Genetic diversity among sublines originating from a single somatic hybrid cell of Duboisia hopwoodii + Nicotiana tabacum

Summary The genetic instability of an intertribal hybrid cell line, Duboisia hopwoodii + Nicotiana tabacum, obtained by mechanical isolation of a single hybrid cell was studied. Ten subclones of calli derived from this hybrid cell line were cultured for 3 years, and their genetic makeup clarified as to nuclear DNA content, chromosome constitution, and peroxidase isozymes. Nuclear DNA content differed in each subclone. In most subclones, mean DNA content was lower than the mean DNA content in the original hybrid cell line determined 1 year after fusion. This decrease in DNA content is partly attributable to the elimination of tobacco chromosomes that occurred in all subclones. The extent to which tobacco chromosomes were eliminated varied among the subclones — evidence that chromosome elimination occurred slowly. Peroxidase isozyme analysis indicated the loss of a tobacco-specific isozyme, thus confirming results obtained by chromosome analysis. Shoots regenerated from two hybrid subclones after 2 years were also heterogeneous in morphology and nuclear DNA content.     

Alkaloid production in cultured roots of three species of Duboisia

Cultured roots were obtained from calluses of Duboisia leichhardtii, D. myoporoides and D. hopwoodii. Cultured roots of all these species produced both tropane and pyridine-type alkaloids. The selected cultured roots of D. leichhardtii showed high contents of tropane alkaloids (hyoscyamine 0.53%, scopolamine 1.16%, on a dry weight basis).     

Isolation and culture of protoplasts from cell suspension cultures of Duboisia myoporoides with subsequent plant regeneration

Protoplasts were isolated from suspension cultures of various cell lines of Duboisia myoporoides R. Br. There were differences among cell lines with respect to optimal conditions for protoplast isolation including the amount and kind of enzymes and the osmoticum concentration. Protoplasts isolated from one cell line were successfully cultured and induced to form cell colonies in liquid modified B5 medium. Addition of conditioned medium, coconut milk and glucose as an osmoticum to protoplast culture medium as well as maintenance of high protoplast density in culture (> 10⁵/ml) were essential to obtain protocolony formation. Reduction of osmoticum concentration and deletion of coconut milk and conditioned medium from the culture medium were necessary to allow further colony development leading to cellus formation. Intact plants regenerated from calli derived from protoplasts were successfully transferred to pots.     

Organogenesis and a general procedure for plant regeneration from callus culture of a commercial duboisia hybrid (D. leichhardtii x D. myoporoides)

Summary The Duboisia hybrid (D. leichhardtii x D. myoporoides) was regenerated from callus derived from shoot tips and young seeds on Murashige and Skoog (MS) medium containing 54M 1-naphthalenacetic acid (NAA) and 1M 6-benzylaminopurine (BA). Callus derived from young seeds was superior to shoot tip callus. Buds were induced from callus on MS medium supplemented with 22M BA. The buds formed shoots when transferred to MS medium with 5 M BA and 0.5M NAA. Shoots were induced to form roots when placed on MS medium containing 25 M indolebutyric acid (IBA). Plantlets so formed were transplanted and subsequently planted out in the open. Approximately 6 months were required for the full regenerative process. Trees so formed have been grown for 3 years, reached a height of 3 metres and flowered and fruited normally.