Activity 6 -- Going for the Globular
Following double fertilization, four complex processes are triggered. The ovary wall and related maternal structures rapidly grow to become the fruit tissue (the pod) surrounding the developing seeds. Each ovule within the fruit enlarges to accommodate the developing endosperm and embryo. The various outer cell layers of the ovule (integuments) eventually become the seed coat. Within the embryo sac, the triploid endosperm nuclei divides very rapidly to form the nutrient-rich, starchy liquid endosperm. The liquid endosperm provides nutrients to the developing embryo.
Since fertilization, the zygote has undergone several mitotic divisions. The first few divisions produced a strand of eight cells known as the suspensor, which is attached to the embryo. The suspensor orients the developing embryo within the ovule and is thought to serve as an "umbilical cord," as it passes nutrients from the endosperm to the embryo cells. The basal cell of the suspensor anchors the developing embryo and orients the embryonic root tip near the micropyle, a hole in the integuments where the pollen tube entered. At the tip of the suspensor, repeated cell divisions give rise to the very young globular embryo (page 64).
Immersed in the nutrient rich endosperm, the embryo develops rapidly. By Day 7, the embryo becomes flattened and
bilaterally symmetric with two lobes which will become the cotyledons. This is the heart stage.
As its development continues, the embryo elongates into the torpedo stage. At this stage the embryo produces chlorophyll and becomes green. Elongation of the embryonic hypocotyl separates the root apical meristem from the shoot apical meristem, which is hidden between the embryonic cotyledons. As the embryo enlarges it consumes space formerly occupied by the endosperm. To package the enlarging embryo, the cotyledons fold around the hypocotyl, now curved within the ovule; this is the walking stick stage. By Day 20, the walls of the ovule (the integuments) harden and become the seed coat (testa) and the embryo within desiccates to become a seed (Stages I and J, page 12). As maturation proceeds within the enlarged folded embryo, the starch reserves within the embryonic cotyledons are converted to lipids as the final form of energy storage.
There are many questions that remain unanswered in developmental embryology - perhaps you and your students can answer some of them.
- What is the normal sequence of embryo development within the ovules of AstroPlants?
- Can your students identify the various stages in the developmental continuum from embryo to mature seed?
- Can your students record, draw to scale and estimate the relative sizes of the embryo at various stages of development?
Normal embryogenesis proceeds rapidly within the developing seed from a singlecelled zygote through recognizable stages of increasing size and complexity ending in a dried seed.
- At specified intervals following pollination, remove embryos from ovules, draw to scale various stages and measure embryo sizes. Construct a developmental chart of embryogenesis.
- Students will record observations and measurements on the Ovule and Embryo Student Data Sheet (page 65).
A period of 36 days from the sowing of seed is required for the growth of the AstroPlants and the completion of Activities 5 and 6. The time required for the embryogenesis activity will vary depending on the amount of class time spent in dissecting embryos. A minimum of two 50 minute class periods is recommended, one for students to practice and develop their dissection and drawing skills and one or more to examine and record the stages of embryogenesis. The number of periods spent on embryo dissection depends on the timing of the dissections students wish to make.
In participating in this activity students will achieve the learning objectives presented for Activity 5 (page 55). In addition, students will:
- learn to dissect embryos from ovules in developing pods, improving their hand-eye coordination;
- learn that embryogenesis is a continuum of development from a very small spherical group of cells to a complex multidimensional, multicellular organism;
- learn how an embryo can enlarge within the limited confines of the ovule and become "packaged" in preparation for desiccation and quiescence as a seed (Stage I of the life cycle, page 12);
- learn to make accurate descriptive observations of specimens under the microscope, draw carefully "to scale," and record and analyze data obtained from the drawings;
- learn to construct a model embryonic development that can be compared to the development that occurs in plants grown in microgravity.
- fresh or fixed pods of AstroPlants at various stages of development
- for a range of embryo stages suitable for initial dissection, ovules sampled between 6 and 12 days after pollination are best, these should provide stages between heart and walking stick
- after developing dissection skills, sample ovules from plants 3 to 6 days after pollination
- see page 98 for instruction on fixing pods
- dissecting microscope with 20 to 40X magnification
- fine-tipped forceps and fine dissecting needles (e.g., tuberculin syringes with #23 or #25 needles)
- dissection strips (page 23)
- 2 cm wide clear adhesive tape
- fine scissors or cutting blade
1. At the desired day after pollination (dap), students should use fine scissors to remove one pod from one of their two plants. Place the pod on the sticky tape of the dissection strip aligning it longitudinally on the scale.
2. Measure and make a drawing of the pod "to scale" in a lab notebook.
3. Using the dissection needles or a sharp blade, cut along one seam of the pod where the two carpels are fused. Pry open the pod to reveal the ovules aligned within the carpel; each ovule is attached to the vascular strands by its funiculus. You will also see a thin paper-like septum separating the ovules in each carpel.
4. Observe the opened pod with a hand lens or under a dissecting microscope. In a lab notebook, make a drawing "to scale" of what you observe.
5. Remove an ovule from the opened pod with fine forceps or dissecting needles, keeping a portion of the funiculus attached to the ovule. Then transfer the ovule onto the sticky tape on a dissection strip. Measure and record the length of the ovule next to the first circle on the Ovule and Embryo Student Data Sheet.
6. With a pipette, transfer a small drop of water to cover the ovule on the sticky tape of the dissection strip. Alternatively a drop of iodine potassium iodide (IKI) staining solution (page 98) can be used in the dissection, in which case any cells or tissues containing starch will turn blue or purple.
7. Place the opened pod with remaining ovules on moist paper toweling in a covered petri dish. This will keep it fresh for further sampling.
8. Place the dissection strip under a dissecting microscope and observe the ovule, noting the funiculus attachment and the micropyle.
- If the ovule is illuminated from below, students may be able to see the indistinct embryo within the ovule. This will depend on the stage of embryo development.
9. With needles make an incision across the ovule at the end opposite the funiculus. As this cut is made, the embryo may float out into the water along with the cloudy starchy liquid endosperm.
- Make a second incision perpendicular to the first and using dissection needles gently pull open the integuments. Embryos at 9 dap will generally be visible once the seed coat is open.
- If the embryo is not visible, slowly and carefully remove small pieces of the integuments, working toward the micropylar end.
- Young embryos in the heart and globular stages are found surrounded by a funnel of aleurone and nucellar tissue from the torn embryo sac. The young embryo is immersed in cellular endosperm and is anchored by the suspensor in the integuments at the base of the funnel.
- Continue to carefully tease out the embryo and, if possible, its attached suspensor.
10. Once the embryo has been removed, students may wish to increase the magnification under the microscope for viewing and drawing.
- Under an appropriate magnification, slide a second dissection strip under the one holding the embryo in the water drop.
- Align the magnified image of the scale of the second dissection strip across the horizontal diameter of the field of view of the microscope.
11. Draw in the magnified scale on the horizontal line of the first circle on the Ovule and Embryo Student Data Sheet. Be as accurate as possible in the spacing between the scale marks.
- Draw a scale bar at the top or bottom of the circle representing the distance of 1 mm or some fraction (0.5, 0.25, 0.1) of the magnified millimeter scaling. Indicate the distance represented by the bar on the drawing.
12. Observe the embryo and accurately draw it to scale within the same circle as the scale bar.
13. Identify and record the stage of embryo development (globular, heart, torpedo, etc.).
- Either from the drawing or directly, measure and record the length of the embryo, excluding the suspensor. Record the magnification of your microscope.
14. Calculate the magnification of the drawing using the following method:
- Measure and record the actual distance in millimeters between the two ends of the scale bar in the circle of the drawing of the embryo (e.g., 21 mm).
- Divide this measurement by the distance in millimeters represented by the scale bar in the circle to give the magnification of the scale bar and drawing (e.g., 21 mm/0.5 mm = 42X).
15. Dissect ovules from pods of different dap making drawings and length measurements of the difference stages of development, using the remaining circles on the Ovule and Embryo Student Data Sheet. If students have "spare" ovules at one developmental stage, they can share them with other students or exchange them for ovules at different stages of development.
Suggested dissection times include 6, 9, 12 and 17 to 20 dap. If dissections are made in a potassium iodide staining solution, note the presence or absence of starch in the ovule at different stages.
Concluding Activities and Questions
In completing Activity 6, students will have taken their AstroPlants through a complete life cycle, from sowing the seed on Day 0 to harvesting the seed for the next generation. Analyze the data taken on the Ovule and Embryo Student Data Sheets. Have students consider the following:
- Students as a group or as a class may construct a developmental graph or chart depicting the dap on the x-axis and the length of the embryo, size and stages on the y-axis, similar to what has been done for the growth of AstroPlants on page 11.
- Students may use the embryo lengths of various developmental stages as a quantitative indicator of development (page 64).
- How much does the embryo enlarge from the time it is a globular until it is mature?
- What are the relative sizes of the various stages in embryogenesis? With what will you compare those sizes?
- In what stages of embryogenesis is the embryo enlarging most rapidly? What is your evidence?
- What becomes of the endosperm? Is there any stage in embryogenesis at which starch is not present in the ovule?
The embryo dissection activity can also be performed using pods that have been harvested and "fixed in an acetic alcohol fixative on specified days after pollination. Fixed pods can be stored for future use. See page 98 for an acetic alcohol fixative recipe and safety information.
Practice makes perfect . . . embryos!
Embryo dissection can be challenging for students, especially when the embryos are at the early stages of development. Prior to beginning the dissection of your experimental embryos, you and your students should practice dissecting embryos at different stages.
Have students plant several film can wick pots of AstroPlants, timed to be different ages the day or two before your class begins its experimental dissections. Pollinate these plants well: the more pods, the more practice!
Germination is the awakening of a seed (embryo) from a resting state. It involves the harnessing of energy stored within the seed and is activated by components in the environment.
In the metaphor of the mission, the seed is pointed in the correct position and ready for launch. Germination, like launch, involves the bringing together of essential chemicals, hydrogen and oxygen, to generate energy that will be directed toward expanding and propelling the two growing points of the germinating seed outward (upward and downward if guided properly).
- What are the main components of the environment necessary for germination?
- How does the germinating seed go about harnessing its stored energy and using the environment?
- How does a germinating seed 'know' which way to grow?
- What developmental events enable the emerging plant to shift its dependency from stored energy to the energy from light?
Germination is the beginning of growth of a plant from a previously dormant seed which contains the embryo. Germination begins when the seed takes up water (imbibition) and the seed coat cracks.
An embryonic root (radicle) emerges from the seed and develops root hairs that bring in water and nutrients. In AstroPlants, an embryonic stem (hypocotyl) elongates, pushing the seed leaves (cotyledons) upwards through the soil. As they emerge from the soil the cotyledons expand. The cotyledons serve as an energy source until true leaves form. These events happen on Days 1, 2 and 3 of the AstroPlants life cycle (see page 11). For germination to take place, water and oxygen are needed and the temperature must be suitable.
With germination water is the "on" switch. Water taken up by the dry seed hydrates the cytoplasm, activating enzymes, solubilizing substrates, loosening the structural fabric of walls and providing pressure that enlarges the cells, resulting in expansion and growth. Oxygen, another essential ingredient, combines with hydrogen from the stored oil reserves in the
embryonic seed leaves and fuels the metabolic combustion that powers life processes. Rapid development of the fine
root hair cells vastly increases the surface area of the root, facilitating the uptake of water that drives the elongation of the hypocotyl, which pushes the seed leaves and shoot meristem upward through the soil.
On Earth, orientation during germination is provided by gravity. Roots respond positively to gravity by growing downward (positive gravitropism), while shoots respond negatively by growing upward (negative gravitropism). Prior to emerging from the soil into the light, the germinating brassica seedling is dependent on the energy reserves stored in the seed leaves. Seeds of other plant species may also store carbohydrates, starch (cereals) or protein (legumes, beans).
Upon emergence from the soil and triggered by the light, the cotyledons expand, casting off the protective seed coat, turn green and become photosynthetically active. At this point the "plant" becomes independent of the stored reserves and dependent on the energy of light. Launch has been successful! The solar panels (cotyledons) have been deployed. The future success of the mission depends on keeping the panels oriented and operational to gather light energy which is converted through photosynthesis to chemical energy for growth, flowering and reproduction.
Activity 7 (Getting Acquainted with a Seed) will provide students with insight into the various stages of germination and give them an introduction to the phenomena associated with orientation. Remember, one of the big questions about the microgravity environment is: how will plants know which way to grow?