In Vitro Propagation Using Transverse Thin Cell Layer Culture and Homogeneity Assessment in Ceropegia bulbosa Roxb.

Ceropegia bulbosa is an endangered medicinal plant used traditionally in the treatment of various diseases. Our aim is to develop a rapid and a competent procedure for direct and indirect organogenesis from transverse thin cell layer (tTCL) explants of C. bulbosa. Optimum response to direct adventitious shoot bud induction from tTCLs was observed on medium augmented with 8.8 µM 6-benzyladenine (BA) producing 15.6 ± 0.31 shoots per responsive explant. Best callusing response (95 %) was observed with tTCL explants in medium containing 4.5 µM 2,4-dichlorophenoxyacetic acid and 2.2 µM BA. High frequency shoot regeneration (75 %) was observed from tTCL derived calli. Medium containing 8.8 µM BA and 0.27 µM α-naphthalene acetic acid produced 22.2 ± 0.64 shoots with shoots acquiring an average length of 4.6 ± 0.12 cm. In vitro rooting was recorded on ½ strength Murashige and Skoog medium, producing 10.9 ± 0.23 roots with a length of 4.24 ± 0.16 cm. Plants were successfully transferred to the field with a survival rate of 89 %. The clonal nature of the regenerants was assessed using Inter-simple sequence repeat markers.     

Multiple shoot regeneration and alkaloid cerpegin accumulation in callus culture of Ceropegia juncea Roxb.

This is the first report of in vitro propagation and alkaloid accumulation in callus cultures of Ceropegia juncea Roxb. a source of “Soma” drug in Ayurvedic medicine. Multiple shoots and callus induction was optimized by studying the influence of auxins [IAA (Indole-3-acetic acid), NAA (2-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid.)] and cytokinins [BA (6-benzyladenine) and Kin (Kinetin)] alone and in combinations. The best response for multiple shoot induction was obtained in nodal explants on MS medium supplemented with 7.5 μM Kin (8.5 ± 3 shoots per explants). The shoots were rooted on half strength MS (Murashige and Skoog’s) medium fortified with either IAA or NAA (0.5–2.0 μM). The plantlets were transferred directly to the field with 100 % success rate. Supplementation of MS medium with auxins and cytokinins enhanced the growth of callus but inhibited the shoot regeneration in nodal explants. Best callus induction and proliferation observed on MS + 1 μM 2,4-D+5 μM BA. However the maximum cerpegin content (470 μg/g dry weight) was recorded in dried callus derived on MS+10 μM IAA+5 μM BA. Quantitative TLC (Thin layer chromatography) studies of the callus revealed a phytochemical profile similar to that of naturally grown plants. The calli were maintained by subculturing at 4 weeks interval on fresh parent medium over a period of 34 months. The optimized in vitro propagation and callus culture protocol offers the possibilities of using organ/callus culture technique for vegetative propagation and production of cerpegin alkaloid.Key words: In vitro propagation, Pyridone alkaloid, Cerpegin, Callus, Ceropegia juncea     

Clonal propagation of Lilium nepalense D.Don, a threatened medicinal plant of Nepal

A protocol for the micropropagation of Lilium nepalense D.Don, a highly prized medicinal plant of Nepal, has been developed. Axillary shoots were regenerated from twin-scale explants prepared from mature bulbs. Multiplication was carried out on MS medium supplemented with 20 µM zeatin using longitudinally split shoot halves. On average, more than seven shoots were obtained from one explant in a 4-week culture period. After rooting the cloned plantlets were successfully hardened to ex vitro conditions. Preliminary trials in Nepal showed that the method can be successfully applied for the production of plants suitable for field cultivation. This may be an alternative to the over-exploitation of the natural resources of the species.     

In vitro propagation, genetic and secondary metabolite analysis of Aconitum violaceum Jacq.: a threatened medicinal herb

Aconitum violaceum Jacq. is an important medicinal species used for various health ailments including renal pain, rheumatism and high fever. In the present report, a reproducible in vitro regeneration system for Aconitum violaceum Jacq. has developed from the nodal segment of the plant. Induction of shoot buds was achieved on basal Murashige and Skoog (MS) medium. The shoots were elongated on MS medium supplemented with 0.5 μM 6-benzylaminopurine (BAP) and 0.1 μM α-napthaleneacetic acid (NAA) and subsequently transferred to rooting medium. In vitro grown microshoots of A. violaceum were encapsulated in the alginate beads. The success rate of their re-growth was found to be approximately 85.43 %. Of the encapsulated microshoots, 39.86 % exhibited formation of multiple shoots following re-growth on plant growth regulator free MS medium. Healthy root formation was observed in all microshoots following 2 weeks of transfer on half-strength MS medium containing 0.1 μM indole-3-acetic acid (IAA) and 1.0 μM α-naphthalene acetic acid (NAA). These plants were subsequently transferred to pots containing a mixture of soil, sand and compost (1:1:1 v/v), and same were then shifted in the greenhouse with 87 % survival rate. The molecular analysis was carried out using 35 random amplified polymorphic DNAs (RAPD) primers and 25 inter simple sequence repeats (ISSR) primers. Cluster analysis of the RAPD and ISSR profile revealed an average similarity coefficient of 0.966 and 0.974, respectively, confirming genetic stability of tissue culture-raised (TR) plants and synthetic seed-derived plants (SR). The phytochemical analysis of tissue culture-raised and synthetic seeds-derived plants showed higher aconitine content than control plant. The propagation protocol developed in this study provides a basis for germplasm conservation and harnessing the medicinally active compounds of A. violaceum.     

An efficient in vitro regeneration of Ceropegia noorjahaniae: an endemic and critically endangered medicinal herb of the Western Ghats

An efficient protocol was developed for the rapid in vitro multiplication of an endemic and critically endangered medicinal herb, Ceropegia noorjahaniae Ans., via enhanced axillary bud proliferation from nodal explants. The effects of phytohormones [6-benzylaminopurine (BAP), kinetin (Kin) thidiazuron (TDZ), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA)] on in vitro regeneration were investigated. The highest number of shoots (18.3 ± 1.3), maximum shoot length (10.1 ± 0.8 cm) and the highest response of shoot induction (95 %) were recorded on MS medium supplemented with 2.0 mg/l BAP. Rooting was best achieved on half-strength MS medium augmented with IBA (1.0 mg/l). Half-strength MS medium supplemented with BAP (4 mg/l) and sucrose (5 %, w/v) produced an average of 5.6 flower buds per microshoots with highest (90 %) flower bud induction response. The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing sterile sand and coco peat (1:1) and grown in a greenhouse with 85 % survival rate. The regenerated plants did not show any detectable morphological variation. The developed method can be successfully employed for large-scale multiplication and conservation of C. noorjahaniae.     

In vitro propagation ofHolostemma annulare (Roxb.) K. Schum., a rare medicinal plant

Summary A rapid micropropagation system was established forHolostemma annulare (Roxb.) K. Schum., (H. ada-kodien R. Br. ex Schult; Asclepiadaceae), a rare medicinal plant. Shoot tips (0.5–0.8 cm) and terminal and basal nodes (1.0–1.5 cm) harvested from actively growing shoots of conventionally raised plants were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). Multiple shoot formation (3.8) was observed in 68% of basal nodes cultured on medium with optimum concentration of 4.43 μM BA and 0.54 μM NAA after 8 wk. Terminal nodes were not suitable for inducing multiple shoots. Irrespective of the orientation (vertical/horizontal), all shoot tip explants responded with a single shoot in all the combinations of plant growth regulators tried. Effects of other cytokinins (kinetin and 2-isopentenyladenine) and auxins [indole-3-acetic acid and indole-3-butyric acid (IBA)] to enhance the regeneration potential of basal nodes were analyzed. Shoots were multiplied by subculture of basal nodes and stumps (the original explant tissue free of shoots, but with remnant axillary, meristem and two or three protruding buds) in a reduced concentration of BA (2.21 μM) and NAA (0.27 μM). Liquid medium for multiplication was found to be ineffective due to a high degree of hyperhydricity. To make the multiplication process cost effective, culture bottles with polypropylene, caps were used for multiplication. The best root induction (75%) and survival (80%) was achieved on 0.5 strength MS medium supplemented with 1.48 μM IBA. Field-established plants had uniform growth habit traits in terms of height of plants and number, length, and weight of the tuberous roots.     

Micropropagation of Allium wallichii kunth, a threatened medicinal plant of Nepal

Summary Bulbs and aerial parts of the Nepalese plant Allium wallichii are widely used for medicinal purposes and as a spice. Due to overharvesting the natural populations of the species have been increasingly reduced and the domestication of the species should be considered. For the purpose of the production of plantlets suitable for field culture, a micropropagation procedure based on multiple shoot culture has been established. Multiplication factors of 4.6 on average were possible on MS medium supplemented with 20 μM zeatin. After rooting on MS medium with 10 μM indolebutyric acid, plantlets were acclimatized to greenhouse conditions and transferred to the field with good success. Part of the PhD thesis of P. R. Malla.     

Direct shoot organogenesis from shoot tip explants of a highly medicinal valued tree Pterocarpus marsupium Roxb.

In Vitro Cellular & Developmental Biology - Plant - An in vitro regeneration system for propagation of a valuable medicinal tree, Pterocarpus marsupium, using shoot tip (ST) explants derived...     

In vitro propagation of Coleus forskohlii Briq., a threatened medicinal plant

In vitro clonai multip1ication of Coleus forskahlii Briq a threatened plant, has been achieved on MS medium supplemented with Kn (2.0 mg/l) and IAA (1.0 mg/l) using nodal segments as explants, Shoots multiplied at a rate of 12 — fold every six weeks. Rooting was achieved upon transfer of shoots onto MS medium containing IAA (1.0 mg/l). The micropropagated plants were successfully established under field conditions. Forskolin content in tubers of plants obtained by micropropagation was found to be 0.1%, the same as that found in wild plants. This micropropagation procedure should be useful for conservation as well as production of this important plantAbbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) basal medium - NAA -naphthalene acetic acid     

Essential oil composition and antimicrobial activity of wild and cultivated Moroccan Achillea ageratum L.: a rare and threatened medicinal species

The essential oils of leaves and flowers of the wild and cultivated Moroccan Achillea ageratum L., a rare and threatened medicinal species, were examined by GC/MS, and their chemical compositions were compared. At least nine components were identified in both wild and cultivated A. ageratum oils, representing more than 95% of the oils. Artemisyl acetate (62.34-78.79%), yomogi alcohol (4.89-12.40%), santolina alcohol (4.86-11.77%), and artemisia alcohol (3.36-7.04%) were the major compounds. Terpene-alcohol proportion was higher in wild A. ageratum than in cultivated A. ageratum. The antibacterial analysis showed that both oils presented high activity against all the studied Gram-positive strains in a range of MIC values from 2.55 to 7.02?mg/ml, but they appeared not effective against the tested Gram-negative ones (MIC values 20.40-41.10?mg/ml). They also exhibited remarkable antifungal activities against Candida species with MIC values ranging from 5.83 to 8.42?mg/ml. From these results, it was concluded that domestication of this threatened medicinal species using clonal propagation did not significantly affect its chemical composition and consequently its antimicrobial properties.     

Urbanization and a threatened freshwater mussel: evidence from landscape scale studies

The inflated heelsplitter, Potamilus inflatus, a federally listed freshwater bivalve, has been eradicated from northern portions of its range in the Amite River in Louisiana, USA. We hypothesized that the remaining populations of Potamilus inflatus in the southern part of the Amite River are being affected by increased urbanization of the watershed caused by growth of the surrounding Baton Rouge metropolitan area. Comparison of catch per unit effort in 2007 with a study conducted in 1994 indicated a significant drop in CPUE from 1.76 heelsplitters per site to 0.87. The size distribution of heelsplitters also had decreased in mean shell length from 116 to 97 mm, owing either to dislodgement of larger individuals in spates, or die-offs of larger males. Logistic regression suggested that site variables like substrate type and current velocity were not as important as landscape scale variables in predicting heelsplitter presence at a site. Heelsplitter presence was positively related to the amount of wetland riparian forest, and negatively related to the amount of residential development at the reach (1 km upstream) scale. Our results are significant because we show (1) that statistical models of GIS-based land use can predict the distribution of threatened mussel species, and that (2) conservation of endangered freshwater mussels will require more emphasis on the integrity of the riparian corridor.     

High frequency in vitro regeneration system for conservation of Coleus forskohlii: a threatened medicinal herb

Present study reports a high frequency regeneration system for in vitro propagation and conservation of an important and threatened medicinal herb Coleus forskohlii (Briq.). Shoot multiplication has been achieved through axillary bud development and direct adventitious shoot formation in nodal explants on Murashige and Skoog (MS) medium containing 6-benzyladenine (BA) (5 μM). Further shoot multiplication was recorded up to third subculture on MS medium containing BA (5 μM) in combination with 1-naphthleneacetic acid (NAA) (0.1 μM). Excised microshoots on transfer to root induction medium consisting of half-strength MS medium (1/2 MS) alone as well as in combination with various auxins, resulted in varied rooting pattern in terms of number, length, and type of roots. Rooted microshoots were acclimatized successfully in earthen pots containing garden manure, garden soil, and sand (1:2:1) as potting mix with survival rate of 70 %. Acclimatized plantlets were studied for the amount of chlorophyll and carotenoid content as well as the net photosynthetic rate (P_N) during subsequent weeks of transfer to ex vitro condition. Histological studies revealed the direct origin and development of shoot buds from basal swollen cut end of nodal explants.     

心心相印的组织培养与快速繁殖

1植物名称心心相印(Ceropegia woodii Schlecht.)。2材料类别茎段。3培养条件(1)芽诱导培养基:MS IBA0.1mg·L-1(单位下同) 6-BA1.0 蔗糖30g·L-1;(2)继代增殖培养基:MS IBA0.1 6-BA0.5 蔗糖3     

Genetic diversity of Stemona parviflora: A threatened myrmecochorous medicinal plant in China

The plant species Stemona parviflora, which is endemically distributed in Hainan and Guangdong Provinces of China, is an important constituent of Chinese traditional medicine “Baibu”. Based on AFLP analysis, at the species level, the percentage of polymorphic bands (PPB), gene diversity H, and Shannon's information index I of S. parviflora are 100%, 0.2826, and 0.4458, respectively. The genetic differentiation among populations is low (G_st = 0.0799), while the gene flow among these populations is high (N_m = 5.754). Actually, no distinguishable genetic structure is inferred according to the AFLP analysis in this study. There was no significant positive correlation between genetic distance and geographic distance either. Based on its medicinal use, seed dispersal pattern (myrmecochory), mating system, fragmentation of the natural habitat by rubber and eucalyptus planting, and the historical formation and transition of Hainan island and Guangdong provinces, we discuss the current threatened condition of the traditional medicinal plant S. parviflora. In/ex situ conservation tactics are recommended to save this important species.     

Evaluation of traditional medicinal plant,Cissus setosa Roxb. (Vitaceae) for antiulcer property

Cissus setosa is an indigenous medicinal herb commonly used for the treatment of gastro ulcers. In the current investigation the aerial methanolic extract of C. setosa was investigated for their antiulcer activity using pylorus ligation and ethanol in experimental rats. The extract was administered at the doses of 200 and 400 mg/kg b.w. orally for 3 days. However, higher dose of the extract subsequently reduced gastric ulcer induced aberrations by pylorus ligation (70.05%) and ethanol (78.16%) as judged by their altered biochemical parameters such as free acidity, total acidity, total carbohydrate, total protein and pepsin activity. Furthermore, macroscopic examination of rat’s stomach also showed that the pretreatment with methanolic extract notably lowered the pylorus ligation and ethanol induced ulcers. As perceived in the present study, evidently, our findings basically supports the potency of the methanol extracts of C. setosa to treat gastrointestinal related disorders, thus lends pharmacological credence to the suggested folklore use.     

In vitro studies of Bacopa monnieri--an important medicinal plant with reference to its biochemical variations

Micropropagation of Bacopa monnieri was achieved on MS and B5 medium supplemented with BAP and NAA using leaf explants and nodal segments. Best results were found on MS medium in both the explants with BAP (2.0 mg/l) showing higher percentage of regeneration. Besides that the biochemical parameters, like chlorophyll, carbohydrate, protein, of leaves both in vivo and in vitro have also been carried out in order to establish the sustainability of plants.     

Thiosugars: new perspectives regarding availability and potential biochemical and medicinal applications

Thiosugars, containing a sulfur atom as heteroatom or a disaccharide linked via a sulfur bridge, possess unique physicochemical properties such as water solubility, which differs from conventional functionalized monosaccharides. The differences in biological activities between thiosugars and their oxygen analogs depend on geometric, conformational, and flexibility differences. They depend also on their electronic differences, the sulfide function being less electronegative and more polarizable than the ethereal moiety. Many functionalized thiosugars occur naturally and are potential targets for the development of carbohydrate-based therapeutics. Among the few new examples of the potential new targets are salacinol and kotalanol, tagetitoxin, thiolactomycin and analogues, mycothiol and analogues, and S-nitrosothiols. These new developments and representative examples of functionalized thiosugar prototypes as potential new targets are presented in this mini review.