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DAPI (4',6-diamidino-2-phenylindole) binds to DNA and labels the nucleus. DAPI staining is a common way to monitor pollen development and look at the effect of various mutants. These images of DAPI stained Arabidopsis pollen were taken on a Zeiss fluorescence microscope in the lab of Dr. Sebastian Bednarek at the University of Wisconsin, Madison (protocol below).
Scale bars = 50 µm.
During the tetrad and microspore stages, the single nucleus is very bright and located in the center of the pollen grain. At the early bicellular stage, just after pollen mitosis I, the larger vegetative nucleus stains very diffusely, whereas the generative nucleus is very bright and compact. This brighter staining is due to the more condensed state of the chromatin in the generative nucleus, probably indicating that it is less transcriptionally active than the vegetative nucleus. As the bicellular stage progresses the vegetative nucleus gradually becomes more distinct, staining as a ring (also seen in the tricellular stage). The generative cell divides during pollen mitosis II to create to two sperm cells, which are very small and bright. The sperm cells have an elongated shape, and associate closely with the (now more distinct) vegetative nucleus to form the "male gametophytic unit" of the tricellular grain.
DAPI Staining Protocol
DAPI Staining Protocol
Pollen Isolation Buffer (PIB)
Pollen Isolation Buffer (PIB)
100 mM NaPO4, pH 7.5
1 mM EDTA
0.1% (v/v) Triton X-100
Mature, Released Pollen:
Mature, Released Pollen:
1) Select 4-6 newly opened flowers (1-2 per infloresence) and pull them off with a pair of forceps
2) Add the flowers to 500 µl Pollen Isolation Buffer (PIB) in a microcentrifuge tube.
3) Vortex for 20 seconds to release the pollen
4) Centrifuge 30 seconds 1,500 g to pellet the pollen
5) Resuspend the pollen in 20-50 µl PIB containing 1 µg/ml DAPI.
6) Place a drop on a glass slide under a coverslip, incubate 5 minutes room temperature, and view with a fluoresence microscope using a DAPI filter set.
Developing Pollen:
Developing Pollen:
1) Dissect the anthers from an unopened bud using a pair of fine-tipped forceps (the same kind one would use for doing crosses). The largest unopened bud in a rosette should contain mature, unreleased tricellular pollen. Successively smaller buds will contain earlier developmental stages; I recommend looking sequentially at each bud in a rosette until you get a feel for the different stages.
2) Place dissected anthers on a glass slide in a drop of PIB containing 3 µg/ml DAPI
3) Cover with a glass coverslip, and use the back of your fingernail to gently squash the anthers between the coverslip and the slide to release the pollen. Wiggle the coverslip around to spread the pollen out. (It may take some practice to learn how to effectively release and spread the pollen without crushing the grains).
4) Incubate 5 minutes room temperature
5) View with a fluoresence microscope using a DAPI filter set.
Note that tricellular pollen stains very brightly and consistently using this protocol, but staining of earlier developmental stages is dimmer and can be spotty (i.e., some sections of the slide will stain okay, but others, particularly near the anther or large clumps of pollen, will be dark). The most difficult stage to stain seems to be the late polarized microspore/early bicellular. Sometimes it is necessary to look around the slide a bit before you can find a nicely stained clump of cells.
Reference: Backues SK, Korasic D, Heese A and SY. Bednarek. (2010) The Arabidopsis Dynamin-Related Protein 2 (DRP2) family is essential for gametophytic development. Plant Cell 22:3218-31.
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