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.     

甘蓝型油菜花粉离体培养

通过对甘蓝型油菜花粉发育阶段和活力的检测确定花粉发育的时期,分离出单核晚期花粉进行离体培养.结果表明,(1)筛选出适合油菜小孢子花粉离体培养的液体培养基为T_1+怀特维生素(White's vitamins)+2%椰子汁+0.5 mol/L麦芽糖,在此培养基上花粉的成熟率可达25.1%,萌发率达6.3%.(2)筛选出适合成熟花粉离体萌发液体培养基为0.6 mol/L麦芽糖+1.6 mmol/L硼酸+2.9 mmol/L硝酸钙+29.6 μmol/L VB_1,在此培养基上,自然成熟花粉的萌发率可达75.2%.将离体培养成熟的花粉培养在萌发培养基,萌发的花粉占成熟花粉的66.3%.     

Maturation of maize pollen in vitro

Summary Maturation of maize pollen was obtained in male reproductive structures cultured in vitro. Immature tassels containing microspores at the mid-uninucleate to late-binucleate stage of development were excised and spikelets, anthers, and/or isolated microspores were cultured on a medium capable of supporting pollen maturation. Microspore mitosis, culminating in the production of starch-filled, trinucleate pollen capable of germination, was observed after 7–15 days, depending on the genotype and stage at which the cultures were initiated. Up to 100%, 70%, and 20% of the cultured spikelets, anthers, and isolated microspores, respectively, produced mature pollen, which germinated, however, at different frequencies (i.e., spikelets, 50–70%; anthers, 5–10%; microspores, <1%). Mature kernels were produced following fertilization with pollen from cultured spikelets and anthers. These procedures provide methods for the in vitro manipulation of a significant phase of the maize life cycle.     

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.     

Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies

Summary Specific stress treatments applied to isolated tobacco (Nicotiana tabacum L.) microspores efficiently induced haploid embryo formation in vitro. A heat shock at 33 or 37°C in the presence of sugar, as well as sucrose-starvation at 25°C, resulted in the formation of embryogenic microspores. A combination of both treatments had an additive effect. Under optimal induction conditions all viable microspores in the culture were embryogenic and developed subsequently into pollen embryos by culture at 25°C in a sugar-containing medium, with induction frequencies of more than 70% with respect to the initial microspore population. A high fraction of the early pollen embryos continued their development in vitro, giving rise to haploid plants. In contrast to other available systems for microspore/pollen embryogenesis, the new protocol allows the production of homogeneous populations of embryogenic microspores and early globular embryos in large-scale cultures, without any purification step, and is therefore well suited for biochemical and molecular work.Abbreviations EDTA ethylenediaminetetraacetate - DAPI 4,6-diamidino-2-phenylindole     

Cell division study in ‘pollen mother cells’ and ‘pollen grains’ of in vivo and ex vitro plants in Drimiopsis botryoides

The various normal and abnormal stages of meiosis and pollen mitosis of Drimiopsis botryoides are described, and a comparison between naturally propagated in vivo and tissue culture derived ex vitro plants in respect to their cytological behaviour presented. We also describe the floral morphology and investigate the relationship between the floral developmental stages and the progression of microgametogenesis. In total, 33 bivalents are observed in diakinesis, which indicate the diploid number 2n = 66 and this number is cross-checked by a haploid set of n = 33 chromosomes in pollen mitosis. Only 6.8% and 4.9% meiotic abnormalities were recorded on in vivo and ex vitro plants, respectively, which led to the formation of non-viable pollen. Finally, the microspores have to develop into tri-cellular male gametophyte. Only 0.2% pollen grains are found with a micro-nucleus. Though the higher pollen viability was recorded on both in vivo (89.3 ± 4.1%) and ex vitro (92.1 ± 4.6%) plants, but surprisingly the pollen germination rate is extremely low with 13.6 ± 1.74% and 21.3 ± 1.55%, respectively. The present study obviously enriches the cytological database of D. botryoides and may help future research on androgenesis and genetic improvement.     

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.     

Improvement in efficiency of microspore culture to produce doubled haploid lines of Ethiopian mustard

Factors affecting microspore embryogenesis of Ethiopian mustard (Brassica carinata A. Braun) were evaluated, including flower bud length, pollen developmental stage, and microspore density. An embryogenic frequency of 300 embryos per Petri plate was observed with NLN (Nitsch-Lichter-Nitsch) medium supplemented with 13% sucrose, 3.0–3.4-mm-long buds, and a plating density of 65,000 microspores/ml. About 65% of the microspores from buds 3.0–3.4-mm long were at the late uninucleate stage. Microspore-derived embryos were successfully transferred to solid medium for germination. After 4 wk, the resulting plantlets were transplanted to a soilless potting mixture and grew well under greenhouse conditions.     

Scanning electron microscopy of microspore embryogenesis inBrassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy     

Effect of gibberellic acid- and water-based pre-soaking treatments under different temperatures and photoperiods on the seed germination of Allium stracheyi Baker: An endangered alpine species of Central Himalaya,India

Allium stracheyi Baker (Alliaceae, 2600–3000 m asl), an endangered species of Central Himalaya, India, has low seed germination in its natural habitat. This study is an attempt to improve seed germination by determining the seed viability with a low mean germination time (MGT) and germination index (GI) under optimum temperature, light, and pre-soaking treatments. The seeds were pre-soaked in hot water (80°C), cold water (10°C), and gibberellic acid (GA₃ at 50 and 100 mg/l) for 24 h and subjected to light (12 h light and 12 h dark) and continuous dark (24 h) conditions with different temperature regimes (10, 15, 20, 25, and 30°C). The viability varied between 66.0% and 69.67% and declined rapidly after 12 months of storage. Our studies suggest that the 100 mg/l GA₃ treatment was beneficial for seed germination and seedling growth. Pre-soaking in a 100 mg/l GA₃ solution and incubation at 20°C under light conditions enhanced the germination significantly (p < 0.05) and resulted in the highest (97.3%) germination with the lowest MGT = 5.7 days, with GI = 8.11. The recommendations of this study support the conservation of alpine A. stracheyi via simple and cost-effective techniques for optimal seed germination.     

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.     

Effects of desiccation and controlled rehydration on germination in vitro of pollen of walnut (Juglans spp.)

Abstract Moisture content and germination percentages on agar-solidified medium were determined for pollen of Juglans regia and J. nigra at the time of release from anther and at 24-h intervals until germination fell to zero. Moisture content of fresh J. regia pollen ranged from 4.6 to 12.1%, and from 3.9 to 4.0% for J. nigra pollen; in vitro germination percentages were uniformly high throughout these ranges. Germination declined as pollen moisture fell below 5% in J. regia with regression analyses predicting 50% germination at 3.9–4.4% moisture. After 4 d at ambient laboratory conditions, all pollen had lost the ability to germinate when incubated directly on medium. This pollen was incubated in chambers where humidity was maintained at saturation and 33% saturation, and retested. Pollen up to 22 d old and having moisture contents as low as 2.6% regained the ability to germinate in vitro when it was incubated in a water-saturated environment before being placed on the agar-solidified medium.     

Factors influencing somatic embryo maturation, high frequency germination and plantlet formation in Terminalia chebula Retz.

The factors influencing somatic embryo maturation, high frequency somatic embryo germination, and plantlet formation were studied in Terminalia chebula Retz. Maturation of somatic embryo were influenced by a number of factors such as in vitro culture passage, concentrations of sucrose, levels of abscisic acid (ABA), basal media and media additive combinations. Maximum frequency of somatic embryo maturation (57.22 ± 2.02), was obtained on MS medium supplemented with 50 g/l sucrose. Different factors such as strengths of MS nutrients, plant growth regulators, media additives and their combinations controlling somatic embryo germination and plantlet formation were studied. High frequency of germination and plantlet formation (58.80 ± 1.47) were achieved by subsequent subculture of mature somatic embryos on MS medium containing 30 g/l sucrose and 0.5 mg/l benzyladenine (BA). However, although duration of in vitro passage of the callus tissue was critical, contribution of the combinations of plant growth regulators and media additives showed nugatory effect on somatic embryo maturation and germination as evident from variable responses.     

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.     

硼酸和蔗糖对‘柔毛齿叶’睡莲花粉萌发的影响

以‘柔毛齿叶’睡莲（Nymphaea tetragona）花粉为试验材料,采用花粉人工培养法研究花粉离体萌发的最适条件。结果表明：蔗糖、硼酸在一定浓度范围内能促进花粉萌发,睡莲花粉萌发的最适蔗糖和硼酸浓度分别为60mg／L和15mg／L,萌发率分别达30．83％和23．73％。     

The in vitro germination and storage characteristics of Keteleeria fortunei var. cyclolepis pollen provide a reference for cross breeding

Keteleeria fortunei var. cyclolepis is an ideal tree species for mountain afforestation, timber forests, and landscaping. Its pollination process can be affected by the rainy season, making it difficult to pollinate the massive female cones, which leads to a high abortion rate and low quality of seeds. Here, we observed the pollen morphology of K. f. cyclolepis using scanning electron and light microscopes, investigated the characteristics of its in vitro germination by the detached method, and explored the effect of different storage temperatures and times on the pollen germination rate and the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Our results indicated that the pollen of K. f. cyclolepis is a five-cell pollen, comprising one noumenon and two air sacs, both of which were oval in polar view. The optimal condition for pollen germination of K. f. cyclolepis was 240 g/L sucrose + 70 mg/L CaCl₂ + 210 mg/L H₃BO₃ at 24 °C and pH 6.0, resulting in a germination rate of 45.0%. The effects of different storage temperature and time on pollen germination rate varied significantly. The best storage temperature was − 80 °C, at which the germination rate was 20.9% after 365 days of storage, and the activity of three protective enzymes remained relatively high, representing relatively strong antioxidation and antiaging activity. Stepwise regression analysis showed that SOD was the main factor affecting the pollen germination rate of K. f. cyclolepis. The function of the three protective enzymes differed under various temperatures, for example, SOD served as a sensitive protective enzyme at room temperature, − 20 °C and − 80 °C, whereas both SOD and CAT served as sensitive protective enzymes at 4 °C.

     

Germination of trinucleated pollen: formulation of a new medium for Capsella bursa-pastoris

Summary In Brewbaker and Kwack's medium (BK) only 16% of the pollen grains germinated, and these produced pollen tubes having a maximum length of 25 m. With a solution based on Monnier's medium 47% germination and 160-mlong pollen tubes were observed. Calcium was shown to be essential for germination; the optimal concentration was 880 mg/l calcium chloride. The optimal concentrations of magnesium sulphate and boric acid were 360 and 50 mg/l, respectively. Germination at pH 4.0 but also pH 8.0 and the presence of vitamins B1 and B6 (1 mg/l each) were stimulatory. Polyethylene glycol (PEG) was superior to sucrose as an osmoticum and germination and tube length were significantly improved using PEG 4000 at a concentration of 120 g/l (0.03 M). Equimolar concentrations of PEG 400 and PEG 600 gave inferior results. Combining PEG with sucrose in the medium did not improve germination or increase tube length.     

The positive effect of arabinogalactan on induction of somatic embryogenesis in Quercus bicolor followed by embryo maturation and plant regeneration

This is the first report on the successful induction of somatic embryogenesis in swamp white oak from leaf and shoot apex explants excised from in vitro shoot cultures derived from 6- to 7-year-old trees. We demonstrated that arabinogalactan from larch wood (2–4 mg/L) promoted embryogenesis in the three genotypes evaluated by increasing the frequency of somatic embryogenesis, the embryogenic sites per explant, and by speeding the onset of embryo initiation. The explants were cultured sequentially on three culture media consisting of Murashige and Skoog (MS) salts and vitamins supplemented with 500 mg/L casein hydrolysate and different concentrations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA). Somatic embryogenesis induction frequencies of up to 12.4, 4.5, and 0.7 % were obtained for the three genotypes. Clonal embryogenic lines were maintained by repetitive embryogenesis following culture on MS medium containing 0.44 μM BA with or without 0.27 μM NAA. Before germination, cotyledonary-stage embryos were cultured for 4 weeks in maturation medium (MS medium with half-strength macronutrients) containing 6 % sorbitol. Germination response was significantly improved by applying a 2-month cold storage as a post-maturation treatment. The mineral formulation and plant growth regulator content of the germination medium influenced the frequency of plantlet conversion with the best results achieved on Gresshoff and Doy medium with BA (0.25–0.44 μM). This procedure resulted in over 50–60 % of germinating embryos exhibiting continuous root growth and either epicotyl elongation or shoot development.     

Development of an improved medium for germination of Cajanus cajan (L.) Millsp. pollen in vitro.

A simple, reliable medium for pollen germination of Cajanus cajan was developed by modifying Brewbaker and Kwack (BK) medium. Past attempts of C. cajan pollen germination in artificial media were not successful. A medium containing polyethylene glycol 4000 (PEG) showed more than 90% germination for C. cajan var. Pusa 33 only when the young buds (36 h before anthesis) were kept in pollen germination medium (PGM) for 36 h before pollen extraction. Supplementation of PGM with epsilon-amino caproic acid (EACA), an amino acid, showed improved pollen germination in Pusa 33 and also helped to avoid preconditioning of young buds before pollen extraction. It was also observed that there is a genotypic difference in the level of EACA required for in vitro pollen germination. Thus a complete medium for C. cajan genotypes consists of 37.5% sucrose+ 15% PEG 4000+250 mg l(-1) boric acid+300 mg l(-1) calcium nitrate+100 mg l(-1) potassium nitrate+ 200 mg l(-1) magnesium sulphate+1% agar+EACA (0, 100, 250, 500, 750 or 1000 mg l(-1)).     

Nuclear pore dynamics during pollen development and androgenesis in Brassica napus

Changes in nuclear pore complex (NPC) densities, NPCs/nucleus and NPCs/μm³, are described using freeze-fractured Brassica napus microspores and pollen in vivo and in vitro. Early stages of microspore- and pollen-derived embryogenic cells were also analysed. The results of in vivo and in vitro pollen development indicate an increase in activity of the vegetative nucleus during maturation of the pollen. At the onset of microspore and pollen culture, NPC density decreased from 15 NPCs/μm² at the stage of isolation to 9 NPCs/μm², under both embryogenic and non-embryogenic conditions. This implies that the drop in NPC density might be a result of culturing the microspores and pollen rather than an indication for microspore and pollen embryogenesis in Brassica napus. However, after 1 day in culture under embryogenic conditions, the NPC density increased again and stabilised around 13 NPCs/μm², whereas under non-embryogenic conditions the NPC density remained about 9 NPCs/μm². This low density of 9 NPCs/μm² was also found in the nuclei of sperm cells, in contrast to the 19 NPCs/μm² found in the vegetative nucleus. It means that, although both the vegetative and sperm nuclei are believed to be metabolically rather inactive in mature pollen, the NPC density of vegetative nucleus is twice as high as the NPC density of the sperm nuclei. In a few cases, embryos formed suspensor-like structures with a NPC density of 9 NPCs/μm², indicating a lower nucleocytoplasmic exchange of the nuclei of the suspensor cells than with the nuclei in the embryo proper. In addition, observations on NPCs and other organelles, obtained by high resolution cryo-scanning microscopy, are presented. Received: 29 December 1999 / Revision accepted: 3 March 2000