Coleus Forskohlii Briq Research Articles

Determination of Metabolic and Genetic Diversity in Coleus forskohlii Briq. Using HPTLC and RAPD Analysis

Coleus (Coleus forskohlii Briq.) belongs to the family lamiaceae and is known for its forskolin content, which has been demonstrated to possess hypotensive, spasmolytic, cardiotonic and antiplatelet activity. The dominant polymorphism between individual samples/races are indicated by the presence or absence of a particular RAPD fragment and extensively applied for assessing genetic diversity and relationship measures in various plant species. However, use of chromatographic fingerprint in analysis of metabolite diversity similar to RAPD in molecular markers is uncommon and can be easily done by developing multiple fingerprints using different solvent systems and analyzing them for determination of metabolic similarity in between species. Simultaneous, metabolic and genetic diversity has been attempted by the authors for first time using HPTLC fingerprints and RAPD markers, respectively. Genetic analysis of 10 different samples of C. forskohlii genotypes was performed using 20 RAPD primers, based on an efficient protocol for isolation of genomic DNA from dried root of coleus. Chromatographic analysis was carried out using three different mobile phases and pre-coated aluminum back TLC sheets as stationary phase. A similarity matrix was constructed using molecular and chromatographic fingerprint data based on Jaccard's coefficient and further subjected to construct dendrogram based on UPGMA. The similarity coefficient for genetic nature ranged from 0.362 to 0.783 while for metabolic profile it was between 0.683 to 0.939. This indicates a genetic diversity in C. forskohlii samples but a lesser chemical diversity among the same samples.

Strategies to Improve the Production of Forskolin from Hairy Root Cultures of Coleus forskohlii Briq.

The aim of this study was to elucidate the effect of elicitors and precursors on the production of forskolin from the hairy root cultures of Coleus forskohlii Briq. Hairy root cultures were established from leaf explants by infecting with Agrobacterium rhizogenes strain A4 on MS basal medium. Suspension cultures of hairy root cultures were initiated in MS medium containing IBA (1.0 mg/L), casein hydrolysate (600 mg/L). We investigated the growth of biomass and forskolin production in suspension cultures of hairy roots. The production of forskolin was parallel to the growth of biomass. The maximum production of forskolin was observed after 5 weeks. With the objective to increase the yield of forskolin, abiotic elicitors such as salicylic acid (100 μM and 500 μM), copper sulphate (100 μM and 500 μM), methyl jasmonate (100 μM and 500 μM) and precursors such as α-ketoglutaric acid (0.2 mM and 1.0 mM), L-phenylalanine (0.2 mM and 1.0 mM) were added to hairy root cultures on different days of incubation period and evaluated their effects on production of forskolin. Elicitor, methyl jasmonate (500 μM) and the precursor, L-phenylalanine (1 mM) on day-14 addition significantly enhanced the production of forskolin over the control hairy root cultures C. forskohlii. Given forskolin’s limited commercial supply, this study provides avenues for improving the production of forskolin in the hairy root culture of C. forskohlii.

Synergy between Glomus fasciculatum and a beneficial Pseudomonas in reducing root diseases and improving yield and forskolin content in Coleus forskohlii Briq. under organic field conditions

Root rot and wilt, caused by a complex involving Fusarium chlamydosporum (Frag. and Cif.) and Ralstonia solanacearum (Smith), are serious diseases affecting the cultivation of Coleus forskohlii, a crop with economic potential as a source of the medicinal compound forskolin. The present 2-year field experiments were conducted with two bioinoculants (a native Pseudomonas monteilii strain and the exotic arbuscular mycorrhizal (AM) fungus Glomus fasciculatum) alone and in combination under organic field conditions in order to evaluate their potential in controlling root rot and wilt. Combined inoculation of P. monteilii with G. fasciculatum significantly increased plant height, plant spread, and number of branches; reduced disease incidence; and increased tuber dry mass of C. forskohlii, compared to vermicompost controls not receiving any bioinoculants. Increase in tuber yields was accompanied by an increase in plant N, P, and K uptake. Co-inoculation of P. monteilii with G. fasciculatum significantly improved the percent AM root colonization and spore numbers retrieved from soil. This suggests P. monteilii to be a mycorrhiza helper bacterium which could be useful in organic agriculture. The forskolin content of tubers was significantly increased by the inoculation treatments of P. monteilii, G. fasciculatum, and P. monteilii + G. fasciculatum.

Analyzing Variability in Coleus forskohlii Briq. Using RAPD Markers

Coleus forskohlii Briq. is an indigenous medicinal plant with high traditional use in India. Genetic analysis of 37 diverse C. forskohlii genotypes was performed using 25 RAPD primers, which yielded 117 bands, of which 60 (51.28%) were polymorphic providing an average of 3.75 bands per primer. There were no genotype-specific products. The number of bands per primer varied from 1 (OPZ 8&16) to 7 (OPZ 11). Similarity matrix was constructed using Jaccard's Coefficient and the data matrix of coefficient of similarity was subjected to cluster analysis using unweighted pair group methodology with arithmetic average (UPGMA). Cluster analysis resulted in grouping of 37 genotypes into two major clusters. The results indicated that RAPD could be used for genetic diversity analysis in C. forskohlii using higher number of primers as it is reliable, easy, rapid and cost-effective.

Studies on Correlation and Path Analysis in Mutants of Coleus (Coleus forskohlii Briq.) for Yield and forskolin Content in V2M1 Generation

The present investigation was carried out during 2003-2007 involving terminal cuttings of coleus ecotype 'Garmai'. Genotypic correlation coefficient between yield and its components in mutants of coleus was studied, viz., plant height, number of branches plant-1, number of leaves plant-1, number of tubers plant-1, tuber length and tuber girth were found to have positive and highly significant correlation with yield. However, forskolin and essential oil content showed negative correlation with yield. Path analysis of component characters on yield of Coleus in V2M1 generation exerted positive direct effect through the characters plant height, number of leaves plant-1 and number of tubers plant-1. Similarly, direct effect was observed to be negative through number of branches plant-1 (-0.930), total amount of alkaloids (-0.066) and forskolin content (-0.026). The current investigation resulted in residual effect of 0.158 indicating the accuracy and appropriate selection of component character for crop improvement programme. Weightage must be given to component characters exhibiting positive attributes towards fresh tuber yield in Coleus. However, some traits with negative attributes are also chosen for getting improved quality, i.e., forskolin content, without much inhibition on fresh tuber yield plant-1.

Potential application of urea-derived herbicides as cytokinins in plant tissue culture

Various urea-derived herbicides and different cytokinin analogues were used to determine their effects on callusing response and shoot regenerating capacity of alfalfa (Medicago sativa L.) and Coleus (Coleus forskohlii Briq.). The herbicides monuron and diuron evoked profuse callusing response from Coleus leaf segments and alfalfa petiole explants on Murashige and Skoog medium. Shoot regeneration by monuron (2.0 mg/l) showed a maximum of 3 multiple shoots both in alfalfa and Coleus with a frequency of 92% and 75%, respectively. Whereas diuron (0.5 mg/l) showed a high frequency of shoot regeneration (89%)with a mean number of 5 shoots in alfalfa, in C.forskohlii, the frequency of regeneration was 90%with a mean number of 6 shoots. Diuron with two chloride groups in the phenyl ring showed significantly higher cytokinin-like activity than single chloride substitution monuron. This study demonstrates the potential use of monuron and diuron as cytokinins in plant tissue culture.

In vitro regeneration and transformation of Coleus forskohlii with glucanase-chitinase gene

Coleus forskohlii Briq. is a threatened medicinal plant was transformed with glucanase-chitinase gene along with npt-II gene. The transformants were obtained on MSO supplemented with 2 mg/l or 3 mg/l BAP and 0.2 mg/l 2,4-D for callus induction using leaf as explants. Multiple shoot production from the leaf-derived transformed callus of Coleus has been standardized using the various concentration of NAA with 2 mg/l BAP. The combination of 2 mg/l BAP and 0.5 mg/l NAA was found to be best for multiple shoot production. Rooting of the transformed shoots was found to be best on MS medium supplemented with 0.7 mg/l NAA. The presence of npt-II genes in the leaves of putative transformants was confirmed by PCR analysis. SDS-PAGE analysis and glucanase assay revealed the over expression of glucanase enzyme in the transgenic plants. The transgenic Coleus plants survived the infection caused by Fusarium chlamydosporium due to the accumulation of glucanase-chitinase enzyme, whereas control plants were susceptible to the disease.

EVALUATION OF COLEUS FORSKOHLII ACCESSIONS FOR TUBER AND FORSKOLIN YIELD

A replicated field evaluation trial was conducted involving 13 accessions of Coleus forskohlii Briq. (Lamiaceae), a forskolin yielding aromatic herb with fasciculate tuberous roots. Forskolin (diterpenoid) is used for treatment of glaucoma, cardiomyopathy and certain cancers. Significant differences were recorded for all the characters except dry mass of roots. Maximum tuber number per plant (12.0) was in IIHR-59. Accession IIHR-59 also produced highest fresh and dry mass of tubers (870 g and 88.75 g, respectively) per plant. The harvest index was maximum (45.1%) in IIHR-80. The forskolin content was estimated using HPLC and was found to vary from 0.025 per cent (IIHR-1) to 0.798 per cent (IIHR-12). Forskolin yield per plant was highest in IIHR-7 (85.00 mg). The accession IIHR-80 with medium tuber yield and higher forskolin content (0.715%) can be promoted for commercial cultivation as the crop is propagated through vegetative means. INTRODUCTION Coleus forskohlii Briq. (Lamiaceae), a tuber bearing, perennial aromatic, Indian herb is an exclusive source of ‘forskolin’ (labdane diterpene). Forskolin is used in the preparation of drugs against glaucoma, congestive cardiomyopathy and certain cancers (deSouza et al., 1986). Apart from their medicinal uses, the tubers are also used as a condiment and pickled. C. forskohlii cultivars grown in India and those collected from wild exhibit a wide diversity in exomorphic and yield characters (Bahl and Tyagi, 1989). The forskolin content in tubers from wild sources is variable and large scale indiscriminate collection of this plant from its natural habitat has contributed to its enlistment as an endangered species (Vishwakarma et al., 1988). Therefore, collection, conservation, and evaluation of available diversity are of utmost important before the valuable genetic diversity is completely lost. In the present study, an evaluation of tuberous collections was undertaken for their tuber and forskolin yielding characters. MATERIALS AND METHODS The plant materials for the present study consisted of thirteen accessions originally collected from diverse regions of India. The experiment was laid out in a completely randomised design with three replications. Polybag rooted terminal shoot cuttings were used for planting. A spacing of 1 m x 1 m was adopted to avoid interplant competition. The roots were harvested 150 days after planting. Two randomly selected plants per replication were used for recording observations. Data on yield was recorded and analysed statistically using analysis of variance technique (Table 1). The forskolin estimation was carried out using HPLC technique as per the procedure described by Inamdar et al. (1984) with little modification. Chromatographic system: Waters HPLC Pump: Waters 575, binary gradient operated under isocratic conditions. Detector: Waters 2487 dual absorbance detector. Software: Millennium 32 chromatography software. Chromatographic conditions: (a) Mobile phase: HPLC acetonitrile grade: Millipore water (50 : 50) Proc. WOCMAP III, Vol. 1: Bioprospecting & Ethnopharmacology Eds. J. Bernath, E. Nemeth, L.E. Craker and Z.E.Gardner Acta Hort 675, ISHS 2005

Molecular cloning, expression and characterization of recombinant 1-deoxy- d-xylulose-5-phosphate reductoisomerase from Coleus forskohlii Briq.

1-deoxy- d-xylulose-5-phosphate reductoisomerase (DXR) catalyses the first committed step in the production of isopentenyl diphosphate (IPP) according to the recently identified plastidial 2 C-methyl- d-erythritol 4-phosphate (MEP) pathway. Forskolin, the major active diterpenoid component in Coleus forskohlii is synthesised via the MEP pathway. Herein, we present the investigation of a cDNA encoding C. forskohlii DXR, its heterologous expression in Escherichia coli and an examination of its properties in vitro. The full-length cDNA sequence of C. forskohlii DXR contains an open reading frame (ORF) of 1407 nucleotides encoding a peptide of 469 amino acids, resulting in an estimated molecular mass of 50.8 kDa. The K m and V max of C. forskohlii DXR were determined to be 147.2 μM and 0.3 units mg protein −1, respectively. Fosmidomycin, a specific inhibitor of DXR, showed dose-dependent inhibition with an IC 50 of 0.45 μM. A gene expression study using a C. forskohlii plant culture showed that DXR is strongly expressed in leaves, less in stems and at the limit of detectability in roots. The presence of fosmidomycin in C. forskohlii plant culture resulted in a decrease in forskolin production in roots. Therefore, we conclude that if C. forskohlii DXR is involved in the biosynthesis of forskolin, it is primarily synthesised in the leaves, and later accumulates in stems and roots.

Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

BackgroundIsopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq.ResultsThe open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots.ConclusionThis investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

Quantitative Analysis of Forskolin in Coleus forskohlii (Lamiaceae) by Reversed-Phase Liquid Chromatography

A rapid method was developed for the evaluation of forskolin in Coleus forskohlii Briq. (Lamiaceae). Forskolin was quantitated in the root and stem of dried C. forskohlii and in 17 market products by reversed-phase liquid chromatography (LC) with a photodiode array detector at 210 nm. The temperature was held constant at 30 degrees C, and the retention time of forskolin was approximately 6.8 min. The samples were extracted with acetonitrile by sonication. The precision of the method was confirmed by a standard deviation < 5.0% (n = 3), and forskolin recovery was 99.1%. Limit of detection was 1.5 microg/mL, and the response was linear through zero from 6.3 to 630 microg/mL with a correlation coefficient (R2) of 0.9998. Identity of the marker compound was confirmed by an LC/mass spectrometry experiment. The method was successful in the qualitative and quantitative evaluation of the marker compound in C. forskohlii plant material and in market products claiming to contain C. forskohlii.

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.

Induction of callus and plant regeneration in Coleus forskohlii Briq.

Induction of callus and plant regeneration in Coleus forskohlii Briq.

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

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

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.

Forskolin production in untransformed root culture of Coleus forskohlii

Untransformed cultures of Coleus forskohlii Briq. were initiated from in vitro micropropagated shoots derived from shoot tip culture. Effects of IAA, IBA and two IAA derivatives, 4-CI-IAA and 5,6-Cl 2-IAA were compared for growth and forskolin production in cultured roots grown on different basal media under different culture regimes. The roots grew well in the presence of all dosages of 4-CI-IAA and 5,6-Cl 2-IAA with 25% MS plus 1% sucrose and produced more forskolin as compared with IAA or IBA. The optimum concentrations needed for forskolin production were 1 μg 1 −1 for 5,6-Cl 2-IAA and 50,μg1 −1 for 4-CI-IAA. The highest amount of forskolin (0.09%) was raised at 50,μg1−1 4-CI-IAA after 60 days of culture. This enhanced level of forskolin was almost equal to the content in the wild plant. However, under similar conditions and with 500,μg 1 −1 IAA or IBA, the amount of forskolin produced was much smaller. IAA derivative-induced production of forskolin indicates the possibility for scaling up of the production of this compound in untransformed root cultur

Production of Forskolin by AxenicColeus forskohliiRoots Cultivated in Shake Flasks and 20-l Glass Jar Bioreactors*

Root cultures of COLEUS FORSKOHLII Briq. were initiated from primary callus or IBA-treated suspension cultures and maintained on Gamborg's B5 medium containing 1 mg/l IBA. Transformed root cultures were established by infecting surface-sterilized leaves with AGROBACTERIUM RHIZOGENES strain 15834. Transformation was confirmed by mannopine detection. These cultures displayed the typical characteristics of hairy root cultures, with the sole exceptions of slow growth in hormone-free medium and accelerated growth on medium containing phytohormones. All root cultures examined formed forskolin and its derivatives in amounts ranging from 500 to 1300 mg/kg dry weight, corresponding to about 4 to 5 mg/l. During cultivation roots could be cut into small pieces without affecting growth and forskolin production. Scale-ups of the cultivation procedure were performed in 20-l glass jars with a working volume of 10 to 13l. Forskolin production in bioreactors was better than in shake flasks. Levels of almost 14 mg/l could be reached after 21 d of cultivation. As in the shake flask experiments cutting the roots did not affect growth or productivity in a negative way.

The Occurrence of Forskolin in the Labiatae

Plants belonging to the Coleus and Plectranthus genera of the Labiatae were semi-quantitatively assayed for the presence of forskolin, a potent cardioactive and hypotensive diterpenoid under development. In the TLC and GLC assay methods employed, forskolin could be detected at levels down to ca. 1 × 10-4 % of the dry plant material. Coleus forskohlii Briq. appears to be unique in that it is the only species among those assayed which was found to contain forskolin.