Name: Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses (Video) | JoVE Journal
Uploaded: 2023-06-30T14:50:00.000+00:00
Duration: 7 min 12 s
Description: Learn to determine pollen compatibility in Citrus maxima using manual pollination, microscopy, and S-genotype analysis. This method helps breeders select compatible pollenizer trees for improved orchard productivity.

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5 minutes

This project was financially supported by the National Natural Science Foundation of China (32122075, 32072523).

1. Preparation for aniline blue staining
<ol>
	<li>Prepare the following reagents and tools for the experiment: a pollinator brush, tweezers, a pencil, sulfate paper, a pollination bag, zip lock bags, paper clips, formaldehyde, glacial acetic acid, absolute ethanol, centrifuge tubes, forceps, glue droppers, glass slides, coverslips, scalpels, and polyethylene glycol.</li>
	<li>Prepare in vitro germination medium containing 0.02% MgSO4, 0.01% KNO3, 0.03% Ca(NO3)2, 0.01% H3BO3, 20% PEG-4000, and 15% sucrose. Adjust the pH to 6.0-6.2 with KOH. Use a magnetic stirrer as PEG-4,000 is difficult to dissolve.</li>
	<li>Prepare Carnoy&#39;s fixative solution, which is absolute ethanol and glacial acetic acid mixed at a ratio of 3:1. Prepare FAA fixation solution, which is 40% formaldehyde, 80% ethanol, and glacial acetic acid (1:8:1). Prepare 4 M sodium hydroxide (NaOH) and aniline blue solution, which is 0.1% aniline blue in 0.1 M K3PO4. Use an amber bottle to store the aniline blue solution because it is light-sensitive.</li>
</ol>
2. Pollen collection
<ol>
	<li>Know the flowering period of the experimental trees (Ma jia pummelo in this study) in advance. Collect mature unopened flowers from the beginning of the flowering period to the peak flowering stage, and put them in a zip lock bag. The flowers can be stored at 4 &#176;C in the refrigerator for 24 h.</li>
	<li>Take the flowers to the lab. Use tweezers to collect the anthers, and place them in a Petri dish containing filter paper. Collect anthers from 20 to 30 flowers.</li>
	<li>Place the Petri dish containing the anthers in a 28 &#176;C oven for 24 h until the pollen grains dry. For the detailed flower organization, see Hu et al.9.</li>
	<li>Put the dried pollen into a 1.5 mL centrifuge tube. Save the pollen in an air-tight zip lock bag containing color-changing silica gel (desiccant). Close the bag, and label the bag with the name of the pollen variety and the date of storage. The dried pollen can be stored in a &#8722;20 &#176;C refrigerator for 96 weeks16.</li>
</ol>
3. In vitro pollen germination test
<ol>
	<li>Pour 300 &#181;L of liquid medium into a cell culture dish or the cap of a 2 mL centrifuge tube, and sprinkle the pollen evenly with the help of a pollination brush. Incubate the pollen at 28 &#176;C in a humidified dark environment for 12 h.</li>
	<li>Remove the top 1 mm of the tip of a 1,000 &#181;L pipette tip. Use the pipette to absorb the pollen with a small amount of culture solution and move it to the center of the microscope slide. Cover it with a coverslip. Observe the specimen with an inverted microscope using a 10x objective.</li>
	<li>Perform three independent replicates using approximately the same pollen density for each replicate17. Visually manage the amount of pollen, making sure that whole of the Petri dish is covered with pollen and that each Petri dish has an almost equal amount of pollen.</li>
	<li>The germinated pollen produces a pollen tube with a length approximately twice its diameter. Calculate the germination rate from 20 visual fields, which gives the percentage of germinated pollen in all pollen fields.</li>
</ol>
4. Pollination
<ol>
	<li>Choose a sunny day without wind for pollination. Select 10 fully developed buds about to open, peel back the petals carefully, and take care not to bruise them.</li>
	<li>Use a pollination brush to spread a sufficient amount of viable pollen onto the surface of the stigma, and take care not to damage the pistil. For self-pollination, use the pollen from the same plant/cultivar. For cross-pollination, use the pollen from a plant with a different genotype.</li>
	<li>Cover the pollinated flowers with a sulfate paper bag. Use a paper clip to seal the bag and to prevent pollination by genotypically distinct pollens.</li>
	<li>Write the name of the species and the number and time of pollination on the label. Hang the label on the branches near the pollinated flowers.</li>
</ol>
5. Sampling, fixation, and preservation
<ol>
	<li>Remove the pollination bags approximately 3-4 days after pollination, and collect the pollinated flowers in zip lock bags.</li>
	<li>Immediately remove the petals, receptacles, and ovaries from the flowers, and immerse the stigmas fused to styles in a centrifuge tube containing a freshly prepared fixative solution. Incubate the stigmas and styles in the fixative solution overnight at 4 &#176;C.</li>
	<li>The next day, discard the fixative solution, and wash the stigmas and styles two to three times in 95% ethanol.</li>
	<li>Transfer the styles to a 70% ethanol solution. Ensure that the sample is completely immersed in the solution. The styles at this stage can be stored at 4 &#176;C for 1-2 months.</li>
</ol>
6. Aniline blue staining
<ol>
	<li>Wash the style samples stored in 70% ethanol with distilled water three to four times. Immerse in 4 M NaOH solution, seal, and incubate in a 65 &#176;C water bath for 60 min. During this step, the color of the style changes from yellow-white to orange-red.</li>
	<li>Soak the styles in distilled water for 30 min. Discard the distilled water, and wash the styles with distilled water three to four times or until the color of the style becomes yellow.</li>
	<li>Place the sample in a 10 mL tube, add the aniline blue until the sample has been dipped, and dye for 12 h in the dark.</li>
	<li>Observe the pollen tube growth with a fluorescence microscope.</li>
</ol>
7. Fluorescence microscopy
<ol>
	<li>Before observing the specimens, place the slide on a flat table, and add two to three drops of polyethylene glycol to the surface of the slide.</li>
	<li>Wash the style to be observed with distilled water. Use a scalpel to divide it into two halves along the longitudinal axis. Place one half of the style on the glass slide, and cover with a coverslip.</li>
	<li>Place the slide on the stage of the microscope above the aperture, and visualize using a 10x objective. Use the DAPI filter (excitation: 325-375 nm; emission: 435-485 nm). Observe five styles for each type of pollination. Observe the pollen tube growth.</li>
</ol>
8. PCR-based S genotype identification
<ol>
	<li>Extract the genomic DNA from the stigma sample using the CTAB method18.
	<ol>
		<li>Put the collected leaves into a 2 mL centrifuge tube, and snap-freeze in liquid nitrogen. Prepare HCl:EDTA:NaCl:H2O buffer at a ratio of 1:1:3:5 and a chloroform isoamyl alcohol mixture at a ratio of 24:1</li>
		<li>Add 10 mL of the prepared buffer, 0.2 g of CTAB, and 200 &#181;L of mercaptoethanol to a 50 mL centrifuge tube, and put them in a water bath at 65 &#176;C for 5 min until the solution becomes clear and transparent.</li>
		<li>Put the blades into a mortar, add the frozen samples, add liquid nitrogen, and grind. Put the ground sample into a 2 mL centrifuge tube, add 600 &#181;L of CTAB mixture, put it in a water bath at 65 &#176;C for 60 min, and mix it upside down every 30 min.</li>
		<li>Add 700 &#181;L of the chloroform isoamyl alcohol mixture (24:1), and mix upside down for 10 min. Centrifuge at 24 &#176;C at 12,000 x g for 10 min, pipette the supernatant, and transfer to a 1.5 mL centrifuge tube.</li>
		<li>Add 60 &#181;L of 5 M NaCl solution and 1 mL of absolute ethanol, and mix upside down. Freeze at &#8722;20 &#176;C for 30 min, and centrifuge at 24 &#176;C and 9,000 x g for 5 min.</li>
		<li>Discard the supernatant, add 1 mL of 70% ethanol solution, and leave at room temperature for 1-2 h. Centrifuge at 24 &#176;C, 9,000 x g for 5 min, discard the supernatant, aspirate the excess ethanol solution, and air-dry for 5 min.</li>
		<li>Add 100 &#181;L of sterile water to dissolve, measure the DNA concentration with a spectrophotometer, and freeze in a -4 &#176;C freezer for long-term storage.</li>
		<li>Configure the RT-PCR reaction system. Prepare the following reaction mix for 10 &#181;L containing 5 &#181;L of 2x PCRMix, 0.25 &#181;L each of forward and reverse primer, 1 &#181;L of DNA (100 ng/ &#181;L), and 3.5 &#181;L of H2O.</li>
	</ol>
	</li>
	<li>Set up the PCR program as per Table 1. The PCR program for all isoforms was 95 &#176;C for 5 min 32x (95 &#176;C for 30 s, 55 &#176;C for 30 s, and 72 &#176;C for 1 min) and 72 &#176;C for 5 min. Separate the products on 1.5% TAE-agarose gels and photograph9. Verify the specified S genotype using genomic DNA.</li>
</ol>

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<li>Min, H. E., Chao, G. U., Juyou, W. U., Shaoling, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+on+self-incompatibility+mechanism+in+fruit+trees%5BTitle%5D)+AND+%22Acta+Horticulturae+Sinica%22%5BJournal%5D)">Recent advances on self-incompatibility mechanism in fruit trees.</a> Acta Horticulturae Sinica. 48 (4), 759-777 (2021).</li>
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<li>Thomas, S. G., Franklin-Tong, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-incompatibility+triggers+programmed+cell+death+in+Papaver+pollen%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Self-incompatibility triggers programmed cell death in Papaver pollen.</a> Nature. 429, 305-309 (2004).</li>
<li>Hu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Downregulated+expression+of+S2-RNase+attenuates+self-incompatibility+in+%26%2334%3BGuiyou+No.+1%26%2334%3B+pummelo%5BTitle%5D)+AND+%22Horticulture+Research%22%5BJournal%5D)">Downregulated expression of S2-RNase attenuates self-incompatibility in &#34;Guiyou No. 1&#34; pummelo.</a> Horticulture Research. 8 (1), 199(2021).</li>
<li>Guo, H., Halitschke, R., Wielsch, N., Gase, K., Baldwin, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mate+selection+in+self-compatible+wild+tobacco+results+from+coordinated+variation+in+homologous+self-Incompatibility+genes%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Mate selection in self-compatible wild tobacco results from coordinated variation in homologous self-Incompatibility genes.</a> Current Biology. 29 (12), 2020-2030 (2019).</li>
<li>Sun, P., Li, S., Lu, D., Williams, J. S., Kao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pollen+S-locus+F-box+proteins+of+petunia+involved+in+S-RNase-based+self-incompatibility+are+themselves+subject+to+ubiquitin-mediated+degradation%5BTitle%5D)+AND+%22The+Plant+Journal%22%5BJournal%5D)">Pollen S-locus F-box proteins of petunia involved in S-RNase-based self-incompatibility are themselves subject to ubiquitin-mediated degradation.</a> The Plant Journal. 83 (2), 213-223 (2015).</li>
<li>Hua, Z., Kao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+characterization+of+components+of+a+putative+petunia+S-locus+F-box-containing+E3+ligase+complex+involved+in+S-RNase-based+self-incompatibility%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Identification and characterization of components of a putative petunia S-locus F-box-containing E3 ligase complex involved in S-RNase-based self-incompatibility.</a> Plant Cell. 18 (10), 2531-2553 (2006).</li>
<li>Entani, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ubiquitin-proteasome-mediated+degradation+of+S-RNase+in+a+solanaceous+cross-compatibility+reaction%5BTitle%5D)+AND+%22The+Plant+Journal%22%5BJournal%5D)">Ubiquitin-proteasome-mediated degradation of S-RNase in a solanaceous cross-compatibility reaction.</a> The Plant Journal. 78 (6), 1014-1021 (2014).</li>
<li>Abdallah, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+self-incompatibility+and+genetic+diversity+in+diploid+and+hexaploid+plum+genotypes%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">Analysis of self-incompatibility and genetic diversity in diploid and hexaploid plum genotypes.</a> Frontiers in Plant Science. 10, 896(2019).</li>
<li>Herrera, S., Lora, J., Hormaza, J. I., Herrero, M., Rodrigo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimizing+production+in+the+new+generation+of+apricot+cultivars%3A+self-incompatibility%2C+S-RNase+allele+identification%2C+and+incompatibility+group+assignment.+%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">Optimizing production in the new generation of apricot cultivars: self-incompatibility, S-RNase allele identification, and incompatibility group assignment. </a> Frontiers in Plant Science. 9, 527(2018).</li>
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</ol>

The authors declare that they have nothing to disclose.

In fruit crops, both parthenocarpy and SI are important traits because they pave the way for seedless fruits - a trait that is highly appreciated by consumers. Self-incompatibility promotes the rejection of self-pollen and, thus, prevents inbreeding20. Among citrus, pummelo is a self-incompatible variety7. Almost 40% of all angiosperm species exhibit SI21. This trait prevents fruit setting, lowers the yield, and brings huge economic losses to growers...

Self-incompatibility (SI) is a genetically controlled mechanism present in approximately 40% of angiosperm species. In this process, the pistil rejects pollen from a plant with the same SI genotype and, thus, prevents self-fertilization1,2. Ma jia pummelo is a local variety in Jinagsu province, China, with the excellent qualities of large, pink fruit, a rich juice content, a sweet and sour taste, and a thick peel3. Although SI promotes outcrossing, it negatively impacts the yield and quality of fruits4 and necessitates suitable pollinizer trees with distinct SI genotypes for reliable fruit-setting rates and high yields. At present, there are two main types of SI, sporophytic self-incompatibility (SSI), represented by Brassicaceae, and gametophytic self-incompatibility (GSI), represented by Rosaceae, Papaveraceae, Rutaceae, and Solanaceae5,6,7,8.
Citrus is one of the most important fruit crops in the world. The S-RNase-based GSI system is found in many citrus accessions and negatively influences the fruit-setting rate9. In this system, SI is controlled by the S locus, a single polymorphic locus with two complex alleles that carry pistil S determinants and pollen S determinants7. The female determinant is the S ribonuclease (S-RNase), and the male determinant is the S locus F-box (SLF)7. The cells of the pistil secrete S-RNase proteins. The non-self S-RNases are recognized by the SLF proteins, which leads to the ubiquitination and degradation of the non-self S-RNases by the 26S proteasome pathway. In contrast, the self S-RNases are able to accumulate and inhibit pollen tube (PT) growth because they evade the SLF proteins and, therefore, are prevented from ubiquitinzation10,11,12,13.
Here, we report an in vivo technique that is useful for identifying S-genotypes and degrees of pollen compatibility and incompatibility. The protocol involves extracting total DNA from leaves and predicting the S genotype using S-specific primers. Moreover, aniline blue staining and fluorescence microscopy followed by hand pollination provide evidence for the degree of compatibility and incompatibility. The semi in vivo pollination procedure, which involves the manual pollination of flowers in the laboratory14,15,&#160;has also been adapted to assess the degrees of self-compatibility and incompatibility. However, we have also used field pollination followed by the bagging of flowers to avoid contamination from undesired pollen to allow the pollen tubes to develop in natural conditions. This protocol is simple and straightforward and provides the information necessary for the successful selection of suitable pollinizer trees.

<table><tbody><tr><td>absolute ethanol</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10009218</td><td></td></tr><tr><td>Aniline blue</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td></td><td></td></tr><tr><td>Boric acid, H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10004818</td><td></td></tr><tr><td>Brown bottle</td><td>Labgic Technology Co., Ltd</td><td></td><td></td></tr><tr><td>Calcium nitrate tetrahydrate, Ca(NO3 )2</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>80029062</td><td></td></tr><tr><td>Centrifugal tube</td><td>Labgic Technology Co., Ltd</td><td></td><td></td></tr><tr><td>centrifuge tubes</td><td>Labgic Technology Co., Ltd</td><td></td><td></td></tr><tr><td>CTAB</td><td>GEN-VIEW SCIENTIFIC INC</td><td>57-09-0(CAS)</td><td></td></tr><tr><td>Dropping</td><td>Jiangsu Songchang Medical Equipment Co., Ltd</td><td></td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid, EDTA</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10009617</td><td></td></tr><tr><td>Forceps</td><td>LUXIANZI Biotechnology Co., Ltd</td><td></td><td></td></tr><tr><td>formaldehyde</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10010018</td><td></td></tr><tr><td>Fully automatic sample fast grinder</td><td>Shanghai Jingxin Industrial Development Co., Ltd</td><td>Tissuelyser-96</td><td></td></tr><tr><td>glacial acetic acid</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10000218</td><td></td></tr><tr><td>Grinding Tube</td><td>Shanghai Jingxin Industrial Development Co., Ltd</td><td></td><td></td></tr><tr><td>Isoamyl alcohol</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10003218</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>80109218</td><td></td></tr><tr><td>label</td><td>M&amp;amp;G Chenguang Stationery Co., Ltd.</td><td></td><td></td></tr><tr><td>Leica DMi8</td><td>Shanghai Leica Co.,Ltd</td><td>21903797</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate, MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10013018</td><td></td></tr><tr><td>MICROSCOPE Cover glass</td><td>Zhejiang Shitai Industrial Co., Ltd</td><td></td><td></td></tr><tr><td>NaCl</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10019318</td><td></td></tr><tr><td>paper clips</td><td>M&amp;amp;G Chenguang Stationery Co., Ltd.</td><td></td><td></td></tr><tr><td>pencil</td><td>M&amp;amp;G Chenguang Stationery Co., Ltd.</td><td></td><td></td></tr><tr><td>pollinator brush</td><td>Shanghai Yimei Plastics Co., Ltd</td><td></td><td></td></tr><tr><td>Polyethylene glycol, PEG 6000</td><td>Beijing Dingguo Changsheng Biotechnology Co., Ltd</td><td>DH229-1</td><td></td></tr><tr><td>Polyethylene glycol, PEG-4000</td><td>Guangzhou saiguo biotech Co., Ltd</td><td>1521GR500</td><td></td></tr><tr><td>Potassium hydroxide, KOH</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10017008</td><td></td></tr><tr><td>Potassium nitrate, KNO3</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10017218</td><td></td></tr><tr><td>Scalpel</td><td>Jiangsu Songchang Medical Equipment Co., Ltd</td><td></td><td></td></tr><tr><td>Slide</td><td>Zhejiang Shitai Industrial Co., Ltd</td><td></td><td></td></tr><tr><td>Sodium hydroxide, NAOH</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10019718</td><td></td></tr><tr><td>Sucrose</td><td>Sinopharm Chemical ReagentCo., Ltd</td><td>10021418</td><td></td></tr><tr><td>sulfate paper</td><td>Taizhou Jinnong Mesh Factory</td><td></td><td></td></tr><tr><td>Thermostat water bath</td><td>Shanghai Jinghong Experimental Equipment Co., Ltd</td><td>L-909193</td><td></td></tr><tr><td>Trichloromethane</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10006818</td><td></td></tr><tr><td>Tripotassium phosphate tribasic trihydrate, K&lt;sub&gt;3&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Shanghai Lingfeng Chemical Reagent Co.,Ltd</td><td>20032318</td><td></td></tr><tr><td>Tris-HCl</td><td>GEN-VIEW SCIENTIFIC INC</td><td>1185-53-1</td><td></td></tr><tr><td>zip lock bags</td><td>M&amp;amp;G Chenguang Stationery Co., Ltd.</td><td></td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>GEN-VIEW SCIENTIFIC INC</td><td>60-24-2(CAS)</td><td></td></tr></tbody></table>

determination of self-(in)compatibility and inter-(in)compatibility relationships in citrus using manual pollination, microscopy, and s-genotype analyses

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.

For the experiments done here, mature flowers were selected, the anthers were collected, dried in an oven, and the pollen was germinated at 28°C for 12 h. The pollen viability and germination rates were quantified as shown in Figure 1.
Citrus was manually pollinated, and the pollen compatibility and incompatibility were assessed using aniline blue staining and fluorescence microscopy. The compatible pollen could germinate on the surface of the stigma and produce a...

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 ...

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Research

JoVE Journal

Biology

We have developed a methodology for performing crosses in Setaria viridis (S. viridis).  The method involves pruning the panicle prior to a hot water treatment to kill viable pollen.  Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

To reveal the pollinator effectiveness of a given plant species, multiple methods of field experiments have been developed. This study demonstrates the basic methods of field experiments for pollination ecology using the case study of Lycoris sanguinea var. sanguinea and the novel pollination mechanism, breaking-bud pollination.

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

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Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

Here, we present a protocol to force flowering in mandarin trees under phytotron conditions. Water stress, high illuminance and a simulated spring photoperiod allowed viable flowers to be obtained in a short period of time. This methodology allows researchers to have several flowering periods in 1 year.

Forced Flowering in Mandarin Trees under Phytotron Conditions

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

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

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.

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

In this work, a robust method for the quantification of mating efficiency in the yeast Saccharomyces cerevisiae is described. This method is particularly useful for the quantification of pre-zygotic barriers in speciation studies.

Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae

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.

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

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

Learn to determine pollen compatibility in Citrus maxima using manual pollination, microscopy, and S-genotype analysis. This method helps breeders select compatible pollenizer trees for improved orchard productivity.

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

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

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