Scanning electron microscopy of microspore embryogenesis inBrassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy     

Nuclear pore dynamics during pollen development and androgenesis in Brassica napus

Changes in nuclear pore complex (NPC) densities, NPCs/nucleus and NPCs/μm³, are described using freeze-fractured Brassica napus microspores and pollen in vivo and in vitro. Early stages of microspore- and pollen-derived embryogenic cells were also analysed. The results of in vivo and in vitro pollen development indicate an increase in activity of the vegetative nucleus during maturation of the pollen. At the onset of microspore and pollen culture, NPC density decreased from 15 NPCs/μm² at the stage of isolation to 9 NPCs/μm², under both embryogenic and non-embryogenic conditions. This implies that the drop in NPC density might be a result of culturing the microspores and pollen rather than an indication for microspore and pollen embryogenesis in Brassica napus. However, after 1 day in culture under embryogenic conditions, the NPC density increased again and stabilised around 13 NPCs/μm², whereas under non-embryogenic conditions the NPC density remained about 9 NPCs/μm². This low density of 9 NPCs/μm² was also found in the nuclei of sperm cells, in contrast to the 19 NPCs/μm² found in the vegetative nucleus. It means that, although both the vegetative and sperm nuclei are believed to be metabolically rather inactive in mature pollen, the NPC density of vegetative nucleus is twice as high as the NPC density of the sperm nuclei. In a few cases, embryos formed suspensor-like structures with a NPC density of 9 NPCs/μm², indicating a lower nucleocytoplasmic exchange of the nuclei of the suspensor cells than with the nuclei in the embryo proper. In addition, observations on NPCs and other organelles, obtained by high resolution cryo-scanning microscopy, are presented. Received: 29 December 1999 / Revision accepted: 3 March 2000     

油菜小孢子胚发生的超微结构和胚状体形态

应用透射电镜和扫描电镜分别研究油菜游离小了包子培养后细胞的超微结构和胚状体的形态。单核晚期小孢子经培养后,具有胚状体发生能力的细胞中央液泡消失,积累淀粉,含丰富细胞器。     

Microspore Culture of Hybrids Between Brassica napus and B. campestris

Embryos and regenerated plants were produced by isolated microspore culture of inter-specific hybrids between Brassica napus and B. campestris. The NLN media with different sucrose concentrations and pH values were tested and a protocol for optimal microspore culture of B. carnpestris was identified. The reciprocal hybrids between UM921 (B. campestris) and 911186 (B. napus) had significant higher embryo yield than other cultured hybrids. Obvious improvement of embryo yield and quality was achieved when hybrid plants of reciprocal UM921 × 911186 were grown under 10 ℃/5 ℃ (day/night) condition. There was significant correlation between embryo yield and seeds per pod on hybrid plants but no correlation between pollen fertility and embryo yield was detected among cultured.hybrids. The majority of microspore-derived plants from the reciprocal B. napus × B. campestris hybrids are aneuploids and 22.8% of the plants observed originated from the microspores with parent′s chromosome numbers, almost all n = 19. The factors affecting the embryogenesis in microspore culture of interspecific hybrids and the possible applications of the technique are discussed.     

Cytoskeletal changes and induction of embryogenesis in microspore and pollen cultures of Brassica napus L.

Microspores and pollen of Brassica napus were cultured under conditions leading to embryo formation. Concomitant changes in cytoskeletal configurations were analysed. The microfilamental cytoskeleton exhibited a loss of polarity in embryogenic cells but cytochalasin treatment revealed that microfilaments do not influence embryogenesis. Two embryogenic pathways started from microspores and were either characterized by turned division planes or by division when the nucleus was in the cell centre. In both cases microtubules clearly exhibited new arrangements and likely played a major role in newly induced symmetrical division. In pollen, embryogenic development started in the vegetative cell provided the generative cell was arrested near the pollen wall. The concomitant disappearance of defined microtubular arrays is likely to be responsible for the positioning of the cell.     

Application of trifluralin to embryogenic microspore cultures to generate doubled haploid plants in Brassica napus

Microspore or anther culture has been used to produce desirable meiotic recombinants in numerous species. However, the utilization of these recombinants relies on inefficient genome doubling procedures to obtain fertile doubled haploid plants. This study presents a simple and rapid procedure to generate fertile doubled haploids in Brassica napus cv. Topas using trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl- p -toluidine), a plant specific microtubule inhibitor. The effects of trifluralin on microtubule depolymerization and chromosome doubling in embryogenic microspore cultures of B. napus were examined and compared with those of colchicine. Indirect immunofluorescence labeling of isolated microspores indicated that microtubules were depolymerized within 30 min of trifluralin treatment and after 3–8 h of colchicine treatment. The direct application of these microtubule inhibitors to microspore cultures resulted in the recovery of fertile doubled haploid plants. Continuous culture in the presence of colchicine, was more effective than 18-h treatments for fertile plant production but resulted in abnormal embryo formation and recalcitrant plant regeneration. The application of 1 or 10 μ M trifluralin during the first 18 h of microspore culture was found to be the superior method for doubled haploid production. The embryos generated after trifluralin treatment developed normally, germinated readily and of the plants produced, close to 60% were fertile. The use of trifluralin to double chromosomes very early in microspore cultures is a simple process requiring minimal manipulation and should be very useful for genetic studies and breeding programs of B. napus and possibly other species.     

Flow Cytometric Characterization of Embryogenic and Gametophytic Development in Brassica napus Microspore Cultures

Microspore cultures are ideal systems for studying plant embryogenesisbecause the resulting embryos are very similar to zygotic embryos,all the stages of development are readily accessible and theprocess can be induced by a simple heat treatment. However,not all microspores are embryogenic and the mixture of cellsthat develops in the cultures complicates the use of this system.Brassica napus microspore cultures cultured at 30°C (induced)and at 25°C (non-induced) were compared by flow cytometryto obtain structure and function information for several typesof cells in the culture. Clear differences in light scatterand fluorescence were found between induced and noninduced culturesthat are related to early stages of embryo development. Viable,round cells that were unique to induced cultures were sortedinto culture media and developed into embryos confirming thatthey were embryogenic. The present study provided flow cytometricidentifiers for embryogenic and gametophytic cells, demonstratedhow flow sorting can be used to isolate specific cell typesand defined benchmarks for assessing the embryogenic potentialof microspore cultures. (Received July 9, 1997; Accepted December 10, 1997)     

Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.     

Stress as the major signal controlling the developmental fate of tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis

Specific stress treatments (sucrose starvation, alone or combined with a heat shock) applied to isolated tobacco (Nicotiana tabacum L.) microspores irreversibly blocked normal gametophytic development and induced the formation of embryogenic cells, which developed subsequently into pollen-derived embryos by culture at 25°C in a sugar-containing medium. A cold shock at 4°C did not inhibit microspore maturation in vitro and did not induce cell division activity, even when combined with a starvation treatment. In the absence of sucrose, microspores isolated in the G1 phase of the cell cycle replicated their DNA and accumulated in G2. Late microspores underwent miotosis during the first day of culture which resulted in a mixed population of bicellular pollen grains and uninucleate microspores, both embryogenic. After the inductive stress treatments the origin of the first multicellular structures, formed in the sugar-containing medium, could be traced to divisions of the microspore cell or divisions of the vegetative cell of bicellular pollen, indicating that the symmetry of microspore mitosis in vitro is not important for embryogenic induction. These results represent a step forward towards a unified model of induction of embryogenesis from microspores/pollen which, within a relatively wide developmental window, are competent to deviate from normal gametophytic development and initiate the alternative sporophytic programme, in response to specific stress signals.Abbreviation DAPI 4,6-diamidino-2-phenylindole We acknowledge the help of Monica Boscaiu and Zarko Hrzenjak with the artwork, and Michaela Braun-Mayer for growing the tobacco plants. This project was financed by the Austrian Fonds zur Forderung der wissenschaftlichen Forschung, grant S6003-BIO.     

The 35S-CaMV promoter is silent during early embryogenesis but activated during nonembryogenic sporophytic development in microspore culture

Summary The cauliflower mosaic virus 35S (35S-CaMV) promoter, which is generally used as a constitutive promoter in plants, is known to be silent during microspore and pollen development. Here we analyzed whether the 35S-CaMV promoter fused to thegus (-glucuronidase) gene can be used as a marker for early sporophytic development in embryogenic microspore cultures of tobacco andBrassica napus. In microspore culture ofB. napus, the 35S-CaMV promoter remained off from the start of embryogenic culture up to the mid-cotyledonary embryo stage. 35S-CaMV promoter activity was only present in those microspores that initiated sporophytic development, but failed to enter embryogenic development. Similar results were also obtained with shed-microspore cultures of tobacco, in which rapid, direct embryogenesis takes place. In isolated-microspore cultures, in which embryogenesis is delayed, an intermitting period of sporophytic development was observed, characterized by extensive 35S-CaMV promoter activity. Therefore, the 35S-CaMV promoter discriminates between two classes of sporophytic development: it is activated in microspores which change fate from gametophytic into (temporarily) nonembryogenic sporophytic development, whereas the promoter is silent in sporophytic microspores that enter embryogenic development directly. This mirrors our observation that the 35S-CaMV promoter is also silent in young zygotic embryos.     

Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies

Summary Specific stress treatments applied to isolated tobacco (Nicotiana tabacum L.) microspores efficiently induced haploid embryo formation in vitro. A heat shock at 33 or 37°C in the presence of sugar, as well as sucrose-starvation at 25°C, resulted in the formation of embryogenic microspores. A combination of both treatments had an additive effect. Under optimal induction conditions all viable microspores in the culture were embryogenic and developed subsequently into pollen embryos by culture at 25°C in a sugar-containing medium, with induction frequencies of more than 70% with respect to the initial microspore population. A high fraction of the early pollen embryos continued their development in vitro, giving rise to haploid plants. In contrast to other available systems for microspore/pollen embryogenesis, the new protocol allows the production of homogeneous populations of embryogenic microspores and early globular embryos in large-scale cultures, without any purification step, and is therefore well suited for biochemical and molecular work.Abbreviations EDTA ethylenediaminetetraacetate - DAPI 4,6-diamidino-2-phenylindole     

Manipulation of division symmetry and developmental fate in cultures of potato microspores

The effect of media composition on microspore culture was investigated in one tetraploid and two diploid potatoes. The viability of microspores isolated from 4.5 to 5 mm buds was in the range of 33 to 52%. In media for anther culture, microspores showed no further development and lost viability within 2 days. In M1 medium containing mineral components, sucrose, uridine, cytidine, myo-inositol, glutamine and lactalbumin hydrolysate, 18 to 37% of microspores underwent mitosis within 14 days. Up to 95% of the divisions were symmetric and produced equal nuclei. Some symmetrically divided microspores eventually produced structures with 3 to 10 nuclei. The proportion of the total microspore population producing multinuclear structures reached 9% in diploid clones responsive to anther culture and 1 to 2% in recalcitrant cv. Borka. Symmetric mitoses in M1 medium were induced in the presence of glutamine and lactalbumin hydrolysate. Nucleosides and myo-inositol had no effect on microspore division. In the absence of all organic components except sucrose, most mitoses were asymmetric, formation of multinuclear structures was reduced and most pollen accumulated starch indicative of gametophytic fate. In complete M1 medium, starch accumulation was suppressed. Suppression also occurred in asymmetrically divided microspores, indicating a direct inhibition of pollen development independent of the mode of microspore division. This inhibitory effect of M1 medium might present a stress which triggers the induction of symmetric microspore division and subsequent formation of multinuclear structures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cytological and ultrastructural changes induced in anther and isolated-microspore cultures in barley: Fe deposits in isolated-microspore cultures

To gain further insight into the role played by sporophytic anther tissues in the early stages of the androgenic process, we have compared the cytology and ultrastructure of barley embryogenic pollen grains obtained by anther culture with those obtained by isolated-microspore culture. The microspores behaved similarly in both culture systems but ultrastructural studies detected a significant difference: the presence of electron-dense deposits on the intine of embryogenic pollen grains generated by isolated-microspore culture compared to their absence in grains generated by anther culture. To discover the nature of these deposits, we applied proteinase K and EDTA treatments to ultrathin sections. We also subjected the deposits to X-ray microanalysis and found that they contained iron. Anthers and isolated microspores were cultured in media containing different concentrations of iron so as to evaluate the presence of these deposits on the intine. Deposits were not found in anther cultures at any iron concentration used or in microspore cultures when concentrations were lower than 40 mg/L. The Fe deposits on the intine appear to derive from an excess of Fe in the isolated-microspore culture medium which, if allowed to pass through the cell wall, could well be toxic to the embryogenic development of the microspores.     

凤仙花花药发育中多糖和脂滴组织化学研究

以不同发育时期的凤仙花花药为实验材料,采用组织化学方法,对花药发育中的结构变化及多糖和脂滴物质分布进行观察。结果表明:(1)凤仙花的花药壁由6层细胞组成,包括1层表皮细胞,2层药室内壁细胞,2层中层细胞和1层绒毡层细胞。其中绒毡层细胞的形态不明显,很难与造孢细胞区分,且在小孢子母细胞时期退化。(2)在小孢子母细胞中出现了一些淀粉粒,但减数分裂后,早期小孢子中的淀粉粒消失,又出现了一些小的脂滴;随着花粉的发育,小孢子形成大液泡,晚期小孢子中的脂滴也消失;小孢子分裂形成二胞花粉后,营养细胞中的大液泡降解、消失,二胞花粉中又开始积累淀粉;接近开花时,成熟花粉中充满细胞质,其中包含了较多的淀粉粒和脂滴。(3)在凤仙花的花药发育中,绒毡层细胞很早退化,为小孢子母细胞和四分体小孢子提供了营养物质;其后的中层细胞退化则为后期花粉发育提供了营养物质。     

Identification of potentially embryogenic microspores in Brassica napus

Studies were undertaken with Brassica napus L. cv. Topas to identify buds containing microspores predisposed to embryogenesis in vitro and to investigate bud and microspore development in relation to this process. No significant correlation was found between the final embryo number and bud components. There appears to be a developmental window of less than 8 h duration during which microspores are very likely to form embryos: over 70% of the microspores can undergo division and up to 70% of these can form embryos. Embryos were mainly obtained from late uninuucleate to early binucleate microspores: the former contained mainly a G2 or M phase nucleus located at the microspore periphery and the latter a generative nucleus (associated with the intine) and a vegetative nucleus. Observations indicated that only the vegetative nucleus contributed to embryo formation. The first embryogenic division occurred between 8 and 16 h for uninucleate- and between 8 and 48 h for binucleate-derived embryos.     

A co-culture system leads to the formation of microcalli derived from microspore protoplasts ofBrassica napus L. cv. Topas

Summary This paper describes a procedure in which protoplasts are obtained from microspores and pollen of rapeseed to induce callus formation aided by a feeder cell system with embryogenic microspores. Microspores at late unicellular stage and pollen at early bicellular stage were isolated and precultured for 24 h at 32 °C before enzymatic treatment. Eleven enzymes were tested in various combinations and concentrations. The optimal enzyme combination was 1.0% cellulase, 0.8% pectinase, 0.3% macerozyme, and 0.02% pectolyase, in which 26.3% of the microspores released protoplasts. A successful co-culture system was set up by employing embryogenic microspores as feeder cells. To this end, microspores were cultured in a medium with high osmotic pressure at 32 °C. Up to 37% of the microspores exhibited cell division and embryos developed to the heart-shape stage without changing medium. Microspore protoplasts were cultured in Millicells surrounded by the embryogenic microspores as feeder. In growth-regulator-free medium 14.5% of the protoplasts divided but only formed budding-like multicellular structures. Only after pretreatment with 4 mg of 2,4-dichlorophenoxyacetic acid and 1 mg of naphthaleneacetic acid per liter protoplasts divided and formed microcalli. Pollen tubes or tubelike structures were not observed. The experiments reveal that selection of the specific developmental stage of microspores, which is a prerequisite for microspore embryogenesis, is also important in microspore protoplast culture. Compared to other methods used before, microculture fed with embryogenic microspores has obvious superiority.Abbreviations CPW basic protoplast washing medium according to Power and Chapman - CPW972 CPW basic medium supplemented with 9% mannitol and 7.2% sorbitol - DAPI 4,6-diamidino-2-phenylindole - NLN nutrient medium according to Lichter modified by Pechan and Keller - NLN13 NLN medium supplemented with 13% sucrose - NLNP NLN13 supplemented with 7.2% sorbitol