Biology Reference
In-Depth Information
pollen grains and tubes used for proteomic analyses. The protocols
were initially optimized for
Lilium
pollen [
1
,
2
] but can be easily
adapted to other pollen species.
2
Materials
The pollen viability, namely, their ability to germinate in appropriate
artifi cial media, depends not only on the plant growth conditions
during pollen development but also very much on the collection
time (state of pollen development) and storage conditions. Note
that optimal conditions may vary between plant species and should
be tested. For instance, the lily pollen is stored at −80 °C whereas
tobacco pollen grains (
Nicotiana tabacum
) “survive” for 1-2 years
at −20 °C. How pollen grains are stored also depends on the subse-
quent studies, e.g., for the identifi cation of pollen allergens, pollen
grains may be stored at room temperature whereas for studies on
pollen germination long-term storage in a freezer is necessary.
2.1 Collection and
Storage of Pollen
Grains for In Vitro
Cultures
1. Collect mature, fully dehydrated anthers (
see
Note 1
) from
fl owers with a pair of tweezers and freeze them immediately in
a tube fi lled with liquid nitrogen.
2. Carefully close the lid (
see
Note 2
) and store the shock-frozen
pollen grains at −80 °C (
Lilium longifl orum
) or −20 °C (
N.
tabacum
,
Arabidopsis thaliana
).
2.2 Collection and
Storage of Pollen
Grains for Allergen
Studies
1. Collect pollen grains from the plants during a dry weather
period with almost no wind into an appropriate plastic cen-
trifugation tube (
see
Note 3
).
2. Sieve the pollen grains through a nylon mesh of appropriate
mesh width to separate the grains from other plant material.
Nylon meshes with standard widths are offered by general lab-
oratory suppliers or can be obtained from special companies
(e.g., Pieper Filter GmbH, Bad Zwischenahn, Germany,
www.
3. Store pollen grains at room temperature in a dry, cool, and dark
place. Avoid direct sunlight as well as temperature and humidity
changes as they may damage the pollen grains (
see
Note 4
).
The following media were tested in the authors' lab for germina-
tion and tube growth of pollen grains from various species. Notice
that pollen grains of
A. thaliana
are diffi cult to cultivate in vitro
and often require specifi c attention concerning the plant ecotype,
developmental state of the fl ower, the temperature regime during
plant growth, and others [
3
]. So far, the best results regarding the
total germination frequency, synchrony of germination, and
2.3 Germination
Media
