Flower Under the Microscope
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The following article was originally published in the journal for educators Biologia w Szkole (eng. Biology in School) (2/2022):
In our discussions about plants, we have repeatedly addressed the flower, its accompanying structures, and its adaptations. We have covered the stamens of barberry Berberis sp., capable of rapid movements; the bracts of strawflower Xerochrysum bracteatum; the huge and magnificent inflorescences of the horse chestnut Aesculus hippocastanum, so commonly seen in Poland; and even the common stork’s-bill Erodium cicutarium, which catapults its seeds over considerable distances relative to its own size [1] [2] [3] [4].
The flower, as the organ responsible for plant reproduction, is a highly complex structure. It is a shortened shoot with limited longitudinal growth that bears elements directly or indirectly involved in sexual reproduction. The flower of angiosperms (Magnoliophyta) — most likely descended from a common ancestor living during the Carboniferous period, i.e., 350–275 million years ago — is homologous to the sporophyll or strobilus found in more primitive representatives of the plant kingdom (Plantae).
The beauty of many flowers has likely inspired admiration since the dawn of human consciousness. They often acquired symbolic or even religious significance. For most of our history, however, we did not understand the purpose and role flowers play in the life of plants; some believed them to be a whimsical ornament of nature, meant to beautify it, yet devoid of deeper meaning. Only in the late 17th century did the German physician and botanist Rudolf Jakob Camerarius experimentally prove that flowers serve reproduction. He also deduced the roles of its major floral components, concluding that the stamens are the male generative organs, while the pistils are female. Subsequent researchers, among them Carl Sprengel, showed that insects frequently participate in pollination and described many phenomena related to plant fertilization, including protogyny and protandry [5].
Because flowers are such complex and fascinating structures, even observing those of readily accessible and ubiquitous plants — often labeled as weeds — can yield many rewarding moments of discovery. This time, I would like to tell you about a modest plant known as the scarlet pimpernel.
Scarlet Pimpernel
The scarlet pimpernel Anagallis arvensis (also called Lysimachia arvensis) belongs to the primrose family Primulaceae [6]. It is quite widespread, occurring naturally throughout Europe, much of Asia, as well as North Africa and Macaronesia [7]. In Poland, it is considered an archaeophyte. It is common and can easily be found in lowland areas as well as in the mountains. Typical habitats for this species include fields, gardens, roadsides, waste grounds, and vineyards. It is often described as a segetal plant appearing in most types of crops, though it does minimal harm. It prefers clay-rich soils with plentiful nitrogen and other nutrients.
The scarlet pimpernel is a small, sprawling herb. Its stem is smooth, prostrate, four-angled, and can reach about 10–20 cm in length. The shoots easily take root wherever they touch the ground. Its leaves are arranged opposite, sometimes in whorls of three or four. They measure up to 3 cm (1.18 in) in length, are sessile and oval, and are either blunt or faintly pointed at the tip, with small dark glands on the underside.
This species is an annual plant, flowering from May to October. The stigma and stamens mature simultaneously, and the flowers have no nectaries, as they are self-pollinating. Seed dispersal occurs via wind (anemochory).
Flowers usually measure about 8 mm in diameter and are brick-red. They grow on fairly long pedicels that emerge from the leaf axils. The corolla’s petals are typically spread out, glandular-hairy along the margins, and slightly serrated at the tip. The calyx segments are lanceolate, only slightly shorter than the corolla petals. The stamens can be observed attached to the throat of the corolla, with haired filaments of equal length in their upper parts. The anthers appear to be roughly three to five times shorter than the filaments.
Its floral formula is *K5[C(5)A5]G5. A floral diagram is shown in Fig.1.
At night and during cloudy weather, these flowers close. Because they open in the morning, scarlet pimpernel flowers are considered by some as a sign of fair weather.
The plant’s fruit is a dehiscent capsule.
The scarlet pimpernel is sometimes described as poisonous, containing various saponins, glycosides, flavonoids (kaempferol C15H10O6 and quercetin C15H10O7), tannins, organic acids (caffeic acid C9H8O4, ferulic acid C10H10O4, sinapinic acid C11H12O5, and p-coumaric acid C9H8O3). If eaten by livestock, it can cause poisoning, with symptoms such as lack of appetite, diarrhea, accelerated breathing (tachypnea), and others [10].
It is worth noting that the scarlet pimpernel is a long-day (obligate) plant. This means it produces flowers only when the night length falls below their critical photoperiod [11]. This must be taken into account if one wishes to cultivate it under artificial conditions.
Petals
The most interesting floral elements in the scarlet pimpernel are the corolla petals. We can observe changes in the shape of their epidermal cells during development, as well as familiarize ourselves with some intriguing preparation techniques.
It is best to begin by examining very young, not yet open or just barely opening buds (Photo.3). Ideally, at this stage the corolla petals are still uncolored and measure under 1 mm in length.
You should cut the petals right at their base. This requires a steady hand and a keen eye — or simply a stereomicroscope — but can be done successfully. The isolated petals can then be processed immediately or preserved in a suitable fixative (for instance, a mixture of 70% ethanol C2H5OH, glacial acetic acid CH3COOH, and 40% formaldehyde CH2O in a volumetric ratio of 90:5:5) and stored for an extended period in a refrigerator [12].
Next, a specific technique for staining cell walls — the PAS method (periodic acid–Schiff) — is applied. It gives the walls a purple color. This hue results from the reduction of colorless leucofuchsin to a red-purple product via aldehyde groups formed by the action of periodic acid on the polysaccharides of the cell wall. The procedure is as follows:
- Treating the petals with periodic acid H5IO6 for 20 minutes (using an aqueous solution at 0.5% concentration).
- Rinsing with distilled water three times (10 minutes each) to remove all traces of periodic acid.
- Staining for 30 minutes in the dark by immersing the samples in Schiff’s reagent (fuchsin C20H20N3 decolorized with sulfur dioxide SO2).
- Removing excess unreacted Schiff’s reagent by transferring the petals to solution — 10 cm3 (approx. 0.34 US fl oz) of 10% sodium disulfite(IV) solution mixed with 10 cm3 (approx. 0.34 US fl oz) of 3.5% hydrochloric acid HCl, then diluted to 200 cm3 (approx. 6.76 US fl oz) with distilled water — for a few seconds (or longer), followed by a rinse in distilled water [13, modified].
If the petals were previously fixed in the mentioned alcohol–acetic acid–formaldehyde solution, you must first remove the fixative and rehydrate the material. To do this, transfer the buds successively into increasingly dilute solutions of the fixative with water (in volumetric ratios of 2:1, 1:1, 1:2) and then into distilled water, determining the immersion time in each solution experimentally.
The stained petals obtained in this manner may then be embedded (after dehydration in organic solvents) in a natural or synthetic resin on microscope slides for further observation — or you can skip embedding and observe them immediately after the procedure.
Although these samples can certainly be viewed under a standard light microscope, the best results come with a fluorescence microscope (Photo.4).
As you can see, the petal is at an early stage of development and is microscopic in size: its total length is under 0.5 mm. We can then observe the beautiful arrangement of cells (or rather their walls) in the petal’s epidermis — they are simply polygonal in shape. One notices a so-called “fountain-like” pattern, in which the columns of cells gradually diverge more and more toward the sides as they move away from the petal’s base.
The PAS method clearly highlights the cell walls. But what if we want to observe the cell nuclei? In that case, different techniques must be used. I would like to propose two relatively simple staining methods — similar to each other, but using different chemical substances, namely gentian violet (a mixture of crystal violet C25H30N3Cl and methyl violet C23H26N3Cl) and safranin C20H19ClN4.
For this purpose, I took freshly collected petals and treated them with 5% sodium hydroxide NaOH for 24 hours. I then rinsed them five times in distilled water, leaving them in each rinse for one hour. Next, I transferred them sequentially to 5%, 15%, and 25% solutions of ethyl alcohol, each time leaving the petals for an hour. In the final solution, the petals can be stored in a refrigerator for a long period.
Staining itself is fairly straightforward: transfer the petals to the dye solution (1% gentian violet in 50% ethanol or, separately, 1.5% safranin in 30% ethanol) for about 15 minutes, then rinse with cold 30% ethanol. Next, the petals are mounted in a 50% aqueous glycerol solution under a coverslip and observed immediately or soon thereafter — these preparations are not suitable for long-term storage.
Whether staining with gentian violet (Photo.5) or safranin (Photo.6), the outcome is similar.
In both cases, the cell nuclei are stained (purple with gentian violet, red with safranin). The peripheral cells appear to have been mechanically or chemically damaged by the NaOH, which is why no nuclei are visible in them.
After examining structures at early developmental stages, it is worth preparing larger petals as well. Buds that have just opened are good for this (Photo.7).
After immersion in sodium hydroxide solution (as described earlier), or even directly by placing the petal in a drop of water on a microscope slide, we can observe it under a bright light with a standard microscope (Photo.8).
When examining the epidermal cells of a mature petal, we see that their shape differs considerably from what we observed at earlier developmental stages — they are more elongated, and their cell walls become significantly undulated, forming a very characteristic, accordion-like pattern.
Explanation
Unlike animal cells, plant cells remain tightly bound to neighboring cells by means of the middle lamella, composed primarily of pectins, located external to the primary cell wall. As a result, plant cells cannot move relative to one another during development, a phenomenon known as symplastic growth. There are few exceptions to this pattern in the plant world. Consequently, the shape and structure of plant organs are closely linked to how the cells that form these organs grow and divide. This holds true for the scarlet pimpernel’s corolla petals: comparing petal cells at various stages of development reveals changes in their shape, yet the overall arrangement remains the same.
I believe these observations show that even with a plant as unassuming as the scarlet pimpernel, we can make many fascinating discoveries and learn new techniques for preparing biological material.
References
- [1] Ples M., Roślinny bokser? Szybkie ruchy pręcików berberysu (eng. A Plant Boxer? The Rapid Stamen Movements of Barberry), Biologia w Szkole (eng. Biology in School), 3 (2020), Forum Media Polska Sp. z o.o., pp. 81-85 back
- [2] Ples M., A jednak się porusza! Ruchy higroskopowe roślin (eng. And Yet It Moves! Hygroscopic Movements of Plants), Biologia w Szkole (eng. Biology in School), 3 (2016), Forum Media Polska Sp. z o.o., pp. 52-56 back
- [3] Ples M., Kasztanowiec - zwyczajny, ale niezwykły (eng. The Common Horse Chestnut: Surprisingly Extraordinary), Biologia w Szkole (eng. Biology in School) w Szkole, 4 (2017), Forum Media Polska Sp. z o.o., pp. 56-61 back
- [4] Ples M., Iglica pospolita - roślinna katapulta i ruchliwe nasiona (eng. Erodium Cicutarium - A Plant Catapult And Moving Seeds), Biologia w Szkole (eng. Biology in School), 5 (2020), Forum Media Polska Sp. z o.o., pp. 54-57 back
- [5] Szafer W., Szaferowa J., Kwiaty w naturze i sztuce, Państwowe Wydawnictwo Naukowe PWN, Warszawa, 1958 back
- [6] Kadereit J. W., Albach D. C., Ehrendorfer F., Galbany-Casals M., Which changes are needed to render all genera of the German flora monophyletic?, Willdenowia, 46, 2016, pp. 39-91 back
- [7] Germplasm Resources Information Network (GRIN), dostępne online: https://www.ars-grin.gov/ [dostęp: 03.02.2022] back
- [8] Kebert T., Floral diagram generator, dostępne online: http://kvetnidiagram.8u.cz/ [dostęp 02.02.2022] back
- [9] De Craene L. P. R., An Aid to Understanding Flower Morphology and Evolution, Cambridge University Press 2010 back
- [10] Mowszowicz J., Przewodnik do oznaczania krajowych roślin trujących i szkodliwych, Państwowe Wydawnictwa Rolnicze i Leśne, Warszawa, 1982, pp. 221 back
- [11] Freyre R., Anagallis monelli plant named 'Wilcat Mandarin' , United States Patent Application, 2007 back
- [12] Broda B., Metody histochemii roślinnej, Państwowy Zakład Wydawnictw Lekarskich, 1971 back
- [13] Mikroskopia - Odczynnik Schiffa (broszura zestawu do barwienia firmy Sigma Aldrich), online: https://www.merckmillipore.com/PL/pl/product/PAS-staining-kit,MDA_CHEM-101646#documentation [02.02.2022] back
All photographs and illustrations were created by the author.
Marek Ples