Hairy root culture of Picrorhiza kurroa Royle ex Benth.: a promising approach for the production of picrotin and picrotoxinin (original) (raw)
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Plant signaling & behavior, 2015
Fast-growing hairy root cultures of Picrorhiza kurroa induced by Agrobacterium rhizogenes offers a potential production system for iridoid glycosides. In present study we have investigated the effects of various nutrient medium formulations viz B5, MS, WP and NN, and sucrose concentrations (1-8%) on the biomass and glycoside production of selected clone (14-P) of P. kurroa hairy root. Full strength B5 medium was found to be most suitable for maximum biomass yield on the 40th day of culture (GI = 32.72 ± 0.44) followed by the NN medium of the same strength (GI = 22.9 ± 0.43). Secondary metabolite production was 1.1 and 1.3 times higher in half strength B5 medium respectively in comparison to MS medium. Maximum biomass accumulation along with the maximum picroliv content was achieved with 4% sucrose concentration in basal medium. RT vitamin and Thiamine-HCl effected the growth and secondary metabolite production of hairy roots growing on MS medium but did not show any effect on other ...
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of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa.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.
Picrorhiza kurrooa: current status and tissue culture mediated biotechnological interventions
Acta Physiologiae Plantarum, 2012
Picrorhiza kurrooa, one of the important plant species among the various medicinal plants, is endemic to Himalaya. As the plant is useful in the treatment of various diseases, e.g., hepatic disorders, gastric troubles, anemia, asthma, etc., illegal collection from the wild is increasing and now this plant is banned for export in any form and listed as 'endangered'. Ecological studies carried out on this species in last few decades suggested that the availability of this species in its specific habitats is comparatively lower than other associate species. Possible factors responsible for this depletion are increasing demand in the pharmaceutical industries, habitat specificity, heavy exploitation from the wild, unorganized cultivation practices etc. Biotechnology is playing a crucial role to conserve this important plant species. The past 23 years have witnessed a progressive biotechnological advances made in P. kurrooa. People have published various reports on establishments of in vitro culture techniques including micropropagation, synthetic seed production, plant regeneration via callus-mediated shoot organogenesis, adventitious shoot regeneration, genetic transformation through Agrobacterium rhizogenes, secondary metabolite analysis etc. This review attempts to focus on present ecological status and provide a comprehensive account on the tissue culture-mediated biotechnological interventions made in P. kurrooa for improvement and conservation of this medicinally important plant. Keywords Picrorhiza kurrooa Á Agrobacterium rhizogenes Á Conservation Á Endangered Á Secondary metabolite Abbreviations MS Murashige and Skoog medium BAP 6-Benzylaminopurine IAA Indole-3-acetic acid NAA a-Naphthaleneacetic acid 2,4-D 2,4-Dichlorophenoxyacetic acid Kn Kinetin IBA Indole-3-butyric acid GA 3 Gibberellic acid m amsl Meter above mean sea level PCR Polymerase chain reaction RAPD Random amplified polymorphic DNA ISSR Inter simple sequence repeat UPGMA Unweighted pair-group method with arithmetic mean Communicated by A. K. Kononowicz.
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
The aim of present investigation was to develop a highly reproducible and well documented protocol for in vitro germination and multiplication of highly valuable medicinal herb, Picrorhiza kurroa via callus mediated organogenesis using the axenic epicotyls originated from in vitro germinated seedlings. Highest germination percentage (88.6) was observed in stratified seeds (4 0 C for 5 days) treated with 100 mg/l GA 3 and maintained at 25 ±1 0 C under continuous light conditions. Out of different combinations of plant growth regulators tested, Murashige and Skoog (MS) medium supplemented with 1.5 mg/l α-naphthalene acetic acid (NAA)+0.75 mg/l kinetin (Kn)+0.75 mg/l 6-benzyladenine (BAP) was found best for callus induction and further proliferation. Addition of gibberellic acid (GA 3) at 1.0 mg/l concentration in hormonal combination of 2.0 mg/l Kn and 0.75 mg/l indole-3-acetic acid (IAA) was found to be optimum to produce highest shooting response (95%) with an average number of 11.40 shoots per calli. The rooting was optimized using 0.5 mg/l IAA with 85% response. The plantlets with well-developed roots were hardened and were successfully acclimatized in field conditions. This protocol may offer the potential to substantially reduce the pressure of commercial need of this valuable herb on wild stock.
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