Plant reproduction & the pollen tube journey--how the females lure the males.
The male gametophyte equals the mature pollen grain that arises in the anther and consists of only three cells (i.e., two sperm cells and a vegetative cell). The female gametophyte corresponds to the embryo sac and covers an egg cell, a central cell, two synergid cells, and several antipodal cells. Embryo sacs are sheltered inside the ovules, which are, in turn, enclosed by the ovary of the pistil (Figure 1). Because embryo sacs are embedded quite deep inside the female reproductive structures, direct fertilization of the egg cell by the immotile sperm cells is prevented. Therefore, flowering plants have evolved a unique and complex fertilization procedure that culminates in double fertilization of egg and central cell and is followed by seed development.
Engagement in flowering plants starts when the pollen is delivered to the stigma of the style. This is achieved by a wide range of mechanisms, such as wind, insects, or self-pollination. After a pollen grain lands on a receptive stigma of a pistil, a pollen tube germinates from the hydrated pollen grain and delivers the immotile sperm cells over a long distance toward the ovule. A pollen tube is a tip-growing cell that grows surprisingly fast. In maize, pollen tubes can grow through a style of as much as 50 cm length at rates close to 1 cm/hour (Barnabas & Fridvalszky, 1984). Similar to axons in the development of the nervous system, the directional growth of pollen tubes is controlled by multifarious interactions with the sporophytic pistil and the female gametophyte (Okuda & Higashiyama, 2010). On its way, the pollen tube penetrates the stigmatic surface, is then guided through the stigma and style and placenta, grows onto the funiculus, and finally enters the micropyle of the ovule for double fertilization (Figures 1 and 3A). This process is known as pollen tube guidance and is based on astonishing molecular communication systems between the pollen tube and the female reproductive structures (reviewed in Shimizu & Okada, 2000; Chapman & Goring, 2010; Chae & Lord, 2011).
* Learning Objectives
This activity aims to prompt students' curiosity toward plant reproduction and provide greater insight into the "real in vivo situation" of this elementary and fascinating process. Though a number of existing teaching activities deal with in vitro pollen tube growth on medium, didactically prepared visualization of pollen tube growth in vivo is more ambitious and usually needs fixed material and advanced equipment (e.g., fluorescence microscopy).
The students set up a semi-in vivo experiment with explants of Fast Plant flowers. The students themselves prepare the growth medium and the plates, dissect and manipulate the flower organs, and see how the pollen tubes penetrate the female pistil. The aim of doing the experiment and evaluating the results is to
* increase students' knowledge about flower anatomy and sexual reproduction of higher plants,
* clarify the beneficial effect of maternal sporophytic tissue and ovules to trigger pollen tube growth,
* demonstrate pollen tube guidance as an example of molecular cell-to-cell communication, and
* improve students' ability to interpret and discuss the outcomes of a biological experiment.
The extentions of the activity can be used to teach soft-skill competences and the understanding of science process skills such as inquiry and experimental design. I have successfully implemented it in undergraduate and graduate courses that cover the molecular biology of plant development.
The activity was inspired by experiments that were published for the model organism Arabidopsis thaliana in open-access journals (Palanivelu & Preuss, 2006; Qin et al., 2009). Because tremendous skill and dexterity are needed to work with the very tiny Arabidopsis flowers, it is impossible to conduct such an experiment with students. Therefore, I extrapolated the experimental setup to Brassica rapa (Wisconsin Fast Plants). Fast Plants are Brassicaceae like Arabidopsis but are substantially larger, display a very similar anatomy, possess the experimental advantage of self-incompatibility, grow very fast, and are especially suited for teaching activities at any educational level. To engage the students, I emphasize the state-of-the-art research that is based on the original experiment in the Arabidopsis model (e.g., applications using transgenic reporter lines that express the green fluorescence protein [GFP] and microarray studies). In advanced courses, I take advantage of supplemental movies about pollen tube guidance that were published as open-source material by Palanivelu & Preuss (2006).
** Rapid-cycling Brassica rapa (Wisconsin Fast Plants; Carolina Biological Supply Company)
** Distilled water
** Agarose (molecular biology grade)
** Table sugar
** Boric acid, calcium nitrate, potassium nitrate, magnesium sulfate
** Petri dishes 90 mm x 14 mm
** Double-face tape
** Precision tweezers (straight; fine tip)
** Object slides
** Analytical balance
** Microwave oven/heating magnetic stirrer
** Stereo magnifying glasses (5x to 10x magnification)
** Optional: student microscopes (you need only 25-50x magnification)
* Safety Concerns
This activity requires working with scalpels and precision tweezers; hence, students must act responsibly during the preparation of flower organs. Surgical blades have to be disposed of properly after use. Eating and drinking must not be authorized in the lab. [H.sub.3]B[O.sub.4] (Hazcard 14); KN[O.sub.3] (Hazcard 82); MgS[O.sub.4] x 7 [H.sub.2]O (Hazcard 59b); Ca[(N[O.sub.3]).sub.2] x 4 [H.sub.2]O (Hazcard 19A).
* Growing Plants
The Fast Plants are cultivated in plastic propagation trays with bottom drain holes, placed in larger trays without holes to achieve continous watering with tap water from below. The plants are grown at 25[degrees]C under continous light until the flowering period is reached (~3 weeks). Do not pollinate accidentally. About 5 to 10 plants should be made available per student. For use in a teaching environment with limited equipment, alternative growth conditions can be found at the official website of Wisconsin Fast Plants (http:// www.fastplants.org/grow.php).
* Growth Medium Preparation
To prepare 2x germination medium (2x GM), dissolve 135 g sugar in a final volume of 500 mL distilled water. After complete dilution of the sugar, add 0.2 mg [H.sub.3]B[O.sub.4], 0.1 g KN[O.sub.3], 0.25 g MgS[O.sub.4] x 7 [H.sub.2]O, and 0.616 g Ca[(N[O.sub.3]).sub.2] x 4 [H.sub.2]O. To prepare 2x agarose (2x AG), dissolve 5 g agarose in 500 mL distilled water and boil (short pulses in a microwave oven or heating stirrer) until agarose is dissolved completely. Let the 2x AG cool to ~60[degrees]C, quickly add the 2x GM solution, mix together both solutions thoroughly by careful shaking, and pour the medium quickly into Petri dishes (~25 mL per plate, sufficient for 20 Petri dishes). Allow the medium to solidify with slightly open lids to prevent excessive water condensation. The plates can be used directly or stored in the refrigerator. If the plates are used within 2-3 days, none of the solutions need to be sterilized. If longer storage is intended, 2x GM should be sterilized by filtration, and 2x AG has to be autoclaved at 121[degrees]C for 15 minutes prior to addition of 2x GM.
Figure 2. Mini-protocol for semi-in vivo pollen tube growth. Preparation of pistil explants and pollination 1. Take a fresh young open flower (*) and carefully remove all parts but the pistil. 2. Take a stamen from a different plant and pollinate the stigma of the pistil. Use 3 stamens from different flowers to increase pollination efficiency. 3. Cut at the border between style and carpel. The length of the style may vary between 1 and 3 mm. 4. Transfer the upper style explant onto a plate. Be careful not to scratch the medium. Push the explant very gently onto the medium. 5. Six to 10 pistils should be arranged equally per plate. 6. Stick a square centimeter of double-face tape onto an object slide. Stick the pistil of a young fresh flower onto the double-face tape such that one of the carpel junctions is pointing upwards. 7. Cut into the junction, open and remove the carpels so that the ovules are exposed. Avoid cutting into the ovules. 8. Transfer the ovule onto the plate and... (see 9.) 9. ... arrange 8 to 10 ovules in a semicircle below the raw edges of the style explants on the plate. Avoid scratching into the medium. Seal plates with parafilm and let stand for at least 15 h. 10. Analyze and evaluate your experimental results.
* Experimental Setup
Tweezers, scalpels, and stereo magnifying glasses should be used for all steps. To be successful, choose only recently opened flowers that are capable to provide and receive pollen (Figure 2, step 1). The developmental stage of the flowers is the most crucial component of the entire experiment. Flowers that are too young (unfolded petals) or too old (petals start to wilt) did not work in the experiment because the developmental stage of the flower and successful fertilization are tightly linked. Teachers who are inexperienced with these plants are encouraged to consult the teaching activities about Fast Plant flower development (e.g., Wisconsin Fast Plant ID: WFP061098: Flowering and Pollination) at the Fast Plants homepage (http://www.fastplants. org/pdf/activities).
Preparation of Pistil Explants & Pollination
Take a flower and carefully remove all parts but the pistil. The pistil is composed of three parts, the stigma (which receives the pollen), the style (the neck below the stigma) and the carpel (or ovary). Take a similar flower from a different plant and tear a fresh pollen-shedding stamen at its filament. Pollinate the excised pistil by pushing the anther slightly onto the stigma (Figure 2, step 2). This stamen has to be from a different plant because Fast Plants are self-incompatible; thus, all flowers of a certain plant are not able to fertilize themselves or each other. Cut at the border between style and carpel and transfer the style explant onto a GM plate (Figure 2, steps 3-5). Be careful not to scratch the medium.
Isolation of Ovules
Stick a square centimeter of double-face tape onto an object slide. Take a flower and prepare a complete pistil (Figure 2, step 6). Brassica flowers have two fused carpels. Stick the pistil onto the double-face tape such that one of the carpel junctions is pointing upward. Cut into the junction and open the carpels so that the ovules are exposed. Avoid cutting into the ovules. Arrange 8 to 10 ovules in a semicircle below the raw edge of a style explant on the plate (Figure 2, steps 7-8). The distance between the raw edge and the ovules should be 0.5 to 1 mm.
Approximately 8 to 10 pistil/ovule units should be arranged per plate. Depending on their skills, students can prepare their own plates or work in pairs. Of course, the quality of the experiment depends on the overall quantity of pistil/ovule units that are laid out in the class. Pollinated pistils without ovules can be used for comparison. Let the plates stand for at least 15 hours at 20-24[degrees]C (dark or light). Incubation time can be extended to 48 hours, or even longer, if microbial contamination of the plates does not get out of control. The tubes may be dry at this point, but they will still be visible. Observe, sketch, and label what you see (Figure 2, steps 9-10).
Results & Discussion
The results obtained demonstrate (1) a part of the route that pollen tubes need to follow to reach the ovules, (2) the strong promotive effect of pistil and ovule tissue on pollen germination and tube growth, and (3) the dependance of proper fertilization on the appropriate developmental stage of Brassicaceae flowers.
Because the developmental stage of pistils and pollen is critical, pollen tube emergence is usually seen in about 60-70% of the pistil/ ovule units that were placed on the plates. Pollen tubes penetrate the stigma papillae (Figure 3B), emerge from the raw edge of the style, and grow toward the ovules (Figure 3C-F). The distance that is covered inside the pistil corresponds to as much as several millimeters. Some tubes reach ovules straight through the air (Figure 3F, white arrows). Because the growth medium was optimized for the pistil experiment, only a few pollen tubes emerge from pollen grains without contacting a pistil (Figure 3D, asterisk).
This is due to the fact that pistil-specific compounds (e.g., specialized proteins) and cell-to-cell interactions are prerequisites for fast tube growth. Sometimes, pollen grains that contact the pistil are triggered to grow rapidly, not into the style but on the medium (Figure 3D, E, black arrows). This shows that contact of the pistil surface is sufficient to promote tube growth in this experimental design. Pollen tubes are lured toward ovules at high frequencies by ovule-specific attractants.
Explore in vitro germination of pollen grains. The described activity can be complemented with studies on in vitro pollen germination. Anthers of Fast Plants were slightly pushed on optimized germination medium (36% sucrose, 0.02% [H.sub.3]B[O.sub.4], 2 mM Ca[Cl.sub.2], 2 mM MgS[O.sub.4] x 7 [H.sub.2]O; 2 mM Ca(N[O.sub.32] x 4 [H.sub.2]O); 0.5% Agarose; according to Ye et al., 2009). Pollen tubes start to evolve after ~30 minutes. Compare your observations at 1 hour and at 24 hours. The experienced observers often see the two sperm cells in the tubes. In my advanced courses, I use DNA staining with DAPI and fluorescence microscopy to visualize the two sperm cells and the vegetative nucleus inside the pollen tubes.
Explore pollen tube growth in fixed styles. Pollinated styles from FastPlants or pistils from Arabidopsis can be fixed, stained with aniline blue using standard methods, and studied by fluorescence microscopy. Alternatively, aniline blue stain can be visualized by light microscopy according to the method described by Motten (1992) if fluorescence microscopy is not available.
Explore the developmental flower clock. To get familiar with flower development, study the developmental gradient of the Fast Plant inflorescence according to activity WFP061098 on the Fast Plants homepage (http://www.fastplants.org/activities.php).
Test different developmental stages. To demonstrate stage dependence of pistil susceptibility, use flowers of different ages for the experiment. Flower stage can be determined (e.g., by measuring pistil length and by digital photography of the used flowers).
Test pollinated pistils. In this activity, pistils are taken from flowers that were hand pollinated one to several days before the experiment and have started to produce seeds. Such pistils do not trigger pollen germination comparably in the assay because pollen susceptibility is turned off after successful fertilization.
This visually appealing hands-on activity fosters students in their science observation skills and their manual dexterity while they acquire the basic principles of plant sexual reproduction. The experiment is close to research, and students gain valuable insight into the setup of experimental designs to address a particular biological question, and into the efforts that usually have to be made.
I thank all students who carried out this experiment in my courses and thereby helped develop and improve this protocol.
Barnabas, B. & Fridvalszky, L. (1984). Adhesion and germination of differently treated maize pollen grains on the stigma. Acta Botanica Hungary, 30, 329-332.
Chae, K. & Lord, E.M. (2011). Pollen tube growth and guidance: roles of small, secreted proteins. Annals of Botany, 108, 627-636.
Chapman, L.A. & Goring, D.R. (2010). Pollen-pistil interactions regulating successful fertilization in the Brassicaceae. Journal of Experimental Botany, 61, 1987-1999.
Motten, A.F. (1992). A simplified experimental system for observing pollen tube growth in styles. American Biology Teacher, 54, 173-176.
Okuda, S. & Higashiyama, T. (2010). Pollen tube guidance by attractant molecules: LUREs. Cell Structure and Function, 35, 45-52.
Palanivelu, R. & Preuss, D. (2006). Distinct short-range ovule signals attract or repel Arabidopsis thaliana pollen tubes in vitro. BMC Plant Biology, 6, 7.
Qin, Y., Leydon, A.R., Manziello, A., Pandey, R., Mount, D., Denic, S. & others. (2009). Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil. PloS Genetics, 5(8), e1000621.
Shimizu, K.K. & Okada, K. (2000). Attractive and repulsive interactions between female and male gametophytes in Arabidopsis pollen tube guidance. Development, 127, 4511-4518.
Ye, J., Zheng, Y., Yan, A., Chen, N., Wang, Z., Huang, S. & Yang, Z. (2009). Arabidopsis Formin3 directs the formation of actin cables and polarized growth in pollen tubes. Plant Cell, 21, 3868-3884.
RENE LORBIECKE is Privatdozent (about the same as an associate professorship at a U.S. university) in the Plant Molecular Biology section at the University of Hamburg, Ohnhorststrasse 18, 22609, Hamburg, Germany. E-mail: email@example.com.
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|Title Annotation:||HOW TO DO IT|
|Publication:||The American Biology Teacher|
|Date:||Oct 1, 2012|
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