scholarly journals Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

2021 ◽  
Author(s):  
Shiori Nagahara ◽  
Tetsuya Higashiyama ◽  
Yoko Mizuta
Keyword(s):  
Pollen Tube ◽  
Germ Cells ◽  
Plasmid Dna ◽  
Genetically Modify ◽  
Pollen Tubes ◽  
Limited Range ◽  
Negative Control ◽  
Male Germ Cells ◽  
Fluorescent Reporter ◽  
Biolistic Delivery

AbstractIn recent years, genome-editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding.Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method is generally applicable to a limited range of plants, such as model species. To overcome this limitation, we developed a method to genetically modify male germ cells without the need forAgrobacterium-mediated transfection and tissue culture, by using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, theArabidopsis UBQ10promoter was found to be the most suitable for driving expression of the delivered gene in pollen tubes. We also evaluated delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9 introduced pollen tubes but were not detected in the negative control. Bombarded pollen germinated pollen tubes on the stigma and produced two sperm cells within the pistil. We also observed ovules showing fluorescence derived from bombarded pollen. The findings of this study provide important insights into the editing of pollen tube genomes and the delivery of genome-modified male germ cells for seed production.

scholarly journals Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

2021 ◽  
Author(s):  
Shiori Nagahara ◽  
Tetsuya Higashiyama ◽  
Yoko Mizuta
Keyword(s):  
Tissue Culture ◽  
Germ Cells ◽  
Plasmid Dna ◽  
Genetically Modify ◽  
Pollen Tubes ◽  
Negative Control ◽  
Fluorescent Reporter ◽  
Biolistic Delivery ◽  
Delivery Efficiency

Abstract Key message Biolistic delivery into pollen. Abstract In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method has general limitations, such as the limited host range of Agrobacterium and difficulties in tissue culture, including callus induction and regeneration. To avoid these issues, we developed a method to genetically modify germ cells without the need for Agrobacterium-mediated transfection and tissue culture using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable promoter for driving the expression of the delivered gene in pollen tubes. We also evaluated the delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9-introduced pollen tubes, but were not detected in the negative control. Bombarded pollen germinated pollen tubes and delivered their contents into the ovules in vivo. Although it is necessary to improve biolistic delivery efficiency and establish a method for the screening of genome-modified seeds, our findings provide important insights for the detection and production of genome-modified seeds by pollen biolistic delivery.

scholarly journals Protein Analysis of Pollen Tubes after the Treatments of Membrane Trafficking Inhibitors Gains Insights on Molecular Mechanism Underlying Pollen Tube Polar Growth

2021 ◽  
Vol 40 (2) ◽  
pp. 205-222
Author(s):  
Monica Scali ◽  
Alessandra Moscatelli ◽  
Luca Bini ◽  
Elisabetta Onelli ◽  
Rita Vignani ◽  
...  
Keyword(s):  
Pollen Tube ◽  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Tip Growth ◽  
Male Gametophyte ◽  
Pollen Tubes ◽  
Structural Features ◽  
Two Dimensional Gel Electrophoresis ◽  
Depth Analysis

AbstractPollen tube elongation is characterized by a highly-polarized tip growth process dependent on an efficient vesicular transport system and largely mobilized by actin cytoskeleton. Pollen tubes are an ideal model system to study exocytosis, endocytosis, membrane recycling, and signaling network coordinating cellular processes, structural organization and vesicular trafficking activities required for tip growth. Proteomic analysis was applied to identifyNicotiana tabacumDifferentially Abundant Proteins (DAPs) after in vitro pollen tube treatment with membrane trafficking inhibitors Brefeldin A, Ikarugamycin and Wortmannin. Among roughly 360 proteins separated in two-dimensional gel electrophoresis, a total of 40 spots visibly changing between treated and control samples were identified by MALDI-TOF MS and LC–ESI–MS/MS analysis. The identified proteins were classified according to biological processes, and most proteins were related to pollen tube energy metabolism, including ammino acid synthesis and lipid metabolism, structural features of pollen tube growth as well modification and actin cytoskeleton organization, stress response, and protein degradation. In-depth analysis of proteins corresponding to energy-related pathways revealed the male gametophyte to be a reliable model of energy reservoir and dynamics.

scholarly journals NANOS2 suppresses the cell cycle by repressing mTORC1 activators in embryonic male germ cells

iScience ◽  
2021 ◽  
pp. 102890
Author(s):  
Ryuki Shimada ◽  
Hiroko Koike ◽  
Takamasa Hirano ◽  
Yuzuru Kato ◽  
Yumiko Saga
Keyword(s):  
Cell Cycle ◽  
Germ Cells ◽  
Male Germ Cells

scholarly journals Hyperoside promotes pollen tube growth by regulating the depolymerization effect of actin-depolymerizing factor 1 on microfilaments in okra

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Biying Dong ◽  
Qing Yang ◽  
Zhihua Song ◽  
Lili Niu ◽  
Hongyan Cao ◽  
...  
Keyword(s):  
Pollen Tube ◽  
Pollen Tube Growth ◽  
Pollen Germination ◽  
Pollen Tubes ◽  
Tube Growth ◽  
Actin Depolymerizing Factor ◽  
Specific Mechanism ◽  
Promoting Effect ◽  
New Research

AbstractMature pollen germinates rapidly on the stigma, extending its pollen tube to deliver sperm cells to the ovule for fertilization. The success of this process is an important factor that limits output. The flavonoid content increased significantly during pollen germination and pollen tube growth, which suggests it may play an important role in these processes. However, the specific mechanism of this involvement has been little researched. Our previous research found that hyperoside can prolong the flowering period of Abelmoschus esculentus (okra), but its specific mechanism is still unclear. Therefore, in this study, we focused on the effect of hyperoside in regulating the actin-depolymerizing factor (ADF), which further affects the germination and growth of pollen. We found that hyperoside can prolong the effective pollination period of okra by 2–3-fold and promote the growth of pollen tubes in the style. Then, we used Nicotiana benthamiana cells as a research system and found that hyperoside accelerates the depolymerization of intercellular microfilaments. Hyperoside can promote pollen germination and pollen tube elongation in vitro. Moreover, AeADF1 was identified out of all AeADF genes as being highly expressed in pollen tubes in response to hyperoside. In addition, hyperoside promoted AeADF1-mediated microfilament dissipation according to microfilament severing experiments in vitro. In the pollen tube, the gene expression of AeADF1 was reduced to 1/5 by oligonucleotide transfection. The decrease in the expression level of AeADF1 partially reduced the promoting effect of hyperoside on pollen germination and pollen tube growth. This research provides new research directions for flavonoids in reproductive development.

scholarly journals Testing of self-(in)compatibility in apricot cultivars from European breeding programmes

2013 ◽  
Vol 40 (No. 2) ◽  
pp. 65-71 ◽  
Author(s):  
D. Milatović ◽  
D. Nikolić ◽  
B. Krška
Keyword(s):  
Fluorescence Microscopy ◽  
Pollen Tube ◽  
Pollen Tube Growth ◽  
Reliable Method ◽  
Pollen Tubes ◽  
Tube Growth ◽  
Self Incompatibility ◽  
Breeding Programmes

Self-(in)compatibility was tested in 40 new apricot cultivars from European breeding programmes. Pollen-tube growth in pistils from laboratory pollinations was analysed using the fluorescence microscopy. Cultivars were considered self-compatible if at least one pollen tube reached the ovary in the majority of pistils. Cultivars were considered self- incompatible if the growth of pollen tubes in the style stopped along with formation of characteristic swellings. Of the examined cultivars, 18 were self-compatible and 22 were self-incompatible. Fluorescence microscopy provides a relatively rapid and reliable method to determine self-incompatibility in apricot cultivars.      

scholarly journals Persistent directional growth capability in Arabidopsis thaliana pollen tubes after nuclear elimination from the apex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kazuki Motomura ◽  
Hidenori Takeuchi ◽  
Michitaka Notaguchi ◽  
Haruna Tsuchi ◽  
Atsushi Takeda ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Pollen Tube ◽  
Sperm Cell ◽  
Pollen Tubes ◽  
Vegetative Nucleus ◽  
Sperm Cells ◽  
Apical Region ◽  
Basal Region ◽  
Specific Expression ◽  
Directional Growth

AbstractDuring the double fertilization process, pollen tubes deliver two sperm cells to an ovule containing the female gametes. In the pollen tube, the vegetative nucleus and sperm cells move together to the apical region where the vegetative nucleus is thought to play a crucial role in controlling the direction and growth of the pollen tube. Here, we report the generation of pollen tubes in Arabidopsis thaliana whose vegetative nucleus and sperm cells are isolated and sealed by callose plugs in the basal region due to apical transport defects induced by mutations in the WPP domain-interacting tail-anchored proteins (WITs) and sperm cell-specific expression of a dominant mutant of the CALLOSE SYNTHASE 3 protein. Through pollen-tube guidance assays, we show that the physiologically anuclear mutant pollen tubes maintain the ability to grow and enter ovules. Our findings provide insight into the sperm cell delivery mechanism and illustrate the independence of the tip-localized vegetative nucleus from directional growth control of the pollen tube.

scholarly journals NETWORKED2‐Subfamily Proteins Regulate the Cortical Actin Cytoskeleton of Growing Pollen Tubes and Polarised Pollen Tube Growth

2021 ◽  
Author(s):  
Patrick Duckney ◽  
Johan T. Kroon ◽  
Martin R. Dixon ◽  
Timothy J. Hawkins ◽  
Michael J. Deeks ◽  
...  
Keyword(s):  
Pollen Tube ◽  
Actin Cytoskeleton ◽  
Pollen Tube Growth ◽  
Pollen Tubes ◽  
Tube Growth ◽  
Cortical Actin

scholarly journals ELECTRON MICROSCOPE STUDIES ON THE DICTYOSOMES AND ACROBLASTS IN THE MALE GERM CELLS OF THE CRICKET

1956 ◽  
Vol 2 (4) ◽  
pp. 123-128 ◽  
Author(s):  
H. W. Beams ◽  
T. N. Tahmisian ◽  
R. L. Devine ◽  
Everett Anderson
Keyword(s):  
Electron Microscope ◽  
Golgi Apparatus ◽  
Electron Micrographs ◽  
Germ Cells ◽  
Light Microscope ◽  
Golgi Body ◽  
Duplex Structure ◽  
Male Germ Cells ◽  
Secondary Spermatocyte ◽  
A Chain

The dictyosome (Golgi body) in the secondary spermatocyte of the cricket appears in electron micrographs as a duplex structure composed of (a) a group of parallel double-membraned lamellae and (b) a group of associated vacuoles arranged along the compact lamellae in a chain-like fashion. This arrangement of ultramicroscopic structure for the dictyosomes is strikingly comparable to that described for the Golgi apparatus of vertebrates. Accordingly, the two are considered homologous structures. Associated with the duplex structure of the dictyosomes is a differentiated region composed of small vacuoles. This is thought to represent the pro-acrosome region described in light microscope preparations. In the spermatid the dictyosomes fuse, giving rise to the acroblast. Like the dictyosomes, the acroblasts are made up of double-membraned lamellae and associated vacuoles. In addition, a differentiated acrosome region is present which, in some preparations, may display the acrosome vacuole and granule. Both the dictyosomes and acroblasts are distinct from mitochondria.

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