This method will facilitate research on sexual reproduction in Arabidopsis by allowing for the observation of gametophyte interactions with high efficiency and with high sample size number per imaging session. The main advantage of this technique is by keeping the ovules on the septum, the amount and receptivity of the ovules is greatly increased over previous methods. The dissection technique can initially be difficult and requires a steady hand.
Therefore, it's recommended to practice a few times on emasculated pistils before getting started with the experiment. Begin by preparing the pollen germination medium as explained in the text. After the agar is completely melted, pipette 130 microliters of the medium into the 14 millimeter well of a 35 millimeter glass bottom dish.
Rotate the dish until the medium covers the well. Aspirate 40 microliters of medium from the center of the dish using a pipette, leaving behind 90 microliters of medium. To ensure even cooling of the medium, cool the dish on an aluminum block in a humidity chamber before storing the plates at four degrees Celsius overnight.
The morning before imaging, choose healthy Arabidopsis plants that have recently begun to produce as septum donors and stigma donors. Remove the older flowers on the inflorescence before emasculating the three-stage 12B septum donor flowers and 12 stage 12B stigma donor flowers by removing their stamens, sepals, and petals. In the morning 24 hours later, pollinate the stigma donors with the pollen donor flowers that are readily shedding pollen.
Pollination is complete when the stigmas are almost completely covered with pollen. 30 minutes after pollination, remove a healthy and mature septum donor pistil at the pedicle and place it into fresh double-sided sticky tape mounted on a glass slide with the stigma sticking just off the edge of the tape. Pin the pistil securely onto the tape by pressing gently on the pedicle and along the ovary using the backside of a new insulin syringe needle.
Remove the sepal, petal, and stamen debris leftover from the emasculation. Under a stereoscope, use an insulin syringe needle to make two cuts per carpel at the style-ovary junction and the ovary-pedicle junction. Then make shallow cuts along the septum of each carpel and peel back the ovary walls onto the tape, exposing the ovules.
Slide the needle gently between the two septa to cut the replum along the entire length of the pistil without disturbing the ovules. Then cut the top septum near the style so the entire septum lifts away. Next, remove the septum from the pedicle using forceps.
Place the septum as flat as possible on the medium with the micropiles of the ovules facing up. Then press the septum gently until the ovules are slightly embedded in the medium. Place the pollinated stigma donor pistils on fresh, double-sided sticky tape mounted on a glass slide such that the ovary-style junction is off the edge of the tape.
Cut the style straight down using a razor blade and lift it away with the stigma attached to the blade. Remove the stigma from the blade with an insulin syringe needle and place it flat approximately 250 to 300 micrometers from the ovules. Transfer up to 12 stigmas, placing two on either side of each septum.
After incubating in a humidity chamber on the microscope maintained at 92%relative humidity in 21 degrees Celsius, the dish is ready for imaging. Bring the samples into focus under brightfield at a low light intensity, then switch to fluorescent light. Next, mark the areas of the sample on the stage overview to be imaged.
Once done, begin imaging using a multi-stage acquisition scheme with an auto-focus function at the beginning of each overview area. Approximately seven to nine hours post incubation, check to see that the pollen tubes were attracted to and received by the ovules. Successful pollen tube reception shows an explosive burst of the pollen tube in the micropile of the ovules.
When done, stop the acquisition. Eight technical replicates of the semi in vitro excised ovules method were conducted and scored for successful pollen tube reception. Green squares show successful reception and red squares show defective reception.
These experiments revealed that around 28%of ovules show explosive pollen tube burst, resulting in an efficiency ranging from approximately 10 to 50%Using the semi in vitro cum septum method, many more ales ovules were able to be scored and up to 40 instances of successful pollen tube reception were recorded in one imaging session as seen in ovules with green squares. Five technical replicates of this method showed that approximately 79%of the ovules attracting pollen tubes show an explosive pollen tube burst, resulting in an efficiency ranging from approximately 70 to 100%Upon pollen tube rupture, the nucleus of the receptive synergid was observed to degrade at the same time with a resolution of one minute. After karyogamy, the migration of the egg cell nucleus toward the micropile was seen.
Both the left and right synergids were able to serve as the receptive synergid. Critical factors to pay attention to for success with this method are staging, using freshly prepared medium, embedding the ovules in the medium, maintaining high humidity, and ensuring low phototoxicity. One can also couple this technique with two-photon excitation imaging for closer observation of physiological and cytological changes that occur during gametophyte interactions.
One can also couple this with electron microscopy by fixing the ovules.
