Yield enhancement strategies for the production of picroliv from hairy root culture of Picrorhiza kurroa Royle ex Benth (original) (raw)
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Acta Physiologiae Plantarum
Picrorhiza kurroa Royle ex Benth. is an endangered plant producing various compounds of medicinal importance. Hairy roots of P. kurroa were obtained following cocultivation of shoot tip explants with Agrobacterium rhizogenes strains A 4 and PAT 405. Bacterial strain A 4 appeared to be better than the strain PAT 405 in terms of both growth of respective hairy root cultures and secondary metabolite production. The optimal growth of both the hairy root cultures occurred on half-strength semisolid medium with 3% sucrose. Picrotin and picrotoxinin from the roots of wild type field grown plants were compared with 8-week-old hairy root cultures induced by the A 4 and PAT 405 strains of A. rhizogenes. Picrotin and picrotoxinin content were evaluated in hairy root cultures as well as roots of field grown plant of P. kurroa. In terms of the production of picrotin and picrotoxinin, the A 4 induced hairy roots appeared to be a better performer than the PAT 405 induced hairy root cultures. The picrotin and picrotoxinin content was highest in 8-week-old A 4 induced hairy roots (8.8 μg/g DW and 47.1 μg/g DW, respectively). Rapid growth of the hairy roots of P. kurroa with in vitro secondary metabolite production potential may offer an attractive alternative to the exploitation of this endangered plant species.
Plant Biotechnology Reports, 2007
A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species.
Hairy root cultures of Rehmannia glutinosa and production of iridoid and phenylethanoid glycosides
Acta Physiologiae Plantarum, 2012
Hairy root lines through the infection of Agrobacterium rhizogenes strain (A4) were established from shoot tips and leaves of Rehmannia glutinosa Libosch. Ten lines of hairy roots were selected on the basis of biomass increase in half-strength Gamborg medium ( 1 / 2 B5). Transgenic status of the roots was confirmed by polymerase chain reaction using rolB and rolC specific primers. Iridoid glycosides (catalposide, loganin, aucubin and catalpol) and phenylethanoid glycosides (verbascoside and isoverbascoside) identified using HPLC-ESI-MS, and their contents were compared with untransformed root culture and roots of 1-year-old field-grown plants of R. glutinosa by RP-HPLC. The growth and production of secondary metabolites in ten hairy root lines varied considerably as to the media. Woody plant (WP) medium displayed higher growth in terms of fresh (FW) and dry weights (DW) compared to 1 / 2 B5 medium. High-yielding hairy root lines produced higher amounts of loganin, catalposide, verbascoside and isoverbascoside in comparison to the untransformed root culture and roots of 1-year-old field-grown plants. The highest amounts of catalposide and loganin in transformed roots were 4.45 mg g -1 DW (RS-2 hairy root line) and 4.66 mg g -1 DW (RS-1 hairy root line), respectively. Aucubin and catalpol were detected in some lines in trace amounts. The highest amounts of verbascoside (16.9 mg g -1 DW) and isoverbascoside (3.46 mg g -1 DW) were achieved in RS-2 root line. The contents of catalposide, verbascoside and isoverbascoside in high-producing lines were several times higher than in untransformed root culture and roots of R. glutinosa plants grown in soil. Loganin and aucubin could not be detected in roots of fieldgrown plants. However, the levels of catalpol were much lower in the in vitro roots.
2021
Hairy roots are obtained from the infection caused by Agrobacterium rhizogenes, a gram negative bacterium and are known to produce different complex molecul es. Various biochemical pathways and physiological aspects in plants can be understood by m eans of hairy roots. Being genetically and biosyntheti cally stable as well as resultant high biomass accumulation and productivity in short period of time, these roots are great alternatives to conventional methods for the production of pharmacologically important compounds. Various biotechnological approaches i.e. culture medium components and their concentration, culture conditions, elicitation etc. are used and optimized to enhance overall yield. To meet up the increasing demand, production on industrial scal e has been considered to be an important where concept of bioreactors is involved. This review presents basic idea of development of hairy roots, requirement of the optimum culture conditions and use of bioreactors to increase yi eld of the bioactive compounds.
Plant Cell, Tissue and Organ Culture (PCTOC), 2009
Establishment of callus cultures and plant regeneration from different explants coupled with estimation of Picrosides in morphogenetically different developmental stages showed that Picroside-I accumulates in shoot cultures of Picrorhiza kurroa with no detection of Picroside-II. The Picroside-I content was 1.9, 1.5, and 0.04 mg/g in leaf discs, stem and root segments, respectively. The Picroside-I content declined to almost non-detectable levels in callus cultures derived from leaf discs, stem segments with no change in Picroside-I content in root segments or calli derived thereof. The biosynthesis and accumulation of Picroside-I started in callus cultures differentiating into shoot primordia and reached to the concentrations comparable to original explants of leaf discs and stem segments in fully developed shoots with contents of 2.0 and 1.5 mg/g, respectively. The shoots formed from root-derived callus cultures were relatively slow in growth as well as the amount of Picroside-I content was comparatively low (1.0 mg/g) compared to shoots derived from callus cultures of leaf and stem segments, respectively. The current study concludes that the biosynthesis and accumulation of Picroside-I is developmentally regulated in different morphogenetic stages of P. kurroa tissue cultures. Keywords Shoots Á Callus Á Regeneration Á Morphogenesis Á Picroside Á Indirect organogenesis Abbreviations IBA Indole-3-butyric acid 2,4-D 2,4 Dichlorophenoxyacetic acid KN Kinetin BA 6-Benzyladenine Picrorhiza kurroa Royle ex Benth (Family: Scrophulariaceae) is a medicinal herb, mainly found in the NorthWestern Himalayan regions of India at altitudes of 3,000-4,300 m. P. kurroa is a well-known herb in the Ayurvedic system of medicine and has traditionally been used to treat disorders of the liver and upper respiratory tract, reduce fevers, and to treat dyspepsia, chronic diarrhea, and scorpion sting. The active constituents are obtained from the shoots, roots and rhizomes of plant. P. kurroa is a high value medicinal herb due to rich source of hepatoprotective metabolites, Picroside-I and Picroside-II and other metabolites like Picroside-III, Picroside-IV, Apocynin, Androsin, Catechol, Kutkoside, etc. (Weinges et al. 1972; Stuppner and Wagner 1989). The medicinal importance of P. kurroa is due to its pharmacological properties like hepatoprotective (Chander et al. 1992), antioxidant (particularly in liver) (Ansari et al. 1988), antiallergic and antiasthamatic (Dorch et al. 1991), anticancerous activity particularly in liver (Joy et al. 2000) and immunomodulatory (Gupta et al. 2006). A hepatoprotective drug formulation, Picroliv has been prepared from the extracts of P. kurroa (Ansari et al. 1991; Dwivedi et al. 1997). Picroside-I is the major ingredient of Picroliv and, therefore, makes this compound a highly valued secondary metabolite of P. kurroa. The P. kurroa plants have been recklessly collected from its natural habitat, thereby, reducing its populations and putting it under the
Electronic Journal of Biotechnology, 2008
The study was undertaken to induce hairy roots in Glycyrrhiza glabra in leaf explants and to optimize the nutritional requirement for its growth kinetics at shake flask and bioreactor level. Pathogenecity of Agrobacterium depends upon transformation ability of strain and age, type, and physiological state of explants. Agrobacterium rhizogenes strain K599 was used to infect leaf explants of G. glabra. Explants of different age groups were obtained from 2 to 5 weeks old in vitro grown cultures. Bacterial strain K599 could induce hairy roots in 3 and 4 weeks old leaf explants cultured on B 5 , MS, NB and WP basal semi-solid medium. Leaf explants of 2 and 5 weeks old culture were not responsive to bacterial infection in terms of hairy root induction. Maximum transformation frequency (TF) of tested bacterial strain was 47% obtained in 3 weeks old *Corresponding author explants after 25 days of incubation on MS basal semi solid medium. NB and B 5 both media composition showed 20% of transformation frequency after 28 and 38 days respectively. WP medium did not support induction of roots in cultured leaf explants infected with A. rhizogenes strain K599even after 50 days of incubation. Further, when all the four media combinations were tested for root growth it was found that though WP was not responsive for hairy root induction, yet all four basal media supported hairy root growth and a gradual increase in fresh weight biomass was observed with an increase in culture duration. However amongst all, the NB medium composition supported best growth of hairy roots followed by MS, B 5 and WP media. About 20 times increase in root biomass on fresh weight basis was recorded after 45days of
Plant Cell Reports, 2006
Hairy root cultures of Gentiana macrophylla were established by infecting the different explants four Agrobacterium rhizogenes strains namely A 4 GUS, R1000, LBA 9402 and ATCC11325, and hairy root lines were established with A. rhizogenes strain R1000 in 1/2 MS + B 5 medium. Initially, 42 independent hairy root clones were maintained and seven clones belongs to different category were evaluated for growth, morphology, integration and expression of Ri T-DNA genes, and alkaloid contents in dry root samples. On the basis of total root elongation, lateral root density and biomass accumulation on solid media, hairy root clones were separated into three categories. PCR and Southern hybridization analysis revealed both left and right T-DNA integration in the root clones and RT-PCR analysis confirmed the expression of hairy root inducible gene. GUS assay was also performed to confirm the integration of left T-DNA. The accumulation of considerable amounts of the root-specific secoiridoid glucosides gentiopicroside was observed in GM1 (T + L and T + R) and the GM2 (T + L and T − R DNA) type clones in considerably higher amount whether as two T − L but T + R callus-type clones (GM3) accumulated much less or only very negligible amounts of gentiopicroside. Out of four media composition the 1/2 MS + B 5 vitamin media was found most suitable. We found that initial establishment of root cultures largely depends on root:media ratio. Maximum growth rate was recorded in 1:50 root:media ratio. The maximum biomass in terms of fresh weight (33-fold) was achieved in 1/2 MS Communicated by A.
Phytochemical studies for quantitative estimation of iridoid glycosides in Picrorhiza kurroa Royle
Botanical Studies, 2016
Background: Picrorhiza kurroa Royle commonly known as 'Kutki or Kutaki' is an important medicinal plant in Ayurvedic system of medicine and has traditionally been used to treat disorders of the liver and upper respiratory tract. The plant is the principle source of iridoid glycosides, picrosides I, II and kutkoside used in various herbal drug formulations mainly as strong hepatoprotective and immune-modulatory compound. The species has become endangered to near extinction due to the unregulated collection from the wild, slower plant growth and ecological destruction of natural habitats. There is a severe shortage of plant material, while the market demand is ever increasing. Hence, it is very important to apply a simple and precise analytical method to determine and validate the concentration of the major bioactive constituents in different populations of this plant species for development of a high yielding chemotype for large scale production and its commercial exploitation on scientific lines. Results: This study assessed and validated a fast and reliable chromatography method for the determination of picroside-I and picroside-II in different populations of this priortized medicinal plant species. Separation and resolution of picrosides was carried out on a reversed phase (C-18) column by using a mobile phase of methanol and water (40:60 v/v). The detection of picrosides was carried out at 270 nm. The average levels of these two major marker compounds in all the seven accessions showed significant quantitative variation (ANOVA, p < 0.05) between mean levels of marker compounds and their accumulation in different parts of the plant viz. roots, rhizomes and leaves. The highest content of pk-I was found in the accession from Gurez altitude (3750 masl) while the highest content of pk-II was found in accession from Keller (Shopian) altitude (3300 masl) demonstrate that picroside accusation is directly correlated with altitudinal variation. The method was validated in terms of linearity, accuracy and precision (within-and betweenassay variation). Conclusion: A simple chromatographic method with the ability to separate both the major chemical constituents effectively in different herbal extracts of P. kurroa and other related species has been standardized and validated, which is more suitable for regular and normal analysis of picrosides in different herbal formulations. The paper accomplish that picroside concentration in different samples showed significant variation based on altitude and other agroclimatic factors, which can be useful in the selection and collection of superior genotypes with higher concentration of these marker compounds.
Plant Cell, Tissue and Organ Culture (PCTOC), 2012
Picrorhiza kurroa and P. scrophulariiflora are two important endangered medicinal plant species of the Indo-China Himalayan region. These species contain several bioactive compounds that have therapeutic properties. In vitro culture studies have been conducted for developing protocols for shoot proliferation via apical/axillary meristem culture, rhizogenesis, acclimatization of plantlets, and nursery establishment. Moreover, successful efforts have been made to induce somatic embryogenesis from callus cultures as well as synchronous maturation of somatic embryos and plantlet conversion. In addition, regeneration has also been achieved via de novo shoot organogenesis, callus-mediated organogenesis, and from synthetic seeds following nutrient-alginate encapsulation. Factors impeding successful in vitro micropropagation have also been investigated. Clonal fidelity of micropropagated plants have been assessed using DNA markers. More recently, genetic transformation of P. kurroa has been reported via Agrobacterium tumefaciens or A. rhizogenes. Hairy root cultures (rhizoclones) containing higher levels of the bioactive compounds kutkoside and picroside I have also been identified. Two genes involved in picroside biosynthesis in P. kurroa have been identified, and these are found to be up-regulated under illumination and low temperature. High throughput de novo transcriptome sequencing has revealed abundance of trinucleotide simple sequence repeat markers associated with temperature-dependent biosynthesis of picrosides. Progress made in developing regeneration, transformation, as well as biochemical and molecular analysis of valuable bioactive compounds present in Picrorhiza species will be reviewed. Keywords Genetic improvement Á In vitro regeneration Á Phytochemistry Á Picrorhiza kurroa Á Picrorhiza scrophulariiflora Á Picrosides An overview Picrorhiza scrophulariiflora Pennell. and P. kurroa Royle ex. Benth. are two highly valued endangered medicinal plant species of the Himalayan region. The former species is restricted to Central through Eastern Himalayas at an altitude of 4,300-5,200 m, while the latter is distributed in Western through Central Himalayas at an altitude of 3,000-4,300 m (Smit 2000). Both the species have various pharmaceutical utilities. Traditionally, native communities of India use these plants to treat several diseases and disorders. Indian pharmaceutical manufacturers such as