Isolation And Purification Of Generative And Sperm Cells In Tomato

The process of male gametophyte development is related to fine regulation. With the help of high throughput omic technology, we wish to learn more about the feature of the cells in each period of male gametophyte development at transcriptome and methyome level in order to have a deeper knowledge of the molecular mechanism related to cell identity and cell differentiation. But the first and the most important step is getting large numbers of highly purified generative cells and sperm cells. For this purpose, we did some research to isolate generative cells and sperm cells in large scale.We chose tomato pollen as the experimental material. After germination cultivating in vitro for20min when germinated pollen grains had a pollen tube shorter than the diameter of the pollen grain, we successfully released generative cells by osmotic shock. And then, we made them purified by Percoll density gradient centrifugation. On another hand, through germination cultivating in vitro for10h, generative cells completed a cell division and gave rise to a pair of sperm cells. We used an enzymolysis approach combined with Percoll density gradient centrifugation to release and purify sperm cells in a large scale. The productivity of isolated generative cells is2.8%～5.6%and the cell density is0.5×107～1×107/mL. Additionally, the productivity of sperm cells is3.7%～5.5%and the sperm cell density is1.0×107～1.5×107/mL. Both types of isolated cells were shown viable by FDA. Our study also showed that the density of pollen grain in the medium is a key factor for pollen tube growth. An osmotic shock buffer without inorganic ion and buffer agent will be beneficial to make the cytoplasm keep diffuse, which is very important for purifying generative cells. Besides, isolating cells in room temperature may be good for keeping both types of cells viable. This is the first time to isolate viable generative cells and sperm cells in tomato for deeper study at omic level, male gametophyte development and cellular function differentiation.