Many aspects of Angiosperm pollen germination and tube growth are discussed including mechanisms of dehydration and rehydration, in vitro germination, pollen coat compounds, the dynamic involvement of cytoskeletal elements (actin, microtubules), calcium ion fluxes, extracellular matrix elements (stylar arabinogalactan proteins), and control mechanisms of gene expression in dehydrating and germinating pollen. We focus on the recent developments in pollen biology that help us understand how the male gamete survives and accomplishes its successful delivery to the ovule of the sperm to effect sexual reproduction.

Chalcone synthase catalyzes the initial step of that branch of the phenylpropanoid pathway that leads to flavonoids. A lack of chalcone synthase activity has a pleiotropic effect in maize and petunia mutants: pollen fertility as well as flavonoid synthesis is disrupted. Both maize and petunia mutants are self-sterile due to a failure to produce a functional pollen tube. The finding that the mutant pollen is partially functional on wild-type stigmas led to the isolation and identification of kaempferol as a pollen germination-inducing constituent in wild-ype petunia stigma extracts. We show that adding micromolar quantities of kaempferol to the germination medium or to the stigma at pollination is sufficient to restore normal pollen germination and tube growth in vitro and full seed set in vivo. Further we show that the rescue ability resides in particular structural features of a single class of compounds, the flavonol aglycones. This rmding identifies another constituent of plant reproduction and suggests that addition or removal of the flavonol signal during pollen germination and tube growth provides a feasible way to control plant fertility.

Ethylene controls fruit ripening. Expression of antisense RNA to the rate-limiting enzyme in the biosynthetic pathway of ethylene, 1-aminocyclopropane-1-carboxylate synthase, inhibits fruit ripening in tomato plants. Administration of exogenous ethylene or propylene reverses the inhibitory effect. This result demonstrates that ethylene is the trigger and not the by-product of ripening and raises the prospect that the life-span of plant tissues can be extended, thereby preventing spoilage.

No abstract

We have developed a general method for electrically introducing DNA into plant cells. Gene transfer occurs when a high-voltage electric pulse is applied to a solution containing protoplasts and DNA. Carrot protoplasts were used as a model system to optimize gene-transfer efficiency, which was measured 24-48 hr after electroporation by the amount of chloramphenicol acetyltransferase activity resulting from the expression of the introduced chimeric plasmids. Gene-transfer efficiency increased with the DNA concentration and was affected by the amplitude and duration of the electric pulse as well as by the composition of the electroporation medium. Our optimized gene-transfer conditions were effective when applied to tobacco and maize protoplasts, demonstrating that the method is applicable to both monocot and dicot protoplasts. Protoplast Isolation. Protoplasts were isolated from rapidly growing carrot suspension cells by incubating 12 ml ofpacked cells in 80 ml of 1% cellulase (Cellulysin, CalbiochemBehring) and 0.5% hemicellulase (Rhozyme, Genencor) in protoplast isolation medium (PIM: 0.2 M mannitol/50 mM CaCl2/10 mM sodium acetate, pH 5.8) for 2 hr at 26°C.Protoplasts were separated from large debris by filtration through 60-gm-mesh nylon screen, centrifuged at 200 x g for 4 min, and washed once either in the same solution without enzymes or in Hepes-buffered saline (10 mM Hepes, pH 7.2/150 mM NaCl/5 mM CaCl2) containing 0.2 M mannitol.Electroporation. Each sample of 3 x 106 protoplasts was resuspended in 1 ml of the desired concentration of phosphate-buffered saline (1 x contains 8 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, and 1.15 g of Na2HPO4 per liter) or of Hepes-buffered saline, containing 0.2 M mannitol with or without plasmid DNA (10 ,g/ml). Electroporation then was carried out according to published procedures (16). The electrical pulse was supplied by an ISCO model 494 power supply set at 2000 V. The solution containing the protoplasts was held at 0°C for 10 min after the electric pulse. The protoplasts then were diluted into MS medium (29) containing 2% sucrose, 0.3 M mannitol, and 2,4-dichlorophenoxyacetic acid (0.1 jig/ml) and incubated at 26°C in a 10-cm plastic Petri dish. Protoplasts subjected to electroporation and then assayed in duplicate produced CAT activities that varied by Abbreviations: NOS, nopaline synthase; CAT, chloramphenicol acetyltransferase; CaMV, cauliflower mosaic virus; kb, kilobase(s). 5824The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.