Production of Dihaploids in Carnation (Dianthus caryophyllus L.)

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  • 1 Dept. de Genètica Vegetal, IRTA, Ctra de Cabrils s/n, 08348 Cabrils, Barcelona, Spain

Callus and shoot organogenesis were obtained from anthers of Dianthus caryophyllus L. `Manon', `Amapola', `Elsy', and `IB212', harboring mid-uninucleated microspores. Significant differences between genotypes were observed on number of responsive anthers (10.4% to 72.1%) and rescued plants per responsive anthers (1.2% to 4.8%). A modified H medium (Nitsch and Nitsch, 1969) with 20 g/L maltose and 0.25% Gelrite, supplemented with 10 μM 2,4-D and 1 μM TDZ, was most appropriate for callus induction. Plants were regenerated after subsequent subculture to the same medium, but amended with 0.1 μM TDZ. Zymogram types for aminopeptidase (AAP) in polyacrilamide gel electrophoresis proved that all 40 plants regenerated from `Amapola', `Elsy', or `IB212' where heterozygous, and consequently not originated from the microspores but from somatic tissue. Alternatively, in situ-induced parthenogenesis through pollination with gamma-irradiated pollen and in vitro embryo rescue was tested. A total of 92 embryos, including normal and no cotyledonary embryos, were rescued from 38 fruits harvested out of 70 crosses between `Scania' and `Amapola'. Embryos were rescued 21 to 28 days after pollination by culture in a modified E20A (Sauton and Vaulx, 1987) medium. Phosphogluco isomerase (PGI) and Shikimic dehydrogenase (SDH) zymograms in starch gel electrophoresis, and AAP in polyacrilamide gel electrophoresis, indicated the parthenogenic origin of three of the regenerated plants. Flow cytometry of nuclei proved the early diploidization, during in-vitro micropropagation, of the parthenogenic carnation haploid plantlets.

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