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.     

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.     

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.     

Specific phosphoproteins in the initial period of tobacco pollen embryogenesis

Several phosphoproteins specifically correlated with the induction of embryogenic cells were detected in immature pollen grains of Nicotiana tabacum L. By regulating the concentration of glutamine in the medium the developmental pathways of immature pollen grains isolated at the mid-bicellular stage could be controlled, resulting in the formation of either mature pollen grains or embryogenic cells. Different phosphoproteins, designated as a-d and as e-i, respectively, were detected when the pollen grains either became embryogenic cells in glutamine-free medium, or when they were allowed to mature in glutamine-containing medium. The formation of embryogenic cells was suppressed by adding glutamine or cytokinin to the glutamine-free medium, nor did it occur with pollen grains at younger or older stages, and in these cases the phosphoproteins a-d were detectable only partially or faintly. The phosphoproteins a-d and e-i thus may be one of the factors necessary to direct the developmental pathway of immature tobacco pollen grains to embryogenic cells and to mature pollen grains, respectively.The authors thank Dr. V.S. Jaiswal (Botany Department, Banaras Hindu University, Varanasi, India) for his valuable suggestion in the preparation of the paper. This work was supported by a Grantin-Aid for special project research from the Ministry of Education, Science and Culture of Japan.     

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.     

Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination

Summary A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L.) pollen from microspores isolated just before entering mitosis. During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition. Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline. Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes. The in vitro-matured pollen grains can be stored at-20° C either suspended in 1.17 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs. The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod. The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection.     

Somatic embryogenesis in Nicotiana tabacum L.: induction by thidiazuron of direct embryo differentiation from cultured leaf discs

Summary Induction of somatic embryogenesis by different growth regulators was examined in leaf disc cultures of Nicotiana tabacum L. Direct differentiation of somatic embryos occurred on media supplemented with naphthaleneacetic acid (NAA) and N⁶-benzylaminopurine (BAP). Thidiazuron (N-phenyl-N-1, 2,3,-thiadiazol-5-ylurea; TDZ) not only substituted for the most effective NAA-BAP combination but also induced a higher frequency of somatic embryogenesis. Regenerated somatic embryos were capable of developing into plants.Abbreviations BAP N⁶-benzylaminopurine - MS Murashige and Skoog (1962) medium - NAA Naphthaleneacetic acid - TDZ N-phenyl-N-1,2,3,- thiadiazol-5-ylurea     

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.     

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.     

In vitro pollen embryogenesis in Nicotiana tabacum L. and its relation to pollen sterility,sex balance,and floral induction of the pollen donor plants

Pollen sterility, sex balance, and floral induction of the pollen donor plants were tested for a possible relation to embryogenesis from in vitro cultured tobacco pollen (Nicotiana tabacum L. var. Badischer Burley). The pollen grains destined to become embryos in culture (P-grains) were sterile for the donor plants as judged by their staining reaction with acetocarmine and fluorescin-diacetate, and by an in vitro germination test. They were produced in high frequency in flowers which exhibited a shift in sex balance towards femaleness. Sex balance could be measured by the relative length of pistil to stamens. High P-grain frequency, high pollen sterility, and a shift in sex balance towards femaleness could be induced by raising the donor plants under short days and/or low temperature (18–15° C) as compared to long days at 24° C. Short days and/or low temperature also reinforced floral induction, revealing that the tobacco variety Badischer Burley is a quantitative short day and low temperature plant and that the variety follows the rule that conditions of strong floral induction shift sex balance towards femaleness. At 12° C and short days, contabescent flowers were formed with completely sterile anthers containing a few and mostly collapsed P-grains. Based on these results, it is now possible to predict conditions by which haploids via pollen embryogenesis might be produced in high frequency from low-yielding and recalcitrant species.Abbreviations DPF dead pollen grain frequency - LD24 long days at 24° C - PD pollen dimorphism - P:S ratio of pistil to stamen length - SD15 short days at 15° C     

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.     

Diphtheria toxin-mediated cell ablation in developing pollen: Vegetative cell ablation blocks generative cell migration

Summary The technique of genetic cell ablation involves the targeted expression of a cell autonomous cytotoxic protein under the control of cell-specific regulatory sequences. This technique allows the investigation of cell-cell interactions by inducing selective death in a precisely controlled and cell autonomous manner. Here, targeted vegetative cell-specific ablation was used to examine the role of the vegetative cell (VC) in controlling generative cell (GC) behaviour and differentiation during pollen development. The tomatolat 52 late-pollen promoter, which has been shown to be activated specifically in the nascent VC immediately following pollen mitosis I (PMI), was used to direct expression of the cytotoxic diphtheria toxin A chain (DTA) in both transient expression assays using microprojectile bombardment and in transgenic tobacco plants. Transient expression of DTA linked to thelat 52 promoter (lot 52-DTA) in pollen dramatically reduced the expression of a co-transfected reporter gene fusion, demonstrating the cytotoxicity of DTA to pollen. Genetic and phenotypic analysis oflat 52-DTA transformants demonstrated that DTA expression led to a pollen-lethal phenotype, recognisable as small acytoplasmic pollen grains at anthesis, which affected 50% of the pollen population in single locus transformants. Detailed cytological analysis using confocal laser scanning microscopy and vital staining using fluorescein diacetate (FDA), showed that the first sign of cell ablation during pollen development was a loss of vital staining of the VC immediately following PMI. In contrast, the GC retained viability for up to several days following VC ablation, but progressively lost viability in the absence of a functional VC. Of particular interest was the observation that in the absence of VC function the generative cell (GC) failed to undergo normal migration away from the pollen grain wall into the VC cytoplasm. These results directly demonstrate the dependence of the GC on VC cell functions and highlight the importance of VC-GC interactions in controlling GC migration.Abbreviations CaMV cauliflower mosaic virus - nos nopaline synthase - DTA diptheria toxin A chain - lat late anther tomato - VC vegetative cell - GC generative cell - PGM pollen germination medium - EtBr ethidium bromide - FDA fluorescein diacetate - FCR fluorochrome reaction - DAPI 4,6-diamidino-2-phenylindole     

Plant transformation by particle bombardment of embryogenic pollen

Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10⁴ pollen grains were GUS⁺. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS⁺ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.Abbreviations GUS ß-glucuronidase - CaMV Cauliflower Mosaic Virus - MCS multicellular structure - NPTII neomycin phosphotransferase - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide - DAPI 4,6-diamidino-2-phenylindole - Tris Tris(hydroxymethyl)aminomethane hydrochloride - EDTA ethylenedinitrilo tetraacetic acid, disodium salt dihydrate     

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.     

Isolation and characterization of a tobacco cDNA clone encoding a putative MAP kinase

We have isolated and sequenced a MAP (mitogen-activated protein) kinase-type cDNA from a tobacco (Nicotiana tabacum L.) cell suspension cDNA library by screening with a PCR fragment amplified from the same library with oligonucleotide primers corresponding to two sequences conserved in yeast and animal MAP kinases. The tobacco sequence, ntf3, shows 45–54% identity to various members of the MAP kinase family at the protein level. Northern experiments showed that ntf3 is expressed in all tobacco tissues tested, including pollen isolated at different developmental stages. Southern analysis indicated that, as in other organisms, there is a family of MAP kinase genes in tobacco. In complementary tests, ntf3 could not substitute the yeast MAP kinase genes fus3 and kss1.     

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.     

Immunogold localization of arabinogalactan proteins,unesterified and esterified pectins in pollen grains and pollen tubes ofNicotiana tabacum L.

Summary The monoclonal antibodies JIM 5 (against unesterified pectin), JIM 7 (against methyl esterified pectin), MAC 207 (against arabinogalactan proteins, AGPs), and JIM 8 (against a subset of AGPs) were utilized singly or in combinations for immunogold labelling of germinated pollen grains and pollen tubes ofNicotiana tabacum. Pectins were localized in the inline of pollen grain, unesterified pectin being more abundant than the esterified one. AGPs were co-localized with pectin in the inline, but were present preferably close to the plasma membrane. In pollen tubes, AGPs, unesterified and esterified pectins were co-localized in the outer and middle layers of the cell wall. The density of the epitopes was not uniform along the length of the pollen tube, but showed alterations. In the pollen tube tip wall esterified pectin was abundantly present, but not AGPs. In the cytoplasm esterified pectin and AGPs were detected in Golgi derived vesicles, indicating their role in the pathway of the cell wall precursors. In the cell wall of generative cell only AGPs, but no pectins were localized. The co-localization of pectins and AGPs in the cell wall of pollen grain and pollen tube might play an important role, not only in maintenance of the cell shape, but also in cell-cell interaction during pollen tube growth and development.Abbreviations AGP arabinogalactan protein - BSA bovine serum albumin - GA glutaraldehyde - MAb monoclonal antibody - NGS normal goat serum - PFA paraformaldehyde