Biology Plant Physiology - Lab Exercise 3 Plant Mineral Nutrition

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1 Biology 3460 Weeks 1, 3, 5, & 7 1 Biology Plant Physiology - Lab Exercise 3 Plant Mineral Nutrition Objectives: This lab exercise is intended to: (1) introduce students to the mineral nutrient requirements of plants, (2) illustrate sand culture as a technique to grow plants under controlled nutrient conditions, (3) provide experience making plant nutrient solutions and calculating nutrient concentrations in different solutions, (4) test of the effect of different sources of nitrogen on growth of rye (Secale cereale) plants. Introduction Mineral nutrition is the study of how plants obtain and use basic mineral nutrients normally taken up by roots from the soil solution. Essential mineral nutrients required by plants are shown in Table 5.1 in your textbook (attached). Access to these nutrients in sufficient supply, along with adequate water and sunlight allows plants to synthesize all the other components they require for growth. An empirical formulation of a complete nutrient solution for plants was developed by Dennis Hoagland, a researcher in California, USA. There have been several modifications of Hoagland s solution over the years with a typical example of a modified Hoagland s solution illustrated in Table 5.3 of your textbook (attached). Hoagland s solutions typically provide nutrient elements in ratios related to the nutrient requirements by plants, but the nutrient concentrations in full strength modified Hoagland s solution can be several times higher than a plant would experience in the natural world. When growing non-agricultural plants it is often necessary to dilute the modified Hoagland s solution in order to prevent injury due to toxicity or salinity stress, particularly with young seedlings. Typical modified Hoagland s solutions provide nitrogen as both ammonium (NH 4 + ) and nitrate (NO 3 - ) ions. Most plants grow best when nitrogen is supplied from both ammonium and nitrate. If nitrogen is supplied as only one type of ion (either nitrate or ammonium) large changes in the ph of the nutrient solution can occur and this can then disrupt the uptake of other required nutrients such as calcium, magnesium or iron. Plants generally grow better when they have access to both ammonium and nitrate because this promotes cation-anion nutrient balances within the plant. Instructions for making the macro-nutrient stock solutions for modified Hoagland s solution are shown below. Three different formulations are illustrated, one with both ammonium and nitrate supplied (typical Hoagland s solution) and then two solutions in which nitrogen only in the form of either nitrate or ammonium is supplied. In this lab experiment, plants will be grown in one of three treatments (both ammonium and nitrate, nitrate only, and ammonium only) to test the effect of nitrogen source on plant growth (biomass (dry weight) production). Note that it is not possible to keep the concentrations of all macro-nutrients at identical concentration in all three versions of the modified

2 Biology 3460 Weeks 1, 3, 5, & 7 2 Hoagland s solution (Calcium concentration is modified in the nitrate-only solution and Sulphur concentration is modified in the ammonium-only solution). In addition to the macro-nutrients shown in the nutrient solution recipes illustrated below, the modified Hoagland s solution also provides micro-nutrients from a prepared stock solution and iron from a chelated-iron solution, which keeps the iron in a soluble form and prevents it from precipitating out of solution and making it unavailable to the plants. Part A. The Nutrient Solutions Modified Hoagland s Solution (both NO 3 and NH 4 provided) Macro-nutrient Stock Solutions: 1M Calcium nitrate [Ca(NO 3 ) 2 4H 2 O] 1M Potassium nitrate [KNO 3 ] 1M Magnesium sulphate [MgSO 4 7H 2 O] 1M Ammonium phosphate [NH 4 H 2 PO 4 ] To make 4 litres, add: 4 ml calcium nitrate 6 ml potassium nitrate 2 ml magnesium sulphate 6 ml ammonium phosphate 2 ml iron + 2 ml micronutrients Top up to 4 L with distilled water Modified Nutrient Solution (only NO 3 provided) Macro-nutrient Stock Solutions: 1M Calcium nitrate [Ca(NO 3 ) 2 4H 2 O] 1M Magnesium sulphate [MgSO 4 7H 2 O] 1M Potassium phosphate (monobasic) [KH 2 PO 4 ] To make 4 litres, add: 10 ml calcium nitrate 2 ml magnesium sulphate 6 ml potassium phosphate 2 ml iron + 2 ml micronutrients Top up to 4 L with distilled water

3 Biology 3460 Weeks 1, 3, 5, & 7 3 Modified Nutrient Solution (only NH 4 provided) Macro-nutrient Stock Solutions: 1M Calcium chloride [CaCl 2 2H 2 O] 1M Potassium phosphate (monobasic) [KH 2 PO 4 ] 1M Magnesium sulphate [MgSO 4 7H 2 O] 1M Ammonium sulphate [(NH 4 ) 2 SO 4 ] To make 4 litres, add: 4 ml calcium chloride 6 ml potassium phosphate 2 ml magnesium sulphate 10 ml ammonium sulphate 2 ml iron + 2 ml micronutrients Top up to 4 L with distilled water Protocol: Work as individuals to complete this part of the lab. 1. Calculate the concentration (mmol/l) of each of the macro-nutrients for the three modified Hoagland s solutions and fill in the table below. A sample calculation is provided. Sample Calculation: For the Hoagland s solution with both ammonium and nitrate present, the concentration of potassium (K+) is determined as follows: 6 ml of 1 M potassium nitrate was added to a bottle and the volume is made up to 4 L. 6 ml x 1 mmol/ml (1 M) potassium nitrate = 6 mmol of potassium nitrate 6 mmol potassium nitrate in 4 L of solution is: 6 mmol/4 L = 1.5 mmol/l Table 1. Concentration of individual macronutrients available in three modified Hoagland s solutions. Macro-Nutrient Both NO & NH 4 (mmol/l) NO - 3 only (mmol/l) NH + 4 only (mmol/l) N P K Ca Mg S

4 Biology 3460 Weeks 1, 3, 5, & 7 4 Part B. The Experimental Design Plants will be grown in sand culture in each of the three nutrient treatments, under the same light conditions. We will measure total dry biomass of the plants at the end of a four-week growing period. Protocol: Work as a group of four students to complete this part of the lab. 1. Send one of your group members to help with mixing the first batches of the three nutrient solutions, according to the recipes outlined in Part A. 2. Prepare one plant pot for each of the three treatments (both NO 3 and NH 4, only NO 3, and only NH 4 ). Be sure to label the pots with the treatment type and a group identifier. Add a layer of polyfilter wool to the bottom of the pot to prevent sand from escaping through the holes. Fill the pot with sand up to about 2 cm below the lip of the pot. 3. Several rye grass seedlings have been germinated in Petri dishes in Promix soil mixture. Carefully choose four seedlings for each treatment (will need 12 seedlings in total), be sure to choose healthy-looking seedlings of about the same stage of germination. Remove the seedlings very gently from the Petri dish, and rinse them under a gentle stream of water to remove as much soil from the roots as possible. 4. Moisten the sand in the prepared plant pots with the appropriate nutrient solution, then make wells in which to plant the four seedlings for each treatment. Poke a well with your finger deep enough to support the roots of the seedling, but not so deep that the shoot will be under sand once planted. Place the seedling into the well then gently push sand around it to fill in the well and support the seedling. Be sure to space the four seedlings evenly throughout the pot. 5. Once you have completed a pot for each treatment, place them in the appropriately labeled plastic tub. Your lab instructor will fill the tubs with the nutrient solutions prepared in Step 1. Nutrient solutions will be changed twice a week. You may be asked in future labs to mix fresh nutrient solutions and refill the tubs. 6. At the end of the four-week growing period, cut the plants off the roots right at the surface of the sand, place them in an envelope for each treatment (label your group names and the treatment type on the envelope). The above-ground plant material will be dried in a drying oven for two weeks. In the next lab period we will collect class data for the above-ground biomass for each treatment.

5 Biology 3460 Weeks 1, 3, 5, & 7 5 Treatment Both NO 3 - & NH 4 + NO 3 - only Above-ground Biomass (g) Bench 1 Bench 2 Bench 3 Bench 4 Bench 5 Mean ± s.d. NH 4 + only Analysis: 1. Use class results to prepare a figure that shows the effect of mineral nutrition treatment on the above-ground biomass of the rye plants. Mean biomass values with standard deviations should be plotted. 2. Using a Kruskal Wallis Test, determine if there is a statistically significant difference between the treatments. 3. What effect did the different nutrient regimes have on plant growth? Why? Explain.

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