Regeneration of anther-derived plants of Hyoscyamus niger L

Abstract

Anthers of Hyoscyamus niger L. were cultured by two different methods. In the first method anthers were cultured in the dark in the late tetrad stage of microspore development on the basal medium of Nitsch and Nitsch (Science 163, 85-87; 1969) supplemented with 5 or 10 mg/l 2,4-dichlorophenoxyacetic acid. The callus which developed was able to produce a large number of plants under photoperiodic conditions of 16 h light from fluorescent tubes at 28° and 20° during the dark cycle. Cytological analysis revealed that about 50% of these plants were haploid. A further imporvement to raise the level of haploids by the use of p-fluorophenylalanine was not achieved. In the second method anthers were cultured in the early mononucleate stage of microspore development on the basal medium of Nitsch and Nitsch which was little modified under various conditions. The largest number of plants which developed directly by embryoids was observed on the medium of Nitsch without indolylacetic acid under photoperiodic conditions of 16 h fluorescent light at 28° and 8 h dark at 20°. Cytological examination determined that approximately 70% of the plants were diploid. A genetic marker was used to ensure homozygoty of the diploid regenerates. 98% of the regenerates which developed via callus as well as by direct formation of embryoids were found to be homozygous and originated therefore from generative cells.