In vitro propagation of Coleus forskohlii Briq. for forskolin synthesis

Shoot multiplication was obtained in vitro within 20–25 d from shoot tip expiants of 30 d old aseptically germinated seedlings of Coleus forskohlii Briq., using 2 mg/1 of 6 benzylaminopurine (BA). Shoot multiplication was further enhanced with the gradual decrease in the level of BA, and its final omission after 4 months. Different auxins supplemented at the level of 0.05 mg/1 with BA did not yield better results. Seven regenerated plants showed only diploid cells in their root tips, while three plants did not. Of these, two were diploid with occasional aneuploid cells. In one plant 32 chromosomes were observed. The potential of shoot culture in vitro and use of micropropagated plants for the production of forskolin has been demonstrated.     

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.     

In vitro multiplication of Coleus forskohlii Briq.: An approach towards shortening the protocol

Summary A protocol has been developed that leads to the development of complete plantlets of Coleus forskohlii within 35–40 d by culturing stem tip explants in MS medium containing 0.57 μM indole-3-acetic acid and 0.46 μM kinetin through direct multiplication at the rate of 12.5 shoots per explant. About 100% shoots rooted and micropropagated plants were successfully established in soil after hardening with a high survival rate. The significance of the present micropropagation protocol of C. forskohlii is the formulation of growth regulators which effected very fast multiplication of the plant (time reduced to one-third of the hitherto known methods).     

In vitro plant development and root colonization of Coleus forskohlii by Piriformospora indica

The present study was conducted for optimization of in vitro substrates under aseptic conditions for interaction of Piriformospora indica with the medicinal plant Coleus forskohlii. It aims to test the effects of different substrates on P. indica colonization as well as growth parameters of the in vitro raised C. forskohlii. Interaction of in vitro C. forskohlii with root endophyte P. indica under aseptic condition resulted in increase in growth parameters in fungus colonized plants. It was observed that P. indica promoted the plant’s growth in all irrespective of substrates used for co-culture study. The growth was found inferior in liquid compared to semisolid medium as well as there was problem of hyperhydricity in liquid medium. P. indica treated in vitro plantlets were better adapted for establishment under green house compared to the non treated plants due to fungal intervention.     

Labdane Diterpenoids from Coleus forskohlii （Willd.） Briq.

in order to investigate the active substances of Coleus forskohlii（Wiiid.） Brlq., a new labdane dlterpenold, 12- hydroxy-8,13E-labdadlen-15.olc acid, together with two 8,13-epoxylabd-14-ene-11-one dlterpenolds, 6-acetyl-1- deoxyforskolln and 6-acetyl-1,9-dldeoxyforskolln, was Isolated on the basis of various chromatographic technlques and spectroscopic methods.     

Forskolin synthesis in in vitro cultures of Coleus forskohlii Briq transformed with Agrobacterium tumefaciens

Agrobacterium tumefaciens mediated tumor tissue and shooty teratomas of Coleus forskohlii were cultured in vitro. Forskolin was detected in tumorous callus (0.002%), rhizogenic callus (0.011%) and root cultures (0.014%), but not in shooty teratomas. Forskolin synthesis and accumulation in tumorous C. forskohlii cultures may permit the elucidation of diterpene metabolism in this species.Abbreviations B₅ medium after Gamborg et al. (1968) - 2,4-D 2,4, dichlorophenoxyaceticacid - MS medium after Murashige and Skoog (1962) - BAP benzylaminopurine - IBA indole-3-butyric acid - Kn kinetin - CH casein hydrolysate - T-DNA transferred DNA     

In vitro propagation of Clerodendrum colebrookianum Walp., a potential natural anti-hypertension medicinal plant

A rapid clonal propagation system for Clerodendrum colebrookianum Walp. (Verbenaceae), a anti-hypertension folk medicinal shrub has been developed. A range of cytokinins has been investigated for multiple shoot induction with shoot apex, axillary shoot, leaf, petiole and root explants. Optimum shoot induction occurred with axillary buds using 6-benzyladenine where an average of 21 shoots were produced per explant in 6 weeks. Subculturing the newly produced shoots, by separating into groups of five shoots, produced an average of 43 new shoots per culture within 4 weeks. In vitro rooting and weaning of over 200 plantlets was completely successful. Cytological studies revealed no visible abnormalities in chromosome number.Abbreviations 2iP 2-isopentenyladenine - BA 6-benzyladenine - LSD Least Significant Difference - NAA 1-naphthaleneacetic acid - TDZ thidiazuron - WPM Woody Plant Medium (Lloyd & McCown 1980) basal medium     

Biological control of root-rot of Coleus forskohlii Briq. using microbial inoculants

The root-rot or wilt of Coleus forskohlii is a very serious soil-borne disease caused by Fusarium chlamydosporum. A field study was undertaken to study the possibility of controlling the disease using three biocontrol agents viz., Glomus mosseae, Pseudomonas fluorescens, Trichoderma viride, singly and in combination. Planting of coleus cutting was done in wilt sick soil. Inoculation with Trichoderma viride + Glomus mosseae gave the best result in controlling the disease. The same treatment also resulted in maximum growth, yield and root forskolin concentration of coleus. Plants treated with T. viride + G. mosseae showed a disease severity index of 33.28% compared to uninoculated control plants, which had a maximum disease severity index of 85.5%. The fungicide Emisan (0.2%) was not as effective as the biocontrol agents in controlling the pathogen.     

In vitro propagation of Leptocarpha rivularis,a native medicinal plant

Leptocarpha rivularis, commonly known as “palo negro”, is a species of the Astereaceae family, endemic in the forests of Southern Chile. The extracts of this plant have medicinal properties attributable to its secondary metabolites, mainly identified as flavonoids and terpenes. Among them, leptocarpine has gained importance as a powerful anticarcinogen. The aim of this study was the in vitro establishment of L. rivularis to develop future alternatives to obtain biomass for medicinal purposes. Young shoots obtained from plants in growth chambers were treated with Captan™ (2.5% (w/v)), subjected to disinfection method with ethanol, commercial chlorine, and antioxidants and tested in different Murashige & Skoog based media to establish in vitro culture. The most effective growth was observed with half strength Murashige & Skoog medium supplemented with 20 g L⁻¹ sucrose and 3.2 g L⁻¹ gelrite (MGS medium) with 95% survival at 30-d and shooting reaching 82% of the explants when Plant Preservative Mixture™ is added to the medium. The addition of 1 mg L⁻¹ of indole-3-butyric acid to the MSG medium was necessary for inducing roots and obtaining well-rooted plants that were acclimatized and successfully established in the greenhouse. Our study represents the first report describing the in vitro culture of L. rivularis, allowing the sustainable use of this plant.

     

In vitro propagation of Rauwolfia micrantha, a rare medicinal plant

Shoot tip and single node explants from young shoots of 1-year old flowering plants of Rauwolfia micrantha Hook. f. were cultured on Murashige & Skoog (MS) medium variously supplemented with 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). A combination of 13.2 M BA and 2.68 M NAA induced high frequency (77%) formation of up to 3 shoots from each node in 8 weeks. The regeneration of shoot tips from the field-grown plants and in vitro shoots placed horizontally differed. Repeated subculturing of the shoot tips and single nodes at 6-week intervals for over a year in combination of 4.4 M BA and 0.27 M NAA enabled mass multiplication of shoots without any evidence of decline. Rooting of the excised shoots on medium containing 2.6 M NAA was preceded by callus formation. The rooted plants were removed off the callus, hardened off and 80% established in pots. Micropropagated plants displayed uniform morphological, growth, flowering, fruiting and seed germination characteristics.Abbreviations BA 6-benzyladenie - IAA indole-3-acetic acid - IBA indole-3-butyrie acid - 2-ip 2-isopentenyladenine - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid     

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.     

In vitro propagation of Acorus calamus Linn.--a medicinal plant

High frequency in vitro propagation protocol was standardized from rhizome explants of A. calamus. Maximum shoot multiplication frequency was obtained on Murashige and Skoog's media supplemented with 4 mg/l 6-benzyl amino purine and 0.5 mg/l indole-3-acetic acid. Regenerated shoots were rooted in vitro or directly transferred to sterile soil and well developed roots were observed within two weeks. The rooted plants were successfully established in the field.     

Higher Production of Forskolin in Genetically Transformed Cultures of Coleus forskohlii Briq Induced by Growth Regulators

Increased forskolin yield was obtained in transformed root, rhizogenic calli and cell suspension cultures of Coleus forskohlii when treated alone with various concentrations of auxins (IAA, IBA, NAA, 2,4-0), auxin conjugates ( IAA-ala, IAA-gly, IAA-phe, IAA-asp), cytokinins (Kn, BAP) and gibberellic acid (GA₃). An 8.9-fold stimulation in forskolin production was achieved in transformed rhizogenic line GCO-RCH-10 in presence of 1.0 mg I^-1 GA₃, 6-fold in cell suspension line GSO-5/7-k in presence of 2 mg I^-1 BAP and 4.3-fold in root line RC-ST -2/16 in presence of 0.5 mg I^-1 GA₃ at the end of a culture period of 4 weeks. Growth and morphology was found to be influenced by the growth regulators studied. A seven fold increase in biomass was obtained in rhizogenic line GCO-RCH-10 with 0.5 mg I^-1 GA₃ In root line RC-ST-2/16, different concentrations of IAA, IAA conjugates and GA3 stimulated growth while cytokinins inhibited growth of roots. The shoot culture line ST -2/51/d, showed prolific growth in the presence of all cytokinins but no forskolin was detected in the shoot cultures treated with any of these hormones.     

Clonal propagation of Arnica montana L., a medicinal plant

Summary Micropropagation of Arnica montana L. using Murashige and Skoog (MS) medium supplemented with N⁶-[2-isopentenyl]adenine (2iP), zeatin and α-naphthaleneacetic acid (NAA) in different concentrations does not ensure the formation of a high number of regenerated plants; a maximum of 3.2 neoplantlets per explant were obtained. After 4 wk of culture on medium with zeatin (4.5 μM) and NAA (5.3 μM), plants were 3.06 cm in length. The following step was to improve the clonal propagation of this species. Micropropagation of Arnica montana L., initiated from nodal segments using semisolid media (4 g l⁻¹ agar), was obtained. Explants were inoculated on MS medium supplemented with NAA (5.3 μM), 2iP (5.0 μM), maize extract (1.0 ml l⁻¹), phloroglucinol (0.6 mM) or adenine sulfate (0.2 mM). Only 3 wk after the inoculation, plant multiplication as well as induction of roots were obtained, the optimal variant being that containing NAA (5.3 μM), 2iP (5.0 μM) and maize extract (1.0 ml l⁻¹). Six weeks after the inoculation plants were transferred to Perlite, with 80% plant survival being obtained. By isoesterase pattern we concluded that we have obtained the clonal propagation of Arnica montana, because the pattern of several individuals belonging to different clones was the same. Only one region with esterase activity that is present in all individuals has been identified.     

In vitro propagation of the Egyptian medicinal plant,Echinops spinosissimus Turra

The in vitro propagation of medicinal species is proving to be one successful approach to addressing the issues raised by increasing global demand, overharvest of wild populations and variability in product quality. In this report we describe a protocol for establishing nodal cultures of an Egyptian medicinal plant, Echinops spinosis-simus Turra and for the induction of regeneration in epidermal peels and leaf explants obtained from the nodal cultures. Kinetin was found to be optimal for induction and proliferation of nodal cultures. Transverse slices of leaf blades produced regenerants resembling somatic embryos in the presence of thidiazuron while regeneration was observed on epidermal peels only when the medium contained elevated auxin concentrations.     

Shoot organogenesis and mass propagation of Coleus forskohlii from leaf derived callus

A high frequency shoot organogenesis and plant establishment protocol has been developed for Coleus forskohlii from leaf derived callus. Optimal callus was developed from mature leaves on Murashige and Skoog (MS) medium supplemented with 2.4 μM kinetin alone. Shoots were regenerated from the callus on MS medium supplemented with 4.6 μM kinetin and 0.54 μM 1-naphthalene acetic acid. The highest rate of shoot multiplication was achieved at the sixth subculture and more than 150 shoots were produced per callus clump. Regenerated shootlets were rooted spontaneously on half-strength MS medium devoid of growth regulators. The in vitro raised plants were established successfully in soil. The amount of forskolin in in vitroraised plants and wild plants was estimated and found that they produce comparable quantity of forskolin. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this plant. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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.