Tissue Culture Studies in Duboisia myoporoides; 1. Plant Regeneration and Clonal Propagation by Stem Node Cultures.

High frequency plant regeneration is reported from nodal explant cultures of DUBOISIA MYOPOROIDES. For the optimal growth of the axillary bud and it subsequent multiplication, fortification of 1.0 mg.l (-1) IAA along with 2.0 mg.l (-1) Kn or BAP was found to be essential. No other auxin could replace IAA since they led to callus formation. Rooting could be induced by auxin alone (0.5 mg.l (-1) NAA), as expected, but only after replacing semi-solid basal medium of Murashige and Skoog (MS) with static liquid MS. No synergism was noticed between cytokinins and adenine sulphate, while GA (3) proved significantly suppressive for the shoot growth. The plantlets thus obtained were successfully raised in large numbers to normal adults under field conditions.     

杜仲带腋芽茎段组织培养的研究

目的研究不同培养条件下杜仲带腋芽茎段愈伤组织的诱导和生长情况,以及腋芽的生长情况。方法以带腋芽的杜仲茎段为外植体,设计9种不同的培养基配方,为了防止褐化,在23.4℃下培养箱中暗培养,30 d后调查出愈率及腋芽的生长情况。结果 NAA浓度从0.05~0.5 mg.L-1,随着浓度增加,腋芽生长受抑制;6-BA浓度从0.3~1.0 mg.L-1,随着浓度增加,愈伤组织块增大,达到1.0 mg.L-1时愈伤组织的生长受到抑制;本实验中最佳的杜仲愈伤组织诱导和生长培养基配方为6号培养基:B5+0.5 mg.L-1NAA+0.3 mg.L-16-BA。腋芽生长最佳培养基配方为4号培养基:B5+0.05 mg.L-1NAA+0.5 mg.L-16-BA。结论本试验的结果对杜仲的生物技术利用有一定的参考价值。     

Micropropagation of Polygonum multiflorum THUNB and quantitative analysis of the anthraquinones emodin and physcion formed in in vitro propagated shoots and plants

An efficient and rapid protocol for in vitro induction and complete plant regeneration of Polygonum multiflorum THUNB has been developed. Nodal explants were grown in vitro on Murashige and Skoog's (MS) basal medium containing different concentrations of alpha-naphthaleneacetic acid (NAA) and benzyladenine (BA). The nodal explants (97%) produced multiple shoots (4.7 shoots per explant) on MS basal medium supplemented with 0.2 mg/l NAA and 2.0 mg/l BA after 6 weeks of culture. Eighty-eight percent to 100% of the shoots (1.0 cm in length) elongated (about 3.02-4.28 cm) and rooted on MS basal medium supplemented with NAA or indole-3-butyric acid (IBA). All the rooted shoots were transferred to pots containing autoclaved soil, vermiculite, and peat moss (1 : 1 : 1). The plantlets were successfully acclimatized under greenhouse conditions with high humidity before transferring to the field. The anthraquinone contents were determined using HPLC. Analysis revealed that the contents of the major medicinal compounds-emodin and physcion in the 6 weeks old in vitro grown shoots and three month old in vitro propagated plants grown in greenhouse were higher than those of the marketed crude drug (processed underground or stem parts of P. multiflorum).     

In vitro propagation of Podophyllum peltatum

The lignan podophyllotoxin, occurring in Podophyllum emodi Wall, ex Royale and Podophyllum peltatum. L., is the starting compound for the semi-synthesis of the anticancer drugs etoposide and teniposide. In this study, we evaluate development of an in vitro propagation protocol to rapidly produce high yielding Podophyllum peltatum plants. Rhizome tips were inoculated on MS medium supplied with 4.4 microM N(6)-benzyladenine and 0.025% (w/v) activated charcoal. These explants formed terminal buds, similar to the ones found in nature. These buds were sources of in vitro bud cultures. These bud cultures were classified as: apical, axillary, and adventitious and the effects of various N(6)-benzyladenine concentrations on the three types of bud cultures were evaluated through bud, leaf, and root inductions. Cultures of axillary and adventitious buds were more proliferous for bud induction. Podophyllotoxin contents of in vitro rooted bud and plantlet cultures were similar to the content found in the wild. Plantlets and buds were acclimatized under controlled environment conditions.     

In vitro plantlet regeneration in Panax sikkimensis

A three-step procedure for complete plantlet regeneration via somatic embryogenesis has been developed in Panax sikkimensis. Somatic embryos (SE) were induced in root callus upon lowering the level of 2,4-D from 1.0 mg/l to 0.25 mg/l in the callusing medium. Maturation of SE occurred on a half-strength MS medium with 0.5 mg/l each of BAP and GA3. An exposure for 15 days of cotyledonary and heart-shaped SE to 1.0 mg/l IBA in liquid shake 1/2 MS medium significantly improved the rate of embryo-to-plantlet conversion and plantlet quality. The procedure has now allowed the retention of high regeneration potential of the root callus for over three years.     

In vitro propagation of Galphimia glauca and content of the sedative compound galphimine-B in wild and micropropagated plants

An in vitro micropropagation protocol is described for Galphimia glauca Cav. (Malpighighiaceae). Multiple shoots were formed in vitro from axillary bud explants inoculated on MS medium supplemented with indole-3-acetic acid (IAA) and kinetin (KN) combinations. A maximum of 20 shoots was obtained from a single bud in a 60-day culture period. In vitro-grown shoots were successfully rooted and transferred to field conditions (90 % survival). The sedative triterpenoid galphimine-B (1) content of micropropagated plants transferred to field conditions was similar to that of wild plants. Our results suggest that the in vitro propagation protocol described here will have positive effects on conservation of natural resources as well as on adequate techniques for multiplication of an important Mexican medicinal plant.     

Effects of fluoroquinolones and magnesium deficiency in murine limb bud cultures

Quinolone-induced arthropathy is probably caused by a lack of functionally available magnesium in immature joint cartilage. We used an in vitro assay to study the effects of fluoroquinolones on cartilage formation in mouse limb buds from 12-day-old mouse embryos in regular and in magnesium-deficient medium. Omission of magnesium from the medium had no adverse effect on the outcome of the culture: limb buds grew and differentiated well in regular and in magnesium-deficient Bigger's medium. Lack of calcium, however, severely impaired the development of the explants; this result was even more enhanced when both minerals (magnesium and calcium) were omitted. Electron microscopy revealed cell necrosis and deposition of electron-dense material in the vicinity of chondrocytes from limb buds after 6 days in a magnesium-free medium. A series of seven fluoroquinolones was tested at 30, 60, and 100 mg/l medium. At a concentration of 30 mg/l sparfloxacin only had a slight effect on limb development. At concentrations of 60 and 100 mg/l sparfloxacin, temafloxacin and ciprofloxacin impaired limb development in␣vitro concentration-dependently. The effects were enhanced in a magnesium-deficient medium (concentration of magnesium <10 μmol/l). Fleroxacin, lomefloxacin and ofloxacin impaired limb development only slightly; no significant differences were recognizable between the outcome in regular and in magnesium-deficient medium. Pefloxacin did not show any effect on limb development in both media. Using electron microscopy, very similar alterations as described above for the limbs cultured in magnesium-deficient medium were observed with ofloxacin at a concentration of 30 mg/l, which had no effect on the growth of the explants when evaluated macroscopically. The affinity of six fluoroquinolones to magnesium was determined by the use of a fluorescence assay. The affinity to magnesium correlated with the activity of the drugs in the limb bud assay. We conclude that fluoroquinolones have no effect on murine limb development in vitro at concentrations that are achieved under therapeutic conditions (peak concentrations approx. 1–5 mg/l in plasma). Effects at higher concentrations (60 and 100 mg/l) are slightly enhanced (factor 2) if the magnesium concentration in the medium is low. Macroscopically, limbs develop regularly in a magnesium-free medium, but ultrastructurally typical alterations are exhibited (e.g. cell necrosis and pericellular deposition of electron-dense material). Received: 7 October 1997 / Accepted: 25 February 1998     

Production of withaferin A in shoot cultures of Withania somnifera

Multiple shoot cultures of Withania somnifera were established from single shoot tip explants and their potential for the production of two principle withanolides, withaferin A and withanolide D was investigated. Shoot tips grown on MS medium supplemented with BA (1 mg l(-1)) induced 10.0 +/- 1.15 microshoots per explants and shoot cultures accumulated both withanolides (withaferin A = 0.04%, withanolide D = 0.06%). Supplementation of MSSM (solid) agar medium with 4% sucrose enhanced accumulation of both withaferin A (0.16%) and withanolide D (0.08%). Reduction of the agar concentration to 0.16% increased the number of microshoots induced per explant to 25.5. MSSM liquid medium containing 10% coconut milk favoured a maximum increase in biomass (27 fold); number of microshoots induced (37.6 +/- 1.45) as well as accumulation of withaferin A (0.14%).     

荷花玉兰组织培养的研究

采用正交设计法考察了４种植物激素对诱导荷花玉兰愈伤组织产生的影响,筛选出最佳的植物激素配比为：ＭＳ培养基＋２,４－Ｄ（２,４－二氯苯氧乙酸,４ｍｇ／Ｌ）＋６－ＢＡ（６－苄基氨基嘌呤,１ｍｇ／Ｌ）＋ＮＡＡ（ａ－萘乙酸,４ｍｇ／Ｌ）;找到最适外植体为花托和叶芽,对比了不同外植体形成愈伤组织的时间、形态和质地的差异。     

Somatic embryogenesis and rhizogenesis of tissue cultures of two genotypes of Papaver somniferum: relationships to alkaloids production

PAPAVER SOMNIFERUM L. tissue cultures, issued from various explants (cotyledons, hypocotyls, roots) derived from plantlets belonging to two genotypes, were established on LS solid medium containing growth regulators (NAA, Kin) in various combinations. Hypocotyls and roots were found to be interesting explants to obtain cellular development. Many roots developed on calli growing on a medium containing NAA (1 mg/l) + Kin (0.1 mg/l) for the PS genotype while somatic proembryos redifferentiated on calli issued from PS 1639 genotype. The same growth substance combination was the most favourable for the production of morphinan alkaloids and papaverine: up to 10 x 10 (-3)% DW in roots redifferentiated from PS calluses.     

林荫银莲花不定芽增殖培养基的优化

为提高林荫银莲花(Anemone flaccida Fr.Schmidt)不定芽繁殖系数,对其组培快繁体系进行优化。以林荫银莲花无菌苗为外植体,研究了培养基中激素、碳源、添加物和矿质元素钙等组分对其不定芽增殖和生长状况的影响,得出最佳的不定芽增殖培养基配方。以1/2 Ca浓度MS为固体培养基,激素为1.0 mg/L 6-BA和1.0 mg/L NAA,蔗糖或食用白糖30 g/L,水解酪蛋白为添加物,浓度为750 mg/L,培养30 d后繁殖系数可达4.01,大幅提高了不定芽的增殖速度和质量,为工厂化生产林荫银莲花种苗奠定了基础。     

Clonal propagation of Isoplexis canariensis

Isoplexis is a plant genus closely related to Digitalis. Members of this genus contain cardenolides considered more "primitive" than those present in Digitalis. Isoplexis plants, tissue cultures, and isolated cardenolides may thus be used to elucidate the biosynthesis of cardenolides in the Scrophulariaceae. Therefore, a method was developed to cultivate and propagate Isoplexis canariensis (L.) Lindl. ex. G. Don in vitro. Seeds were germinated in liquid modified MS medium and shoot cultures were established and propagated in liquid modified MS medium containing 0.1 mg/l IAA and 1 mg/l BAP. Shoot cultures were also established from excised axillary buds and propagated on solid culture medium containing 0.1 mg/l IAA and 1 mg/l BAP. Shoots of either origin were rooted in medium containing 1 to 5 mg/l IAA and 0.5 to 4 mg/l IBA. Rooted plantlets were cultivated for 2 to 3 weeks in hormone-free modified MS medium and then transferred to the greenhouse, where they developed into healthy plants.     

Multi-pumping flow system for the spectrophotometric determination of dipyrone in pharmaceutical preparations

A novel flow system for the spectrophotometric determination of dipyrone with p-dimethylaminobenzaldehyde exploiting the multi-pumping approach was developed. The proposed methodology utilises several micro-pumps for propelling the involved fluids under improved mixing conditions, introducing sample/reagent aliquots and providing commuting facilities. As a consequence the multi-pumping system presents high versatility and manifold simplicity, as well as a straightforward operational control and enhanced analytical capabilities. Linearity of the analytical curve was observed within 10 and 400 mg l(-1) dipyrone (r=0.9997; n=6), results were precise (r.s.d.<0.12%; n=20) and sampling rate was 50 h(-1). Detection limit was estimated as 1 mg l(-1) dipyrone. The method was applied to pharmaceutical preparations and the results were in agreement with those obtained by the reference procedure with relative deviations within -1.7 and +2.2%.     

Production of the sedative triterpene galphimine B in Galphimia glauca tissue culture

A tissue culture method is described for callus formation from Galphimia glauca (Mapighiaceae) in MS (Murashige & Skoog) medium supplemented with various growth regulators. Best induction was achieved when using hypocotyls as explants in medium supplemented with 2 mg/l of 2,4-dichlorophenoxyacetic acid (2,4-D). Major cellular growth of calluses was obtained with naphthaleneacetic acid (2 mg/l) + kinetin (1 mg/l). Subcultivation of calluses using various concentrations of 2,4-D allowed the production of the sedative nor-seco-triterpenoid, galphimine B and a new related compound. The structure of the new constituent was elucidated as 6-acetoxygalphimine B. The highest accumulation of active constituent 1 (1.5 x 10(-2) dry weight) was achieved when 4 mg/l of the hormone were used, and this experimental condition allowed the detection of only galphimine B. A preliminary screening of the methanolic extracts prepared from calluses, using the isolated guinea-pig ileum as a general test system for pharmacological effects, demonstrated that the most active material was the one with the highest concentration of galphimine B. Total accumulation of this sedative triterpene, in the optimized tissue culture conditions, was in the same order of magnitude as the value quantified for wild plants (4.5 x 10(-2) dry weight).     

Tropane alkaloid production and shoot regeneration in hairy and adventitious root cultures of Duboisia myoporoides-D. leichhardtii hybrid

Co-culture conditions for Duboisia myoporoides-D. leichhardtii hybrid hairy root induction were investigated using leaf explants and Agrobacterium rhizogenes ATCC 15834. The bacteria density and duration of co-culture greatly affected the induction rate; the highest rate of 50% was obtained when the leaf explants were co-cultured for 2 d with 10(6) bacteria. One hairy root clone that showed the fastest root growth was selected and used for comparison study with adventitious roots cultured with 0.5 mg/l indole-3-acetic acid (IAA). The hairy roots cultured in Murashige and Skoog (MS) liquid medium grew well and yielded much more tropane alkaloids (35 mg/l scopolamine and 17 mg/l hyoscyamine) than adventitious roots cultured in 0.5 mg/l IAA after 6 weeks of culture at 25 degrees C in the dark. The hairy and adventitious roots (2.5 cm) grown in liquid media were divided into 5 parts (each 0.5 cm) along the root axis. Distribution of scopolamine and IAA was then determined by enzyme-linked immunosorbent assay (ELISA). Inverse relationship between contents of scopolamine and IAA was observed in the hairy roots; increase of scopolamine and decrease of IAA were proportional to the distance from the root meristem. In contrast, the contents of scopolamine and IAA were relatively constant in the adventitious roots. In shoot regeneration experiments, the hairy and adventitious root segments (1 cm) were placed onto 1/2 MS solid medium containing various concentrations of IAA and BA cultured at 25 degrees C under 16 h light. In adventitious roots, the shoots regenerated on media containing 6-benzyladenine (BA) (0.5 to 5 mg/l), and 100% regeneration was observed in medium with 0.1 mg/l IAA and 2 mg/l BA. On the other hand, shoot regeneration was only observed in 33% of hairy roots cultured on medium containing 5 mg/l BA.     

Isolation and quantitative analysis of cryptotanshinone,an active quinoid diterpene formed in callus of Salvia miltiorrhiza BUNGE

Effect of N(6)-benzyladenine (BA) on tanshinone formation in callus cultures of Salvia miltiorrhiza was examined in an attempt to increase the productivity of the medicinal compound, cryptotanshinone. Primary callus was induced by culturing leaf explants on Murashige and Skoog's (MS) basal medium supplemented with 1.0 mg l(-1) of 2,4-dichlorophenoxyacetic acid (2,4-D) in darkness. The callus proliferated further on MS basal medium containing 1.0 mg l(-1) 2,4-D and 0.5 mg l(-1) BA and was analyzed for cryptotanshinone by high performance liquid chromatography (HPLC). The HPLC results indicated that it contained small amounts of cryptotanshinone (0.26+/-0.05 mg/g dry wt). Omission of 2,4-D from the medium resulted in a marked increase in the content of cryptotanshinone in callus. The HPLC analysis revealed that the content of cryptotanshinone in the callus cultured on the MS basal medium supplemented with 0.1, 0.2, 0.5, 1.0, and 2.0 mg l(-1) of BA was significantly higher than the marketed crude drug (processed underground parts of S. miltiorrhiza). Maximum yield of cryptotanshinone (4.59+/-0.09 mg/g dry wt) was observed in the callus cultured on MS basal medium supplemented with 0.2 mg l(-1) BA for 60 d. Cryptotanshinone was isolated from callus through silica gel column chromatography followed by preparative TLC and characterized based on NMR and mass spectral data.     

Direct determination of metformin in urine by adsorptive catalytic square-wave voltammetry

A new adsorptive catalytic voltammetric method for voltammetric determination of metformin based on the catalytic hydrogen evolution reaction at a hanging mercury drop electrode was developed. The electrode reaction was analyzed under conditions of linear sweep voltammetry (LSV), differential pulse voltammetry (DPV) and Osteryoung-type square-wave voltammetry (SWV). The peak current depends on pH of the medium, concentration and chemical composition of the buffer solution, and instrumental parameters. The optimal conditions for quantitative determination were obtained in an acetate buffer at pH 4.7. The voltammetric procedure was characterized with respect to the repeatability, precision and the recovery. The detection and quantification limits were found to be 1.8 x 10(-8) and 5.9 x 10(-8) mol l(-1) for SWV, 3.2 x 10(-8) and 1.0 x 10(-7) mol l(-1) for DPV, and 7.7 x 10(-8) and 2.5 x 10(-7) mol l(-1) for LSV, respectively. The SW voltammetric method, as the most sensitive one, was applied for determination of metformin in human urine. The voltammetric method has been validated by using HPLC with UV detection.     

Standard operating procedure for immunuslotblot assay for analysis of DNA/sulfur mustard adducts in human blood and skin

A standard operating procedure has been developed for an immunoslotblot assay of sulfur mustard adducts to DNA in human blood and skin for use in a field laboratory. A minimum detectable level of exposure of human blood in vitro (> or = 50 nM) sulfur mustard is feasible with the assay. In the case of human skin, a 1 s exposure to saturated sulfur mustard vapor (830 mg/m(-3)) could still be detected.     

Callus cultures of Annona squamosa for the production of annonaceous acetogenins

Callus cultures of Annona squamosa were induced using different explants including petals, seed contents (megagametophyte and embryo) and fruits (mesocarp). Growth of the calli induced from the explants was found to be influenced by the type, concentration and ratio of auxin vs. cytokinin. The content of squamocin (67.8 μg g -1 dry weight) in calli cultured on Gamborg B-5 medium containing 5.0 mg l -1 naphthalene acetic acid and 4.0 mg l -1 zeatin was nearly seven times higher than that in intact fruits.     

粗毛牛膝带腋芽茎段的植株再生研究

目的:研究粗毛牛膝带腋芽茎段植株的再生。方法:以粗毛牛膝带腋芽茎段为外植体诱导腋芽生长,继代诱导生根成完整小植株,考察基本培养基、萘乙酸(NAA)和6-苄基嘌呤(6-BA)浓度3个因素对茎段和腋芽生长的影响。结果:基本培养基MS、6-BA浓度对腋芽的生长有显著影响,最佳的培养基组合为MS+0.1mg·L-1NAA+0.5mg·L-16-BA;而在1/2MS+0.5mg·L-1NAA培养基上100%生根。结论:该培养基组合可使粗毛牛膝腋芽诱导生根能力强,且幼根生长健壮,值得推广。