Autotrophic plants generally require 13 elements which they absorb as inorganic ions in addition to carbon, hydrogen, and oxygen which are obtained from carbon dioxide and water. Six of these elements are required in greater amounts than the others and are called macronutrients. The other seven are needed in very small amounts and are called micronutrients or trace elements. See Table 1 below for the elements.

There are other elements that are required by some species but not by others. For example, sodium is required for certain blue-green algae and halophytes. Cobalt is a micronutrient for some microorganisms and symbionts, although it has not been demonstrated to be essential for green plants. Silicon is indispensable for diatoms, and vanadium is reported to be essential for one green algae (Scenedesmus obliquus).

Except for nitrogen, which is obtained from molecular nitrogen (N₂) in the earth's atmosphere, all the essential elements are derived from parent rock and are therefore called "mineral elements." However, aside from those plants which fix atmospheric nitrogen, either by themselves or symbiotically, nitrogen is absorbed as an inorganic ion (nitrate or ammonium ion) by autotrophic plants. Thus nitrogen is included in discussions of the "mineral nutrition" of plants.

One approach to determining the metabolic role of an essential element is to determine the consequence of its deficiency. In the case of all the macronutrient elements, there are characteristic symptoms which develop in the shoots of deficient plants. This type of approach often yields sufficient information to suggest metabolic blocks and thus specific sites where a particular element may function. The common method of implementing this approach is to grow plants hydroponically in solutions of precisely known chemical composition. Hydroponics can also be used to investigate the consequence of an excess of micronutrients. Many of the micronutrients can be toxic to plants if present in excessive amounts. The same is true of heavy metals, many of which are by-products of industrial manufacturing.

The purposes of this experiment are: (1) learn deficiency symptoms associated with inadequate supplies of particular essential elements, and (2) investigate some consequences of ion uptake by plants from unbalanced nutrient solutions.

Part #1: Culture of Mineral Deficient Plants

Part #1: METHODS and MATERIALS
 

Table 3: Preparation of Nutrient Solutions
Type of nutrient solution	ml of D D H2O	ml stock solution / liter of nutrient solution
		A	B	C	D	E	F	G	H	I	J
Complete	985	5.0	5.0	2.0	1.0				1.0	1.0
?	938.5	7.5		2.0		50				1.0	1.0
?	968.5	7.5		2.0			20			1.0	1.0
?	727.5			0.5		50	20	200		1.0	1.0

One or Two Weeks Before the Laboratory Period

Each team will be provided with Sunflower seeds, 4 three-section plant pots, vermiculite, four 1-liter storage jars, assorted graduated cylinders and pipettes. Place labeling tape on each plant pot. Label the plant pots with your group name and either C (for complete), -N, -K, or -P. On the front laboratory table are a series of flasks containing solutions A,B,C ...J listed in Table 3 above as well as a supply of distilled-deionized water (D D H₂O). Each laboratory group will need to make 1 liter of the different nutrient solutions that you will use to water your plants. Prepare the solutions by first obtaining the D D H₂O. Then add the solutions in the order listed.

Fill each labeled plant pot about three fourths full with vermiculite. Place five Sunflower seeds on the top of the vermiculite in each plant pot. Cover the seeds with a thin layer of vermiculite. Water each plant pot with a sufficient amount of the appropriate nutrient medium. Place your plant pots in the plant pot tray. Place all the plants in the growth chamber. The growth chamber will be set for 16 hours of full light, 95% relative humidity, and 25^oC.

IMPORTANT!!!!! EACH DAY A STUDENT FROM YOUR LAB GROUP WILL NEED TO OBTAIN THE GROWTH CHAMBER ROOM KEY FROM THE DEPARTMENTAL SECRETARY (SHERRY) IN ORDER TO WATER THE PLANTS. ARRAIGNMENTS WILL BE MADE FOR THE WEEKENDS. YOU REALLY NEED TO CHECK YOU PLANTS EACH DAY! WATER THE PLANTS WITH THE APPROPRIATE NUTRIENT MEDIUM.

We will finish this laboratory exercise at a later date.

Part #2: Plant Tissue Testing for Nutrient Deficiencies

Observations, Shoot Length, Root Length, Fresh Weights, and Dry Weights

Obtain four sheets of white paper and label the sheets as Control, -N, -P, or -K as well as your group name. As you remove plants from their treatments, place them on these pieces of paper. This will help keep the plants labeled.

For each treatment (control, -N, -P, -K) gently remove three Sunflower plants from the pot. Gently rinse off the roots to remove the vermiculite. Using a ruler, obtain the stem length in centimeters of each of these plants and record the data in the table provided. Measure the stem from the first root to the first set of true leaves (located just above the cotyledons). Next, measure the root length in centimeters of each of these plants and record the data in the table provided. The root length should be measured from the first root to the tip of the tap root.

Using the balance provided, obtain the fresh weight of each plant in grams and record the data in Table #1. Calculate the average fresh weight from the individual weights. Fold these plants inside the labeled sheets of paper and dry them over night in the oven. You will need to reweigh the individual plants the following day to obtain the dry weights. Again, determine the average dry weight of these plants and calculate the ratio between the dry weight and the fresh weight for the different treatments.

NITRATE: Caution! The Nitrate Detection Reagent contains concentrated H₂SO₄, do not get it on your hands or cloths.

Obtain a petri dish, one control treated plant, and one minus nitrogen treated plant. Place the top and bottom of the petri dish side by side. Remove two of the very top leaves from the control plant and place them at the top of one of the petri dishes. Do the same for the very top leaves of the minus nitrogen treated plant but place these leaves in the other petri dish. In the middle of each petri dish, put the next series of plant leaves. Place the cotyledons from each of the plants at the bottom of each petri dish. This will give you a control and minus nitrogen series of leaves.

Now place one drop of the Nitrate Detection Reagent in the center of each leaf. The presence of nitrates is indicated by a blue color. A dark color indicates an abundant or excessive nitrogen supply; a pale blue or green color, an adequate supply; no color accompanied by a pale green color of the foliage, a limiting supply. Compare the colors of both the normal and the unknown plant, record observations, and indicate the level of nitrate nutrition in the plant.

PHOSPHATE:

Obtain four test tubes and label them: Stem -P, Stem Control, Leaf -P, Leaf Control. Obtain a fifth test tube that will contain no plant material - set this tube up along with the plant material test tubes (this tube will be used to blank the spectrophotometer).

Using the stem from just below the first leaves of a minus phosphate plant, slice the stem into 15 uniform pieces, about 1 mm in thickness. Do the same to a stem from a control plant. Place the slices in separate test tubes labeled -P or C (control). For the leaf tissue use two leaves from a minus phosphate plant and two leaves from a control plant. Cut the leaves up into fine pieces and place them in two separate test tubes.

To each test tube add 10 ml of Phosphate Detection Reagent No. 1, shake vigorously for 1 minute, and add an amount of Phosphate Detection Reagent No. 2 approximately equal in size to a pin head. Mix thoroughly and at once observe the color. Add another portion of reagent No. 2 to make certain that a sufficient amount of reagent has been added. A dark blue color indicates an abundant supply of phosphorus: medium blue, a non-limiting supply; light blue, a slightly limiting supply; a green or bluish-green, a moderately limiting phosphate level; a colorless or yellow, a very limiting supply.

Blank the spectrophotometer to air. Then measure the absorbance of each tube at 627 nm. Record your observations in Table #3

POTASSIUM:
 

Obtain four test tubes and label them: Stem -K, Stem Control, Leaf -K, Leaf Control. Obtain a fifth test tube that will contain no plant material - set this tube up along with the plant material test tubes (this tube will be used to blank the spectrophotometer).

Using the stem from just below the first leaves of a minus potassium plant, slice the stem into 15 uniform pieces, about 1 mm in thickness. Do the same to a stem from a control plant. Place the slices in separate test tubes labeled -K or C (control). For the leaf tissue use two leaves from a minus potassium plant and two leaves from a control plant. Cut the leaves up into fine pieces and place them in two separate test tubes.

Place the plant tissues in the labeled test tubes and add 5 ml Potassium Detection Reagent No. 1 then shake vigorously for 1 minute. Add 2.5 ml Potassium Detection Reagent No. 2 and mix. After 3 minutes note the amount of precipitate or turbidity. A dense precipitate indicates an adequate or abundant supply of potassium: a medium precipitate, a slightly limiting supply; a light precipitate, a moderately limiting supply; no precipitate or only a trace indicates a very limiting potassium supply. Make comparisons with the plant grown on complete nutrient. Transfer the liquid content of the test tubes to plastic spectrophotometer tubes. Obtain spectrophotometer readings at 600nm on these samples to obtain a more accurate account of the amount of precipitate. Use the Blank sample to blank the spectrophotometer at 600 nm. Take readings on each of the four plant samples.

Student ______________________ Date _____________________

Name

Team Mates _____________________________________________________

Graph the ratios as bar graphs using a computer graphing package. Staple the graphs onto the back of this sheet.

Table #3: Observations from mineral analysis
 

	Observations From Mineral Analysis
Tissue Test For	Control Plant	Deficient Plant	Conclusions
Nitrate
Phosphate Stems
Phosphate Leaves
Potassium Stems
Potassium Leaves

Discussion: Describe in a short essay how the mineral nutrients you analyzed would effect the growth of a plant. Relate this information to the results you obtained. Your discussion question must be typed and stapled onto the back of this sheet.