thereby making them visible under UV light inside maternal pistil
tissues and ovules.
In contrast to pollen grains and tubes, the female gametophyte
or embryo sac is less accessible, as it develops inside the ovules that
are enclosed themselves by two fused carpels. Embryo sacs consist
of one egg cell surrounded by two synergid cells at the micropylar
region of the ovule, one central cell and three antipodal cells at the
chalazal pole [8]. Marker lines have now been developed showing
each of these cells simultaneously [9], and a number of protocols
that can be used to recognize and characterize embryo sac mutants
[10]. During the process of double fertilization one sperm cell fuses
with the central cell to produce the endosperm and the other one
fuses with the egg cell to form the embryo [11]. Life-cell imaging
procedures are now available to visualize the entire fertilization
process [12–15] and thus allowing to identify fertilization failures.
Gametophytic and fertilization mutants prevent ovule development
resulting in small aborted ovules. This is in contrast to mutants
being affected during seed development. Here, mutant phenotypes
are usually correlated to an arrest of embryo and/or endosperm
development at a stage when the affected gene function is required
[16, 17] leading to seeds that are often smaller than wild type and
whitish. Many essential genes including so-called housekeeping
genes are embryo lethal and are thus often identified in mutant
screens [1, 18].
Although a number of protocols have been reported to study
and visualize individual reproductive processes, in this chapter we
present a simple and structured step-by-step guideline describing
all basic protocols routinely used to quickly investigate in a logical
order the origin of reproductive alterations in a given reproductive
mutant.
2 Materials
2.1 Silique
Dissection
1. Mature green siliques.
2. Two high-precision tweezers (tip width 0.1 mm  length
110 mm) and fine-tip scissors.
3. 1.5 mL Microcentrifuge tubes.
4. Standard stereomicroscope equipped with a 5:1 zoom
minimum.
5. Standard glass microscope slides.
6. Disposable medical hypodermic syringe needles (0.4 mm diameter and 13 mm length).
7. Micropipette of 1 mL capacity.
8. Fixing solution: !99.8% ethanol, p.A., absolute, acetic acid,
(3:1) (v/v).
374
Marı ´a Flores-Tornero et al.
tissues and ovules.
In contrast to pollen grains and tubes, the female gametophyte
or embryo sac is less accessible, as it develops inside the ovules that
are enclosed themselves by two fused carpels. Embryo sacs consist
of one egg cell surrounded by two synergid cells at the micropylar
region of the ovule, one central cell and three antipodal cells at the
chalazal pole [8]. Marker lines have now been developed showing
each of these cells simultaneously [9], and a number of protocols
that can be used to recognize and characterize embryo sac mutants
[10]. During the process of double fertilization one sperm cell fuses
with the central cell to produce the endosperm and the other one
fuses with the egg cell to form the embryo [11]. Life-cell imaging
procedures are now available to visualize the entire fertilization
process [12–15] and thus allowing to identify fertilization failures.
Gametophytic and fertilization mutants prevent ovule development
resulting in small aborted ovules. This is in contrast to mutants
being affected during seed development. Here, mutant phenotypes
are usually correlated to an arrest of embryo and/or endosperm
development at a stage when the affected gene function is required
[16, 17] leading to seeds that are often smaller than wild type and
whitish. Many essential genes including so-called housekeeping
genes are embryo lethal and are thus often identified in mutant
screens [1, 18].
Although a number of protocols have been reported to study
and visualize individual reproductive processes, in this chapter we
present a simple and structured step-by-step guideline describing
all basic protocols routinely used to quickly investigate in a logical
order the origin of reproductive alterations in a given reproductive
mutant.
2 Materials
2.1 Silique
Dissection
1. Mature green siliques.
2. Two high-precision tweezers (tip width 0.1 mm  length
110 mm) and fine-tip scissors.
3. 1.5 mL Microcentrifuge tubes.
4. Standard stereomicroscope equipped with a 5:1 zoom
minimum.
5. Standard glass microscope slides.
6. Disposable medical hypodermic syringe needles (0.4 mm diameter and 13 mm length).
7. Micropipette of 1 mL capacity.
8. Fixing solution: !99.8% ethanol, p.A., absolute, acetic acid,
(3:1) (v/v).
374
Marı ´a Flores-Tornero et al.