The following points highlight the six experiments on mineral nutrients in plant body. Some of the experiments are on: 1. Preparation of Different Culture Solutions 2. Determination of Mineral Deficiency by Foliar Diagnosis 3. Influence of Aeration of Culture Solutions on Growth of Plants 4. Detection of Nutrient Deficiency Occurring Under Field Conditions and Others.
Contents
Mineral Nutrients in Plant Body
Experiment # 1
Preparation of Different Culture Solutions:
Several early methods of studying plant nutrition are still found to be useful today. The analysis of plant ash and the use of liquid and sand cultures are techniques used for the study of plant nutrition in laboratories throughout the world. These methods, however, have been improved much.
A. Solution Cultures:
This technique is useful because:
(i) It provides an excellent means for controlling the quantity and relative proportions of mineral salts given to a plant in any experiment,
(ii) Of the good solvent characteristics of water, and
(iii) Of the relative ease with which water can be freed from contaminating influences.
Some of the sources of contamination, which is the main drawback of this technique, are the rooting medium, reagents used, and containers, water, cutting implements, seeds and the dust in the surrounding atmosphere. Though it is impossible to eliminate contaminating influences, yet they can be kept to a minimum.
The first step is to prepare a stock solution from inorganic salts containing the necessary elements for normal plant growth. Once the stock solutions are prepared, nutrient solutions may be prepared by simply adding in the correct proportions the necessary inorganic salts from the stock solutions.
For ready use some of the satisfactory formulae for nutrient culture solutions are given below. These solutions may be directly used for the experimental purpose.
The roots of the plant to be grown are submerged in the nutrient solution, the stem projecting through an opening of the lid of the container. To give a more rigid system, the stem is generally held stationary in this opening by cotton padding.
The container is to be covered in order to eliminate, as much as possible, the contamination caused by atmospheric dust. For good root growth and mineral salt absorption it is often necessary to provide some means of aeration.
B. Sand Cultures:
Solid media, such as sand or crushed quartz or vermiculite (any of a number of micaceous minerals that are hydrous silicates derived usually from alteration of mica whose granules expand greatly at high temperatures to give a light-weight highly water absorbent material) are generally easier to work with than with a liquid medium.
Today it is possible to get highly purified silica sand or crushed quartz which is very low in trace elements. One important aspect of sand culture is that the roots are growing in a natural medium and no means of support needs to be provided.
Nutrient solutions are added to the solid culture by three different ways:
(i) Slop culture:
Pouring over the surface,
(ii) Drip culture:
Allowing to fall in drops over the surface, and
(iii) Sub-irrigation:
Forcing solution up from the bottom of the container. In all the three systems, the added nutrient solutions drain out through an opening at the bottom of the container. In sub-irrigation, the solution is collected in a reservoir and used repeatedly.
Experiment # 2
Determination of Mineral Deficiency by Foliar Diagnosis:
The effects of deficiency of various essential mineral elements can be demonstrated by means of sand culture or water culture techniques. But the water culture technique is cumbersome for practical purposes because of the problem of aerating the culture solution every day and the possibility of frequent contamination.
The sand culture technique is, therefore, more suitable where artificial aeration is not necessary. The culture solutions should be replaced daily to avoid danger from evaporation and contamination. The following stock solution will be required for this experiment. The molecular weight of each salt is given within parentheses.
The solutions are prepared for studying the effects of various elements according to the following table. The number of millilitres of each one molar single salt solution needed for one litre of the designated solutions is given below.
The required volumes of stock solutions obtained from the Table 2 are added in serial order to about 500 ml distilled water and the volume is made up to one litre.
If the stock solutions are mixed together before dilution, precipitate will appear which can be dissolved only with difficulty. Seeds are allowed to germinate aseptically in sand pots using distilled water.
After the seedlings have grown to a uniform size, the distilled water is replaced with culture solutions. It will not be necessary to change the culture solution during the first two weeks, but thereafter at intervals of a week, the culture solution (excepting the trace element solutions) in each pot should be replaced with fresh solution. The sand may be kept moistened with distilled water.
Observation:
The deficiency symptoms are observed according to the key given below:
Inference:
The essential major elements are necessary for normal growth and physiological function of plants. Trace elements function in plants as constituent of specific enzymes and coenzymes. Moreover, some trace elements are also required in the biological electron transport system. The deficiency of a particular essential elements results in the derangement of the complicated mechanism of the vital activities of plants.
Experiment # 3
Influence of Aeration of Culture Solutions on Growth of Plants:
Experiment:
Equal quantities of culture solutions (Hoagland’s) are taken in two wide-mouthed bottles of three litre capacity, according to the Expt. 1 (a) and their pH is recorded. In one set-up aeration arrangement by bubbling through rubber tubing is attached and the others lacking such arrangement.
Some freshly grown pea seedlings are transferred to each bottle. The bottle is aerated 6 hours every day till the end of the experiment. The growth data (average length of shoot and root, fresh and dry weights) are taken at an interval of 3 days. The pH of the solutions of the aerated and non-aerated bottles is recorded after the experiment.
Results:
Percentage increase in length of shoot and root and fresh and dry weights are recorded and graphically plotted.
Discussion:
The aeration of the solution is one of the most important aspects of solution culture techniques. In order to carry on the processes of growth and absorption of nutrients and water, it is essential that the living cells of the root have a supply of dissolved free oxygen for respiration.
Another factor involved is the number of plants per culture bottle. When relatively few plants are present, the roots develop better owing to less competition for oxygen. There is also a slower depletion of essential elements and a slower change in pH.
Growth of plants, particularly in earlier stages, is usually better when air is forced to the culture solution mechanically, regardless of the type of container or composition of the solution.
Experiment # 4
Detection of Nutrient Deficiency Occurring Under Field Conditions:
There are perhaps 4 general methods which are used for ascertaining the soil condition with respect to immediate plant nutrient requirements.
They are:
(i) Plant diagnosis (already described),
(ii) Biological soil test (described below),
(iii) Chemical soil test, and
(iv) Chemical test for plant material.
The last two chemical tests are beyond the scope of the present book and hence omitted.
Biological Soil Test:
A. Aspergillus Niger method for determining Mg, Cu, Fe, Mn, Mo and Zn in soils and plant materials (Nicholas, 1945).
(a) Principle of the method:
This method of bioassay of mineral elements is based on the fact that Aspergillus Niger requires specifically a number of mineral elements for its growth. These include the macronutrients —N, P, Ca, Mg, K and S and the micronutrients—Cu, Fe, Mn, Mo and Zn.
When any one of these nutrients is omitted from the culture medium, the growth of the fungus is reduced much, as may be shown by dry weight yields of the felts and by the intensity and type of sporulation.
A progressive increase in any one of these essential nutrients from deficiency to sufficiency level, provided others are present in optimum amounts, results in significant and quantitative increase in growth. A standard growth series may thus be prepared for each of the essential nutrients listed above.
A bioassay of test material for any given nutrient is made by adding a known amount of the material to a culture solution containing all the nutrients except that to be determined.
The growth of the fungus under these conditions will depend on the amount of the test nutrient derived from the material added. The growth response is determined by comparison with a standard series for the elements prepared at the same time.
(b) Experimental Procedure:
(i) Type of glassware:
Hard glass 500 ml Erlenmeyer’s flask and 150 ml beakers thoroughly cleaned by soaking in H2SO4dichromate mixture or in dilute HNO3 followed by washing with dieionised water.
A simple test to detect the presence of metals on glass ware is to rinse with a 0.01 per cent solution of dithizone in chloroform. The presence of metal is shown by a change of colour from reddish brown of dithizone to red of the metal-dithizonate.
(ii) Purification of water:
Good grade deionised water, checked by means of a conductivity bridge, is suitable for dispensing media.
(iii) Composition of Basal Culture Medium:
Deionised water is added to one litre giving a solution of pH 3.8. For each culture 50 ml of solution is dispensed in a 500 ml Erlenmeyer’s flask.
(iv) Purification of the medium:
Refer— “The diagnosis of mineral deficiencies in plants by visual symptoms” by T. Wallace, 1961.
(v) Choice of strain of fungus:
Aspergillus Niger M. has been found to be; the most suitable strain.
(vi) Preparation of spore inoculum:
The fungus is sub cultured weekly on nutrient agar slopes by spore transfer technique. This is cultured at 30°G for four days.
The composition of nutrient agar medium is as follows:
This medium is autoclaved at a pressure of 10lb per square inch for 10 minutes and then dispensed in culture tubes and plugged with non-absorbent cotton wool and stored in a refrigerator at 4°C.
(vii) Transfer:
The spores are transferred into 10 ml sterile deionised water and 0.1 ml of the inoculum is used for each 50 ml culture solution.
(viii) Preparation of standard series for the test metals:
The sterile culture solution is transferred to clean 500 ml Erlenmeyer’s flask in 50 ml lots. Necks of the flasks are covered with 50 ml inverted beakers.
The fungus is grown at 30°C for four days. The standard series may be assessed, visually by taking into account the thickness of the mycelia growth, intensity of sporulation and quantitatively by dry weight yield of the felts. A standard series for a particular metal (different concentrations of the element) is to be prepared when an assay is to be made on test materials.
In the absence of Fe, Zn and Mo, thin mucoid mycelium devoid of spores are formed yielding between 20 and 50 mg dry weight compared with 900 mg for a normal mycelium grown in complete culture. The reduction in yield of felts grown without Cu or Mn is less striking than those for deficiencies of Fe, Zn and Mo although visually there is a marked difference.
(c) Procedure for Assay:
(i) Soils:
Soils are air-dried and passed through a sieve of 2 mm mesh and following amounts of soil are used for assay: 1 and 0.5 gm for Mg and 0.5 and 0.25 gm for Cu, Mn, Mo and Zn.
The interference of micro-organisms in the soil may be overcome by adding 1 ml of 85 per cent redistilled ethanol per 0.5 gm of soil, removing the alcohol completely on a water bath.
The fungus grows well between pH 1.8 and 8. For the assay of manganese in the soil, the best pH value is 7.5.
The optimum pH for assay of Mo in soil is 2. The effect of pH on assay of Mg, Cu or Zn is less striking so that these can be done at the usual pH of the culture medium.
(ii) Plant materials:
The trace metal contents of plant material may be determined directly or by ashing. The ashing may be done in platinum crucible at 400°C after which the aliquots of the cooled residue dissolved in dilute HCL and transferred to the assay flask.
B. Soil plaque method:
The method is based upon the assumption that species of the nitrogen fixing bacterium Azotobacter commonly found in soils have nutrient requirements similar to those of higher plants so far as N, P, K are concerned.
The extent of growth of Azotobacter on the surface of the moistened variously treated soil samples are considered to indicate the soil requirements for these elements.
Experiment # 5
Demonstration of Unequal Absorption of Cations and Anions by Plant:
Experiment:
Twenty-day-old seedlings of rice or wheat are suitable for this experiment. The following nutrient solutions are first prepared. The molecular weight of each salt is given within parentheses.
Two lots of Solution-A are prepared and pH of one lot is adjusted to 4 and the other to 7 with 1 % HCL and 1 % KOH respectively. Solution-B is similarly adjusted to pH 5 and 7.5.
The solutions are taken in separate Erlenmeyer’s flasks and their levels are marked. Seedlings are taken out and roots are washed well without damaging them. Five such seedlings arc placed in each flask.
Bubbling of the solution may be done if possible. Distilled water should be added from time to time to make up the loss due to evaporation and uptake by plants.
Observation:
pH of each of the solutions is recorded at two-day intervals with the help of a pH metre or Universal indicator paper.
Discussion:
Plants absorb cations and anions of different salts at different rates. It is difficult to measure these rates directly. But it can be demonstrated by an indirect method.
Unequal uptake of cations and anions results in changes in the pH of the nutrient solution. If the cation of a salt is absorbed in excess of the anion, the pH will decrease (acidic) and if the anion is absorbed in excess, the pH of the medium will increase (alkaline).
Experiment # 6
Quantitative Determination of Accumulation of Chloride Ions in Plant Cells:
Experiment:
Weighed amount of Hydrilla plants is taken in a mortar and crushed with a little neutral sand and water. The extract is diluted to 50 ml with water after filtration. In order to remove chlorophyll pigment the solution may be treated with activated charcoal, if necessary 10 ml of the extract is titrated against 0.02 AgNO2, solution (0.34gm AgNO2per 100 ml solution) using 5% K2CrO4solution as an indicator.
As the end point reaches, a faint reddish brown colour of silver chromate (AgCrO4) remains permanent when all AgCl has been precipitated. Before the experiment, 0.02 N AgNO3is to be standardized against a standard NaCl solution (329.6 mg dry NaCI per 1000 ml solution) following the above procedure.
If the volume of 0.02 N AgNO3 to titrate 10 ml of plant extract be z then the amount of chloride ion is 0.02 × y × z/x mg per 10 ml of the sap. The amount of chloride ion per gm plant material may be calculated.
Discussion:
In plant cell the vacuolar and plasma membranes are differentially permeable allowing water to pass freely but not the solutes. But under most conditions solutes (both electrolytes and non-electrolytes) may enter the cells passively.
The electrolytes, on the other hand, may be actively accumulated within the plant cells. This active accumulation of ions requires energy which is supplied by respiratory breakdown of foods.
N.B. For determination of the chlorine per ml of the medium in which the plant grows, the medium (water) is taken and similarly titrated against 0.02 N AgNO3. The difference of the chlorine content of the medium and that of the plant extract indicates the actual amount of chlorine accumulated by the plant.