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 ...

This technique can efficiently identify the S genotype and accurately identify citrus self-incompatibility, providing necessary information for selecting suitable pollenizer trees. This is a time-efficient, simple, and reliable technique to select the appropriate parents trees in breeding programs. This technique is simple and easy to do and doing experiment for the first time needs patience and care.The S genotype identification and S-specific primer designing needs more attention and care. Demonstrating the procedure will be Yu Hu, a master graduate student from my laboratory. Begin pollen collection by taking the Majia pomelo, Citrus maxima, flowers to the laboratory.Collect the anthers using tweezers and place them in a Petri dish containing filter paper. Dry the pollen grains by placing the Petri dish containing the anthers in a 28-degree-Celsius oven for 24 hours. After drying, save the pollen in a Micro Centron airtight zip lock bag containing color-changing silica gel.Close the bag and label the bag with the name of the pollen variety and the date of storage. Pour 300 microliters of the liquid medium into the cap of a two-milliliter centrifuge tube and sprinkle the pollen evenly with the help of a pollination brush. Place the pollen in a humidified black box and incubate at 28 degrees Celsius for 12 hours.After incubation, cut the top one-millimeter tip off a 1, 000 microliter pipette tip and aspirate the pollen with a small amount of culture solution. Place the pollen at the center of the microscope slide and cover it with a cover slip. Once done, observe the specimen with an inverted microscope using a 10X objective.Perform three independent replicates using approximately the same pollen density for each replicate. For pollination, use a pollination brush to spread a sufficient amount of viable pollen onto the surface of the stigma, ensuring not to damage the pistil. Cover the pollinated flowers with a sulfate paper bag and prevent pollination by genotypically-distinct pollens by sealing the bag using a paperclip.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. Remove the pollination bags approximately three to four days after pollination and collect the pollinated flowers in zip lock bags.Immediately remove the petals, receptacles, and ovaries from the flowers and immerse the stigma fused to style in a centrifuge tube containing a freshly-prepared fixative solution. Incubate the stigma and style in the fixative solution overnight at four degrees Celsius. The next day, discard the fixative solution before washing the stigma and style two to three times in 95%ethanol.Then, add 70%ethanol solution to the style, ensuring the sample is completely immersed in the solution. The styles at this stage can be stored at four degrees Celsius for one to two months. For aniline blue staining, wash the style samples stored in 70%ethanol with distilled water three to four times.Immerse it in a four molar sodium hydroxide solution and seal it before incubating it in a 65-degree-Celsius water bath for 60 minutes. During this step, the color of the style changes from yellow-white to orange-red. Next, soak the styles in distilled water.After 30 minutes, discard the distilled water and wash the styles with distilled water three to four times or until the color of the style becomes yellow. Then, add the aniline blue until the sample is submerged and incubate for 12 hours in the dark. Observe the pollen tube growth under the fluorescence microscope.Place the slide on a flat table. Divide the style into two halves along the longitudinal axis using a scalpel. Place one half of the style on a glass slide and add two to three drops of polyethylene glycol to the surface of the slide.Then, cover it with a cover slip. Once done, place the slide on the microscope stage above the aperture and visualize using a 10X objective. Use the DAPI filter and observe the pollen tube growth.Observe five styles for each type of pollination. The pollen viability of Citrus maxima and germination rates were quantified using fluorescent microscopy. The compatible pollen successfully germinated on the surface of the stigma and produced a normal pollen tube, which grew and ultimately led to fertilization in the ovary.In contrast, incompatible pollen tubes grew through approximately 2/3 of the style and then stopped growing. Agarose gel electrophoresis detected the DNA product amplified using the S locus-specific primers. It showed the amplification of the DNA product between 500 to 1, 000 base pairs.The corresponding S genotype was identified. 21 S haplotypes in different citrus species were identified using this method. The pollination step is critical.Only the successful pollination can ensure the subsequent observation of pollen tubes. The technique was used to analyze the self-incompatibility traits and to explore and identify the female determinants of the self-incompatibility. This method is important for self-incompatibility research and breeding programs.It can also facilitate the farming community for selecting pollenizer trees for their orchards.

Research

JoVE Journal

Biology

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

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

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 ...