23. Study the morphology of wild-type ovules (Fig. 5i) (see Note
21).
24. Check the presence of embryo sac cells by identifying the nuclei
of the central cell (CCn), egg cell (ECn) (Fig. 5j), and the two
synergids (SYn) and their positions within the embryo sac
(Fig. 5k, l). When you are familiar with ovule and embryo sac
structures of the wild type, study your mutants. Take care to
use the same ecotype for comparative analyses.
3.4 Embryo
Development
If seed development has been initiated, but mutants generate
aborted seeds recognized by a smaller size, aberrant shape, or
white or brown color, respectively, embryo or endosperm development might be affected. Green siliques at the bottom of the inflorescence should be analyzed first because they contain embryos at
later stages. Because embryo development in Arabidopsis follows a
stereotypic pattern [23], it is easy to determine at which stage seeds
are affected (see Note 22).
1. Take a green silique from the base of the inflorescence (see Note
23) and follow steps 1–9 described above in Subheading 3.1.1
(see also Note 24).
2. Detach seeds by scraping them carefully off with a needle with
its bevel facing downward to avoid damaging seeds (see Note
25).
3. Add 7 μL of clearing solution on top, place a coverslip (see Note
26), and incubate in a humid chamber for at least 2 h to obtain
good results (see Note 2).
4. Observe embryo and seed developmental stages under a microscope equipped with DIC optics (Fig. 6a–i).
4 Notes
1. Germination rate and tube growth can be significantly accelerated by the addition of brassinosteroids as described in [15].
2. After fertilization, clearing success strongly depends on the
developmental stage of the seed. Therefore, for ovules 2 days
after fertilization, 2 h of incubation is sufficient, while overnight clearing is recommended from 10 days after fertilization.
Alternatively, Hoyer’s solution could be used for seeds 10 days
after fertilization, as described [18].
3. Do not use the first or last siliques after flowering as these are
often sterile and could lead to wrong interpretations.
4. Reducing the distance between the holding disposable medical
hypodermic syringe needle and the one used to perform the
dissection makes the process more accurate. For long siliques,
Identifying Reproductive Mutations in Arabidopsis
385
21).
24. Check the presence of embryo sac cells by identifying the nuclei
of the central cell (CCn), egg cell (ECn) (Fig. 5j), and the two
synergids (SYn) and their positions within the embryo sac
(Fig. 5k, l). When you are familiar with ovule and embryo sac
structures of the wild type, study your mutants. Take care to
use the same ecotype for comparative analyses.
3.4 Embryo
Development
If seed development has been initiated, but mutants generate
aborted seeds recognized by a smaller size, aberrant shape, or
white or brown color, respectively, embryo or endosperm development might be affected. Green siliques at the bottom of the inflorescence should be analyzed first because they contain embryos at
later stages. Because embryo development in Arabidopsis follows a
stereotypic pattern [23], it is easy to determine at which stage seeds
are affected (see Note 22).
1. Take a green silique from the base of the inflorescence (see Note
23) and follow steps 1–9 described above in Subheading 3.1.1
(see also Note 24).
2. Detach seeds by scraping them carefully off with a needle with
its bevel facing downward to avoid damaging seeds (see Note
25).
3. Add 7 μL of clearing solution on top, place a coverslip (see Note
26), and incubate in a humid chamber for at least 2 h to obtain
good results (see Note 2).
4. Observe embryo and seed developmental stages under a microscope equipped with DIC optics (Fig. 6a–i).
4 Notes
1. Germination rate and tube growth can be significantly accelerated by the addition of brassinosteroids as described in [15].
2. After fertilization, clearing success strongly depends on the
developmental stage of the seed. Therefore, for ovules 2 days
after fertilization, 2 h of incubation is sufficient, while overnight clearing is recommended from 10 days after fertilization.
Alternatively, Hoyer’s solution could be used for seeds 10 days
after fertilization, as described [18].
3. Do not use the first or last siliques after flowering as these are
often sterile and could lead to wrong interpretations.
4. Reducing the distance between the holding disposable medical
hypodermic syringe needle and the one used to perform the
dissection makes the process more accurate. For long siliques,
Identifying Reproductive Mutations in Arabidopsis
385