Isolation of sperm cells, egg cells, synergids and central cells from Solanum verbascifolium L

Sperm cells of Solanum verbascifolium L. were isolated by using an in vivo/in vitro method. Hand-pollinated styles were first grown in vivo for several hours, then cut off from their base and cultured in vitro until pollen tubes grew out from the cut end. When the pollen tubes were transferred to broken solution, the sperm cells were released from broken tubes. For isolation of egg cells and associated cells, ovules were digested in an enzymatic solution for 10–20 min and then transferred to an isolation solution without enzymes for the dissection. The digested ovules were cut transversely and the micropyle end was compressed to release the egg apparatus and central cell. Typically, 7–10 egg cells could be isolated in 1 h and batches of cells could be collected together and concentrated using a micromanipulator. The protocol reported here for the isolation of sperm cells and egg cells of S. verbascifolium potentially provides a basis for in vitro fertilization of this dicotyledonous plant, and for the provision of materials for molecular-biological investigations of the mechanisms of fertilization.