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

This protocol provides a way to assess the involvement of a certain factor in double fertilization of Arabidopsis. The fertilization status can be judged by the morphology of the sperm nuclei at a glance, so the quick obtaining of the data for statistical analysis is possible. This method is specific to assessment for fertilization phenotype in Arabidopsis.

There are still unknown fertilization regulators, even if in Arabidopsis. This method would be useful for functional analysis of future unknown fertilization factors. To master the skill of quick and intact handling of the ovules are important for the success of this analysis.

To begin, grow Arabidopsis thaliana at 22 degrees Celsius under a 16-hour light and eight-hour dark cycle in a growth chamber. Cut and remove the first developed flower stalk with scissors to promote development of axillary buds. Two to three weeks after cutting off the first stalk, measure the plant height.

Plants with height above 25 centimeters are considered vigorously growing plants and are suitable for analysis. To emasculate, look for buds at stage 11 with bits of petals seen at the top. Use a number five forceps to remove sepal, petal, and stamen of flower buds.

15 to 18 hours after emasculation, take the stamen of a DMP9-knockdown line with background of HTR10-mRFP flower at stage 13 by pinching the filament with forceps. To pollinate, gently pat the stigma of an emasculated pistil several times with a dehiscent anther. Seven to eight hours after pollination, collect the pistil and place it on the double-sided tape.

Then, press gently with forceps to fix the pistil on the tape. Under a dissecting microscope, cut off the upper and lower ends of the ovary using an injection needle. Move the tip of the injection needle to slit the ovary wall along both sides of the replum.

Cut carefully so as not to separate the funiculus and the transmitting tract. Evert the ovary wall. Pinch the base of the septum to which ovules are connected, and lift it up carefully with forceps.

Transfer the ovules into a drop of water on a slide glass, and gently cover with a coverglass for observation under a fluorescence microscope. On an epifluorescence microscope equipped with a fluorescence filter cube, adjust the objective lens to 20x or 40x. Acquire images of the ovules containing sperm nuclei labeled with mRFP.

Confirm the number of mRFP-labeled sperm nuclei in an embryo sac. Confirm the shape and position of each mRFP-labeled sperm nucleus in an embryo sac. In this study, fertilization phenotypes were judged by sperm nuclear signal morphology in ovule.

Most ovules contained two decondensed mRFP-labeled sperm nuclei at the egg cell micropylar side and central cell charazal end side. This indicates successful double fertilization. In addition, ovules containing a decondensed mRFP-labeled sperm nucleus at the central cell nucleus plus a condensed mRFP-labeled sperm nucleus outside the egg cell were observed, indicating single fertilization.

In the case of failed double fertilization, two condensed mRFP-labeled sperm nuclei were observed at the boundary of the egg cell and the central cell. In the analysis using DMP9-knockdown line with background of HTR10-mRFP pollen, ovules containing a single condensed mRFP-labeled sperm nucleus or three or more mRFP-labeled sperm nuclei were rarely observed. Combining with the use of female marker lines, more accurate fertilization status can be analyzed.

For example, the fertilization status at after gamete fusion and before the karyogamy can be judged. Yes, the fertilization process is divided into several steps. The questions that each fertilization regulator involved in which steps can be answered with this improved method.

It would provide deeper insight into the sexual reproduction of the plant.