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

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.