A Micromanipulation Method for Artificial Fertilization in Torenia

156

182

Electrofusion-mediated in vitro fertilization of maize using single sperm and egg cells was performed. Sperm cells were released from pollen grains after rupture of the latter by osmotic shock in the fusion medium (0.55 M mannitol). Egg cells were isolated by enzyme treatment (pectinase, pectolyase, hemicellulase, and cellulase) followed by mechanical isolation. The conditions generally used for the electrical fusion of protoplasts of somatic cells were also applied to the protoplasts of gametic cells of maize. Electrofusion was performed with single pairs of gametes under microscopic observation. The mean fusion frequency was 79%. Isolated egg cells of maize showed protoplasmic streaming during 22 days of culture, but they did not divide. However, after fusion of the sperm with the egg cells, these fused cells did develop, with a mean division frequency of 83%, and grew to multicellular structures. Egg cells and fusion products were cultivated with a maize feeder-cell system.

Section: Introductionmentioning

confidence: 99%

In vitro fertilization of single, isolated gametes of maize mediated by electrofusion

Kranz¹,

Bautor²,

Lörz³

1991

156

182

“…The fact that sperm cells have a haploid genome could be advantageous in in vitro culture experiments (McConchie and Knox 1986). Another use of isolated sperm cells could lay in their fusion with isolated female gametes in order to achieve in vitro fertilization (Keijzer 1988).…”

Section: Discussionmentioning

confidence: 99%

Isolation of sperm cells from mature pollen grains of Spinacia oleracea L.

Theunis¹,

Went²

1989

“…Zhou et al 1988). Subsequent purifications have been performed manually (Keijzer et al 1988) or by centrifugation on a sucrose gradient (Russell 1986; Cass and Fabi 1988) or a percoll gradient (Dupuis et al 1987; Nielsen and Olesen 1988; Tanaka 1988; Theunis and Van Went 1989). In Brassica campestris sperm cells have been collected on a membrane filter (Hough et al 1986).…”

Section: Methodsmentioning

confidence: 99%

Isolation of male and female gametes in higher plants

Theunis

Pierson

Cresti

1991

The availability of generative cells, sperm cells, embryo sacs and egg cells from angiosperm plants in isolated conditions has opened up many prospects: study of the mechanism of recognition and fusion between gametes of opposite sex and detailed observation of the process of fertilization, biochemical and genetic analysis of gamete-specific components and genetic engineering in combination with in vitro fertilization. This review provides a list of about ninety publications, in which the isolation of male or female angiosperm gametes and the closely related generative cells and embryo sacs is reported. The species used are summarized in two tables. A description is given of the diverse isolation techniques, which consist of enzymatic digestion, bursting of pollen by osmotic shock, squashing, grinding and micro-dissection. Viability of isolated cells and yield, two important aspects of biotechnological manipulation, are emphasized. A critical evaluation of the most significant results obtained so far with isolated material is presented together with notes on prospects for the future.