Induction of embryogenesis from isolated apple microspores

 We report, for the first time, the induction of embryogenesis and plant formation from isolated apple (Malus domestica Borkh.) microspores in vitro. Different isolation techniques were tested and an optimized protocol was elaborated. Furthermore, the influence of the induction medium and starvation treatment, using different starvation material, temperatures and time, were studied. In addition to embryo induction, the number of multicellular structures per divided microspores was found to be a suitable parameter of assessment and could be used in earlier stages during microspore culture. Although the number of embryos induced in these first experiments is low, the best frequency of embryo induction was shown to be at least twice as efficient as that obtained by anther culture. Received: 9 September 1998 / Revision received: 22 December 1998 / Accepted: 12 January 1999     

Temperature controls both gametophytic and sporophytic development in microspore cultures of Brassica napus

Summary Temperature controls the developmental fate of isolated Brassica napus microspores in vitro. Culture at 32.5°C leads to sporophytic development and the formation of embryos. Here we show that culture at 17.5°C leads to gametophytic development, and the formation of pollen-like structures at high frequencies (up to 80% after 7 days in culture). Early stages of both developmental pathways are observed in culture at 25.0°C, and embryos are produced at low frequencies (0.7%) at that temperature. Culturing B. napus microspores at 32.5°C versus 17.5°C brings the switch from gametophytic to sporophytic development under simple experimental control and provides a convenient tool for investigating the cellular and molecular mechanisms controlling this developmental switch.     

Effects of aluminum on DNA synthesis, cellular polyamines, polyamine biosynthetic enzymes and inorganic ions in cell suspension cultures of a woody plant, Catharanthus roseus

Individual buds of Brassica napus cv. Topas, near the first pollen mitosis, were used for microspore culture. Bud and petal lengths were recorded. Microspores isolated from the individual buds were plated and small samples were fixed for cytology. Following embryo induction and three weeks of culturing, numbers of embryos were scored. Bud and petal lengths did not accurately indicate which buds would supply microspores that would form embryos at high frequencies. Fluorescence microscopy was used to examine nuclei stained with Hoechst 33258 and vacuolar morphology of microspores was revealed by the weaker fluorescence due to glutaraldehyde fixation. Following isolation, nuclear and vacuolar characteristics were used to stage the microspores as miduninucleate, late uninucleate vacuolate, late uninucleate, mitotic, or binucleate. The relationship of developmental stage to the frequency of microspore-derived embryos was evaluated. A classification scheme was developed which uses the relative proportions of microspores at each of the stages to identify microspore isolations that would form embryos at high frequencies. It was found that when 1 to 87% of the isolated microspores were binucleate, 21.4 ± 3.0% of the viable microspores developed into embryos. This was a significant ( P < 0.001) increase over the other 3 classes. The ability to select highly embryogenic microspore isolations is of great advantage for developmental cell biology studies.     

Effects of culture conditions on isolated microspore response of barley cultivar Igri

Pretreatment of anthers in mannitol prior to isolation of microspores by glass rod homogenization was effective for in vitro induction of embryogenesis in barley cv. Igri. A procedure for separation of viable microspores using centrifugation on 20% maltose was developed. The concentration of microspores was important and greatly increased the number of developing structures. Initial culture of microspores on FHG medium containing 62 g l^-1 maltose, 4.4 M (1 mg l^-1) BA and 200 g l^-1 Ficoll-400 resulted in high frequencies of plant regeneration. Albino plant frequency was correlated to length of time in culture. Stock plant condition appeared to be a major factor influencing induction frequency. From 868 to 1738 green plants per 100 anthers were produced. The number of calli and embryos obtained and the number of green plantlets regenerated were improved by increasing the Ficoll concentration from 100 g l^-1 to 400 g l^-1 during the culture period compared to continuous culture on FHG Ficoll 200 g l^-1.Abbreviations BA benzyladenine     

Development of microsporesin vivo andin vitro inBrassica napus L.

Summary Populations of highly homogeneous uninucleate and binucleate microspores ofBrassica napus cv. Topas were obtained by bud selection and percoll fractionation. The development of the uninucleate and the binucleate microspores in culture was compared to thosein vivo using the fluorochrome DAPI to stain DNA. The major developmental pathway of the uninucleate microsporesin vitro resulted in embryo formation. The characteristic of this pathway was that the first division produced two diffusely stained nuclei and subsequent divisions gave rise to a multinucleate embryoid. The second pathway which occurred in a small number of the uninucleate microspores led to callus formation. The majority of the binucleate microsporesin vitro followed the developmental pattern of their counterpartsin vivo and were not embryogenic. The embryogenic binucleate microspores produced embryos through the divisions of the vegetative nucleus.Plant Research Centre Contribution # 1147     

Isolated microspore culture overperforms anther culture for green plant regeneration in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Barley isolated microspore culture (IMC) was compared to anther culture (AC) for its efficiency in green plant (GP) regeneration. With six cultivars investigated, IMC resulted in significantly more GPs (3.6–287 per 100 anthers) than AC (0–29.6), which was on average 9.3-fold more efficient (113.7 vs 12.2). GPs were produced via IMC from all the genotypes tested, whereas no green shoot was generated by AC in two of the cultivars. In spite of genotype dependency for regeneration rates, the average GP percentage of IMC was just slightly higher than that of AC. Effects of microspore developmental stages and medium-sterilization methods on IMC were examined with the aim of optimizing culture conditions. We found that the optimum stage for cold pretreatment of spikes was different from that for mannitol starvation of anthers. Significant variations in microspore embryogenesis and regeneration were observed among five stages tested. Optimal stages for the two pretreatments were accordingly determined. Percentages of viable microspores were strongly influenced by protocols of medium-aseptisation. Although filter-sterilized media yielded two-time higher frequencies of living microspores and significantly more GPs than autoclaved ones, the filtration protocol was rather labor-intensive and time-consuming. Therefore a new procedure by combining filtering with autoclaving was subsequently developed as it was more effective than autoclaving and more convenient than filter-sterilization. The method described here could be useful for large-scale preparation of culture media.     

Development of asparagus microspores in vivo and in vitro is influenced by gametogenic stage and cold treatment

Summary Development of asparagus microspores in cold-treated buds of varying sizes and shed microspores from these buds in in vitro culture were observed cytologically for the G459 genotype. Before cold pretreatment, more than 75% of the microspores in flower buds of the 1.4–1.6, 1.7–1.9, 2.0–2.2, 2.3–2.5, and 2.6–2.8 mm size classes were at the early-, mid-, late-uninucleate, early-, and late-binucleate stages, respectively. After 7 d in cold treatment, percentages of microspores at different stages changed in all flower buds. Most notable was the appearance of binucleate microspores resulting from symmetric rather than asymmetric division. For flower buds of 1.7–1.9, 2.0–2.2, and 2.3–2.5 mm size classes, 4.9%, 27.2%, and 11.4% of the microspores had divided symmetrically, respectively. When microspores from buds of each size category were cultured in androgenesis induction medium, only microspores completing symmetric pollen mitosis I during cold treatment were observed to divide further, and calluses were only obtained from microspores of flower bud size classes where symmetric divisions were observed after several days of cold treatment. Significant correlations existed among microspore callus yield, the percentage of late-uninucleate microspores in vivo before cold treatment, and the frequency of symmetric pollen mitosis I after 7 d of cold treatment. Consequently, asparagus microspore androgenesis may occur through one developmental pathway, where a symmetric first mitotic division is a prerequisite for continued development.     

Isolated microspore culture of Chinese flowering cabbage (Brassica campestris ssp. parachinensis)

Microspores of several genotypes of Brassica campestris ssp. parachinensis have been cultured in vitro and induced to undergo embryogenesis and plant formation. Conditions favourable for embryogenesis in this species include a bud size of 2–2.9 mm, NLN-13 culture medium (Nitsch and Nitsch 1967; Lichter 1981, 1982; Swanson 1990), and an induction through exposure to 32°C for a period of 48 h. Longer periods of an elevated temperature for induction of embryogenesis resulted in embryo abortion at early developmental stages. With the protocol developed here, microspores of 60–80% of donor plants could be induced to produce embryos, although embryo yields were low, i.e. 2–5 embryos per 10 buds. Some genotypes responded to culture conditions with high numbers of embryo formation (100–150 embryos per 10 buds) but most of these subsequently failed to mature. The pattern of cell division and morphological changes of the microspores in culture were studied using various microscopic techniques.     

Pro-embryos induction from <Emphasis Type="Italic">Olea europaea</Emphasis> L. isolated microspore culture

Studies were undertaken with one olive (Olea europaea L.) cultivar to identify buds with microspores competent to embryogenesis in vitro. Isolated microspore cultures were performed for the induction of gametic embryogenesis. Different pollen development stages and stress conditions (heat or cold shock) were evaluated. The correlation of inflorescence, anther morphology and the suitable stage of microspore development were analysed. The morphology of responsive buds was identified which corresponded with microspores from the late uni-nucleate to early bi-nucleate pollen stages. Symmetrical divisions of microspores as well as resulting multinucleate structures and pro-embryos were observed. In this paper, a new method of isolated microspore culture that leads to cell division and pro-embryos in olive, is reported.     

Effect of microspore stage and media on anther culture of peanut (Arachis hypogaea L.)

This study was designed to study the effects of stage of microspore development and culture medium on androgenic response in peanut (Arachis hypogaea L.). Anthers of various developmental stages were cultured for 7 days, then fixed and observed cytologically. Three sets of media, involving different basal media, growth regulators, sucrose levels and glutamine concentrations, were tested. In all experiments, the stage of development of the microspores at the time of culture was highly significant. The early uninucleate microspores stage was identified as producing the highest anther response rating. The effect of media was nonsignificant in all experiments. However, the stepwise modification of the media through the course of the study resulted in an almost 8 x increase in anther response rating. Numerically, the best media tested was N₆ basal medium with 1 mg 1^-1 NAA, 0.1 mg 1^-1 BA, 5.5% sucrose, and 3.5 g 1^-1 glutamine. While no haploids were obtained, four-nucleate cells were observed, indicating the potential in peanuts for an androgenic reponse.     

An efficient method for flow cytometric analysis of pollen and detection of 2n nuclei in Brassica napus pollen

A simple and reliable method was developed for isolating pollen nuclei from Brassica napus and Triticum aestivum for DNA analysis using flow cytometry. The nuclei were released from pollen by ultrasonic treatment. The isolated nuclei following filtration through nylon mesh and a purification procedure were suitable for flow cytometric analysis as well as for isolating genomic DNA. Ultrasonic treatment time was optimized for B. napus pollen at different developmental stages. The method is effective and suitable for the preparation of many samples. We analyzed the nuclear DNA levels in pollen of B. napus at three major developmental stages as well as in mature wheat pollen. Only a single 1C peak representing the haploid DNA level was detected in the nuclei isolated from Brassica uninucleate microspores as well as in mature Triticum pollen. Interestingly, diploid nuclei were detected in both binucleate and mature pollen of B. napus. The possible origins of the diploid nuclei are discussed.Abbreviations DAPI 4,6-Diamidino-2-phenylindole - NIB Nuclear isolation buffer     

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter--glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.     

Microsporogenesis and pollen formation in cassava

This article describes the complete microsporogenesis and pollen formation in cassava (Manihot esculenta Crantz) at the various developmental stages (pollen mother cell, meiosis, tetrads, early free spore, mid uninucleate, late uninucleate, binucleate and mature pollen grain). Light microscopy, transmission electron microscopy and confocal laser scanning microscopy were used for the study. Floral bud size and other inflorescence characteristics were correlated with specific stages of the microspore development. This association allows a rapid selection of floral buds with similar microspore developmental stages, useful when a large number of homogeneous cells are needed for analysis and for in vitro induction of androgenesis. This article also compares methods for digestion the exine wall in microspores.     

Colchicine-induced microspore embryogenesis in coffee

A protocol for the induction of androgenesis and plant regeneration from C. arabica cv. Caturra isolated microspores in vitro using colchicine pretreatment has been developed. Microspores were mechanically isolated and then carefully purified. Before colchicine pretreatment, microspores were cultured in a semi-solid medium for further develop and regeneration. Different times of colchicine exposure as well as different concentrations were tested. The best androgenic response was found when microspores were precultured in 100 mg l^–1 colchicine for 48 h. The microspore developmental stages responsive to colchicine were late-uninucleated and early binucleated pollen. Flow cytometry and morphological analyses revealed that 95% of regenerated plants were dihaploids (2n=2x=22). However, some doubled dihaploid plants (2n=4x=44) were also obtained, suggesting that not only androgenic induction but also chromosome duplication could be expected as result of colchicine exposure of coffee microspores. This report represents a new approach in the coffee pollen culture, as well as a major step forward to the utilization of haploid technology in coffee breeding.     

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

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

Factors affecting in vitro maturation of isolated maize microspores

Summary An in vitro method to simulate pollen development was developed in maize (Zea mays L.). Microspores at the late uninucleate to early binucleate stage were isolated and cultured under various conditions. Cell viability, starch content and the formation of the three nuclei as found in normal mature pollen were monitored during the course of the culture. Media composition was modified in order to promote starch accumulation and frequency of mitosis, while maintaining the viability of the microspores. Under the best conditions, up to 12% of the microspores matured in vitro into trinucleate, starch-filled viable pollen grains which were unable to germinate or produce seeds. At different stages during in vitro maturation, proteins patterns were analyzed and compared with their in vivo equivalent and the patterns were only partially similar.     

Isolated microspore culture of wheat (Triticum aestivum L.) in a defined media I. Effects of pretreatment,isolation methods,and hormones

Significant improvements were achieved in the production of haploid and doubled haploid plants from isolated microspore culture of wheat c.v. Chris on a defined media. Procedures found to be of benefit included: A 7-day pretreatment of anthers in 0.4M mannitol plus the macronutrients from FHG medium; the inclusion of 4.5 mg/liter abscisic acid in the pretreatment solution; the isolation of microspores from pretreated anthers by vortexing; and the use of phenylacetic acid (PAA) as the auxin source in MS medium. The best response was achieved with 4.0 mg/liter PAA in MS medium containing 90 g/liter maltose as the sugar source. Under these conditions, 68% of viable microspores underwent division, and an average of 93 embryos and 92 green plants were regenerated per 100 anthers used. The root-tip chromosome number and the fertility of 114 regenerating green plants revealed that 75% were completely fertile spontaneously doubled haploids.     

The conditions required to isolate and maintain viable cotton (Gossypium hirsutum L.) microspores

Cultural systems developed for the successful production of haploid plants from anther and microspore culture of a number of species are lethal to cotton microspores. Components of these systems were examined individually and in simple combinations to determine their specific effect on cotton microspore viability during isolation and culture. An organically buffered pH of 7.0 was critical for survival. The addition of potassium salts, near standard concentrations used for other species, enhanced survival and the cytoplasmic appearance of isolated microspores. Severe toxicity resulted from ammonium, calcium and magnesium salts, and these ions were tolerated only at very low concentrations. Iron, glutamine, serine, inositol, vitamins, and trace minerals were generally not detrimental to microspores at standard concentrations. An isolation and cultural maintenance system was developed that yields large quantities of healthy, viable cotton microspores. This initial step allows for further research in inducing cotton microspores to divide and undergo embryogenesis.Abbreviations ACES N-2-Acetamido-2-aminoethanesulfonic acid - ADA N-2-Acetamidoiminodiacetic acid - BES N,N-bis-(2-hydroxethyl)-2-aminoethanesulfonic acid - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - MOPS 3-(N- Morpholino) propanesulfonic acid - TES 2- tris-(hydroxymethyl) methyl-aminoethanesulfonic acid - PIPES Piperazine-N,N-bis(2-ethanesulfonic acid) dipotassium salt USDA-ARS, in cooperation with the New Mexico Agricultural Experimental Station, Las Cruces, NM 88003, USA. This paper is published as Journal Article No. 1012     

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.