Leaf Elongation Bioassay in Zea mays
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Introduction

A drive through Missouri, particularly throughout the major river valleys, gives a visual demonstration of the importance of corn and other crops to the local economy. In both St. Louis and St. Charles Counties there are many farms growing corn. We are very fortunate to have a fairly regular rainfall, although, some years, or even months can be very dry compared to others. Farmers in our area solve the problem of irregular rainfall with irrigation. Because of the Missouri and Mississippi rivers, they can be assured that water will always be available to them for this purpose. Not all farmers are so lucky. For instance, many countries in Africa have great difficulty feeding their populations because of drought. They do not have dependable rainfall or a source of fresh water for irrigating crops. In these poor nations most people cannot afford food that has been imported from far away and without the ability to grow their own food they may starve. Scientists all over the world, including Monsanto and the Donald Danforth Plant Science Center in St. Louis, are conducting research to find a solution to this problem. While the basic mechanism of water movement through plants is known it isn’t well understood how some plants are better able to survive under droughted conditions.

Plants have several responses to drying soil. One thing they do is close the stoma on their leaves. This is the opening, or pore that allows plants to take in carbon dioxide and release oxygen during photosynthesis. Unfortunately, much water is also lost from the leaves in this way. So when water availability decreases the stoma close to conserve water and the leaves of the plant slow their growth rate. Also, they may have an increase in root growth as the shoot growth decreases. If scientists can figure out the mechanism for these responses they might be able to increase the drought resistance of some plants making it easier to grow them in areas without consistent rainfall.

How do the shoots or leaves of the plants know that the soil is getting dry? Is the signal just the leaves receiving less water, which is a hydraulic signal? Or is there another way that roots can communicate with shoots? Are there chemical signals being sent from the roots via the xylem sap? Does pH of the sap change to signal the shoots that water is becoming limited? To conduct a test to try and answer some of these questions, we must be able to eliminate the roots so that we control what signals get sent and how they arrive at the shoot (unmodified).

A bioassay is a test to determine the impact of a certain substance on living organisms. By using different concentrations of the substance, you can find out which concentration has the greatest effect or what is the greatest concentration that has no effect. Since we specifically want to know how some factors affect leaves, we can separate the shoots from the roots; and thereby eliminate the possibility that the roots are sending, or modifying signals unknown to us.

Purpose

1. To learn a method for measuring affects of different substances fed to corn seedlings.

2. To determine whether corn seedlings will grow more in a 24 hr period when fed an artificial sap that contains nutrients or when fed distilled water.

Methods

In order to simplify both methods and materials we will use seedlings rather than large plants, and test tubes to both contain our seedlings and to deliver treatments. We will then measure growth of the third leaf after 24 hours to determine the affect of different conditions applied to the seedling on leaf growth or elongation.

Part 1

• Your first step is to grow seedlings with an extended mesocotyl. As you know, young seedlings gets nutrients and water from the roots through the mesocotyl (pictured below at number 2.), so by growing an extended mesocotyl we can use it as a delivery system without the added variable of roots.

• The other job of the mesocotyl is to push the coleoptile (#1, notice the green color) up towards the light, so the deeper you plant the seed, the longer will be your mesocotyl.

• For this bioassay you will want mesocotyls that are approximately 6-8 cm long. So plant about 12 seeds to a depth of 8 cm in a 25 cm deep pot and add enough water to moisten the soil throughout the pot. These will be kept in the dark cabinet for approximately 5 days or until several seedlings emerge.

• Once seedlings emerge you should transfer the pot to the light stand. Plants will be harvested when the third leaf on a majority of plants has emerged to at least two centimeters.

Part 2

• Number and label one set of tubes 1-6 and add 5 ml of distilled water. A second set 7-12, should be numbered and labeled and then filled with 5 ml of the artificial sap (see below). Fix stoppers with holes into the tubes. See figure below.

• Harvesting must be done with great care so you don’t tear any roots or cause breaks in the mesocotyls because this could cause distress signals to be sent from the roots.

• Gently separate the seedlings from the soil and each other and lay them in a pan of water so the roots are submerged but the leaves are not.

• Using a razor blade cut each mesocotyl as close to the kernel, or seed, as possible. Keep the entire mesocotyl/root system under water when you make this cut. Now gently insert the end of the mesocotyl through the hole in the stopper. Your mesocotyl should be submerged in solution to a depth of at least 2cm. Refer to the figure above.

• Now take an initial measurement of the third leaf. Measure from the top edge of the stopper to the tip of the leaf. Have everyone in your group measure each leaf to check for accuracy of the measurements. Take an average if necessary. Record this initial length in your lab notebook.

• Now place the tubes under the grow light. Subsequent measurements should be made after 24 hrs and then again after 48 hrs. Measure the leaf in the same manner as before and record total growth each time. Calculate an average growth rate that includes data from the entire class for each condition and make a graph of the results.

• Write a discussion of the results in your lab book. Be sure to include difficulties you encountered and all possible sources of error in your results.