Origin of Multicellular Pollen and Pollen Embryos in Cultured Anthers of Pepper (Capsicum Annuum)

Anthers of Capsicum annuum L. were cultured on Murashige and Skoog (MS) medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ kinetin. Inoculated anthers were subjected to 31 °C and development of microspores in anthers of varying stages was observed cytologically using 4′-6-diamidino-2-phenylindol-2HCl (DAPI). Pepper was characterized by a strong asynchrony of pollen development within a single anther. Percentage of pollen at different stages changed with the culture period, and the proportion of dead pollen increased drastically from day 2 after culture. Microspores that were cultured at the late-uninucleate stage followed one of two developmental pathways. In the more common route, the first sporophytic division was asymmetric and produced what appeared to be a typical bicellular pollen. Embryogenic pollen was formed by repeated divisions of the vegetative nucleus. In the second pathway, which occurred in fewer microspores, the first division was symmetric and both nuclei divided repeatedly to form embryogenic pollen. In early-bicellular pollen, sporophytic pollen was produced through division of the vegetative nucleus. In mid-bicellular pollen, the generative nucleus may undergo division to produce two or more sperm-like nuclei. However, division of the generative nucleus alone to form the embryo was never observed. The anther stage optimal for embryo production contained a large proportion (>75%) of early-binucleate pollen. Associations were found among the percentage of early-binucleate pollen, the frequency of embryogenic multinucleate pollen, and the yield of pollen embryos.     

Pollen from cold-treated Nicotiana tabacum buds: Embryogenic capacity,peroxidase activity and partitioning in aqueous two-phase systems

Pollen isolated from fresh and cold treated tobacco (Nicotiana tabacum L. ew. Wisconsin 38) flower buds were separated using aqueous polymer two-phase partitioning and analysed with respect to embryogenic capacity, peroxidase activity and isoperoxidase pattern. Pollen with embryogenic capacity from cold-treated flower buds were enriched in fractions with higher partitioning than those from fresh flower buds, but the amounts were the same. Cold led to a general increase in specific peroxidase activity in pollen fractions enriched in embryogenic pollen, and also to specific changes in the isoperoxidase pattern. Neutral peroxidase species (pI around 7) and alkaline species (pIs around 9) could be related to pollen fractions enriched in embryogenic pollen. The data agree with earlier data showing that the amount of pollen with the potential to form embryos is established at an early stage in flower development, whereas if they really do so depends on how they are pretreated, e.g. by cold treatments of the buds. The latter is also reflected by quantitative and qualitative differences in peroxidase.     

Pollen dimorphism in relation to pollen plant formation

Induction of haploid plants from pollen grains on culture of anthers has been possible in a number of angiosperms but it is not yet understood why only a fraction of pollen responds to form haploids. Noteworthy in this connection are the recent experiments on anther culture of barley, tobacco and wheat, where it has been pointed out that pollen populations are basically dimorphic. Pollen capable of forming haploids occur in a low frequency, arc smaller, and different from the majority of pollen destined to form gametes. Particularly in tobacco it has been possible to increase the frequency of pollen capable of forming embryos by subjecting plants to low temperature prior to flowering, and to achieve differentiation of embryogenic pollen by subjecting young buds from such plants to a prolonged cold treatment. On selective isolation, from the rest of the pollen, the embryogenic pollen from such buds readily form embryos at high frequency on a simple mineral-sucrose medium. The embryogenic pollen are repressed for gametophytic differentiation and in culture they differentiate to produce embryos. These experiments provide evidence that only certain pollen are capable of forming haploids.     

Pollen Protoplast Culture Leading to Embryogenic Divisions in Hemerocollis fufva

Pollen protoplasts were isolated from the cold-pretreated flower buds containing uninucleate microspores and were inoculated in K3 basic medium supplemented with various additives. A part of the pollen protoplasts were triggered to embryogenic divisions and further to formation of proembryos and clusters. Microscopical observations showed that the first cell division might be equal or unequal and the subsequent growth might be organized or unorganized. This is the first example of pollen protoplast culture in which a sporophytic, or embryogenic, pathway is triggered     

Pollen embryogenesis in anther cultures of Solanum carolinense L.

The direct differentiation of bicellular pollen grains of Solanum carolinense L. (Horse-nettle; Solanaceae) into embryoids and plantlets was induced by culturing whole anthers on Murashige and Skoog's medium supplemented with IAA. The highest frequency of embryogenic induction occurred at 10 mg/l IAA. Developmentally, both the generative and vegetative cells of the pollen grain contributed to embryoid formation whose pattern of development was similar to that of zygotic embryos. In a previous study, it was show that 2,4-D promoted callus formation by pollen grains in cultured anthers of S. carolinense. It appears then that there are two distinct pathways of androgenesis in this species that are determined by the type of auxin present in the medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - BA benzyladenine - KIN kinetin - MS Murashige and Skoog     

Studies on Conditions for Cell Division and Embryogenesis in Isolated Pollen Culture of Nicotiana rustica

A method for the induction of a high rate of cell division and embryogenesis of Nicotiana rustica pollen was developed. Binucleate pollen grains were fractionated by Percoll density gradient (35/45%) centrifugation and cultured in 0.4 molar mannitol at 30°C (the first culture). After 3 days in culture pollen was recollected by a second Percoll fractionation (0/30%) and transferred to and cultured in a medium containing the Murashige-Skoog macro-elements, 0.4 molar mannitol, 40 millimolar galactose, 3 millimolar glutamine, and 5 micromolar ABA for 10 days (the second culture). The cell population consisting of about 80% dividing pollen was transferred to a Murashige-Skoog medium containing 0.4 molar mannitol, 3 millimolar glutamine, and no phytohormone (the third culture), where about 40% of dividing pollen developed into embryos or embryogenic calli.     

Pollen Cryopreservation and Pollen Protoplast Isolation in Brassica carnpestris var.purpurea

The authors have investigated the factors affecting pollen cryopreservation in Brassica campestris var. purpurea, such as pollen development stages cryoprotectant and the process of freezing. A suitable procedure was established as follows :Pollen grains suspended in B5 medium containing 10X DMSO and 1SM sucrose were frozen by a three- –1℃/min – 1 ℃/min step method(0℃———→–10 ℃ ,standing for 15 min———→–40 ℃ , standing for 1 hr→liquid nitrogen)and later thawed in 40℃ water bath. During a period of 60, 90 days′preservation, the relative survival percentage of mature (at the day of anthesis)and nearly mature(2 days before anthesis, trinucleate stage)pollens maintained at ca. 91% that of young pollens(7-8 days before anthesis, late uninucleate stage to early binucleate stage)slightly declined from the original 91.6% to 84. 3%. Culture. experiment showed that the cryopreserved young pollen could be induced to cell division just as well as the fresh pollen. The method of isolating protoplasts from fresh mature pollen developed previously was improved and simplified. As a result, protoplasts were isolated more conveniently from mature pollen and young pollen for the first time. The protoplasts from cryopreserved mature and young pollen could be obtained as well with an isolation rate of 77.4% and 35.9% respectively. However, for isolation of protoplasts from preserved young pollen, an incubation in NLN medium at 35℃ after thawing was necessary.     

Specific phosphoproteins in the initial period of tobacco pollen embryogenesis

Several phosphoproteins specifically correlated with the induction of embryogenic cells were detected in immature pollen grains of Nicotiana tabacum L. By regulating the concentration of glutamine in the medium the developmental pathways of immature pollen grains isolated at the mid-bicellular stage could be controlled, resulting in the formation of either mature pollen grains or embryogenic cells. Different phosphoproteins, designated as a-d and as e-i, respectively, were detected when the pollen grains either became embryogenic cells in glutamine-free medium, or when they were allowed to mature in glutamine-containing medium. The formation of embryogenic cells was suppressed by adding glutamine or cytokinin to the glutamine-free medium, nor did it occur with pollen grains at younger or older stages, and in these cases the phosphoproteins a-d were detectable only partially or faintly. The phosphoproteins a-d and e-i thus may be one of the factors necessary to direct the developmental pathway of immature tobacco pollen grains to embryogenic cells and to mature pollen grains, respectively.The authors thank Dr. V.S. Jaiswal (Botany Department, Banaras Hindu University, Varanasi, India) for his valuable suggestion in the preparation of the paper. This work was supported by a Grantin-Aid for special project research from the Ministry of Education, Science and Culture of Japan.     

鱼腥草花粉母细胞减数分裂特征及其育性研究

为深入了解鱼腥草花粉母细胞的减数分裂特征与花粉育性的关系,该研究采用卡宝品红染色法对2个鱼腥草居群花粉母细胞的减数分裂过程进行观察,并采用氯化三苯基四氮唑(TTC)染色法、I2-KI染色法、B-K培养基培养法及荧光显微镜观察法来检测鱼腥草花粉的活力及萌发率。结果发现:(1)鱼腥草减数分裂的进程与花序大小、花药颜色、花药长度均有密切的关系。(2)2个居群的鱼腥草中花粉母细胞减数分裂过程正常占88.2%,有11.8%的花粉母细胞减数分裂异常。(3)减数分裂异常表现在减数分裂过程中出现微核、落后染色体、染色体桥、不均等分离、多分体等现象,并发现在二分体阶段及单核花粉发育过程中存在细胞融合。(4)2个居群的鱼腥草花粉活力均不超过1.5%,花粉几乎不萌发。研究认为,鱼腥草花粉育性低的主要原因是单核花粉的发育过程异常,而非鱼腥草花粉母细胞减数分裂异常所致。     

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.     

Electrotropism of Nicotiana Pollen Tubes

Electrotropism of tobacco pollen tubes towards the anode wasanalysed. The threshold and saturation values for the electrotropismwere less than 50 mV mm^–1 and 200 mV mm^–1, respectively.The tropic response gradually increased with increasing durationto exposure, but no further increase in the tropic responsewas observed when exposure of the electric field was terminated.Pollen tubes growing towards the cathode had a tendency to burstin a strong electric field. These results suggest that an externallyapplied electric field acts as a motive force for electrotropismbut not as a trigger and that endogenous currents play a rolein tip growth of pollen tubes. Possible mechanisms responsiblefor the electrotropism of pollen tubes are discussed. (Received July 9, 1993; Accepted September 18, 1993)     

AN ULTRASTRUCTURAL AND STEREOLOGICAL ANALYSIS OF POLLEN GRAINS OF HYOSCYAMUS NIGER DURING NORMAL ONTOGENY AND INDUCED EMBRYOGENIC DEVELOPMENT

Selected nuclear and cytoplasmic changes of pollen grains of Hyoscyamus niger during normal gametophytic development and embryogenic development, induced by anther culture, were analyzed and compared ultrastructurally using stereological methods. Potentially embryogenic, uninucleate pollen could be identified within 6 hr of culture by an increased ratio of the volume density of the nucleolar granular zone to the volume density of the fibrillar zone and an increased ratio of dispersed to condensed chromatin in the nucleoplasm. Nonembryogenic pollen in vitro and in vivo possessed prominent nucleolar fibrillar zones and low ratios of dispersed to condensed chromatin. These differences may reflect changes in nuclear activity in potentially embryogenic pollen grains during early stages of culture. Following the first haploid mitosis, in potentially embryogenic pollen the generative cell maintained its large granular nucleolus and high ratio of dispersed to condensed chromatin through its first division to form a proembryoid. The volume fraction of the cytoplasm occupied by mitochondria and plastids and the area fraction occupied by RER and Golgi cisternae differed in the generative cells of potentially embryogenic and nonembryogenic pollen. Those changes only detected in generative cells of potentially embryogenic pollen include: increased area and complexity of cytoplasmic membranes, increased mitochondrial volume, and the presence of plastids at all stages of development. These results support the idea that embryogenic induction of H. niger takes place at the uninucleate stage of development and that subsequent nuclear and cytoplasmic changes are essential for continued sporophytic development.     

Daily Ambrosia Pollen Concentration in the Air of Ankara,Turkey (1990-1999)

The airborne ragweed pollen spectrum was investigated in the air of Ankara, Turkey for aperiod of ten years (1990-1999) using a Burkard seven-day volumetric recording trap. In our study period,long distance transported Ambrosia pollen has been registered. Daily pollen levels varied from low to highin Burge's system. In last three years, the pollen concentration of Ambrosia showed a clear increasingtendency. Our results prove that ragweed pollen may be an important threat for ragweed sensitive patientsin Ankara city in near future.     

Pollen as a marker for migration of Helicoverpa armigera and H. punctigera (Lepidoptera: Noctuidae) from western Queensland

Abstract Pollen carried on the probosces of Helicoverpa punctigera (Wallengren) and H. armigera (Hübner) trapped in western Queensland and in cropping areas of eastern Australia in September 1989 and 1990 was identified by scanning electron microscopy. Ninety-five per cent of moths carried pollen. A total of 19 morphological pollen species’, representing 14 plant families, was found. Up to six pollen species were found on individual moths, and 61% carried more than one. Pollen from plants unsuitable for larval survival was common. Pollen loads generally reflected the abundance of locally flowering plants, but there were exceptions which suggested migration. Pollen of Ptilotus (Amaranthaceae), Velleia (Goodeniaceae) and Eremophila (Myoporaceae), and the Asteraceae (Tubuliflorae) were found on moths trapped in the east. These plants either did not occur in the areas where the moths were caught, or did not flower there at the time the moths were caught. However, they were abundant in possible source areas such as western Queensland. Among moths caught in eastern regions, 30% of H. punctigera and 18% of H. armigera carried pollen from such plants. The value and limitations of moth-borne pollen as a marker for migration are discussed.     

莴苣花粉发育过程中ATP酶的分布

对莴苣花粉发育过程中ATPase的分布特征做了研究。四分体早期的小孢子细胞质中开始出现ATPase反应颗粒。之后,小孢子在发育过程中,花粉内壁聚集大量体积较大的ATPase反应颗粒,并一直保持到花粉即将成熟。在小孢子发育晚期,在花粉萌发孔处和小孢子大液泡中也特异性地聚集了较多ATPase颗粒。二胞花粉刚形成的生殖细胞表面呈现大量的ATPase反应颗粒,当生殖细胞脱离花粉内壁移入营养细胞,ATPase反应颗粒基本消失。生殖细胞分化过程中生殖细胞的ATPase反应颗粒逐渐低于营养细胞中的。在成熟花粉中,精细胞中的ATPase反应颗粒比营养细胞中的少,且主要集中在细胞核中。结果显示花粉发育过程中ATPase的特异分布与花粉发育的一些生物学事件密切相关。     

华北落叶松花粉发育过程中的钙动态分布

运用焦锑酸钾沉淀法研究了华北落叶松(Larixprincipis-rupprechtiiMayr)小孢子发育过程中不同阶段Ca2 的分布情况。减数分裂时期,小孢子囊壁表皮和中层细胞的细胞壁及细胞间隙Ca2 分布较多,绒毡层只有外切向面的细胞膜有Ca2 分布,小孢子母细胞的各部位则很少有Ca2 ;四分体时期,包围四分小孢子的胼胝质壁上有大量的Ca2 分布,在四分孢子壁上也有较多沉淀;游离小孢子时期,钙离子在小孢子壁的分布较四分体时期有所减少,而到花粉成熟时又逐渐增多;从四分体到花粉成熟,乌氏体周围的Ca2 有增多的趋势。对四分体外壁Ca2 的大量分布与花粉壁的形成及信号物质在花粉表面贮存的关系,以及小孢子囊的外壁、绒毡层和乌氏体在Ca2 向花粉运输中所起的作用进行了讨论。     

植物花粉培养研究进展

花粉培养又称为游离小孢子培养,指将发育到一定阶段的花粉从花药中游离出来成为分散或游离状态,通过培养使花粉粒脱分化,进而发育成完整植株的过程。花粉培养的主要目的是获得单倍体植株,进而得到双单倍体(double haploid,DH)植株,最终获得纯合系物种。本文对花粉培养形成植株的物种信息进行了收集整理,概述了国内外花粉培养的一些最新研究进展,包括影响花粉培养形成胚的因素以及提高花粉胚产量的措施,并对花粉培养的前景进行了展望。