Pollen exine development precedes microtubule rearrangement inVigna unguiculata (Fabaceae): A model for pollen wall patterning

Summary Although patterns on pollen exines are highly conserved, heritable traits, there is no prevailing explanation for control of pattern development. InVigna unguiculata (Fabaceae), the exine reticulum is unusually coarse so that development of the reticulum can be followed by light microscopy. Developing exine patterns were compared with the arrangement of microtubules in the microspore using immunofluorescence labeling of microtubules. Exine pattern developed in microspores at stages with a regular microtubule pattern. At later stages of exine formation, microtubules were arranged in patches under the lumina of the reticulum. We conclude that microtubules do not determine exine pattern. The developing exine appears to rearrange microtubules. We interpret this as evidence for the selfpatterning of exine based on intrinsic properties of the matrix between the microspore and the callose wall.Abbreviations DIC differential interference contrast - ECM(s) extracellular matrix(ces) - MT(s) microtubule(s) - PBS phosphate buffered saline - SEM scanning electron microscopy     

POLLEN MORPHOLOGY IN THE GENUS ERYTHRONIUM (LILIACEAE) AND ITS SYSTEMATIC IMPLICATIONS

Pollen morphology was examined in 40 samples from 19 species in the genus Erythronium (Liliaceae) using scanning (SEM) and transmission electron microscopy (TEM). Pollen grains are monosulcate monads, except for a plant of E. oregonum which has dyad grains. North American and European species are characterized by reticulate exine sculpture composed of various degrees of wavy muri and variably-sized lumina. The most distinctive feature of exine sculpture in the genus is exhibited by the Asian species E. japonicum, which has a unique reticulate pattern composed of striate muri and a distinctive exine structure without columellae. The distinctness of E. japonicum pollen suggests that it has specialized in isolation from species in North America and Europe.     

凤仙花属棒凤仙亚属(凤仙花科)植物花粉形态的分类学意义

棒凤仙亚属代表着凤仙花属最先分化出的一支,以多年生草本,多于5朵花的总状花序,侧生萼片4枚,3沟花粉,心皮四室且每室具1枚种子,果实棒锤状,种子椭圆形以及三沟花粉其极面观为三角形而区别于其它种类。该亚属主要分布于中国南部,少数种类辐射分布至华中和中南半岛地区。已有报道证实花粉的形态特征对凤仙花属植物的属下分类具有重要意义,但对棒凤仙亚属的种类少有涉及。为了进一步探讨该亚属花粉的形态特征及其分类学价值,该研究应用扫描电镜技术对国产棒凤仙亚属植物的14种以及凤仙花亚属的窄萼凤仙花的花粉进行了观察。结果表明：棒凤仙亚属的花粉以单粒存在,具3条萌发沟,辐射对称,极面观为三角形或三角状圆形,花粉粒外壁具均匀的网状纹饰,网脊平滑或具波状边缘,网眼中具不同密度的颗粒状突起。棒凤仙亚属的花粉形态与水角属的花粉形态相似,表现出了其原始性;其花粉形态特征与花部形态性状等相关性差,对该亚属的组级分类意义较小,但对于该亚属的种间界定具有重要意义;而其花粉的极面观形状、赤道面观形状以及网状纹饰等变异相对稳定,对于界定该亚属具有重要的分类学价值。     

Pollen exine ornamentation in the F2 generation of an interspecific hybrid of Chorisia (Bombacoideae,Malvaceae) in relation to inheritance pattern

Pollen morphology in ten plants of F₂ progeny of an interspecific tree hybrid, Chorisia insignis H. B.& K.×C. speciosa St. Hil (Bombacoideae, Malvaceae) has been studied with a view to have an insight in to the effect of hybridization on the pollen morphological features in F₂ generation. The study is a sequel to an earlier study on the pollen morphology of Chorisia species and their F₁ hybrid, in which case the hybrid pollen uniformly exhibited the apertural features of the male parent and exine features of the female parent. In the F₂ progeny the pollen grains display the apertural feature of the male parent in all the plants. However, with regard to exine ornamentation, variability has been observed. Of the ten plants, two plants exhibit the exine features of the male parent showing empty lumina (without bacula), five plants have their pollen exactly like that of the F₁ plant (showing prominent columellar heads in the lumina of the apocolpium region) and the remaining three plants showing columellae in lumina of both apocolpium and mesocolpium region being different from the above types. It has been inferred that the variability in the exine pattern in F₂ pollen indicates that the pollen exine pattern in hybrids, perhaps, is not unequivocally controlled by sporophytic or gametophytic genomes.     

Pollen Morphology and Its Taxonomic Significance of Caragana Fabr. (Fabaceae) from China

Pollen grains of thirty one species and two varieties of the genus Caragana Fabr. were observed under LM and SEM.The results revealed that the pollen exine ornamentation of the species examined could be divided into two types: perforate and reticulate.The later type consists of two subtypes according to lumina size and muri width. In the first subtype the lumina is round with d iameter equal to muri width,and the exine is perforate in two polar areas,while in the second subtype lumina is irregular or polygonal,with diameter sm aller than muri width.The pollen volume was divided into four types.The genus Caragana is a natural group which was indicated by the general resemblance of pollen morphology. In this genus the pollen morphological classification was not in concordance with the arrangement of the sections and series except Ser.Pygmeae and some species. The pollen variation at infraspecific level was obvious,especially the species distributed in Qinghai Xizang Plateau, such as C.jubata, C.bicolor and C. erinacea . The evolutionary trend of pollen morphology is from perforate to reticulate, which is congruous with the evolution from the pinnate leaf group to the almate leaf group in the genus.     

中国东北牻牛儿苗科（Geraniaceae）花粉形态与系统分类学研究

本文对中国东北牛儿苗科Ｇｅｒａｎｉａｃｅａｅ老鹳草属Ｇｅｒａｎｉｕｍ和牛儿苗属Ｅｒｎｄｉｕｍ植物计２属,１４种,２变种及１变型的花粉形态进行了较系统的研究。并对本科花粉形态进行了分类处理,编制了花粉形态科种检索表。结果表明,本科花粉如按萌发孔类型、外壁雕纹式样可以分为二大类：即三孔沟,外壁雕纹瘤状（Ｇｅｒａｎｉｕｍ）三沟,外壁雕纹网状（Ｅｒｏｄｉｕｍ）。在老鹳草属种及种下等级的分类处理中,花粉粒大小、萌发沟长度、外壁覆盖层网眼均匀性、直径大小、网脊上纹饰及外壁雕纹瘤纹突起均匀性、大小及瘤上纹饰都具有较大的分类学意义。花粉形态分类与经典分类对本科分类群的处理意见相吻合。大大丰富了本科分类微形态的证据。     

Pollen morphology inParis and its related genera

Pollen grains ofParis, Medeola, andScoliopus have been examined with light, scanning electron, and transmission electron microscopies.P. quadrifolia L.,P. verticillata Bieb.,P. delavayi Franch. andP. polyphylla Smith have monosulcate pollen characterized by psilate, foveolate or reticulate exine sculpture. In contrast to the former species,P. japonica (Fr. et Sav.) Franch. andP. tetraphylla A. Gray have monosulcate pollen with gemmate (rarely rugulate) exine.Medeola has monosulcate pollen with reticulate exine that is distinct from that ofParis. Scoliopus has monosulcate pollen characterized by a peculiar reticulate exine pattern. The palynological evidence suggests thatParis andTrillium are closely related to each other, andMedeola andScoliopus should be separated fromParis andTrillium.     

Pollen wall development in Vigna vexillata I. Characterization of wall layers

Light and electron microscope observations characterized the layers that comprise Vigna vexillata L. pollen walls, and identified the timing of their development. Exine sculpturings form an unusually coarse ektexinous reticulum. The structure of the ektexine is granular; this differs from the columellate/tectate type of structure typical of most angiosperm pollen. The ektexine overlies a homogeneous-to-lamellar, electron-dense endexine, which in turn surrounds a thick, microfibrillar intine. Pollen grains are triporate and operculate, with Zwischenkörper and thickened intine underlying the apertures. The ektexine forms during the tetrad period of microspore development, the endexine and Zwischenkörper during the free microspore stage, and the intine during the bicelled (pollen) stage. Coarsely reticulate exine sculpturings and the granular structure of the patterned exine wall of the pollen grains are features that make this species suitable for detailed studies of pollen wall pattern formation.     

The pollen morphology ofOdontites (Scrophulariaceae) and its taxonomic significance

The pollen morphology of 29 species ofOdontites and related genera was examined by light and scanning electron microscopy. Three major pollen types differing fundamentally in exine sculpturing were found. In the parasitic tribePedicularieae retipilate sculpturing is the most widespread type representing a plesiomorphic character state from which the two other types are derived. Of these, reticulate sculpturing is confined toOdontites, whereas a complex retirugulate pattern was found only in the monotypic Near East genusBornmuellerantha. Within the retipilate and the reticulate major types eight minor types were distinguished based on the differential correlation of exine surface morphology, size, shape and amb form. The pollen data are generally well correlated with macromorphological features and furnish important taxonomic characters at the genus—as well as at the specieslevel.     

锦鸡儿属花粉形态及其分类意义

在光学显微镜和扫锚电镜下对锦鸡儿属31种2变种植物花粉形态进行了观察。根据外壁表面纹饰,可分：类型I,表面具小穿孔;类型II, 具网状纹饰。 在类型II中据网眼大小和网脊宽度特征又分为两个亚类型。 根据花粉体积大小,可分4个类型。本属植物花粉形态较为一致,表明是一个自然类群。组、 系的花粉形态特征与一般形态变异不一定相对应,即形态上有明显区别的种类,在花粉形态上未必有很大区别,仅一些种类及矮锦鸡儿系 Ser. Pygmaeae的花粉形态相近。 本属花粉形态在种下等级的变异, 特别是青藏高原分布的种类的变异较为明显,例如 C.bicolor,C. jubata,C. erinacea 与各自的近缘种类。花粉形态为分类处理提供了参考。本属植物花粉形态是从表面具小穿孔向具网状纹饰演化,对应于羽状叶类群向掌状叶类群演化。     

Microspore development inBrassica napus and the effect of high temperature on division in vivo and in vitro

Summary Brassica napus cv. Topas microspores isolated and cultured near the first pollen mitosis and subjected to a heat treatment develop into haploid embryos at a frequency of about 20%. In order to obtain a greater understanding of the induction process and embryogenesis, transmission electron microscopy was used to study the development of pollen from the mid-uninucleate to the bicellular microspore stage. The effect of 24 h of high temperature (32.5 °C) on microspore development was examined by heat treating microspore cultures or entire plants. Mid-uninucleate microspores contained small vacuoles. Late-uninucleate vacuolate microspores contained a large vacuole. The large vacuole of the vacuolate stage was fragmented into numerous small vacuoles in the late-uninucleate stage. The late-uninucleate stage contained an increased number of ribosomes, a pollen coat covering the exine and a laterally positioned nucleus. Prior to the first pollen mitosis the nucleus of the lateuninucleate microspore appeared to be appressed to the plasma membrane; numerous perinuclear microtubules were observed. Microspores developing into pollen divided asymmetrically to form a large vegetative cell with amyloplasts and a small generative cell without plastids. The cells were separated by a lens-shaped cell wall which later diminished. At the late-bicellular stage the generative cell was observed within the vegetative cell. Starch and lipid reserves were present in the vegetative cell and the rough endoplasmic reticulum and Golgi were abundant. The microspore isolation procedure removed the pollen coat, but did not redistribute or alter the morphology of the organelles. Microspores cultured at 25 °C for 24 h resembled late-bicellular microspores except more starch and a thicker intine were present. A more equal division of microspores occurred during the 24 h heat treatment (32.5 °C) of the entire plant or of cultures. A planar wall separated the cells of the bicellular microspores. Both daughter cells contained plastids and the nuclei were of similar size. Cultured embryogenie microspores contained electron-dense deposits at the plasma membrane/cell wall interface, vesicle-like structures in the cell walls and organelle-free regions in the cytoplasm. The results are related to embryogenesis and a possible mechanism of induction is discussed.Abbreviations B binucleate - LU late uninucleate - LUV late uninucleate vacuolate - M mitotic - MU mid-uninucleate - RER rough endoplasmic reticulum - TEM transmission electron micrograph     

Scanning electron microscopy of microspore embryogenesis inBrassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy     

Sporoderm development inNymphaea mexicana (Nymphaeaceae)

Mature pollen grains ofNymphaea mexicana have a verrucate proximal surface, a psilate distal surface and an anazonasulculus (encircling-sulcate aperture). The developmental events of microspores and tapetal cells were observed with TEM and SEM. Radially oriented substructural elements are seen in the microspore surface coating ofNymphaea mexicana from the early tetrad stage through the whole exine development. These elements, being the structural units of the microspore surface matrix (glycocalyx), are associated with sporopollenin precursor accumulation. In young free microspores, radially oriented elements are observed at both proximal and distal poles as a palisade between the endexine and plasmalemma.—Several points are discussed: (1) the initial and mature forms of exine substructure elements; (2) the significance of exine substructure for realisation of morphogenetic processes; (3) the ways by which verrucate and psilate sculpture patterns are developed.     

Cell cycle dependent distribution of phosphorylated proteins in microspores and pollen ofBrassica napus L., detected by the monoclonal antibody MPM-2

Summary The monoclonal antibody MPM-2, which interacts with a mitosis-specific phosphorylated epitope, has been used to study phosphorylation of proteins in microspores and pollen ofBrassica napus. One- (1-D) and two-dimensional (2-D) immunoblots revealed that MPM-2 recognized a family of phosphorylated proteins in freshly isolated microspores and pollen. The same set of phosphorylated proteins was found after 8 h of culture at embryogenie (32 °C) and non-embryogenic (18 °C) conditions. Two major spots were observed on 2-D immunoblots, one of which (M_r75 kDa, pI5.1) co-localized with the 70 kDa heat shock protein. Immunolabelling of sectioned microspores and pollen showed that MPM-2 reactive epitopes were predominantly observed in the nucleoplasm from G₁ until G2-phase, and in the cytoplasm during mitosis. This may be due to a cell cycle related translocation of phosphoproteins from the nucleus to the cytoplasm, or alternate phosphorylation and dephosphorylation in nucleus and cytoplasm. Detectability of epitopes on sections depended on the embedding procedure. Cryo processing revealed epitope reactivity in all stages of the cell cycle whereas polyethylene glycol embedded material showed no labelling in the cytoplasm during mitosis. Processing might reduce the antigenicity of cytoplasmic MPM-2 detectable proteins, probably due to dephosphorylation. The MPM-2 detectable epitope was observed in all cells investigated, irrespective of culture conditions, and its intracellular distribution depended on the cell cycle stage and was not related to the developmental fate of the microspores and pollen.     

Cortical ultrastructure of freeze-substituted protonemata of the mossFunaria hygrometrica

Summary The ultrastructural organization of the cortical cytoplasm has been examined in caulonemata, branches and buds of the mossFunaria hygrometrica, which were prepared by rapid freeze-fixation and freeze-substitution (FS). The same structural components occur in the cortex of all three cell types: microtubules (MTs), endoplasmic reticulum (ER), coated and uncoated vesicles, coated pits, and dictyosomes. However, the configuration and density of the cortical ER varies between the three. Caulonemata have an open, polygonal network of ER associated with long MTs oriented mostly parallel to the length of the cell. Lamellar ER, covered with polysomes, is interspersed in the network. Branches have a more tightly arranged ER network, at places occurring in a thick layer, and occasional polysome-decorated lamellae. MTs, which extend to the tip of the branch, are oriented mainly parallel to the cell's long axis and are associated with the cortical ER. Buds have the tightest ER network, which is frequently arranged in a thick layer. Tubules in the polygonal ER of buds are densely covered with ribosomes, whereas tubules in the ER network of caulonemata and branches range from nearly smooth to moderately rough. Closely-spaced ER lamellae, with many polysomes, occur in some buds. The MTs of buds extend into the apical dome and are associated with the cortical ER, but are more randomly oriented than in caulonemata or branches. Close appositions between the ER and PM are observed in all three cells, but are more frequent in buds.Abbreviations DiOC₆(3) 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - FS freeze-substitution - MT microtubule - MF microfilament - PM plasma membrane     

A Study on Pollen Morphology of Potamogeton,Zannichellia and Triglochin from China

The pollen morphology of 19 species in the genera Potamogeton, Zannichellia and Triglochin from China was examined under light and scanning electron microscopes. 1. Pollen characteristics (1) Potamogeton L. (Plate 1, 2:1-8) Pollen grains spheroidal, subspheroidal, rarely oblate or ovoid, (17-35)24.41×27.76(18-45)μm in size; anaperturate; exine 1-2μm thick, 2layered or obscure; crass-reticulate, reticulate or finely-reticulate under LM, distinctly reticulate under SEM; muri uniform or not uniform, smooth or granular, rarely with processes; bacula thicker or thinner at the intersection of muri. (2) Zannichellia L. (Plate 2:9) Pollen grains spheroidal, ,(20-26)23.1×24.15(21-27) μm in size; anaperturate; exine 1 μm thick, stratification obscure; nearly reticulate under LM, shallowly reticulate under SEM; luminae shallow, large and irregular, muri very thin and not uniform; bacula absent. (3) Triglochin L. (Plate 2:10-11) Pollen grains spheroidal or subspheroidal; (20-30)23.65×23.7(20-30) μm in size; anaperturate; exine about 1μm thick, stratification obscure, finely reticulate under LM, distinctly reticulate under SEM; luminae rather large or minute; muri uniform, with 1-2 lines of granules, bacula present or absent. 2. Pollen types of Potamogeton L. (1) Thin muri--thick bacula: Potamogeton pectinatus, P. intramongolicus, P. filiformis, P. pamiricus; (2) Uniform muri and bacula: 1) Large pollen subtype: Potamogeton crespus; 2) Medium-sized pollen subtype: Potamogeton heterophyllus, P. perfoliatus, P. distinctus, P. malaianus; 3) Minute pollen subtype: Potamogeton oxyphyllus, P. obtusifolius, P. lucens, P. cristatus, P. gramineus, P. octandrus, P. pusillus     

The HKM gene, which is identical to the MS1 gene of Arabidopsis thaliana, is essential for primexine formation and exine pattern formation

A male-sterile mutant of Arabidopsis thaliana was isolated by T-DNA tagging screening. Using transmission electron microscopy analysis, we revealed that the microspores of this mutant did not have normal thick primexine on the microspore at the tetrad stage. Instead, a moderately electron-dense layer formed around the microspores. Although microspores without normal primexine failed to form a proper reticulate exine pattern at later stages, sporopollenin was deposited and an exine-like hackly structure was observed on the microspores during the microspore stage. Thus, this mutant was named hackly microspore (hkm). It is speculated that the moderately electron-dense layer was primexine, which partially played its role in sporopollenin deposition onto the microspore. Cytological analysis revealed that the tapetum of the hkm mutant was significantly vacuolated, and that vacuolated tapetal cells crushed the microspores, resulting in the absence of pollen grains within the anther at anthesis. Single nucleotide polymorphism analysis demonstrated that the hkm mutation exists within the MS1 gene, which has been reportedly expressed within the tapetum. Our results suggest that the critical process of primexine formation is under sporophytic control .     

Significance of the cytoskeleton for cytoplasmic organization and cell organelle dynamics in epithelial cells of fresh-water sponges

Summary The cytoskeleton in epithelial cells ofSpongilla lacustris is constructed of microtubules radiating from the nuclear region and terminating at the cell periphery as well as microfilaments forming a cortical layer beneath the plasma membrane and distinct fibers in the cytoplasmic matrix. Single frame analysis and in vivo labeling of mitochondria with Rh 123, endosomes or lysosomes with TRITC-BSA, endoplasmic reticulum (ER) with DiOC₆ (3) and dictyosomes with C₆-NBD-ceramide points to the microtubular system as a candidate for controlled cytoplasmic organization and active transport of these cell organelles. In epithelial cells with an intact microtubular system, mitochondria and endosomes or lysosomes show a regular shuttle movement between the nucleus and the cell periphery with a velocity of 1.3–1.4 m/s; the ER forms a more or less dynamic two-dimensional network in the entire cytoplasmic matrix, and dictyosomes are arranged in a ring-like manner around the nucleus. In epithelial cells treated with colchicine or colcemid, mitochondria and endosomes or lysosomes gather in the perinuclear region and become immobile; the ER accumulates near the cell center, whereas most dictyosomes distribute randomly over the whole cytoplasm. Transformation of the microfilament system with cytochalasin D has no influence on cell organelle distribution and dynamics but impedes cell locomotion and cell surface activities.Abbreviations BSA bovine serum albumin - C₆-NBD-ceramide 6-[(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)caproyl]sphingosine - DiOC₆(3) 3,3-dihydroxyloxacarbocyanine jodide - DMSO dimethylsulfoxide - EGTA ethylenediaminetetraacetic acid - GTX glycerol-Triton-X-100 - PBS phosphate buffered saline - PEG polyethylene glycol - PIPES 1,4-piperazine-N,N-bis-(2-ethanesulfomc) acid - Rh 123 rhodamine 123 - TRITC tetramethylrhodamine isothiocyanate     

Mitosis and microtubule organizational changes in isolated generative cells of Allemanda neriifolia

Summary The organizational changes of the microtubules of isolated generative cells of Allemanda neriifolia during division were followed using anti--tubulin and immunofluorescence microscopy. Generative cells were isolated from the pollen tubes after osmotic shock treatment. Immediately after isolation most of the cells remain either in early or late prophase. The shape of the cell changes from spindle to spheroidal. In early prophase the nuclear membrane of the cell appears intact and the cytoplasm full of reticulate microtubules of different shapes and thicknesses. Later, the nuclear membrane breaks up. After the nuclear membrane has broken up, the chromosomes scatter into the cytoplasm and mix with the microtubules. When cells enter metaphase, spindle microtubules form. Afterwards, in anaphase, sister chromatids separate and the spindle disappears. A new array of longitudinally oriented cage microtubules appears. As the cells enter early telophase, the cage microtubules disappear and an array of interpolar microtubules begins to form. Later, in some telophase cells the interpolar microtubules become highly elongated, but in others they soon disappear and become replaced by a thick band(s) (or sheet(s)) of microtubules in the midplane between the two clusters of chromosomes and the cell shape reverts back to spheroidal. In culture no phragmoplast junctions appear in any of the late telophase cells although they are present under the in situ condition (i.e. in pollen tubes).     

Pollen Morphology of Guttiferae in China

The present paper deals with the pollen morphology of 23 species belonging to 6 genera of Guttiferae occurred in China. All pollen grains have been observed under light microscope, and those of 11 species in 5 genera have been examined with SEM as well. The pollen grains of Guttiferae are subspheroidal, spheroidal or prolate, (17–43) × (15–26)μ in size. Mostly 3-eolporate, less frequently 4-colporate or 5-colporate. The exine is 2-layered, but the occassionally demarcation of these layers is generally indistinct. The exine is 0.8–2.6 μ in thickness. The oramentations of all the pollen grains is generlly finely reticulate under the light microscope. Pollen morphology of the 11 species of 5 genera observed under the SEM is stressed. Their exine oramentations may be classified into four major groups, ie. reticulate, finely reticulate, baculate and perforate. Calophyllum: pollen grains reticulate and rugulo-reticulate. Cratoxylon: pollen grains reticulate, with finely and densely granulate over entire muri. Gareinia: pollen grains baculate, reticulate, and exine with perforate oramentation; in reticulate grains, muri with spinulation. Hypericum: pollen grains reticulate, perforate and finely reticulate. Mesua: pollen grains finely reticulate. From the comparison of pollen morphology, Hypericaceae has much connection with Guttiferae (sensu stricto). Because their pollen grains are all 3-eolporate and the exine is reticulate or oerforate under light mieroseope, but it is different between the pollen grains of Hypericum and those of Guttiferae.