.36 mg/L (mg/kg of sand) FeCl3·6H2O; 26.3 – MnSO4·5H2O; 13.2 – ZnSO4·7H2O; 1.96 – CuSO4·5H2O; 8.58 – H3BO3; 0.126 – Na2MoO4·2H2O; 0.238 – CoSO4·7H2O.

Pryanishnikov's medium in Treyman's modification (wheat): 0.480 g/kg of sand NH4NO3;,0344 – CaH2PO4·2H2O; 0.570 – KCl; 0.49 – MgSO4·7H2O; 0.688

– CaSO4·2H2O; 1.11 3)2·4H2O; 14.52 mg/kg of sand FeCl3·6H2O; 8.78 – MnSO4·5H2O; 6.60 – ZnSO4·7H2O; 1.180 – CuSO4·5H2O; 8.580 – H3BO3; 0.075 – Na2MoO4·2H2O; 0.143 mg/kg of sand CoSO4·7H2O.

Micronutrient solutions for universal nutrient mixtures:

Braunor–Bukach solution: 350 mg/L MnCl2·4H2O; 500 – H3BO3; 50 – ZnSO4·7H2O; 50 – CuSO4·5H2O; 50 – Al2(SO4)3·18H2O; 50 – NiSO4·7H2O; 50 – Co(NO3)2; 50 – TiO2; 25 – KBr; 25 – KJ; 25 mg/L SnCl2·2H2O. Add 1 ml of this solution to each liter of nutrient solution.

Berthelot solution in Gautheret's modification: 50 g/L Fe2(SO4)3; 2.0 – MnSO4; 0.5 – KCl; 50 – NiCl2; 50 – CoCl2; 200 – TiSO4; 100 – ZnSO4; 50 – CuSO4; 100 – BeSO4; 50 – H3BO3; 50 g/L H2SO4. Add one drop of this solution to the nutrient solution.

Hoagland and Snyder solution (A-Z solution): two solutions, A and B, of micronutrient mixtures are prepared. Add 1 ml of each of these solutions per 1 L of nutrient solution. To prepare solution A for 18 L, take the following amounts of salts: 1.0 g Al2(SO4)3; 0.5 – KJ; 0.5 – KBr; 1.0 – TiO2; 0.5 – SnCl2·2H2O; 0.5 – LiCl2; 7.0 – MnCl2·4H2O; 11.0 – H3BO3; 1.0 – ZnSO4·7H2O; 1.0 – CuSO4·5H2O; 1.0 – NiSO4·6H2O; 1.0 g Co(NO3)2·6H2O. When preparing solution B for 18 L, add: 0.1 g As2O3; 0.5 – BaCl2; 1.0 – CdCl2; 0.1 – Bi(NO3)2; 0.1 – Rb2SO4; 0.5 – K2C2O4; 0.5 – SrSO4; 0.1 – VCl3; 0.1 – KF; 0.1 – HgCl2; 0.425 – MoO2; 0.1 g H2SeO4.

When selecting a series of containers for a particular sand culture scheme, ensure that they have approximately the same capacity and mass. Container capacity should be determined by the amount of water they can hold. A 1–2% discrepancy between the extreme values of any series of containers is permissible both in terms of their volume and mass. To set an equal mass, calibrate the containers with broken glass. Place 200–300 g of pre-washed and dried broken glass into the heaviest container selected for the experiment. Afterward, bring the other containers to the same mass by adding glass pieces. Push the drainage glass toward the wall into a pile so that it occupies no more than 2/3 of the container floor area, then cover it with a cheesecloth disc. Make a hole in the cheesecloth to insert a tube for irrigation. Place the tube into the drainage pile and spread the cheesecloth so that it covers the drainage glass and part of the free floor of the container (Fig. 94; Shestakov A.G., 1954).

The nutrient mixture is applied based on 1 kg of sand. The composition of the mixtures is determined by the biological characteristics of the experimental plants and the research objective. All water-soluble salts are introduced into sand cultures in the form of solutions. Insoluble ones are added as finely ground powder samples.

Fig. 94. Drainage in vegetation containers

Pour a measured amount of sand onto a polyethylene film or into a basin, add nutrients, mix thoroughly, moisten to 60% of the total water-holding capacity, and place everything into the prepared container. The total water-holding capacity should be determined in advance. River sand with a particle size of 0.5–0.7 mm has a water-holding capacity of 25%. For 1 kg of dry sand, take 150 ml of water, which constitutes 60% of the total water-holding capacity; the volume of the nutrient mixture solutions must be included in the calculated volume of water. Pack the container tightly with sand; the surface of the sand should not reach the edges of the container by 2–4 cm. After packing, attach a corresponding label to the container wall. Place the container in its designated spot and cover it with a sheet of cardboard.

Sowing in sand cultures is performed using both germinated and dry seed. Before sowing the seed, the surface of the sand in the vessels is leveled and moistened, and holes for the seed are made using templates. The number and depth of the holes depend on the size of the seed and the quantity of plants to be left in the vessel. Typically, 1.5–2 times more seed is planted than is intended to be left after thinning.

After sowing, the vessels are covered with cardboard or placed in a shaded area until emergence. The vessels are watered daily in the morning and evening at a rate of 50–100 ml of water. After the emergence of seedlings, the vessels are moved to an open area and the cardboard is removed from their surface.

Sometimes it happens that the number of germinated seed in a vessel is less than the quantity intended to be left after thinning. In such cases, it is necessary to perform replanting of seedlings. The plants for replanting must be of the same age and stage of development as those growing in the vessel. It is best for these purposes to set up an experiment with an additional replication.

On the 5–6th day after emergence, the vessels are weighed and the plants are watered, ensuring that the amount of water does not exceed 15% of the sand's mass. During the period of intensive plant growth, irrigation is conducted twice a day, and the soil moisture of the sand is maintained at a level of 16–17% of the sand's mass, which corresponds to 70% of the total water-holding capacity of the sand.

To obtain reliable results in a growing season experiment, it is important to form an optimal plant density in a timely manner. Thinning begins when the seedlings transition from feeding on seed reserves to absorbing nutrients from the external environment. At this moment, a strictly defined number of evenly spaced plants are left in the vessels.

The number of plants left after thinning depends on the volume of the growing vessel and the biological characteristics of the crop. For standard vessels with a diameter of 25 cm, specific density rates are provided. Large crops are grown using an individual regime.

Large crops such as potatoes, beet, corn, grapes, and cotton require a larger nutrient area. They are grown in increased-size containers. After thinning, only one plant is left in each vessel — similar to the methodology for soil cultures.

For high-stalk plants, it is mandatory to install support frames during thinning. These structures help the stems maintain a strictly vertical position as they grow. Furthermore, all vessels in the experiment must be rotated daily during irrigation.

Observations during the growing season and harvesting

From the moment of emergence, continuous observation of the plants is conducted. The experimental log records development dynamics: height is measured, the number of leaves is counted, and the dates of phenological phases are noted. If plants in different vessels ripen unevenly, harvesting is also carried out at different times, as ripeness is reached.

• Standard vessel diameter — 25 cm
• Cessation of irrigation before harvesting — 3–5 days prior
• Stem cutting height — 1–2 cm

Harvesting and preparation of samples for analysis are performed in a strict sequence. This allows for the avoidance of biomass losses and an accurate assessment of the effect of the nutrient medium on the development of plant organs. All obtained data are recorded for subsequent calculations.

1. Stop irrigation of the growing vessels 3–5 days before the planned harvesting.
2. Cut the above-ground mass with scissors at a distance of 1–2 cm from the surface of the sand substrate.
3. Clear the root system from the sand by carefully washing it with water on a sieve.
4. Dry the above-ground organs and roots of the plants, weigh them, and send the dry mass for chemical analysis to determine the nutrient content.