Abstract: An attempt has been made to manipulate the cytological processes regulating the switch from gametophytic to sporophytic development induced by culturing the microspores of higher plants. Previous studies have indicated that sporophytic development, which leads to the formation of haploid embryos, normally follows the symmetrical division of the microspore rather than the asymmetric mitosis characteristic of normal development. To determine whether symmetry of division is a key factor in the determination of subsequent development, cells were supplied with the antimicrotubule drug colchicine to disrupt elements of the microtubular cytoskeleton believed to be involved in nuclear positioning. The treatment resulted in a highly significant increase in the numbers of cells turning to sporophytic development; further, timed applications indicated that the cells were sensitive to the drug over a 12-h period immediately prior to pollen mitosis. The results suggest that alteration of division symmetry is sufficient to switch the developmental pathway from gametophytic to sporophytic. These findings are discussed in the perspective of current models proposed for the regulation of development in eukaryotic cells.

Abstract: Brassica napus L. microspores at the late uninucleate to early binucleate stage of development can be induced in vitro to alter their development from pollen to embryo formation. High temperatures or other stress treatments are required to initiate this redirection process. The critical period for induction of microspore embryogenesis is within the first 8 h of temperature-stress imposition. During this period, which precedes the first embryogenic nuclear division, the process regulating the induction and sustainment of microspore embryogenesis is activated. A number of mRNAs and proteins, some of them possibly heat-shock proteins, appear in microspores during the commitment phase of the induction process.

Abstract: In the present experiment, different chromosome reduplication techniques were applied to microspore-originated Triticum aestivum L. (cv ‘Ciano’) haploids. In addition to the conventional treatment (whole plant exposure to colchicine solution), spontaneously redoubled haploids were also examined. As an experimental treatment, different concentrations (0.01, 0.02, 0.04%) of colchicine were added directly to the induction media. Colchicine did not affect the anther response or the plant regeneration capacity. The success and stability of genome redoubling was estimated on the basis of the fertility of the regenerated (R0) plants and their progeny (R1). Chromosome doubling produced by colchicine before the first microspore mitosis was significantly more efficient than the conventionally used techniques.

Abstract: The pollen-stigma interaction of self-incompatibility in crucifers is correlated with glycoproteins localized in the cell wall of the stigmatic papillae that are encoded by the S locus glycoprotein (SLG) gene. When fused to the [beta]-glucuronidase (GUS) reporter gene, the 5[prime] upstream regulatory region of SLG directed high level expression in the papillae of transgenic Brassica plants. Histochemical and fluorometric assays revealed that, in addition to its primary site of expression in the stigmatic papillae, the SLG-GUS fusion was also expressed in the transmitting tissue of stigma, style, and ovary, and in anthers. This conclusion was verified by the detection of transgene-encoded GUS transcripts and endogenous SLG-homologous transcripts by RNA gel blot analysis. Significantly, in anthers, the SLG promoter was active not only sporophytically in the nurse cells of the tapetum, but also in the haploid microspores. Because self-incompatibility systems exhibiting sporophytic control of pollen phenotype are thought to have evolved from systems with gametophytic control, we suggest that sporophytic control was acquired without loss of gametophytic function.

Abstract: The ultrastructure of the secretory, binucleate tapetum of Brassica oleracea in the micro spore mother cell (MMC) stage through to the mature pollen stage is reported The tapetal cells differentiate as highly specialized cells whose development is involved in lipid accumulation in their final stage They start breaking down just before anther dehiscence Nuclei with dispersed chromatin, large nucleoli and many ribosomes in the cytoplasm characterize the tapetal cells The wall-bearing tapetum phase ends at the tetrade stage The dissolution of tapetal walls begins from the inner tangential wall oriented towards the loculus and proceeds gradually along the radial walls to the outer tangential one The plasmodesmata transversing the radial walls between tapetal cells persist until the mature microspore, long after loss of the inner tangential wall After wall dissolution, the tapetal protoplasts retain their integrity and position within the anther locule The tapetal cell membrane is in direct contact with the exine of the microspores/pollen grains and forms tubular evaginations that increase its surface area and appear to be involved in the translocation of solutes from the tapetal cells to the microspores/ pollen grains The tapetal cells exhibit a polarity expressed by spatial differentiation in the radial direction

Abstract: Cultured microspores of Brassica napus L. cvs Topas and Reston initiated cell divisions within 3 to 4 days, and globular, heart and torpedo shaped embryos were prevalent after approximately 6, 8, and 10 days, respectively. Embryos with rudimentary cotyledons were evident within 2 weeks, but those that reached this stage of development represented only 1–5% of the original microspore population. The fresh weight of microspore-derived embryos at all stages of development was significantly greater than that for zygotic embryos, but the pattern of change in fresh weight and fatty acid accumulation was similar in developing zygotic and microspore embryos. In freshly isolated microspores of both Topas (low erucic acid) and Reston (high erucic acid), the predominant fatty acid was 18:3, while 18:1 comprised less than 15% of total fatty acids. During development in both zygotic and microspore embryos, the level of 18:3 declined markedly while 18:1 rapidly increased. Erucic acid (22:1) was not detected in the early stages of embryogenesis in Reston. However, small amounts of 22:1 appeared by early cotyledonary stage and the level gradually increased in both zygotic and microspore embryos through the later stages of development. The fatty acid compositions of mature embryos was nearly identical to that of dry seed, except the level of 22:1 in Reston embryos was consistently less than in the seed. Triacylglycerols comprised only 15% of total lipids in freshly isolated microspores, but increased to more than 90% by 4 weeks. The fatty acid composition of the triacylglycerol fraction was generally similar to that of total lipids at all stages of development of microspore-derived embryos.

Abstract: A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L) pollen from microspores isolated just before entering mitosis During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes The in vitro-matured pollen grains can be stored at-20° C either suspended in 117 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection

Abstract: The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. ‘Hella’. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.

Abstract: Abstract The fossil record of the heterosporous lycopsids (lsoetales and Selaginellales) and water ferns (Marsileales and Salviniales) is reviewed. Fertile whole plants and dispersed megaspores with attached microspores or microspore massulae are the crucial fossils for understanding the evolution, diversification, and phylogeny of these plants. Salviniales are particularly well represented with fertile fossil plants substantiating an extensive dispersed spore record. Azollaceae (Azolla) and Salviniaceae (Salvinia) are distinct, and the modern genera clearly identifiable, from the earliest Tertiary onwards. Azolla and extinct relatives are also known in the late Cretaceous. A mosaic of characters is presented by these late Cretaceous fossils most of which is lost during the early Tertiary. Marsileales are represented by one sporocarp species in the early Tertiary but otherwise only by a patchy record of dispersed spores from the early Cretaceous onwards. Isoetales and Selaginellales have a longer history.

Abstract: Cooling treatments at different stages of pollen development lowered the percentage of fertilized spikelets through decreasing the number of engorged pollen grains per anther at anthesis. Cooling during the period from anther differentiation to the tetrad phase decreased the number of engorged pollen grains mainly by decreasing the number of differentiated microspores. Cooling in the period from the early microspore phase to the late microspore phase, however, decreased the number of engorged pollen grains primarily by increasing the number of aborted microspores. Cooling at the young microspore stage, which consists of the two phases of the tetrad and the early microspore phase, caused the largest decrease in the number of engorged pollen grains, resulting in the largest decrease in the percentage of fertilization. Cooling at the tetrad phase caused the largest decrease in the number of differentiated microspores, resulting in the largest decrease in the number of engorged pollen grains. On the other hand, cooling at the early microspore phase caused the largest increase in the number of aborted microspores, resulting in the largest decline in the number of engorged pollen grains. These results indicate that the highest susceptibility to coolness of anthers at the young microspore stage, which has been estimated as the percentage of fertilized spikelets, is caused by the high susceptibility to coolness of the differentiation and development of microspores.

Abstract: A study was made of the differential effects of specific chromosomal deficiencies on the development of the maize pollen grain. Twenty-six B–A translocations involving 17 of the 20 chromosome arms were used to produce hypoploid plants in which one half of the microspores had a predictable chromosomal deletion. Breakpoints of the translocations were proximally located in most cases, although some were more distal. Deficient and normal male gametophytes from these hypoploids were studied cytologically to characterize developmental changes. Generally, loss of part of a chromosome arm caused abnormal microspore development, a slowing of the normal mitotic or developmental processes in the male gametophyte, or a termination of development. Slowing of development was observed as early as the quartet stage in deficient microspores from TB-1Sb and TB-9Sd hypoploids, while in others the developmental delay occurred later, mostly during the first pollen mitosis. The abnormal or blocked development associated with o...

Abstract: Effect of age of donor plants and age of inflorescence on embryogenesis in microspore culture of B. napus was examined. Microspores isolated from buds of older plants had a higher embryo yield than those of younger ones. The effect of the age of inflorescence showed a different pattern. In older plants, a higher embryogenesis response was observed in microspores isolated from buds of new inflorescences, while in young plants, microspores isolated from buds of old inflorescences showed high embryo yield. These different responses were considered to be attributable to a difference in the developmental stage of pollen at the time of microspore isolation. Our results indicated that microspores collected from older inflorescences and older plants have sufficient embryogenic potential when the optimum developmental stage of pollen was used. Frequency of embryo to plant conversion was influenced by the size of embryos subcultured, but not by donor plant age or the age of the inflorescence.

Abstract: In wheat, plants may be regenerated from microspores via direct embryogenesis or organogenesis or embryogenesis from callus. Light and scanning electron microscopy were used to carefully study morphogenesis of microspore-derived plants from anther culture on modified 85D12 starch medium and to determine whether the plants were formed via organogenesis or embryogenesis. Our results indicate that plants are formed via embryogenesis from microspores. Evidence for embryogenesis included the formation of the epidermis and a suspensorlike structure (21 days after culture), followed by initiation of an apical meristem, differentiation of the scutellum, and embryo elongation. At 28 days in culture, the embryo possessed a well-developed scutellum and axis with suspensor. Embryogenesis was further confirmed by coleoptile and radicle elongation during germination when the embryos were cultured on medium supplemented with kinetin with or without coconut water. In this system, an average 67 microspores per responsive anther began cell division but only 3.69 embryos were formed per responsive anther after 6 wk. Adventitious embryos could be induced if the embryos, once formed, remained on initiation medium for 10 wk instead of being transferred to regeneration medium. Developmental stages which may be amenable to changes that could enhance plant production were identified. The potential to use this information to enhance plant production is discussed.

Abstract: Recently identified conditions for in vitro perithecia formation made it possible to demonstrate the existence of previously suggested heterothallism in Phaeosphaeria nodorum. Of the 28 possible pairwise crosses between the eight cultures derived from the spores of a single ascus, 9 were fertile, whereas none of the cultures ever produced fertile perithecia on their own. This result is smaller than the one obtained in classical bipolar heterothallism and suggests the existence of another incompatibility mechanism. The growth and the colour of mycelia derived from eight single ascospores from the same ascus seem to cosegregate with the factor for heterothallism. The formation of microspores in colonies from single ascospores indicates that they may play a role in the formation of perithecia. Used for fertilization, these microspores showed that they were able to bring the complementary nucleus, thus enabling the differentiation of perithecia. However, they are able to germinate and do not behave strictly a...

Abstract: Twenty-five inbred lines, including grain and forage types from the USA and China, two hybrids, one Sorghum almum, and one Parasorghum (S. versicolor) were tested for their response to anther culture. Three nutrient media were effective in inducing anther calli from six cultivars (Xin White, TX 403-TSB, DDY Sommer Milo, TX 2779, Brawley, and Spur Federal) and one was effective for plant regeneration for one cultivar, Xin White. Averaged over media, callus induction frequency (number of calli per 100 anthers) was highest in cultivars Xin White and TX 403-TSB (6.7 and 3.9%, respectively). The means of cultivars for media C17-2 and Ms-t-z-2, 4.3 and 3.2%, respectively, were superior to that for medium 85D3-2 (0.1%). Expressed as an average of the six cultivars and three media the mean calli induction frequency was 2.6%; however, differential responses of genotype and medium were noted. Among the 10 regeneration media tested, medium MS-d-4 containing Murashige and Skoog basal components plus 2.0 mg/l indole-3-acetic acid (IAA) and 2.5 mg/l kinetin was the most effective for plant regeneration. Numbers of albino plants and calli developing only roots increased directly with callus-induction time, whereas the frequency of plant regeneration decreased. Regenerated plants had varied numbers of chromosomes in root tip cells: 10, 15, 20, 40, and 60. The 29 regenerated plants that reached maturity, however, were highly fertile and contained only 10 bivalents in pollen mother cells. Normal chromosome number and behavior for the regenerated plants suggest that induced calli originated from cells other than microspores. However, spontaneous chromosome doubling in microspore-derived haploids may occur. The appearance of albinos also implies that haploids may have been produced from anther culture.

Abstract: Using an amylose-free (amf)mutant of diploid potato (Solanum tuberosum), diploid and tetraploid clones with different genotypes at the amf-locus were produced. In order to make use of the diploid material in analytic breeding of amf-potatoes, clones were selected that produced a considerable frequencies of 2n-pollen and 2n-eggs. Successful attempts were made to select normal synaptic as well as desynaptic clones producing 2n-gametes. Based on the phenotype of starch in the microspores, tetraploid clones with nulliplex, simplex and duplex genotypes at the amf-locus were selected. Prospects of using this material in conventional as well as in analytic breeding of potato are discussed.

Abstract: Following EMS, DES and gamma ray treatment in Arkel and Bonneville pea varieties, three male sterile mutants were induced in Pisum sativum. Sterility in each of these is conditioned by single recessive genes, the three genes being non-allelic. Whereas in one mutant, the ms gene acts during pre-meiosis, in the other two the genes act during post-meiosis. In the premeiotic mutant, the PMCs either degenerate before attaining genetic autonomy or after separating out from one another. Their chromatin either compacts or fragments to degenerate completely. Thus, no meiosis occurs in this mutant.In both the post-meiotic mutants, male meiosis is normal. In one, the microspores are released from the PMCs either in free or in adhered state. They develop thick walls, their chromatin condenses centrally and finally they degenerate. In the other mutant, monads are released from the PMCs. In 93% microspoes, the nuclear degeneration is faster than cytoplasmic degeneration, in 7% the reverse occurs. In both the post-meiotic mutants, microspores degenerate fully. Male sterility in all the three mutants is complete while female fertility is normal.

Abstract: Androgenic plants have been obtained via anther culture in four natural populations of Hordeum spontaneum. Microscopic observations revealed that androgenesis started with the formation of two vegetative-type nuclei derived from the mitotic division of the uninucleate microspores. In this species androgenesis was affected by the type and concentration of the sugars added to the culture medium: the highest response (17% of callusing anthers) was observed on media containing 80 g l−1 maltose. The highest production of androgenic plants (per 100 anthers, 5.9 green and 4.3 albino plants) was obtained from callus grown on these same media. About half of the green plants regenerated were haploid, while the others were diploid and set seed.

Abstract: Abstract The development of the tapetum and orbicules in Catharanthus roseus (L.) G. Don has been examined using transmission electron microscopy (TEM). Precursors of orbicules are formed as spheroidal vesicles within the tapetal cytoplasm and are associated with the endoplasmic reticulum. The pro-orbicules are extruded through the cell membrane of the tapetal cell and this coincides with the dissolution of the ta petal cell wall and the callosic wall of the microspores. The orbicules are developed by irregular deposition of sporopollenin on the pro-orbicules. A thin membranous layer is developed between the orbicules, forming the orbicular membrane. At free microspore stage globular bodies are seen in the cytoplasm of the tapetal cells. Some of these bodies are large and coated with a thin electron-dense layer, others are small bodies, electron-dense and gathered in groups. At vacuolate stage the tapetal cells extend into the anther locule, acting as a periplasmodial tapetum. At late vacuolate stage the tapetal cells retreat from the anther locule and possess an organized and apparently functional structure. The tapetal cells start to degenerate just before an thesis of the pollen grains. The diversity in the structure of orbicules and tapetum may suggest different functions at different stages of development and may have arisen by remarkable processes of adaptation.

Abstract: This study was designed to study the effects of stage of microspore development and culture medium on androgenic response in peanut (Arachis hypogaea L.). Anthers of various developmental stages were cultured for 7 days, then fixed and observed cytologically. Three sets of media, involving different basal media, growth regulators, sucrose levels and glutamine concentrations, were tested. In all experiments, the stage of development of the microspores at the time of culture was highly significant. The early uninucleate microspores stage was identified as producing the highest anther response rating. The effect of media was nonsignificant in all experiments. However, the stepwise modification of the media through the course of the study resulted in an almost 8 x increase in anther response rating. Numerically, the best media tested was N6 basal medium with 1 mg 1-1 NAA, 0.1 mg 1-1 BA, 5.5% sucrose, and 3.5 g 1-1 glutamine. While no haploids were obtained, four-nucleate cells were observed, indicating the potential in peanuts for an androgenic reponse.

Abstract: A mutant causing partial desynapsis (dissociation of paired chromosomes), and consequently a high frequency of univalents at metaphase 1, was found in Rhoeo spathacea among the selfed progeny of a wild-collected ring-forming complex interchange heterozygote. All the plants were diploid, 2n = 2x = 12. The mutant formed univalents in all microsporocytes (range 2–12; average 7.56/cell), and 23.69% of the pollen mother cells contained the maximum of 12 univalents at metaphase I. There was no significant difference in pollen fertility between the mutant and parental plants. The desynaptic mutant produced microspores with chromosome numbers of n = 6–14 in the same anther. Unreduced pollen was formed at telophase II by second division restitution and comprised 52.74% of the pollen grains. By selfing, diplandrogynous tetraploid progeny (2n = 4x = 24) of 12 seedlings were obtained. The results demonstrate that both male and female gametes are unreduced.Key words: Rhoeo, desynapsis, univalent, second division resti...

Abstract: To clarify at what stage of development flower buds are subject to blasting by high temperature, a correlation between the length of perianths and the stage of pollen formation in a miniature Cymbidium orchid (C. × Sazanami 'Haru-no-umi') was investigated.1. High correlations between the variables were obtained during the stages of sporogenous cells and pollen mother cells (PMC) regardless of the position of the flower buds. Flower buds exposed to high temperatures (30°C day/25°C night) at sporogenous cell stage became blasted without entering into the meiotic stage. Similarly, buds which were heated at premeiotic stage became blasted at various stages after chromosomes underwent meiosis.2. Flower buds reaching the meiotic prophase of PMC never responded to the high temperature and bloomed normally.3. The application of gibberellic acid (GA3) reduced the low temperature requirement for bud development. GA3-treated buds entered the meiotic cell differentiation stage and proceeded to develop normally with mature viable pollen, even when the GA3 spray was combined with the high temperature treatment.4. The results indicate that buds at premeiotic stage are very sensitive to blasting by exposure to high temperature, and that temperatures below 20°C are required for cells to undergo meiosis, form mature pollen, and develop normal flowers.5. Different patterns of microspore arrangement were observed even in the same anther.Most uninucleate microspores of blasted flower buds were diads, fewer progressed to tetrads.On the other hand, binucleate microspores of both blasted and normal flower buds were mainly in the tetrad stage. The patterns and kinds of tetrads were not changed by the GA3 treatment, whether the buds were blasted or normal.

Abstract: The development of sporogenous and tapetal cells in the anthers of male-fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.) plants was studied using light and transmission electron microscopy. In general, male-sterile anthers showed a much greater variability in developmental pattern than male-fertile anthers. The earliest deviation from normal anther development was observed to occur in sterile anthers at meiotic early prophase: there was a degeneration or irregular proliferation of the tapetal cells. Other early aberrant events were the occurrence of numerous small vesicles in the microspore mother cells (MMC) and a disorganized chromatin condensation. Deviations that occurred in sterile anthers at later developmental stages included: (1) less distinct inner structures in the mitochondria of both MMC and tapetal cells from middle prophase onwards. (2) dilated ER and nuclear membranes at MMC prophase, in some cases associated with the formation of protein bodies. (3) breakdown of cell walls in MMCs and tapetal cells at late meiotic prophase. (4) no massive increase in tapetal ER at the tetrad stage. (5) a general dissolution of membranes, first in the MMC, then in the tapetum. (6) abortion of microspores and the occurrence of a plasmodial tapetum in anthers reaching the microspore stage. (7) no distinct degeneration of tapetal cells after microspore formation. Thus, it seems that the factors that lead to abortive microsporogenesis are structurally expressed at widely different times during anther development. Aberrant patterns are not restricted to the tetrad stage but occur at early prophase.

Abstract: An immunological approach has been used to identify embryo-specific products that can be used as molecular markers of embryogenesis. Immunoadsorption of antisera to remove antigens common to embryos, meristematic cells and callus, revealed one major embryo-specific antigen, a polypeptide of 17 kDa. The antigen appeared at mid-stages of zygotic embryo formation and remained at similar levels up to six days post-germination of the seedling. The polypeptide could not be detected by protein staining, suggesting it is a non-abundant product. Appearance of the antigen could be induced by culture of zygotic embryos in vitro on abscisic acid (1 pM) or mannitol YO mass/vol.). Cross-reactive products of near-identical molecular mass were observed in embryos of wheat, rye and oats but not of distantly related cereals, nor embryos from dicotyledonous species. The timing of the appearance of the antigen was different in embryos formed from microspores during anther culture in vitro. In the cultured material, the 17-kDa polypeptide preceded the appearance of morphologically distinct embryonic structures.