Three pumpkin embryogenic lines were initiated on wounded zygotic embryos cultured on medium with or without 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryo development was controlled by the availability of various compounds in the medium: presence/absence of 2,4-D, nitrogen sources. The highest rate of DNA methylation was in the early embryo stages, predominantly on MSC medium with 2,4-D and on auxin-free medium supplemented with 1.0 m M NH(4)Cl. DNA methylation was correlated with early embryo development in a manner that was not exclusively dependent on the presence/absence of exogenous auxin. DNA methylation decreased during embryo maturation on auxin-free MSC medium and on auxin-free MSC supplemented with 12.3 micro M 5-azacytidine (5-azaC). The embryogenic features of the pumpkin tissue were preserved, even after a 2-month treatment with 5-azaC.

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Anther culture was performed on two local cultivars, Ljubljansko and Vara~dinsko, and the F lcv. Krautman (Bejo-Zaden). The effects on androgenesis of hot and cold temperature treatments and different dissections of anthers were evaluated. In contrast to cv. Krautman, cvs. Ljubljansko and Vara~dinsko produced more embryos after cold pretreatment of flower buds (4°C, 48 h) than after standard treatment (35°C, 24h). Simultaneous cutting of the anther tip and removal of the filament gave the best results in comparison to other tested dissections. Microscopical observations of sectioned anthers revealed enhanced embryo development near the cut ends of the anthers. Ploidy analysis revealed the presence of haploids among embryos resulting from cold treatment (4°C, 48 h), treatment at elevated temperature (35°C, 24 h), and among embryos resulting from dissections of anther tips.

Embryogenic callus was induced by culturing explants of pumpkin hypocotyls on Murashige‐Skoog‐medium with the addition of 3% glucose and one of the following growth substances (or combinations of them): β‐indolylbutyric acid, 2,4‐dichlorophenoxyacetic acid, β‐indolylacetic acid, α‐naphthyl‐acetic acid, adenine (natural), kinetin, autoclaved water‐melon sap and yeast extract (Difco). A large number of embryoids and adventive buds were produced. These were able to develop to normal plants. The 17 strains of embryogenic tissue obtained have maintained their embryogenic characteristics for more than 3 years. The induction of embryogenic callus in pumpkin seems to be strongly dependent on the genetic constitution of each individual plant.