Begin by stratifying A.thaliana seeds in sterile Milli-Q water for three days at four degrees Celsius. Transfer the stratified seeds to the compost pots by pipetting the stratified seeds. Place the pots in an environmentally controlled growth chamber for approximately six weeks until the inflorescences are well established.
To ensure synchronous flowering, sow the pollen donor and recipient plants and any other appropriate control plant lines together. One day prior to the bioassay, select these stage 12 floral buds from the recipient plant, Having unopened flower buds that will show the opening of the flower and the anther dehiscence on the following day. Next, place a glass slide under a stereo dissecting microscope.
Tape the region of the stem close to the selected recipient flower on that glass slide. Using a pair of fine-tipped forceps, carefully tease open the flower bud and remove all the flower petals and anthers. Once done, ensure that the pistil is undamaged and the stigma is free of contaminating pollen.
