Microspore Nucleus Research Articles

Transcriptomics reveals a core transcriptional network of K-type cytoplasmic male sterility microspore abortion in wheat (Triticum aestivum L.)

BackgroundCytoplasmic male sterility (CMS) plays a crucial role in hybrid production. K-type CMS, a cytoplasmic male sterile line of wheat with the cytoplasms of Aegilops kotschyi, is widely used due to its excellent characteristics of agronomic performance, easy maintenance and easy restoration. However, the mechanism of its pollen abortion is not yet clear.ResultsIn this study, wheat K-type CMS MS(KOTS)-90-110 (MS line) and it’s fertile near-isogenic line MR (KOTS)-90-110 (MR line) were investigated. Cytological analysis indicated that the anthers of MS line microspore nucleus failed to divide normally into two sperm nucleus and lacked starch in mature pollen grains, and the key abortive period was the uninucleate stage to dinuclear stage. Then, we compared the transcriptome of MS line and MR line anthers at these two stages. 11,360 and 5182 differentially expressed genes (DEGs) were identified between the MS and MR lines in the early uninucleate and binucleate stages, respectively. Based on GO enrichment and KEGG pathways analysis, it was evident that significant transcriptomic differences were “plant hormone signal transduction”, “MAPK signaling pathway” and “spliceosome”. We identified 17 and 10 DEGs associated with the IAA and ABA signal transduction pathways, respectively. DEGs related to IAA signal transduction pathway were downregulated in the early uninucleate stage of MS line. The expression level of DEGs related to ABA pathway was significantly upregulated in MS line at the binucleate stage compared to MR line. The determination of plant hormone content and qRT-PCR further confirmed that hormone imbalance in MS lines. Meanwhile, 1 and 2 DEGs involved in ABA and Ethylene metabolism were also identified in the MAPK cascade pathway, respectively; the significant up regulation of spliceosome related genes in MS line may be another important factor leading to pollen abortion.ConclusionsWe proposed a transcriptome-mediated pollen abortion network for K-type CMS in wheat. The main idea is hormone imbalance may be the primary factor, MAPK cascade pathway and alternative splicing (AS) may also play important regulatory roles in this process. These findings provided intriguing insights for the molecular mechanism of microspore abortion in K-type CMS, and also give useful clues to identify the crucial genes of CMS in wheat.

Comparative transcriptome analysis provides insight into the important pathways and key genes related to the pollen abortion in the thermo-sensitive genic male sterile line 373S in Brassica napus L.

The thermo-sensitive genic male sterility (TGMS) system plays a key role in the production of two-line hybrids in rapeseed (Brassica napus). To uncover key cellular events and genetic regulation associated with TGMS, a combined study using cytological methods and RNA-sequencing analysis was conducted for the rapeseed TGMS line 373S. Cytological studies showed that microspore cytoplasm of 373S plants was condensed, the microspore nucleus was degraded at an early stage, the exine was irregular, and the tapetum developed abnormally, eventually leading to male sterility. RNA-sequencing analysis identified 430 differentially expressed genes (298 upregulated and 132 downregulated) between the fertile and sterile samples. Gene ontology analysis demonstrated that the most highly represented biological processes included sporopollenin biosynthetic process, pollen exine formation, and extracellular matrix assembly. Kyoto encyclopedia of genes and genomes analysis indicated that the enriched pathways included amino acid metabolism, carbohydrate metabolism, and lipid metabolism. Moreover, 26 transcript factors were identified, which may be associated with abnormal tapetum degeneration and exine formation. Subsequently, 19 key genes were selected, which are considered to regulate pollen development and even participate in pollen exine formation. Our results will provide important insight into the molecular mechanisms underlying TGMS in rapeseed.

Cytogenetics, genome size and anther anatomy inBulbostylis(Cyperaceae)

AbstractCyperaceae display diverse and unusual cytogenetical characteristics, but are still sparsely documented and poorly understood in this respect. Six new chromosome counts are presented for Bulbostylis. Absolute DNA content was close to 1 pg in all species and was independent from chromosome number. Despite variations in chromosome number from 2n = 30 to 2n = 102, DNA content only varied from 0.932 pg to 1.198 pg. The studies presented in this work showed that species possess holokinetic chromosomes. The size of stomata and pollen grains did not vary. These results would confirm that the chromosome evolution of these species was via agmatoploidy and symploidy rather than by polyploidy. Abnormalities during meiotic spindle formation in microsporogenesis followed by simultaneous cytokinesis resulted in four microspores, of which only one was functional, resulting in a pseudomonad maturing into pollen grains with the usual vegetative and generative nuclei in addition to three degenerated microspore nuclei.

Resolvase OsGEN1 Mediates DNA Repair by Homologous Recombination.

Yen1/GEN1 are canonical Holliday junction resolvases that belong to the RAD2/XPG family. In eukaryotes, such as budding yeast, mice, worms, and humans, Yen1/GEN1 work together with Mus81-Mms4/MUS81-EME1 and Slx1-Slx4/SLX1-SLX4 in DNA repair by homologous recombination to maintain genome stability. In plants, the biological function of Yen1/GEN1 remains largely unclear. In this study, we characterized the loss of function mutants of OsGEN1 and OsSEND1, a pair of paralogs of Yen1/GEN1 in rice (Oryza sativa). We first investigated the role of OsGEN1 during meiosis and found a reduction in chiasma frequency by ∼6% in osgen1 mutants, compared to the wild type, suggesting a possible involvement of OsGEN1 in the formation of crossovers. Postmeiosis, OsGEN1 foci were detected in wild-type microspore nuclei, but not in the osgen1 mutant concomitant with an increase in double-strand breaks. Persistent double-strand breaks led to programmed cell death of the male gametes and complete male sterility. In contrast, depletion of OsSEND1 had no effects on plant development and did not enhance osgen1 defects. Our results indicate that OsGEN1 is essential for homologous recombinational DNA repair at two stages of microsporogenesis in rice.

Nuclear Fusion during Early Stage of Microspore Embryogenesis Indicates Chromosome Doubling in Wheat (Triticum aestivum)

Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat (Triticum aestivum) remains undetermined. The purpose of this study was to determine the relative time during induction culture when chromosome doubling may occur in wheat, and to identify early indicators for doubled haploid microspores. Microspore nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescent microscope on the day of isolation, three days after isolation, and six days after isolation. The change in the percentage of microspores containing a single small nucleus, two small nuclei, a single enlarged nucleus, and three or more nuclei was then tracked throughout the six-day period. Ploidy levels were estimated by determining the cross-sectional area and number of nucleoli in microspores containing small and large nuclei then comparing the results of each respective cell-type. The percentage of microspores containing enlarged nuclei increased throughout the six-day test period, and the percentage of binucleated microspores containing small nuclei decreased. Comparison of the changes in average percentage of microspores containing a single small nucleus, binucleated microspores, microspores containing a single large nucleus, and multinucleate microspores on days 0, 3, and 6 indicates that nuclei classified as “small” are likely haploids and nuclei classified as “large” are doubled haploids. The percentage of microspores with enlarged nucleus (nuclei) during the first six days of induction culture could be used as an early indicator for the frequency of chromosome doubling in wheat microspore culture.

3H-thymidyne incorporation into the microspores and pollen grains nuclei in excised Tradescantia stamens

The development of microspores and pollen grains lasts in <i>Tradescantia bracteata in vivo</i> from the tetrad stage to pollen shedding about 14 days. This including 7 days of the microspore life cycle. In stamens excised and placed on a medium the microspores and pollen grains develop normally for at least 3 days. <sup>3</sup>H-thymidine is added into medium culture. DNA synthesis m the microspore nucleus is demonstrated 6 days after tetrad formation so at the end of microspore interphase. During synthesis the nucleus lies at one end of the long axis of the vacuolated microspore. Synthesis ends before migration of the nucleus to the proximal pole of the microspore where mitosis begins. Incorporation of <sup>3</sup>H-thymidine into the generative nucleus is noted in two-celled pollen grains as early as about 24h after the end of microspore division. During DNA synthesis the generative cell is rounded and is still adjacent to the pollen grain wall. DNA synthesis ends before separation of the generative cell from the sporoderm, before the generative nucleus starts to elongate. <sup>3</sup>H-thymidine is not incorporated into the vegetative nucleus in stamens developing <i>in vitro</i>.

Characterization of polarity development through 2- and 3-D imaging during the initial phase of microspore embryogenesis in Brassica napus L

Isolated microspores of B. napus in culture change their developmental pathway from gametophytic to sporophytic and form embryo-like structures (ELS) upon prolonged heat shock treatment (5 days at 32 °C). ELS express polarity during the initial days of endosporic development. In this study, we focussed on the analysis of polarity development of ELS without suspensor. Fluorescence microscopy and 3-D confocal laser scanning microscopy (CLSM) without tissue interfering enabled us to get a good insight in the distribution of nuclei, mitochondria and endoplasmic reticulum (ER), the architecture of microtubular (MT) cytoskeleton and the places of 5-bromo-2′-deoxy-uridine (BrdU) incorporation in successive stages of microspore embryogenesis. Scanning electron microscopy (SEM) analysis revealed, for the first time, the appearance of a fibrillar extracellular matrix-like structure (ECM-like structure) in androgenic embryos without suspensor. Two types of endosporic development were distinguished based upon the initial location of the microspore nucleus. The polarity of dividing and growing cells was recognized by the differential distributions of organelles, by the organization of the MT cytoskeleton and by the visualization of DNA synthesis in the cell cycle. The directional location of nuclei, ER, mitochondria and starch grains in relation to the MTs configurations were early polarity indicators. Both exine rupture and ECM-like structure on the outer surfaces of ELS are supposed to stabilize ELS's morphological polarity. As the role of cell polarity during early endosporic microspore embryogenesis in apical–basal cell fate determination remains unclear, microspore culture system provides a powerful in vitro tool for studying the developmental processes that take place during the earliest stages of plant embryogenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00709-013-0530-y) contains supplementary material, which is available to authorized users.

DsDNA and protein co-delivery in triticale microspores

The efficiency of cell-penetrating peptide (CPP)-mediated dsDNA transfection in triticale microspores was investigated through transient and stable integration of the β-glucoronidase (GUS) reporter gene and expression assays in microspore-derived embryos and plantlets. The RecA protein, usually associated with prokaryote homologous recombination, was also tested for its capacity to protect the linear transgene from degradation. Transfections mediated by the CPP nanocarriers Tat2 and Pep1 reduced the number of regenerated embryos from 158 in the control to 122 and 100, respectively. The co-delivery of CPP-dsDNA with RecA protein also resulted in fewer embryos, 87 and 104 for Tat2 and Pep1, respectively. Delivery of dsDNA with Tat2 or Pep1, without RecA, resulted in the highest frequencies of GUS activity in regenerated embryos, at 26%. Co-delivery with RecA decreased the percentage of GUS-positive embryos to 16%. Interestingly, co-delivered RecA-dsDNA reduced the loss of integrity of inserted genetic construct, as observed by polymerase chain reaction (PCR) amplification of the 5′ and 3′ ends. GUS activity was also detected in mature haploid and diploid plants. Of all treatments, 31 T0 plants tested positive for the GUS gene by quantitative PCR, although 50% were derived from the single treatment dsDNA-Tat2. The estimated copy number of the GUS transgene varied between four and eight. This study provides the foundations for CPP-mediated co-delivery of dsDNA and protein RecA in haploid microspore nuclei for functional genomic studies in crop species.

Spatial and Temporal Expression of Actin Depolymerizing Factors ADF7 and ADF10 during Male Gametophyte Development in Arabidopsis thaliana

The actin cytoskeleton plays a crucial role in many aspects of plant cell development. During male gametophyte development, the actin arrays are conspicuously remodeled both during pollen maturation in the anther and after pollen hydration on the receptive stigma and pollen tube elongation. Remodeling of actin arrays results from the highly orchestrated activities of numerous actin binding proteins (ABPs). A key player in actin remodeling is the actin depolymerizing factor (ADF), which increases actin filament treadmilling rates. We prepared fluorescent protein fusions of two Arabidopsis pollen-specific ADFs, ADF7 and ADF10. We monitored the expression and subcellular localization of these proteins during male gametophyte development, pollen germination and pollen tube growth. ADF7 and ADF10 were differentially expressed with the ADF7 signal appearing in the microspore stage and that of ADF10 only during the polarized microspore stage. ADF7 was associated with the microspore nucleus and the vegetative nucleus of the mature grain during less metabolically active stages, but in germinating pollen grains and elongating pollen tubes, it was associated with the subapical actin fringe. On the other hand, ADF10 was associated with filamentous actin in the developing gametophyte, in particular with the arrays surrounding the apertures of the mature pollen grain. In the shank of elongating pollen tubes, ADF10 was associated with thick actin cables. We propose possible specific functions of these two ADFs based on their differences in expression and localization.

The SIDECAR POLLEN gene encodes a microspore-specific LOB/AS2 domain protein required for the correct timing and orientation of asymmetric cell division

Cellular patterning and differentiation in plants depend on the balance of asymmetric and symmetric divisions. Patterning of the male gametophyte (pollen grains) in flowering plants requires asymmetric division of the microspore followed by a symmetric division of the germ cell to produce three highly differentiated cells: a single vegetative cell and two sperm cells. In Arabidopsis sidecar pollen (scp) mutants a proportion of microspores first divide symmetrically, and then go on to produce 'four-celled' pollen with an extra vegetative cell; however, details of the timing and origin of phenotypic defects in scp and the identity of the SCP gene have remained obscure. Comparative analysis of the original hypomorphic scp-1 allele and a T-DNA-induced null allele, scp-2, revealed that in the absence of SCP, microspores undergo normal nuclear positioning, but show delayed entry into mitosis, increased cell expansion and alterations in the orientation of nuclear division. We identified the SCP gene to encode a male gametophyte-specific LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES 2-like (LBD/ASL) protein that is expressed in microspore nuclei in a tightly regulated phase-specific manner. Therefore, our study demonstrates that the correct patterning of male gametophyte depends on the activity of a nuclear LBD/ASL family protein that is essential for the correct timing and orientation of asymmetric microspore division.

Cryptic dioecy in Mussaenda pubescens (Rubiaceae): a species with stigma-height dimorphism.

Evolutionary transitions from heterostyly to dioecy have been proposed in several angiosperm families, particularly in Rubiaceae. These transitions involve the spread of male and female sterility mutations resulting in modifications to the gender of ancestral hermaphrodites. Despite sustained interest in the gender strategies of plants, the structural and developmental bases for transitions in sexual systems are poorly understood. Here, floral morphology, patterns of fertility, pollen-tube growth and floral development are investigated in two populations of the scandent shrub Mussaenda pubescens (Rubiaceae), native to southern China, by means of experimental and open-pollinations, light microscopy, fluorescence microscopy and scanning electron microscopy combined with paraffin sectioning. Mussaenda pubescens has perfect (hermaphroditic) flowers and populations with two style-length morphs but only weak differentiation in anther position (stigma-height dimorphism). Experimental pollinations demonstrated that despite morphological hermaphroditism, the species is functionally dioecious. The long-styled (L) morph possesses sterile pollen and functions as a female, whereas the short-styled (S) morph is female sterile and functions as a male. Self- and intra-morph pollinations of the S-morph were consistent with those expected from dimorphic incompatibility. The two populations investigated were both S-morph (male) biased. Investigations of early stages of floral development indicated patterns typical of hermaphroditic flowers, with no significant differences in organ growth between the floral morphs. Meiosis of microspore mother cells was of the simultaneous type with tetrads isobilateral in shape. The tapetal cells in anther walls of the L-morph became vacuolized during meiosis I, ahead of the uninucleate microspore stage in the S-morph. In the L-morph, the microspore nucleus degenerated at the tetrad stage resulting in male sterility. Microsporogenesis and male gametophyte development was normal in the S-morph. Failure in the formation of megaspore mother cells and/or the development of megagametophytes resulted in female sterility in the S-morph, compared with normal megasporogenesis in the L-morph. In M. pubescens, cryptic dioecy has evolved from stigma-height dimorphism as a result of morph-specific sterility mutations.

Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores

Microspore culture is contributing significantly in the field of plant breeding for crop improvement in general and cereals, in particular. In the present study, we investigated the uptake of fluorescently labeled cell-penetrating peptides (CPP; Tat, Tat(2), M-Tat, peptide vascular endothelial-cadherin, transportan) in the freshly isolated triticale microspores (mid-late uninucleate stage). We demonstrated that Tat (RKKRRQRRR) and Tat(2) (RKKRRQRRRRKKRRQRRR) are able to efficiently transduce GUS enzyme (272 kDa) in its functional form in 5 and 14% of the microspores, respectively, in a noncovalent manner. Pep-1, a synthetic CPP, was able to transduce GUS enzyme in its active form in 31% of the microspores. The effect of various endocytic and macropinocytic inhibitors on Tat(2)-mediated GUS enzyme delivery was studied and revealed a preferred micropinocytosis entry. DNase I protection assay and confocal laser microscopy was carried out to recommend a ratio of 4:1 Tat(2)-linear plasmid DNA (pActGUS) in complex preparation for microspore transfection. We further show that Tat(2) can successfully deliver GUS gene in near to 2% triticale microspores. The negative control mutated Tat (M-Tat: AKKRRQRRR) failed to transducer the GUS protein and transfect the GUS gene in microspore nucleus. The ability of CPPs to deliver macromolecules (protein as well as linear plasmid DNA) noncovalently has been demonstrated in triticale isolated microspores. It further confirms potential applications of CPPs in developing simple, time saving, cost effective plant genetic engineering technologies.

Nuclear bodies in Douglas fir (Pseudotsuga menziesii Mirb.) microspores

The identification of nucleolar proteins and immunocytochemical localization of small nuclear ribonucleoprotein (snRNP) elements revealed the presence of three types of nuclear bodies in Douglas fir microspore nuclei. One type consists of structures resembling Cajal bodies (CBs) and contains nucleolar proteins as well as snRNPs and U2 snRNA. The second type is bizonal bodies, which are nuclear bodies also linked with the splicing system. The bizonal body comprises two parts: the first contains Sm proteins and stains strongly with silver stain, and the second resembles CBs in terms of the degree of silver staining and molecular composition. Douglas fir is the second species after larch where the presence of bizonal bodies has been demonstrated. Pseudotsuga menziesii Mirb and Larix decidua Mill are species with one of the longest microsporogenesis processes known in plants. The presence of bizonal bodies in both species may be linked to the intensification of the splicing processes in microspores with an exceptionally long cell cycle. The third type of structure is dense bodies, whose morphology and degree of silver staining strongly indicate their functional and spatial relationship to the dense part of bizonal bodies.

Microspore development of three coniferous species: affinity of nuclei for flavonoids

The nuclear localization of blue-staining flavanols was investigated histochemically throughout microsporogenesis in yellow cypress (Callitropsis nootkatensis (D. Don) Oerst., formerly Cupressus nootkatensis), juniper (Juniperus communis L.) and yew (Taxus baccata L.). During meiotic development, both the cytoplasm and nuclei of microspores of all species contained varying amounts of flavanols; however, the flavanols were largely confined to the nuclei in microspores just released from tetrads. Quantification by HPLC analysis indicated that, in all species, catechin and epicatechin were the dominant nuclear flavanols. At the early free microspore stage, the nuclear flavanols were barely detectable in all species, but they increased fivefold on incubation in the presence of 0.1 mM benzylaminopurine (BA) or zeatin. Histochemical studies revealed that, in addition to non-fluorescing flavanols, microspores contained yellow-fluorescing flavonoids, which yielded a distinct HPLC flavonoid profile for each species. In yellow cypress, the hydrolyzed flavonoids were identified as quercetin, apigenin, kaempferol and luteolin, whereas only quercetin and myricetin were found in microspores of juniper and in anthers of yew. Application of a UV-VIS titration technique revealed that the aglycone quercetin seems to interact more strongly with histone H3 than either glycoside rutin or kaempferol.

Haploidy in tomato (Lycopersicon esculentum Mill.): a critical review

Results on the induction of haploidy in tomato via both gynogenesis and microspore embryogenesis in vitro are far from satisfactory. The number of reports available on the gynogenic induction via in vitro non-fertilized ovary culture, wide hybridization and the use of irradiated pollen are limited. The main reason for this may be the difficulty experienced in working with this species. Therefore, many failed attempts have not been reported. Non-fertilized ovary culture and wide hybridization using Solanum sisymbriifolium Lam. as the male parent seem to be promising (Bal and Abak, Pak J Biol Sci 6:745–749, 2003a, b). Further efforts in this line may improve results obtained earlier. Several reports (Gresshoff and Doy, Planta 107:161–170, 1972; Sharp et al., Planta 104:357–361, 1972; Zamir et al., Plant Sci Lett 17:353–361, 1980; Chlyah and Taarji, Proc. Int. Symp. Plant tissue and cell culture application to crop improvement. 24–29 Sept. 1984; Jaramillo and Summers, J Amer Soc Hort Sci 115:1047–1050, 1990, HortScience 26:915–916, 1991; Summers et al., HortScience 27:838–840, 1992) are available on anther culture of tomato but a working protocol is yet to be developed. For the induction of anther callus, anthers carrying microspores at the meiotic stages appear to be the most responsive. However, the callus and the regenerants obtained were mainly of somatic origin. Somatic tissues of tomato anthers carrying the meiotic stages are highly responsive to tissue culture manipulations in comparison to anther tissues of the later stages. Therefore, reports on the induction of callus from anthers carrying early microspore stages should be met with caution. If culturing young anthers is of any help then it may be that the anther tissues are nursing the microspores and bringing them to the responsive uninucleate stage. Following the first report by Sharp et al. (Planta 104:357–361, 1972) on the induction of microspore embryogenesis, using a modified version of the microspore culture, reports concentrated only on anther culture (reviewed by Chlyah et al., Haploids in crop improvement I. Biotechnology in agriculture and forestry 12. Springer-Verlag, Berlin, 1990). Based on findings reported by Yinnan et al. (J Agric Biotechnol, http://www.cau.edu.cn/ agrocbi/periodical/ nyswjsxb/ nysw99/ nysw9901/ 990115.htm , 1999) and Bal and Abak (Biotechnol Biotechnol Equip 19:35–42, 2005) on the induction of symmetrical division of microspore nuclei from uninucleate microspores, the formation of multicellular structures and globular embryos, it is likely that the future of tomato haploidy lies in the technique of isolated microspore culture.

Extra divisions and nuclei fusions in microspores from Brassica allohexaploid (AABBCC) × Orychophragmus violaceus hybrids

Abnormal meiosis and microspore development and related defective mutants have often been reported in plants and wide hybrids. Here extra divisions and nuclei fusions were observed to occur in microspore nuclei of partial hybrids between synthetic Brassica hexaploid (2n = 54, AABBCC) and another crucifer Orychophragmus violaceus (2n = 24). Abnormal spindle were formed and chromosomes were separated into several nuclei of variable sizes after bi-, or multi-polar divisions in the four cells of tetrads. As a consequence, more than eight mini-microspores of different sizes were produced by one tetrad. Genomic in situ hybridization results indicated that no chromosome replication occurred during such divisions. In some tetrads, the four nuclei were fused to form one large cell with increased chromosome number. The extra divisions or fusions appeared only in some flower buds of one plant, some anthers in the same buds, or even in individual cells of tetrads. The possible mechanisms behind these cytological phenomena are discussed.

Correlation of sequential floral and male gametophyte development and preliminary results on anther culture in Opuntia ficus-indica

Before approaching anther culture as a tool to trigger an androgenic response in a new species, it is advisable to characterize and correlate flower and male gametophyte development to enable reproducible identification of the appropriate starting material. Buds and flowers of Opuntia ficus-indica cv. Gialla were classified in eight stages according to their total length at the earlier stages and the length of the corolla in flowers with emerging sepals. Due to the low condensation of chromatin in the microspore nucleus as well as in the vegetative nucleus of the bi- and tricellular pollen along with the high autofluorescence of the intricate exine, DAPI staining turned out not to be feasible in this species. Therefore an approach based on light-microscopy observation of semithin sections was used. These sections were stained with toluidine blue for general structure recognition and I2KI to study starch deposition. Correlations were made between the sequential floral and male gametophyte development. Using this approach we determined the timing of pollen formation and observed that pollen development is impaired in plants producing seedless fruits. Furthermore, anther culture was carried out with anthers collected from flower buds at stages 2 and 3. Most of the anthers produced callus, however no regeneration was obtained.

Induction of Symmetrical Nucleus Division and Multicellular Structures from the Isolated Microspores of Lycopersicon Esculentum Mill

ABSTRACTEffects of lactalbumin hydolysate (LH), casein hydrolysate (CH) and a starvation pretreatment using mannitol in the induction of symmetrical nucleus division of uninucleate microspores and formation of multicellular structures from the isolated microspores of tomato were studied. Flower buds containing uninucleate microspores were incubated on media for the pretreatment containing either mannitol or sucrose at 10 °C for seven days. Subsequently microspores were isolated and cultured on NLN basic media in the presence of BAP, NAA, LH and CH. Microspores from the cv. Fantastic, cultured in the presence of BAP at 0.5 mg l−1 and NAA at 0.5, 1.0 and 1.5 mg l−1 together with LH at 2 or 4 ml l−1 proceeded in the sporophytical pathway, microspore nuclei dividing symmetrically. Formation of multicellular structures were induced which was also the case for other cultivars cultured in the NLN medium containing 0.5 mg l−1 BAP and 0.5 mg l−1 NAA together with CH either at 5 or 10 g l−1. Mannitol pretreatment induced microspore starvation and stress therefore microspores from the mannitol pretreatment were more responsive to the symmetrical division than those from the sucrose pretreatment.

Distribution of splicing proteins and putative coiled bodies during pollen development and androgenesis in Brassica napus L.

Small nuclear ribonucleoprotein particles (snRNPs) are subunits of splicing complexes, which show a transcription-dependent localization pattern. We have analyzed the labelling pattern of snRNPs during pollen development and microspore and pollen embryogenesis in Brassica napus with an antibody which recognizes protein D of U1, U2, U4, U5, and U6 snRNPs. It was found that nuclei were labelled almost uniformly for snRNPs in microspores and young bicellular pollen. In the generative nuclei of late-bicellular pollen and in the vegetative nuclei and sperm nuclei of mature pollen no snRNPs could be detected. The snRNP-positive nuclei contained mostly one or two brightly labelled nuclear bodies, most likely coiled bodies, often closely related to the nucleolus. These nuclear bodies increased in size from 0.5 micron in nuclei of young microspores up to 2 microns in nuclei of late microspores and the vegetative nucleus of early-bicellular pollen. Also their number increased during these developmental stages. After induction of embryogenesis the size of the coiled bodies decreased to about 0.5 micron and in several occasions the coiled body was found free in the nucleoplasm, away from the nucleolus. The results support the idea that the size and number of coiled bodies coincide with changes in general nuclear activity. They also indicate that, in nuclei of Brassica napus, at least assembly and disassembly of coiled bodies takes place in the nucleoplasm, whereas mature coiled bodies are located adjacent to the nucleolus.

Unique positioning of mitochondria in developing microspores and pollen grains inPharbitis nil: mitochondria cover the nuclear surface at specific developmental stages

Changes in the number and distribution of mitochondria in microspores and pollen grains during male gametogenesis inPharbitis nil were examined with Technovit sections stained with 3,3′-dihexyloxacarbocyanine iodide. The number of mitochondria per microspore or pollen grain ofP. nil increased constantly and dramatically during male gametogenesis. During this process, mitochondria exhibited characteristic localizations: subpopulations of mitochondria covered the surface of the microspore and vegetative nuclei before and again just after postmeiotic mitosis I (9 and 5 days before flowering, respectively). The mitochondria also surrounded the generative nucleus 2 days after postmeiotic mitosis I (5 days before flowering), although the density of mitochondria on the nuclear surface was lower. Electron microscopy showed that the mitochondria were about 30 nm from the nuclear envelope and that each mitochondrion was located near a nuclear pore. The characteristic localization of mitochondria inP. nil pollen may serve as a model to analyze the mechanisms that control mitochondrial positioning within a cell and interactions between mitochondria and nuclei.