PLANT REPRODUCTIVE SYSTEMS

Contributed by: Brenda L. Young, Department of Natural Sciences, Daemen College, Amherst, NY 14226

SPECIES THAT COULD BE USED IN THIS LAB:

Impatiens capensis. Large flowers, protandrous, should be fairly easy to work with. Could be crossed with I. pallida. Lab would have to be done very early in the semester.

Campanula americana. Abundant, large flowers. Proximity of anthers to stigma might complicate manipulations. No close relatives available, but could be crossed with Lobelia.

Gentiana crinita. Large flowers, fairly open. Could be crossed with closed gentian species. But nearest population is 45 min drive.

In zoology, the term "reproductive system" refers only to the set of organs used for sexual reproduction. This makes sense because animal reproductive biology is generally pretty simple: female individuals are fertilized by male individuals, producing offspring. Usually, any 2 members of a species that are of opposite sex can breed together. The anatomical distinctions between male and female are thus the major point of interest. In contrast, plant reproduction is complicated by the potential for reproductive modes beyond mating of distinct male and female individuals. For example, most flowering plants have flowers that are perfect or bisexual (male and female organs combined). This allows the possibility, at least, of self-fertilization. In many cases, however, a plant cannot be fertilized by its own pollen. Nor can any two plants within a species necessarily be crossed together successfully, even though each has perfect flowers. The usual cause of this is genetic incompatibility between the individuals. Such incompatibility cannot be determined from simply looking at the plants involved, but must be tested experimentally. Finally, many plants can reproduce asexually. In some cases, the nature of the asexual reproduction is clear -- for instance, runners or stolons grow into new plants. But in some species, asexual reproduction mimics sexual reproduction with fruits and seeds produced without fertilization.

Patterns of intraspecific variation are linked to reproductive systems. Sexual reproduction tends to mix genes throughout a population or even an entire species. Such mixing might lead to relative uniformity -- the members of the population or species are, genetically speaking, all pretty much alike. Where sexual reproduction is restricted or absent, local populations can begin to diverge from one another. Unusual individuals can propagate themselves, leading to considerable (and sometimes confusing) phenotypic variety. The way in which variation is produced helps to determine the evolutionary possibilities open to a population or species. Also, it strongly influences the ways in which we identify and classify taxa. In order to understand variation and evolution in a given plant taxon, therefore, we have to understand the taxon's reproductive system.

Reproductive systems are also important to understanding how plant species maintain themselves as (more or less) distinct entities. Typically, breeding between species goes on at a much lower rate than within species because of barriers to interspecific reproduction. Barriers to crossing may occur at several points in the life cycle. Hybridization may be blocked by prezygotic (such as different flowering times, different pollinators, etc.) or postzygotic barriers (such as failure of fertilized ovules to develop, viable but sterile offspring, etc.). See Stace, pp. 138-40 for a more detailed review. In this section of the lab, we will look for one particular form of incompatibility between related species, namely the failure of pollen tubes to grow through the style of another species.

Flower structure

Look for adaptations for animal pollination: A conspicuous, brightly colored flower probably has evolved to attract pollinators. Are all the flowers perfect? How many anthers are there? How are they located with relation to the stigma? Does the flower provide a source of nectar or another conspicuous food source? (Look at the base of the petals or within the receptacle for the nectary. What flower parts might provide a food source?) Summarize your observations with a sketch of the flower. Collect a flower, return it to the lab and count the number of ovules.

Flower visitors

Observe a known number of flowers for 15 min or more and determine whether potential pollinators visit the flowers. Use the field guides provided to identify each visitor as closely as possible. (You may need to collect some visitors.) Record the number of visits by each type of visitor and express in units of (visits/flower/time unit).

Asexual reproduction

Check the plant for obvious signs of asexual reproduction. Dig it up and look for runners and stolons. (You may find the bud of next year's shoot at or below ground level, near the base of this year's shoot. Runners and stolons, on the other hand, will extend a considerable distance from the current stem base.)

Experimental pollinations

Pick another two plants. Mark each with your name. They will be used to examine reproduction via flowers. In all treatments, be careful not to damage the gynoecium. Mark and treat 6 unopened flower buds on each plant in the following ways:

      A. Control. Marked, no treatment.

      B. Bagged. Enclose a bud in a nylon net bag to exclude pollinators.

      C. Selfed. Open a bud and use a brush, toothpick, or forceps to transfer pollen from the anthers to the stigma. Enclose the flower in netting after treatment. (Why is the bag needed?)

      D. Bagged, no stamens. Open a bud and remove the stamens. Enclose in netting.

      E. Outcrossed, near neighbor. Transfer pollen from an adjacent plant. (If you found evidence of asexual reproduction, we might need to discuss what "adjacent plant" really means.) Enclose in netting.

      F. Outcrossed, distant neighbor. Transfer pollen from a plant in as distant a part of the population as possible. Enclose in netting.

      G. (Optional) Foreign pollen. Transfer pollen from a related species, if available. 4. We will return next week or the week after, collect the developing fruits, and determine numbers of seeds produced in each fruit.

Pollen tube growth (optional)

Some crosses will not set seed because genetically inappropriate pollen is not allowed to penetrate the style. To test for this, we will set up three treatments and examine the group of pollen tubes into the stigma. The treatments to be used are C, E, F and G (if done) above.

      1. Approximately 24 hrs after pollination, collect the flowers and remove the styles by slicing them off the top of the ovary.

      2. Place the styles (separated by pollination treatment) in a small vial and fix them in ethanol-lactic acid for at least 15 min. (Longer is fine.)

      3. Rinse the styles with DW and place them on a slide. Add a drop of stain and allow the preparation to sit for 5-20 minutes. (You may have to avoid more stain to avoid drying.)

      4. Add a drop of 40 % acetic acid to destain.

      5. Rinse with DW.

      6. Mount in a drop of lactic acid and cover. Apply slight pressure to the coverslip to crush the styles.

      7. Examine under a compound or dissecting scope. Pollen tubes contain large amounts of the carbohydrate callose, which is selectively stained by aniline blue. They will show up as dark streaks against a light background.

      8. Count the number of pollen grains on the stigma. Count the number of pollen tubes growing into at least halfway down the style section.

Movement of fluorescent dyes (optional)

The sections of the lab above will tell us something about the reproductive system of our plant. Can it self, or must it receive pollen from another individual? Is reproductive success improved by receipt of pollen from a distant plant? But how far does pollen actually go? Does a plant usually receive its own pollen, or that of neighbors, distant or near? This is a very difficult question to answer satisfyingly in the field. It is generally not possible to look at the pollen grains on a stigma or on the body of a pollinator and tell what plant they came from. (Note that molecular biology techniques can provide useful information, but are beyond what we can do in this course.)

One method for following pollen flow is to use a tracer that will, we hope, mimic the movement of pollen. We will add a fluorescent to marked flowers and come back later to find out where it has gone. The advantage of the fluorescent material is that it will glow when exposed to UV ("black") light. If this section is done, we will return to the population after dark to determine which flowers have received dye from the dosed, marked flower. Since we have dyes in various colors, several flowers in different parts of the population can be marked. Measure the distance from the starting point to each flower receiving the tracer.

ASSIGNMENT

Experimental pollination

1. What features of the flower might promote self-fertilization? Outcrossing? Include your drawing and refer to your field notes to back up your statements. 2. Explain the structure of this experiment -- why was each of the treatments done? In particular, for each treatment explain what you would conclude if you had found no seeds vs. large numbers of seeds produced.

3. For each treatment, calculate mean and standard deviation of the numbers of seeds per fruit. Summarize in a table. Instructions on statistical testing of these numbers will be provided.

4. From these data, what do you conclude about the reproductive system of this species? Does it require outside pollen for successful reproduction? Is it capable of self-fertilization? Agamospermy? How does the distance between parent plants affect seed set? Be sure to support your conclusions with data.

5. Does this species appear to be capable of vegetative reproduction? Explain.

Pollen tube growth

1. For each treatment calculate mean and standard deviations of the percent of pollen grains growing into the style. Summarize in a table. Instructions on statistical testing will be given.

2. Does failure of pollen to germinate or grow through the style constitute a significant barrier to interspecific reproduction in these species, or is the barrier or another kind? Explain, and support your conclusions with data.

Tracer movement

1. On the average, how far does pollen appear to travel in this population? What was the greatest distance observed?

2. In this population, could seed set be limited by the pattern of pollen flow? Explain.

3. What are some of the strengths and weaknesses of the tracer method?