Biology 164 Laboratory

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1 Biology 164 Laboratory Artificial Selection in Brassica, Part I (Based on a laboratory exercise developed by Professor Bruce Fall, University of Minnesota) I. Objectives 1. Gain familiarity with the process of artificial selection. 2. Gain familiarity with some plant varieties in the genus Brassica that are the products of artificial selection. 3. Conduct an artificial selection experiment involving two cultivars of Brassica rapa in which you will: a. quantify the variability of a specific trait in the first generation of the two cultivars, b. attempt to change the genetic makeup of the next generation with respect to this trait so that, on average, the next generation exhibits the trait to a substantially greater degree than the initial generation, and c. compare the performance between the two cultivars with respect to the expression of the trait to determine if an upper limit has been reached in the expression of the trait. II. Introduction A visit to a farm, supermarket, pet store or plant nursery will offer many examples of selective breeding of plants and animals by humans. Over hundreds and even thousands of years, humans have altered various species of plants and animals for our own use by selecting individuals for breeding that possessed certain desirable traits. This selective breeding process was continued for generation after generation. Often the products of such selective breeding are remarkable. Quite diverse domestic dog breeds, from chihuahuas and miniature poodles to Newfoundlands and Irish wolfhounds are all related to a common wild ancestor, the wolf (Canis lupus). Domestic chickens are all derived from the wild Jungle Fowl (Gallus gallus). The success of plant and animal breeders in dramatically changing the appearance of various lineages of organisms in a relatively short period of time is an obvious yet profound fact. This fact did not escape Charles Darwin as the first chapter of The Origin of Species concerns artificial selection by humans ( Variation under Domestication ). Darwin used many examples of artificial selection by humans to help support the case for his proposed mechanism for the evolution of natural populations natural selection. For example, Darwin was particularly taken with the number of pigeon varieties such as tumblers, pouters, fantails and many others. All of these pigeon varieties were derived from wild Rock Doves (Columba livia) over the past 5,000 years. One can find similar examples of selective breeding among plants, including those humans have bred for food. To gain a better understanding of selection and inheritance, biologists have experimented with artificial selection involving a variety of traits in many different species of bacteria, yeast, plants and animals. The results, obtained in a relatively short period of time, are often impressive. Artificial Selection in Brassica, Part I Page 1

2 A general finding of these studies is that most variable traits in organisms respond to artificial selection. In other words, it is usually possible to increase or decrease the frequency or average value of a trait in a lineage through careful selective breeding. We will begin a study today to see if you can accomplish the same thing over the course of the semester. How artificial selection differs from natural selection In contrast to natural selection, artificial selection 1) favors traits that for some reason are favored by humans; 2) has a goal or direction toward which the selection process is directed; and 3) generally is much faster than natural selection because the next generation can be absolutely restricted to offspring of parents that meet the desired criteria (natural selection is rarely so absolute). In artificial selection, humans are doing the selecting, intentionally restricting breeding to individuals with certain characteristics. In natural selection, the environment does the selecting individuals that survive and reproduce better in a given environment, are naturally selected. The environment can include a large number of factors such as predators, food supply, weather, to name just a few. Some important plant cultivars that have arisen by artificial selection A specific plant type or variety that is cultivated for human use is known as a cultivar (cultivated variety). The genus Brassica of the mustard family (Brassicaceae) includes many cultivars that are important in agriculture. A number of nutritious and tasty vegetable cultivars have originated from three species of Brassica (Brassica rapa, B. oleracea and B. juncea). Some cultivars have been bred for root production, others for leaves, flower buds or oil production. You may be surprised to learn that these familiar vegetables, though very different in appearance, are actually descendents of the same ancestral species. Centuries of artificial selection have produced greatly divergent cultivars within these species (Fig. 1). The following cultivars originate from the three wild species indicated in italics: Brassica oleracea kale, cauliflower, broccoli, cabbage, Brussels sprouts, kohlrabi, collards, Savoy cabbage Brassica juncea leaf mustard, root mustard, head mustard and lots of other mustard varieties Brassica rapa turnip, Chinese cabbage, pak choi, rapid-cycling Fig. 1: Examples of four cultivars of the species, Brassica rapa. Artificial Selection in Brassica, Part I Page 2

3 III. Experimental Materials and Methods A. Screening of the First Generation of Cultivars You and the other members of your lab section will participate as plant breeders in an effort to artificially select for a particular variable trait in two rapid-cycling cultivars of Brassica rapa. The experiment will begin today but will not be completed for several weeks. The Brassica rapa cultivars you will be using are the products of intense artificial selection over the past 20 years for the following traits: rapid flowering and maturation; high seed production; short stature; ability to thrive under artificial light. The result of these efforts (conducted at the University of Wisconsin) is a valuable research and educational tool. The generation time (from seed to maturation to fertilization to mature seed) is 6-7 weeks, short enough to allow us to study one complete generation this semester. The life cycle of these so-called Wisconsin Fast Plants is shown below. Figure 2. Life cycle of rapid-cycling cultivars of Brassica rapa. Artificial Selection in Brassica, Part I Page 3

4 The two cultivars of rapid cycling plants used in the study include a commercially available standard cultivar and an experimental cultivar that was previously selected from the standard cultivar. We established populations of the two cultivars by planting seeds approximately 17 days prior to the lab. All plants were grown identically, at 23 C, using commercial potting soil, a constant feeding regime, and continual light of 200µE. brightness. As you begin this study, these plants will have begun flowering (see Figure 2 above). These plants represent the initial populations (the first generations). Your challenge as a class is to select, from these initial populations, the top ten per cent of the plants that exhibit an extreme form of a specific variable trait, and to use these selected lineages as parents of the succeeding generations. The study will be divided into two parts. In the first part of the study you will determine whether or not the standard and experimental cultivars differ significantly from one another with respect to the specific variable trait in question. In the second part of the study you will determine whether or not the standard and experimental cultivars respond similarly to artificial selection to increase the expression of this specific trait, and decide whether or not an upper limit is being reached in the expression of the trait. Variable traits: continuous or discreet? There are a number of fairly obvious variable traits that one can observe in a large population of Fast Plants. A brief list might include: number of flowers, color of hypocotyl, number of leaves, type of petiole, size of first true leaf, and plant height. In contrast, other traits usually don t vary at all: number of petals per flower (four), petal color (yellow), number of cotyledons (two), and leaf color (green). Refer to Fig. 2 if you are unfamiliar with any of these terms. Your lab instructor will give each student a container with ten plants. Treat these plants gently; they are young and tender, and easily damaged! Examine the small sample of plants you have at your table and note the variability you can see. Remember, the plants are all the same age so differences you see (such as height or leaf number) are not due to differences in age. Note how some traits vary continuously over a range of possibilities, while others fall into discreet (either/or) categories. For the traits below indicate whether they vary continuously or discreetly. Number of flowers: Color of hypocotyl: Number of leaves: Type of petiole: Size of first true leaf: Plant height: One variable that you might not have noted is hairiness of leaves, petioles and stems. Look at some plants more closely using a hand lens, and see if you can observe these hairs, also known as trichomes. Artificial Selection in Brassica, Part I Page 4

5 Does the hairiness trait vary continuously or discreetly? Trichomes have been demonstrated to have specific functions, which differ from species to species. In the space below, hypothesize what these functions might be. What do you speculate are some functions of trichomes? Counting hairs As you may have guessed, the variable trait we will investigate in this study is hairiness. In order to compare plants to one another we need a way to quantify hairiness. For this measurement we could count all the trichomes on all parts of the plant. This task would be quite time-consuming and unnecessary. We know that the hairiness of one part of a plant is strongly correlated with hairiness on other parts. In other words, a plant s hairiness in general can be quantified by assessing hairiness of a specific structure. The structure we will use in this exercise is the petiole where the trichomes are large, conspicuous and easily counted. The petiole is relatively small with an easily defined starting and ending point. To be consistent, 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. See Figure 3 below. Figure 3. Young Brassica rapa showing first true leaf, petiole and trichomes. Note that the two lowermost, squarish, two-lobed and rather thick leaves are actually cotyledons (seedleaves) and not true leaves. The first true leaf is just above the cotyledons. You now need 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 other students in this laboratory section. Artificial Selection in Brassica, Part I Page 5

6 Use a hand lens and desk lamp. The trichomes are most conspicuous if strongly illuminated against a dark background, such as the black surface of the lab table. If present, the trichomes will generally be concentrated on the lower side of the petiole, but some can occur on the top or sides as well. Count a second time for verification. Use lab tape and indelible marker to record on each cell of your planter the number of trichomes for each plant. Enter your raw data in the data box below, and determine the median number of trichomes per petiole for your sample. The median is the middle value in a distribution, above and below which lie an equal number of values. Enter your data into the computer spreadsheet for the class. To avoid confusion, we will collect data for the standard cultivar first. Once that data has been recorded, we will repeat the process for the experimental cultivar. Enter the number of trichomes on the petiole of the first true leaf, per plant: Standard cultivar: Experimental cultivar: Median: Combined data for original populations When all students have finished quantifying the trichome number of individual plants and entering data into the class spreadsheet, your lab instructor will use statistical software to generate a frequency histogram of the class data. Your instructor will make the summary available to you and you should copy the data into the frequency table below. Next, plot a frequency histogram of these data using the axes provided below. Use open columns for standard cultivar, use hatched columns for experimental. Frequency Table for Class Data Number of Trichomes Per Petiole # of Individuals Category for # of Trichomes Standard Experimental Total # of Individs. Median Trichome. # # of Petiole Trichomes Artificial Selection in Brassica, Part I Page 6

7 B. Selecting the Parents of the Next (Second) Generation Only a small fraction (about 10%) of this original population of plants will be selected to be parents of the next generation. These will not be randomly chosen, but instead will be the hairiest plants in the population. As a class, we need to identify the plants that had the highest petiole trichome counts by referring to the label you created for each plant s container Record the trichome values of the selected individuals (parents-to-be) in the appropriate data box below. Data Box for STANDARD Cultivar Selected parents (number of trichomes on petiole of first true leaf) Selected individuals median value Data Box for EXPERIMENTAL Cultivar Selected parents (number of trichomes on petiole of first true leaf) Selected individuals median value Comparison of selected parents-to-be to original population In the data box below, record the medians and their difference for the selected individuals and for the original population. Data Box STANDARD cultivar EXPERIMENTAL cultivar Selected individuals, median Original population, median Difference Artificial Selection in Brassica, Part I Page 7

8 C. Pollination of Selected Parental Plants Brassica rapa plants need assistance in sexual reproduction because in nature they are totally dependent on certain insects for transferring sperm-bearing pollen from the male part of the flowers of one plant to the female part of the flowers of another plant. In these plants, the most conspicuous floral structures are the yellow petals. These petals enclose the male sexual structures (stamens, terminating in the pollen-producing anthers) and female structures (pistil, with the pollen-receiving stigma, style and eggproducing ovary), as shown in Figure 4 below. Figure 4. Cross-section of flower of Brassica rapa. Although each flower has both male and female parts, sperm (pollen) from one plant usually do not fertilize the eggs for the same plant. This self-incompatibility ensures that out-crossing (mating between different individuals) normally occurs. The insect pollinators do not transfer pollen out of courtesy. Rather, the insects are lured to the flowers by the reward of nectar and edible pollen. The insects inadvertently pick up the sticky pollen on various body parts and then carry it with them to the next flower. Honeybees are common pollinators of Brassica in the field, and we will use honeybees (dead ones) to help us pollinate the plants. Artificial Selection in Brassica, Part I Page 8

9 Figure 5. Pollen on a bee. Bee-sticks have been prepared for you from the thoraxes of dead honeybees (collected from beekeepers after the bees died naturally worker bees are short-lived). Glued to the end of a toothpick, these bee thoraxes make quite efficient pollinating devices; pollen grains cling to the many fine hairs on the bee s body and are easily transferred to the stigmas of other flowers. Performing pollination (a.k.a. group sex) Each student should obtain one of the selected parental plants. Your objective is to transfer pollen from each plant to every other plant at your table. This process can be done in the following way. Using a single bee-stick, each student will lightly rub and twirl the bee end for several seconds on the anthers and stigma of each open flower of her/his plant. Each then passes the bee-stick, now loaded with yellow pollen, to the neighboring student to the right, who will repeat the process. After each bee stick has made a complete round of the table (all open flower on all plants have been visited by all bee-sticks), the pollination process is completed. When finished discard the used bee-sticks; do not return the bee-sticks to their original container where other students might confuse them with fresh, unused ones. Fertilization will result in the development of the ovules (each containing an embryo) into mature seeds, which will be contained in the fruit or seedpod (the elongated ovary). The length of time from fertilization to mature seed is about 3-4 weeks, sometimes a little longer. In subsequent weeks we will prune away the apical meristem plus additional flowers and axillary buds that may form in order to concentrate plant resources into the developing fruits and to hasten 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 will be nearly complete. You will harvest and plant these seeds (the second generation) in five weeks. Continuing pollination of new flowers as they develop during the week To insure that enough viable seed will be produced from the selected parental plants, the crosspollination procedure will need to be continued for one week. Your lab instructor will devise a lab pollination schedule for which you will sign up to come in once during the week to pollinate the plants for your lab section. Your lab section s plants will be grown in the Arey greenhouse. Please note: always use a fresh bee stick for performing pollinations to insure that unwanted pollen is not transferred during the pollination. Artificial Selection in Brassica, Part I Page 9

10 IV. Paper Assignment A scientific paper on the artificial selection study will be due in lab during the week of March 4, The paper should be written in standard scientific format and include all of the sections outlined in A Guide to Writing Scientific Papers. For the Materials and Methods you can simply cite the lab handout unless you deviated from the instructions. It is important to keep in mind that this study is being conducted in two parts: the first part of the study compares the hairiness of the experimental cultivar to the standard cultivar (the results of which are available at the end of the first lab); the second part of the study compares how the two cultivars respond to artificial selection, and attempts to determine if an upper limit is being approached in the expression of the hairiness trait (the results of which will only be available later in the semester). Even though the study is being conducted in two parts, your Introduction should address both components of the study. After the second part of the study is completed, you will append your paper with the additional findings. You will need to cite at least three papers from the primary scientific literature 1 on artificial selection. It is not necessary to find only papers on artificial selection in Brassica; appropriate articles covering artificial selection in other organisms are acceptable. Your paper may cite pertinent references from other reliable sources not part of the primary scientific literature, but they cannot be counted toward the three-paper minimum mentioned above. Your paper must include an appropriately labeled graphical summary of the results. In addition you must include a summary of the results of a statistical analysis that compares the experimental cultivar to the standard cultivar. Graphical and statistical summaries must conform to the guidelines outlined in the Style Guide for Papers and Lab Reports. Here are some questions to consider as you prepare your paper: 1) Was there a difference in median phenotype between the standard cultivar and the experimental cultivar? If so, what was the magnitude and direction of the difference? 2) Was there a difference in the frequency distribution of the phenotypes between the two cultivars 3) Were the differences mentioned in questions 1 and 2 statistically significant? 4) How did your results compare to findings reported in the scientific literature? 5) Given that the experimental cultivar originated through artificial selection of the standard cultivar, is there evidence that evolution occurred during the creation of the experimental cultivar? 1 Primary scientific literature includes articles in which scientists first publish their original research findings in authoritative peer-reviewed scientific journals. Artificial Selection in Brassica, Part I Page 10