Abstract:

Pollen of Nicotiana tabacum L. is bicellular type, a generative cell and a vegetative cell in a mature pollen grain. To isolate sperm cells pollen tube should be induced in advance. Using an in vivo-in vitro technique, the style was pollinated and kept in vivo growing for 37 h and then cut about 3.5-4.0 cm long to be cultured in a medium containing 15% (W/V) sucrose, 0.01% (W/V) boric acid, 0.01% (W/V) CaCl₂, 0.01% (W/V) KH₂PO₄, at pH 5.0. A numerous pollen tubes grew out of the cut end of the style after being cultured for 3-5 h. When the pollen tubes were transferred to a broken solution just containing 9% mannitol some tubes broke and released the tube content into the broken solution including two sperm cells. Two sperm cells just released from the tube are connected with vegetative nucleus (VN) of pollen tube consisting of a male germ unit (MGU). Two sperm cells of tobacco are dimorphism: one is big and the other small. The small one (Svn) connects with vegetative nucleus and the big one only associates with the small one. Two isomorphic sperm cells might be selective fusion with egg cell and central cell during in vivo fertilization (preferential fertilization). When two associated sperm cells were transferred into a solution containing 0.01% cellulase, 0.008% pectinase and 9% mannitol, the association between two brother sperm cells disappeared, and both cells could be easily separated using a micromanipulator. To probe the mechanism of preferential fertilization two sperm cells from one pollen tube have to be separated each other to find the differences at the molecular level. Two sperm cells were respectively collected into two individual populations, each containing over thousand big sperm cells or small one, using a micromanipulator. The two sperm populations will offer a possibility to find the differences between two sperm cells in genes and proteins by using molecular methods, which will help us to understand the mechanism of preferential fertilization and gametic recognition of higher plants, especially in the species with bicellular pollen.

Key words: sperm cell, sperm dimorphism, preferential fertilization, Nicotiana tabacum