[Plants regeneration from genetically transformed root and callus cultures of periwinkle Vinca minor L. and foxglove purple Digitalis purpurea L].

Plants regenerated from hairy roots and calluses of foxglove purple and periwinkle have been obtained. It was found that organogenesis in hairy root culture occurs spontaneously on hormone-free medium but with different efficiencies. The frequency of direct shoot formation from root cultures was up to 60% in Digitalis and 3.7% in Vinca. Addition of 1 mg/l BA, 0.1 mg/l NAA and 5% sucrose to B5 medium increased regenerative capacity of Vinca roots up to 19.1%. Regenerated plants showed morphological features typically seen in Ri-transgenic plants. They include growth and plagiotropism of the root system, increased shoot formation, changed leaf morphology and short internodes.

Biotransformation of 21-O-acetyl-deoxycorticosterone by cell suspension cultures of Digitalis lanata (strain W.1.4).

Cell cultures of Digitalis species are known to accept exogenous substrates for biotransformation reactions. We here report the biotransformation of 21-O-acetyl-deoxycorticosterone (1) by cell suspension cultures of Digitalis lanata strain W.1.4. Nine derivatives of 1 were obtained and their chemical structures determined by spectroscopic methods. 2ß-Hydroxylation and C-21-glucosylation of the steroidal nucleus were described for the first time in suspension-cultured plant cells. Steroid 5α- and 5ß-reduction products were also observed. Among the compounds isolated and structures elucidated were 2ß,3ß,21-trihydroxy-4-pregnen-20-one, 2ß,3α,21-trihydroxy-4-pregnen-20-one and 3ß,21-dihydroxy-5α-pregnan-20-one-3ß-O-ß-glucoside.

Ultrastructural evidence for a dual function of the phloem and programmed cell death in the floral nectary of Digitalis purpurea.

BACKGROUND AND AIMS: The floral nectary of Digitalis purpurea is a transitory organ with stomatal exudation of nectar. In this type of nectary, the nectar is thought to be transported to the exterior via intercellular ducts that traverse the nectariferous tissue. The latter is also traversed by a ramified system of phloem strands from which prenectar sugar is most probably unloaded. The aims of this study were to provide some of the basic information needed to evaluate the possible mechanism involved in nectar secretion and to discover the fate of the nectary. METHODS: The ultrastructure of the nectary was investigated at different stages of development by analysis of a series of ultrathin (7 x 10(-8) m) sections 7 x 10(-7) m apart from one another. Proportions of the cells typical of the nectary were documented by 3D-reconstruction and morphometry. KEY RESULTS: The phloem consisted of variably shaped sieve elements and companion cells which, as a rule, were more voluminous than the sieve elements. Direct contact between the phloem strands and intercellular ducts was observed. In contrast to the phloem, which remained structurally intact beyond the secretory phase, the nectariferous tissue exhibited degenerative changes reminiscent of programmed cell death (PCD), which started as early as the onset of secretion and progressed in a cascade-like fashion until final cell death occurred in the exhausted nectary. Hallmarks of PCD were: increased vacuolation; increase in electron opacity of individual cells; progressive incorporation of plasmatic components into the vacuole reminiscent of autophagy; degradation of plastids starting with hydrolysis of starch; deformation of the nucleus and gradual disappearance of chromatin; loss of tonoplast integrity and subsequent autolysis of the rest of cellular debris. Degeneration of the cells occurred against a background of increasing cell size. CONCLUSIONS: The cytological and anatomical evidence presented here, and calculations of the solute fluxes necessary for accumulation of starch and for the production of nectar support the view that: (a) in the foxgloves' nectary, apoplastic phloem unloading dominates, at least during exudation of nectar; (b) the obsolete nectary may be dismantled by PCD; and (c) at least the products of late nectary degradation are loaded via the apoplast into the unchanged phloem and exported to sinks elsewhere in the plant for reallocation.

Haploid plants regenerated from androgenic cell cultures of Digitalis lanata.

Androgenic callus was obtained from cold treated anthers and pollen of Digitalis lanata. The callus was mixoploid and contained haploid, diploid and tetraploid cells as shown by impulse cytophotometry. Haploid cell lines were selected by colony cloning. They were unstable and selection had to be repeated every 1-2 months. Mixoploid shoot cultures were derived from embryogenic haploid cell lines via somatic embryos. Haploid shoots were selected by explanting shoot tips. The shoots showed wide variability in cardenolide content and profile. Rooting of the haploid shoots resulted in haploid plants. These plants were smaller in size than diploid plants. Often the flowers were morphologically abnormal and showed male sterility due to crippled anthers.

Extracellular polysaccharides produced by suspension-cultured cells from Digitalis lanata.

Extracellular polysaccharides (ECP) were isolated in yields of up to 4 mg/ml from the culture media of suspension-cultured cells from Digitalis lanata Ehrh. ECP content was increasing continuously over the first ten days of cultivation and then stayed constant until day 20. ECP were fractionated by ion-exchange chromatography into two neutral and one acidic fractions. Further fractionation was achieved by gel-permeation chromatography (GPC). One neutral fraction was separated into two distinct fractions with average molecular weights of 160 and 70 kDa, respectively. The second neutral fraction was hetero-disperse in GPC with average molecular masses of 112, 32, and 8 kDa. Polysaccharides of all neutral fractions consisted of glucose, xylose, galactose, and arabinose. Methylation analysis indicated these fractions to contain xyloglucans besides minor amounts of highly branched arabinogalactans. Xyloglucans were, using endo-beta-(1-->4)glucanase, fragmented into subunits which were identified mainly as tri- and pentasaccharides. The acidic fraction eluated as a single peak during gel-permeation chromatography with an average molecular weight of 56 kDa. Analysis of carbohydrate composition and linkage analysis indicated that this polysaccharide is an acidic arabinogalactan. 2,6-Dideoxysugars, the typical carbohydrate components of cardiac glycosides in Digitalis lanata, were not detected in ECP.

21'-Di-dehydro-deacetyllanatoside C, a biotransformation product of deacetyllanatoside C from senescent shoot cultures of Digitalis lanata.

Feeding deacetyllanatoside C to senescent shoot cultures of Digitalis lanata resulted in the formation of a new product, which was isolated by semi-preparative HPLC. The molecular structure was elucidated by means of HPLC-mass spectrometry and NMR as 21'-di-dehydro-deacetyllanatoside C.

Electrofusion of plant cell protoplasts under microgravity--a D-2 spacelab experiment.

Plant cell protoplasts, derived from sexually incompatible plant species, have proved to be a good system for somatic hybridization by electrofusion. Under microgravity, an increase in fusion yield can be expected, especially if the parental cells differ markedly in size or specific density. On the D-2 spacelab mission flown in 1993, electrofusion experiments were performed with three different objects, i.e. tobacco as model system, Helianthus as an important crop, and Digitalis as a plant of pharmacological interest. The resulting fusion products were cultivated (along with parental cells) for 10 days under microgravity, and subsequently regenerated on ground for biochemical analysis. Results are presented on the observation of the fusion process during flight, heterofusion yields, ultrastructural investigation of fusion products immediately after fusion, and characterization of the resulting hybrids. The results are interpreted on the background of earlier microgravity-experiments on sounding rockets or parabolic flights.

12 beta-Hydroxylation of digitoxin by suspension-cultured Digitalis lanata cells: production of digoxin in 20-litre and 300-litre air-lift bioreactors.

A biotransformation process for the production of digoxin was developed using Digitalis lanata cell suspension cultures. Digitoxin was used as the substrate for biotransformation. Digoxin production was carried out in a variety of vessels, including 1-l exsiccators, 20-l glass reactors and a 300-l air-lift bioreactor. A culture volume of 200 l was established after 28 d and the cells were then cultured semi-continuously in a 300-l bioreactor employing the draw-fill cultivation method. Maximal digoxin production was achieved in an 8% glucose medium with a production optimum after 40-60 h of incubation in the presence of 0.65-0.8 mmol digitoxin per l. Levels of 0.52, 0.53 and 0.60 mmol digoxin per l suspension were achieved in 1-l, 20-l and 300-l vessels, respectively. About 80% of the digoxin produced was found in the bathing medium.

Two-stage cultivation of Digitalis lanata cells: semicontinuous production of deacetyllanatoside C in 20-litre airlift bioreactors.

A two-stage cultivation method was employed to develop a semicontinuous biotransformation process for the production of deacetyllanatoside C, a cardenolide of the important digoxin series. Digitoxin was used as the substrate for biotransformation. The process was optimized in 1-l shake flasks and then established on the 20-l scale using two airlift bioreactors, one for cell growth (working volume 12 litres) and another for deacetyllanatoside C production (working volume 18 litres). Growth and production phases were synchronized and the process finally ran semicontinuously in 7-d cycles. Six consecutive production runs were performed yielding a total of 43.8 g deacetyllanatoside C.