Method Article

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

DOI:

10.3791/65056

⸱

June 30th, 2023

 ,  ,  ,  ,  ,  , 
1National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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 g....

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

1. Preparation for aniline blue staining

  1. 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.
  2. 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....

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.......

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.......

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors declare that they have nothing to disclose.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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

....

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
absolute ethanolSinopharm Chemical ReagentCo., Ltd10009218
Aniline blueSinopharm Chemical Reagent Co.,Ltd
Boric acid, H3BO3Sinopharm Chemical ReagentCo., Ltd10004818
Brown bottleLabgic Technology Co., Ltd
Calcium nitrate tetrahydrate, Ca(NO3 )2Sinopharm Chemical ReagentCo., Ltd80029062
Centrifugal tubeLabgic Technology Co., Ltd
centrifuge tubesLabgic Technology Co., Ltd
CTABGEN-VIEW SCIENTIFIC INC57-09-0(CAS)
DroppingJiangsu Songchang Medical Equipment Co., Ltd
Ethylenediaminetetraacetic acid, EDTASinopharm Chemical Reagent Co.,Ltd10009617
ForcepsLUXIANZI Biotechnology Co., Ltd
formaldehydeSinopharm Chemical ReagentCo., Ltd10010018
Fully automatic sample fast grinderShanghai Jingxin Industrial Development Co., LtdTissuelyser-96
glacial acetic acidSinopharm Chemical ReagentCo., Ltd10000218
Grinding TubeShanghai Jingxin Industrial Development Co., Ltd
Isoamyl alcoholSinopharm Chemical Reagent Co.,Ltd10003218
Isopropyl alcoholSinopharm Chemical Reagent Co.,Ltd80109218
labelM&G Chenguang Stationery Co., Ltd.
Leica DMi8Shanghai Leica Co.,Ltd21903797
Magnesium sulfate heptahydrate, MgSO4Sinopharm Chemical ReagentCo., Ltd10013018
MICROSCOPE Cover glassZhejiang Shitai Industrial Co., Ltd
NaClSinopharm Chemical Reagent Co.,Ltd10019318
paper clipsM&G Chenguang Stationery Co., Ltd.
pencilM&G Chenguang Stationery Co., Ltd.
pollinator brushShanghai Yimei Plastics Co., Ltd
Polyethylene glycol, PEG 6000Beijing Dingguo Changsheng Biotechnology Co., LtdDH229-1
Polyethylene glycol, PEG-4000Guangzhou saiguo biotech Co., Ltd1521GR500
Potassium hydroxide, KOHSinopharm Chemical ReagentCo., Ltd10017008
Potassium nitrate, KNO3Sinopharm Chemical ReagentCo., Ltd10017218
ScalpelJiangsu Songchang Medical Equipment Co., Ltd
SlideZhejiang Shitai Industrial Co., Ltd
Sodium hydroxide, NAOHSinopharm Chemical Reagent Co.,Ltd10019718
SucroseSinopharm Chemical ReagentCo., Ltd10021418
sulfate paperTaizhou Jinnong Mesh Factory
Thermostat water bathShanghai Jinghong Experimental Equipment Co., LtdL-909193
TrichloromethaneSinopharm Chemical Reagent Co.,Ltd10006818
Tripotassium phosphate tribasic trihydrate, K3PO4Shanghai Lingfeng Chemical Reagent Co.,Ltd20032318
Tris-HClGEN-VIEW SCIENTIFIC INC1185-53-1
zip lock bagsM&G Chenguang Stationery Co., Ltd.
β-MercaptoethanolGEN-VIEW SCIENTIFIC INC60-24-2(CAS)

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Matsumoto, D., Tao, R. 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. Plant Molecular Biology. 100 (4-5), 367-378 (2019).
  2. Goldberg, E. E., et al.

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Citrus Self IncompatibilityS Genotype AnalysisManual PollinationPollen CompatibilityAniline Blue StainingAgarose Gel ElectrophoresisFluorescence MicroscopyPollen Tube GrowthPCR GenotypingPollinizer Selection

Related Articles