Maintenance of gametophytic development after symmetrical division in tobacco microspore culture

Isolated tobacco (Nicotiana tabacum L.) microspores maturing in vitro can be induced to undergo symmetrical divisions, instead of the normal asymmetrical first pollen mitosis, by addition of anther extracts to the culture medium. The two daughter cells in symmetrically divided pollen resemble vegetative pollen cells in cytological characteristics, nuclear size and chromatin condensation, are separated by a cell wall and remain viable during in vitro maturation. After transfer to a germination medium, only one of the two vegetativelike cells forms a pollen tube in vitro. Therefore, apparently normal gametophytic development can be maintained after symmetrical microspore division. These results are discussed in relation to current models for induction of microspore embryogenesis.     

Methotrexate is a new selectable marker for tobacco immature pollen transformation

We report here a new selectable marker for tobacco immature pollen transformation based on the expression of dihydrofolate reductase (dhfr) gene which confers resistance to methotrexate (Mtx). Two immature pollen transformation approaches, i.e., male germ line transformation and particle bombardment of embryogenic mid-bicellular pollen have been used for the production of stable transgenic tobacco plants. In the first method, two methotrexate-resistant plants were selected from a total of 7161 seeds recovered after transformation experiments. In the second method, four methotrexate-resistant plants were obtained from 29 bombardments using 3.7×10⁵ pollen grains per bombardment. Southern analysis confirmed the transgenic nature of T₀ and T₁ candidate transgenic plants, and a genetic analysis showed that the transgenes are transmitted to subsequent generations.     

Derepression of the cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis

Summary Microspectrophotometry following Feulgen staining and autoradiography following (³H)-thymidine labelling were used to study cell-cycle events during pollen development in tobacco (Nicotiana tabacum L.). During normal gametophytic pollen development in the anther and in vitro the generative nucleus passes through the S phase to the G₂ phase soon after microspore mitosis, while the vegetative nucleus remains arrested in G₁ (=G₀). During embryogenie induction by an in vitro starvation treatment of immature pollen ongoing DNA replication in the generative nucleus is completed and followed by DNA replication in the vegetative cell in a large fraction of the pollen grains. Addition of the DNA replication inhibitor hydroxyurea to the starvation medium postpones S phase entry until the pollen is transferred to a rich medium and does not affect embryo formation. These results demonstrate that one of the crucial events of embryogenic induction is the derepression of the G₁ arrest in the cell cycle of the vegetative cell.     

In-situ seed production after pollination with in-vitro-matured,isolated pollen

Immature tobacco (Nicotiana tabacum L.) pollen has been isolated from anthers in three distinct stages of development, including the microspore stage. In in-vitro cultures, fully functional, mature pollen was obtained. In a germination medium, this pollen produced pollen tubes. After application to stigmas in situ, the in-vitro-matured pollen fertilized ovules, and seeds were produced. Genetic tests with seedlings obtained from pollinations with in-vitro-matured pollen from a transgenic plant revealed normal Mendelian segregation of two marker genes, the neomycin-phosphotransferase II gene and the nopaline-synthase gene. These results are of interest with respect to the control of self-incompatibility, cytoplasmic male sterility and pollen-allergen formation, and it offers an alternative route for gene transfer in those plants which cannot be regenerated in vitro.Abbreviations cms cytoplasmic male sterility; AMGLU, MS, M2S, MR26 - GK culture media, see Material and methods     

Effects of high humidity and temperature stress on pollen membrane integrity and pollen vigour in Nicotiana tabacum

Summary The prolonged exposure of pollen Nicotiana tabacum to high humidity at both room temperature and 38° C did not affect membrane integrity as revealed by the fluorochromatic reaction (FCR) test, but did affect pollen vigour. At room temperature germination was not affected, although tube growth was reduced; at 38° C, there was both a reduction in tube growth and delayed germination. When the pollen was subjected to 1 h hydration followed by 1 h desiccation (up to a maximum of four cycles) at room temperature, a reduction in the FCR, germination and tube length after each desiccation treatment was observed. Subsequent hydration fully restored the FCR, but only partially restored germination and tube growth. At 38° C, however, FCR, germination, and tube growth were drastically reduced. The implications of these results on the relationship between FCR and germinability, the responses of pollen exposed to humidity and temperature stress in the field, and on pollen storage are discussed.     

Oleosin-like proteins are not present on the surface of tobacco pollen

Pollen-stigma interactions on wet- and dry-type stigmas involve similar processes: the hydration of the pollen, followed by pollen tube growth and penetration of the stigma. Furthermore, in some species, identical molecules, namely lipids, are used to achieve this. In addition to lipids, oleosin-like proteins of the pollen coat of dry-type stigma plants have been shown to be involved in pollen-stigma interactions. However, little information is present about the proteins on the surface of pollen of wet-type stigma plants, in particular that of the Solanaceae. To analyze proteins from the surface of pollen of Nicotiana tabacum (tobacco), a solanaceous plant, we used an antiserum raised against Brassica pollen coat, a dry-type stigma plant of the Brassicaceae. In addition we used a molecular approach to identify tobacco homologues of oleosin-like genes. Our results show that no proteins similar to Brassica oleracea pollen coat proteins are present on the surface of tobacco pollen, and that oleosin-like genes are not expressed in tobacco anthers or stigmas.     

Environmental control and evidence for predetermination of pollen embryogenesis inNicotiana tabacum pollen

Summary The effect of daylenght and temperature for the donor plants (Nicotiana tabacum var. Badischer Burley) on the formation of pollen competent for embryogenesis (P-pollen) by the three possible routes (during normal flower developmentin situ (pollen dimorphism), during cold-treatment of excised flower buds, in cultured anthers) was studied. In all three routes, P-pollen frequency (premitotic pollen, before 1. sporophytic division, PPF) was affected in essentially the same way. At 24 °C and long days, PPF was low and short days had only a slightly increasing effect. At 18 °C and long days, PPF was higher and short days further increased it. Correlated with PPF under the different growth regimes was the percentage of units with more than one vegetative-type nucleus (normal embryos + abortive embryos + multinucleate pollen) in 3 weeks old anther cultures. Under greenhouse conditions, PPF was generally higher than at 24° in growth rooms and showed a maximum in the winter months. Plant age did not affect PPF. These results give further evidence that pollen embryogenesis is predetermined before excision and culture of the pollen or anthers.     

Genotypic control of pollen plant formation in Nicotiana tabacum L.

Summary Tobacco plants (Nicotiana tabacum L.) of four varieties (Badischer Burley, White Burley, Techne, Kupchunos) were raised at different temperatures and daylengths and the effect of genotype on embryogenic pollen grain formation in situ and on pollen plant formation in anther and pollen cultures from these plants was studied. Genotype controlled embryogenic pollen grain and pollen plant formation by defining productivity under standard growth conditions (long days at 24 °C). Kupchunos was the most productive variety, followed by White Burley, Techne, and Badischer Burley. Furthermore, genotype defined which environmental factor was able to affect embryogenic pollen grain and pollen plant formation and also to which degree. In anther cultures, in addition to these effects, genotype controlled the formation of (an) inhibitory substance(s) in the anther wall in interaction with the plant growth conditions. In Badischer Burley and Techne, inhibitor action could be prevented by isolation of the pollen after one week of anther culture. Finally, direct pollen cultures in Badischer Burley and Techne produced embryos were only when the pollen was isolated from nearly mature anthers, while in White Burley and Kupchunos, embryos also produced at earlier stages and at higher yields. This indicated that genotype controls the time when the embryogenic pollen grains become ready to divide. The results are discussed in relation to strategies to overcome recalcitrance of species and genotypes.     

Distribution of calcium in the stigma and style of tobacco during pollen germination and tube elongation

Potassium antimonate was used to locate loosely bound calcium in the stigma and style of tobacco. The tobacco stigma is wet and covered by a thick layer of glycoprotein exudate at anthesis. The exudate contains abundant vesicles, which are densely labeled with calcium precipitates. When pollen grains arrive at the stigma, become hydrated, and as the pollen swells, Ca²⁺ precipitates accumulate at the aperture. Calcium precipitates that accumulate in pollen cytoplasm are initially concentrated within small vacuoles, but as germination proceeds these appear to fuse, forming prominent, densely labeled vesicles that preferentially accumulate near the proximal region of the growing tube. Although the stigma has abundant particles, few calcium precipitates are observed in the transmitting tissue from anthesis to 11 h after pollination. However, at 22 h after pollination, accumulation of calcium increases distally from the stigmatic interface with the transmitting tissue through the length of the style to the ovary. An examination of flowering plants with differing floral biology will be needed to understand the role of loosely bound calcium accumulation and its relationship to tissue-level changes in calcium uptake, maintenance of other calcium pools, including [Ca²⁺]_cyt, and in pollen and style maturation during the progamic phase.     

Nonradioactive in situ localization of poly(A)+ RNA during pollen development in anthers of tobacco (Nicotiana tabacum L.)

Summary A method is described for non-radioactive labeling of total mRNA [poly(A)+ RNA] in plastic-embedded plant tissue sections. Oligo-deoxythymidylic acid (oligo-dT) labeled with digoxigenin-conjugated dUTP was used for in situ hybridization to poly(A)+ RNA in sections of tobacco (Nicotiana tabacum) anthers. The digoxigenin was immuno-stained using antidigoxigenin IgG and gold-labeled protein-A, followed by silver enhancement of the gold label. Reproducibly similar positive staining patterns were obtained with digoxigenin-labeled oligo-dT and polyuridylic acid [poly(U)], but not with a similarly labeled sense probe, poly(A). In the developing anthers, from the onset of meiosis to the production of pollen grains, labeling patterns were compatible with a gradual depletion of nuclear and chromosome-associated sporophytic mRNA molecules during prophase of meiosis, followed by postmeiotic production of gametophytic mRNA in microspore nuclei and the vegetative nuclei of the pollen grains.Abbreviations BSA bovine serum albumin - DIG digoxigenin - IgG immunoglobulin-G - oligo-dT oligo-deoxythymidylic acid - PAS-ABB periodic acid Schiff-aniline blue black - PBS phosphate buffered saline - poly(A) polyadenylic acid - poly(U) polyuridylic acid - SSC standard saline citrate     

A methionine synthase homolog is associated with secretory vesicles in tobacco pollen tubes

Seven isoforms of 85 kDa polypeptides (p85) were identified as methionine synthase (MetE) homologs by partial aminoacid sequencing in tobacco pollen tube extracts. Immunocytochemistry data showed a localization of the antigen on the surface of tip-focussed post-Golgi secretory vesicles (SVs), that appear to be partially associated with microtubules (Mts). The chemical dissection of pollen tube high speed supernatant (HSS) showed that two distinct pools of MetE are present in pollen tubes, one being the more acidic isoforms sedimenting at 15S and the remaining at 4S after zonal centrifugation through a sucrose density gradient. The identification of the MetE within the pollen tube and its possible participation as methyl donor in a wide range of metabolic reactions, makes it a good subject for studies on pollen tube growth regulation.     

Regulation of developmental pathways in cultured microspores of tobacco and snapdragon by medium pH

The regulation of developmental pathways in cultured microspores of tobacco (Nicotiana tabacum L) and snapdragon (Antirrhinum majus L) by medium pH is described for the first time. Unicellular tobacco and snapdragon microspores developed into normal, fertile pollen when cultured in media T1 and AT3 at pH 7.0 and 25°C for 6 and 8 days, respectively. First, pollen mitosis was asymmetric and mature pollen grains were filled with starch granules and germinated upon transfer to a germination medium. However, when tobacco and snapdragon microspores were cultured in media T1 and AT3, respectively, at pH 8.0–8.5 for 4–6 days at 25 °C, the frequency of symmetric division increased significantly with the formation two nuclei of equal size, and the gametophytic pathway was blocked, as seen by the lack of starch accumulation and the inhibition of pollen germination. The transfer of these microspores to embryogenesis medium AT3 at pH 6.5 resulted in the formation of multicellular structures in both species and, in tobacco, in the formation of embryos and plants. In order to understand the possible mechanisms of the action of high pH, sucrose metabolism was analysed in isolated microspores of tobacco cultured at various pH values. Invertase (EC 3.2.1.26) activity in microspores was maximal at pH 5.0 and strongly decreased at higher pH, leading to a slow-down of sucrose cleavage. At the same time the incorporation of ¹⁴C-labelled sucrose from the medium into microspores was drastically reduced at high pH. These data suggest that isolated microspores are not able to metabolise carbohydrates at high pH and thus undergo starvation stress, which was shown earlier to block the gametophytic pathway and trigger sporophytic development.     

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.     

Dynamics of inorganic ions in microspore and pollen grain during development of the tobacco male gametophyte

We studied the dynamics of mobile potassium, chloride, and nitrate ions during development of the microspore and differentiation of the pollen grain inNicotiana tabacum L. by measuring their concentration in aqueous extracts from cells destroyed by freezing-thawing using ion-selective electrodes. Stage-specific changes in the ion content and intracellular concentration in the male gametophyte were found. A relationship of the dynamics of ions to growth processes and changes in metabolic activity during gametophytogenesis has been discussed. The changes in the potassium and chloride ion concentrations have been interpreted as regulatory changes controlling protein synthesis in the pollen grain vegetative cell. Deceased.     

Auxin levels,antiauxin(s) and androgenic plantlet formation in isolated pollen cultures ofNicotiana tabacum

Summary Anthers ofNicotiana tabacum var. Badischer Burley contain endogenous auxins, one of these was identified as indoleacetic acid. At the developmental stage shortly after the first pollen mitosis the anthers contain equivalents of 0.1 mg IAA per kg fresh weight. This endogenous auxin level is maintained during the eight-day preculture of the anthers prior to isolated pollen culture. However, in anthers of short-day plants, which are characterized by a high proportion of embryogenic pollen at the end of preculture (Heberle-Bors andReinert 1979), an increase of the auxin level till the fourth day of culture is detectable.Preculture of anthers in the presence of an inhibitor of auxin synthesis (7-azaindole) and an antiauxin (-(o-chlorophenoxy)-isobutyric acid) results in enhanced plantlet yield by pollen cultures. The significance of these observations for androgenesis is discussed.     

Non-symmetrical cytosine methylation in tobacco pollen DNA

We have detected sequence-specific non-symmetrical cytosine methylation within a 140 bp region of the promoter for the tobacco auxin-binding protein gene T85 in pollen DNA. Direct sequencing of the population of bisulphite reaction products showed that, in this region, 10 out of a possible 49 cytosine residues were methylated at a high frequency in pollen whereas the corresponding region from somatic cells (leaf DNA) did not show a detectable level of methylation. The context of these sites was 1×m⁵CpTpC, 1×m⁵CpGpT, 1×m⁵CpCpT, 2×m⁵CpTpT, 2×m⁵CpGpG, and 3×m⁵CpApT of which only m⁵CpGpG and m⁵CpGpT fitted the consensus sequence for symmetrical methylation in plants.     

Protein synthesis in tobacco pollen tubes: preferential synthesis of cell-wall 69-kDa and 66-kDa glycoproteins

One- and two-dimensional electrophoresis of Nicotiana tabacum pollen and pollen tube proteins confirmed that a new protein is preferentially synthesized during pollen germination and tube growth and becomes the most abundant protein in pollen tubes. Analysis of proteins extracted with sodium dodecyl sulfate (SDS) from different pollen tube fractions showed that it is the most abundant non-covalently bound wall protein, characterized by molecular mass of 69 kDa, pI between 7.9 and 8.2, and glycosylation with glucose and/or mannose. Amino acid analysis revealed relative abundance of serine, glutamic acid and glycine, but did not show the presence of hydroxyproline. According to all these characteristics, it cannot be classified as an extensin-like protein. Another prominent wall-bound glycoprotein has a molecular mass of 66 kDa and the same pI as the 69 kDa glycoprotein. These two glycoproteins are similar also in ConA binding, rate of synthesis, and rapid incorporation into pollen tube walls. Their synthesis is strongly reduced by tunicamycin and this inhibition results in the occurrence of new polypeptides in the range of 57–61 kDa. Tunicamycin also inhibited pollen tube growth. At 10 ng ml^-1 and 50 ng ml^-1 the inhibitor reduced pollen tube mass after 24 h of culture by 30% and 85%, respectively. This indicates that tobacco pollen presents a system highly sensitive to tunicamycin and that cotranslational N-linked glycosylation on the rough endoplasmic reticulum is required for 66 and 69 kDa glycoprotein formation and for pollen tube growth. Although other proteins appear during pollen germination and tube growth, the new proteins occur at low levels and seem to originate through modifications of preexisting polypeptides. In contrast to 69 and 66 kDa proteins, most proteins detected by [¹⁴C]amino acid incorporation and fluorography of gels were not revealed by Coomassie blue staining.     

Cell cycle dependent distribution of phosphorylated proteins in microspores and pollen ofBrassica napus L., detected by the monoclonal antibody MPM-2

Summary The monoclonal antibody MPM-2, which interacts with a mitosis-specific phosphorylated epitope, has been used to study phosphorylation of proteins in microspores and pollen ofBrassica napus. One- (1-D) and two-dimensional (2-D) immunoblots revealed that MPM-2 recognized a family of phosphorylated proteins in freshly isolated microspores and pollen. The same set of phosphorylated proteins was found after 8 h of culture at embryogenie (32 °C) and non-embryogenic (18 °C) conditions. Two major spots were observed on 2-D immunoblots, one of which (M_r75 kDa, pI5.1) co-localized with the 70 kDa heat shock protein. Immunolabelling of sectioned microspores and pollen showed that MPM-2 reactive epitopes were predominantly observed in the nucleoplasm from G₁ until G2-phase, and in the cytoplasm during mitosis. This may be due to a cell cycle related translocation of phosphoproteins from the nucleus to the cytoplasm, or alternate phosphorylation and dephosphorylation in nucleus and cytoplasm. Detectability of epitopes on sections depended on the embedding procedure. Cryo processing revealed epitope reactivity in all stages of the cell cycle whereas polyethylene glycol embedded material showed no labelling in the cytoplasm during mitosis. Processing might reduce the antigenicity of cytoplasmic MPM-2 detectable proteins, probably due to dephosphorylation. The MPM-2 detectable epitope was observed in all cells investigated, irrespective of culture conditions, and its intracellular distribution depended on the cell cycle stage and was not related to the developmental fate of the microspores and pollen.     

Thin-alginate-layer technique for protoplast culture of tobacco leaf protoplasts: Shoot formation in less than two weeks

Summary Regeneration of plants from protoplasts is regarded a difficult and lengthy procedure which requires well developed skills on the side of the experimenter. Therefore, where alternative procedures for genetic engineering of plants are available, protoplast-based techniques are frequently avoided. Here, we demonstrate, that by our newly developed thin-alginate-layer technique it is possible to regenerate shoots from leaf protoplasts ofNicotiana tabacum L. at very high efficiency and very rapidly, with the first shoots appearing within less than two weeks. Root formation is induced on a third medium with first roots being found after only 10 more days of culture.