Presence of the protruding oncus is affected by developmental stage and acetolysis technique

Abstract: A protruding oncus is a projection of the intine in the aperture region. The ubiquitous use of acetolysis in palynological research has led to the presence of a protruding oncus being underreported. Controlled experiments with pollen samples collected from undehisced and dehisced anthers demonstrate that the presence of a protruding oncus is affected by the state of the anther at maturity: dehisced or undehisced and by the preparation technique. In investigating the occurrence of onci, particular attention should be paid both to the dehiscence state of the anthers and the effect of the preparation technique on the intine. Although it has been suggested that protruding onci and pollen buds can be distinguished based on three criteria (size, presence of a large vacuole, separation of the protrusion from the grain), most of these distinctions break down when information is included from more recent studies. Additional study of protruding intinous structures may help clarifing the difference between pollen buds and protruding onci. pollen grain | intine | acetolysis | anther | oncus | pollen bud | Rubiaceae | biology | Keywords: botany | plant evolution Article: The oncus (pl. onci) is an intinous structure occurring beneath the apertures of many types of pollen grains The term 'protruding oncus' was proposed by As intinous structures, onci are not resistant to acetolysis In this paper, we draw attention to the presence of protruding onci in the Rubiaceae and other angiosperms, investigate factors that may have caused the presence of protruding onci to be underreported and discuss the distinctions between pollen buds and protruding onci based on our current knowledge of these structures. Material and methods Unopened flowers of Uncaria hirsuta Havil. with polliniferous anthers were collected from living plants growing in the South China Botanical Garden, Guangzhou, Guangdong Province, China. Uncaria hirsuta was chosen for study because of its possession of pollen with obvious protruding onci and because sufficient living material was available for developmental study. Collected anthers were air-dried at room temperature for 24 h, then wrapped in absorbent filter paper, sealed in a clear Ziploc bag, and stored at -4 °C. Although the flowers had not opened, they contained both dehisced and undehisced anthers. A voucher specimen (KuangYan-Feng 1002) is preserved at South China Botanical Garden Herbarium IBSC. To prepare unacetolysed samples, dehisced and undehisced anthers were dissected out of unopened flowers, separated and put into separate plastic vials in 70% ethanol. The anthers were then squeezed with forceps to release the pollen. The separation of the pollen grains from the remaining anther material was accomplished with a steel sieve (mesh diameter 100 μm). The separated pollen was rinsed in 70% ethanol with ultrasonic vibration for 30 min and centrifuged (4000-5000 RPM, 5 min) to create a pellet. The ethanol was decanted and fresh 70% ethanol was added. The samples were stored in 70% ethanol prior to decanting onto stubs, air drying and observation with a scanning electron microscope. For acetolysis, undehisced anthers were placed in plastic vials, covered with glacial acetic acid, squeezed to release the pollen and filtered as described earlier. The pollen was subdivided into four subsamples, all of which were centrifuged separately to create pellets. The glacial acetic acid was decanted and a mixture (9:1) of acetic anhydride and concentrated sulphuric acid was added to each subsample. The four subsamples were acetolysed in a hot water bath at c. 90 °C for 1.5 min, 3 min, 5 min and 10 min, respectively. After acetolysis, the samples were cooled at room temperature, and centrifuged again at 4000-5000 RPM for 5 min. The residue of the chemical mixture was decanted and the pollen samples were rinsed in an ultrasonic bath with two changes of 70% ethanol, for 30 min each. The treated samples were stored in 70% ethanol prior to decanting onto stubs, air drying, and observation. For scanning electron microscopy (SEM), the acetolysed and unacetolysed grains were mounted on small pieces of cover slip that were attached to SEM stubs with a strip of double-stick conductive tape. The pollen suspension was removed from the bottom of the plastic vials with a Pasteur pipette, placed on the cover glass and air-dried. Colloidal silver paint (#12630, Electron Microscopy Sciences, Fort Washington, PA, USA) was used to paint strips from the edges of the cover slip to the side of the stub. These strips conduct electrons from the top of the cover slip to the stub. Each sample was then sputter-coated with gold for 4 min (two coatings of 2 min each) in a PELCO Model 3 Sputter Coater 9100. A tilted stub holder was used for the second coating. Digital images were captured with a Hitachi S-4800 scanning electron microscope. For light microscopy (LM) and transmission electron microscopy (TEM), fresh flowers of Uncaria hirsuta at different stages were collected from living plants growing in the South China Botanical Garden and the Dinghushan National Natural Reserve (Zhaoqing, Guangdong Province, China). Plants at both sites are cultivated. Voucher specimens (Kuang Yan-Feng 1002 from South China Botanical Garden, Kuang Yan-Feng 002 from Dinghushan) are deposited at IBSC. The fresh flowers were immediately fixed in 2.5% glutaraldehyde in 0.1 mol/l phosphate buffer, pH 7.2, in the field. When back in the laboratory, the materials were placed under vacuum for 2 h and stored at 4 °C for several days. After removal from storage, the anthers were extracted, placed in the same fixation fluid under vacuum for 2 h, rinsed in 0.1 mol/l phosphate buffer for 2 h and postfixed in 1% osmium tetroxide overnight. Following postfixation, the anthers were washed in phosphate buffer, dehydrated in an acetone series, embedded in Spurr's resin and cured at 70 °C. Semi-thin sections (1-2 μm) were cut with glass knives on a LKB-11800 microtome, stained with 0.1% toluidine blue and observed and photographed with an Olympus-AX70 light microscope equipped with an Olympus-DP50 digital camera. Ultrathin sections (80 nm) were cut using a Leica-Ultracut S ultramicrotome with a diamond knife and stained with uranyl acetate and lead citrate. Transmission electron micrographs were taken with a JEM-1010 transmission electron microscope at 100 KV. Results Morphology of pollen and protruding onci at different developmental stages Pollen grains of Uncaria hirsuta are oblate spheroidal in equatorial view, semi-circular in polar view, tricolporate with of long ectocolpi and lolongate to (sub)circular mesopori. The exine ornamentation might either be described as striate-reticulate with interwoven muri or as rugulate with slender, long striae ( The number of grains with protruding onci is much lower in dehisced ( In undehisced anthers, the protruding onci are usually irregularly hemispherical, with a varying number of contortions on their surface ( Acetolysis effects on protruding onci Protruding onci are destroyed and the mesopori become conspicuous, following acetolysis treatments of 1.5 min or more ( Microspore and protruding oncus development At the tetrad stage, the microspores are enclosed by callose walls ( At the early microspore stage, the callose wall dissolves and the free microspores are released into the anther locule. As the pollen grain increases in size, the electron-lucent areas broaden and become lens-shaped ( At the middle microspore stage, the electron-lucent areas flatten even more and the endexine thickens into costae around the aperture region ( During the vacuolated microspore stage, the electron-lucent area disappears and intine deposition starts ( In pollen grains from dehisced anthers, the intine beneath the aperture is thinner and the core of the protruding oncus is more electron-lucent ( Discussion Development of the protruding oncus In this paper, we have elucidated the main stages of the formation of protruding onci in Uncaria hirsuta. At the tetrad stage, a lens-shaped area forms beneath the future aperture and determines the site of the protruding oncus. During the vacuolated microspore stage, the intine forms, the lens-shaped area flattens and the initine thickens beneath the aperture and protrudes outwards, forming the protruding oncus. At anther anthesis, the aperture intine is much thinner than at earlier stages and the protruding onci may hardly be visible. These changes are likely due to the harmomegathic effect, which occurs after anther anthesis. The time of the onci formation is dependent on the taxon. The onci of Mitriostigma axillare Hochst. form at the tetrad stage, protrude through the aperture when the grains separate, contain only minimal cytoplasmic contents and are not shed from the grain The effects of acetolysis and anther dehiscence on the protruding oncus The acetolysis method introduced by Erdtman (1943) is a widely employed, successful technique in palynology. However, acetolysis destroys all pollen contents with the exception of the sporopollenin The presence of protruding onci is also affected by the stage of anther dehiscence. Before anther dehiscence, protruding onci are almost universally present and conspicuously visible in the grains. At anther dehiscence, protruding onci are usually absent, and when present, are small and undistinguished. Hence, it is important to identify the developmental status of anthers (whether dehisced or undehisced) from which pollen grains are collected in order to verify the presence or absence of protruding onci. Pollen buds versus protruding onci The term 'pollen buds' was introduced to describe the spherical buds occurring outside of the germ pores of developing pollen grains in Ophiorrhiza mungos L. Weber and Igersheim (1994) suggested (in a footnote) that the term 'pollen buds' is inappropriate, but did not suggest an alternative. In Weber's (1996) following paper, she referred to the apertural intine protrusions of Geranium (Geraniaceae) as 'apertural chambers', but this term has not been used extensively in the later literature. The term 'protruding oncus' was initially proposed to replace the term 'pollen bud' In the Rubiaceae, The most comprehensive investigation of protruding intinous structures in the Rubiaceae is in the Naucleea