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ALGAL ORIGINS of LAND PLANTS Introduction The LAB 1: ALGAL ORIGINS OF LAND PLANTS Introduction The Class Charophyceae contains mostly freshwater green algae and is believed to be the line containing the ancestral stock from which land plants evolved. Multigene DNA phylogenetic analysis has shown that the Charales (Chara, Nitella) are the closest living relatives of green land plants (McCourt et al, 2004). Green algae classified in the Class Charophyceae have certain traits (features) also found in land plants. Such algae are believed to be in the evolutionary lineage from which land plants evolved. Land plants have uninucleate cells as their basic unit. The cells are arranged in tissue known as parenchyma; which is formed by cell division in three planes resulting in a solid mass of tissue. Typical land plants have functional differentiation with aboveground, green photosynthetic structures or shoots (differentiated into stems and leaves) and belowground anchoring structures (rhizoids or roots) for absorption of water and soil nutrients. We will look at examples of algae that are representative of the sister lineage to land plants. Morphological traits, combined with molecular phylogenetics, have helped to determine the algal lineage that is the closest relative of land plants, the Charophyceae. Shared features include a pattern of nuclear and cell division (mitosis and cytokinesis) similar to that of extant land plants and details of the ultrastructure of motile cells. In Charophyceae motile cells are asymmetrical with subapical flagella that extend at right angles from the cell; and nuclear and cell division combining an open spindle, a phragmoplast at nuclear separation and the cell plate type of cytokinesis (Fig. 1.1). Figure 1.1 Mitosis and cytokinesis showing the open spindle, phragmoplast and cell plate that are shared by land plants and Charophyceae Figure 1.2: Chara. A) “plant” showing nodes and internodes, B) node with whorl of branches, C) fertile branch, D) gametangia, oogonium above (female), antheridium below (male) McCourt, R.M., Delwiche, C.F., and Karol, K.G. (2004). Charophyte algae and land plant origins. Trends in Ecology & Evolution 19, 661–666 3 Laboratory Exercise Goals: I. Become familiar with the use of the dissecting and compound microscopes. II. Observe representatives of the green algal lineage most closely related to land plants. III. Start spore cultures of the fern Ceratopteris richardii for Lab. 5. IV. Investigate key innovations of land plants. I. INTRODUCTION TO MICROSCOPY: DISSECTING MICROSCOPE 1. Adjust seat height. Always begin a lab by adjusting seat height. Place the dissecting scope on the bench in front of you and adjust your seat height so you are looking comfortably into the eyepieces without stretching your neck up or bending over too much. 2. Fresh plant material may not be placed directly on the stage. If you are examining fresh plant material it will gum up the stage and will also tend to quickly dry up. Work on a damp piece of towel paper or kimwipe. Or, in some cases, you may wish to place small specimens in a Petri dish with a few drops of water. 3. Adjust the light. For thin specimens which light will pass through, you can use both transmitted light (from below) and reflected light (from above). For opaque objects, the light must be from above. 4. Adjusting the eyepieces. Start by roughly focusing and then make the following adjustments. This is an important step to reduce fatigue and eyestrain. a. Interocular distance: The eyepieces can be moved closer together or farther apart to accommodate the distance between your eyes, which varies from person to person. You may also want to experiment with how close you want your eyes to the eyepieces. Sometimes it helps to move back a bit. b. Focusing the 2 eyepieces separately: The vision in your two eyes is probably not identical and, in any case, whoever used the scope before you will have adjusted the eyepieces to suit themselves - and it won't suit you. Proceed as follows: i. Look through the right eyepiece (close left eye or simply ignore what it sees or put your hand over it). Focus with the focusing knob. ii. Now look through the left eyepiece, closing the right eye. Focus using the eyepiece itself (it should have a knurled ring which you can turn). If the image is not sharp when you have finished the above, you may have to make some more minor adjustments or try cleaning the eyepieces and objective lens (see T.A. for how to proceed). Additional suggestions. When studying microscope slides, it is a good idea to examine them with the dissecting scope before using the compound scope. The reason for doing this, particularly with large specimens, is that you may see only a small part of the specimen with the compound scope. Looking at the entire thing first will help in getting oriented. Use transmitted light (from below) when looking at a slide on the dissecting scope. 4 COMPOUND MICROSCOPE 1. You will be using an Olympus microscope, so the basic set of instructions refers to the CH-2 Olympus compound scope. 2. Adjust seat height. Ideally, you will not be stretching your neck or bending your back excessively to look through the eyepieces. 3. Starting light intensity. Most of the Olympus scopes have a light switch on the base of the scope in front. Turn the light on. On the right side of the base, you will find voltage control dial that sets the brightness. For starters set it about in the middle. 4. Substage condenser. The substage condenser focuses the light on your slide. There is a knob (condenser height adjustment knob) that drives the condenser up and down. Move it so that the condenser is all the way up, then back off slightly. The substage condenser should remain in this position. 5. Bright field illumination. On Olympus phase microscopes, the substage condenser has a plate that rotates displaying different numbers. The number (which shows in front) should always be in the zero position, which is the bright field position. 6. Using the revolving nosepiece rotate the 10X objective into place. Always start with the lowest magnification. Place the slide on the compound scope stage and roughly center the tissue section under the objective. With the lowest magnification only, you can start by moving the stage all the way up with the coarse focus knob. For a practice session, the T.A. will assign you to look at either a root tip (Allium) or a shoot tip (Salvia or Coleus). 7. Initial light adjustment. Look in the eyepieces. If the light is too bright, use the substage condenser iris diaphragm lever to adjust it. Always make your initial light adjustment with the condenser diaphragm. Use the rheostat adjustment (dial at base) only if the condenser iris diaphragm does not suffice. 8. Find the tissue section and roughly focus on it. Use the coarse focus knob (the larger one) to slowly move the specimen into focus. If you don't see the tissue section, you will have to move the stage around as well to get the tissue section under the objective. 9. Adjusting the eyepieces. Once you have the light adjusted and have focused on the tissue, it is time to adjust interpupillary distance and individual eyepieces a. Interpupillary distance: The eyepieces slide closer together and farther apart to accommodate the distance between your eyes. Make this adjustment while looking at the tissue section. Sometimes it helps to move your head back slightly so that your eyes are further away from the eyepieces. You will have to experiment to find the best position for you. b. Focusing eyepieces: The vision in your two eyes is probably not identical and, in any case, whoever used the scope before you will have adjusted the eyepieces to suit themselves - and may not suit you. Proceed as follows: i. Look through the right eyepiece (close left eye or simply ignore what it sees or put your hand over it). Focus with the focusing knob. 5 ii. Now look through the left eyepiece, closing the right eye. Focus using the eyepiece itself (using knurled ring which you can turn). If the image is not sharp when you are done, you may have to make minor adjustments or perhaps try cleaning the eyepieces or objective lens. These steps are very important for avoiding fatigue and eyestrain, so be sure to do them. 10. Now switch to the 43X objective and practice adjusting the light, something you will have to do each time you change objectives. In general, the light should not be too bright. Bright light produces light scatter and a fuzzy image. It would be better to have the light too dim than too bright. Always start the light adjustment with the substage condenser iris diaphragm. Only adjust the rheostat current if the iris diaphragm does not allow enough latitude. Never lower the condenser itself to get more light. The best optics are obtained with the condenser nearly all the way up. Lab policy on microscope slides. Most of the time you will be examining commercially prepared microscope slides. Sometimes we have only a few slides of each type for the entire class. It creates a problem when students collect more than one slide at a time and have them piled up waiting to look at. So take only one slide at time and return it as soon as you are done. 6 BIOL 441: MICROSCOPE PARTS 7 II. ALGAL ORIGIN OF LAND PLANTS You will examine the freshwater algae Chara and Nitella, which are evolutionarily closely related to land plants. While you won’t be able to observe their microscopic traits, they do have the unilateral type of motile cell organization and the land plant pattern of mitosis and cytokinesis.
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