INS 2007-08 Lab V An Introduction to Plant Vascular Systems and Reproductive Systems Based on: Thompson, L. K. 2000. Introduction to plant vascular systems. Pages 180-194, in Tested studies for laboratory teaching, Volume 21 (S. J. Karcher, Editor); and Thompson, L. K. 2000. Plant reproductive systems: An investigative approach. Pages 198-217, in Tested studies for laboratory teaching, Volume 22 (S. J. Karcher, Editor). Introduction Two of the most important developments shown by plants which make them suited to life on dry land are: (1) reproductive systems which do not depend upon standing water for gamete or population dispersal, and (2) vascular systems which distribute water, nutrients and sugars throughout the plant from their disparate sources (leaves, roots, atmosphere, soil, etc.). In today's lab you will investigate various aspects of plant vascular systems, flower and pollen structures in angiosperms, as well as adaptations that aided the increasing use of wind and animals for gamete and population dispersal. I. MICROSCOPIC ANATOMY OF THE XYLEM SYSTEM IN DICOT STEMS Background: Xylem System The xylem system of plants is that portion of the vascular system that transports water and nutrients from the roots to the leaves. The function of this system is dependent primarily upon the "negative pressure" generated in the xylem tube system by the evaporation of water from the upper parts of the system, including the leaves. Such water loss "pulls" water upward in the xylem system by the cohesive forces between water molecules within the xylem tubes. The tubes of the xylem system need structural support to keep from collapsing and this support is provided by lignin. Lignin is, after cellulose, the most abundant organic substance in most plants. It is a highly branched polymer of phenylpropane units that can be covalently bound to cellulose. The subunits of lignin are joined together by the action of the enzyme peroxidase, forming a physically rigid, largely indigestible polymer matrix that branches in three dimensions. Lignin is found in the cell walls of various types of supporting and conducting plant tissue. Lignin is stained by Safranin O, while cellulose is stained by Methyl Green (two stains you will be using on the dicot stem). You will determine which anatomical regions of a dicot stem in cross-section contain cell walls composed of lignin and which regions contain the tubes of the xylem system. Experimental Protocol: You will produce hand-made sections (thin slices) through a dicot stem, the xylem system of which you have stained with a fluorescent dye, Acridine Orange (caution: it is a mutagen). You will observe one of the sections under ultraviolet light to observe and document the regions of the stem stained with Acridine Orange. You will stain another section with Safranin O and Methyl Green to observe and document the regions of the stem which have walls containing lignin, as opposed to only cellulose. You will then compare the distribution of the xylem system and the distribution of lignified cell walls. Read the entire procedure (steps 1-16) carefully before you begin. Divide the steps among your group. Be prepared for steps which are timed and must be done immediately following one another. With label tape, mark five Pasteur pipets: "ethanol solutions," "Safranin O," "Methyl Green," "water," and "glycerol-water." Staining the stem's xylem system with the fluorescent dye: 1. Obtain a fresh 1-cm length of a dicot stem from the instructor. Be careful to note and remember which end is the base of the stem. Carefully cut the base of the stem at a 45o angle with a razor blade. Place the stem in a tube containing a small amount (only about 2-3 mm depth) of 3% Acridine Orange. Let the stem sit in the tube for ten minutes. During this time, the dye-containing solution will be transported up the xylem system of the stem. Preparing sections of the stem: 2. Loosen the nut from the "nut and bolt microtome" so it is barely secured to the end of the bolt. This makes the "well" formed by the interior of the nut as deep as possible. Stand the bolt up so that its head is on the table. Remove the stem from the Acridine Orange. Add a small amount of melted wax to the bottom of the "well" inside the nut and immediately stand the stem piece up in the melted wax. When the stem can stand up on its own, add more melted wax to fill the well completely and to form a mound of melted wax around the base of the protruding stem. Let the wax harden completely (about ten minutes).3. Hold the head of the bolt flat on the table with one hand. Hold the razor blade with the other hand (Figure 9.1). With the razor blade, carefully shave off the mound of wax and stem from above the top of the nut. Run the blade over the nut's surface to make the wax completely smooth. 4. Turn the bolt clockwise about 1/12th of a turn (see Figure 9.2) to make the wax cylinder protrude slightly. Again holding the microtome and blade as shown in Figure 9.1, slice the blade over the nut's surface to make a shaving of the wax surface through the stem. A smooth, quick slicing motion works best. 5. With the needle probe, gently remove the stem section from the wax shaving. If the section is thin and not torn, give it to your lab partner who will immediately proceed with the fluorescent viewing of the stem section (steps 6-8). (An otherwise good section that is too thick for staining is quite suitable for fluorescent viewing.) Cut the wax block smooth with the nut's surface again and return to step 3 to cut a section you will stain for lignin and cellulose (steps 9-15). This section needs to be as thin as possible. If you leave some stem remaining in the wax block of the microtome for a while, place a drop of water on it to keep it moist in case you want to cut more sections later. If you make so many unsuccessful shavings that the wax block becomes too loose in the ever-shallowing well of the nut, you will need to remove the wax from the well and start again with another piece of stem. Figure 9.1 Observing the fluorescently stained xylem system: 6. With the needle probe, carefully transfer the stem section to a clean microscope slide. Be careful not to let the section fall off the slide when your transport it. 7. Observe the slide under long-wave ultraviolet illumination with the high power of the dissection