VARIATIONS IN MICROSPORANGIA AND MICROSPORE DISPERSAL IN SELAGINELLA

Fifty-three species of Selaginella were examined for variations in microsporangium anatomy and microspore dispersal strategies. Five anatomically different kinds of microsporangia were observed, displaying three different strategies for microspore dispersal. Xeric species, including all isophyllous species, have microsporangia that only dry and shrink as they dehisce, resulting in microspores being passively dispersed. Among anisophyllous species from tropical and subtropical habitats some species actively eject their microspores, while others of the series Articulatae eject the entire microsporangium. These two types of active ejection both utilize a mechanism similar to that found in fern leptosporangia.     

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 .     

Regulation of developmental pathways in cultured microspores of tobacco and snapdragon by medium pH

The regulation of developmental pathways in cultured microspores of tobacco (Nicotiana tabacum L) and snapdragon (Antirrhinum majus L) by medium pH is described for the first time. Unicellular tobacco and snapdragon microspores developed into normal, fertile pollen when cultured in media T1 and AT3 at pH 7.0 and 25°C for 6 and 8 days, respectively. First, pollen mitosis was asymmetric and mature pollen grains were filled with starch granules and germinated upon transfer to a germination medium. However, when tobacco and snapdragon microspores were cultured in media T1 and AT3, respectively, at pH 8.0–8.5 for 4–6 days at 25 °C, the frequency of symmetric division increased significantly with the formation two nuclei of equal size, and the gametophytic pathway was blocked, as seen by the lack of starch accumulation and the inhibition of pollen germination. The transfer of these microspores to embryogenesis medium AT3 at pH 6.5 resulted in the formation of multicellular structures in both species and, in tobacco, in the formation of embryos and plants. In order to understand the possible mechanisms of the action of high pH, sucrose metabolism was analysed in isolated microspores of tobacco cultured at various pH values. Invertase (EC 3.2.1.26) activity in microspores was maximal at pH 5.0 and strongly decreased at higher pH, leading to a slow-down of sucrose cleavage. At the same time the incorporation of ¹⁴C-labelled sucrose from the medium into microspores was drastically reduced at high pH. These data suggest that isolated microspores are not able to metabolise carbohydrates at high pH and thus undergo starvation stress, which was shown earlier to block the gametophytic pathway and trigger sporophytic development.     

Cryopreservation of isolated micropores of spring rapeseed (Brassica napus L.) for in vitro embryo production

Microspore cryopreservation is a potentially powerful method for long-term storage of germplasm for in vitro embryo production in plant species. In this study, several factors influencing embryo production following the ultra-low temperature (–196 °C in liquid nitrogen) storage of isolated microspores of rapeseed (Brassica napus L.) were investigated. Microspores were prepared in cryogenic vials and subjected to various cooling treatments before immersion in liquid nitrogen for varying periods. Efficiency of microspore cryopreservation was reflected by in vitro embryo production from frozen microspores. Of all the cooling treatments, microspores treated with a cooling rate of 0.25% °C/min and a cooling terminal temperature of –35 °C before immersion in liquid nitrogen produced the highest embryo yields (18% and 40% of unfrozen controls in two genotypes, respectively). Fast thawing in a 35 °C water bath was necessary to recover a high number of embryos from microspore samples being frozen at a higher cooling rate, while thawing speed did not affect samples after freezing at a slower cooling rate. The storage density of cryopreserved microspores affected embryo production. Storage at the normal culture density (8×10⁴ microspores/ml) was less efficient for embryo production than at high densities (4×10⁶ microspores/ml and 1.6×10⁷ microspores/ml), although no significant difference was found between the high densities. Evaluation of plant lines derived from frozen microspores indicated no variation in isozyme pattern and no enhanced cold tolerance of these lines. Isolated microspores of B. napus could be stored for extended period for in vitro embryo production.     

Octad pollen formation in Cymbopetalum (Annonaceae): the binding mechanism

Our recent study of tetrad pollen formation in Annona (Annonaceae) revealed that after meiosis the callose-cellulose envelope forms a special conjugation with individual microspores and the forthcoming callose digestion is incomplete. The undigested part forms a central binder holding the four microspores of the tetrad together. This process causes the microspores to rotate 180 degrees. In this paper we describe pollen formation in another annonaceous genus, Cymbopetalum, in which the pollen is shed in octads, through use of light microscopy, epifluorescence microscopy, and TEM. In Cymbopetalum, two meiocytes, connected by abundant cytomictic channels, are produced in each sporangium. Octad pollen formation in Cymbopetalum is shown to be comparable to the synchronized formation of two connected Annona tetrads, which then integrate into a single octad. Unique features of Annona polyad formation, e.g. special binding between the callose-cellulose envelopes and microspores, incomplete callose digestion, and microspore rotation, also occur in Cymbopetalum. In addition, formation of the Cymbopetalum octad involves development of a cushion-like structure that binds the distal pronexine of all eight microspores, and there is the production of intine protrusions. The evolutionary origin of the callose-cellulose binding mechanism within the family is discussed.     

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     

Biosystematic studies on the genusIsoetes (Isoetaceae) in Japan. III. Variability within qualitative and quantitative morphology of spores

Qualitative and quantitative characteristics of mega- and microspores from all the cytotypes of JapaneseIsoetes are described based on the voucher specimens whose chromosome numbers were known. InI. japonica, the hexaploid possessed reticulate megaspores and levigate microspores, while the octaploid and the heptaploid had echinate microspores. Mega- and microspores of the hexaploid and the octaploid were of normal appearance, while those of the heptaploid displayed polymorphism. The tetraploid and the hexaploid ofI. sinensis resembled each other, since they both possessed cristate megaspores and echinate microspores. Echinate megaspores and levigate microspores characterized the diploidI. asiatica. The spore size was largely variable within each cytotype, while the size of the megaspores varied more than that of the microspores. The microspore length was closely correlated with polyploid level. InI. sinensis, the mean microspore length of the tetraploid was 27.6 μm while that of the hexaploid was 31.9 μm, hence these two cytotypes were easily distinguishable. In the hexaploidI. japonica, variations in mega-and microspore size displayed geoclinal variation showing a positive correlation (r=0.43–0.55) with the longitude and the latitude of the populations. A palynological key for cytotypes is presented.     

Cytological analysis of early microspore divisions leading to gametic embryo formation in <Emphasis Type="Italic">Quercus suber</Emphasis> L. anther cultures

The correlation between the phenologic stage of the inflorescence and the microspore development stage was studied. Cytological examinations of the development of microspores during in vitro anther culture of cork oak (Quercus suber L.), were carried out during the first four weeks of culture. To observe the division occurring in the microspores, anthers were taken randomly from the cultures after heat shock treatment and were stained with DAPI. Most of the anthers responding to a heat stress treatment contained 91 % vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in cork-oak microspores. After the heat shock treatment some cork-oak microspores were induced and initiated the embryogenic pathway with the occurrence of numerous symmetric mitosis, producing structures with two to ten or more nuclei. These lead to the formation of high numbers of multicellular cork-oak microspores (pro-embryos). Twenty-forty days after induction, small white globular and cotyledonal embryos were observed, which further developed root and shoot, regenerating plantlets.     

海南七仙岭7种卷柏属植物的微观形态与生境间的关系分析

为探讨生境对卷柏属(Selaginella)植物微观形态的影响,利用扫描电子显微镜对海南七仙岭采集的7种卷柏属植物的侧叶、中叶、孢子叶的叶表皮形态以及小孢子形态进行全面观察分析,并计算气孔器大小、气孔密度、孢子大小等,比较分析其微观形态的区别及微形态与生境间的关系,为卷柏属植物的分类提供依据。结果显示:(1)同种卷柏属植物的侧叶、中叶与孢子叶在叶表皮形态上具明显差异,尤其是孢子叶上的气孔与营养叶相对较小且稀疏,与孢子叶的繁殖功能相符合。(2)不同种卷柏属植物的叶表皮特征也明显不同,主要表现在叶缘刺、气孔和瘤状突起上,表明这些特征可以作为卷柏属植物种间区分的依据。(3)卷柏属植物的小孢子形态稳定,纹饰多样;部分种间的小孢子形态相似,但可通过纹饰类型、裂缝的曲直进行区分。(4)琼海卷柏的小孢子具有独特的网状纹饰,暗示其具有独特的演化途径。(5)卷柏属植物叶表皮的气孔特征、瘤状突起特征,小孢子的颜色、纹饰,与海拔、生境的湿度有一定的相关性,但其形成机理有待进一步研究。     

Development of microsporesin vivo andin vitro inBrassica napus L.

Summary Populations of highly homogeneous uninucleate and binucleate microspores ofBrassica napus cv. Topas were obtained by bud selection and percoll fractionation. The development of the uninucleate and the binucleate microspores in culture was compared to thosein vivo using the fluorochrome DAPI to stain DNA. The major developmental pathway of the uninucleate microsporesin vitro resulted in embryo formation. The characteristic of this pathway was that the first division produced two diffusely stained nuclei and subsequent divisions gave rise to a multinucleate embryoid. The second pathway which occurred in a small number of the uninucleate microspores led to callus formation. The majority of the binucleate microsporesin vitro followed the developmental pattern of their counterpartsin vivo and were not embryogenic. The embryogenic binucleate microspores produced embryos through the divisions of the vegetative nucleus.Plant Research Centre Contribution # 1147     

凤仙花花药发育中多糖和脂滴组织化学研究

以不同发育时期的凤仙花花药为实验材料,采用组织化学方法,对花药发育中的结构变化及多糖和脂滴物质分布进行观察。结果表明:(1)凤仙花的花药壁由6层细胞组成,包括1层表皮细胞,2层药室内壁细胞,2层中层细胞和1层绒毡层细胞。其中绒毡层细胞的形态不明显,很难与造孢细胞区分,且在小孢子母细胞时期退化。(2)在小孢子母细胞中出现了一些淀粉粒,但减数分裂后,早期小孢子中的淀粉粒消失,又出现了一些小的脂滴;随着花粉的发育,小孢子形成大液泡,晚期小孢子中的脂滴也消失;小孢子分裂形成二胞花粉后,营养细胞中的大液泡降解、消失,二胞花粉中又开始积累淀粉;接近开花时,成熟花粉中充满细胞质,其中包含了较多的淀粉粒和脂滴。(3)在凤仙花的花药发育中,绒毡层细胞很早退化,为小孢子母细胞和四分体小孢子提供了营养物质;其后的中层细胞退化则为后期花粉发育提供了营养物质。     

Fern-associated arbuscular mycorrhizal fungi are represented by multiple Glomus spp.: do environmental factors influence partner identity?

Symbioses involving arbuscular mycorrhizal fungi (AMF) are among the most important ecological associations for many plant species. The diversity of AMF associated with ferns, however, remains poorly studied. Using recently designed Glomus-specific primers, we surveyed the AMF community associated with ferns from deciduous, broad-leaved second-growth forest habitats at the eastern edge of the piedmont region of central Virginia, USA. Results indicate that this molecular approach may be a useful tool for detecting AMF in ferns compared to traditional techniques based on morphology. Over 30 potential fungal ribotypes were identified from eight fern species using denaturing gradient gel electrophoresis. Fungal ribotypes were found to differ widely in terms of (1) the number of fern partners with which they interact and (2) their relative frequency within each fern. Sequence analysis of fungal isolates from three species of fern indicated that the primers were generally highly specific for Glomus species but some non-target DNA was also amplified. Cloned polymerase chain reaction (PCR) products from Polystichum acrostichoides and Osmunda regalis revealed several phylogenetically distinct Glomus species. A single Glomus species was identified in the cloned PCR products from Botrychium virginianum. These findings challenge the hypothesis that the extent or degree of fern–fungal symbiosis is somehow tied to root complexity. Environmental factors appear to influence the suite of AMF that form partnerships with ferns. Some species of fern from similar habitats associated with dissimilar fungal partners (e.g., P. acrostichoides and Athyrium filix-femina var. asplenioides), whereas others harbored uniform fungal communities (e.g., Asplenium platyneuron). The significance of these data in terms of ecological and evolutionary dynamics of the AMF–fern symbiosis is discussed. Brittany West, Jessica Brandt, and Kay Holstien contributed equally to this work.     

Manipulation of division symmetry and developmental fate in cultures of potato microspores

The effect of media composition on microspore culture was investigated in one tetraploid and two diploid potatoes. The viability of microspores isolated from 4.5 to 5 mm buds was in the range of 33 to 52%. In media for anther culture, microspores showed no further development and lost viability within 2 days. In M1 medium containing mineral components, sucrose, uridine, cytidine, myo-inositol, glutamine and lactalbumin hydrolysate, 18 to 37% of microspores underwent mitosis within 14 days. Up to 95% of the divisions were symmetric and produced equal nuclei. Some symmetrically divided microspores eventually produced structures with 3 to 10 nuclei. The proportion of the total microspore population producing multinuclear structures reached 9% in diploid clones responsive to anther culture and 1 to 2% in recalcitrant cv. Borka. Symmetric mitoses in M1 medium were induced in the presence of glutamine and lactalbumin hydrolysate. Nucleosides and myo-inositol had no effect on microspore division. In the absence of all organic components except sucrose, most mitoses were asymmetric, formation of multinuclear structures was reduced and most pollen accumulated starch indicative of gametophytic fate. In complete M1 medium, starch accumulation was suppressed. Suppression also occurred in asymmetrically divided microspores, indicating a direct inhibition of pollen development independent of the mode of microspore division. This inhibitory effect of M1 medium might present a stress which triggers the induction of symmetric microspore division and subsequent formation of multinuclear structures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microspore culture of swede (Brassica napus ssp. rapifera) and the effects of fresh and conditioned media

Microspore culture was successfully carried out with four cultivars of swede (Brassica napus ssp. rapifera). Embryo yields increased markedly if the culture medium was replaced by fresh medium after a culture period of three days, with more than 200% increase with the highly responding cultivar Gry and more than 800% with the poorly responding cultivar Stenhaug. If the medium was changed at a later time, embryo production declined sharply. Transfer of microspores after 3 days of culture to a medium in which microspores of a different cultivar had been cultured for 3 days had no effect on embryogenesis. Bud size had a large effect on embryo production, particularly with the least responsive cultivar, Stenhaug.     

Pollen ontogenesis in Oenothera: a comparison of genotypically normal anthers with the male-sterile mutant sterilis

Summary A 12-stage normal table of anther development in Oenothera, is presented. The stages are characterized by developmental steps in the reproductive cells and the tapetum, including waves of amylogenesis and lipogenesis as well as the production of the sporoderm layers. This is compared to a corresponding table for the male-sterile (mst) mutant sterilis (ster). Differences between the development of fertile and mst anthers appear after the liberation of the microspores from the tetrads. Male sterility results from a malfunction of the tapetum in the production of ektexine sporopollenin precursors, which aggregate in the tapetal cells. The consequence is the absence of ektexine from the microspores. The endexine is then dissolved, presumably by an enzyme. This process leads to naked microspores whose unprotected cytoplasms are attacked by hydrolytic enzymes present in the thecal fluid. At anthesis the anthers contain only undefined remnants of microspores and tapetum.     

Microspore development during <Emphasis Type="Italic">in vitro</Emphasis> androgenesis in triticale

Microspore division was monitored in three triticale (× Triticosecale Wittmack) genotypes over 21 d of in vitro anther culture, on two media differing in their 2,4-dichlorophenoxyacetic acid content. After low temperature (4 °C) pre-treatment for two weeks, all the microspores were still alive, but they began to die from day one of culture. Both genotype and culture medium affected the number of microspores that aborted over time (82 – 97 % by day 21), the number of microspores that underwent the first symmetrical division (> 82 % over all), the number of microspores that attained four or more nuclei, and the number of divisions per 100 alive microspores after 21 d of culture.     

Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperature

We have established an efficient method to induce embryo formation from isolated wheat (Triticum aestivum L.) microspores. Culture of excised anthers under starvation and heat shock conditions induced the formation of embryogenic microspores at high frequency in nine Austrian winter wheat genotypes, including cultivars that had been considered as recalcitrant in anther culture. Percoll gradient centrifugation of the mechanically isolated microspores allowed us to obtain homogeneous populations of embryogenic microspores in all genotypes which, after transfer to a rich medium containing immature ovaries for conditioning, divided and produced globular embryos. Thousands of embryos were produced in one petri dish. Many of these embryos developed into plantlets after transfer to a solid medium without ovaries.     

Phylogenetic relationships of ferns deduced from rbcL gene sequence

Part of the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) gene (rbcL) was sequenced from three fern species: Adiantum capillus-veneris, Botrypus strictus, and Osmunda cinnamomea var. fokiensis. This region included 1,333 base pairs, about 90% of the gene. Maximum likelihood analysis of the deduced amino acid sequences indicated that (1) Botrypus (Ophioglossaceae) clustered monophyletically with other ferns (Adiantum, Angiopteris, Osmunda); the closest relative to Botrypus among the three species was Osmunda, which did not support the hypothesis that the Ophioglossaceae are linked to the progymnosperm-seed plant lineage. (2) Eusporangiate ferns containing Botrypus (Ophioglossaceae) and Angiopteris (Marattiaceae) were a paraphyletic group. (3) Seed plants and the four fern species examined formed a monophyletic group, but ferns and bryophytes (liverwort) did not. Variations in rates of substitution for synonymous and nonsynonymous codons were found in fern lineages.Correspondence to: M. Hasebe     

Metabolism of maltose and sucrose by microspores isolated from barley (Hordeum vulgare L.)

The aim of this work was to discover why barley (Hordeum vulgare L.) microspores die when cultured on media containing 40 mM sucrose but undergo embryogenesis on 40 mM maltose. Freshly isolated microspores were cultured for 6–24 h on media containing either [U-¹⁴C]maltose or [U-¹⁴C]sucrose at 40 mM, and the detailed distribution of ¹⁴C was determined. The amounts of glycolytic intermediates, ATP, ADP and AMP, in microspores were also measured. Cultures on sucrose differed from those on maltose in that the initial rate of metabolism was faster but declined rapidly, less ¹⁴C was recovered in polymers and more in alanine, there was extensive leakage of assimilated carbon, significant accumulation of ethanol and a lower adenylate energy charge. It is argued that microspores cultured on 40 mM sucrose die because they metabolize the sugar rapidly, become hypoxic and, as a result, accumulate large quantities of ethanol within the cells. Metabolism of maltose is slower and there is sufficient oxygen available to allow cells to survive in culture. Consequently some of the cultured cells undergo embryogenesis.P.S. thanks the Science and Engineering Research Council and Shell Research Ltd., Sittingbourne, for a Cooperative Award in Science and Engineering studentship.     

Factors affecting in vitro maturation of isolated maize microspores

Summary An in vitro method to simulate pollen development was developed in maize (Zea mays L.). Microspores at the late uninucleate to early binucleate stage were isolated and cultured under various conditions. Cell viability, starch content and the formation of the three nuclei as found in normal mature pollen were monitored during the course of the culture. Media composition was modified in order to promote starch accumulation and frequency of mitosis, while maintaining the viability of the microspores. Under the best conditions, up to 12% of the microspores matured in vitro into trinucleate, starch-filled viable pollen grains which were unable to germinate or produce seeds. At different stages during in vitro maturation, proteins patterns were analyzed and compared with their in vivo equivalent and the patterns were only partially similar.