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.     

Cloning of cDNA encoding NtEPc, a marker protein for the embryogenic dedifferentiation of immature tobacco pollen grains cultured in vitro

We partially purified three Nicotiana tabacum L. embryogenic pollen-abundant phosphoproteins (NtEPa to c) which appeared in the cells undergoing a dedifferentiation process from immature pollen grains to embryogenic cells, caused by glutamine-deficiency in vitro. All the NtEPs had a highly conserved N-terminal amino acid sequence. Using degenerate oligonucleotide probes designed from the amino acid sequences, the cDNA for NtEPc was isolated from a cDNA library of pollen cultured in glutamine-free medium The cDNA sequence showed moderate homology with several type-1 copper-binding glycoproteins and with a kind of early nodulin though its function could not be predicted. Expression analysis revealed that the level of mRNA for NtEPc was high during the dedifferentiation and also in the very early period of pollen embryogenesis but it was low in the developmental process of microspores/pollen in anthers, in the in vitro maturation process and both in the stational and logarithmic growth phases of tobacco BY-2 cells. Furthermore, an acidic medium pH, which promoted the induction of dedifferentiation increased the level of mRNA for NtEPc, whereas the presence of 6-benzylaminopurine, which inhibited it, decreased the level. These results suggest that the expression of NtEPc gene is correlated with the dedifferentiation but not with pollen development or cell division.     

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.     

The frequency of embryogenic pollen grains is not increased by in vitro anther culture in Nicotiana tabacum L.

The numbers of embryogenic (S) grains present in in-situ mature anthers of Nicotiana tabacum L. were compared to the numbers of embryos and plantlets produced in cultured anthers excised at the optimal mitotic stage of development for anther culture. The Feulgen technique of staining embryos caused a considerable loss of grains from cultured anthers but this did not seriously affect the determination of the percentage of embryos present. In no instance did the numbers of embryos produced exceed the maximum number of S grains found, and the distributions of S grain and embryo frequencies in anthers were similar. In rare instances S grains which had undergone the first embryogenic division were observed in situ. The results indicate that all grains capable of embryogenesis are determined during early flower formation and that their number is not increased by in vitro culture.     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Comparative analysis of callus formation and regeneration on cultured immature maize embryos of the inbred lines A188 and A632

Induction, maintenance, differentiation and embryogenic capacity of callus obtained from immature embryos by culture on induction medium, proliferation medium, maturation medium and regeneration medium, respectively, were compared for two inbred lines of maize, i.e. A188 and A632. The callus of inbred line A188 was embryogenic and maintained embryogenic capacity for at least 1 year. Immature embryos of inbred line A632 formed callus that was not embryogenic. It only produced roots. When sucrose was replaced by sorbitol to induce or improve embryogenesis, again only A188 formed embryogenic callus. Subculture of this callus, however, allowed 4 week intervals in stead of 2 week intervals without loss of embryogenic capacity. When A188 was pollinated with A632 pollen, embryogenic callus was obtained from cultured immature "F₁" embryos, showing that embryogenic capacity was inherited, maternally. The callus did not differ from the embryogenic callus generated on selfed A188 embryos. When A632 was pollinated with A188 pollen, embryogenic callus was obtained too, showing that embryogenic capacity was also inherited paternally, though the embryogenic capacity diminished quickly, and the stability of the callus was lower than in the reciprocal cross. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Separation of precultured pollen from Nicotiana tabacum by aqueous polymer two-phase partition

Pollen isolated from cold treated and precultivated anthers of tobacco (Nicotiana tabacum L. var. Wisconsin 38) were separated into different fractions with counter-current distribution using an aqueous Dextran-polyethylene glycol two-phase system. It was possible to distinguish among eight pollen classes differing in developmental stage and in partitioning. A part of each fraction was cultivated for analysis of embryo formation. This was highest in a fraction with an intermediate to high partition in the phase system. Enriched in this fraction were also pollen that were fairly well stained with acetocarmine, contained several nuclei and had a relatively low mitochondrial activity. The enrichment of embryogenic pollen offers several advantages especially to physiological studies on embryogenesis.     

Histology of somatic embryogenesis in cultured immature embryos of maize (Zea mays L.)

Summary The developmental histology of somatic embryo (=embryoid) formation in cultured immature embryos of hybrid maize cultivars (Zea mays L.) is described. Embryos cultured on media containing 2% sucrose formed distinct globular embryoids. These embryoids arose either directly by divisions confined to the epidermal and the subepidermal cells at the coleorhizal end of the scutellum or from a soft and friable embryogenic callus produced by them. On media containing 6% sucrose divisions were initiated in the cells adjacent to the procambium of the cultured embryos. Subsequently, zones of meristematic cells also were observed in the region of the node and in the basal portion of the scutellum. Mature, well organized somatic embryos as well as a compact nodular type of embryogenic callus were produced as a result of localized meristematic activity along the tip of the scutellum toward the coleorhiza. Some embryos formed only the compact type of callus, and shoot primordia were organized later in the surface layers of this callus.Abbreviations CH casein hydrolysate - MS Murashige and Skoog's nutrient medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant regeneration from embryogenic callus initiated from immature inflorescences of several high-tannin sorghums

A procedure was established for the induction of regenerable calli from immature inflorescence segments of high-tannin cultivars of sorghum (Sorghum bicolor (L.) Moench). Murashige & Skoog's medium with several components altered was utilized for inducing, maintaining, and regenerating the cultures. Embryogenic calli formed at a frequency of 8–70% depending on the genotype. During a ten-month period, 3600 plants were regenerated from eight genotypes tested. Among the developmental stages of immature inflorescence tested (from differentiation of secondary branch primordia to floret formation) no critical differences were found in potential for callusing, embryogenesis or regeneration. Genotypic differences were observed in pigment production, embryogenic callus formation, shoot differentiation, and in maintenance of regeneration capacity.Abbreviations 2,4-D dichlorophenoxyacetic acid This is Journal Paper Number 11972 from the Purdue University Agricultural Experiment Station     

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.     

Selection of embryogenic pollen from cold-treated buds ofNicotiana tabacum var. badischer burley and their development into embryos in cultures

Summary By using density gradient centrifugation, employing 55% percoll and 4% sucrose as suspension medium, it is possible to select embryogenic pollen from buds after cold treatment at 10 °C for 8 or more days. These buds at the uninucleate stage of pollen were collected from plants grown in 8 hours photocycles at 18 °C and supplied with mineral salts. The embryogenic pollen are small, starch-free with a clear cytoplasm whereas large starch-filled ones are nonembryogenic. The embryogenic pollen regularly form embryos at a frequency of 2% on a mineral medium supplemented with glutamine, asparagine and sucrose at pH 6.5.These results demonstrate, for the first time, that it is possible to have embryos in appreciable frequencies in ab initio pollen cultures raised from cold treated anthers.On leave from University of Delhi.     

Induction of somatic embryogenesis and adventitious shoots from immature leaves of cassava

Direct somatic embryogenesis was successfully achieved from immature leaves of cassava (Manihot esculenta Crantz) cultured on induction medium containing 2,4-dichlorophenoxyacetic acid or naphthaleneacetic acid. Changing the duration of induction or changing plant growth regulators resulted in differences in regeneration of somatic embryos or adventitious shoots. The results showed that auxin was a key factor for inducing embryogenic cells. The embryogenic cells were mainly induced within 4–12 days. Only if the embryogenic cells were induced, the auxin enhanced formation of somatic embryo whereas 6-benzylaminopurine stimulated development of adventitious shoots. Histological examinations supported the conclusion.     

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     

Callus formation and plant regeneration through direct culture of isolated pollen of Hordeum vulgare cv. ‘Sabarlis’

Summary Pollen calli and plantlets of Hordeum vulgare cv. Sabarlis were obtained through direct pollen culture without pretreatment of spikes or preculture of anthers. Isolated immature pollen grains were cultured first in a 0.3 M mannitol solution or a C1 basal medium (Chen et al. 1979) supplemented with 0.3 M mannitol but without sucrose for 5–7 days, then transferred into a C1 medium containing 6% sucrose, 3 mM glutamine and 5 mM m-inositol. After a 3 week culture period small pollen calli derived from the pollen grains were transferred into a growth medium comprising C1 basal medium supplemented with 250 mg/1 lactalbumin hydrolysate and 0.5 mg/1 kinetin. For shoot regeneration, vigorously growing calli were transferred onto agarsolidified MS medium (Murashige and Skoog 1962) containing 3% sucrose, 2 mg/1 benzyladenine and 0.5 mg/1 indole-3-acetic acid. The ratio of green plants to albino was approximately 12.2.     

Isolation of Allium pollen protoplasts

Studies on protoplasts isolation were carried out with mature pollen grains of 29 samples of species of Allium aflatunense, A. cepa, A. fistulosum, A. karataviense, A. longicuspis, A. nutans, A. odorum, A. sativum and A. schoenoprasum. Surface sterilized pollen grains drifted from crushed anthers were incubated in an enzyme solution containing 1% (w/v) cellulase Onozuka R-10, 1% (w/v) Macerozyme R-10, 0,5 mol l^-1 sucrose and the basal salts of Nitsch medium. Protoplasts were released within 3 to 120 min, either from the pollen grain, through a slightly disturbed germination pore (narrow aperture), or through a wider aperture, when the exine surrounding the germination pore was disturbed. For the first time, protoplasts were obtained from 13 genotypes of 6 Allium species, at a rate of 1 to 30% of the digested intact pollen grains, depending on the genotype.     

Immature pollen-derived doubled haploid formation in barley cv. Golden Promise as a tool for transgene recombination

Barley transformation mediated by Agrobacterium tumefaciens is routinely performed in a number of laboratories. However, elimination of selectable marker genes and formation of plants homozygous for the transgene via conventional segregation is laborious and time-consuming. Here we suggest a concept that includes the production of primary transgenic plants via infection of immature embryos with A. tumefaciens followed by androgenetic generation of a segregating population of entirely homozygous plants. Selectable marker-free, truebreeding plants carrying a single-opy transgene integrant may thus be efficiently and rapidly obtained. However, amenability to Agrobacterium-mediated transformation as well as androgenetic potential is genotype-dependent. Efficient genetic transformation by infection of immature embryos is so far confined to the spring type cultivar ‘Golden Promise’ which, however, turned out to be recalcitrant in pollen embryogenesis. To facilitate androgenetic generation of homozygous segregants from primary transformants, we have established a method for embryogenic pollen culture in cv. Golden Promise that includes conventional cold-treatment and subsequent preculture of immature pollen under starvation conditions prior to transfer to complete nutrient medium. Further we show that conditioning of the pollen culture medium by co-culture of immature wheat pistils as well as addition of pistil-preconditioned medium considerably support androgenetic development. Employment of the established method using immature pollen of primary transgenic plants demonstrates that selectable marker-free, true-breeding transgenic progeny can be rapidly obtained pursuing the concept proposed. The protocol presented will be useful in functional genomics as well as in molecular breeding approaches.     

Cellular changes during the acquisition of embryogenic potential in isolated pollen grains ofNicotiana tabacum

Summary We used electron microscopical techniques to study ultrastructural changes during the acquisition of embryogenic competence in immature pollen grains ofNicotiana tabacum, isolated at the early- or mid-bicellular stage and cultured in vitro under starvation conditions. Cytoplasmic and nuclear changes during the starvation treatment are reported. Dedifferentiation of plastids, dilation of the wall of the generative cell, the appearance of a large vacuole, loss of nuclear pores in the vegetative nucleus, changes in chromatin and nucleolar structure, and a decrease in the size of the nucleolus were observed. We suggest that these events are the first step in the switch from generative to vegetative generation during pollen embryogenesis.