How to Make Glass Microtools for the Injection of Isolated Plant Sperm Cells into Embryo SAC Cells Using a Microforge

1993 ◽  
pp. 11-17
Author(s):  
C. J. Keijzer
Keyword(s):  
Embryo Sac ◽  
Sperm Cells

scholarly journals Calcium: A Critical Factor in Pollen Germination and Tube Elongation

2019 ◽  
Vol 20 (2) ◽  
pp. 420 ◽  
Author(s):  
Ren Zheng ◽  
Shun Su ◽  
Hui Xiao ◽  
Hui Tian
Keyword(s):  
Calcium Ion ◽  
Pollen Germination ◽  
Higher Plants ◽  
Male Gametophyte ◽  
Embryo Sac ◽  
Critical Factor ◽  
Sperm Cells ◽  
Critical Element ◽  
Major Function ◽  
Spatial And Temporal Characteristics

Pollen is the male gametophyte of higher plants. Its major function is to deliver sperm cells to the ovule to ensure successful fertilization. During this process, many interactions occur among pollen tubes and pistil cells and tissues, and calcium ion (Ca2+) dynamics mediate these interactions among cells to ensure that pollen reaches the embryo sac. Although the precise functions of Ca2+ dynamics in the cells are unknown, we can speculate about its roles on the basis of its spatial and temporal characteristics during these interactions. The results of many studies indicate that calcium is a critical element that is strongly related to pollen germination and pollen tube growth.

Immunofluorescent Localization of Myosin on the Sperm Cells of Plumbago Zeylanica

1997 ◽  
Vol 3 (S2) ◽  
pp. 183-184
Author(s):  
Zhaojie Zhang ◽  
Scott D. Russell
Keyword(s):  
Immunofluorescence Microscopy ◽  
Embryo Sac ◽  
Indirect Evidence ◽  
Sperm Cells ◽  
Immunofluorescent Localization ◽  
Plumbago Zeylanica ◽  
Actomyosin Interaction ◽  
Rabbit Igg ◽  
Sigma Chemical ◽  
Phosphate Buffered Saline

Sperm cells in flowering plants are non-motile and are passive participants in their movement to the female reproductive cells. It is believed that actomyosin interaction may play a key role for sperm cell transmission in the pollen tube as well as in the embryo sac. However, indirect evidence has shown that the surface of sperm cells lacks amounts of myosin sufficient to support movement. Immunofluorescence microscopy was used in this study to further assess the presence of myosin on the surface of sperm cells of Plumbago Zeylanica.Sperm cells of Plumbago Zeylanica were isolated according to published methods. Isolated sperm cells were blocked 20 min with 1% bovine serum albumin and 2% normal goat serum in phosphate buffered saline (PBS) (pH 7.3, in 15% sucrose), incubated 2 hrs in anti-myosin antibody (M-7648, Sigma Chemical Co., St. Louis, MO) diluted 1:10 with blocking solution, washed three times (5 min) with blocking solution and incubated 1 hr in 1:30 FITC-conjugated anti-rabbit IgG as secondary antibody (EY Labs, Inc., San Mateo, CA) in blocking solution. Samples were rinsed in PBS and mounted in an anti-fading solution with 1:1 PBS:glycerol with 3% n-propyl gallate.

scholarly journals Isolation of living sperm cells and in vitro fusion of Torenia fournieri gametes

2006 ◽  
Vol 12 (4) ◽  
Author(s):  
P. Vági ◽  
K. Imre ◽  
Z. Kristóf
Keyword(s):  
Male Gametophyte ◽  
Embryo Sac ◽  
Pollen Tubes ◽  
Sperm Cells ◽  
Torenia Fournieri ◽  
Isolation Medium ◽  
Vitro Fertilization ◽  
Crucial Part

In contrast to most angiosperms, Torenia contains a naked embryo sac and therefore has been considered since many years as an exciting model plant to study the double fertilization process of flowering seed plants. It is thus not surprising that the isolation of protoplasts from the female gametophyte has been reported already 20 years ago by Mol, the isolation of megaspores and megagametophytes has been published by the authors of this manuscript in 1996 and in 1999. The isolation of the male gametophyte and of sperm cells was published by the authors in 2004. The isolation of viable Torenia sperm cells is a crucial part of the elaboration of an in vitro fertilization system. Torenia sperm cells were isolated from in vivo — in vitro cultured pollen tubes. In this system pollen tubes first grow inside a cut style then follow their elongation in a solid isolation medium. The medium contained agarose in order to detain pollen tube contents. Released sperm cells and enzymatically isolated egg cells were collected and handled using glass micropipettes and transmitted to an electrofusion apparatus or polyethylene glycol containing media for fusion probes.

Cytoskeletal organisation and modification during pollen tube arrival, gamete delivery and fertilisation in Plumbago zeylanica

Zygote ◽  
1993 ◽  
Vol 1 (2) ◽  
pp. 143-154 ◽  
Author(s):  
Bing-Quan Huang ◽  
Elisabeth S. Pierson ◽  
Scott D. Russell ◽  
Antonio Tiezzi ◽  
Mauro Cresti
Keyword(s):  
Pollen Tube ◽  
Embryo Sac ◽  
Central Cell ◽  
Egg Cell ◽  
Target Cells ◽  
Sperm Cells ◽  
Plumbago Zeylanica ◽  
Rhodamine Phalloidin ◽  
Male Gametes ◽  
Pollen Tube Penetration

The cytoskeletal organisation of the isolated embryo sac and egg cells of Plumbago zeylanica was examined before, during and after pollen tube penetration into the embryo sac to determine the potential involvement of microtubules and actin filaments in fertilisation. Material was singly and triply stained using Hoechst 33258 to localise DNA, fluorescein isothiocyanate (FITC)-labelled anti- α-tubulin to detect microtubules and rhodamine-phalloidin to visualise F-actin. Microtubules in the unfertilised egg cell are longitudinally aligned in the micropylar and mid-lateral areas, aggregating into bundles near the filiform apparatus. In the perinuclear cytoplasm of the egg cell, microtubules become more or less randomly aligned. F-actin bundles form a longitudinally aligned mesh in the chalazal cytoplasm of the egg cell. In the central cell, microtubules and F-actin are distributed along transvacuolar strands and are also evident in the perinuclear region and at the periphery of the cell. During pollen tube penetration, sparse microtubule bundles near the pathway of the pollen tube may form an apparent microtubular ‘conduit’ surrounding the male gametes at the delivery site. Actin aggregates become organised near the pathway of the pollen tube and at the delivery site of the sperm cells. Subsequently, actin aggregates form a ‘corona’ structure in the intercellular region between the egg and central cell where gametic fusion occurs. The corona may have a role in maintaining the close proximity of the egg and central cell and helping the two sperm cells move and bind to their target cells. The cytoskeleton may also be involved in causing the two nuclei of the egg and central cell to approach one another at the site of gametic fusion and transporting the two sperm nuclei into alignment with their respective female nucleus. The cytoskeleton is reorganised during early embryogenesis.

Microtubule-organelle associations during generative cell polarization in plumbago zeylanica

1986 ◽  
Vol 44 ◽  
pp. 280-281
Author(s):  
S. D. Russell ◽  
T. W. Mislan
Keyword(s):  
Sexual Reproduction ◽  
Sperm Cell ◽  
Generative Cell ◽  
Embryo Sac ◽  
Cell Polarization ◽  
Precursor Cell ◽  
Flowering Plant ◽  
Sperm Cells ◽  
Plumbago Zeylanica ◽  
Preferential Fertilization

Sexual reproduction in the flowering plant Plumbago zeylanica is characterized by pollen in which the two sperm cells differ significantly with respect to size, shape and organellar content. The sperm cell with the majority of mitochondria (ave. 256) typically fuses with the central cell to form the nutritive endosperm in which the embryo will develop. The sperm cell containing essentially all of the numerous plastids (ave. 24) and few mitochondria (ave. 40) fuses with the egg to form the embryo. This system of preferential fertilization results in each sperm cell having a predetermined fate upon arrival at the embryo sac. Features determining the distribution of organelles in the sperm cells originate in the polarity of their precursor cell, the generative cell.

scholarly journals Isolation of Sperm and Egg Cells from Pepper

2018 ◽  
Vol 143 (4) ◽  
pp. 310-315 ◽  
Author(s):  
Wei Deng ◽  
Yunling Xie ◽  
Yilan Qiu
Keyword(s):  
Generative Cell ◽  
Embryo Sac ◽  
Pollen Tubes ◽  
Enzymatic Digestion ◽  
Sperm Cells ◽  
Free Solution ◽  
In Vitro Method ◽  
Viable Embryo

Pepper (Capsicum annuum) pollen is bicellular and contains a vegetative cell and a generative cell, which divides in pollen tubes to form two sperm cells. Sperm cells of pepper were isolated using an in vivo–in vitro method. Hand-pollinated styles were first grown in vivo for several hours, then cut from their base and cultured in vitro until pollen tubes grew from the cut end. When the pollen tubes were transferred to a breaking solution, sperm cells were released from broken tubes. Viable embryo sac cells of pepper were isolated using enzymatic digestion and mechanical dissection. Isolated ovules were digested using cellulase and pectinase for 40 minutes and then transferred to an enzyme-free solution for mechanical dissection. Three cells of the egg apparatus and a central cell were released from a cut at the chalazal end of each ovule by pressing on the micropylar area of the ovule with a microneedle. Optimal isolation conditions included 11% mannitol, 0.04% CaCl2, 1% bovine serum albumin (BSA), 1% cellulase, 1% pectinase, and 0.3% pectolyase. Using this protocol, populations of pepper egg cells, synergids, and central cells were isolated.

Ultrastructure of Testicular Spermatozoon of the Fish Oreochromis Niloticus

1988 ◽  
Vol 46 ◽  
pp. 278-279
Author(s):  
T. Guha ◽  
A. Q. Siddiqui ◽  
P. F. Prentis
Keyword(s):  
Electron Microscope ◽  
Oreochromis Niloticus ◽  
Osmium Tetroxide ◽  
Sperm Cells ◽  
Adult Fish ◽  
Testicular Sperm ◽  
Thin Sections ◽  
Important Fish ◽  
Testicular Spermatozoon ◽  
Diamond Knife

Tilapia, Oreochromis niloticus, is an economically important fish in Saudi Arabia. Elucidation of reproductive biology of this species is necessary for successful breeding program. In this paper we describe fine structure of testicular sperm cells in O, niloticus.Testes from young adult fish were fixed in gluteraldehyde (2%) and osmium tetroxide (1%), both in cacodyl ate buffer. Specimens were processed in the conventional way for electron microscopy and thin sections of tissues (obtained by cutting the blocks with a diamond knife) were stained by ura- nyl acetate and lead citrate. These were examined in a Carl Zeiss electron microscope operated at 40 kV to 60 kV. Sperm cells were obtained from testes by squeezing them in cacodyl ate buffer. They were fixed in gluteraldehyde (2%) in the same buffer, air dried, gold coated and then examined in a Philips scanning electron microscope (SEM) operated at 25kV.The spermatozoon of O. niloticus is consisting of head, midpiece and tail (Fig. 1).

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