This research was funded by Ministerio de Ciencia, Innovaci&#243;n y Universidades-European Regional Development Fund, European Union (AGL2016-77267-R, and AGL2015-74071-JIN); Instituto Nacional de Investigaci&#243;n y Tecnolog&#237;a Agraria y Alimentaria (RFP2015-00015-00, RTA2017-00003-00); Gobierno de Arag&#243;n-European Social Fund, European Union (Grupo Consolidado A12_17R), Fundaci&#243;n Biodiversidad, and Agroseguro S.A.

1. Self-(in)compatibility determination
<ol>
	<li>Sample the flowers in the field. It is necessary to collect the flowers at balloon stage (Figure 1A), corresponding to stage 58 on the BBCH scale for apricot28, to avoid unwanted previous pollination.</li>
	<li>Self- and cross-pollinations in the laboratory
	<ol>
		<li>Remove the anthers of the flowers at balloon stage and place them on a piece of paper to dry at laboratory temperature.</li>
		<li>After 24 h, sieve the pollen grains by using a fine mesh (0.26 mm) (Figure 1B).</li>
		<li>Emasculate a group of 30 flowers at the same balloon stage for each self-pollination and cross-pollination and place the pistils on florist foam in water at laboratory temperature (Figure 1C).</li>
		<li>Hand pollinate the pistils with the help of a paintbrush with pollen from flowers of the same cultivar 24 h after emasculation. In addition, pollinate another set of pistils of each cultivar with pollen from flowers of a compatible pollinizer as control (Figure 1D).</li>
		<li>After 72 h, fix the pistils in a fixative solution of ethanol/acetic acid (3:1) for at least 24 h at 4 &#176;C29. Then discard the fixative and add 75% ethanol ensuring that the samples are completely submerged in the solution. Samples can be conserved in this solution at 4 &#176;C until use8,17,30,31,32.</li>
	</ol>
	</li>
	<li>Evaluating pollen viability through in vitro pollen germination
	<ol>
		<li>To prepare the germination medium, weight 25 g of sucrose, 0.075 g of boric acid (H3BO3) and 0.075 g of calcium nitrate (Ca(NO3)2)33.</li>
		<li>Add the components of the medium in 250 mL of distilled water and dissolve completely.</li>
		<li>Solidify the medium adding 2 g of agarose and mix by swirling.</li>
		<li>Check the pH of the medium using a pH meter and adjust the value to 7.0 with NaOH or HCl solution.</li>
		<li>Autoclave the mixture to sterilize the medium.</li>
		<li>After autoclaving, cool down the medium and distribute it into Petri dishes in a sterile laminar flow hood.</li>
		<li>Scatter the pollen grains of the same cultivars used for the controlled pollinations in the solidified pollen germination medium and observe them under the microscope after 24 h6. 
		NOTE: To sterilize the laminar flow hood, clean the surface with 70% ethanol and switch on the UV lamp during 10 min.</li>
		<li>Store the Petri dishes in a refrigerator at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>Microscopy observations
	<ol>
		<li>Wash the pistils three times for 1 h with distilled water and leave them in 5% sodium sulphite at 4 &#176;C. After 24 h, autoclave them at 1 kg/cm2 during 10 min in sodium sulphite to soften the tissues34.</li>
		<li>Place the autoclaved pistils over a glass slide and, with the help of a scalpel, remove the trichomes around the ovary to get a better visualization of the pollen tubes. Then, squash the pistils with a cover glass.</li>
		<li>Prepare 0.1% (v/v) aniline blue stain: mix 0.1 mL of aniline blue in 100 mL of 0.1 N potassium phosphate tribasic (K3PO4). Apply a drop of aniline blue over the preparations to stain callose depositions during pollen tube growth.</li>
		<li>Observe the pollen tubes along the style by a microscope with UV epifluorescence using 340-380 bandpass and 425 longpass filters.</li>
	</ol>
	</li>
</ol>
2. DNA extraction
<ol>
	<li>Sample 2-3 leaves in the field in spring. It is recommended to sample the leaves at young stages since DNA obtained is of higher quality and lower levels of phenolic compounds compared to old leaves.</li>
	<li>Extract Genomic DNA following the steps described in a commercially available kit (see Table of Materials).</li>
	<li>Analyze the quantity and quality of DNA concentrations using UV-vis spectrophotometer (260 nm).</li>
</ol>
3. S-allele identification
<ol>
	<li>Setting up of the PCR Reactions
	<ol>
		<li>Prepare a 50 ng/&#956;L dilution in distilled water of each DNA extraction sample.</li>
		<li>Thaw out the PCR reagents slowly and keep them on ice. Leave the DNA polymerase in the freezer until needed.</li>
		<li>Prepare the amplification reactions using the different combinations of primers. Create the PCR reaction mix by combining the components in Table 1. Vortex the PCR reaction mix well and distribute the volume indicated for the different combinations of primers to each well of the PCR plate. Then, add 1 &#956;L of the DNA dilution in each well.</li>
		<li>Place the PCR plate in the thermocycler and run the corresponding PCR program shown in Table 1.</li>
	</ol>
	</li>
	<li>Analyze the amplified fragments. There are mainly two different ways to analyze the PCR amplified fragments: capillary electrophoresis (CE) with fluorescent-labelled primers or as visualize amplicons of agarose gel electrophoresis with not-labelled primers.
	<ol>
		<li>Capillary Electrophoresis
		<ol>
			<li>To prepare the loading buffer, mix 35 &#956;L of deionized formamide with 0.45 &#956;L of labeled sizing standard. Vortex the reagent to mix well, and then dispense 35.5 &#956;L into the well of the reader plate.</li>
			<li>Add 1 &#956;L of the PCR product into the well. In addition, add a drop of mineral oil to prevent water evaporation.</li>
			<li>Prepare the separation plate adding separation buffer.</li>
			<li>Use the commercial software included with the gene analyzer (see Table of Materials). Create a new sample plate and save the sample names for all wells on the plate.</li>
			<li>Select the method of analysis. In this case, denature the samples at 90 &#176;C for 120 s, inject at 2.0 kV for 30 s, and separate at 6.0 kV for 35 min.</li>
			<li>Insert the two plates into the gene analyzer. Fill the capillary array with distilled water.</li>
			<li>Load the patented linear polyacrylamide (LPA) gel. Finally, click Run.</li>
		</ol>
		</li>
		<li>Gel Electrophoresis
		<ol>
			<li>Prepare a 1% agarose gel adding 1.5 g of molecular biology grade agarose in 150 mL of 1x TAE (Tris-acetate-EDTA) electrophoresis running buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA at pH 8.0). Dissolve the agarose by microwave heating for 2-3 min.</li>
			<li>To visualize the DNA, add 4 &#956;L of a nucleic acid stain (see Table of Materials) and mix gently.</li>
			<li>Add a gel comb, with sufficient wells for ladders, controls and samples, into a gel tray. Then, pour slowly the mix into the middle of the gel tray and avoid bubbles.</li>
			<li>Let the gel cool down for 30-45 min at room temperature until the gel has completely solidified. Introduce the gel in the electrophoresis chamber, remove the gel comb and fill the chamber with enough 1x TAE buffer to cover the gel. 
			NOTE: Check the placement of the gel. The wells should be placed close to the negative pole since negatively charged DNA migrates towards the cathode.</li>
			<li>Add 5 &#956;L of loading buffer (0.1% (v/v) bromophenol blue) to the PCR products and mix well.</li>
			<li>To estimate the size of the bands, load 5 &#956;L of DNA molecular weight ladder (see Table of Materials).</li>
			<li>Load the samples into the additional wells of the gel.</li>
			<li>Once all the samples and the DNA molecular weight ladder are loaded, run the gel at 90 V for 1-1.5 h, until the blue dye line is approximately at 75% the length of the gel.</li>
			<li>Visualize the bands in a transilluminator for nucleic acids.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Traditionally, most commercial apricot European cultivars were self-compatible36. Nevertheless, the use of North American self-incompatible cultivars as parents in breeding programs in the last decades has resulted in the release of an increasing number of new self-incompatible cultivars with unknown pollination requirements7,8,37. Thus, the determination of self- and inter-(in)compatibility relationships in apricot cultivars is increasingly important. This is accentuated in those areas where winter chilling is decreasing, since high year to year variations in the time of flowering are preventing the coincidence in flowering of cultivars and their pollenizers in many cases, especially in cultivars with high chilling requirements38. The methodology described herein, combining hand-pollination, microscopy and genetic analyses has been very useful to determine the self(in)compatibility of each cultivar and to establish its potential pollinizer cultivars.
Pollination requirements can be determined through field-control experiments in orchard conditions11,39. However, the exposition to external factors including meteorological adverse conditions can cause pollination failure10, which may result in erroneous diagnoses of self-incompatibility. The methodology described herein allows to evaluate self-(in)compatibility more accurately by microscopy observations of hand-pollinated flowers in laboratory-controlled conditions, avoiding environmental influence. Moreover, this approach allows analyzing a higher number of cultivars per year, since only a small number of flowers is required instead of several adult trees for each cultivar that are required in field experiments40.
Incompatibility relationships can be established combining hand-pollinations and microscopy14. However, pollinations can only be performed for a short period during the flowering season in spring, and adult trees near the laboratory are needed, since the lifespan of the flowers collected is very short. Thus, the number of incompatibility relationships that can be analyzed by controlled hand-pollinations in each season is very low. The characterization of the genes encoded by the S-locus has enabled the development of PCR-based methods for S-allele genotyping18,41. This approach accelerates S-allele identification since it does not require flowers, and the experiments can be carried out with any vegetative tissue42. This extends the period during which plant material, usually young leaves, can be collected43. Furthermore, the leaves can be lyophilized or frozen, so that the analysis can be done at any time of the year, unlike pollinations that can only be done on fresh flowers during the flowering season44. An additional benefit is that leaves can be collected from young trees even before entering flowering age, facilitating the collection of samples and the early obtaining of results45.
The genetic analysis allows a better differentiation of self-incompatibility alleles since it provides precise results of amplified fragment sizes21,46. To date, thirty-three S-alleles have been identified in apricot12,18,20,21,22,23,24, which has allowed to establish 36 incompatibility groups based on S-genotype8,9,17,25,26,27. On the other hand, a drawback of this methodology is that different alleles in the same range size or mutations can be erroneously identified as the same allele. Thus, Sc and S8 alleles are identical for the RNase sequence but a 358-bp insertion is found in the SFB gene of Sc19. Likewise, the first intron region of the alleles S1 and S7 are identical and are indistinguishable using the primers SRc-F/SRc-R. In addition, several homologies, such as S6 and S528 or S20 and S55, and S7, S13 (EF062341) and S4617, have been found because some of these alleles have been partially sequenced or by failures during PCR amplification and, consequently, further work is needed to distinguish them correctly.
PCR analysis and S-RNase sequencing are adequate for establishing incompatibility relationships through the identification of S-alleles and the allocation of cultivars in their corresponding Incompatibility Group8,17,26,27. However, this methodology has the limitation of preventing the determination of the self-(in)compatibility for particular apricot cultivars. Self-compatibility (SC) has been associated to particular S-alleles in other Prunus species47, as almond (Sf)48,49 or sweet cherry (S4&#8217;)50,51. However, in apricot, the Sc allele, which has been associated to SC21, can be erroneously identified as S8, a self-incompatible allele19,22, and possible mutations not linked to the S locus, as the M-locus12,52, conferring SC have been identified. Recently, the M-locus has been genotyped using SSR markers12. Therefore, the genetic identification of SC for apricot genotypes needs further research and, in order to avoid mistakes due to factors not linked to the S locus, in this work the characterization of self-(in)compatibility has been determined also by phenotyping the behavior of the pollen tubes through the pistil of self-pollinated flowers.
The methodology described herein combining the determination of self-(in)compatibility by hand-pollinations in laboratory conditions with the subsequent observation of the behavior of pollen tubes in the pistil of controlled self-pollinations under the fluorescence microscopy and the identification of the S-genotype by PCR analysis allows establishing the pollination requirements of apricot cultivars. This provides a valuable information for growers and breeders, since it allows establishing the incompatibility relationships between cultivars to choose suitable pollinizers in the design of new orchards as well as to select appropriate parents to design new crosses in apricot breeding programs.

Self-incompatibility is a strategy of flowering plants to prevent self-pollination and promote outcrossing1. In Rosaceae, this mechanism is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S2. In the style, the RNase gene encodes the S-stylar determinant, a RNase3, while a F-box protein, which determines the S-pollen determinant, is codified by the SFB gene4. The self-incompatibility interaction takes place through the inhibition of pollen tube growth along the style preventing the fertilization of the ovule5,6.
In apricot, a varietal renewal has taken place worldwide in the last two decades7,8. This introduction of an important number of new cultivars, from different public and private breeding programs, has resulted in the increase of apricot cultivars with unknown pollination requirements8.
Different methodologies have been used to determine pollination requirements in apricot. In the field, self-(in)compatibility may be established by controlled pollinations in caged trees or in emasculated flowers and subsequently recording the percentage of fruit set9,10,11,12. In addition, controlled pollinations have been carried out in the laboratory by semi-in vivo culture of flowers and analysis of the pollen tube behavior under fluorescence microscopy8,13,14,15,16,17. Recently, molecular techniques, such as PCR analysis and sequencing, have allowed the characterization of incompatibility relationships based on the study of the RNase and SFB genes18,19. In apricot, thirty-three S-alleles have been reported (S1 to S20, S22 to S30, S52, S53, Sv, Sx), including one allele related with self-compatibility (Sc)12,18,20,21,22,23,24. Up to now, 26 incompatibility groups have been stablished in this species according to the S-genotype8,9,17,25,26,27. Cultivars with the same S-alleles are inter-incompatible, whereas cultivars with at least one different S-allele and, consequently, allocated in different incompatible groups, are inter-compatible.
To define the pollination requirements of apricot cultivars, we describe a methodology that combines the determination of self-(in)compatibility by fluorescence microscopy with the identification of the S-genotype by PCR analysis in apricot cultivars. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars.

<table><tbody><tr><td>Agarose D1 Low EEO</td><td>Conda</td><td>8010.22</td><td></td></tr><tr><td>BIOTAQ DNA Polymerase kit</td><td>Bioline</td><td>BIO-21060</td><td></td></tr><tr><td>Bright field microscope</td><td>Leica Microsystems</td><td>DM2500</td><td></td></tr><tr><td>CEQ System Software</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>DNeasy Plant Mini Kit</td><td>QIAGEN</td><td>69106</td><td></td></tr><tr><td>dNTP Set, 4 x 25 &amp;micro;mol</td><td>Bioline</td><td>BIO-39025</td><td></td></tr><tr><td>GenomeLab DNA Size Standard Kit - 400</td><td>Beckman Coulter</td><td>608098</td><td></td></tr><tr><td>GenomeLab GeXP Genetic Analysis System</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>GenomeLab Separation Buffer</td><td>Beckman Coulter</td><td>608012</td><td></td></tr><tr><td>GenomeLab Separation Gel LPA-1</td><td>Beckman Coulter</td><td>391438</td><td></td></tr><tr><td>HyperLadder 100bp</td><td>Bioline</td><td>BIO-33029</td><td></td></tr><tr><td>HyperLadder 1kb</td><td>Bioline</td><td>BIO-33025</td><td></td></tr><tr><td>Image Analysis System</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Molecular Imager VersaDoc MP 4000 system&amp;nbsp;</td><td>Bio-Rad</td><td>170-8640</td><td></td></tr><tr><td>NanoDrop One Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>13-400-518</td><td></td></tr><tr><td>pH-Meter BASIC 20</td><td>Crison</td><td></td><td></td></tr><tr><td>Phusion&amp;nbsp;High-Fidelity PCR Kit</td><td>Thermo Fisher Scientific</td><td>F553S</td><td></td></tr><tr><td>Power Pack P 25 T</td><td>Biometra</td><td></td><td></td></tr><tr><td>Primer Forward</td><td>Isogen Life Science</td><td></td><td></td></tr><tr><td>Primer Reverse</td><td>Isogen Life Science</td><td></td><td></td></tr><tr><td>Quantity One Software</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Stereoscopic microscope</td><td>Leica Microsystems</td><td>MZ-16</td><td></td></tr><tr><td>Sub-Cell GT</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>SYBR Safe DNA Gel Stain</td><td>Thermo Fisher Scientific</td><td>S33102</td><td></td></tr><tr><td>T100 Thermal Cycler</td><td>Bio-Rad</td><td>1861096</td><td></td></tr><tr><td>Taq DNA Polymerase</td><td>QIAGEN</td><td>201203</td><td></td></tr><tr><td>Vertical Stand Autoclave</td><td>JP Selecta</td><td></td><td></td></tr></tbody></table>

determination of self- and inter-(in)compatibility relationships in apricot combining hand-pollination, microscopy and genetic analyses

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. In apricot, the determination of self- and inter-(in)compatibility relationships is increasingly important, since the release of an important number of new cultivars has resulted in the increase of cultivars with unknown pollination requirements. Here, we describe a methodology that combines the determination of self-(in)compatibility by hand-pollinations and microscopy with the identification of the S-genotype by PCR analysis. For self-(in)compatibility determination, flowers at balloon stage from each cultivar were collected in the field, hand-pollinated in the laboratory, fixed, and stained with aniline blue for the observation of pollen tube behavior under the fluorescence microscopy. For the establishment of incompatibility relationships between cultivars, DNA from each cultivar was extracted from young leaves and S-alleles were identified by PCR. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars, which provides a valuable information to choose suitable pollinizers in the design of new orchards and to select appropriate parents in breeding programs.

Watch this Scientific Journal Video about Determination of Self- and Inter-incompatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses at JoVE.com

Determination of Self- and Inter-incompatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

We present a methodology to establish the pollination requirements of apricot (Prunus armeniaca L.) cultivars combining the determination of self-(in)compatibility by fluorescence microscopy with the identification of the S-genotype by PCR analysis.

Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. ...

determination-self-inter-compatibility-relationships-apricot

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.2K Views.  Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA). Deze methode kan helpen bij het bepalen van bestuivingsvereisten bij verschillende soorten en het tot stand brengen van onverenigbaarheidsrelaties tussen cultivars. Het belangrijkste voordeel van deze aanpak is dat het mogelijk maakt om verschillende onverenigbaarheid onder laboratoriumomstandigheden te evalueren. Het vermijden van de vele problemen die kunnen optreden in experimenten uitgevoerd onder veldomstandigheden.Dit maakt identificatie mogelijk samen met toewijding van een aant...

Video: Determination of Self- and Inter-incompatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4&#8242;,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. This morphological alteration is also recognized as epithelial to mesenchymal transition (EMT). Dysregulation of EMT is associated with fibrosis and cancer metastasis. There is emerging evidence that EMT is controlled by a number of molecular mechanisms. As such, many key genes that control apical constriction are also known to be important factors in the EMT observed in cancer metastasis. Like EMT during Drosophila gastrulation, epithelial cells can be induced to change their shape and be reprogrammed to redirect cell fate towards various other cell types. Here we provide a robust imaging method of Drosophila gastrulation to assay the initiation of morphogenetic cellular movements and cell fate identification during this stage of embryonic development. Using this method, we identify cell rearrangement at the time of gastrulation and demonstrate the importance of apical constriction during gastrulation using GFP labeled DE-cadherin.

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes&#8217; nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...

Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping

The Japanese plum cultivars commonly grown are interspecific hybrids derived from crosses between the original Prunus salicina with other Prunus species. Most hybrids exhibit gametophytic self-incompatibility, which is controlled by a single and highly polymorphic S-locus that contains multiple alleles. Most cultivated hybrids are self-incompatible and need pollen from a compatible donor to fertilize their flowers. Establishing pollination requirements in Japanese plum is becoming increasingly important due to the high number of new cultivars with unknown pollination requirements. In this work, a methodology for the determination of pollination requirements in Japanese plum-type hybrids is described. Self-(in)compatibility is determined by hand-pollinations in both the field and in the laboratory, followed by monitoring pollen tube elongation with fluorescence microscopy, and also monitoring fruit maturation in the field. Selection of pollinizer cultivars is assessed by combining the identification of S-genotypes by PCR analysis with the monitoring of flowering time in the field. Knowing the pollination requirements of cultivars facilitates the selection of cultivars for the design of new orchards and allows the early detection of productivity problems related with pollination deficiency in established orchards.

A methodology for the determination of pollination requirements in Japanese plum-type hybrids is described, which combines field- and laboratory-pollinations and observations of pollen tubes under the fluorescence microscopy with the identification of S-genotypes by PCR and the monitoring of flowering for the selection of pollinizers.

The Japanese plum cultivars commonly grown are interspecific hybrids derived from crosses between the original Prunus salicina with other Prunus species. ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Biology

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and fertilization. However, identifying suitable varieties to serve as pollinizers is a time-consuming process. To solve this problem, we have developed a rapid method for identifying pollination-compatible citrus cultivars that utilizes agarose gel electrophoresis and aniline blue staining. Pollen compatibility is determined based on the identification of S genotypes by extracting total DNA and performing PCR-based genotyping assays with specific primers. Additionally, styles are collected 3-4 days after manual pollination, and aniline blue staining is performed. Finally, the growth status of the pollen tubes is observed with a fluorescence microscope. Pollen compatibility and incompatibility can be established by observing whether the pollen tube growth is normal or suppressed, respectively. Due to its simplicity and cost-effectiveness, this method is an effective tool for determining the pollen compatibility and incompatibility of different citrus varieties to establish incompatibility groups and incompatibility relationships between different cultivars. This method provides information essential for the successful selection of suitable pollinizer trees and, thus, facilitates the establishment of new orchards and the selection of appropriate parents for breeding programs.

Determination of Self-Incompatibility and Inter-Incompatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

This protocol provides a rapid method for determining pollen compatibility and incompatibility in citrus cultivars.

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and ...

https://cloudfront-jove-com.remotexs.ntu.edu.sg/CDNSource/teasers/65056.jpg

An Improved Method to Measure Pollen Hydration Profiles in Arabidopsis thaliana

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

Author Spotlight: A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...

Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video