Isolation of male and female gametes, zygotes and proembryos of leek (Allium tuberosum Roxb)

SummaryThe isolation of male and female gametes is an effective method to study the fertilization mechanisms of higher plants. An osmotic shock method was used to rupture pollen grains of Allium tuberosum Roxb and release the pollen contents, including generative cells, which were mass collected. The pollinated styles were cut following 3 h of in vivo growth, and cultured in medium for 6–8 h, during which time pollen tubes grew out of the cut end of the style. After pollen tubes were transferred into a solution containing 6% mannitol, tubes burst and released pairs of sperm cells. Ovules of A. tuberosum were incubated in an enzyme solution for 30 min, and then dissected to remove the integuments. Following transfer to a dissecting solution free of enzymes, each nucellus was cut in the middle, and squeezed gently on the micropylar end, resulting in the liberation of the egg, zygote and proembryo from ovules at selected stages. These cells can be used to explore fertilization and embryonic development using molecular biological methods for each cell type and development stage.

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Some permeability properties of angiosperm pollen grains, pollen tubes and generative cells

Sexual Plant Reproduction ◽

10.1007/bf00189264 ◽

1988 ◽

Vol 1 (2) ◽

Cited By ~ 1

Author(s):

J. Heslop-Harrison ◽

Y. Heslop-Harrison

Keyword(s):

Pollen Grains ◽

Pollen Tubes ◽

Angiosperm Pollen ◽

Generative Cells

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Embryological studies of reciprocal crosses between Vicia faba and Vicia narbonensis

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1998.004 ◽

2014 ◽

Vol 67 (1) ◽

pp. 37-43 ◽

Cited By ~ 4

Author(s):

Maciej Zenkteler ◽

Mechtild Tegeder ◽

Otto Schieder

Keyword(s):

Vicia Faba ◽

Pollen Grains ◽

Pollen Tubes ◽

Flower Buds ◽

Reciprocal Crosses ◽

Globular Embryos ◽

Vicia Narbonensis ◽

Whole Number

A study was undertaken to asses the reciprocal crossability between Vicia faba and Vicia narbonensis. Flower buds or only ovaries of several varietes and genotypes were cross-pollinated in vivo (green house and field) and in vitro. Only few pollen tubes passed the style and entered into the ovary. On the whole number of 5320 cross pollinated in vivo and in vitro flowers and ovaries of Vicia narbonensis only 78 globular hybrid embryos were observed. After cross pollination in vivo of 3860 flower buds and ovaries of Vicia faba globular embryos developed in 124 ovules. The highest number of globular embryos were obtained when the Vicia faba line 1/33 was pollinated with Vicia narbonensis lines P3, P5, 150, SE.Embryogenesis proceeded till the 6-10 day after pollination, however, karyological disturbances in the cells of embryos and endosperm were often noticed at earlier stages. In vitro pollen grains of Vicia faba germinated on stigmas and ovaries of Vicia narbonensis, a significant increase in the growth of pollen tubes was noticed after ovary pollination. The technique of in vitro pollination was not suitable for Vicia faba as the inoculated explants died shortly after transferring onto the medium. The results indicate that finding a more suitable genotype for crossing may give a chance to obtain higher number of embryos (example line 1/33) - thus sufficient number for culturing them on media.

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Myosin associated with the surfaces of organelles, vegetative nuclei and generative cells in angiosperm pollen grains and tubes

Journal of Cell Science ◽

10.1242/jcs.94.2.319 ◽

1989 ◽

Vol 94 (2) ◽

pp. 319-325

Author(s):

J. HESLOP-HARRISON ◽

Y. HESLOP-HARRISON

Keyword(s):

Pollen Tube ◽

Pollen Grains ◽

Giant Cells ◽

Pollen Tubes ◽

Microfilament Bundles ◽

Hyacinthus Orientalis ◽

Actin Fibrils ◽

Angiosperm Pollen ◽

Muscle Myosin ◽

Generative Cells

Myosin, detected by immunofluorescence using an antibody to bovine skeletal and smooth muscle myosin, has been localised on individual identifiable organelles from the grasses Alopecurus pratensis and Secale cereale, and on the surfaces of vegetative nuclei and generative cells from pollen and pollen tubes of Hyacinthus orientalis and Helleborus foetidus. Taken in conjunction with recent evidence showing that the growing pollen tube contains an actin cytoskeleton consisting of numerous mainly longitudinally oriented microfilament bundles, and that isolated pollen-tube organelles show ATP-dependent movement along the actin bundles of the giant cells of the characeous algae, this finding suggests that an actomyosin motility system is present in pollen tubes, and indicates that the movements of the different classes of inclusions are driven by interaction of the surface myosin with the actin fibrils at the zones of contact.

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Characterization of self-incompatibility in Campanula rapunculoides (Campanulaceae) through genetic analyses and microscopy

Botany ◽

10.1139/b07-100 ◽

2008 ◽

Vol 86 (1) ◽

pp. 1-13 ◽

Cited By ~ 8

Author(s):

S. V. Good-Avila ◽

D. Majumder ◽

H. Amos ◽

A. G. Stephenson

Keyword(s):

Genetic Basis ◽

Male Gametophyte ◽

Single Gene ◽

Pollen Grains ◽

Diallel Analysis ◽

Pollen Tubes ◽

Self Incompatibility ◽

The Family

In this paper, we seek to identify the genetic basis of self-incompatibility (SI) in Campanula rapunculoides L. through diallel analysis of full siblings; to characterize the growth of pollen tubes in vivo after incompatible and compatible pollination; and to determine whether the SI system is based on pistil S-RNases. Pollinations were performed among individuals from five diallel crosses and scored for both fruit set and pollen-tube growth to determine the genetic basis of SI. On a subset of these individuals with known cross-(in)compatibility relationships, additional crosses were performed and pistils collected 1, 3, 6, 12, and 24 h after pollination to assess both the percentage of pollen grains that had germinated on the stigma, and the number of pollen tubes that had grown 20%, 40% 60%, 80%, and 100% of the distance down the pistil over five time intervals. Finally, total pistillate proteins were extracted and subjected to isoelectric focusing and RNase activity staining to find evidence of a highly basic S-RNases associated with SI in the Solanaceae. We found evidence that the SI system was based on the haplotype of the male gametophyte, and was not sporophytic. Protein analyses showed that SI was not based on a pistillate S-RNase. The existence of modifiers of SI and possible polyploidy at the S-locus complicated the expression of SI in this species, and single-gene inheritance could not be determined. This represents the first published characterization of incompatibility in the family Campanulaceae.

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Pollen tube growth of Paeonia tenuifolia L. (Paeoniaceae) in vitro and in vivo

Bangladesh Journal of Botany ◽

10.3329/bjb.v40i1.8003 ◽

1970 ◽

Vol 40 (1) ◽

pp. 93-95 ◽

Cited By ~ 1

Author(s):

Feruzan Dane ◽

Nuran Ekici

Keyword(s):

Pollen Tube ◽

Pollen Germination ◽

Pollen Viability ◽

Pollen Grains ◽

Pollen Tubes ◽

In Vivo Studies ◽

In Vivo Growth ◽

Paeonia Tenuifolia

In vitro and in vivo studies on pollen germination of Paeonia tenuifolia L. (Paeoniaceae) revealed that pollen grains are shed at two-celled stage. Normal and abnormal pollens were observed. Pollen viability was recorded between 55 and 75%. In vitro studies revealed 85% germination and usually the germination was monosphonic. Some pollen tubes with swollen tube tip and undulations were found. Styles and stigma were found to contain many pollen tubes 24 hrs after pollination. Key words: Paeonia tenuifolia; Pollen tube; In vitro growth; In vivo growth DOI: http://dx.doi.org/10.3329/bjb.v40i1.8003 Bangladesh J. Bot. 40(1): 93-95, 2011 (June)

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Expression of the cell cycle in sperm of Arabidopsis: implications for understanding patterns of gametogenesis and fertilization in plants and other eukaryotes

Development ◽

10.1242/dev.126.5.1065 ◽

1999 ◽

Vol 126 (5) ◽

pp. 1065-1075 ◽

Cited By ~ 2

Author(s):

W.E. Friedman

Keyword(s):

Cell Cycle ◽

Higher Plants ◽

Generative Cell ◽

Pollen Grains ◽

Pollen Tubes ◽

Flowering Plants ◽

S Phase ◽

Flowering Plant ◽

Sperm Nuclei

The relationship between developmental events and the cell cycle was examined in sperm of Arabidopsis thaliana. Sperm of Arabidopsis rapidly enter the S (synthesis) phase of the cell cycle after inception from mitosis of the generative cell. Sperm in pollen grains within anthers continue to synthesize DNA, and at the time of pollination, contain approximately 1.5C DNA. Following pollination, sperm continue through the S phase of the cell cycle during pollen tube growth. By the time pollen tubes reach the ovary, sperm nuclei contain approximately 1.75C DNA. Just prior to double fertilization, sperm nuclei within embryo sacs contain the 2C quantity of DNA. These data indicate that molecular programs associated with the G1-S transition and the S phase of the cell cycle are expressed in sperm cells of developing pollen grains and pollen tubes in Arabidopsis. This pattern of prefertilization S phase activity in the sperm of a flowering plant stands in marked contrast to all other non-plant eukaryotes (from ciliates to yeast to sea urchins to mammals) where sperm remain in G1 during development, prior to the initiation of gametic fusion. In addition, when patterns of cell cycle activity in sperm of Arabidopsis and other flowering plants are compared, developmental analysis reveals that heterochronic alterations (changes in the relative timing of ontogenetic events) in cell cycle activity are a central cause of the diversification of patterns of gametogenesis in higher plants. Finally, comparative analysis of the patterns of cell cycle activity in Arabidopsis and other angiosperms may be used to predict which flowering plants will be amenable to development of successful in vitro fertilization techniques.

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In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083035 ◽

1978 ◽

Vol 36 (2) ◽

pp. 434-435

Author(s):

D. Reis ◽

B. Vian ◽

J. C. Roland

Keyword(s):

Cell Wall ◽

Self Assembly ◽

Plant Cell Wall ◽

Higher Plants ◽

Three Dimensional ◽

Cytochemical Study ◽

Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

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