In vitro differentiation of embryogenic pollen,control by cold treatment and embryo formation inab initio pollen cultures ofNicotiana tabacum var. badischer burley

Summary InNicotiana cold treatment causes differentiation of embryogenic pollen. This differentiation initiates on the plant and is completed in culture. Differentiation on the plant results in pollen dimorphism and differentiation in culture leads to pollen embryogenesis. An increased number of pollen capable of embryogenesis is possible on plants induced to flower in short days and low temperature (8 hours light, 18 °C). These embryogenic pollen on selective isolation, from buds at a petal length of 3.4±0.1 cm, fail to form embryos but do so in the cultures which receive cold treatment at 10 °C for 10 days. To some extent the differentiation of embryogenic pollen can be completed on plants induced to flower at 15 °C and embryogenic pollen from such plants form embryos at a low frequency which can be substantially increased on giving the cultures a cold treatment. The frequency of embryogenesis is higher in cultures of 15 °C plants than those of 18 °C plants. Low temperature requirements at two stages—to the plant and to the culture—are essential and complimentary for embryogenesis inab initio pollen cultures.Cold treatment causes repression of gametophytic differentiation and this results in the differentiation of embryogenic potential. The embryogenic pollen, unlike gametophytic pollen, are not fully differentiated structures. They are unable to divide and form embryos in presence of metabolic inhibitors such as actinomycin-D and cycloheximide.     

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.     

Differentiation of embryogenic pollen in cold-treated buds ofNicotiana tabacum var. Badischer burley and nutritional requirements of the isolated pollen to form embryos

Summary Embryogenic pollen were selectively isolated from buds after cold treatment at 10 °C for 10 days; it was immaterial whether the buds were taken from short day and low temperature (SD and LT; 8 hours light, 18 °C) or long day and high temperature (LD and HT; 16 hours light, 24 °C) plants. However, in buds from SD and LT plants the differentiation of embryogenic pollen could be detected as early as 7 days after the cold treatment, and pollen from these plants formed embryos at higher frequency (up to 4% of cultured pollen) than those from LD and HT plants (up to 1% only).The embryogenic pollen, in isolated buds, differentiated by way of pollen dimorphism. During cold treatment a fraction of pollen remained small, retained clear cytoplasm and was capable of embryogenesis in comparison to gametophytic pollen which enlarged and acquired granular cytoplasm. In our experiments cold treatment was a key factor in the induction of pollen dimorphism. This aspect of cold treatment in pollen embryogenesis is reported for the first time and was possible on the basis of selection of embryogenic pollen by density gradient centrifugation. The ratio of embryogenic pollen was about one fifth of the total population.The nutritional requirements of isolated pollen for embryogenesis were rather simple. These pollen formed embryos which readily developed into plantlets on a mineral medium supplemented with sucrose provided the pH was 6.8.     

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

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

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.     

Stage-related expression of mRNAs during pollen development in lily and tobacco

Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h^–1 in young binucleate cells, 138 fg · h^–1 in late binucleate cells and 56 fg · h^–1 in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.Abbreviations cDNA copy DNA - pI isolectric point To whom correspondence should be addressed.     

Environmental control and evidence for predetermination of pollen embryogenesis inNicotiana tabacum pollen

Summary The effect of daylenght and temperature for the donor plants (Nicotiana tabacum var. Badischer Burley) on the formation of pollen competent for embryogenesis (P-pollen) by the three possible routes (during normal flower developmentin situ (pollen dimorphism), during cold-treatment of excised flower buds, in cultured anthers) was studied. In all three routes, P-pollen frequency (premitotic pollen, before 1. sporophytic division, PPF) was affected in essentially the same way. At 24 °C and long days, PPF was low and short days had only a slightly increasing effect. At 18 °C and long days, PPF was higher and short days further increased it. Correlated with PPF under the different growth regimes was the percentage of units with more than one vegetative-type nucleus (normal embryos + abortive embryos + multinucleate pollen) in 3 weeks old anther cultures. Under greenhouse conditions, PPF was generally higher than at 24° in growth rooms and showed a maximum in the winter months. Plant age did not affect PPF. These results give further evidence that pollen embryogenesis is predetermined before excision and culture of the pollen or anthers.     

Induction of embryogenic pollen grains in situ and subsequent in vitro pollen embryogenesis in Nicotiana tabacum by treatments of the pollen donor plants with feminizing agents

Tobacco plants ( Nicotiana tabacum L., var. Badischer Burley) were treated with chemicals (sprays and soil drenches) known to affect sex expression in other species. Their effect was tested on sex balance, pollen sterility, embryogenic pollen grain (P-grain) formation in situ, and on pollen plant formation in anther and pollen cultures after anther preculture. Napthalene acetic acid (NAA) increased the length of pistils and stamens and shifted sex balance towards femaleness when the plants were raised in long or short days at 24 or 15°C. In parallel, pollen sterility, P-grain frequency in situ and pollen plant production from anther and pollen cultures were increased by NAA. Alar 85 redueed the length of pistils and stamens and shifted sex balance towards femaleness when the plants were raised in long days at 24°C, but shifted it towards maleness in short days and/or at 15°C. In parallel, pollen sterility, P-grain frequency in situ, and pollen plant production in vitro were increased when plants in long days at 24°C were treated with Alar 85, but decreased when plants in short days and/or at 15°C were treated. Ethrel, Cycocel, and GA₃ applied in a similar manner, were ineffective. Water sprays and nitrogen starvation shifted sex balance towards femaleness in long days at 15°C and increased pollen sterility, P-grain frequency in situ and pollen plant production in vitro. At 24°C, water sprays and nitrogen starvation had no effect.     

Ultrastructure of embryogenic pollen ofNicotiana tabacum var. Badischer burley

Summary Pollen grains capable of embryogenesis were selectively isolated from (a) near-mature buds from plants induced to flower in short days and low temperature (8 hours light and 18 °C) and (b) young buds from these plants with an additional low temperature treatment (10 °C for 10 days) and fixed for electron microscopy. The pollen from the former formed embryos at a very low frequency in culture, and at the subcellular level showed different degrees of regression of cytoplasm and mitochondria. On the contrary, cold-treated pollen were characterized by a high frequency of embryogenesis, up to 25% of the cultured pollen. They did not show regression of cytoplasm or organelles but had an attenuated cytoplasm which was not rich in ribosomes. Another noteworthy feature of embryogenic grains was the condensed nature of mitochondria. These characteristics of embryogenic grains indicate that they are repressed for gametophytic differentiation. The embryogenic pollen did not differentiate from gametophytic pollen which were very distinctive, having a thick exine, and dense cytoplasm rich in ribosomes. The close similarity of embryogenic grains with young microspores in terms of thin exine and sparse cytoplasm is suggestive of an indeterminate state and that determination into gametophytic or sporophytic (embryogenic) type is probably the function of differential gene activity. Of interest, in this context, is the condensation of mitochondria in embryogenic grains. The relationship, if any, between mitochondrial condensation and embryogenesis remains to be resolved.     

A study of Quercus pollen in the Derby area, UK

The major allergenic pollen prevalent in the Derby air in May is Quercus pollen which has been monitored volumetrically from 1970–1997. Quercus pollen levels in Derby are increasing, showing an established long term trend, with 1995 being an exceptionally high year. There is now an earlier start date and a longer seasonal duration. The mean Quercus diurnal periodicity for 1991–1997 shows a peak at 15.00 hours.A detailed study of the 1990–1997 seasons established that a maximum temperature of 20 °C or above, at the usual time of flowering, occasions the start of the Quercus pollen season. Average May temperature and drought in the previous June and July are important factors in determining Quercus pollen totals. Predictions for the forthcoming seasons were produced which compared favourably with the actual pollen totals.     

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.     

Microspore embryogenesis in anther cultures of two Indian cultivars of Brassica juncea (L.) Czern.

Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained. Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis. Embryogenesis was also stimulated by an initial culture at 5°C for 3 days. Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos. All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets. The androgenic plants were haploid (2n=18).     

Airborne pollen data of Platanaceae in Santiago de Compostela (Iberian Peninsula)

The pollen count of the Platanus genus in the atmosphere of Santiago de Compostela (Galicia, Spain) was carried out from 1993 to 1998, with the pollen of this taxon representing 6% of total identified pollen. The principal pollination period (P.P.P.) was centred on the months of March and April, with an average duration of 23 days.During the study period we obtained negative correlations with precipitation and humidity and positive ones with temperature and hours of sunshine, with a confidence index of more than 90%. On the basis of the six-year study period, we also calculated the average accumulated temperature required to trigger the onset of flowering, which oscillated between 330 and 456 °C, with this result being confirmed for 1999 (437 °C). We also observed that the values of maximum pollen concentration of this taxon in the last 7 years (1993–1999), were obtained on days during the P.P.P. with maximum temperatures above 18 °C. The model of intradiurnal variation reflects, for the majority of years, a greater representation during the central hours of the day.     

The isolation and culture of lily pollen protoplasts

Methods for the enzymatic isolation of lily protoplasts and their successful culture are described. When pre-anthesis binucleate pollen (immature pollen grains) was treated in enzyme solution containing macerozyme and cellulase, up to 80% lost their exine and gave rise to intact protoplasts within 1 h. These pollen protoplasts were uniform in size and densely cytoplasmic with two prominent generative and vegetative nuclei. The isolated pollen protoplasts regenerated a cell wall within 1 day of culture and produced a structure resembling a pollen tube after 10–12 days of culture. During this culture period, dividing generative nuclei or 2 sperm nuclei were observed in many protoplasts with regenerated cell walls.     

Induction of embryos in pollen cultures of Nicotiana sylvestris

Induction of embryos in pollen cultures was possible when young buds from plants induced to flower at 15°C were given a cold treatment at 10°C for 15 days. Following selective isolation of embryogenic pollen the cultures raised were given cold treatment at 10°C for 12 or more days. In such cultures the embryos appeared at a frequency of 1.2% of the cultured pollen on mineral-sucrose medium at pH 6.8.     

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     

The coexistence of binucleate and trinucleate pollen in Mitrephora macclurei Weerasooriya & R. M. K (Annonaceae)

Pollen of angiosperms may be either binucleate or trinucleate at maturity. According to Schürhoff–Brewbaker law, the binucleate state is pleisomorphic occuring in about 70% of all angiosperms, while the trinucleate state is advanced occurring in about 30% of the angiosperms. The derivation of trinucleate pollen from binucleate pollen is irreversible, thus trinucleate or mixed pollen was considered uncommon in early lineage. In the present work, using conventional paraffin section method, the coexistence of both binucleate and trinucleate pollen in the same intact anther were observed in Mitrephora macclurei, a species belonging to the eumagnoliid family Annonaceae. The increasing number of plants with trinucleate or mixed pollen discovered in early lineages, as well as climatic introduced changes in nuclear number challenge analyses of ancestral state and evolution of pollen nuclear number in angiosperms.     

Susceptibility of pollen to UV-B radiation: an assay of 34 taxa

Much of the ultraviolet-B radiation (UV-B) research on plants has concentrated on vegetative plant parts, and only a small fraction has dealt with the reproductive system. The present study analyzed pollen grains of 34 taxa germinated and grown under two levels of UV-B radiation (187 and 460 mW/m2) or no UV-B (control group). Visible radiation at 260 mmol/m/s was present in all treatments. Taxa included those with binucleate and trinucleate pollen types. We detected differences among species. A significant reduction in pollen germination occurred in only five species. Pollen tubes of >50% of the species showed significant reduction in length. Trinucleate pollen types were more likely to exhibit tube length reduction than the binucleate types. Proportionately more monocotyledonous species were sensitive to UV-B treatment than dicotyledonous species, and proportionately more wild species were sensitive than cultivated species and pollen collected from plants growing in the field were somewhat more sensitive than pollen collected from plants grown in the greenhouse. Species in which pollination occurred earlier in the season were more likely to be susceptible to UV-B radiation than those for which anthesis took place later in the season, suggesting a possible adaptation to UV-B radiation.     

Airborne Pinus pollen in the atmosphere of Brisbane, Australia and relationships with meteorological parameters

Relationships between weather parameters andairborne pollen loads of Pinus inBrisbane, Australia have been investigated overthe five-year period, June 1994–May 1999.Pinus pollen accounts for 4.5% of the annualairborne pollen load in Brisbane where thePinus season is confined to the winter months,July–early September. During the samplingperiod loads of 11–>100 grains m³ wererecorded on 24 days and 1–10 grains m³ on204 days. The onset and peak dates wereconsistent across each season, whereas the enddates varied. The onset of the Pinuspollen season coincided with the coolestaverage monthly temperatures (< 22°C),lowest rainfall (< 7mm), and four weeks afterdaily minimum temperatures fell to 5–9°Cin late autumn. Correlations obtained betweendaily airborne Pinus pollen counts andtemperature/rainfall parameters show thatdensities of airborne Pinus pollen arenegatively correlated with maximum temperature(p < 0.0001), minimum temperature (p < 0.0001)and rainfall (p < 0.05) during the mainpollination period. The mean duration of eachpollen season was 52 days; longer seasons wereshown to be directly related to lower averageseasonal maximum temperatures (r² = 0.85,p = 0.025). These results signify that maximumand minimum temperatures are the majorparameters that influence the onset andduration of the Pinus pollen season inthe environs of Brisbane. Respiratory allergyis an important health issue in Brisbane,Australia, but it remains unknown whether ornot airborne Pinus pollen is acontributing factor.     

BA对大麦花药培养中药壁的衰退和植株再生频率的影响

用含20ppm 6-BA的0.1％吐温-80溶液喷施花粉为单核前期的大麦上部叶片和穗部,明显影响大麦花药培养效率。实验结果表明:1)BA处理可明显延缓培养花药的药壁衰退进程。2)BA处理后的花药,在培养期间,其死亡的花粉数比对照大大减少,相反其双核或多核的花粉数比对照明显增加。3)BA处理虽然没有促进大麦花粉愈伤组织的诱导率,但显著地促进愈伤组织的生长。提高愈伤组织成长率,增加可转入分化培养的愈伤组织块数。4)BA处理促进愈伤组织的再分化,尤其是绿苗的分化。