A cytoskeletal system predicts division plane in meiosis ofSelaginella

Summary An ultrastructural investigation of the monoplastidic microsporocytes ofSelaginella arenicola revealed a unique cytoskeletal array that predicts the future division plane before nuclear division takes place. By midprophase of the first meiotic division, the single plastid has divided once and the two plastids lie on opposite sides of the nucleus which is elongated in the plane of the incipient metaphase I spindle. A cytoplasmic structure, the procytokinetic plate (PCP), predicts the division plane of of both plastid and cytoplasm. The PCP consists of a distinct concentration of vesicles lying in the future division plane and an elaborate system of microtubules aligned parallel to the long axis of plastids and nucleus. Microtubules of the axially aligned system appear to terminate in clusters of vesicles in the central zone of the PCP. The PCP with axially aligned microtubules is as predictive of the division plane in these meiotic cells as is the girdling preprophase band of microtubules in mitotic cells.     

Ultrastructural analysis of sporoderm development in megaspores ofSelaginella galeottii (Lycophyta)

Structural members within the exospore ofSelaginella galeottii suggestive of those present at maturity are first detectable when the exospore is approximately 5 µm in thickness. Subsequent changes in successively larger sporangia involve a gradual size increase of the component units simultaneously throughout the exospore. Concomitantly, non-membrane bound material present at the inner surface of the tapetum (and the persistent megasporocytes) and throughout the sporangium locule changes from primarily droplets and weftlike material (including beaded wefts) to coarse fibrous material. The taxa which possess this unusual wall pattern cut across presently accepted taxonomic schemes. This is not the case with the other wall ultrastructural types in the genus. The possibility exists that this megaspore wall type defines a separate lineage within the genus which, by virtue of its large megaspores, was able to compete well and radiate to produce a variety of life forms.     

Division polarity,development and configuration of microtubule arrays in Bryophyte meiosis

Summary Immunofluorescence and TEM studies of meiosis in two mosses (Bryophyta) provide evidence that the prophasic tetrahedral system of microtubules contributes directly to the metaphase I spindle. Intense staining of tubulin, conspicuously absent around the nuclear envelope, is first seen associated with plastids. By mid-prophase, microtubules radiate from the plastids to the nuclear envelope and become organized into six bands that interconnect the four plastids, forming a tetrahedral cytoskeleton surrounding the nucleus. During transition of prophase to metaphase, the four poles of the tetrahedral microtubule system converge in pairs toward opposite cleavage furrows. Opposite furrows occupy mutually perpendicular planes and the pair of microtubule focal points straddling one furrow lies at right angles to the pair straddling the opposite furrow. Additional microtubules terminate in numerous small clusters in the concave polar regions arching over the cleavage furrows. By early anaphase, the microtubule focal points lie very close to the division axis. We conclude that microtubules recruited from the prophasic quadripolar system are incorporated into the mature metaphase I spindle and the two principal focal points at each pole are those derived from poles of the prophasic quadripolar system.     

Comparative karyomorphology and interrelationships ofSelaginella in Japan

Karyomorphological comparisons were made of 16 native and cultivated species ofSelaginella in Japan. The somatic chromosome numbers are 2n=16 inS. boninensis; 2n=18 inS. doederleinii, S. helvetica, S. limbata, S. lutchuensis, S. nipponica, S. selaginoides, S. tama-montana, andS. uncinata; 2n=20 inS. biformis, S. involvens, S. moellendorffii, S. remotifolia, andS. tamariscina; 2n=30 inS. rossii; and 2n=32 inS. heterostachys. The interphase nuclei of all species examined are uniformly assigned to the simple chromocenter type. The metaphase karyotype of 2n=16 (x=8) is 8 m (=median centromeric chromosomes)+8(st+t)(=subterminal and terminal). The group of the species having 2n=18 (x=9) is heterogeneous karyomorphologically: The karyotype ofS. nipponica is 2n=18=6 m+12(st+t),S. tama-montana 10 m+2 sm(=submedian)+6(st+t), andS. uncinata 6 m+7 sm+5(st+t). Although the remaining five species have the common karyotype 8 m+4 sm+6(st+t), the values of mean chromosome length are variable. Another group of the specles having 2n=20 (x=10) is homogeneous, since all species have the same karyotypes 8 m+4 sm+8(st+t) and have similar chromosome size. The karyotype of 2n=30 is 12 m+6 sm+12(st+t) and is suggested to be a triploid of x=10, and 2n=32=16m+16(st+t), a tetraploid of x=8. Thus, three kinds of basic chromosome numbers, x=8, 9, 10 are present in JapaneseSelaginella examined, and their karyomorphological relationships are discussed.     

Plastid apportionment and preprophase microtubule bands in monoplastidic root meristem cells ofIsoetes andSelaginella

Summary Ultrastructural observations on monoplastidic root tip cells ofIsoetes andSelaginella demonstrate two important phenomena associated with preprophasic preparation for mitotic cell division, 1. the preprophase band and 2. precise orientation of the dividing plastid relative to the preprophase band. Both of these phenomena accurately predict the future plane of cell division. The plastid divides in a plane parallel to the spindle and each cell inherits a single plastid which caps the telophase nucleus. When succesive transverse divisions occur, the plastid migrates prior to prophase from a position near an old transverse wall to a lateral position in the cell. The plastid is oriented with its median constriction precisely intersected by the plane of the preprophase band. When a longitudinal division follows a transverse division, the plastid remains in its position adjacent to an old transverse wall where it is bisected by the plane of the longitudinally oriented preprophase band microtubules.     

Preprophasic microtubule systems and development of the mitotic spindle in hornworts (Bryophyta)

Summary Studies of monoplastidic mitosis in hornworts (Bryophyta) using transmission electron microscopy and indirect immunofluorescence staining of microtubules have revealed that two mutually perpendicular microtubule systems predict division polarity in preprophase. Events of cytoplasmic reorganization in preparation for division occur in the following order: migration of the single plastid to a position perpendicular to the division site, constriction of the plastid where its midpoint intersects the division site, development of an axial system of microtubules parallel to the elongating plastid isthmus, and appearance of an atypical preprophase band of microtubules (PPB). The PPB is asymmetrical with a tight band of microtubules on the side over the plastid isthmus and a broad band of widely spaced microtubules over the nucleus. The axial system contributes directly to development of the spindle. In prometaphase, the axial system separates at the equator and additional microtubule bundles project from polar regions, creating two opposing halfspindles. The PPB is still present during asymmetrical organization of the spindle and microtubules extending from the broad portion of the PPB to poles appear to be incorporated into the developing spindle. Dynamic changes in the microtubular cytoskeleton demonstrate (1) intimate relationship of plastid and nuclear division, (2) contribution of preprophase/prophase microtubule systems to spindle development in monoplastidic cells, and (3) dynamic reorientation of microtubules from one system to another.     

Plastid differentiation during Cucurbita pepo (Cucurbitaceae) pollen grain development

The development of plastids in the pollen of Cucurbita pepo was followed from meiosis to pollen maturation by quantitative light and electron microscopy. Plastids are initially undifferentiated, then divide, and at late microspore stage differentiate into amyloplasts containing starch. Later the amyloplasts form lobes and divide. Amyloplasts containing a single starch grain are present from the early bicellular stage. Plastid development is considered in relation to such cytoembryological features as the pollen does not dehydrate at anthesis and germination begins 3 min after pollination.     

Application of confocal microscopy to exospore ultrastructure in megaspores ofSelaginella convoluta andS. marginata

Information pertaining to the arrangement of the rodshaped units that form the exospore ofSelaginella convoluta (Walk. Arn.) Spring andS. marginata (Humb. & Bonpl.) Spring megaspores was obtalned using both a confocal laser scanning microscope and a transmission electron microscope. Units are helically coiled, as we interpret them. The highest levels of fluorescence with confocal microscopy were in the places where the coiled exospore units that contain the fluorochrome dye overlap. These sites of overlap occurred in a close packed (hexagonal or pentagonal) arrangement. A more-or-less circular central nonfluorescent area was contiguous between overlapping exospore units. We conclude that the space between exospore units is a continuous channel (conduit). Each conduit is embraced by five or six helical-units that interdigitate.     

Anther development, microsporogenesis and microgametogenesis in Heliconia (Heliconiaceae, Zingiberales)

Anther development, microsporogenesis and microgametogenesis in several species of Heliconia were investigated as part of a complementary embryological study of the Heliconiaceae. All studied Heliconia species present bithecate and tetrasporangiate anthers with fertile pollen grains; only H. rivularis, a natural hybrid, presented sterile pollen grains of variable size and no content. The anther wall has an uniseriate epidermis and endothecium, the latter with helicoidal thickenings, although some cells of the middle layers also showed thickenings; the biseriate tapetum is of amoeboid non-syncytial type, since the tapetum cells did not fuse together forming a true plasmodium. The microsporogenesis is successive leading to isobilateral tetrads. The inaperturate pollen grains had a very reduced exine consisting of a thin, more or less continuous layer with small spines upon; the pollen grain shape is variable among the species, all of them presenting heteropolar pollen, except H. angusta with isopolar ones. Most of these characteristics were shared with other studied Zingiberales, although more studies need to be done.     

Changes in calcium and calmodulin levels during microsporogenesis,pollen development and germination in Gasteria verrucosa (Mill.) H. Duval

Summary The distribution of membrane calcium and calmodulin (CaM) has been fluorimetrically determined in the anther of Gasteria verrucosa with particular attention to sporogenous cells, meiocytes, microspores, pollen and stages of pollen germination and tube growth using chlortetracycline (CTC) and fluphenazine (FPZ). CTC and FPZ fluorescence in sporogenous cells is relatively higher than in the adjacent tapetal cells, indicating higher membrane calcium and CaM levels in the former cell type. However, during meiosis there is a significant increase in membrane calcium and CaM levels in the meiocytes compared to that found in the young microspores. CTC and FPZ fluorescence in the sporogenous cells, meiocytes and young microspores is punctate and slightly diffused throughout the cytoplasm. In the microspores of the tetrad and the young released microspores CTC fluorescence (CTCf) is polarized and mainly associated with the area opposite the future colporal region. FPZ fluorescence (FPZf) becomes polarized in the young microspore. Subsequently, there is a shift in the polarity, and most of the CTCf and FPZf in the old microspores and pollen is regionalized towards the colporal region, and the fluorescence is more diffused, indicating a change in the organellar-bound calcium and CaM. This final graded distribution of CTCf is maintained during pollen germination in that the growing pollen tubes invariably show a tip to base membrane-calcium gradient. In the tapetal cells a high level of Ca²⁺ is present during the microspore stage. During the preparation for anthesis the endothecium differentiation is marked by the presence of Ca²⁺. Post-treatment of labelled cells with a Ca²⁺ chelator such as EGTA resulted in a substantial decrease in diffuse and punctate CTCf. Alternatively, treatment of cells with non-ionic detergent Nonidet P-40 resulted in the total elimination of CTCf, suggesting that the observed CTC fluorescence was due to membrane-associated calcium. The cytological specification of CTC as a probe for calcium is discussed. From cytofluorometric measurements and atomic absorption, it became clear that the level of Ca²⁺ in the anther is high during the sporogenous and meiotic phases. An increase in CTCf and FPZf occurred after microspore mitosis. An interaction of Ca²⁺ transport from tapetum to the young pollen is postulated. These findings suggest that the level of Ca²⁺ in the anther during meiosis is generally relatively higher than at the sporogenous or young microspore stage. These findings are discussed in the light of available information on the role of Ca²⁺ and CaM-mediated processes such as cell division, callose synthesis and pollen-tube tip growth.     

Developmental anatomy of the shoot apical cell,rhizophore and root ofSelaginella uncinata

The developmental anatomy of the shoot apex, rhizophore and root ofSelaginella uncinata was examined by the semi-thin section method. The shoot apex has a single, lens-shaped apical cell with two cutting faces. Rhizophore primordia are initiated exogenously at the branching point of the second youngest lateral shoot. The rhizophore apex has a tetrahedral apical cell with three cutting faces. A pair of root primordia is initiated endogenously from inner cells of the rhizophore apex, after the rhizophore apical cell becomes unidentifiable losing its activity, and subsequently a root cap is formed from the distal face of the root apical cell. During the course of successive root branching the apical cell in an original root apical meristem becomes unidentifiable and then a new apical cell is initiated in each of the bifurcated root apical meristems. The root branching mode seems to be equivalent to the described dichotomous branching mode of fern shoots. Our results demonstrate a distinct morphogenetical difference between the rhizophore and the root, and confirm the exogenous origin of the rhizophore, as described for other species ofSelaginella. This evidence indicates that the rhizophore is not an aerial root but a leafless, root-producing axial organ.     

Plastid marker gene excision by the phiC31 phage site-specific recombinase

Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.     

Morphogenetic plastid migration and microtubule organization during megasporogenesis inIsoetes

Summary The large megasporocytes ofIsoetes provide an exceptional system for studying microtubule dynamics in monoplastidic meiosis where plastid polarity assures coordination of plastid and nuclear division by the intimate association of MTOCs with plastids. Division and migration of the plastid in prophase establishes the tetrahedrally arranged cytoplasmic domains of the future spore tetrad and the four plastid-MTOCs serve as focal points of a unique quadripolar microtubule system (QMS). The QMS is a dynamic structure which functions in plastid deployment and contributes directly to development of both first and second division spindles. The nucleation of microtubules at discrete plastid-MTOCs is compared with centrosomal nucleation of microtubules in animal cells where growth of microtubules involves dynamic instability.Abbreviations AMS axial microtubule system - MTOC microtubule organizing center - N nucleus - QMS quadripolar microtubule system - P plastid - PPB preprophase band of microtubules     

Plastid ultrastructure and photosynthesis in greening petaloid hypsophylls

The ultrastructural and biochemicalphysiological aspects of postfloral greening have been studied in hypsophylls of Heliconia aurantiaca Ghiesbr., Guzmania cf. x magnifica Richter and Spathiphyllum wallisii Regel. In all three species the greening of the hypsophylls is due to plastid transformation, chloroplast formation proceeding from the initially different types of plastids. The degradation process of the original plastid structures and the mode of thylakoid formation are distinct in each case. In none of the species do the transformed plastids look identical to the chloroplasts of the corresponding foliage leaves. On a chlorophyll basis, the rate of photosynthesis of the greened hypsophylls surpasses the rate of the leaves considerably in Spathiphyllum, but is much lower in Heliconia (no data for Guzmania). In all species, anatomy, plastid structure, pigments, 77° K-fluorescence emission, ribulose-1,5-bis-phosphate carboxylase activities and short-term photosynthesis ¹⁴CO₂-assimilation patterns prove the greened hypsophylls to be capable of providing additional carbon to the developing fruits, thus supplementing the import of organic matter from the foliage leaves.Abbreviations MDH malate dehydrogenase (EC 1.1.1.37) - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)     

Sequence of the Tomato Chloroplast DNA and Evolutionary Comparison of Solanaceous Plastid Genomes

Tomato, Solanum lycopersicum (formerly Lycopersicon esculentum), has long been one of the classical model species of plant genetics. More recently, solanaceous species have become a model of evolutionary genomics, with several EST projects and a tomato genome project having been initiated. As a first contribution toward deciphering the genetic information of tomato, we present here the complete sequence of the tomato chloroplast genome (plastome). The size of this circular genome is 155,461 base pairs (bp), with an average AT content of 62.14%. It contains 114 genes and conserved open reading frames (ycfs). Comparison with the previously sequenced plastid DNAs of Nicotiana tabacum and Atropa belladonna reveals patterns of plastid genome evolution in the Solanaceae family and identifies varying degrees of conservation of individual plastid genes. In addition, we discovered several new sites of RNA editing by cytidine-to-uridine conversion. A detailed comparison of editing patterns in the three solanaceous species highlights the dynamics of RNA editing site evolution in chloroplasts. To assess the level of intraspecific plastome variation in tomato, the plastome of a second tomato cultivar was sequenced. Comparison of the two genotypes (IPA-6, bred in South America, and Ailsa Craig, bred in Europe) revealed no nucleotide differences, suggesting that the plastomes of modern tomato cultivars display very little, if any, sequence variation. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Rüdiger Cerff]     

Down the Slippery Slope: Plastid Genome Evolution in Convolvulaceae

Cuscuta (dodder) is the only parasitic genus found in Convolvulaceae (morning-glory family). We used long PCR approach to obtain large portions of plastid genome sequence from Cuscuta sandwichiana in order to determine the size, structure, gene content, and synteny in the plastid genome of this Cuscuta species belonging to the poorly investigated holoparasitic subgenus Grammica. These new sequences are compared with the tobacco chloroplast genome, and, where data are available, with corresponding regions from taxa in the other Cuscuta subgenera. When all known plastid genome structural rearrangements in parasitic and nonparasitic Convolvulaceae are considered in a molecular phylogenetic framework, three categories of rearrangements in Cuscuta are revealed: plesiomorphic, autapomorphic, and synapomorphic. Many of the changes in Cuscuta, previously attributed to its parasitic mode of life, are better explained either as plesiomorphic conditions within the family, i.e., conditions shared with the rest of the Convolvulaceae, or, in most cases, autapomorphies of particular Cuscuta taxa, not shared with the rest of the species in the genus. The synapomorphic rearrangements are most likely to correlate with the parasitic lifestyle, because they represent changes found in Cuscuta exclusively. However, it appears that most of the affected regions, belonging to all of these three categories, have probably no function (e.g., introns) or are of unknown function (a number of open reading frames, the function of which, if any, has yet to be discovered).[Reviewing Editor: Dr. Debashish Bhattacharya]     

Plastid development in seedlings of Echinochloa crus-galli var. oryzicola under anoxic germination conditions

Plastid development in the primary leaf of Echinochloa crus-galli (L.) Beauv. var. oryzicola (Vasing.) Ohwi was followed during 5 d of anoxic germination and growth. Plastids develop slowly from simple spheroidal proplastids into larger pleomorphic plastids with several stromal membranes and many peripheral membrane vesicles. A small prolamellar body is present at 96 h with perforated (pro)thylakoids extending into the stroma. Changes in starch grains and plastoglobuli are evidence of carbohydrate and lipid metabolism. Plastid division is indicated by dumbbell plastid profiles after 4 d of anoxia. These results demonstrate that plastids not only maintain their integrity during anaerobic germination but also show developmental changes involving an increase in internal membrane complexity, although to a lesser extent than in etiolated shoots.Abbreviation PLB prolamellar body Scientific paper No. 6167. College of Agriculture, Washington State University, Pullman     

A new type of microtubular cytoskeleton in microsporogenesis of Lavatera thuringiaca L.

Summary. In Lavatera thuringiaca, kariokinesis and simultaneous cytokinesis during the meiotic division of microsporogenesis follow a procedure similar to that which takes place in the majority of members of the class Angiospermae. However, chondriokinesis occurs in a unique way found only in species from the family Malvaceae. Chondriokinesis in such species is well documented, but the relationship between the tubulin cytoskeleton and rearrangement of cell organelles during meiosis in L. thuringiaca has not been precisely defined so far. In this study, the microtubular cytoskeleton was investigated in dividing microsporocytes of L. thuringiaca by immunofluorescence. The meiotic stages and positions of cell organelles were identified by staining with 4′,6-diamidino-2-phenylindole. We observed that, during prophase I and II, changes in microtubular cytoskeleton configurations have unique features, which have not been described for other plant species. At the end of prophase I, organelles (mostly plastids and mitochondria) form a compact envelope around the nucleus, and the subsequent phases of kariokinesis take place within this arrangement. At this point of cell division, microtubules surround the organelle envelope and separate it from the peripheral cytoplasm, which is devoid of plastids and mitochondria. In telophase I, two newly formed nuclei are tightly surrounded by the cell organelle envelopes, and these are separated by the phragmoplast. Later, when the phragmoplast disappears, cell organelles still surround the nuclei but also move a little, starting to occupy the place of the disappearing phragmoplast. After the breakup of tetrads, the radial microtubule system is well developed, and cell organelles can still be observed as a dense envelope around the nuclei. At a very late stage of sporoderm development, the radial microtubule system disappears, and cell organelles become gradually scattered in the cytoplasm of the microspores. Using colchicines, specific inhibitors of microtubule formation, we investigated the relationship between the tubulin cytoskeleton and the distribution of cell organelles. Our analysis demonstrates that impairment of microtubule organization, which constitutes only a single component of the cytoskeleton, is enough to disturb typical chondriokinesis in L. thuringiaca. This indicates that microtubules (independent of microfilaments) are responsible for the reorganization of cell organelles during meiotic division. Correspondence: D. Tchórzewska, Department of Plant Anatomy and Cytology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland.     

Stable Plastid Transformation in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes—aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)—in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species. Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable to investigations of the interactions between plastid and nucleus in N. benthamiana.     

Microtubules are involved in maintaining the cellular polarity in pollen tubes ofNicotiana sylvestris

Summary The polarity of a growing pollen tube is clearly reflected by a distinct zonation of the cytoplasmic content. The vegetative nucleus and the generative cell (GC) are located in the tip region of the tube, and the basal cytoplasmic portion is highly vacuolated. Using pollen tubes ofNicotiana sylvestris Spegazz. & Comes grown in vitro, we examined the effects of varying concentrations of the microtubule inhibitors colchicine and propham. The depolymerization of the cortical microtubules by 25 M colchicine led to a disorganization of the cytoplasm, i.e., vacuolization of the tip region, and to a deranged position of both the vegetative nucleus and the generative cell. The same concentration of colchicine inhibited tube growth by 10–20% of the control. Mitosis of the GC was not affected. Only from concentrations of 200 M the configuration of the GC's microtubules was altered and an inhibition of mitosis was observed. At this concentration the disorganization of the cytoplasm was always reversible, but neither inhibition of mitosis nor derangement of the nuclear positioning was. At 1,800 M colchicine, pollen tube growth was inhibited by 50% of the control. Using propham, the same three steps of action were observed, although propham proved to be about a hundred times more effective than colchicine. We conclude that the cortical microtubules of the pollen tube are involved in maintaining cellular polarity, probably as a part of a heterogeneous cytoskeletal network including also microfilaments and membranous elements. Nuclear positioning seems to be dependent on both, the tube's cortical and the GC's microtubules. A possible involvement of the extracellular matrix in maintaining intracytoplasmic polarity is suggested.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EGTA ethyleneglycol-bis-(aminoethyl ether) tetraacetic acid - GC generative cell - MF microfilament - MT microtubule - PEM-buffer 50 mM PIPES, 1 mM EGTA, 2 mM MgSO₄, pH 6.9 - PBS phosphate buffered saline - PIPES piperazine-bis-ethanesulphonic acid - PTG-test pollen tube growth test - VN vegetative nucleus Dedicated to Professor Peter Sitte on the occasion of his 65th birthday