Solasodine production by immobilized cells and suspension cultures of Solanum surattense

Summary Immobilized cells ofSolanum surattense Burm release far more solasodine into the medium than free cell suspension cultures. This enhancement is probably due to stabilization of cells after immobilization as well as the effect of growth hormones in the medium.     

Antifungal stress metabolites from Solanum aviculare

The steroidal alkaloids 5β-solasodan-3-one and solasodenone were isolated as antifungal stress metabolites from leaves of Solanum aviculare stressed by vacuum infiltration with water. The constitutive leaf alkaloids, solasodine and solamargine, also showed antifungal activity.     

Solasodine production in rapidly proliferating tissue cultures of Solanum laciniatum ait

Callus and suspension cultures, established from seedling and leaf explants of Solanum laciniatum Ait were analysed for solasodine using a spectrophotometric assay. Solasodine concentration in both types of culture ranged from 0.5 – 1.0 mg/g dry wt., with a small number of callus explants containing higher concentrations. There was no overall fall in concentration as a result of serial subculture, and in suspension cultures the level remained constant throughout a single passage. Solasodine concentration was enhanced by the induction of organogenesis in both primary leaf explants and callus. ABA, added at 0.04 mg 1^?1, increased solasodine yield in callus cultures whilst CEPA, at concentrations of 10 mg 1^?1 and higher, inhibited production. Dark grown callus contained significantly more solasodine than light grown.     

Solasodine production in transformed organ cultures (roots and shoots) of Solanum eleagnifolium Cav.

Summary Transformed organ cultures of Solanum eleagnifolium Cav. (roots and shoots) were obtained by infection with different Agrobacterium species (A. rhizogenes and A. tumefaciens). The growth index and the solasodine productivity of both kinds of cultures were determinated for several clones. Transformed roots (clone 5) shows a Growth Index of 39.07 and a solasodine yield of 1.90 ± 0.08 mg g^-1DW . Solasodine content in clone 5 of transformed roots (1.9 ± 0.08 mg g^-1 DW) is similar to those obtained by hormonal manipulation (1.00 – 1.68 mg g^-1DW) but higher than those found in shoots and leaves of the wild-grown plant (0.3–0.4 mg g^-1DW).     

Biotransformation of (−)-limonene using Solanum aviculare and Dioscorea deltoidea immobilized plant cells

The biotransformation course of S-(–)-limonene by Solanum aviculare and Dioscorea deltoidea plant cells was studied using 5 different immobilization methods (entrapment into alginate, carrageenan and pectate gels, binding to polyphenyleneoxide and permeation into polyurethane foam). All techniques, with exception of permeation into pre-formed matrix, affected the product ratio. The main products were cis- and trans-carveol and carvone.     

Effect of cell-cycle inhibition on synthesis of steroidal alkaloids by Solanum aviculare plant cells

Summary Addition of 40 mg l^-1 hydroxyurea to Solanium aviculare cultures resulted in complete inhibition of growth and an increase in the levels of steroidal alkaloids in the biomass. These results demonstrate that solasodine synthesis and growth can be uncoupled, and suggest that steroidal-alkaloid production can be significantly enhanced by inhibiting growth at high cell density. Treatment with 1 mg l^-1 abscisic acid had a negligible effect on growth and product synthesis.     

Plant growth regulator-induced xylogenesis in cotyledons of Solanum aviculare

Treatment of explanted cotyledons of Solanum aviculare with2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine resultsin the transformation of the mesophyll cells into tracheary,sclereid-like and vessel xylem elements. The initial programmingfor xylogenesis can be achieved with as little as 10 h exposureto the plant growth regulators when endogenous auxin levelsremain relatively high. The externally applied growth regulatorswould appear to enter the cotyledons both from the cut end andvia the adaxial epidermis. Key words: 4-D, BAP, Solanum aviculare, cotyledons, xylogenesis, vessels, carboxylesterase     

Oxygen transfer and culture characteristics of self-immobilized Solanum aviculare aggregates

Oxygen transfer characteristics of self-immobilized Solanum aviculare cells were measured using aggregates 3.0 to 12.5 mm in diameter. Apparent specific oxygen uptake rates in the absence of external boundary layers varied from 5.9 x 10(-11) to 8.5 x 10(-7) kg kg(-1) s(-1) dry weight, but did not decline continuously with increasing particle size. The effective diffusivity of oxygen in deactivated aggregates increased with particle diameter, varying from 5.0 x 10(-11) to 1.0 x 10(-9) m(2) s(-1) or between 2% and 40% of the molecular diffusivity in water at the same temperature. Gas spaces detected in the larger aggregates were confined to the central core and were not distributed throughout the tissue to facilitate oxygen transfer. Oxygen consumption rates in the absence of diffusional limitations were estimated using the relationship between the observable Thiele modulus and effectiveness factor for zero-order reaction. The calculated results indicated severe oxygen limitations in the aggregates, but were inconsistent with the observation that relatively large S. aviculare aggregates contained a high fraction of viable cells and were capable ofactive growth and steroidal alkaloid synthesis. This work suggests that oxygen delivery is facilitated in living plant cell aggregates by mechanisms which depend on metabolic activity and which do not function in deactivated cells. (c) 1995 John Wiley & Sons Inc.     

Solasodine production from cell culture of <Emphasis Type="Italic">Solanum hainanense</Emphasis> Hance

Stem explants of Solanum hainanense Hance plantlets were cultured on Murashige and Skoog solid medium, containing 3% (w/v) sucrose, supplemented with 0.1 mg/L benzylaminopurine (BAP) and 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) for callus production. To establish the cell suspension culture, 3 g of fresh callus were cultured in 50 mL of the same medium, but without a solid agent, at an agitation speed of 120 rpm. Every 15 mL of culture was sub-cultured in fresh MS liquid medium for maintenance. The cell biomass of S. hainanense reached a maximum value of 18.47 g after 4 weeks of culture on the same MS medium, but with the sucrose content increased to 4%, at an agitation speed of 150 rpm, with 20 mL of inoculum. Analysis via high performance liquid chromatography (HPLC) showed that the solasodine content in the cell suspension after 4-weeks old (121.01 mg/g) was higher than that of in planta 1-year old roots (20.52 mg/g) by approximately 6-fold.     

In Vitro Culture and Regeneration of Solanum khasianum and Extraction of Solasodine

Leaf explants of Solanum khasianum regenerated on MS medium containing 2, 4-D (3 mg/l) and kinetin (1 mg/l). Shoots could be induced from these calli on medium containing BAP (3 mg/l) alone. Rhizogenesis of these shoots occurred when transferred to medium containing 2 mg/l NAA. The yield of solasodine — a pharmaceutically important compound, from 4-month-old callus tissue was remarkable at 2 per cent of dry weight.     

Solasodine Yield in Callus Cultures of Solanum Iaciniatum Ait. Selected for Growth on Hormone-free Medium

Callus autotrophic for both auxin and cytokinin was selectedfrom a hormone-requiring culture of Solanum laciniatum Ait.The control of solasodine yield, callus growth and adventitiousshoot initiation by some exogenous growth regulators and/orlight in the habituated culture was determined. Solasodine concentrationvaried from 0.3 to 1.4 mg g^–1 d.wt. Solanum laciniatum, callus, habituation, solasodine     

Occurrence of betulinic acid in different callus cultures of Solanum aviculare

Betulinic acid (3β-hydroxy-lup-20(29)-en-28-oic acid) was obtained from in vitro cultures of Solanum aviculare in yields up to 3% of the dry weight. This is a further example of overproduction by cells cultured in vitro of a product not found in the original parent plant.     

Enhanced production of scopolin bySolanum aviculare cells immobilised within Ca-alginate gel beads

Summary Different matrices, obtained by varying calcium (0.1 to 1.5M) and alginate (1 to 1.5%) concentrations, were used to study the influence of immobilisation parameters on the behaviour ofS. aviculare. A significant modulation of cell growth, cell release, and scopolin production and excretion has been observed. Physiological and morphological characteristics ofSolanum aviculare cells immobilised within Ca-alginate beads were notably different from those of suspended cells. ImmobilisedS. aviculare have accumulated scopolin (up to 120 g·g^–1 FWB) within beads and excreted it into the culture medium (up to 8 g·g^–1 FWB). Contrary to suspended cells which have accumulated only traces of this metabolite within intracellular compartments (1 g·g^–1 FWB), no scopolin has been found into the culture medium.Abbreviations ANA -Naphthaleneacetic acid - HPLC high performance liquid chromatography - FWB fresh weight biomass - LS medium Linsmaier and Skoog medium - MS mass spectroscopy - NMR nuclear magnetic resonance - r² coefficient of determination - s standard deviation     

Some Accounts on Culture Conditions of Callus Tissues of Solanum laciniatum Ait. for Producing Solasodine

Production of solasodine in callus cultures of Solanum laciniatum Ait. was examined under several culture conditions. The steroidal alkaloid was produced more actively in rapidly proliferating callus tissues cultured on PN medium. The alkaloid concentration in the tissue was about 0.05% (dry weight basis) during the first 5 weeks’ culture. The highest accumulation of the alkaloid per culture was obtained with 2,4-d concentration in the medium at 1~2 ppm. It is noteworthy that the alkaloid production was not inhibited by such high concentration of 2,4-d as up to 10 ppm in the medium. Supplementation of kinetin slightly increased the alkaloid production.     

Early Cellular Events During Direct Somatic Embryogenesis in Cotyledon Explants of Solanum aviculare Forst.

A detailed light and electron microscope study of early cellularevents at the onset of somatic embryogenesis in cotyledon explantsof Solanum aviculare Forst., cultured on MS medium supplementedwith 1 mg l^–1 2,4-dichloro-phenoxyacetic acid (2,4-D)for periods of 0–12 d in darkness, is described. Examinationsof longitudinal sections in a plane offset from the centralveins indicated that the earliest embryogenic events in explantstook place within the first 3–4 d of culture in the parenchymacells associated with the vascular traces closest to the cutbasal ends of cotyledons. Thereafter, parenchyma associatedwith more distal vascular traces became active in an apparentlysequential manner such that, by the second week of culture,progressive stages of embryogenesis could be observed alongthe lengths of cotyledon sections. Despite the fact that epidermalcells and palisade tissues were exposed directly to the 2,4-Dmedium, initiation of embryogenic development was never observedin cells other than those directly associated with vasculartraces. None of the embryogenic events characterized at theultrastructural level were observed in cotyledons cultured onMS medium in the absence of 2,4-D with the exception that starchaccumulated in decreasing amounts from the wounded basal endto the distal end of each cotyledon. This system provides a valuable model with which to study earlybiochemical and molecular events occurring in explants duringthe onset of somatic embryogenesis because they occur in a predictablefashion at sequentially situated sites along the explant tissues. Somatic embryogenesis, Solanum aviculare, cotyledon explants, cellular changes     

Solasodine glycoside production by hairy root cultures of Physalis minima Linn.

Hairy root cultures of Physalis minima L. were developed using Agrobacterium rhizogenes, strain ATCC 15834 mediated transformation and grown in half strength of Murashige and Skoog medium containing 8% (w/v) sucrose. Media supplementation with 1 mg naphthalenacetic acid l(-1) and 1 mg benzyladenine increased solasodine glycoside up to 900 g dry wt, which was 20 times higher than that in the native root.     

Cytokinin-induced apoptotic nuclear changes in cotyledons of Solanum aviculare and Lycopersicon esculentum

Cytokinin-induced nuclear changes have been observed in epidermal and sub-epidermal layers of cells in cotyledons of Lycopersicon esculentum and Solanum aviculare which compare closely to those observed during apoptosis in mammalian, insect and nematode species. The nuclei, which show invagination of the nuclear membrane, margination and eventual breakdown of the chromatin, are positive with the TUNEL reaction. Such changes are not induced by treatment of the cotyledons with 2,4-dichlorophenoxyacetic acid. In contrast, mesophyll cells induced to form vessels (programmed cell death) by treatment with 2,4-dichlorophenoxyacetic acid together with either 6-benzylamino-purine or zeatin, retain their diploid nuclei until the end of differentiation.     

Production of solasodine by hairy root,callus, and cell suspension cultures of Solanum aviculare Forst.

The production of the steroidal alkaloid solasodine, an alternative to diosgenin as a precursor for the commercial production of steroid drugs, was studied in hairy root, callus, and cell suspension cultures of Solanum aviculare Forst. through manipulation of culture medium. The individual and combined effects of medium components on the growth index and the production of solasodine were analyzed using factorial analysis of variance. Solasodine content was optimized to 6.2 mg g⁻¹in the hairy root, 1.4 mg g⁻¹callus, and 0.7 mg g⁻¹in cell suspension cultures (dry weight). An improved isocratic reversed phase high performance liquid chromatographic method provided selective determination of the solasodine content of these samples. Analysis of growth and solasodine content of hairy root cultures and callus cultures demonstrated that the production of solasodine was shown to be growth-dependent in hairy root cultures but not in callus cultures. This revised version was published online in June 2006 with corrections to the Cover Date.