Abstract: An experiment was conducted to determine critical factors in the recovery of embryos from cultured anthers of broccoli (Brassica oleracea var. ‘italica’) and to unambiguously distinguish whether embryos were of gametophytic origin. Among factors tested, genotype, genotype x anther developmental stage, and method of anther culture had a distinct impact on embryo recovery, whereas length of anther exposure to the culture medium did not. However, extreme heterogeneity of embryo emergence within and among replications precluded statistical contrasts. Among 762 plants derived from embryos of four independent cultivars, only one was determined to be of sporophytic origin by use of heterozygous codominant isozyme markers. Two of the cultivars tested were heterozygous at two or more loci. While segregation among loci was consistent with previously published linkage data, segregation of alleles was consistently non-random. In all of seven separate cases involving four cultivars, a significant over-representation of the fast-migrating class was observed. It appears, therefore, that populations of plants derived from microspores within cultured anthers of broccoli do not necessarily represent a random gametic array, and that care must be exercised in breeding and genetic applications.

Abstract: Solatium tuberosum L diploid strains with superior androgenetic capacity have been selected for from androgenetic progenies of unselected diploid material The paper also demonstrates that the use of a liquid medium for culturing potato anthers, instead of the conventional solid agar plates, improves the yield of androgenetic embryoids The new method, associated with two successive cycles of selection for superior androgenetic response, allows the induction and regeneration of microspore derived plants on a large scale The best genotype (clone 21 in this paper) regenerates androgenetic plants with a frequency around 30 per each anther plated Over 80% of the regenerated plants are diploid It is suggested that the androgenetic embryoids mainly originate from unreduced microspores by a mechanism which maintains a heterozygous or a partly heterozygous genetic situation

Abstract: The synthesis of beta-galactosidase during Brassica campestris pollen development results from the transcription of the haploid genome. A quantitative cytochemical method has been developed in which 5-bromo-4-chloro-3-indoxyl-beta-d-galactoside is used as substrate giving a blue-green final reaction product. We have recently detected oilseed rape plants which are heterozygous for the beta-galactosidase locus, in which 50% of the pollen grains produced are Gal (having enzyme activity), while the other 50% are gal (enzyme deficient). The gal pollen grains served as a built-in control during microspectrophotometric determinations of enzyme activity. The present study has identified the developmental phase at which synthesis of the enzyme commenced. Activity is absent in microsporocytes, tetrads, and at microspore release. Enzyme activity is first detected in the young microspores and, by early vacuolate period, there is an increase in the rate of enzyme activity. A second period of increased enzyme synthesis occurred prior to generative cell division, although the rate is reduced in mature pollen.

Abstract: During male gametophyte development the synthesis of several proteins occurs from transcripts of the haploid genome. Alcohol dehydrogenase (ADH1), a developmentally regulated protein, was chosen for study to determine the stage at which its synthesis was initiated and the pattern of its synthesis during microsporogenesis. The ability of ADH to reduce p-nitro blue tetrazolium chloride in situ was used as an indicator of enzyme activity. Maize strains heterozygous for adh1 were utilized to provide an internal control, 50% of the grains being adh1(+) and 50% being adh1(-). No ADH activity was detectable when tetrads were first formed after meiosis. Activity was initially detected soon after the tetrads began to break apart but before the microspores in the tetrads had completely separated. The transcription of the adh1 gene from the haploid genome must thus occur very soon after meiosis is completed. ADH activity increases at a constant rate thereafter until microspore mitosis when an increase in the rate takes place which lasts until generative cell division. Thereafter, there is a marked decrease in the rate of accumulation of ADH activity.

Abstract: Papaya (Carica papaya L.) anther containing microspores in tetrad to early-binucleate stages were successfully cultured on 1/2 strength MS salts and vitamins with full strength Na-Fe-EDTA supplemented with 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus initiation and formation. Highest frequencies of callus induction were obtained when anthers at the uninucleate stage were cultured in the dark. Haploid plantlets and pollen-derived embryoids were obtained from anthers cultured at the uninucleate stage on solidified MS medium containing 3% sucrose without any growth regulators under a low light intensity (1,500 lux). Large quantities of embryoids were obtained when the original embryoids were transferred to MS medium with 3% sucrose and no growth regulators. Cytology of root tips of embryoid-derived plants confirmed the haploid chromosome number of 9 indicating that the embryoids originated from pollen.

Abstract: This paper describes the development of pollen grains ofGasteria verrucosa from the late microspore to the mature two-cellular pollen grain. Ultrastructural changes and the distribution of plastids as a result of the first pollen mitosis have been investigated using light and electron microscopy. The microspores as well as the generative and the vegetative cell contain mitochondria and other cytoplasmic organelles during all of the observed developmental stages. In contrast, the generative cell and the vegetative cell show a different plastid content. Plastids are randomly distributed within the microspores before pollen mitosis. During the prophase of the first pollen mitosis the plastids become clustered at the proximal pole of the microspore. The dividing nucleus of the microspore is located at the distal pole of the microspore. Therefore, the plastids are not equally distributed into both the generative and the vegetative cell. The possible reasons for the polarization of plastids within the microspore are briefly discussed. The lack of plastids in the generative cell causes a maternal inheritance of plastids inGasteria verrucosa.

Abstract: A medium and replenishment technique for the production of large numbers of calli from anthers of the barley cultivar Elrose is described. The barley anther culture basal medium supplemented with 8 mg/L 2,4-D and 9% sucrose and replenished at 10-day intervals with basal medium containing 1 mg/L indole acetic acid and 3% sucrose was optimal for anther response (21.0%) and productivity (67 calli per 100 anthers plated).

Abstract: Factors favouring pollen callus proliferation, induction of embryogenesis and plant regeneration from cultured anthers of Digitalis obscura L were determined The presence of auxins was essential for cell proliferation and morphogenesis, and incubation in darkness singificantlyincreased these responses Callus proliferation usually preceded embryo development, although sometimes direct embryogenesis was observed On the other hand, bud differentiation was achieved only when callus was transferred to media containing cytokinin or several auxin/cytokinin combinations Different ploidy levels] were observed in the regenerated plants, with approximately 50% being haploid

Abstract: Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis Embryogenesis was also stimulated by an initial culture at 5°C for 3 days Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets The androgenic plants were haploid (2n=18)

Abstract: Normal meiosis and pollen maturation occurred in vitro in anthers of cultured tassels of Zea mays L. (cv. Seneca-60). The meiotic figures observed in propiocarmine squashes of these anthers were comparable to those of anthers which developed in situ, not only in terms of the end product, pollen, but also in the intermediate stages. Meiotic prophase stages, quartets and microspores, and binucleate pollen regularly appeared within 7, 10, and 14 days of culture, respectively. This timing was 3–4 days behind that found in situ. Cytoplasmic and nucleolar staining, chromosomal configurations, and cell size were similar to that found in situ.

Abstract: Cytologically verified ândrogenetic haploid plants were re generated from anthers of "Sarhad Yellow", a maize variety of Pakistani origin, cultured on the modified "yp" medium of GENOVESI and COLLINS /1982/. A 14-day pretreatment of the tassels with liquid Ng medium at 8 C in the dark -proved to be favourable to the ândrogenetic callus and embryoid formation. In pretreated anther cultures the highest induction frequency was shown by uninucleate microspores synchronized before the pollen mitosis I /2.38%/.

Abstract: Publisher Summary The chapter presents a review based on ultrastructure and histochemistry of cellular changes during microsporogenesis The chapter briefly describes the recent information: (1) the ultrastructure of MMCs, (2) nuclear and cytoplasmic changes during microsporogenesis, (3) the ultrastructure of microspores, (4) formation and ultrastructure of vegetative and generative cells, and (5) DNA, RNA, and protein synthesis during microsporogenesis, vegetative, and generative cell formation The MMCs are formed from the archesporial cells The latter cells divide to form a primary parietal layer toward the outside and a primary sporogeneous layer toward the inside The primary sporogeneous cells either function directly as MMCs or undergo further divisions to form a large number of cells The chapter describes the structural and ultrastructural details of MMCs during meiotic prophase The MMCs,at the onset of meiosis, are linked by plasmodesmata In Cucurbita maxima and Lycopersicum esculentum plasmodesmatacontaining cytoplasmic inclusions are observed The mitochondria undergo a few changes during microsporogenesis At prometaphase I, when the nuclear envelope breaks down, the mitochondria intrude into the nuclear area The spherosomes undergo two cycles of aggregation and disaggregation as the meiotic divisions progress in Lilium longiflorum The microspores divide to form pollen grains which consist of two cells of unequal size The two cells are the vegetative and the generative cell The far-reaching changes in the cytoplasm and nucleus of microspore mother cells are presumably manifestations of the restandardization associated with sexual reproduction

Abstract: The histochemical constitution and relationship between the sporogenous tissue and tapetum in the anther of Euphorbia pulcherrima Wilid. is studied. The substances localized are insoluble carbohydrates, RNA and proteins. The sporogenous tissue until it differentiates into PMCs, stores abundant PAS positive grains. This is a distinctive and a rare feature. RNA and proteins are also high in it. In PMCs all these substances reduce to low level in the cytoplasm. From microspore stage to mature pollen consistent increase in RNA and proteins is seen. But the PAS positive grains are noticed only for a short period in the freed spores and they are again accumulate when pollen matures. The differentiation and chemical nature of orbicular bodies based on azure B stain in the tapetum is given.Orbicules from the beginning to end exhibit green colour. Therefore, they are These chemically similar to pollen wall. Extensive deposition of PAS positive callose thickening around the PMCs and its persistence until meiosis is noticed. The probable implications of all these events are discussed in relation of male gametophyte development.

Abstract: Meiotic configurations were studied in pollen mother cells of a tertiary trisomic of rye. Chains of five and chains of three, in alternate orientation, were the most frequent configurations. Assumi...

Abstract: Total polysaccharides, RNA, proteins and ascorbic acid (AA) were localized histochemically in the developing sporogenous tissue and periplasmodium tapetum of Parthenium. Storage polysaccharides are not observed in the sporogenous tissue, meiocytes and in microspore tetrads. High contents of RNA and proteins present in the sporogenous tissue decline in the cytoplasm of the meiocytes. Young microspores show reduced contents of RNA and proteins which increase later when the microspores mature. The microspore wall, the exine and the diffused material accumulated around the microspores, all react green with azure B. AA content is rich in the sporogenous tissue, but it declines in the meiocytes. The microspores within the tetrad show varied quantity of AA. The tapetum exceptionally stores PAS positive and AA granules. RNA and proteins are also high in it. In the plasmodium the stored PAS positive and AA granules decline and lost whereas the RNA and proteins persist considerably in it. In the plasmodium all these histochemical constituents concentrate more around the microspores. The present study clearly recognized the storage nature of the periplasmodial tapetum.

Abstract: Male gamete development in the heterosporous water fern Marsilea vestita is a highly synchronous process which is completed within 5 to 6 hours at 30°C. The morphogenetic and ultrastructural changes which take place during spermatogenesis have been well characterised in light and electron microscope investigations (Sharp 1914; Hepler 1976) but few studies exist on any biochemical aspects of spermatogenesis in this system. To obtain a more comprehensive overview of the molecular events occurring during microspore development, we have investigated the pattern of some of the biochemical processes by a variety of methods. Results from experiments in which inhibitors of RNA, DNA and protein synthesis and microtubule function were applied to developing microspores at various times during the 6 hour period have been described previously (Hyams et al. 1983). They have enabled us, with certain limitations, to determine at what times during spermatogenesis specific molecular syntheses or functions are important. Protein synthesis has been studied further in some detail. Polyacrylamide gel electrophoresis of extracts of Marsilea microspores during development reveals that there is an overall increase in the amount of protein present as spermatogenesis proceeds. In addition to tubulin, the major flagellar protein and constituent of the microtubule ribbon, major polypeptides are detected at 15 k, 26 k, 31 k and 90 k on Coomassie Blue stained gels. Few qualitative changes over the 6 hour period can be detected, even when very sensitive staining methods are used to detect minor proteins. This would appear to show that few proteins are synthesised specifically at particular stages of microspore development. We have investigated this question further by labelling the microspores with 14C leucine and 35S methionine, and performing fluorography of SDS polyacrylamide gels run with the radiolabelled samples. Fluorographs reveal more detail than it is possible to resolve with ordinary staining methods, and several polypeptides have been detected which appear to be heavily labelled at the onset of spermatogenesis but which incorporate progressively less radioactive amino acid as development proceeds. As yet, the identity of these proteins is unknown. We have initiated a study into the presence of individual proteins during spermatogenesis, using specific antibodies reacted against Western blots of polyacrylamide gels. A monoclonal antibody against tubulin (Kilmartin et al. 1982) reveals that this protein is present in small amounts from the onset of microspore development but that the amount increases greatly between 3 and 4 hours, the time when sperm flagella are observed to assemble. We have recently raised a polyclonal serum in rabbit which cross reacts with a high molecular weight polypeptide on Western blots of Marsilea microspore extracts. This protein appears to be present in large amounts at the beginning of the 6 hour period but decreases slightly as development proceeds. The identity of this protein is currently under investigation.

Abstract: The development of gametophytes, initiation of integuments and embryogeny are described inMicrostylis cylindrostachya. The anther wall consists of an epidermis, fibrous endothecium, one middle layer and secretory tapetum with uninucleate cells. Its development corresponds to the Monocotyledonous type. Cytokinesis is simultaneous. The microspore tetrads are decussate, isobilateral and tetrahedral. At shedding, the pollinia are 2-celled. The ovules are anatropous, bitegmic and tenuinucellate. Both the integuments are dermal in origin. Development of the female gametophyte is of the Monosporic type. Double fertilization occurs. The primary endosperm nucleus degenerates. Development of the embryo corresponds to the Asterad type. The mature embryo is undifferentiated. The seed is non-endospermic and the seed coat is formed entirely by the outer layer of the outer integument.

Abstract: Both diploid and tetraploid experimental interspecific hybrids betweenRanunculus silerifolius (2x) andR. chinensis (2x) exhibit normal bivalent pairing. However, microspores of diploid hybrids do not undergo mitosis and their pollen grains are highly sterile, whereas tetraploid hybrids form good pollen grains after microspore division. Evidence is forwarded for the assumption thatR. cantoniensis (4x) has originated by hybridization between these two diploid parental species and by polyploidization of the diploid hybrids. Parallelisms between the different karyotypes ofR. cantoniensis (4x) andR. silerifolius (2x) suggest that the former is a species of polyphyletic origin.

Abstract: Microsporogenesis in cultured material of Azolla microphylla was studied with the light and transmission electron microscopes. The first formed sporangium, a megasporangium, aborts and several microsporangia develop below. Initially, a single sporogenous cell is present, surrounded by a single layered tapetum and the microsporangial wall. Subsequently, several sporogenous cells are connected by plasmodesmata. The microspore mother cells are less densely cytoplasmic than the tapetal cells. Callose-like material is deposited around the microspore mother cells, but disappears before meiosis. The tetrads of microspores contain well defined organelles but less dense cytoplasm than the surrounding periplasmodium. Electron dense material deposited on the plasma membrane of the microspores eventually forms the endospore. The unornamented exospore develops by continued deposition of electron dense material. Degeneration of the periplasmodium gives rise to membranous material which appears to form a template for the massulae.