Sperm dimorphism in terms of nuclear shape and microtubule accumulation in Cyrtanthus mackenii

Pollen tubes of Cyrtanthus mackenii, a species with bicellular pollen, were cultured in vitro to investigate nuclear phase changes during generative cell division and male germ unit (MGU) formation, using flow cytometric analysis. Results revealed that sperm cells were formed after 12 h of culture. During sperm maturation, the nuclei of sperm cells were not associated with the vegetative nucleus (unassociated sperm cells; Sua) and became longer than those of sperm cells associated with the vegetative nucleus (Svn). These findings indicate that the pair of sperm cells in the C. mackenii MGU is dimorphic in terms of nuclear shape. Dimorphism coincides with anti-α-tubulin antibody immunofluorescence, which was higher in the Sua than in Svn. Following treatment with oryzalin, triggering microtubule depolymerization, differences between nuclear shapes in the two sperm nuclei disappeared, suggesting that microtubule accumulation between sperm cells in the MGU correlates with differences in the nuclear shape.     

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.     

Epigenetic marks in the <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L. mature pollen grain and during in vitro pollen tube growth

During the sexual reproduction of flowering plants, epigenetic control of gene expression and genome integrity by DNA methylation and histone modifications plays an important role in male gametogenesis. In this study, we compared the chromatin modification patterns of the generative, sperm cells and vegetative nuclei during Hyacinthus orientalis male gametophyte development. Changes in the spatial and temporal distribution of 5-methylcytosine, acetylated histone H4 and histone deacetylase indicated potential differences in the specific epigenetic state of all analysed cells, in both the mature cellular pollen grains and the in vitro growing pollen tubes. Interestingly, we observed unique localization of chromatin modifications in the area of the generative and the vegetative nuclei located near each other in the male germ unit, indicating the precise mechanisms of gene expression regulation in this region. We discuss the differences in the patterns of the epigenetic marks along with our previous reports of nuclear metabolism and changes in chromatin organization and activity in hyacinth male gametophyte cells. We also propose that this epigenetic status of the analysed nuclei is related to the different acquired fates and biological functions of these cells.     

Flow Cytometric Determination of Relative Nuclear DNA Contents in Bicellulate and Tricellulate Pollen

Nuclear DNA content in mature pollen was measured with a flowcytometer Pollen of Lilium longiflorum, Dendranthema grandiflora(syn Chrysanthemum monfolium) and Zea mays was chopped and stainedwith the DNA fluorochrome DAPI DNA levels, expressed as arbitraryC values, were compared with those of nuclei isolated from leafor root material of the same plants In mature tricellulate pollen the generative cell is dividedafter second pollen mitosis into two sperm cells Tricellulatepollen from maize and chrysanthemum gave rise to one large 1Cpeak and, only in the case of chrysanthemum, a much smallerone at the 2C level These results suggest that the haploid nucleiof the vegetative as well as both sperm cells in tricellulatepollen are arrested in the G₁ stage of nuclear division Thesmall 2C peak in the case of chrysanthemum probably arose froma fraction of pollen with the sporophytic chromosome number(2n pollen) In contrast to this, mature bicellulate lily pollengave rise to two identical peaks at the 1C and the 2C levelFrom this result it was concluded that in bicellulate pollen,the 1C peak is caused by the signal of the haploid vegetativenucleus arrested in the G₁ stage of nuclear division, whereasthe 2C peak originates from the haploid generative nucleus whichhas already undergone DNA synthesis and is arrested in G₂ Lilium longiflorumThunb, lily, Dendranthema grandiflora Tzelev (syn Chrysanthemum morifolium Ramat ), chrysanthemum, Zea maysL, maize, male gametophytic cells, vegetative cells, generative cells, sperm cells, unreduced pollen, sporophytic cells, relative nuclear DNA contents, replication stage     

烟草精细胞两个群体的分离

高等植物的倾向受精是一个非常吸引人的研究课题,目前对其机理还不清楚.要想探索高等植物倾向受精现象,前提之一是要分离出一定数量的两个精细胞群体作为分子生物学研究方法的材料.以前的研究表明,烟草(Nicotiana tabacum L.)花粉管中的两个精细胞体积差异明显.这种异型性的精细胞可能与倾向受精有关.烟草是二胞型花粉,生殖细胞只在体内生长的花粉管中才分裂形成两个精细胞.用体内/体外技术培养出花粉管后,爆破花粉管即可释放出花粉管内含物,其中包括两个精细胞.用微量酶液可使两个精细胞分开.然后用显微操作器可挑选出两个大小不同、数量上千的精细胞群体.这种单一纯化的精细胞群体为用分子生物学方法区分两个精细胞的DNA和蛋白质差异打下基础.本研究是高等植物的第二例、二胞花粉植物中的第一例分离两个特定精细胞群体的尝试,为构建烟草两个精细胞的cDNA文库创造了条件.     

烟草雌、雄配子DNA含量变化

采用显微分光光度法测定了烟草（Nieotiana tabacum）精细胞和卵细胞的DNA含量。烟草是二胞花粉,花粉萌发后生殖细胞在花粉管中分裂形成精细胞。授粉后45h花粉管到达子房,在花粉管内的精细胞DNA含量为1C。当花粉管在退化助细胞中破裂,释放出的两个精细胞开始合成DNA。在与卵细胞融合前,两个精细胞DNA含量接近2C。随着精细胞的到达及合成DNA,卵细胞也开始合成DNA,融合前的卵细胞DNA含量也接近2C。精、卵细胞融合后,合子DNA含量为4C。烟草雌、雄配子是在细胞周期的G2期发生融合,属于G2型。     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

Changing patterns of nucleic acids, basic and acidic proteins in generative nuclei during pollen germination and pollen tube growth in Hippeastrum belladona

Generative and vegetative nuclei of mature and germinated pollen grains from Hippeastrum belladonna were separated in a continuous Ficoll gradient. Less than 3% contamination was observed between the generative and vegetative nuclear fractions. The vegetative nuclei were composed of two populations; the larger population consisted of nuclei with 1C levels of DNA and the smaller with 2C levels. The generative nuclei consisted of a homogeneous population composed of nuclei possessing 2C levels of DNA. Histone synthesis did not occur in vegetative nuclei. Changes appeared in the gel-electrophoretic banding patterns of the F1 histones of vegetative nuclei during germination. Changes were not observed in the generative nuclei. A reduction of general proteins and RNA was observed in vegetative nuclei by 20 h of germination. The phenol-soluble nuclear proteins of vegetative nuclei revealed transitions in electrophoretic banding patterns during pollen germination that were greater than those shown by the histones. These changes in the PSNP primarily involved reduced concentrations of certain proteins rather than synthesis of new ones. However, a new band was observed in the electrophoretic pattern of the PSNP of vegetative nuclei after 12 h of pollen tube growth. No transition was seen in the PSNP of generative nuclei during pollen germination and tube growth. The regulatory role of the PSNP in cell differentiation is discussed in the light of these findings.     

烟草精细胞两个群体的分离

高等植物的倾向受精是一个非常吸引人的研究课题,目前对其机理还不清楚。要想探索高等植物倾向受精现象,前提之一是要分离出一定数量的两个精细胞群体作为分子生物学研究方法的材料。以前的研究表明, 烟草(Nicotiana tabacum L.)花粉管中的两个精细胞体积差异明显。这种异型性的精细胞可能与倾向受精有关。烟草是二胞型花粉,生殖细胞只在体内生长的花粉管中才分裂形成两个精细胞。用体内/体外技术培养出花粉管后,爆破花粉管即可释放出花粉管内含物,其中包括两个精细胞。用微量酶液可使两个精细胞分开。然后用显微操作器可挑选出两个大小不同、数量上千的精细胞群体。这种单一纯化的精细胞群体为用分子生物学方法区分两个精细胞的DNA和蛋白质差异打下基础。本研究是高等植物的第二例、二胞花粉植物中的第一例分离两个特定精细胞群体的尝试,为构建烟草两个精细胞的cDNA文库创造了条件。     

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.     

Semi in vivo pollen tube growth of Aechmea fasciata

With semi in vivo pollen tube growth assays, stigmas are pollinated in vivo and, after a fixed time interval, the styles are isolated from the ovary and placed on culture medium in vitro. Semi in vitro pollination includes isolation of the stigma and style complex, followed by pollination and placing the stylar end on nutrient medium. After semi in vivo pollination more and longer pollen tubes protruded from the cut end of the styles into medium, in comparison to semi in vitro pollination. Medium with 3 g l^–1 agar was better than that with 6 g l^–1 agar for pollen tube growth after the tubes emerged from the cut style. Semi in vitro pollination of the reversed style indicated that pollen tube growth was not influenced by the direction of the style. Fructose and glucose inhibited pollen tube growth compared to sucrose. Swollen tips characterized tube growth inhibition. After semi in vivo pollination all generative nuclei had divided to give two sperm nuclei. The average distance between the last sperm nucleus and the pollen tube tip as well as the distance between the two sperm nuclei diminished in growing pollen tubes between 24 and 48 h after pollination. The arrangements between the vegetative and the generative nuclei did not differ in semi in vivo and in vitro cultured pollen tubes of Aechmea fasciata. This information is important to explain why fertilization rate is low after placental pollination in comparison to placental grafted style pollination of Aechmea fasciata. The data may also contribute to the improvement of in vitro fertilization methods in Bromeliaceae and other higher plants.     

Untersuchungen zur Pollenentwicklung und Pollenschlauchbildung bei höheren Pflanzen

Zusammenfassung In reifen Pollenkörnern der beiden Sommergerstensorten Amsel und Wisa sowie der F₁-Pflanzen, die aus den Sorten-Kreuzungen Impala X Wisa und Union X Wisa hervorgegangen sind, wurde die DNS-Menge der Kerne cytophotometrisch bestimmt. Die Messungen wurden zugleich bei Spermakernen und vegetativen Kernen eines Pollenkorns vorgenommen. Außerdem wurde der DNS-Gehalt von Kernen von Wurzelspitzen-Zellen der Sorten Amsel und Wisa ermittelt.Amsel und Wisa unterscheiden sich signifikant im DNS-Gehalt der Kerne von Wurzelspitzen-Zellen.Die Befunde der Messungen des DNS-Gehalts von vegetativen und Sperma-Kernen bei vier Gerstenformen zeigen, daß zum Zeitpunkt der Anthese die DNS-Replikationsphase bei vegetativen und Sperma-Kernen noch nicht abgeschlossen ist. Der DNS-Gehalt vegetativer Kerne von Wisa ist signifikant niedriger als die entsprechenden Werte der übrigen drei Gerstenformen. Der Verlauf der DNS-Replikation erfolgt bei beiden Spermakernen synchron. Hingegen verläuft die DNS-Replikation bei vegetativen und Sperma-Kernen mit großer Wahrscheinlichkeit nicht gleichsinnig.Im Diskussionsteil wird erstens erläutert, daß bei allen bisher analysierten Pflanzenarten des zwei- oder dreikernigen Pollenkorn-Typs zum Zeitpunkt der Pollenreife die DNS-Replikation der generativen bzw. Sperma-Kerne eingesetzt hat, aber je nach Pflanzenart noch nicht beendet sein muß. Zum gleichen Zeitpunkt der Pollenkornentwicklung kann der vegetative Kern in Abhängigkeit von der Pflanzenart auf dem C-Niveau verharren, eine teilweise oder bereits abgeschlossene DNS-Replikation erfahren haben oder schon teilweise oder ganz degeneriert sein, ohne zuvor eine DNS-Replikation vollzogen zu haben. Zweitens wird in diesem Abschnitt diskutiert, daß mit großer Wahrscheinlichkeit im Ablauf der DNS-Replikation zwischen zwei- und dreikernigen Pollenkorn-Typen keine Unterschiede bestehen. Drittens wird die Hypothese vertreten, daß nur auf einem sehr frühen Stadium die normale Pollenkornentwicklung einschließlich des Ablaufs der DNS-Replikation insbesondere des vegetativen Kerns so abgewandelt werden kann, daß aus Pollenkörnern haploide Pflanzen erzeugt werden können.

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The development of pollen grains and formation of pollen tubes in higher plantsIII. DNA-replication of vegetative and sperm nuclei in mature pollen grains of barley

Summary The DNA-content of vegetative and sperm nuclei in mature pollen grains of the barley varieties Amsel and Wisa and the F₁-plants of crossings of the barley varieties Impala X Wisa and Union X Wisa was determined by cytophotometry. In addition, the DNA-content of nuclei of root tips of Amsel and Wisa was cytophotometrically measured.The DNA contents of the nuclei in root tips of Amsel and Wisa differed significantly.The data obtained from the measurements of the vegetative and sperm nuclei of the four types of barley show that DNA-replication continues in the nuclei of mature pollen grains. The DNA values of vegetative nuclei of Wisa are significantly lower than the values of Amsel and of the F₁ plants. The DNA values of the different nuclei indicate that DNA replication of both types of sperm nuclei is synchronous, whereas it probably is not synchronous in vegetative and sperm nuclei respectively.In the discussion it is pointed out that a survey of the literature shows that in all of the plant species having binucleate or trinucleate pollen DNA replication of generative and of sperm nuclei has started at the time of pollen grain maturation. Depending on the plant species, replication may or may not be completed in the mature pollen grain. At a given stage of development of the pollen grain the vegetative nucleus may be arrested at the C-stage, may have partially or completely finished its DNA replication or may be partially or completely degenerated without prior replication of DNA.In the second part of the discussion it is stated that the course of DNA replication is likely to be similar in binucleate and trinucleate pollen grains. Thirdly, the hypothesis is discussed that in order to get haploid plants from pollen grains, changes in the normal development of the pollen grain and in the pattern of DNA replication must occur at a very early stage of pollen grain development.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Angenommen durch F. Mechelke

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Mein Dank grit Herrn Prof. Dr. F. Mechelke fiir die Anregung zu diesen Untersuchungen sowie fiir die Unterstfitzung und die kritischen Diskussionen w/ihrend ihres Verlaufs und Fr/iulein H. Nagel fiir zuverl/issige technische Hilfe.     

Migration of sperm cells during pollen tube elongation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: behavior during transport,maturation and upon dissociation of male germ unit associations

The promoter sequence of sperm-expressed gene, PzIPT isolated from the S_vn (sperm associated with the vegetative nucleus) of Plumbago zeylanica, was fused to a green fluorescent protein (GFP) reporter sequence and transformed into Arabidopsis thaliana to better visualize the live behavior of angiosperm sperm cells. Angiosperm sperm cells are not independently motile, migrating in a unique cell-within-a-cell configuration within the pollen tube. Sperm cells occur in association with the vegetative nucleus forming a male germ unit (MGU). In Arabidopsis, GFP was expressed equally in both sperm cells and was observed using a spinning disk confocal microscope, which allowed long duration observation of cells without bleaching or visible laser radiation damage. Pollen activation is reflected by conspicuous movement of sperm and pollen cytoplasm. Upon pollen germination, sperm cells enter the forming tube and become oriented, typically with a sperm cytoplasmic projection leading the sperm cells in the MGU, which remains intact throughout normal pollen tube elongation. Maturational changes, including vacuolization, general rounding and entry into G2, were observed during in vitro culture. When MGUs were experimentally disrupted by mild temperature elevation, sperm cells no longer tracked the growth of the tube and separated from the MGU, providing critical direct evidence that the MGU is a functional unit required for sperm transmission.     

Division of the generative nucleus in cultured pollen tubes of the Bromeliaceae

Pollen germination, division of the generative nucleus and position of the generative nucleus in the pollen tube during in vitro germination were examined for six bromeliad cultivars. The influence of mixed amino acids (casein hydrolysate) and individual amino acids (Arg, Asn, Asp, Glu, Gly, Met, Phe, Orn, Tyr) were tested. Aechmea fasciata and A. chantinii pollen tubes showed more generative nuclear division in cultured pollen tubes than the other four cultivars tested. Casein hydrolysate did not stimulate generative nuclear division. In general arginine (1 mM) improved division of the Aechmea generative nucleus and to a lesser extent this of Vriesea `Christiane', Guzmania lingulata and Tillandsia cyanea. A concentration of 2 mM arginine reduced pollen tube growth of Aechmea. The vegetative nucleus was ahead of the generative nucleus in approximately 50% of the pollen tubes of all cultivars studied. In about 25% of the pollen tubes, the generative nucleus was ahead and in ±25% pollen tubes the vegetative and generative nuclei were joined together. The distance between the two generative nuclei and the distance from the generative nuclei to the pollen tube tip differed significantly for Aechmea fasciata and A. chantinii. The influence of different amino acids for Aechmea fasciata and A. chantinii varied with respect to pollen germination and generative nuclear division. Arg and Met improved nuclear division of both Aechmea cultivars. Pollen germination and sperm cell production were not linked. This information is important to ameliorate in vitro pollination methods used to overcome fertilization barriers in Bromeliaceae and other higher plants.     

Pollen vegetative cell-specific expression of Bra r 1: useful tool for observation of the vegetative nucleus and identification of transgenic pollen by nuclear-targeted GFP

Bra r 1 encodes a novel Ca²⁺-binding protein specifically expressed in pollen and is localized in cytoplasm of pollen and pollen tubes. In this study, we demonstrated the expression of green fluorescent protein (GFP) with a nuclear localization signal under the control of Bra r 1 promoter in tobacco pollen. A fluorescent signal was detected in the vegetative nucleus (VN) but not in generative and sperm cell nuclei, indicating pollen vegetative cell-specific expression of Bra r 1. The fluorescent signal in elongating pollen tubes was stronger than that in mature pollen, indicating that the expression of Bra r 1 was more activated during pollen tube growth. This result suggests that Bra r 1 protein might be necessary for pollen tube growth. The pattern of green fluorescence in the VN revealed that VN chromatin is dispersed during the mid-bicellular pollen stage and condensed at the mature stage. This suggests that the level of chromatin condensation might be linked with gene expression in pollen vegetative cells. We also found that the expression of GFP and its targeting of the VN have no detrimental effect on pollen maturation and pollen tube growth. Expression of GFP in pollen thus makes rapid non-destructive monitoring of transgenic pollen and pollen tubes possible. The GFP which moved into the VN was found to be a convenient tool for observation of the VN and could be useful as a selectable marker of transgenic pollen for the analysis of pollen-specific genes. Received: 6 December 2000 / Revision accepted: 20 March 2001     

烟草雌、雄配子DNA 含量变化

采用显微分光光度法测定了烟草( Nicotiana tabacum) 精细胞和卵细胞的DNA 含量。烟草是二胞花粉, 花粉萌发后生殖细胞在花粉管中分裂形成精细胞。授粉后45 h 花粉管到达子房, 在花粉管内的精细胞DNA 含量为1C。当花粉管在退化助细胞中破裂, 释放出的两个精细胞开始合成DNA。在与卵细胞融合前,两个精细胞DNA 含量接近2C。随着精细胞的到达及合成DNA, 卵细胞也开始合成DNA, 融合前的卵细胞DNA 含量也接近2C。精、卵细胞融合后, 合子DNA 含量为4C。烟草雌、雄配子是在细胞周期的G2 期发生融合, 属于G2 型。     

Microphotometric Determination of DNA Contents of Early Developmental Pollen Grains in Tobacco Anther Culture

The Feulgen-DNA contents of microspores, vegetative and generative nuclei of tobacco pollen grains in vivo and in anther culture have been determined by microphotometry. 1. The values of DNA content of vegetative and generative nuclei of the pollen grains selected at definite developmental stages vary between 1C and 2C levels, which coincide with the role of the dynamics of DNA in haploid cell cycle. This method applied in the study of androgenesis in anther culture is proved successful and valid. 2. By the cytomorphological investigation on androgenesis, the pollen embryoid in this experiment results from repeated divisions of the vegetative cell within the pollen grains. 3. In mature pollen grains of the same variety of tobacco in vivo, DNA replication has not occured in vegetative nuclei, in which the level of DNA remains in 1C. 4. In the cultured anthers after 8 days innoculation, 30% of the total pollen grains measured indicate that the vegetative nuclei have completed DNA replication and show 2C level. The pollen grains which have the potential to differentiate into the embryogenie pollen grains, may be distinguished from non-embryogenie ones by this method before any cytomorphological sign appears. The significance of this method in the study of the mechanism of androgenesis is discussed.     

Mitosis and Amitosis of Generative Cell Nuclei in Artificially Germinated Pollen Tubes in Zephyranthes candida(Lindl.)Herb.

This paper deals with the comportmem of the vegetative nucleus and its spatial association with the generative cell and sperm cells in the artificially germinated pollen tubes of Zephyranthes candida (Lindl.) Herb. before and after generative cell mitosis with the use of DNA-specific fluochrome 4′,6-diamidino-2-phenylindole (DAPI). The induction of amitosis and abnormal mitosis of generative cell nuclei by cold-pretreatment of the pollen prior to germination was studied in particular. In normal case, the generative cell, after appressing to the vegetative nucleus for certain time, underwent mitosis to form two sperms, while the vegetative nucleus became markedly elongated, diffused, and exhibited blurring of its fluorescence. After division, a pair of sperms remained shortly in close connexion with the vegetative nucleus. Then the vegetative nucleus returned to its original state. In the pollen tubes germinated from cold-pretreated pollen, amitosis of some generative cell nuclei were frequently observed. Amitosis took place via either equal or unequal division with a mode of constriction. During amitosis, the dynamic change of vegetative nucleus and its intimate association with generative cell afore described did not occur. Sperm nuclei produced from amitosis could farther undergo amitisis resulting in micronnclei. Factors affecting the amitosic rate of generative cells, such as pollen developmental stage, temperature and duration of cold-pretreatment, were studied. Besides amitosis, cold-pretreatment also induced some abnormal mitotic behavior leading to the formation of micronuclei. Based on our observations and previously reported facts in other plant materials, it is inferred that the vegetative nucleus plays an important role in normal mitosis of generative cell and development of sperms.