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.     

In vitro chemotropism of pearl millet pollen tubes to stigma tissue: a response to glucose produced in the medium by tissue-bound invertase

Summary Water-homogenized stigma pellets of pearl millet and precipitates resulting from dialysis of their salt extracts were observed to: (1) chemotropically attract pearl millet pollen tubes on a sucrose-containing pollen germination and growth medium, (2) have acid invertase activity as assayed by the arsenomolybdate method, (3) hydrolyze sucrose in the pollen germination and growth medium to glucose as assayed by coupled glucose oxidation with Nitro Blue Tetrazolium, and (4) lose chemotropic and invertase activities upon heat treatment. The results indicate that the in vitro chemotropic attraction of pearl millet pollen tubes to water-homogenized stigma pellets is a response to glucose produced by homogenate-pellet-bound invertase hydrolyzing the sucrose present in the pollen germination and growth medium. Yeast and tomato invertases used as controls verified this conclusion. Water extracts of whole stigmas contained water-soluble acid invertase. The results are discussed in relation to the identification of possible in vivo chemotropic factors of pearl millet and other plants by in vitro assays.Abbreviations dH₂O Deionized, house-distilled water - NBT Nitro Blue Tetrazolium, NBT-medium - PGG medium, pollen germination and growth medium (10% sucrose, 1 mM H₃BO₃, and 1% agarose); - WHS pellet, water-homogenized stigma pellet On Specific Cooperative Agreement 58-6612-8-002 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

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.     

Increased frequency of dividing microspores and improved maintenance of multicellular microspores of Coffea arabica in medium with coconut milk

The number of dividing microspores of Coffea arabica L. cv. Catuai and Catimor could be drastically increased in microspore media containing 16% (w/v) coconut milk, allowing cell divisions to continue in the microspore and multicellular microspores to survive until day 60. After a cold treatment, the microspores were mechanically isolated prior to cultivation in Murashige and Skoog medium supplemented with sucrose and maltose and (mg l^-1) 2,4-d: 2, BAP: 1 or a combination of kinetin: .5, 2,4-d: .5 and NAA: .5 as stationary suspension at a density of 1,200/ml. The crucial stage during microsporogenesis suitable for in vitro androgenesis proved to be mid uninucleate till early binucleate in flowerbuds with the size of 13–15 mm two to three days before anthesis. The initial steps of androgenesis were determined.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxy-acetic acid - NAA 1-naphthaleneacetic acid     

Profilin inhibits pollen tube growth through actin-binding,but not poly-<Emphasis Type="SmallCaps">l</Emphasis>-proline-binding

Previously, we have shown that excess profilin inhibits pollen tube growth at significantly lower concentrations than it blocks cytoplasmic streaming. To elucidate the mechanism by which profilin achieves this function, we have employed mutant profilins from Schizosaccharomyces pombe [J. Lu and T.D. Pollard (2001) Mol Biol Cell 12:1161–1175], which have defects in actin-binding, ability to inhibit polymerization, and poly-l-proline (PLP)-binding. Using Lilium longiflorum L. pollen and S. pombe profilins as wild-type (wt) standards, mutant profilins have been injected into pollen tubes of Lilium, and examined for their effects on growth rate and cell morphology. Our results show that mutant Y5D (68% actin-binding; 1.1% PLP-binding) is indistinguishable from wt-standard profilins. However mutant K81F (2.7% actin-binding; 77% PLP-binding) and especially mutant K67E (<1% actin-binding; 100% PLP-binding) are significantly less effective than wt-standard profilins in their ability to inhibit pollen tube growth. PLP also inhibits pollen tube growth. However, PLP is not different from K67E/PLP combined, which has no actin-binding, suggesting that PLP does not function by binding to profilin. In addition, there are differences in the morphology and F-actin organization in cells injected with PLP versus wt-profilin. Whereas wt-profilin causes a fragmentation and marked reduction in the amount of F-actin [L. Vidali et al. (2001) Mol Biol Cell 12:2534–2545], PLP generates an extensive disorganization without any apparent reduction in the amount of F-actin. We conclude that along with actin-binding activity of profilin, PLP-containing proteins also participate in the growth control process, and can do so independently of binding to profilin.Abbreviations 3D Three-dimensional - PLP Poly-l-proline - RMS Root mean square - wt Wild type     

Storage of pollen grains of Crotalaria retusa in oils

Summary Attempts were made to store pollen grains of Crotalaria retusa L. in a mineral oil (paraffin oil) and two vegetable oils (soybean oil and olive oil). Under laboratory conditions pollen grains not stored in oil lost in vitro germinability within 15–30 days, while those stored in oils maintained some degree of germinability even after 60 days. Pollen samples stored in oils at –20° C did not show any decline in germinability or pollen tube vigour even after 6 months of storage. The results amply demonstrate the feasibility of using oils for short- and long-term pollen storage.     

Proline production by regulatory mutants of Serratia marcescens

Summary Proline-producing strains of Serratia marcescens Sr41 were constructed by three rounds of mutagenesis. A strain SP103 which did not degrade l-proline carried the putA mutation leading to lack of proline oxidase. A 3,4-dehydroproline-resistant mutant SP105, derived from strain SP103, carried the dpr-1 mutation which resulted in desensitization of the feedback inhibition of glutamate kinase. Strain SP103 produced 5.5 mg of l-proline per ml of fermentation medium containing sucrose and urea. Growth inhibition by proline analogs was enhanced when succinate was used as a carbon source in the medium. A thiazolidine-4-carboxylate-resistant mutant SP126 derived from strain SP105 produced 20.5 mg of l-proline per ml of medium. The mutation carried by strain SP126 might be distant from dpr-1 and putA mutations on the chromosome. Pyrroline-5-carboxylate reductase was not repressed by proline in S. marcescens Sr41.     

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.     

Growth and development of conifer pollen tubes

Conifer pollen tubes are an important but underused experimental system in plant biology. They represent a major evolutionary step in male gametophyte development as an intermediate form between the haustorial pollen tubes of cycads and Ginkgo and the structurally reduced and faster growing pollen tubes of flowering plants. Conifer pollen grains are available in large quantities, most can be stored for several years, and they grow very well in culture. The study of pollen tube growth and development furthers our understanding of conifer reproduction and contributes towards our ability to improve on their productivity. This review covers taxonomy and morphology to cell, developmental, and molecular biology. It explores recent advances in research on conifer pollen and pollen tubes in vivo, focusing on pollen wall structure, male gametophyte development within the pollen wall, pollination mechanisms, pollen tube growth and development, and programmed cell death. It also explores recent research in vitro, including the cellular mechanisms underlying pollen tube elongation, in vitro fertilization, genetic transformation and gene expression, and pine pollen tube proteomics. With the ongoing sequencing of the Pinus taeda genome in several labs, we expect the use of conifer pollen tubes as an experimental system to increase in the next decade.     

Optimization of conditions for germination of cold-stored <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> pollen

One of the rare weak points of the model plant Arabidopsis is the technical problem associated with the germination of its male gametophyte and the generation of the pollen tube in vitro. Arabidopsis pollen being tricellular has a notoriously low in vitro germination compared to species with bicellular pollen. This drawback strongly affects the reproducibility of experiments based on this cellular system. Together with the fact that pollen collection from this species is tedious, these are obstacles for the standard use of Arabidopsis pollen for experiments that require high numbers of pollen tubes and for which the percentage of germination needs to be highly reproducible. The possibility of freeze-storing pollen after bulk collection is a potential way to solve these problems, but necessitates methods that ensure continued viability and reproducible capacity to germinate. Our objective was the optimization of germination conditions for Arabidopsis pollen that had been freeze-stored. We optimized the concentrations of various media components conventionally used for in vitro pollen germination. We found that in general 4 mM calcium, 1.62 mM boric acid, 1 mM potassium, 1 mM magnesium, 18% sucrose at pH 7 and a temperature of 22.5°C are required for optimal pollen germination. However, different experimental setups may deviate in their requirements from this general protocol. We suggest how to optimally use these optimized methods for different practical experiments ranging from morphological observations of pollen tubes in optical and electron microscopy to their bulk use for molecular and biochemical analyses or for experimental setups for which a specific medium stiffness is critical. F. Bou Daher and Y. Chebli contributed equally to this study.     

In vitro maturation and germination of <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> microspores

Microspores cultured in vitro can be regarded as a system to study gene regulation, cell fate determination and cell differentiation during pollen development as well as an alternative method of genetic transformation in plants. In our study, pollen development and viability in Orychophragmus violaceus in vivo were determined and then pollen from the late unicellular stage was cultured in vitro. MS liquid medium + White vitamins + 2% (V/V) coconut milk + 0.5 M maltose, pH = 7.0 was the most appropriate for in vitro culture of Orychophragmus violaceus microspores. With this medium, the rates of in maturation and germination were 19.3% and 4.7%, respectively. Liquid medium with 0.6 M maltose + 1.6 mM boric acid + 2.9 mM Ca(NO₃)₂ + 29.6 μM vitamin B1, pH = 7.0 was optimal for germination of pollen matured in vivo. The rate of germination was 70.7%. Pollen matured in vitro cultured in similar medium exhibited a rate of germination of 62.7%. Hence, the experimental study showed that in vitro maturation of microspores is feasible and this experimental system can be applied to further theoretical and practical research.     

Isolated pollen culture: plant reproductive development in a nutshell

Abstract

Isolated pollen can develop into two different directions when cultured in vitro. In a rich medium, microspores and young pollen grains develop into mature pollen that is fertile. When pollen is treated by a stress treatment such as a hunger treatment in a sucrose - and/or nitrogen - free medium, embryogenic pollen is formed that, after transfer to a rich medium, develops into embryos and haploid plants. This system of isolated pollen culture offers an opportunity to study two developmental processes, i.e. pollen development and embryogenesis, as well as a basic regulatory event, i.e. the transition from the gametophytic to the sporophytic phase in the alternation of generations in higher plants. In addition, both systems offer various application-oriented possibilities, such as production of doubled haploids, to overcome self-incompatibility, to rescue sterile pollen, pollen selection and pollen transformation. An understanding of the cell biological and molecular events during embryogenic induction may promote a wider application of doubled haploid breeding and the use of such plants for gene transfer.     

Seed set after pollination with in-vitro-matured,isolated pollen of Triticum aestivum

Summary Immature pollen of two varieties of Triticum aestivum, at the stage right after the first pollen mitosis, was isolated from individual anthers and cultured in microcultures of microliter droplets. In a specifically designed medium, some of the pollen grains developed to maturity. These were applied to excised stigmas on agar, where they produced pollen tubes. Application to flowers in vivo led to seed set. Pollen was matured in vitro from a variety that produced a different protein banding pattern on SDS-PAGE as compared to the variety that was pollinated. The protein banding in the produced seeds showed the hybrid pattern, demonstrating that the seeds were not produced by self-pollination in this in-breeding species but by pollination with the in-vitro-matured pollen.     

Induction of pollen callus in anther cultures of Feijoa sellowiana Berg. (Myrtaceae)

Summary Anthers of Feijoa sellowiana Berg. (feijoa) produced pollen callus when cultured in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine or in nurse cultures. Somatic callus was also formed in large amounts from the connective and from the cut end of the filament. Anthers containing microspores at the stage immediately prior to the first pollen mitosis cultured in the presence of 3% sucrose, presented the highest frequencies of induction. Androgenetic divisions were initiated by the formation of two morphologically equal cells, the so-called B-pathway. Attempts to regenerate pollen plants were unsuccessful but leaf-like structures could be obtained in regeneration media containing combinations of gibberellic acid and benzyladenine.Abbreviations 2,4-D 2,4-diclorophenoxyacetic acid - BA benzyladenine - FDA fluorescein diacetate - GA₃ gibberellic acid - Kn kinetin - MS Murashige and Skoog (1962) medium     

Metabolism ofl-proline toN-acetyl-d-glucosamine during germ tube formation ofCandida albicans

The metabolism ofl-proline toN-acetyl-d-glucosamine (GlcNAc) during germ tube formation ofCandida albicans (C. albicans) ATCC 1002 was studied. In uptake experiments, 6.9 nmol ofl-[¹⁴C]proline were taken up by 1×10⁶ cells during 3 h of incubation at 37°C. The percentage of germ tube formation was 94 under the same condition. The presence of GlcNAc reduced the uptake ofl-proline to 3.0 nmol. The percentage of germ tube formation was 95 in the presence and absence of GlcNAc. The [³H]GlcNAc uptake was 3.0 nmol and was constant whetherl-proline was present or not. After the preparation of a chitin fraction from germ tubes that were labeled withl-[¹⁴C]proline, the radioactivity froml-proline was detected in the glucosamine (GlcN) fraction by thin-layer chromatography (TLC). The metabolism ofl-proline to GlcNAc in chitin during germ tube formation was confirmed in this experiment.     

Control of the developmental pathway of tobacco pollen in vitro

We developed a new method for culture of isolated pollen. Using highly homogeneous populations of immature pollen grains of Nicotiana tabacum L. prepared by means of Percoll density gradient centrifugation, we could direct their developmental pathway by regulating certain culture conditions. When the pollen population was cultured in basal medium with glutamine, most pollen grains underwent normal maturation. On the other hand, when first cultured in basal medium without glutamine, most pollen grains did not mature but after transfer to medium with glutamine and sucrose began to divide. This method for inducing pollen cell division was possible only with midbinucleate pollen grains which are characterized by having no central vacuole and no or only a few starch grains. Evidently, some essential changes necessary for the embryogenic response can be induced by glutamine starvation only in pollen grains at a specific stage.     

Studies on heteromorphic self-incompatibility systems: Physiological aspects of the incompatibility system of Primula obconica

Summary In Primula obconica, a species with a heteromorphic self-incompatibility system, the distinction between compatible and incompatible pollen tubes takes place on the stigma surface in thrum flowers, self tubes growing randomly over the papillar cells. No differences were seen between self and cross tube behaviour on the pin stigma surface, but self tubes were inhibited within the stigmatic tissue with differences in tube length evident after 24 h. The stigma surface bears a proteinaceous pellicle and binds the lectin Concanavalin A. Removal of the stigma removes the incompatibility barrier in mature gynoecia. Bud pollination shows that pollen tubes cannot grow in a normal manner on immature stigmas; the random growth of tubes over the stigma surface resembles that of mature thrum selfs. Fewer compatible tubes reach the style base of young gynoecia and smaller numbers of seeds are set than in mature flowers. Pin and thrum pollen grains germinate and grow in aqueous media, thrum tubes growing longer than pin. The presence of H₃BO₄ and CaCl₂ in the growth medium promotes tube elongation and lengths equivalent to compatible styles can be obtained. The pollen grains have proteinaceous materials in their walls which diffuse out on moistening. Prolonged washing in aqueous media removes these materials but the incompatibility reaction remains unchanged. Thus the incompatibility reaction is between pollen tubes and stigmatic tissue and differs from the homomorphic, sporophytic system where pollen wall proteins elicit the incompatibility response.     

The reversible inhibition of pollen germination of Nicotiana tabacum L.: an entry into a transformation system

Summary The hydrodynamics of mature pollen rehydration in Nicotiana tabacum was used to study reversible inhibition of pollen germination in vitro. Tobacco pollen was incubated for various times in media containing calcium, potassium and magnesium salts, boric acid, and exhibiting different osmotic pressures as a function of sucrose concentration. Total inhibition of germination with complete viability preservation was achieved for 56 h by keeping the grains in medium with 80% sucrose, since typical percentages of germination and pollen tube lengths were recovered after this treatment. These effects were considered as consequences of natural osmoregulation of rehydration/germination in mature pollen. The possibility of applying these findings to the incubation of pollen with Agrobacterium tumefaciens to develop a pollen transformation method is discussed.     

Somatic embryogenesis and plant regeneration of pepper in liquid media

A protocol was developed for regeneration of pepper (Capsicum annuum var. Ace) through somatic embryogenesis in liquid media. For embryogenic callus formation, mature zygotic embryo explants were used on basal Murashige and Skoog medium with 9.05 M 2,4-dichlorophenoxyacetic acid and 3% sucrose. Embryogenic callus was transferred to liquid basal Murashige and Skoog medium with 4.52 M 2,4-dichlorophenoxyacetic acid and 3% sucrose in order to increase the mass of the embryogenic culture. After pretreatment with potassium citrate, cells were placed into embryo initiation medium with 6 g l^-1 l-proline and a decreased (10 mM) ammonium concentration. Embryos were matured in 1.89 M abscisic acid containing half-strength Murashige and Skoog medium and converted into plants bothin vivo andin vitro at up to a 97% efficiency.