Optimized methods for the isolation of Arabidopsis egg cell, synergid cell and developing embryos

Abstract

Arabidopsis female gametophyte, so called embryo sac, contains seven cells with eight haploid nuclei. An egg cell fertilized with a sperm cell generates a diploid zygote which becomes an embryo while a diploid central cell fertilized with the other sperm cell forms an endosperm. Synergid cell is known to play an important role for pollen tube entry so that the two sperm cells can be successfully delivered into the ovules for the double fertilization. After fertilization an embryo develops within the seeds along with several distinctive stages; globular, heart, torpedo, bending torpedo and mature green stage. Except central cells, epigenetic states of the female gametophytic cells are still largely unknown mainly due to the technical difficulties of isolation of that specific cells that are deeply buried within the several layers of the other maternal tissues. To explore the epigenetic state, isolation of pure egg cells and synergid cells is prerequisite for the construction of DNA methylome. Therefore, here I present the optimized conditions for the isolation of egg cells and synergid cells using cell-specific markers. This method involves in degradation of the cell walls and making protoplasts from the ovules to take out the egg cell and synergid cell within the embryo sac. Then, those cells can be picked up using microcapillary under the microscope. For the developing embryos, the sample enrichments are optimized depending on the embryo developmental stages for the methylome construction. The methods I developed will help to understand the epigenetic states and the cell fate between egg cell and synergid cell that are originated from the same mitotic products of the megaspore. My optimized method for the developing embryos, especially for the earlier embryos, will also help to broaden our knowledge for the epigenetic reprogramming during plant reproduction