Steroidal alkaloids in tissue cultures and regenerated plants of Solanum dulcamara

Callus and cell suspension cultures were established with shoots of the soladulcidine variety of the bittersweet Solanum dulcamara L. Plantlets were regenerated from undifferentiated callus. From mixotrophic callus as well as mixotrophic suspension cultures soladulicidine, solasodine and the corresponding neutral spirostanes tigogenin and diosgenin were isolated and identified by thin layer chromatography and mass spectrometry. Total alkaloid concentrations were about 0.2 mg/g dry weight (callus) and 0.1 mg/g dry weight (green suspension cultures). In the heterotrophic cell line only the neutral sapogenins could be detected. Alkaloid accumulation in callus of Solanum dulcamara could be enhanced by the induction of organogenesis. The shoots of the regenerated plants from the mixotrophic callus contained soladulcidine (1.6 mg/g dry weight) and tigogenin. Thus, in concentration and composition the regenerated plants equalled the source plant.Abbreviations 2.4-D 2.4-dichlorophenoxyacetic acid - NAA naphthylacetic acid - HPLC high performance liquid chromatography - TLC thin layer chromatography     

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 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     

Production of solasodine by Solanum laciniatum using plant tissue culture technique

Leaf and hypocotyl explants of 15 days old aseptically grown seedlings of Solanum laciniatum were cultured on MS medium supplemented with NAA (2 mg/l) and kinetin (0.5 mg/l) for callus initiation. For maintenance and proliferation of callus MS medium supplemented with 2,4-D (1 mg/l) and kinetin (0.5 mg/l) was used. The growth of the calli derived from hypocotyls increased with time of incubation and remained almost constant after 45 days. The solasodine content in callus culture was maximum after 30 days of incubation. Addition of L-arginine in the medium (50-150 mg/l) increased growth as well as chlorophyll content in the callus culture. The solasodine content also increased up to 1.2 to 1.4 times in these cultures. High frequency shoot regeneration was obtained in MS medium having BA (4 mg/l) and IBA (0.25 mg/l). For shoot multiplication, MS medium having BA (4 mg/l) was used. Shoots rooted on the same medium. Organogenesis promoted solasodine accumulation in the cultures. Regenerated shoots yielded higher solasodine content than undifferentiated as well as organogenic callus. Solasodine contents in the regenerated shoots was found to be 10 times higher than the callus culture and approached towards the field grown plants. Thin layer chromatography revealed the presence of three compounds. The most predominant spot (Rf 0.789) corresponded to the reference solasodine.     

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.     

The effect of phosphate,nitrogen and sucrose on the production of phenolics and solasodine in callus cultures of solanum laciniatum

In leaf derived callus cultures of Solanum laciniatum Ait. both phenolics and solasodine concentrations increased when medium phosphate or nitrogen concs. were reduced to one-eighth or when sucrose concentration was increased from 3 to 4–8 %. Under these conditions growth was reduced and final FW:DW fell. Growth was inhibited by sucrose depletion and nitrogen supple -mentation. On additional nitrogen the concentrations of phenolics and protein significantly increased, FW:DW was reduced and solasodine concentration was unaffected. In seedling derived cultures phosphate depletion resulted in a significant increase in phenolics concentration, an inhibition of growth and a rise in solasodine concentration.     

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.     

An NAD(P)H Model Reaction: Reduction of α-Keto Esters by the Hantzsch Ester Accelerated by Zinc Complex in the Reformatsky Mixture

Aiming to get useful steroidal alkaloids by tissue culture of Solanum laciniatum Ait., indefinitely growing callus tissue was prepared from the mother plant. Some nutritional requirements for the growth of the callus tissue were studied. By examining steroidal compounds in callus culture, cholesterol, stigmasterol, β-sitosterol, lanosterol, squalene, diosgenin and a new steroidal alkaloid were found to be formed in the callus culture. The new steroidal alkaloid was found to be solasodine derivative containing rhamnose and other unidentified sugars.     

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.     

Effect of l-arginine,casein hydrolysate,banana powder and sucrose on growth and solasodine production in shoot cultures of Solanum laciniatum

The addition of l-arginine, casein hydrolysate, banana powder and the reduction of the concentration of sucrose in the media could increase the solasodine content in the shoot cultures of Solanum laciniatum. No linear correlations between growth index, chlorophyll content and solasodine content were observed, however a positive linear correlation between solasodine productivity and chlorophyll content occurred in these shoot cultures.Abbreviations Ch chlorophyll content - DW dry weight - fl flask - FW fresh weight - GI growth index - LOD limit of detection - LOQ limit of quantitation - SDc solasodine content - SDp solasodine productivity - w week     

Root, Callus, and Cell Suspension Cultures, from Atropa belladonna, L. and Atropa belladonna, Cultivar lutea Doll

Root, callus, and cell suspension cultures have been establishedfrom seedlings of Atropa belladonna, L. and Atropa belladonna,cultivar lutea Döll. The growth of these cultures is described.Callus cultures transferred to auxin (-naphthaleneacetic acid)-freemedium initiated roots and shoots. Excised root cultures havebeen established from such roots and plants from such shoots.Extracts of the cultures have been submitted to the Vitali—Morinreaction and following chromatography, to the Dragendorff reaction.Cultured excised roots and plants raised from shoots initiatedon cultured callus were shown to contain atropine (hyoscyamine)and reactive substances corresponding in Rf to hyoscine andcuscohygrine. These alkaloids were absent from cultured callusand cultured cell suspensions and from leaves when initiatedwithout roots on callus. The cultured calluses and cell suspensionscontained choline (0.022–0.027 g per 100 g dry weightof root callus). The growth of cell suspension cultures wasnot inhibited by incorporating atropine sulphate, L-hyoscyamine,L-hyoscine hydrobromide, or DL-scopoline nitrate in the culturemedium at 250 mg/I. These alkaloids were absorbed by the cells,a high proportion of the added alkaloid could be recovered fromthe cultures even after 4 weeks' growth and no evidence wasobtained of the presence of degradation products of the alkaloids.The suppression of alkaloid formation in actively growing callusand cell suspension cultures is discussed.     

Biosynthesis and accumulation of a medicinal compound,Picroside-I,in cultures of <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis> Royle ex Benth

Establishment of callus cultures and plant regeneration from different explants coupled with estimation of Picrosides in morphogenetically different developmental stages showed that Picroside-I accumulates in shoot cultures of Picrorhiza kurroa with no detection of Picroside-II. The Picroside-I content was 1.9, 1.5, and 0.04 mg/g in leaf discs, stem and root segments, respectively. The Picroside-I content declined to almost non- detectable levels in callus cultures derived from leaf discs, stem segments with no change in Picroside-I content in root segments or calli derived thereof. The biosynthesis and accumulation of Picroside-I started in callus cultures differentiating into shoot primordia and reached to the concentrations comparable to original explants of leaf discs and stem segments in fully developed shoots with contents of 2.0 and 1.5 mg/g, respectively. The shoots formed from root-derived callus cultures were relatively slow in growth as well as the amount of Picroside-I content was comparatively low (1.0 mg/g) compared to shoots derived from callus cultures of leaf and stem segments, respectively. The current study concludes that the biosynthesis and accumulation of Picroside-I is developmentally regulated in different morphogenetic stages of P. kurroa tissue cultures.     

Behaviour of mlo evoking potato witches’ broom in callus tissue culture ofSolatium laciniatutn ait. andNicotiana tabacum L. cv. samsun

The growth of callus tissue cultures and the infectivity of twenty fiveSolanum laciniatum Ait. plants and of sixteenNicotiana tabacum L. cv. Samsun plants were investigated. The plants were obtained from callus tissue cultures derived from stem pieces of the respective plants infected with a mycoplasma-like organism (MLO) evoking potato witches’ broom. The tissues were cultivated on synthetic nutrient medium with kinetin and IAA. Allde novo obtainedS. laciniatum plants were healthy. On the contrary twelve of the sixteen reconstituted tobacco plants showed MLO presence. Summarizing these and previous results, the authors suppose that the most important factor influencing MLO persistence in callus tissues cultivated on the applied nutrient medium may be the callus growth rate and the organogenesis set. Both these conditions are determined by the metabolism of the investigated plant species.     

Biotransformation of Externally Added Vanillin Related Compounds by Multiple Shoot Cultures of Vanilla planifolia L

The multiple shoots and callus cultures of Vanilla planifolia obtained from the nodal explant on MS medium supplemented with 6-benzylaminopurine (BAP) 2 mg l^?1 and α-naphthalene acetic acid (NAA) 2 mg l^?1 were maintained by regular subculturing every 30 days and also cultured liquid MS medium of the same hormonal combination. Shoots were transferred to the MS basal medium for rooting. Different explants along with vanilla pods and in vitro cultures were analyzed using HPLC for the presence of vanillin and related compounds. When the amount of these compounds was determined in explants and in in vitro cultures after precursor feeding and curing process, explants showed different profile after precursor feeding and after undergoing curing process. During further investigations we have applied a novel approach for curing in vitro tissues as done for vanilla beans. Curing of in vitro shoots resulted in a significant change in the aromatic compound profile.     

In Vitro Preservation of Asparagus Officinalis

A simple systems for in vitro storage of health asparagus germplasm was developed. High percent (90 %) of shoots cultured in a standard multiplication medium were maintained viable in vitro at 5 °C in darkness for 12 months. This percent was decreased to 60 % when cultures were stored for 18 months. At normal temperature, shoots and callus cultures also survived for 1 year under osmotic stress on medium containing 40 g dm^-3 mannitol.     

Stevioside biosynthesis by callus,root, shoot and rooted-shoot cultures in vitro

Leaf explants of Stevia rebaudiana Bertoni (Compositae), an herb which produces the sweet ent-kaurene glycoside stevioside, were cultured in Murashige and Skoog medium with vitamins, sucrose (30 g l^–1), agar (0.9% w/v) and supplemented with naphthaleneacetic acid (NAA, 0.5 mg l^–1) and benzylaminopurine (BAP, 0.5 mg l^–1). These conditions yielded friable callus cultures. Differentiation of the callus tissue was then achieved by eliminating the agar and modulating the medium's hormone concentrations. Thus, medium containing increased auxin concentration (1.0 mg l^–1) and no cytokinin or increased cytokinin (1.0 mg l^–1) and no auxin yielded root or shoot cultures respectively. Supplementation of the shoot medium with NAA (1.0 mg ml^–1) induced shoot cultures to grow roots thereby differentiating into rooted-shoot cultures. Only the rooted-shoot cultures tasted sweet. Feedings of [2-¹⁴C]acetic acid to callus, shoot or rooted-shoot cultures demonstrated that only the rooted-shoot cultures are capable of de novo biosynthesis of the aglycone moiety of stevioside (steviol). In addition, [methyl-³H(N)steviol feedings to shoot or rooted-shoot cultures illustrated that both types of cultures are capable of the glycosylation reaction. The ability of these tissues to glycosylate steviol to stevioside was also demonstrated employing crude enzyme preparations derived from shoot or rooted-shoot cultures. These results suggest that stevioside biosynthesis is a function of tissue differentiation since both roots and leaves are required for cultured S. rebaudiana to biosynthesize stevioside from acetate, while the final biosynthetic steps can be performed at all levels of differentiation.     

The effect of 2,4-dichlorophenoxyacetic acid and donor plant environment on plant regeneration from immature inflorescence-derived callus of Lolium perenne L. and Lolium multiflorum L.

Callus cultures were obtained from immature inflorescences of perennial ryegrass (Lolium perenne) and Italian ryegrass (Lolium multiflorum). Inflorescence segments were cultured on Murashige and Skoog medium (MS) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The response in culture with regard to compact callus induction, embryogenesis and plant regeneration was determined for different varieties. The in vitro response was compared for explants from field-grown plants and explants from greenhouse-grown plants. The effect of different 2,4-D concentrations on the in vitro response was also investigated in one L. perenne variety and one L. multiflorum variety. The percentage of explants that formed compact callus and embryogenic callus differed strongly with the cultivar. There was no consistent effect of the growth conditions of the donor plants or the 2,4-D concentration of the medium on this response. Green plants were regenerated from all the cultivars tested. Explants from field-grown plants showed a higher tendency to form albino shoots than explants from greenhouse-grown plants. In the L. perenne variety tested higher 2,4-D concentrations (up to 15 mg/l) resulted in a lower regeneration frequency of green shoots and a higher regeneration frequency of albino shoots (up to 12.5 mg/l). In the L. multiflorum variety tested the effect of 2,4-D on regeneration was less pronounced.     

Production of glycyrrhizin in callus cultures of licorice

Licorice plants, Glycyrrhiza glabra, G. uralensis, and G. inflata, were investigated for callus induction using Murashige and Skoog (MS) medium combined with auxins and cytokinins. After 4 weeks of culture, 33-100% of leaf or stem explants formed calli. Maximum of shoot induction from callus cultures was achieved by G. inflata stem explants cultured on MS medium supplemented with 1 mg/l alpha-naphthaleneacetic acid (NAA) and 0.5 mg/l 6-benzyladenine (BA) (67%) which also gave maximum shoot formation per explant (two shoots per explant). These results indicated that all three Glycyrrhiza species regenerated shoots from callus cultures on MS medium combined with NAA and BA or only thidiazuron (TDZ; 0.1 and 0.5 mg/l). Glycyrrhizin contents of G. uralensis calli induced using MS medium in combination with NAA and BA [(27.60 +/- 8.47) microg/g DW] or TDZ alone [(36.52 +/- 2.45) microg/ g DW] were higher than those found in other combinations.     

Effect of reverse photoperiod on in vitro regeneration and piperine production in Piper nigrum L.

In this study, a novel approach for in vitro regeneration of Piper nigrum L. has been applied in order to increase healthy biomass, phytochemicals and piperine production via reverse photoperiod (16hD/8hL). Leaf portions of the seed-derived plants were placed on an MS-medium fortified with different PGRs. Under 16hD/8hL, thidiazuron (TDZ; 4.0 mg L⁻¹) and BA (1.5 mg L⁻¹) was found to be the most effective (< 90%) in callus induction. Two concentrations (1.5, 2.0 mg L⁻¹) of the IBA produced > 80% shoots from callus cultures. Healthy shoots were transferred to rooting medium and higher percentage of rooting (< 90%) was observed on IBA (1.5 mg L⁻¹). These in vitro tissues were subjected to amino acid analysis, spectrophotometry, and HPLC. ARG, SER, THR, and TYR were the most abundant components out of 17 amino acids. Higher amino acid production was observed under normal photoperiod (16hL/8hD) than under reverse photoperiod (16hD/8hL). The highest total phenolic content (TPC; 9.91 mg/g-DW) and flavonoid content (7.38 mg/g-DW) were observed in callus cultures incubated under 16hL/8hD than other tissues incubated under 16hD/8hL photoperiod. Higher DPPH and PoMo activities were observed in tissues incubated under 16hL/8hD photoperiod, while ABTS and Fe²⁺ chelating activities were found higher in tissues incubated under reverse photoperiod. Significant quantities of piperine content were observed in all tissues except callus cultures. These results suggest that reverse photoperiod is a promising approach for callus induction, phytochemicals and piperine production for commercial applications.     

Accumulation of anthocyanin in callus cultures of red-pod okra [<Emphasis Type="Italic">Abelmoschus esculentus</Emphasis> (L.) Hongjiao] in response to light and nitrogen levels

Nitrogen and light are critical determinants of biomass accumulation and secondary metabolite production under in vitro culture conditions. In this study, we analyzed the effects of varied concentrations of total nitrogen in Murashige and Skoog (MS) medium and light intensity on the production of biomass, anthocyanin pigments, and bioactive antioxidants in callus cultures of Abelmoschus esculentus cv. ‘Hongjiao’. Maximum callus biomass accumulation (3 g FW) was achieved when calluses were cultured on MS medium containing 60 mM nitrogen under 40 μmol m^??2 s^??1 light intensity. In contrast, maximum values of total anthocyanin accumulation (TA; 7.3 CV/g FW), total phenolic content (TP; 12.07 mg/100 g FW), total flavonoid content (TF; 2.47?±?0.15 mg/100 g FW), and total antioxidant activity (TAA; 56.10 μmol Trolox/g FW) were observed when calluses were cultured on MS medium containing 40 mM total nitrogen under 80 μmol m^??2 s^??1 light intensity. In addition, callus grown under same culture condition exhibited high flavonoid content along with increased phenolic content and antioxidant activity. High performance liquid chromatography (HPLC) was performed for qualitative and quantity analysis of callus cultures. Most of the pigments from the callus extracts were identical with pod anthocyanins, and appeared on the ODS-column HPLC with lower concentration than the main pigments of the pod tissues. These findings indicate that callus cultures of red-pod okra represent a potential source of bioactive compounds with antioxidant properties for industrial applications.