Microspore Developmental Stage Research Articles

The influence of pretreatment on cell stage progression and the time of DNA synthesis in barley ( Hordeum vulgare L) uninucleate microspores.

The objective of this study was to correlate the time that DNA synthesis first occurs in haploid microspores of barley with cell cycle and plant morphological stages and to subsequently assess the influence of pretreatments on DNA synthesis at different stages of microspore development. Spikes with microspores in early, mid, and late uninucleate stages of the two-rowed barley cultivars Manley and Igri were subjected to two commonly used pretreatments. First, during cold pretreatment for 28 days there was a slow increase in relative DNA values as well as asymmetric nuclear divisions in some microspores. Second, during a 4-day cold plus 0.3 M mannitol pretreatment, there was very little change in the microspore stage or DNA values indicating that for the duration of this pretreatment the progression of the cell cycle was essentially suspended at all stages, both in Igri and Manley. The results are discussed relative to the potential for genetic transformation of microspores.

Low temperature induced spikelet sterility in rice. II. Effects of panicle and root temperatures

Low temperatures impose restrictions on rice (Oryza sativa L.) production at high latitudes. This study is related to low temperature damage that can arise mid-season during the panicle development phase. The objective of this study was to determine whether low temperature experienced by the root, panicle, or foliage is responsible for increased spikelet sterility. In temperature-controlled glasshouse experiments, water depth, and water and air temperatures, were changed independently to investigate the effects of low temperature in the root, panicle, and foliage during microspore development on spikelet sterility. The total number of pollen and number of engorged pollen grains per anther, and the number of intercepted and germinated pollen grains per stigma, were measured. Spikelet sterility was then analysed in relation to the total number of pollen grains per spikelet and the efficiency with which these pollen grains became engorged, were intercepted by the stigma, germinated, and were involved in fertilisation. There was a significant combined effect of average minimum panicle and root temperatures on spikelet sterility that accounted for 86% of the variation in spikelet sterility. Total number of pollen grains per anther was reduced by low panicle temperature, but not by low root temperature. Whereas engorgement efficiency (the percentage of pollen grains that were engorged) was determined by both root and panicle temperature, germination efficiency (the percentage of germinated pollen grains relative to the number of engorged pollen grains intercepted by the stigma) was determined only by root temperature. Interception efficiency (i.e. percentage of engorged pollen grains intercepted by the stigma), however, was not affected by either root or panicle temperature. Engorgement efficiency was the dominant factor explaining the variation in spikelet sterility. It is concluded that both panicle and root temperature affect spikelet sterility in rice when the plant encounters low temperatures during the microspore development stage.

Mammalian neurohormones: potential significance in reproductive physiology of St. John's wort (Hypericum perforatum L.)?

Melatonin and serotonin are indoleamine neurohormones that function as photoperiod signals in many species and have recently been found in St. John's wort, a medicinal plant used in the treatment of depression. There is no known role for melatonin in higher plants but melatonin functions as a signal of changes in photoperiod in other species. In the current study, serotonin and melatonin were quantified during flower development. Higher concentrations of serotonin were found in flower buds at the tetrad stage of microspore development and higher melatonin concentrations were detected during uninucleate mircosporogenesis. Additionally, the regeneration potential of isolated anthers was highest in the same stage that had elevated melatonin concentrations. These data provide the first evidence of the presence of melatonin during flower development and raise many questions about the potential roles of serotonin and melatonin as regulatory molecules in the reproductive flexibility of higher plants.

A chimeric ecdysone receptor facilitates methoxyfenozide-dependent restoration of male fertility in ms45 maize.

A mutation in the maize Ms45 gene results in abortion of microspore development and a male-sterile phenotype. MS45 protein has been localized to the tapetum and maximally expressed in anthers at the early vacuolate stage of microspore development. Molecular complementation analysis determined that a transformed copy of the gene fully restored fertility to ms45 maize. In this report, using phenotypic complementation as an assay, chimeric transcriptional activators were expressed to regulate a gal:MS45 gene and test the ability of a multi-component system to restore male fertility. A high frequency of phenotypic complementation was observed when either C1-GAL4 or VP16-GAL4 activators were transcribed by promoters that expressed at a stage of anther development that precedes the early vacuolate stage of microsporogenesis. For the conditional regulation of male fertility, these transcriptional activators were modified by the addition of regions that include the ligand-binding domain from the European corn borer ecdysone receptor to generate the nuclear receptors C1-GAL4-EcR (CGEcR) and VP16-GAL4-EcR (VGEcR). These chimeric receptors were introduced with the gal:MS45 gene into ms45 maize, and in the absence of ligand, these plants were male sterile. In contrast, application of the ecdysone agonist, methoxyfenozide, to plants containing either a constitutive (Ubiquitin1) or anther-specific (maize 5126) VGEcR resulted in the restoration of fertility to ms45 plants grown in either the greenhouse or in the field.

Colchicine-induced microspore embryogenesis in coffee

A protocol for the induction of androgenesis and plant regeneration from C. arabica cv. Caturra isolated microspores in vitro using colchicine pretreatment has been developed. Microspores were mechanically isolated and then carefully purified. Before colchicine pretreatment, microspores were cultured in a semi-solid medium for further develop and regeneration. Different times of colchicine exposure as well as different concentrations were tested. The best androgenic response was found when microspores were precultured in 100 mg l−1 colchicine for 48 h. The microspore developmental stages responsive to colchicine were late-uninucleated and early binucleated pollen. Flow cytometry and morphological analyses revealed that 95% of regenerated plants were dihaploids (2n=2x=22). However, some doubled dihaploid plants (2n=4x=44) were also obtained, suggesting that not only androgenic induction but also chromosome duplication could be expected as result of colchicine exposure of coffee microspores. This report represents a new approach in the coffee pollen culture, as well as a major step forward to the utilization of haploid technology in coffee breeding.

An efficient androgenic embryogenesis and plant regeneration method through isolated microspore culture in timothy ( Phleum pratense L.)

A method employing isolated microspore culture was established for the androgenic embryogenesis of timothy (Phleum pratense L). Embryos/calli were obtained and green plants regenerated. The induction medium was PG-96 (1.0 mg l-1 2,4-D, 0.1 mg l-1 Kinetin) supplemented with 6% maltose monohydrate. Timothy microspore culture was genotype-dependent, among 12 genotypes, 6 produced embryos/calli and 4 produced green plants. Macerating the spikes with a blender and purifying the microspores at a mannitol/maltose monohydrate interface gave a relatively high percentage of cell vitality. The optimum microspore developmental stage was from the very late uninucleate stage to the binucleate stage. Heat shock promoted the initiation of microspore culture. Over 150 regenerated green plants were obtained; in a random sample of 32 of these 65.6% were doubled haploids (6n=42). Albinism was a problem in plant regeneration (9.3-22%). This paper is the first to describe timothy androgenic embryogenesis by isolated microspore culture.

Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers.

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

Pleiotropic effects of a nuclear restorer-of-fertility locus on mitochondrial transcripts in male-fertile and S male-sterile maize.

Cytoplasmic male sterility (CMS) is encoded by the plant mitochondrial genome and can be reversed by nuclear restorer-of-fertility(Rf) alleles. In the CMS-S system of maize, reproductive failure and fertility restoration are gametophytic, occurring during the starch-filling stages of pollen development. Transcripts of the CMS-S-associated mitochondrial open reading frames (orf355 and orf77) are present from the early stages of microspore development through the aborted pollen stage. To investigate the molecular basis of fertility restoration, we compared mitochondrial-transcript accumulation in aborting CMS-S pollen and in CMS-S pollen restored to fertility by the Rf3 nuclear allele. In the presence of the Rf3 allele, novel, shorter transcripts of the orf355-orf77, cob and atp6 mitochondrial genes were created, and the relative abundance of larger transcripts was decreased for each of these loci. The altered transcript patterns cosegregated with male fertility conditioned by the Rf3 allele. The novel cob and atp6 transcripts were also observed in leaf-tissues of both normal and S-cytoplasm plants carrying the Rf3 allele. These observations support the hypothesis that the Rf3 allele encodes, or regulates, a modifier of mitochondrial transcript (Mmt) activity that affects both CMS and essential mitochondrial gene transcripts.

Mitochondrial gene expression in developing male gametophytes of male-fertile and S male-sterile maize

Mitochondria play a critical role in the normal development of the plant male gametophyte and in the disruption of normal gametophyte development associated with cytoplasmically inherited male sterility (CMS). To investigate the role of mitochondria in these processes, the accumulation of mitochondrial gene transcripts and the accumulation of nuclear gene transcripts encoding mitochondrial proteins were investigated through male gametophyte development in normal maize and through the course of pollen abortion in CMS-S maize. Male gametophytes differing in developmental stage were isolated from male-fertile or male-sterile plants by sucrose density gradient centrifugation. Mature pollen was collected from dehiscent anthers of male-fertile plants. Aborted pollen, which collapsed during starch accumulation, was isolated from emergent tassels of CMS-S male-sterile plants. Microspores, developing pollen and mature pollen exhibited striking differences in mitochondrial transcript accumulation. Mature pollen lacked detectable mitochondrial transcripts. Aborted pollen of CMS-S plants contained abundant, intact transcripts of all mitochondrial genes studied, but prematurely degraded transcripts of several nuclear genes. Transcripts of the CMS-S associated mitochondrial open reading frames (orf355 and orf77) were detected from the early stages of microspore development through the aborted pollen stage. The implications of these findings are discussed in terms of the mitochondrial requirements for pollen function and the mechanism of pollen abortion in CMS-S maize.

Characterization of an anther-expressed protein kinase gene in the potato Solanum berthaultii and its antisense inhibition in transgenic plants

A cDNA clone (1.7 kb) isolated from a cDNA library of in-vitro germinated pollen from the potato species Solanum berthaultii contains an open reading frame of 465 amino acid residues. The predicted protein sequence has typical sequence characteristics of the catalytic subunit of the cyclic-nucleotide dependent protein kinases isolated from yeasts and animals, and the gene has been provisionally termed SBPK, (Solanum berthaultii protein kinase) A transcript of about 1.9 kb was detected on a northern blot in developing anthers, mature pollen, and in-vitro germinated pollen, but not in other plant tissues. The expression in anthers corresponds to the late uninucleate stage of microspore development. Isolation and nucleotide sequencing of a SBPK genomic clone revealed the kinase transcribed region to be interrupted by two introns, both close to the 5′end of the open reading frame, one upstream and one downstream of the start codon. Chimeric genes were constructed by fusing the coding sequence in the antisense orientation to the promoter of a strong late-expressed pollen gene, LAT52, and introduced in potato. Transformants fell into two classes, one displaying a typical antisense phenotype, and a second type arising from a cosuppression event. Both types of transformant gave rise to increased proportions of defective pollen, although the cosuppression effect was more severe. The results indicate that this anther-expressed protein kinase is essential for pollen development in potato.

Expression of tandem invertase genes associated with sexual and vegetative growth cycles in potato.

The organisation of two invertase genes (invGE and invGF) linked in direct tandem repeat within the potato genome is detailed. The genes exhibit a similar intron/exon structure which differs from previously described plant invertase genes; while intron locations are conserved between the genes, minor differences in exon length are seen. Both genes encode enzymes with putative extracellular location. Biochemical analysis of gene expression showed expression in floral tissues for both genes, with expression of the upstream gene (invGE) also detected in leaf tissue. Promoter sequences from both genes have been fused to the beta-glucuronidase (GUS) reporter gene (uidA) and transformed into potato. One promoter-GUS reporter construct was also transformed into tobacco. Histochemical analysis of transgenic lines defined specific expression from the downstream (invGF) promoter in potato and tobacco pollen, with expression first detected in the late uninucleate stage of tobacco microspore development. The invGE promoter determined expression in pollen and other floral tissues, but also at lateral nodes in stem, root and tuber. An association of invertase expression with generative tissue, both in vegetative and sexual modes of growth, is indicated.

Impact of microspore developmental stage on induction and plant regeneration in rye anther culture

Rye anthers grouped into eight length classes, containing microspores from early uninucleate to binucleate developmental stage, were cultured to study the effect of microspore development on induction of embryogenesis and regeneration of green and albino plants. Three rye cultivars were monitored in two tests. Callus and embryoid induction increased in all cases beyond first pollen mitosis. Embryo quality was influenced by microspore age, and the ability of embryoids to develop into plants decreased as induction rate increased. Microspore developmental stage did not influence the proportion of albino plants of all regenerants. These findings have consequencies for studies on regeneration and in determining the optimum microspore developmental stage for efficient regeneration of green rye plants.

`Isubgol' as an alternative gelling agent in plant tissue culture media.

`Isubgol', the mucilaginous husk derived from the seeds of Plantago ovata, was successfully used as a gelling agent in tissue culture media for in vitro seed germination, shoot formation and rooting in Syzygium cuminii and anther culture in Datura innoxia. For seed germination, Knop's basal medium supplemented with 1% sucrose was employed, whereas for the development of shoots the epicotyl segments excised from in vitro-developed seedlings were cultured on MS basal medium supplemented with 4% sucrose and 1 mg/l 6-benzyladenine. Shoots that developed from the epicotyl segments were rooted on Knop's medium enriched with 2% sucrose and 1 mg/l indole-3-acetic acid. The anthers of D. innoxia excised at the late uninucleate to early binucleate stages of microspore development were cultured on Nitsch's basal medium containing 2% sucrose. Media were either gelled with 0.9% agar or 3% `Isubgol'. The response on media gelled with `Isubgol' in each of the cases was similar to that on media solidified with agar.

Improved Nuclear Staining of Asparagus Microspores for Cytological Analysis

Clear visualization of asparagus (Asparagus officinalis L.) microspore nuclei with common stains such as acetocarmine or DAPI is difficult, hindering cytological analyses. The addition of saturated aqueous ferric chloride solution to Carnoy's I fixative (30 μL·mL-1) resulted in clear visualization of nuclei. A distinct nucleus was observed in uninucleate cells and the vegetative and generative nuclei were clearly visible in binucleate microspores. This method can be used reliably for determination of asparagus microspore developmental stage. Chemical name used: 4′,6-diamidino-2-phenylindole-2HCL (DAPI).

PRODUCTION OF EMBRYOS FROM MICROSPORE CULTURES OF PORTUGUESE TRONCHUDA CABBAGE LANDRACES

Twelve accessions of tronchuda cabbage landraces were tested for their ability to produce embryos through microspore culture in NLN-13 medium. A sample from all the isolations was stained with DAPI for determination of microspore developmental stage. The relationship between the microspore developmental stage and the production of microspore-derived embryos was evaluated. Embryos were obtained from all the accessions. Considerable variation was observed between isolations with different developmental stage of microspores, accessions and plants within the same accession. The best embryogenic responses occurred in microspore populations with approximately 10 to 89% of binucleate pollen. The highest embryo yield was obtained with one of the ‘Couve de Valhascos’ accessions (0.41 embryos / 1 000 microspores) and the lowest yields with the accessions of ‘Couve Portuguesa’ (less than 0.07 embryos / 1 000 microspores).

Induction of embryogenesis with colchicine instead of heat in microspores ofBrassica napus L. cv. Topas

Prior to this report, heat treatment (32.5°C, 24 h) was the method used to induce embryogenesis fromBrassica napus microspores. Continuous culture at 25°C results in pollen development. This study shows that colchicine alone, at the non-inductive temperature of 25°C, can induce embryogenesis, thus demonstrating that heat shock is not required for embryogenic induction inB. napus cv. Topas. Embryogenic frequencies of over 15% were obtained by culturing isolated microspores with 25 μM colchicine for 42 h at 25°C. The microspore developmental stages responsive to colchicine were unicellular vacuolate and late unicellular, somewhat earlier stages than the population responsive to heat induction. Other groups have reported that heat-shock proteins are essential to the induction of embryogenesis. The present study offers a method of embryogenic induction without the use of heat which will allow discrimination between the factors associated with response to heat shock and those involved with changing cell development.

Callus and Somatic Embryogenesis from Cultured Anthers of Service Tree (Sorbus domestica L.)

The effects of stage of microspore development, cold pretreatment, and growth regulators on the embryogenic response of anthers from service tree (Sorbus domestica L.) are reported. Callus proliferation required the presence of growth regulators in the culture medium, best results being obtained with combinations of auxins and BA. Microspore embryogenesis was only induced when anthers containing tetrads or uninucleate pollen were cultured on BA-supplemented media containing IBA or IAA. A cold pretreatment before anther culture elicited a significant decrease in callus formation and inhibited embryogenesis. Chemical names used: benzyladenine (BA), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA).

Induced androgenesis in alfalfa (Medicago sativa L.)

Anthers of 10 alfalfa (Medicago sativa L.) lines were used as initial material for the production of androgenic haploids. More than 30 variants of nutrient media were tested. Twenty five different treatments with low temperatures and gamma rays were tried in order to find optimal conditions for callus induction and organogenesis.The genotype, stage of microspore development, phytohormonal composition of the nutrient media and pretreatment with physical agents, alone or in combination, affected the efficiency of organogenesis and regeneration in anther cultures of alfalfa.Plants exhibited a high degree of variability in their chromosome number. Haploids, dihaploids and mixoploids were obtained.Cytological studies of in vitro pollen development revealed the origin of the regenerants from microspores.

Deep floodwater protects high-nitrogen rice crops from low-temperature damage

A field experiment was conducted to investigate the interaction of nitrogen (N) status, sowing date, and water depth on rice yield in the Murray Valley, New South Wales, where low minimum temperatures often lead to pollen sterility and low yield in high N status crops. The experiment combined 4 N rates (0, 75, 125, 250 kg N/ha) applied as urea just before permanent flood; 3 cultivars (Jarrah, early maturing; Amaroo and Doongara, midseason); 2 sowing dates (26 September, 31 October 1991); and 2 water depths (5, 20 cm) at the microspore stage. The early-sown crops and the later sown Jarrah crop encountered minimum air temperatures of 19�C at the cold-sensitive microspore stage, compared with 15�C for the other 2 later sown crops. Total dry matter production was little affected by either water depth or sowing date, but increased from 16 to 22 t/ha with N application. Shallow water depth and delayed sowing date affected yield through reduced harvest index. Grain yield response to N fertiliser was dependent on sowing date, water depth, and variety. For the early-sown crops grown in deep water, yields of Amaroo and Jarrah-increased from 7 to 13 t/ha with increasing N supply, while the yield response of Doongara plateaued at 9 t/ha. With shallow water, the yields of all varieties decreased from 7 to 3 t/ha with increasing N. For the later sowing date, Jarrah growing in deep water yielded up to 13 t/ha at high N, but yields of Amaroo and Doongara decreased from 7 to 2 t/ha with increasing N supply. For the later sowing date, yields of all varieties growing in shallow water decreased to <2 t/ha with applied N. In the deep water crops, developing microspores were submerged or partially submerged and so avoided low air minimum temperatures. In the early-sown crops, the microspore stage occurred during a period of relatively warm nights. Harvest index was successfully modelled using panicle temperature and N content at early pollen microspore. The experiment shows that high N applications can lead to high rice yields provided the microspores are protected from low temperatures by the use of deep water at the stage of microspore development. As a result, recommendations for N fertiliser application need to be adjusted for sowing date and expected water depth at early pollen microspore

The proteins encoded by two tapetum-specific transcripts, Satap35 and Satap44, from Sinipis alba L. are localized in the exine cell wall layer of developing microspores

By differential screening of a copy DNA (cDNA) library from flowering Sinapis alba L. apices against cDNAs from vegetative apices, two cDNA clones were isolated representing transcripts that are expressed transiently at an early stage of tapetum development. The Sa tap35 cDNA encodes a polypeptide with a predicted molecular weight of 12.7 kDa and an isoelectric point of 10.4. The sa tap44 cDNA codes for a putative 12.4-kDa polypeptide with an isoelectric point of 7.5. The deduced amino-acid sequences display 76% sequence identity and contain an N-terminal stretch of hydrophobic amino acids which has characteristics of secretory signal sequences. In-vitro transcription of the cDNAs and translation of the resulting RNAs in the presence of canine pancreatic microsomes demonstrates that the two proteins are translocated into the microsomes and that the putative preproteins are proteolytically processed to the mature forms. By immunoelectron microscopy the Sa TAP35 and Sa TAP44 proteins were detected at the developing peritapetal membrane between the tapetal cytoplasm and the adjacent middle layer of the anther wall. Furthermore, labelling was observed within the locule in association with globules resembling pro-Ubisch bodies which appeared at the tetrad stage. During the early vacuolate stage of microspore development the young exine was strongly labelled. The exine and the peritapetal membrane both are composed of sporopollenin, and the pro-Ubisch bodies are thought to contain sporopollenin precursors. Thus, Sa TAP35 and Sa TAP44 might be involved in sporopollenin formation and/or deposition.