Terrific trichomes (and other specialised cells) in African violets: how to get a lot from one plant in the classroom

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1 Terrific trichomes (and other specialised cells) in African violets: how to get a lot from one plant in the classroom Vicki M. Cottrell ABSTRACT African violet (genus Saintpaulia) was identified as a particularly suitable genus for the study of specialised plant cells in the classroom using microscopes. The techniques described here involve simple preparation without staining. The cells and structures that can be investigated include: trichomes (hairs); stomata; guard cells and their subsidiary cells; xylem with lignified spirals; petal cells; and pollen grains. The techniques were written with the curriculum for those aged in mind, although there is no reason why they could not be used with younger students, or extended for use with post-16 students. Introduction Plant cell studies in the classroom at key stages 3 (age 11 14) and 4 (age 14 16) are often limited to cells of onion epidermis. While these cells are easy to observe, they lack chloroplasts (a characteristic of a typical plant cell) and do not have an obvious specialised function related to their structure. Stomata are often studied indirectly using nail varnish peels that leave an impression of the pores and their guard cells. Obviously, the chloroplasts present in guard cells will not be seen from these impressions. African violet (genus Saintpaulia) plants are easy to obtain and to maintain in a school environment. They possess a range of easily viewable specialised cells, making them particularly useful in the classroom. Using Saintpaulia to see specialised plant cells Saintpaulia spp. are native to Tanzania and Kenya, and so need to be able to survive in high temperatures where water may be in short supply. They have large, easily visible stomata on the underside of the leaves. The chloroplasts inside the guard cells can be viewed easily using a basic light microscope. The plant is also covered in multicellular trichomes (hairs) that are easily viewed. The cells that make up the trichomes 54 SSR June 2013, 94(349) are large, may contain chloroplasts, and contain small granules (possibly starch grains although the author has not been able to confirm this with plant scientists) that can be seen moving around inside the cells. Trichomes and cytoplasmic movement The cells making up the trichomes are large and clearly visible at all magnifications on a standard microscope. Movement of particles, suspected to be starch grains, is visible using a 40 objective lens. Students are able to obtain good-quality samples from the leaves by using a sharp scalpel. The trichomes are large, protrude from the plant surface, and so do not require the sample to be particularly thin. Stomata and guard cells Using plants with natural pink pigment on the underside of leaves works particularly well, as it is easy to see the stomata, guard cells and subsidiary cells standing out as white areas against the surrounding pink cells. Students do need to make sure samples are fairly thin for this to work well, although at the edge of most thicker samples there are normally easily identifiable stomata. If the sample is thin enough, it is just as reliable and fast as a stomatal peel. Additionally, the chloroplasts are clearly visible in the guard cells using a 40 objective lens, something which is obviously not possible to see using stomatal peels.

Cottrell Specialised cells in African violets: how to get a lot from one plant in the classroom Other structures to investigate Xylem vessels with spirals of lignin can be seen in the leaf veins,

2 Cottrell Specialised cells in African violets: how to get a lot from one plant in the classroom Other structures to investigate Xylem vessels with spirals of lignin can be seen in the leaf veins, petioles (leaf stalks), pedicels (flower stalks) and flower petals themselves. When petal cells are torn and the ragged edge looked at, it is apparent that the petal cells are dome-shaped and completely filled with pigment. Trichomes are also visible on the petals and movement of particles within these cells is visible. The pollen is easily visible without the need for staining. As the leaves are thick it is fairly easy to take cross sections (of leaves that have been removed from the plant) using a sharp scalpel to show the epidermal cells, palisade layer (very dense; appears black), spongy mesophyll cells and lower epidermal cells. Method for obtaining samples Trichomes and stomata Take small sections of tissue from the underside of a leaf using a sharp blade or scalpel (Figure 1). To remove the tissue in one piece, after slicing underneath the tissue (and before removing the tissue completely), the scalpel can be withdrawn, used to cut across the end of the slice, and then placed back under the slice, which will now easily lift off. Figure 1 A razor blade cutting into the underside of a leaf See Box 1 for safety information on handling scalpels, blades and glass slides. Have a microscope slide ready with a drop of water on it onto which to place the slice of tissue, and then place a cover slip over the sample. On low magnifications, stomata, guard cells and subsidiary cells stand out as white patches against a pink background. Trichomes, typically made of two to five elongated large cells (although they can be up to 12 cells long), can be seen easily and clearly at all magnifications. BOX 1 Risk assessment for Saintpaulia microscopy practical session Hazard Risk Reducing risk Emergency action Scalpel/blades Glass microscope slides and cover slips (these are thin and can break producing sharps) Cuts Infection in cuts Cuts/splinters from broken glass Students shown how to hold and use scalpels and scalpel blades. If blades are not new, ensure they are disinfected, cleaned and dried before use. Emphasise to students that they should not pick up broken glass. A dust pan and brush should be used by them or the teacher in charge. Show students how to handle cover slips and not to press down with fingers unless there is a thick layer of paper towel over the cover slip Wash cuts thoroughly in clean running cold water. Dress wound if required, avoiding sticking plasters where allergic, etc. Seek first aider if cut is deep/ problematic/possiblility of infection who can advise on whether medical attention is required. Cuts treat as above. Splinters seek first aider to remove splinter hygienically. First aider will advise if medical attention is required. SSR June 2013, 94(349) 55

Specialised cells in African violets: how to get a lot from one plant in the classroom Cottrell Petal cells Tear a petal so that you create a ragged edge with some thin areas.

Examination of the ragged edge should reveal that the cells are cone-shaped with a thick and slightly iridescent cell wall.

What you should see All images in this article were taken with a Canon S90 digital compact camera that was manually held to the eyepiece of the micorscope (no adapter was used).

3 Specialised cells in African violets: how to get a lot from one plant in the classroom Cottrell Petal cells Tear a petal so that you create a ragged edge with some thin areas. Mount a small section of the petal (with the thin ragged edge) onto a microscope slide in a drop of water and cover with a cover slip. Examination of the ragged edge should reveal that the cells are cone-shaped with a thick and slightly iridescent cell wall. Spirals of lignified xylem are also often seen if the tear crosses the vascular tissue of the petals. What you should see All images in this article were taken with a Canon S90 digital compact camera that was manually held to the eyepiece of the micorscope (no adapter was used). The photographs have been cropped and labelled using a commonly available software programme (in this case, Microsoft Paint). The microscope was a standard school monocular microscope with built-in illumination. Trichomes and stomata Figures 2 4 show how stomata may look through the microscope. Figure 2 shows that by choosing leaves with natural pink pigment the stomata are easily identified as pale/white structures against a pink background. Figure 3 Saintpaulia stoma, guard cells and subsidiary cells, taken using a 40 objective lens; the stoma appears to be open and the guard cells are turgid; this was a very thin sample, allowing for good focus; s = stoma, gc = guard cell, sc = subsidiary cell, c = chloroplast Figure 2 Saintpaulia lower leaf epidermis; s = stoma surrounded by pale subsidiary cells (many can be seen but only three have been labelled), t = multicellular trichome*, gt = shorter trichomes with rounded ends (similar to glandular trichomes in species such as tomato); photo taken using 10 objective lens. *when viewed using a 40 objective lens, the cell contents (as dark or light granules) can often be seen moving round the cell Figure 4 Saintpaulia stoma, guard cells, subsidiary cells and surrounding epidermal cells with pink pigment, taken using a 40 objective lens; the stoma appears to be partially closed and the guard cells less turgid than in Figure 3 although the stoma is at a slight angle; s = stoma, gc = guard cell, sc = subsidiary cell, c = chloroplast 56 SSR June 2013, 94(349)

Cottrell Specialised cells in African violets: how to get a lot from one plant in the classroom Petals Figure 5 shows what you may see if you tear a petal and view the cells at the edge of the tear.

Xylem and spirals of lignin Vascular tissue is easy to spot by eye in the leaves, leaf stalks (petioles) and petals. Sectioning across this can lead to images such as those in Figure 6.

save time if a good discussion about the different layers is required. Pollen grains Figure 8 shows that pollen grains can be seen without the need for staining.

[prevent water loss via evaporation, insulation, defence] l Why are there no stomata on the upper side of the leaf? [conserve water by reducing evaporation] l Why are petal cells dome-shaped?

4 Cottrell Specialised cells in African violets: how to get a lot from one plant in the classroom Petals Figure 5 shows what you may see if you tear a petal and view the cells at the edge of the tear. Notice the dome shape of the cells that is mentioned below in the Possible discussions section. Xylem and spirals of lignin Vascular tissue is easy to spot by eye in the leaves, leaf stalks (petioles) and petals. Sectioning across this can lead to images such as those in Figure 6. Leaf cross sections As Saintpaulia leaves are fairly thick and stiff, slicing off thin cross sections of the leaf (Figure 7) is fairly easy although possibly best done by a technician beforehand to save time if a good discussion about the different layers is required. Pollen grains Figure 8 shows that pollen grains can be seen without the need for staining. Possible discussions arising from the practical work Adaptations of these plants to their environment l Possible reasons for the Saintpaulia having so many trichomes. [prevent water loss via evaporation, insulation, defence] l Why are there no stomata on the upper side of the leaf? [conserve water by reducing evaporation] l Why are petal cells dome-shaped? [better grip for pollinators (Glover, 2012)] Guard cell function and photosynthesis l Why do guard cells have chloroplasts when other epidermal cells do not? [Simply Figure 5 Saintpaulia petal cells, taken using a 40 objective lens; the dome shape can be seen in cells along the torn edge explained, energy is required for opening/closing mechanisms. This can lead to some in-depth discussions at post-16 level. In order for a stoma to open, the guard cells need to be turgid. In the Figure 6 Saintpaulia petiole xylem tissue with spirals of lignin, taken using a 40 objective lens: (left) the microscope needle is pointing at a spiral of lignin in the xylem tissue; (middle) the spirals of lignin are clearly visible running down the centre of the picture, and green chloroplasts in the petiole cells are also clearly visible; (right) two spirals of lignin have been torn out of the xylem tissue during the section SSR June 2013, 94(349) 57

Specialised cells in African violets: how to get a lot from one plant in the classroom Cottrell Figure 7 Saintpaulia leaf cross section, taken using a 40 objective lens; v = vacuole in mesophyll

As the guard cells become more turgid, their shape changes and the stoma opens.] Cytoplasmic streaming l Why do cell contents need to move around the cells?

5 Specialised cells in African violets: how to get a lot from one plant in the classroom Cottrell Figure 7 Saintpaulia leaf cross section, taken using a 40 objective lens; v = vacuole in mesophyll cell, s = stoma (out of focus but visible along the lower epidermis) cells reduces their water potential, causing water to flow into the guard cells from surrounding epidermal cells. As the guard cells become more turgid, their shape changes and the stoma opens.] Cytoplasmic streaming l Why do cell contents need to move around the cells? [transport proteins, nutrients and organelles around the cell] l What could the granular contents of the cells be? [from reading articles on other species with trichomes, these are likely to be lipid droplets, mitochondria and/or polysaccharides such as starch granules] Leaf structure l The reasons for the different layers in the leaf. [transparent epidermis to allow light through, etc.] Figure 8 Saintpaulia pollen grains, taken using a 40 objective lens light, proton pumps force hydrogen ions out of the guard cells. Potassium ions are then pumped into the guard cells via voltage-gated channels using adenosine triphosphate (ATP) in the process. The increase of potassium ions in the guard 58 SSR June 2013, 94(349) Cloning Saintpaulia can be used in class to demonstrate cloning. Leaves can be removed from the plant and planted in soil. After a few weeks they should start to grow into new plants (this can take a while and the most likely reason for them to fail is overwatering).

6 Cottrell Specialised cells in African violets: how to get a lot from one plant in the classroom Plant defence Trichomes such as the multicellular hair-like ones seen on Saintpaulia act as a physical barrier to herbivorous insects. Many plants (including Saintpaulia) also have shorter, glandular trichomes that produce chemicals. These may be toxic to insects and may slow down their growth and development (Stipanovic, 1983). Maintaining Saintpaulia at school Saintpaulia are easily obtained from garden centres and large supermarkets. They can be grown and maintained easily in the classroom on a windowsill. With enough bright light they can flower all year round (leaves will become scorched if left in direct sunlight). They prefer room temperature conditions. They should be watered sparingly (from below in a tray seems to work best to keep the top of the compost fairly dry). They can be susceptible to overwatering. If leaves are removed for practical work, ensure the entire leaf stalk is removed so that the stub does not rot back into the main stem of the plant (Huxley and Gilbert, 1991). When removing small slices from leaves, the whole leaf does not necessarily need to be removed from the plant. Propagation Saintpaulia are fairly easily propagated by removing the leaves and placing them stalk downwards into moist potting compost. They can take several months to develop into plantlets and care needs to be taken not to overwater. Acknowledgements This article was developed by the author while on secondment as an Education Fellow to the Nuffield Foundation and Science & Plants for Schools. References Glover, B. (2012) Gilding the lily: understanding angiosperm diversity through petal evolution and development. Lecture at Cambridge University Botanic Garden. Huxley, A. and Gilbert, R. (1991) Success with Houseplants. Reader s Digest Association. Stipanovic, R. D. (1983) Function and chemistry of plant trichomes and glands in insect resistance. In Plant Resistance to Insects, ed. Hedin, P. A. American Chemical Society Symposium Series, 208, chapter 5, Vicki Cottrell was a science teacher for 10 years, including 4 years as Head of Science at Didcot Girls School. In September 2011, she took up a 6 month secondment as an Education Fellow with the Nuffield Foundation and Science & Plants for Schools. During that time she researched the use of microscopes and plants in the classroom and produced resources for teachers that are available at vickicottrell1@aol.com SSR June 2013, 94(349) 59

stomata means mouth in Greek because they allow communication between the internal and

stomata means mouth in Greek because they allow communication between the internal and Name: Date: Period: Photosynthesis Lab #1: Leaf Structure and Function Purpose: The purpose of this lab is to explore the structure of a leaf, specifically the stomata and guard cells. Once we have an