Activity -- Making Beesticks, Pollinating and Observing Pollen
As their plants come into flower, students need to be prepared to pollinate. The act of pollination is the prelude to the beginning of a new generation that starts with double fertilization. In preparation for pollination of their plants, students will need to understand the developmental biology leading to the production of male and female gametes (sperm and egg) and the concepts associated with the coevolutionary relationships between flowers and their pollinators.
It is unknown whether natural pollen vectors on Earth, such as bees, flies hummingbirds and bats, are capable of flight or transit between and among flowers in conditions of microgravity. For this reason, in the B-STIC experiment, the CUE mission's Payload Specialist will be simulating the flight of a bee using an artificial pollination device to transfer pollen among the AstroPlants flowers.
How is effective pollination carried out?
The transfer of adequate numbers of viable, compatible pollen from the anthers ofone plant to the stigmas of another plant will result in effective pollination.
- With a beestick as the vector, pollen is collected from the anthers of various plants and transferred to the stigmas of other plants.
- Students will record data on their Floral Clock Student Data Sheet (page 51).
- It is important for each class to have at least two extra sets of four film can wick pots (16 plants) in PGCs to serve as unpollinated control plants. The extra PGCs should have been planted along with and maintained in the same manner as the experimental PGCs.
A period of 16 days from the sowing of seed is required for the growth of the AstroPlants and the completion of the activity. Making beesticks will require about 15 minutes and should be done one to two days prior to pollination. Observation of the bee and a lesson on the relationship of bees and the AstroPlants flower in pollination could take one 50 minute class period. The pollination will require one 50 minute class period.
In participating in this activity students will:
- understand flowering as the sexually mature stage of plant development;
- understand where and how ovules and pollen originate (male and female gamete formation);
- explore the parts of the flower and the role that each part plays in reproduction;
- observe the reproductive tissues of plants, including pollen and stigma, under magnification;
- understand the interdependent coevolutionary relationship of bees and brassicas; and
- begin the process of reproduction in their AstroPlants by performing a pollination using a beestick, setting the stage for future developmental events.
- flowering AstroPlants (Day 14 to 16)
- two dissection strips (page 23)
- 2 cm wide clear adhesive tape
- glue (e.g., DucoR Cement)
Procedure: Making Beesticks
1. One to two days prior to pollination, students should make beesticks. While making beesticks the teacher may wish to have students observe the anatomy of a bee, focusing on the legs and proboscis, to reinforce an understanding of the design (role and function) of the bee in relation to the flower.
2. Carefully remove the legs, head and abdomen of the dried bee, leaving the fuzzy thorax. Pollination can be performed
with "whole bee" beesticks as well.
3. Place a drop of fast-drying glue on the tip of a toothpick. Carefully push the toothpick into the top of the thorax of the
bee. Remove the wings. Let the beesticks dry overnight.
Procedure: Setting the Floral Clock
1. At a time between Day 14 and Day 16 when five or more flowers are open on each plant in the PGC, cross-pollinate all open flowers on each plant with a beestick by gently rolling the bee thorax back and forth over the anthers of flowers of several plants until yellow pollen can be observed on the hairs.
Moving to other plants and repeating the rolling motion over the anthers and the stigma of each pistil, students should make
sure to deposit pollen collected on the beestick on to the stigma of each flower. Students from one group may wish to "fly" their bees to flowers of other groups. Buzz!
2. Take the top open flower of the first plant and carefully remove it with a forceps. Place it on the sticky tape of a dissection strip.
3. While observing with a hand lens or microscope, carefully remove the flower parts, noting their relative positions on the receptacle, making sure not to damage the pistil. Refer to the illustration on page 43 for help in identifying the floral parts. You and your students may want to test the nectaries for the presence of glucose (write to Wisconsin Fast Plants for the activity "The Hunt for Glucose - A Flower's Treasure").
4. Slip the ruler on a second dissection strip (without tape on it) under the first strip. Each student should measure the length of the pistil from the receptacle to the top of the stigma and record the pistil length on his/her Floral Clock Student Data Sheet (page 51).
5. Remove the top open flower of the second plant, measure the length of the pistil and record the data on the Floral Clock Student Data Sheet in the column under the number of the second plant.
- With higher powered microscopes students could observe magnified views of pollen trapped on the stigmas of dissected flowers or on a beestick.
- While pollinating, students might also observe the pollen trapped on the tape of a dissection strip.
- Alternatively, look at the pollen on the stigmas or on the beestick with a hand lens.
- A beestick with pollen can be rolled over the sticky tape on the dissection strip. Observation under a magnifier will reveal pollen attached to the bee setae.
6. Note that flowers on the plants are produced and open in a sequence spiraling up the flower stem. Starting with the next flower down from the one that you removed, number the remaining flowers as 4, 3, 2 and 1 as shown in the illustration on page 49, with number 1 being the oldest flower.
- With a sharp pair of fine scissors, carefully snip off all additional remaining flowers below flower number 1 (including side shoots), leaving only the four open flowers that have been numbered.
- Snip off the developing apical flower shoot and buds above the four remaining open flowers. This is known as terminalizing the plant.
7. Returning to the four remaining flowers, note the position of the stigmas relative to the tall anthers. Is the stigma below (-1), equal to (0) or above (+1) the tall anthers? Record this information for each flower of both plants on the Floral Clock Student Data Sheet.
- Is there a relationship between flower age and the position of the stigma relative to the level of the four tall anthers?
8. Be sure to measure the length of the uppermost open flower of each plant in the set that was grown for the unpollinated controls. Data for the unpollinated control sets should be recorded on a separate Floral Clock Student Data Sheet.
9. Before leaving the plants be sure that each of the remaining stigmas has been adequately pollinated by the beestick. Can you see any pollen on the stigmas? Check with a hand lens.
Concluding Activities and Questions
"People from a planet without flowers would think we must be mad with joy ... to have such things." - Iris Murdoch
The completion of pollination sets the floral clock at "0 dap" (days after pollination). Activities 5 and 6 in the section "Double Fertilization and Post-Fertilization Events" involve observing, recording and analyzing pistil development as an indication of successful pollination. In participating in these activities students will complete their Floral Clock Student Data Sheets. After completing Activity 4, have students consider the following:
- What is the relative importance of each flower part in relation to pollination and sexual reproduction? Are some parts more important than others? Why?
- Does the pistil continue to elongate after the flower is functionally open? Even if the flower is not pollinated?
- How many flowers on average open each day once the first flower has opened?
- What is the average amount of time (in hours) between the development of one flower and its nearest neighbor?
For activities on observing and experimenting with pollen germination in vitro under the microscope and on observing compatible and incompatible pollen-stigma interactions and pollen tube growth in the style and ovaries, write to Wisconsin Fast Plants for the activities "Pollen Germination" and "Pollen-Stigma Interactions and Pollen Tube Growth."
With a separate third set of plants, try self-pollinating and compare the amount of seed produced with the seed produced by the cross-pollinated plants.
Double Fertilization and Post - Fertilization Events
Fertilization is the event in sexual reproduction which follows pollination. In higher plants, two sperm are involved in fertilization, reaching the ovule via a pollen tube from the germinating pollen grains. One sperm fertilizes the egg cell within the embryo sac to produce the zygote and begin the new generation. The other combines with the fusion nucleus to produce the endosperm, a special tissue that nourishes the developing embryo. Fertilization also stimulates the growth of the maternal tissue (seed pod or fruit) supporting the developing seed. In AstroPlants the fertilized egg cell develops through various stages over the next 20 days until it becomes a mature quiescent embryo, the seed.
- What is the effect of microgravity on fertilization and embryo development?
- Will the maternal parent be affected by microgravity? Will fruits develop?
What happens between fertilization and seed harvest? After pollination, each compatible pollen grain adhering to the stigma sends through the style a pollen tube which carries two male gametes (sperm) to the ovule, where the egg and other cell nuclei are housed in the embryo sac. One sperm unites with the egg cell (n) to produce a zygote (2n) which
becomes the embryo. The second sperm (n) unites with the diploid fusion nucleus (2n) to form the triploid endosperm (3n), the energy source for the developing embryo. This process is known as double fertilization.
Within two to three days after fertilization, the pistil begins to elongate and swell to accommodate the enlarging ovules. The sepals and petals wither and drop off, having completed their functions.
Within the ovules, the embryos differentiate and enlarge through a series of developmental stages, known collectively as embryogenesis. Enlarging also within the ovule is the endosperm. In the latter stages of embryo development in brassicas, the nutrient reserves in the endosperm are used by the embryo and the space that was filled by the endosperm is occupied by the enlarging embryo.
In cereal crops, such as wheat, rice and corn, endosperm is not used by the enlarging embryo and remains a major portion of the seed as a starchy energy source for the germinating seedling.
Through the development of the seed, the plant has solved the problem of packaging its new generation to survive until favorable conditions for growth return. As the seed matures, the walls of each ovule develop into a protective seed coat and the entire ovary becomes a fruit (seed pod). In AstroPlants, embryos mature into seeds in 20 days after successful pollination.