This protocol describes how to properly dissect flower buds, flowers and young siliques. And the subsequent whole-mounting clearing for diverse staining and observation techniques. This method can help answer key questions in the sexual plant production field.
Such as a steadily mutant analysis or any environmentally and experimentally induced failure in flower development and function. The main advantage of this technique is to allow for easy and quick screening of many developmental stages simultaneously. Visual demonstration of this method is critical, as flower organ dissection steps are difficult to learn because they require prior knowledge of flower anatomy and dissection skills.
To begin, use small scissors to remove flowers from synchronized plants. And immediately place them into individual microcentrifuge tubes, containing an appropriate fixative. Next, remove the fixative and add enough 70%ethanol to completely cover the samples.
Return the samples to four degree Celsius for at least 24 hours. Then fill a watchmaker's glass with freshly made 70%ethanol and place it into a small Petri dish for support. Then place the inflorescence into the watchmaker's glass and ensure it is totally covered by the ethanol.
Using forceps and a syringe equipped with a needle, dissect the flower under the stereo microscope by tearing apart the sepals, petals and stamen. Then place the watchmaker's glass aside and use a glass slide for the final dissection. Move the sample to slide and add 10 microliters of freshly made 70%ethanol.
Make cuts on each side of the carpal, rotating it as needed. Then gently remove the valves to expose the ovules. Make a sharp small cut to detach half of the transmitting tissue from the receptacle and use forceps and a needle to tear apart the two halves.
Remove the stigma style and the peduncle with sharp cuts. Using the forceps and needle gently flip and arrange the still attached ovules to obtain two parallel rows. Use a small piece of blotting paper to remove ethanol from the sample on the glass slide.
Then add 20 microliters of clearing solution to the slide. Using a pair of syringes with hypodermic needles position the specimens and remove any remaining air bubbles. Next, gently place a cover slip onto the slide and wait until the clearing solution fills the space between the cover slip and the sample.
Place the slide in a slide holder and leave it under the fume hood. So let's freshly harvested flower buds that are about to open with non-dehiscent anthers. Then prepare a 96 well plate with enough Alexander solution to fully submerge the flower buds.
Remove the sepals and petals to expose the anthers and dip them into the Alexander solution. Allow the plate to sit under the fume hood for one to three hours. Then replace the Alexander solution with Herr's four and a half solution and incubate the plate under the fume hood overnight.
The next day use forceps to carefully move the cleared buds to a new slide. Next, dissect the stamens and remove all other tissues from the samples. Then proceed with chloral hydrate based clearing and combined clearing staining using Herr's four and a half solution.
First move the flower from the watchmaker's glass to the slide and use a blotting paper to remove excess ethanol. Add five to 10 microliters of DAPI solution. Using two syringes with hypodermic needles, dissect the stamens and remove all other flower organs.
Then release the pollen grains into the DAPI solution. Remove all debris from the stamen from the slide ensuring that only the pollen grains are left on the slide. Removing all the stamen debris from the slide is the most critical step for exine removal.
Only pollen grains should be left between the slide and cover slip. Next, place a cover slip on the specimen bearing slide and add the minimum amount of DAPI solution needed to fill the space between the slide and the cover slip. Place an index finger gently on the cover slip and make a quick short sliding movement.
Place the slide in a human box in the dark at four degrees Celsius for at least 15 minutes. Then observe the slide under wide field florescence or confocal microscopy within 24 hours. Move the dissected sample from the watchmaker's glass to a glass plate with cavities filled with 1%SDS and a 0.2 sodium hydroxide solution.
Incubate overnight at room temperature. The SDS sodium hydroxide solution will quickly clear the samples, making them appear transparent within a few minutes. Carefully rinse the sample with water and place it onto a clean slide.
Then add 20 to 40 microliters of staining solution to the slide. Depending on the floral organ to be analyzed and on the researcher's skills this DS treatment can be carried out before, for experienced researchers or after the dissection step for less experienced researchers. Next place a cover slip onto the slide taking care not to crush the preparation.
Then place all the prepared slides into a human box covered with aluminum foil and incubate at four degrees Celsius for one hour. Finally observe the specimen under wide field florescence or confocal microscopy. Following the dissection procedure, described in the protocol, unnecessary tissues that might hinder observations are removed and more detailed images are achieved.
Depending on the objective, chloral hydrate staining can be used to characterize early flower development at the whole flower bud level, meiosis and micro-gomedal genesis in the anther, early ovule development and the specification of the megaspore mother cell, female go-med-ified development, or even pollen tube invasion of the center grid. Combined Alexander staining and Herr's four and a half clearing, enables the evaluation of pollen abortion within a given locule or an anther as a whole. While viable pollen grains show a red cytoplasm, aborted ones show an empty cytoplasm and eventually they're irregularly shaped and totally crushed.
Removing the exine results in an increased staining intensity of nuclei and higher details of chromatin organization. Using and SDS sodium hydroxide clearing prior to staining made inflorescence transparent. And resulted in higher staining of callose within floral tissues.
It revealed usually difficult to see callose accumulations. Such as the Callose layer that separates the generative cell from the vegetative cell. Even when pollen grains were still contained within the anther.
Once mastered, these techniques can be done in less than 48 hours, if they are performed properly. While attempting this procedure, it's important to remember that the results largely depend upon a good sample preparation, such as using the proper fixative, performing the dissection properly and isolating the tissue of interest on the slide.
