The overall goal of this procedure is to isolate young plant embryos. This is accomplished by first dissecting the seeds. The second step is to gently crush the seeds in an isolation buffer.
Next embryos are screened for and then collected by micro aspiration under an inverted microscope. The final step is to recollect all embryos in a minimal volume after washing and to transfer them to the destination buffer. Ultimately, embryos can be used for RNA or DNA extraction to analyze the transcriptome or epigenome, or used for cytological analyses, such as gene reporter assays, or fluorescent in situ hybridization.
The main advantage of this technique is to be able to isolate plant embryos in bulk and at specific developmental stages. Generally, individuals new to this method will have no problems to quickly and efficiently apply it. To begin, select the best shaped capillaries under a stereo microscope that were previously siliconized pulled and cut.
As described in the text protocol, the opening should be smooth and around 50 to 100 microns. Next, wash the siliconized multi-well glass microscope slides in coplin jars for 10 minutes in 10%SDS following detergent wash. Wash the slides twice for two minutes.
In nuclease free water for two minutes in 70%ethanol for two minutes in 100%ethanol and then air dry. Then spread about 0.5 microliters of 10 milligram per milliliter, bovine serum albumin, or BSA with a pipette over the whole surface of each well and air dry. Next, prepare the embryo isolation microscope.
Use an inverted microscope with a 10 x and 20 x magnification objective. Connect the glass capillary to a micro injector to coat the micro capillary. Take up about six microliters of BSA and release it.
Collect sleeks at the desired stage of development, either following crosses or self fertilization sole's. 2.5 days after pollination are used here and contain two to four cell embryos. Remove the seeds from 10 to 15 LICs under a stereo microscope with forceps and insulin.Needles.
Immerse the seeds in 20 microliters of isolation buffer in a two milliliter round bottom eend tube placed on ice. Gently crush the seeds with a plastic pestle pre-cleaned as described in the text protocol to release the embryos until the seed extract is cloudy. The force to apply is to be determined by every user upon trial.
Rinse the pestle with 300 microliters of isolation buffer to wash the pestle and dilute the sample. Spin down the extract at 5, 000 chi for five seconds following centrifugation. Gently resuspend the pelleted extract by pipetting up and down two to three times.
Filter the extract with a 30 micron nylon mesh mounted on tube adapters and swing the tube. Rinse the mesh with an additional 200 microliters of isolation, buffer, and swing.Again. To prepare slides for embryo isolation, place a clean BSA coated and siliconized multi-well slide on the stage of the inverted microscope.
Place the capillary above the slide and adjust the position to have the opening in the field of view. After Resus, suspending the filtered seed extract by pipetting gently up and down pipette two droplets of 40 to 50 microliters of seed extract into one or two wells. Screening only one or two drops at a time, prevents evaporation of the sample place 50 microliters of fresh isolation buffer in a well of a different slide prepared as before.
To serve as the first wash drop. Keep this slide in a covered humid chamber to prevent evaporation. Screen the droplets of seed extract for embryos at the desired stage with the 10 x magnification objective if necessary.
Confirm the stage with the 20 x magnification. The embryos usually sink to the bottom of the slide. The most important aspect of this procedure is to collect embryos with minimal debris.
This can be achieved by cleaning with a tungsten needle, by carefully controlling the wash drop for potential carry debris and by repeating wass if necessary. Manually remove debris around the embryo with a tungsten needle, an insulin needle, or similar equipment. Move the glass capillary near the embryo using the micro manipulator and take up the embryo with as little solution as possible.
Collect several embryos and release them in the first wash.Drop. For molecular applications for cytological applications, the embryos can go directly to the destination buffer. Each collecting round should be kept within five to 10 minutes, and embryos should be collected in a minimal volume.
Repeat the screening and collection until the desired amount of embryos is gathered in the first wash drop. If debris are carried over into the wash drop, remove them with a needle before recollecting all embryos at once from the drop. Release the embryos into a second wash drop of 50 microliters and repeat the recollection.
Finally, release the embryos in the destination buffer. The transfer should involve only a contact and not immersion of the capillary tip shown. Here are several embryos in the last drop following washes.
The isolated plant embryos can now be used for various downstream applications to control the embryonic CD NA.Libraries prior to sequencing. Embryo specific transcripts were amplified shown. Here are the results of PCR amplification on CD NA.Libraries from two to four cell embryos washed one time, and on genomic DNA embryo isolation has proven very useful in reporter gene assays with low embryonic expression and where the maternal seed coat confounds detection shown here is an embryo expressing a GUS reporter gene under the control of the Madea promoter.
Finally fluorescent in C two hybridization was applied to study the nuclear architecture in isolated embryos embedded in acrylamide pads on slides. An example using probes against the centromere repeats and nuclear lower organizing regions is shown here. Once mastered, this technique allows the isolation of 30 embryos in less than an hour for cytological applications, and in less than three hours for molecular applications, including all the washing steps.
Actually, this technique will enable the plant research community to explore genetic and epigenetic mechanisms in embryogenesis.
