Name: determination of self-(in)compatibility and inter-(in)compatibility relationships in citrus using manual pollination, microscopy, and s-genotype analyses
Uploaded: 2023-06-30T14:50:00
Description: Watch this Scientific Journal Video about Determination of Self-Incompatibility and Inter-Incompatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses at JoVE.

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)...

<ol>
	<li>Matsumoto, D., Tao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+S-RNases+by+an+S+locus+F-box+like+protein+and+an+S+haplotype-specific+F-box+like+protein+in+the+Prunus-specific+self-incompatibility+system.">Recognition of S-RNases by an S locus F-box like protein and an S haplotype-specific F-box like protein in the Prunus-specific self-incompatibility system.</a> Plant Molecular Biology. 100 (4-5), 367-378 (2019).</li><li>Goldberg, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+selection+maintains+self-incompatibility.">Species selection maintains self-incompatibility.</a> Science. 330 (6003), 493-495 (2010).</li><li>Zhang, L., Wang, R., Zhao, G., Wang, A., Lin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+on+fruit+quality+of+Guangfeng+Ma+jia+pummelo+and+Pinghe+red+pummelo.">Comparative study on fruit quality of Guangfeng Ma jia pummelo and Pinghe red pummelo.</a> China Agricultural Science Bulletin. 37 (22), 126-130 (2021).</li><li>Min, H. E., Chao, G. U., Juyou, W. U., Shaoling, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+on+self-incompatibility+mechanism+in+fruit+trees.">Recent advances on self-incompatibility mechanism in fruit trees.</a> Acta Horticulturae Sinica. 48 (4), 759-777 (2021).</li><li>Fujii, S., Kubo, K., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-self-+and+self-recognition+models+in+plant+self-incompatibility.">Non-self- and self-recognition models in plant self-incompatibility.</a> Nature Plants. 2 (9), 2-9 (2016).</li><li>Meng, X., Sun, P., Kao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-RNase-based+self-incompatibility+in+Petunia+inflata.">S-RNase-based self-incompatibility in Petunia inflata.</a> Annals of Botany. 108 (4), 637-646 (2011).</li><li>Liang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+self-compatibility+by+a+mutant+Sm-RNase+in+citrus.">Evolution of self-compatibility by a mutant Sm-RNase in citrus.</a> Nature Plants. 6 (2), 131-142 (2020).</li><li>Thomas, S. G., Franklin-Tong, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-incompatibility+triggers+programmed+cell+death+in+Papaver+pollen.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulated+expression+of+S2-RNase+attenuates+self-incompatibility+in+&amp;#34;Guiyou+No.+1&amp;#34;+pummelo.">Downregulated expression of S2-RNase attenuates self-incompatibility in &amp;#34;Guiyou No. 1&amp;#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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mate+selection+in+self-compatible+wild+tobacco+results+from+coordinated+variation+in+homologous+self-Incompatibility+genes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pollen+S-locus+F-box+proteins+of+petunia+involved+in+S-RNase-based+self-incompatibility+are+themselves+subject+to+ubiquitin-mediated+degradation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-proteasome-mediated+degradation+of+S-RNase+in+a+solanaceous+cross-compatibility+reaction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+self-incompatibility+and+genetic+diversity+in+diploid+and+hexaploid+plum+genotypes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+production+in+the+new+generation+of+apricot+cultivars:+self-incompatibility,+S-RNase+allele+identification,+and+incompatibility+group+assignment.+.">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><li>Yuan, S. C., Chin, S. W., Lee, C. Y., Chen, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phalaenopsis+pollinia+storage+at+sub-zero+temperature+and+its+pollen+viability+assessment.">Phalaenopsis pollinia storage at sub-zero temperature and its pollen viability assessment.</a> Botanical Studies. 59 (1), 1 (2018).</li><li>Liang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+evolution+of+genes+related+to+self-incompatibility+in+citrus.">Identification and evolution of genes related to self-incompatibility in citrus.</a> , (2019).</li><li>Cheng, Y. J., Guo, W. W., Yi, H. L., Pang, X. M., Deng, X. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+protocol+for+genomic+DNA+extraction+from+Citrus+species.">An efficient protocol for genomic DNA extraction from Citrus species.</a> Plant Molecular Biology Reporter. 21 (2), 177-178 (2003).</li><li>Wei, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+S-genotypes+of+63+pummelo+germplasm+resources.">Identification of S-genotypes of 63 pummelo germplasm resources.</a> Acta Horticulturae Sinica. 49 (5), 1111-1120 (2021).</li><li>de Nettancourt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incompatibility+in+angiosperms.">Incompatibility in angiosperms.</a> Sexual Plant Reproduction. 10, 185-199 (1997).</li><li>Igic, B., Lande, R., Kohn, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+self-incompatibility+and+its+evolutionary+consequences.">Loss of self-incompatibility and its evolutionary consequences.</a> International Journal of Plant Sciences. 169 (1), 93-104 (2008).</li><li>Guerrero, B. I., Guerra, M. E., Rodrigo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+pollination+requirements+in+Japanese+plum+by+phenological+monitoring,+hand+pollinations,+fluorescence+microscopy+and+molecular+genotyping.">Establishing pollination requirements in Japanese plum by phenological monitoring, hand pollinations, fluorescence microscopy and molecular genotyping.</a> Journal of Visualized Experiments. (165), e61897 (2020).</li><li>Herrera, S., Lora, J., Hormaza, J. I., Rodrigo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+self-+and+inter-(in)compatibility+relationships+in+apricot+combining+hand-pollination,+microscopy+and+genetic+analyses.">Determination of self- and inter-(in)compatibility relationships in apricot combining hand-pollination, microscopy and genetic analyses.</a> Journal of Visualized Experiments. (160), e60241 (2020).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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, H3BO3</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&#38;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, MgSO4</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&#38;G Chenguang Stationery Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pencil</td>
			<td>M&#38;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, K3PO4</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&#38;G Chenguang Stationery Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-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

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&#160;were collected, dried in an oven, and the pollen was germinated at 28&#176;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...

Watch this Scientific Journal Video about Determination of Self-Incompatibility and Inter-Incompatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses at JoVE.

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.

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 ...

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In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically ...