Shoot Tip Culture of Arnica montana for Micropropagation

Multiple shoots were regenerated from shoot tips of ARNICA MONTANA on MS and B5 media supplemented with BA (1 mg/l) and NAA (0.1 mg/l). Sections of 1-2 mm in length cultured from IN VITRO germinated seedlings regenerated 7.7 (mean) shoots on the MS medium, whereas sections cultured from greenhouse plants regenerated 9.0 (mean) shoots on the B5 medium within 6 weeks. Subsequent subcultures of shoots on the same media but without NAA resulted in similar or lower multiplication rates (1.6 to 3.1 in 3 weeks). Shoot development was promoted, whereas shoot initiation was simultaneously inhibited by the addition of activated charcoal to the media. Rooting was induced by culturing shoots from seedling as well as from greenhouse plant shoot tips on MS or B5 medium supplemented with NAA. The plantlets were transplanted into soil and grown successfully under greenhouse and field conditions.     

Regeneration of Stevia Plant Through Callus Culture

Stevia rebaudiana Bertoni that conventionally propagated by seed or by cuttings or clump division which has a limitation of quality and quantity seed material. In present study, callus culture technique was tried to achieve rapid plant multiplication for quality seed material. Callus induction and multiplication medium was standardized from nodal as well as leaf sagments. It is possible to maintain callus on Murashige and Skoog medium supplemented with 6-benzyl amino purine and naphthalene acetic acid. Maximum callus induction was obtained on Murashige and Skoog medium incorporated with 6-benzyl amino purine (2.0-3.0 mg/l) and naphthalene acetic acid (2.0 mg/l) treatments. However, Murashige and Skoog medium containing 2.0 mg/l 6-benzyl amino purine+2.0 mg/l naphthalene acetic acid was found to be the best for callus induction. Higher regeneration frequency was noticed with Murashige and Skoog medium supplemented with 2.0 mg/l 6-benzyl amino purine+0.2 mg/l naphthalene acetic acid. Regenerated plants were rooted better on ¼ Murashige and Skoog strength supplemented with 0.1 mg/l indole-3-butyric acid. The rooted plantlets were hardened successfully in tera care medium with 63 per cent survival rate. The developed protocol can be utilized for mass production of true to type planting material on large scale independent of season, i.e. external environmental conditions.     

脱病毒生姜同源四倍体的诱导和鉴定

采用组织培养技术，运用正交试验设计，进行了脱病毒生姜试管苗快速繁殖技术的优化和多倍体的诱导与鉴定，并对试管苗进行了生理生化指标的考察。结果表明MS＋BA（6-苄氨基嘌呤）2．0mg／L＋NAA（α-萘乙酸）0．1mg／L＋KT（激动素）1．0mg／L的培养基组成最适合于丛生芽的繁殖，MS＋BA0．2mg／L＋NAA1．0mg／L＋KT0．1mg／L的培养基组成最适合于生姜根的生长。多倍体诱导试验表明，秋水仙碱浸泡生姜芽12h的诱导率最高。并且发现检测的多倍体生姜试管苗的蛋白含量升高，SOD酶活升高，POD酶活升高，APX酶活升高，叶绿素含量升高，预示多倍体可能具有较好的生长势，对提高生姜的产量有利。     

In vitro Propagation of Valeriana wallichii

A tissue culture procedure has been developed for the rapid multiplication of VALERIANA WALLICHII D C. through shoot tip and axillary bud explants. MS medium containing Kn or BAP (5.0 mg/l (-1)) in combination with IAA (1.0 mg/l (-1)) induced an optimal growth of shoots within 6-8 days from both apical and axillary bud explants. The roots developed on the same medium within 2-3 weeks. Hardening of IN VITRO grown plantlets in pots under glass-house conditions was dependent upon the temperature and humidity. A cold-temperate climate favoured early establishment. Following the given procedure, a large number of plants have been established under field conditions at two locations. The method has implications in the early introduction of an elite population as well as its improvement.     

Helenalin Acetate in in vitro Propagated Plants of Arnica montana

Propagated "IN VITRO" shoots and plantlets of ARNICA MONTANA L. (Asteraceae) have been shown to produce sesquiterpene lactones, i.e. helenalin and 11,13-dihydrohelenalin esters. The compounds were detected in green organs only; roots of the plantlets contained no sesquiterpene lactones. The helenalin acetate content in leaves of the plantlets (0.073% dry wt) was 4-times higher than in proliferated shoots (0.016% dry wt). The best rate of shoot multiplication was achieved on MS medium, supplemented with NAA 0.5mg/l and Kn 2.5 mg/l (formation of 22 shoots within 8 weeks).     

The effect of creosote on the growth of an axenic culture of Myriophyllum spicatum L

Due to the importance of submersed, rooted macrophytes to the aquatic ecosystem and the use of creosote impregnated structures adjacent to or within water bodies, a study was conducted using an axenic culture of Myriophyllum spicatum to determine the effect of creosote on this aquatic macrophyte. Four plants were cloned and exposed to nominal creosote concentrations ranging from 0.16 to 200 mg/l for 14 days. A variety of response parameters were assessed, including shoot and root length, number of roots and nodes, and dry weight biomass, as well as visual observations on plant colouring and morphology. Regression and ANOVA analyses were conducted to determine EC50s and significant differences. Biphasic responses were observed for shoot length, node production and biomass, with shoot length showing statistically significant stimulation (hormesis) at creosote concentrations below 13.3 mg/l. EC50 values of 55.1 (CI 40-60) mg/l, 33.4 (CI 26-48) mg/l and 86 (CI 70-120) mg/l were determined for shoot length, dry weight and node production, respectively. Root number was significantly higher at 3.6 mg/l and root length was significantly reduced at 4.5 mg/l creosote, within the concentration range that stimulated shoot growth. Visual changes, including an increase in pink colouration and changes in the location of root initiation, were also observed in the same creosote concentration range that affected root length and numbers. Therefore, it appears that changes in root growth and location of root initiation may be the most sensitive endpoints for creosote effects on Myriophyllum.     

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.     

In vitro propagation of Stevia rebaudina plants using multiple shoot culture

A multiple shoot culture was induced from nodal segments on MS medium containing half concentration of macroelements, 1% sucrose, and supplemented with NAA (0.01 mg/l). A bioreactor with hormone-free MS medium (300 ml) was inoculated with 1.5 g of the multiple shoot culture and cultivated for a month. The cultivating process of the multiple shoot culture in the bioreactor and the transfer into ex vitro conditions took about 8-9 weeks and produced approx. 600 new seedlings, that could be transferred from greenhouse to field conditions.     

Rutin and Scopoletin Content and Micropropagation of Fabiana imbricata

An HPLC method for the simultaneous quantitative determination of rutin and scopoletin in the aerial parts of FABIANA IMBRICATA is presented. The results showed high variability in the scopoletin (240-2,400 mg%) and rutin (195-1,950 mg%) content in the populations surveyed. A micropropagation method for F. IMBRICATA plants was established by culturing shoot tips on Murashige-Skoog (MS) medium containing 1.0 mg/l benzylaminopurine (BAP), 0.01 mg/l naphthaleneacetic acid (NAA), and 0.1 mg/l gibberellic acid (GA3). Auxin addition to the rooting medium, especially 0.5 mg/l indolebutyric acid (IBA) or 0.5 mg/l IBA and 0.1 mg/l NAA, enhances root formation. The micropropagation method presented allowed the obtention of regenerated plantlets in six weeks from shoot tips.     

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.     

Growth and asiaticoside production in multiple shoot cultures of a medicinal herb,Centella asiatica (L.) Urban,under the influence of nutrient manipulations

Growth and in vitro asiaticoside accumulation in multiple shoot cultures of Centella asiatica (L.) Urban was studied as a function of nutrient manipulations in the culture media. Shoot cultures raised in liquid Murashige and Skoog medium supplemented with 2.5 mg/l kinetin attained a growth index (GI) of 6.06 along with the highest asiaticoside content of 3.8 mg/g dry weight on the 35th day of the culture cycle. The shoot growth and asiaticoside accumulation were found to be influenced by the relative proportions of NH₄ ⁺-N:NO₃ ⁻-N or Cu²⁺ concentration in the medium. Asiaticoside content in shoots increased from 5.3 to 8.9 and 8.7 mg/g dry weight when total nitrogen concentration of 60 mM in the control medium was reduced to 50 and 40 mM with a corresponding change in NH₄ ⁺:NO₃ ⁻ ratio from 20:40 to 20:30 or 20:20, respectively. Total nitrogen level higher than 60 mM drastically reduced the asiaticoside concentration in these in vitro shoot cultures. Medium devoid of Cu²⁺ significantly favored higher asiaticoside accumulation in the cultured tissue (7.05 mg/g dry weight) along with an improved biomass production (GI = 7.7) when compared with shoots reared on the control medium with 0.10 μM Cu²⁺ (GI = 5.8; asiaticoside content = 4.4 mg/g dry weight). Carbohydrate enrichment of the medium by increasing the sucrose concentration from 3.0 to 5.0 or 7.0% was also beneficial for biomass and asiaticoside production with GI = 17.1 and 16.9 and asiaticoside content = 7.2 and 5.2 mg/g dry weight, respectively, in comparison to control cultures maintained on medium containing 3.0% sucrose. The procedure described here provides a viable production platform for generating clean and quality material from Centella with high bioactive content.     

In vitro plant regeneration from leaf-derived callus of Cimicifuga racemosa

Cimicifuga racemosa (L.) Nutt., also known as Black Cohosh, is among the top 10 selling medicinal herbs in the United States. The rhizomes have been used to relieve menopausal discomfort. This plant is wild crafted and conservationists have expressed concerns with the sustainability of C. racemosa. Excised tissues from young leaves of C. racemosa were cultured on Murashige and Skoog's medium (MS) supplemented with various concentrations of NAA and TDZ for production of callus. The optimum callus growth and maintenance was in 1.0 microM NAA plus 0.5 microM TDZ. Two-month-old calli were sub-cultured on different concentrations of cytokinins (BA, kinetin, 2ip, TDZ) or in combination with GA(3) for shoot induction. The rate of shoot induction and proliferation was higher in MS media supplemented with 2.0 or 4.0 microM of TDZ. Concentrations of TDZ greater than 4.0 microM suppressed shoot growth. Adding 3.5 microM of GA(3) into media containing BA increased shoot growth. The presence of GA(3) with kinetin or TDZ did not affect shoot production. For rooting, shoots were transferred to MS medium with activated charcoal supplemented with various auxins (IAA, IBA and NAA), roots were noticed 20 days after transference. Activated charcoal was an essential component for vigorous rooting formation. Our results suggest that conservation of C. racemosa is possible through in vitro multiplication of leaf-derived callus.     

The effect of sulbactam on the in vitro activity of mezlocillin, piperacillin and cefotaxime]

The in vitro activity of mezlocillin (MZL, CAS 51481-65-3), piperacillin (PIP, CAS 61477-96-1) and cefotaxim (CTX, CAS 63527-52-6) alone and in combination with sulbactam (SBT; CAS 68373-14-8) against mezlocillin-resistant pathogens was determined in a multicenter study. A total of 870 strains were investigated (481 Enterobacteriaceae, 57 Pseudomonas aeruginos, 41 Acinetobaster spp., 194 Bacteroides fragilis, and 97 Staphylococcus spp.). Determinations of MIC were performed according to DIN-guidelines (agar-dilution method for aerobes and microbroth-dilution method for anaerobes). Sulbactam was added in fixed concentrations of 5 mg/l and 10 mg/l. In all sulbactam-combinations examined mean MIC as well as MIC50 and MIC90 were reduced compared to the respective values for the antibiotics alone. Consequently, percentages of susceptible strains increased significantly: i.e. for Enterobacteriaceae: MZL 1% vs. MZL + 10 mg/l SBT 53%; PIP 4% vs. PIP + 10 mg/l SBT 54%; CTX 52% vs. CTX + 10 mg/l SBT 68%. The effect of sulbactam was most pronounced in Bacteroides spp. with an increase in susceptible strains from 2% to 97% for MZL, from 6% to 95% for PIP and from 7% to 98% for CTX. The results indicate that by adding sulbactam the in vitro activity of mezlocillin, piperacillin and cefotaxim against resistant pathogens is augmented significantly. In addition, the spectrum of antibacterial activity is extended to anaerobic pathogens such as Bacteroides spp. The availability of sulbactam as a monosubstance for combination with various beta-lactam-antibiotics thus represents a useful improvement of therapeutic options in bacterial infections.     

Effect of 3-hydroxymethylene-2-thioxopyrrolidine on growth of two species of mutans streptococci and their in vitro plaque formation

3-Hydroxymethylene-2-thioxopyrrolidine (HMTP), the major product derived from radish mustard oil, was studied for its activity to inhibit the growth of mutans streptococci, their in vitro plaque formation and their glucan production. The minimum inhibitory concentration (MIC) (800-1600 mg/l) of HMTP at pH 7.0 was reduced to 200 mg/l by lowering the medium pH to 5.0. A dose-dependent inhibition of in vitro plaque formation was observed at 200-800 mg/l dose of HMTP. Production of water-insoluble glucan (WIG) was effectively inhibited by 45-98%, depending on HMTP dose (200-800 mg/l), while only 22% inhibition of water-soluble glucan (WSG) production was observed at an 800 mg/l dose.     

Furanocoumarins in Pastinaca sativa L. in vitro culture

Callus cultures of Pastinaca sativa L. (parsnip), Apiaceae, were cultivated on variants of Linsmaier-Skoog's medium, containing varying quantities (0.1-10.0 mg/l) of phytohormones: NAA-BAP and IBA-BAP which allowed to obtain 1.5-3-fold fresh biomass growth during 6-week subcultures. HPLC analyses showed that tissues cultured in vitro produced psoralen, bergapten, xanthotoxin, isopimpinellin and umbelliferone which are well known metabolites in plants growing under natural conditions. Total content of coumarins depended on the nature and quantity of phytohormones present in the medium, and ranged from 115.7 to 408.5 mg/100 g of the dry weight, isopimpinellin being the metabolite which dominated quantitatively (maximum content of 238.9 mg/100 g). Psoralen was also accumulated in callus tissues at considerable amounts (maximum content of 108.8 mg/100 g). This metabolite dominated in vegetative plant parts that have been analysed in our study (leaves, stems, roots) but its contents were lower than in the material from in vitro culture (48.9 mg/100 g 10.6 mg/100 g and 14.9 mg/100 g, respectively). Imperatorin was not detected in callus tissues although it dominated in the analysed fruits of the studied plant (200.0 mg/100 g). The best of the tested media in respect of promoting tissue biosynthetic capabilities was that which contained 3 mg/l NAA and 1 mg/l BAP. The studies showed that in vitro cultures of Pastinaca sativa L. can be a convenient model to study the biosynthesis of furanocoumarins and also a potential rich source of these compounds, particularly isopimpinellin.     

The effects of humic substances on copper toxicity to Ceriodaphnia silvestrii Daday (Crustacea, Cladocera)

A major question in the field of ecotoxicology is how DOM affects copper accumulation and toxicity in planktonic organisms; copper acute toxicity and bioaccumulation in Ceriodaphnia silvestrii were investigated in the presence and absence of humic substances (HS) under controlled laboratory conditions. Copper was determined as free Cu2+ ions in the media and total copper in the animals; metal ion buffers were used for ion selective electrode calibration, extending the lower detection limit to 10(-11) mol l(-1). Groups of 20 adult females of similar sizes were exposed (24 h) to a range of nominal copper concentrations. Based on total added copper, LC50 was 4.4 x 10(-8) mol l(-1) without HS, whereas with 20 mg l(-1) HS, it was 25 times higher (1.1 x 10(-6) mol l(-1)). Based on free Cu2+ ions LC50 was statistically similar either with (2.8 x 10(-8) mol l(-1)) or without HS (3.3 x 10(-8 )mol l(-1)). The present results showed that natural DOM reduced copper toxicity and that free Cu2+ ions correlates to the bioavailable fraction to zooplankton. Nevertheless, copper bioaccumulation by C. silvestrii was similar either in the presence or absence of humic substances, suggesting that C. silvestrii regulates its body copper content up to 3.0 x 10(-8) mol l(-1) free Cu2+ ions in the media. The organisms were not able to deal with higher free Cu2+ ions concentrations in the media.     

In vivo performance of parenteral theophylline-loaded polyisobutylcyanoacrylate nanoparticles in rats.

Theophylline-loaded polyisobutylcyanoacrylate (PICA) nanoparticles were prepared by emulsifier-free polymerization in aqueous media at ambient conditions. PICA nanoparticles were shown (in vitro) to be a promising controlled delivery system for theophylline. Therefore, this study was conducted to investigate the feasibility of PICA nanoparticles as a parenteral controlled drug delivery system in rats. Wistar rats were given intraperitoneal (i.p.) injections of theophylline solution (4 mg/kg) and theophylline nanospheres suspension (8 mg/kg) on two different occasions. Theophylline serum concentrations were measured by an HPLC assay. The drug solution was rapidly absorbed, distributed, and eliminated. The peak concentration (Cmax), 5.34+/-1.9 mg/l, was achieved 20 min following administration. The mean residence time was 2.94 h, and the apparent clearance was 0.31 (l/h)/kg. After nanospheres administration the mean Cmax, 2.53+/-1.1 mg/l, was attained at 3 h. The drug was successfully maintained around this elevated serum drug concentration up to 11 h in rats. The drug concentration was only reduced to 1.43+/-0.98 mg/l (i.e. reduced by 43.5%) after 20 h of administration. This present study provides evidence that the sorption of theophylline to PICA nanoparticles could control the drug release in rats.     

Micropropagation of Hydrastis canadensis: Goldenseal a North American endangered species

An in vitro propagation protocol for rapidly producing Hydrastis canadensis L., Goldenseal, plantlets from disk tissue of young leaves was developed. Leaf explants were inoculated on MS medium supplemented with various concentrations of NAA and TDZ for production of callus. Two-month-old calli were sub-cultured on MS media containing cytokinins (BA, kinetin, TDZ) in different concentrations for shoot initiation. The optimum level of callus induction and maintenance was in 5.3 microM NAA in combination with 2.2 microM of TDZ. Shoot multiplication was achieved on MS medium with 2.2 microM TDZ in combination with 0.5 microM NAA. The alkaloid profile of micropropagated plantlets was similar to the profile of the mother plants. These results suggest that our in vitro propagation protocol will produce a positive impact in the conservation of H. canadensis.     

莫邪菊的离体培养研究初报

目的 为建立莫邪菊的离体培养体系.方法 以成熟果实中的种子为外植体,探讨不同生长调节剂对离体培养中愈伤组织诱导、不定芽的分化和试管苗生根的影响.结果 培养基配方为MS+2,4-D 2.0 mg/L+6-BA0.2 mg/L时,种子破壳萌发率为70%,胚轴处愈伤组织诱导率为100%;MS+6-BA 2.0 mg/L为合适的丛生芽诱导和增殖培养基配方;培养基为MS0时生根率为95%,生根效果好且配方简单,移栽的试管苗成活率可达95%.结论 该试验建立的离体培养体系可用于莫邪菊的离体培养.     

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.