Evolution and Ecology (BIOL 3113)

Spring 2003


Artificial Selection and Evolution, Part 2

Introduction

Two weeks ago, we established for each lab section a population of about 100 plants, all from the same basic stock of "wild type" Fast Plant seeds, initially obtained from a commercial source. Each population at this point represents the initial generation of a separate lineage or line of descent.

The goals of this lab are to: 1) quantify the variability of a particular trait in this generation; and, 2) attempt to change the genetic makeup of the population, so that the next generation, on average, exhibits the trait to a substantially greater degree than does the present one. If successful, you will have accomplished artificial selection, resulting in evolution within this particular lineage from one generation to the next.

What trait will you attempt to artificially select? There are a number of fairly obvious variable traits that one can observe in a large population of mature Fast Plants. A brief list could include: total number of flowers, total number of leaves, length of the lower leaf, surface area of the lower leaf, plant height, total number of seeds per plant, total mass of plant, stem length between first and second leaves, etc. In contrast, there are other traits that don't appear to vary at all, such as: number of petals per flower (4); number of sepals (green petal-like structures just below the petals) per flower (4); petal color (bright yellow); and number of cotyledons ("seed-leaves") (2).

Click here to see a diagram of the key parts of a Brassica plant.

Each bench group will receive a sample of 18 plants. Treat these plants gently; they are young and tender, and easily damaged! When your group obtains your sample, look carefully at the plants and note the variability you can see. Remember, all your plants are almost exactly the same age, so differences you see (such as height, leaf size, etc.) are not due to differences in age.

One variable trait that you might not have noticed is "hairiness" of leaves, petioles (leaf stalks) and stems, but if you look more closely (especially with a hand lens) you should see these trichomes (hairs) on some plants. Trichomes have been shown to have a specific function. What might this function be?

Counting "hairs"

As you have probably guessed, the variable trait that you are going to attempt to alter in this lineage is "hairiness." In this "wild type" population, some plants should be noticeably hairy, many slightly hairy, and others apparently hairless. But such an observation is too general, and needs further quantification.

To accomplish this, you could count all hairs on all parts of each plant, but this would be a time-consuming task, and an unnecessary one. Fortunately, hairiness of one part of a plant (such as the leaf surface) tends to be strongly correlated with hairiness of other parts (such as the leaf margin). In other words, a plant's hairiness in general can be estimated by measuring the hairiness of a specific structure.

The structure we will use for this "hairiness index" is the petiole, or leafstalk, where trichomes are large, conspicuous and rather easily counted, and the structure is relatively small with a defined starting and ending point. For consistency, we will use the petiole of the first (lowermost) true leaf, and we will define the limits of the petiole as follows: from its junction with the main stem (usually marked by a small bulge or ridge, often differently colored) to its junction with the lowermost leaf vein. Click here for a picture.

An important note: the two lowermost squarish, two-lobed and rather thick leaves are actually cotyledon ("seed leaves"), not true leaves. The first true leaf is just above these two cotyledons. What you need to do now is to count the total number of trichomes on the petiole of the first true leaf of each plant in your sample. Your data will then be combined with data from the rest of the class.

Use the hand lens and desk lamp, if available; otherwise use a dissecting microscope. The trichomes are most conspicuous if strongly illuminated against a dark background, such as the black tabletop. If present, the trichomes will generally be concentrated on the lower side of the petiole, but some could occur on the sides or top as well. Be sure to rotate the plant so that you can see all aspects of its petiole. Count a second time for verification, then record the data on your data sheet. Also record this number on the plant's ID label. As a reminder: handle these plants gently. They are easily damaged if treated roughly, and bent stems may result in death of the plant.

When all groups have finished assessing trichome number of individual plants, we will combine the separate data into a single data table, which you should copy into a frequency table. Next, plot a frequency histogram (bar graph) of these data. What is the average "hairiness index" for this population?

Selecting the parents of the next (second) generation

Only a small fraction (about 10%) of this population of plants will be selected to be parents of the next generation. These won't be randomly chosen. As a class, we will identify the 10 hairiest plants, and mark each label. The unselected plants will not be permitted to breed, and thus will not contribute any offspring to the subsequent generation. Record the trichome values of the selected individuals (parents-to-be), as well as the average value. Now calculate the difference between the average number of petiole hairs of the selected parents-to-be and that of the population as a whole. How hairy do you think individuals in the second generation will be? Why?

Pollination of Selected Plants

Note: Fast Plants grow very quickly, but the exact timing of specific events (seed germination, first flower, seed maturation, etc.) depends on local environmental conditions. We are not entirely sure whether the selected plants will be ready for this next step this week or next. We will adjust our schedules to those of the plants!

When the 10 plants you selected to be parents of the second generation in your section's Fast Plant lineage are in full flower, you will assist them in the process of sexual reproduction. In nature, Brassica rapa plants depend on insects to transfer sperm-bearing pollen from the male part of the flowers (stamens, terminating in the pollen-producing anthers) of one plant to the female part of the flowers (pistil, with the pollen-receiving stigma, style and egg-producing ovary) of another plant. Although each flower has both male and female parts, sperm from one plant are incapable of successfully fertilizing eggs of the same plant. This self- incompatibility ensures outcrossing (mating between different individuals). The plants lure and reward the insect pollinators with nutrients (nectar and edible pollen), and the insects inadvertently pick up the sticky pollen on various body parts and then carry it with them to the next flower. Using specially-designed pollination wands, you will play the role of pollinator, with a successful experiment rather than a sweet treat as your reward.

Each bench group should obtain two of the selected plants. The object is to transfer pollen from each plant to every other plant. This can be done in the following way. Using a single pollination wand, one group will lightly rub and twirl the brush end for several seconds onto the anthers and stigma of each open flower on their plant(s). Then pass the wand, now loaded with yellow pollen, to the next group, who will repeat the process. After the wand has made one complete round (all open flowers on all plants have been "visited" by the wand), make a second round in the same manner. When finished, place the wand in the designated "Used wand" tub. Finally, return all plants to the watering tray.

Fertilization will result in the development of the ovules (each containing an embryo) into mature seeds, which will be contained in the fruit or seed pod (the elongated ovary). The length of time from fertilization to mature seed is about 3-4 weeks. We don't need to do anything more with these plants until it is time to harvest the seeds except to remove them from water in 3 weeks, which hastens the seed-maturation process. Next week, you should easily be able to see the elongating fruits. In two weeks, they will have become even longer (2-5 cm) and the individual seeds inside (perhaps 5-20 per pod) will have become visible. In three weeks, the maturing process is nearly complete. You will plant these seeds (generation 2) in 4 weeks, during lab 6.

Acknowledgements: This lab is closely derived from one developed by Dr. Bruce A. Fall of the University of Minnesota.


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