hormones, affect seed germination and/or dormancy. A germination test can be performed as follows:
1. Label a 10 cm diameter Petri dish with name and date.
2. Place four layers of 9 cm filter paper in the bottom of the dish
and moisten with distilled water. Remove excess water.
3. Distribute 100 seeds evenly on the surface of the filter paper.
Seal the dish with Parafilm to prevent drying.
4. Place dish at 4
C for 3 days (see Note 22).
5. Move the dish to an illuminated shelf or to a growth chamber
(see Note 47).
6. Record germination percentage after 3–14 days by dividing the
number of seedlings by the total number of seeds, and multiplying by 100.
Germination tests can also be performed on solid media, such
as MS.
3.5 Genetic Crosses
Some species of Arabidopsis, particularly A. thaliana, are mostly
self-pollinating, especially in a growth chamber or greenhouse
setting where insect populations are minimized [18]. It should be
noted that the pollen of Arabidopsis does not disperse through the
air. Therefore, crossing Arabidopsis is mainly conducted through
manual emasculation of flowers just prior to flower opening, followed by transfer of pollen from the desired male parent to the
stigma of the emasculated flower.
Species, such as Arabidopsis halleri and Arabidopsis lyrata, have
natural self-incompatibility mechanisms, which prevent the plant
from self-pollinating and result in obligate outcrossing [19]. For
such species, simple maintenance of a genetic stock cannot easily be
accomplished from a single plant, and it is most convenient to start
with a small population of founders and perform cross-pollination.
The manual techniques for performing genetic crosses of
A. thaliana can be generalized to the related species. The method
for genetic crosses of Arabidopsis have been previously
described [7].
3.6 Floral Dip
Transformation
of Arabidopsis
with Agrobacterium
tumefaciens
In the “floral dip” transformation method, the need for vacuum
infiltration was replaced by the use of Silwet L-77
® , a surfactant
that aids the entry of bacteria into plant tissues [20]. The use of this
protocol revolutionized the field of Arabidopsis functional genomics, by enabling high throughput generation of T-DNA mutants
and other resources that show stable inheritance of the mutations
and other modifications caused by transformation events. The floral
dip has become the most widely used protocol in most research
labs. The method for floral dip transformation was previously
described in Arabidopsis Protocols Methods in Molecular Biology
vol. 1062 [7].
Handling Arabidopsis Plants
15
1. Label a 10 cm diameter Petri dish with name and date.
2. Place four layers of 9 cm filter paper in the bottom of the dish
and moisten with distilled water. Remove excess water.
3. Distribute 100 seeds evenly on the surface of the filter paper.
Seal the dish with Parafilm to prevent drying.
4. Place dish at 4
C for 3 days (see Note 22).
5. Move the dish to an illuminated shelf or to a growth chamber
(see Note 47).
6. Record germination percentage after 3–14 days by dividing the
number of seedlings by the total number of seeds, and multiplying by 100.
Germination tests can also be performed on solid media, such
as MS.
3.5 Genetic Crosses
Some species of Arabidopsis, particularly A. thaliana, are mostly
self-pollinating, especially in a growth chamber or greenhouse
setting where insect populations are minimized [18]. It should be
noted that the pollen of Arabidopsis does not disperse through the
air. Therefore, crossing Arabidopsis is mainly conducted through
manual emasculation of flowers just prior to flower opening, followed by transfer of pollen from the desired male parent to the
stigma of the emasculated flower.
Species, such as Arabidopsis halleri and Arabidopsis lyrata, have
natural self-incompatibility mechanisms, which prevent the plant
from self-pollinating and result in obligate outcrossing [19]. For
such species, simple maintenance of a genetic stock cannot easily be
accomplished from a single plant, and it is most convenient to start
with a small population of founders and perform cross-pollination.
The manual techniques for performing genetic crosses of
A. thaliana can be generalized to the related species. The method
for genetic crosses of Arabidopsis have been previously
described [7].
3.6 Floral Dip
Transformation
of Arabidopsis
with Agrobacterium
tumefaciens
In the “floral dip” transformation method, the need for vacuum
infiltration was replaced by the use of Silwet L-77
® , a surfactant
that aids the entry of bacteria into plant tissues [20]. The use of this
protocol revolutionized the field of Arabidopsis functional genomics, by enabling high throughput generation of T-DNA mutants
and other resources that show stable inheritance of the mutations
and other modifications caused by transformation events. The floral
dip has become the most widely used protocol in most research
labs. The method for floral dip transformation was previously
described in Arabidopsis Protocols Methods in Molecular Biology
vol. 1062 [7].
Handling Arabidopsis Plants
15