Isolation of Egg and Sperm Cells from Bauhinia blakeana

Abstract

Isolated sperm cells and egg cells provide useful materials for the study of fertilization in higher plants, under controlled conditions, in the absence of somatic tissue. Viable egg cells from Bauhinia blakeana were isolated using enzymatic digestion and mechanical dissection. Ovules were digested with enzymes for 40–50 min and then transferred to a solution without enzymes for dissection. The three cells of the egg apparatus (i.e., the egg cell and two synergids) were released from a cut ovule by pushing the micropylar region of the ovule with a dissecting needle. Typically, 7–10 egg cells were isolated within 1 h. B. blakeana pollen is bicellular and contains only a vegetative and generative cell at anthesis. Sperm cells were isolated using an in vivo–in vitro method. The isolation of B. blakeana egg and sperm cells potentially provides a basis for in vitro fertilization of this woody dicotyledonous plant and materials for molecular biological investigations of fertilization mechanisms.