Abstract: The cytological development of male cells and the tapetum of male fertile and combined cytoplasmic triazine-resistant cyto-plasmic-genetic male sterile (ctr) lines of B. napus L. was studied using light, scanning electron, and transmission electron microscopy. Development of the cytoplasmic-genetic male sterile anther was similar to the normal anther up to and including meiotic prophase I. After this stage, degeneration of the microspore mother cells occurs within the callose walls, and tetrads of microspores are not formed. These degenerating microspore mother cells appear to develop numerous endoplasmic reticulum derived vesiculated structures, which may be involved in lysis of organelles. Degeneration occurs simultaneously with a proliferation of the tapetum, which eventually fills the anther locule. It is not clear whether the abortion of the microspore mother cells during meiosis stimulates proliferation of the tapetum or whether the proliferating tapetum actually interferes with microspore developme...

Abstract: From an F1 hybrid between the two barley (Hordeum vulgare L.) cultivars ‘Golden Promise’ and ‘Mazurka’ a series of doubled haploid (DH) lines were generated both from microspores by anther culture and from immature zygotic embryos after hybridization withH. bulbosum. The DH lines from both sources were used to monitor the segregation of the five major genes, rachilla hair length, DDT susceptibility, height, C hordein polymorphism and mildew resistance. Whereas the microspore-derived samples showed significant departures from the expected 1∶1 ratio for three of the five genes, theH. bulbosum lines showed deviation for only one gene. Analysis of linkage data also showed differences between the two series of DH lines. Cytogenetic analysis revealed a mean chiasma frequency in theH. bulbosum lines which was very similar to the F1 hybrid. In contrast, four of the ten microspore derived lines examined showed a reduced chiasma frequency. One showed evidence of translocation heterozygosity.

Abstract: The development of haploid embryoids from microspores of Datura innoxia by anther culture was first reported in 1964 (Guha and Maheshwari). Since then many scientists in China and other countries have paid great attention to the induction of androgenetic haploids, mainly because anther culture provides an excellent means for the production of large numbers of haploids which are useful in fundamental genetic research and plant breeding (see H. Hu 1978, Bajaj 1983 c). The first research work on wheat anther culture was by Fujii (1970) in which six species were tested; calli were obtained in Triticum aegilopoides, T. dicoccides, but anthers of Triticum aestivum did not respond. Pollen plants of wheat were first obtained by J. Ouyang et al. (1973). At that time the frequency of induction of green plantlets was only 0.7%.

Abstract: The male gametophyte of higher plants is metabolically active; during microspore maturation, grain germination and tube growth, the most important physiological processes are those related to respiration, reserve mobilization, nutrient uptake, biosynthesis first of intine and then of pollen tube wall (3). Pollen is also able to respond to environment; this ability is expressed both in physiological interaction with stylar tissues during tube development, and in relation to environmental stresses, toxic products, pathogens (8). The responses are generally correlated to sporophytic behaviour (7,9).

Abstract: Microsporogenesis in the parthenogenetic species Houttuynia cordata Thunb. (Saururaceae) was studied microscopically. Cytokinesis failure after meiosis results in the formation of coenocytic microspores that show a wide range of variation in shape and size. Although tapetal cells do not show any demonstrable abnormalities, the microspores degenerate at the vacuolate microspore stage some time after dissolution of the callose. At the last stage of microsporogenesis, ghosts of the coenocytic microspores, which consist of only ektexine, remain in the loculus. The species is completely, or nearly completely, male-sterile.

Abstract: Nuclear growth, microspore cell growth and cell cycle stage were examined in microspores of anthers of Hordeum vulgare L. cv. Klages taken from florets of the middle of the spike as per anther staging methods. Although there was wide variation in nuclear size at all stages of the cell cycle, mean nuclear size appeared to be a good indicator of cell cycle stage for microspores within anthers. Microspore cell size increased considerably during Gl of the cell cycle. Anthers bearing microspores cytologically characterized as in the mid-uninucleate stage, which have proven to yield high levels of callus production, were determined to be in G1 of the cell cycle and were regularly found in spikes taken from tillers in which the base of the flag leaf had emerged 0 to 3 cm above the penultimate leaf.

Abstract: Several techniques have been described recently for germinating spores of aquatic species of Isoetes which grow in lakes, ponds, and streams in eastern North America. Boom (1980) used glass vials containing sterile sand and pond water in which he successfully raised sporelings of I. engelmannii, I. flaccida, I. macrospora, and I. riparia. Kott and Britton (1982) germinated spores of I. acadiensis, I. echinospora, I. macrospora, I. riparia, and I. tuckermanii in small vials half filled with distilled water. However, many of their vials became contaminated with fungi that destroyed the viability of the megaspores. Sam (1982) germinated megaspores of I. engelmannii in an inorganic nutrient medium supplemented with Nystatin to inhibit fungi. Webster (1979) described a technique for germinating spores of Selaginella that is similar to the following procedure we use for Isoetes. We have been culturing plants from spores of Isoetes acadiensis, I. echinospora, I. engelmannii, I. hieroglyphica, I. macrospora, I. riparia, and I. tuckermanii for use in germination and hybridization experiments for several years. The aquatic species of Isoetes are ideal pteridophytes for germination and hybridization experiments. Their sporangia are large and contain many spores. Megaspores and microspores are borne in different sporangia on separate leaves. If necessary, unopened sporangia can be excised from sporophylls intact and surface cleansed to eliminate extraneous spore contamination. Thus, megaspores and microspores can be completely isolated for controlled breeding experiments. Also, their spores readily germinate in demineralized water and gametophytes and sporophytes develop normally for several months without supplemental nutrients. Spores are obtained from plants collected in September and October, since plants harvested earlier in the year may not have mature, viable spores. Isoetes hieroglyphica and I. macrospora retain their spores through the winter, so mature spores of these species can also be gathered in the spring. Megaspores are cleansed of microspores in a sieving apparatus made of plastic tubing approximately 10 cm long and 2.5 cm in diameter. An 8 cm square of 0.27 mm sifter mesh (available from Carolina Biological Supply Company) is fitted to one end of the tubing with a rubber band. The sieving apparatus, containing megaspores, is attached to a water faucet using plastic strapping tape. Spores are washed with a steady flow of cold water for 30 minutes, followed by a one minute rinse with sterile, demineralized water. Although megaspore surfaces appear clean after the above treatment, unopened sporangia are preferred for crossing experiments because there is less chance of contamination from microspores. Intact sporangia can be excised from

Abstract: Abstract Pollen of plants of Phaseolus vulgaris L. ‘Red Mexican’ is affected adversely by water stress. The critical stress period occurs 10-13 days after emergence, during the transition from the vegetative to the reproductive phase of development. The transition phase is characterized by the presence of tiny green buds (>0.5-2.0 mm in length), bearing anthers that contain tetrads or free microspores. Pollen abortion of plants stressed during the transition phase is significantly higher than that of pollen from nonstressed plants or from those stressed during later phases of reproductive development. The percentage of pollen germination and total pollen production also are reduced in transition phase-stressed plants. Exine development is abnormal. This abnormal development is particularly interesting because the exine is regarded as so genetically stable that it is regularly used for species identification. Results of these experiments raise questions about the infallibility of identifications, since an environmental stress (such as water) imposed at a critical time of gametophytic development can markedly alter exine patterning.

Abstract: SUMMARY Ultrastructual development from microspores at the tetrad stage up to the bicellular stage of pollen of diploid Euphorbia dulcis L. was investigated. At the tetrad stage the microspores are enclosed by a thick callose wall. The nucleus occupies a central position in the microspore, is nearly spherical and shows a distinct nucleolus. The thin primexine varies in thickness. Large plastids with starch grains are present. The generative cell becomes separated from the intine and changes into two short “tails”. Many long rough endoplasmic reticulum (RER) cisternae are arranged parallel to the plasmalemma in the tail parts. Plastids are not present. Microtubules are parallel to the plasma membrane of the generative cell. In the vegetative cell, the plastids undergo two amylogeneses. The first occurs at the tetrad stage and the second after the first pollen mitosis, when the generative cell starts to move to the central position in the vegetative cell. Many lipid droplets surround the generative cell. RER is arranged in aggregations near the generative cell. After the first mitotic division the intine forms a network of microfibrillar substances around the pores. The cell wall, separating the vegetative and generative cell, is regularly interrupted by plasmodesmata or plasma channels.

Abstract: Androgenetic doubled haploid spring barley lines (DH-lines) were compared in field experiments with their pedigree-selected sister lines (PD-lines). The PD-lines resemble a selected proportion of 1 % (10−2) or less of the original complete F2-population. By comparison, an average of 10 DH-lines were recovered from 10,000 anthers X > 1,000 microspores, i.e. at least 107 microspores per cross. The average success rate was therefore approximately 10−6. Both PD- and DH-families showed similar levels of performance with regard to all the characters studied, although no conscious selection had been applied in deriving doubled haploid progeny. Therefore, it is concluded that the regenerated microspore-progenies may be the result of unconscious selection among the large numbers of microspores cultured.

Abstract: Cultural systems developed for the successful production of haploid plants from anther and microspore culture of a number of species are lethal to cotton microspores. Components of these systems were examined individually and in simple combinations to determine their specific effect on cotton microspore viability during isolation and culture. An organically buffered pH of 7.0 was critical for survival. The addition of potassium salts, near standard concentrations used for other species, enhanced survival and the cytoplasmic appearance of isolated microspores. Severe toxicity resulted from ammonium, calcium and magnesium salts, and these ions were tolerated only at very low concentrations. Iron, glutamine, serine, inositol, vitamins, and trace minerals were generally not detrimental to microspores at standard concentrations. An isolation and cultural maintenance system was developed that yields large quantities of healthy, viable cotton microspores. This initial step allows for further research in inducing cotton microspores to divide and undergo embryogenesis.

Abstract: Cytochemical studies of androgenic anthers of Oryza sativa picked from the culture at 2 day intervals from 0 to 40 days have been carried out. Glutaradehyde-OsO4-fixed and plastic-embedded sections were stained with TBO, SBB and PAS for acidic polymers, lipids and polysaccharides respectively. Among the population only 4% of microspores, which accumulate abundant amorphous lipid in the first few days of culture, are androgenic. Less than 30%, which have many lipid granules and some amorphous lipid, become nutritive microspores. Starch grains also accumulate in these nutritive microspores which degenerate at the stage when the androgenic multicellular microspores are in rapid development. The remaining microspores, which have no or little lipid, degenerate early. At about the 100-cell stage, each multicellular unit consists of two cell types, large and small. The large cells contain abundant amorphous lipid and starch grains which the small ones stain intensely with TBO.

Abstract: Isolated uninucleate microspores of tomato,Lycopersicon esculentumMill, were cultured in defined, liquid nutritive media. The microspores developed to haploid embryoids with or without an attached suspensor or into calli with compactly or loosely arranged cells.

Abstract: Results are reported on the induction of Phytophthora and Fusarium resistance in potato and on the production and screening of doubled haploid barley plants resistant to barley yellow mosaic virus. In potato, calli and protoplasts are used as in vitro system, while in barley the work concentrates on microspores either cultured within or in isolation from the anther. In conditioned media the regeneration rate of isolated microspores was high enough to use this system for selection. Up to 0.2M Fusaric acid was applied as selective agent and calli could be selected which survived this toxic concentration. The different strategies of in vitro selection for resistances are discussed.

Abstract: SUMMARY Protoplast yield, survival, and regeneration was investigated in dihaploid (2n = 2x = 24) clones of potato. These clones consisted of unselected interdihaploids and of microspore or protoplast derived clones, particularly doubled haploids regenerated from the same anther donor clone, androgenetic clones regenerated in a second cycle from androgenetic clones, and protoclones. Significant differences were observed for all characters studied between donor plants without an in vitro history and the other groups, but also within the androgenetic clones, which underlines that they originated from different gametes. Non-significant differences among clones having passed twice through anther culture are indicative of the stability and the homozygosity of the androgenetic diphaploids. Differences between second cycle anther derived clones observed as well, indicated the induction of mutational events during microspore culture. Such variation of the regeneration parameters was also observed between protoplast cultures of an interdihaploid clone and of its protoclone. As in most experiments such variation is undesired, experiments were conducted to reduce the somaclonal variation. By optimizing the culture procedure, the undifferentiated callus phase could be reduced thus far, that the majority of the regenerants proved to be phenotypically uniform at the diploid or tetraploid ploidy level.

Abstract: Total polysaccharides, RNA and proteins were localized cytochemically in the developing pollinium of Calanthe masuca (Orchidaceae) The sporogenous cells show thin PAS-positive cell walls and high contents of RNA and proteins to begin with and later PAS-positive granules accumulate in them In the meiocytes, the PAS-positive granules disappear and RNA and protein contents decline Following meiosis, the microspores do not separate from the tetrad The microspores syn-thesize PAS-positive granules, RNA and proteins The thick cell wall material around the meiocytes and tetrads reacts red purple with PAS and toluidine blue The tapetum possesses high RNA and proteins, but does not store polysaccharides Significantly from the tapetum, toluidine blue-positive green material extrudes and accumulate around the pollinium to begin with, and later, on the periphery of the microspores As a consequence, the microspore wall develops green tinge with toluidine blue The anther wall layers store abundant PAS-positive granules, RNA and proteins The endothecial thickenings react red-purple, green and blue with PAS, toluidine blue and amido black lOB tests, respectively

Abstract: Desynapsis, induced by water and temperature stress, was recorded in two varieties of Hemerocallis. Average ‘metaphase I’ association per cell during de-synapsis was 0.76II+20.48 I in ‘Love Song’ and 0.80II+20.39 I in ‘Sleeping Beauty’ whereas during normal meiosis 11 II were invariably observed in the same plants. Equatorial congression in desynaptic meiosis was lacking, but majority of cells at ‘anaphase I’ showed clustering of chromosomes into 2-4 groups. Chromosome decondensation following ‘metaphase I’ was non-synchronous. Second division of meiosis was completely blocked so that the microspores were produced directly from telophase I products.

Abstract: This thesis presents an analysis of the structural and histochemical aspects of cytoplasmic male sterility (cms) in Petunia hybrida . In petunia and in other crops, cms is the most commonly used tool for hybrid seed production. Application of the trait makes hybrid seed production possible without the need of emasculation of the maternal line. However, in spite of its economic importance, little is known on the primary causative factor and the initial step of pollen abortion in cms plants. Insights in the initial effects of cms may lead to a more comprehensive understanding of the regulation and expression of male sterility controlling genes, and, additionally, may possibly provide strategies for the introduction or induction of male sterility in crops in which cms systems are not available. In the first Chapter, some molecular aspects of cms are evaluated. Several lines of evidence indicate that the genetic determinants responsible for cms are carried by the mitochondrial genome. The mitochondrial involvement is found in a variety of plant species, including Petunia hybrida . Most of the mitochondrial encoded polypeptides are components of complexes which are responsible for key steps in the process of oxidative phosphorylation and the generation of ATP. Correspondingly, mitochondria isolated from tissues of cms plants, may code for an aberrant polypeptide composition of components of one of these complexes. Nevertheless, more information on the expression of mitochondrial genes in different anther tissues at various stages of development is necessary before we can conclude whether or not the deviations in mitochondrial DNA are functionally associated with the non-formation of viable pollen. The initial abnormalities in anther development of cms plants are generally found in the tapetal tissue. Also in the cms form of Petunia hybrida cv. Blue Bedder (BBS), the first symptoms of deviation are found in the tapetum (Chapter 2). Light microscopical analysis shows, that in BBS anthers, the tapetal breakdown begins at the prophase stage of the meiocytes. At the preceding stages of development, microsporogenesis in BBS anthers is normal and indistinguishable from the development in the male fertile counterpart (BBF). At the ultrastructural level, the initial aberration of BBS anthers is represented by the presence of large vacuoles in the cytoplasm of the tapetal cells (Chapter 3). At the leptotene stage of the meiocytes, these vacuoles are the first symptoms of degeneration. At later stages, the tapetal and sporogenous cells are highly distorted, the nucleus is disrupted and the cytoplasm disorganized. Mitochondria and plastids degenerate and many lipid droplets are present. Chapter 4 describes the way in which the biochemical and histochemical aspects of an enzyme system are influenced by the degeneration of the tapetal and sporogenous tissues. The Chapter gives information on the isoenzyme pattern, the activity, and the localization of esterases in anther tissues of cms and male fertile petunia cultivars. Esterases are rather unspecific, nuclear encoded enzymes occuring in all plant parts. The biochemical data show that, from the early meiosi S onward, esterase activity in cms-type anthers remains at a low level and hardly any new isoenzyme bands show up as compared to the situation in the male fertile counterpart. The histochemical determinations reveal, that in male fertile-type anthers, esterase activity is concentrated in the outer tapetal layer at late prophase and that it accumulates there till the early microspore stage. In anthers of cms plants, esterase accumulation in the tapetal cells ceases at the moment that tapetal breakdown becomes evident. These results suggest that the differences in total esterase activity and esterase isoenzyme patterns are an effect rather than a cause of the failing pollen formation. In cms forms of different species, aberrations in cytochrome c oxidase activity and other mitochondrial redox processes are associated with the cms plasmatype. A biochemical determination of the cytochrome c oxidase activity in anthers of Petunia hybrida and Zea mays is given in Chapter 5. The biochemical analysis is combined with a cytochemical localization of enzyme activity in mitochondria of sporogenous and tapetal tissues in both species. The data show that in anthers of different cms maize strains, the cytochrome c oxidase activity is reduced in comparison with the level found in male fertile-type anthers. Additionally, there are consistent cytochemical differences in the mitochondrial organization of cytochrome c oxidase activity between pollen of cms- S and male fertile maize plants. The aberrations in enzyme activity are observed at stages of development at which the structural aspects of degeneration are not yet evident. In fact, the deviation in cytochrome c oxidase may represent the initial symptom of male sterility in this maize type. Contrarily, in petunia, the first detectable differences in the mitochondrial enzyme activity occur only after the initial effects of tapetal degeneretion are apparent. Hence, in petunia, the decline in cytochrome c oxidase activity is the result rather than the cause of the proceeding process of degeneration. In Chapter 7 it is postulated that the cms specific deviations in the mitochondrial genome induce alterations in protein complexes which are essential for energy generating processes. Possibly, these aberrations adversely affect the energy status of cms cells. However, BBF and BBS plants possess similar growth characteristics, and, apparently, the viability of plants with cms plasmatype, is not diminished by the mitochondrial defects. In fact, abnormalities in the development of cms plants are only observed in particular anther tissues. These results may suggest that the aberrations in the mitochondrial genome are only expressed in the tapetal or sporogenous tissues at certain moments of development. However, this assumption is inconsistent with the fact that deviations in mitochondrial products are sometimes found in organelles isolated from vegetative parts of the plant. An alternative explanation for the tissue specific character of cms is, that the degeneration of the anther tissues is initiated by the specific metabolism of the cells. The adenylate energy charge ratios of petunia anther tissues is discussed in Chapter 6. As compared with petunia leaf tissue, the results give evidences for the particular metabolic state of the tapetal and sporogenous tissues. Examples of the metabolic activity in anthers of other plant species are evaluated in Chapter 7. Furthermore, the structural analyses as presented in the second and the third Chapter of this thesis, reveal that the cms petunia anther development is distorted at the moment at which there is a considerable rise in the metabolic activity of the tapetal cells of the male fertile counterpart. Possibly, during moments of energetic stress, the mitochondrial synthesis of energyrich products in tapetal cells of cms petunia is insufficient to meet the energetic demands for the normal functioning of the cells at that stage. Hence, as a result of defects in the mitochondrial genome, the tapetal, and consequently, the sporogenous tissues degenerate.

Abstract: Genetically balanced and unbalanced microspores of an interchange heterozygote of Allium triquetrum are shown to behave differently with respect to their first pollen grain mitosis (PGMI). Two type...

Abstract: InHabenaria edgeworthii, Habenaria elisabethae andHabenaria galeandra the young anther wall consists of epidermis, endothecium, single middle layer and glandular tapetum. Tapetal cells are uninucleate and show dual origin. The microspore tetrads are linear, tetrahedral, decussate and isobilateral. At shedding the microspores are 2-celled. Ovules are anatropous, bitegmic and tenuinucellate. The development of embryo sac is of the Polygonum type. The primary endosperm nucleus degenerates. Development of the embryo in all the 3 species corresponds to the Onagrad type. The seed coat is formed entirely from the outer layer of outer integument.

Abstract: The anther wall is 4-layered thick. Its development is of the Monocotyledonous type. Simultaneous cytokinesis results in decussate, isobilateral, linear and tetrahedral tetrads. At anthesis, the microspores are 2-celled. The mature ovules are anatropous, bitegmic and tenuinucellate. Both the integuments are dermal in origin and 2-layered. The inner integument alone forms the micropyle. Development of the female gametophyte is of the Monosporic type. Double fertilization occurs but the primary endosperm nucleus degenerates without any division. Development of embryo corresponds to the variation of the Onagrad type. The mature embryo lacks differentiation. The seeds are minute and non-endospermic. The seed coat is formed entirely by the outer layer of outer integument. There are three sterile and three fertile valves in the ovary. In the prefertilization stages valves consist of parenchymatous cells. After fertilization, the sterile valves become sclerenchymatous whereas the fertile valves remain parenchymatous.

Abstract: The behaviour of plastids and mitochondria during the formation and development of the male gametophyte of Chlorophytum comosum has been investigated using electron microscopy. During first pollen mitosis an intracellular polarization of plastids occurs in that the plastids are clustered in the centre of the microspore. The originating generative cell normally lacks plastids. Only in a small number of microspores have plastids been observed near the dividing nucleus of the microspore and later on in the generative cell. These observations agree with the genetic investigations of Collins (1922) on the mode of plastid inheritance which demonstrated a small amount of biparental plastid inheritance in Chlorophytum. The cytological mechanisms underlying plastid polarization during the first pollen mitosis are discussed.