Anti-neuroinflammatory potential of Tylophora indica (Burm. f) Merrill and development of an efficient in vitro propagation system for its clinical use.

Neuroinflammation is a major risk factor associated with the pathogenesis of neurodegenerative diseases. Conventional non-steroidal anti-inflammatory drugs are prescribed but their long term use is associated with adverse effects. Thus, herbal based medicines are attracting major attraction worldwide as potential therapeutic candidates. Tylophora indica (Burm. f) Merrill is a valuable medicinal plant well known in Ayurvedic practices for its immunomodulatory, anti-oxidant, anti-asthmatic and antirheumatic activities. The present study aimed to elucidate the anti-neuroinflammatory potential of water and hydroalcoholic leaf extracts of micropropagated plants of T. indica using BV-2 microglia activated with lipopolysaccharide as an in vitro model system and development of an efficient reproducible protocol for its in vitro cloning. Non cytotoxic doses of the water and hydroalcoholic extracts (0.2µg/ml and 20µg/ml, respectively) were selected using MTT assay. α-Tubulin, Iba-1 and inflammatory cascade proteins like NFκB, AP1 expression was studied using immunostaining to ascertain the anti-neuroinflammatory potential of these extracts. Further, anti-migratory activity was also analyzed by Wound Scratch Assay. Both extracts effectively attenuated lipopolysaccharide induced microglial activation, migration and the production of nitrite via regulation of the expression of NFκB and AP1 as the possible underlying target molecules. An efficient and reproducible protocol for in vitro cloning of T. indica through multiple shoot proliferation from nodal segments was established on both solid and liquid Murashige and Skoog's (MS) media supplemented with 15µM and 10µM of Benzyl Amino Purine respectively. Regenerated shoots were rooted on both solid and liquid MS media supplemented with Indole-3-butyric acid (5-15µM) and the rooted plantlets were successfully acclimatized and transferred to open field conditions showing 90% survivability. The present study suggests that T. indica may prove to be a potential anti-neuroinflammatory agent and may be further explored as a potential therapeutic candidate for the management of neurodegenerative diseases. Further, the current study will expedite the conservation of T. indica ensuring ample supply of this threatened medicinal plant to fulfill its increasing demand in herbal industry.

Micropropagation and genetic transformation of Tylophora indica (Burm. f.) Merr.: a review.

KEY MESSAGE: This review provides an in-depth and comprehensive overview of the in vitro culture of Tylophora species, which have medicinal properties. Tylophora indica (Burm. f.) Merr. is a climbing perennial vine with medicinal properties. The tissue culture and genetic transformation of T. indica, which has been extensively studied, is reviewed. Micropropagation using nodal explants has been reported in 25 % of all publications. Leaf explants from field-grown plants has been the explant of choice of independent research groups, which reported direct and callus-mediated organogenesis as well as callus-mediated somatic embryogenesis. Protoplast-mediated regeneration and callus-mediated shoot organogenesis has also been reported from stem explants, and to a lesser degree from root explants of micropropagated plants in vitro. Recent studies that used HPLC confirmed the potential of micropropagated plants to synthesize the major T. indica alkaloid tylophorine prior to and after transfer to field conditions. The genetic integrity of callus-regenerated plants was confirmed by RAPD in a few reports. Tissue culture is an essential base for genetic transformation studies. Hairy roots and transgenic T. indica plants have been shown to accumulate tylophorine suggesting that in vitro biology and transgenic methods are viable ways of clonally producing valuable germplasm and mass producing compounds of commercial value. Further studies that investigate the factors affecting the biosynthesis of Tylophora alkaloids and other secondary metabolites need to be conducted using non-transformed as well as transformed cell and organ cultures.

Transgenic mimicry of pathogen attack stimulates growth and secondary metabolite accumulation.

Plant secondary metabolites, including pharmaceuticals, flavorings and aromas, are often produced in response to stress. We used chemical inducers of the pathogen defense response (jasmonic acid, salicylate, killed fungi, oligosaccharides and the fungal elicitor protein, cryptogein) to increase metabolite and biomass production in transformed root cultures of the medicinal plant, Withania somnifera, and the weed, Convolvulus sepium. In an effort to genetically mimic the observed effects of cryptogein, we employed Agrobacterium rhizogenes to insert a synthetic gene encoding cryptogein into the roots of C. sepium, W. somnifera and Tylophora tanakae. This genetic transformation was associated with stimulation in both secondary metabolite production and growth in the first two species, and in growth in the third. In whole plants of Convolvulus arvensis and Arabidopsis thaliana, transformation with the cryptogein gene led, respectively, to increases in the calystegines and certain flavonoids. A similar transgenic mimicry of pathogen attack was previously employed to stimulate resistance to the pathogen and abiotic stress. In the present study of biochemical phenotype, we show that transgenic mimicry is correlated with increased secondary metabolite production in transformed root cultures and whole plants. We propose that natural transformation with genes encoding the production of microbial elicitors could influence interactions between plants and other organisms.

[Effect of sugars, gibberellic acid and abscisic acid on somatic embryogenesis in Tylophora indica (Burm. f.) Merrill].

The effect of sugars, gibberellic acid (GA3) and abscisic acid (ABA) on somatic embryogenesis from internodal explant-derived callus of Tylophora indica (Burm. f.) Merrill has been investigated. Embryogenic calli were produced from internodal explants and the best result was achieved by using MS medium supplemented with 4micromol/L 2, 4-Dichlorophenoxyacetic acid (2, 4-D). Up to 69% of such embryogenic calli differentiated into somatic embryos with an average of 25 embryos per explant (per gram of the calli) on Murashige and Skoog (MS) medium containing 6micromol/L kinetin (Kn). The individual effect of sucrose and glucose together with 6micromol/L Kn was evaluated. There was a significant difference among concentrations of sugar and among kinds of sugar tested in somatic embryogenesis. Sucrose at 200mmol/L with 6micromol/L Kn gave rise to a maximum embryogenesis (71%) with an average of 49 embryos per explant. However, glucose together with 6micromol/L Kn or a combination of glucose, sucrose and 6micromol/L Kn reduced the percentage of embryogenesis culture and the number of embryos per explant. The presence of GA3 and ABA at particular concentrations promoted somatic embryogenesis in T. indica. The addition of 10mol/L GA3 into the 200mmol/L sucrose-containing medium gave a 98% embryogenesis response with an average of 51 embryos per explant. Somatic embryogenesis was significantly enhanced by the addition of 2micromol/L ABA to 200mmol/L sucrose-containing medium. On this medium 95% embryogenesis with an average of 44 embryos per explant was observed. The study reported here indicates that 200mmol/L sucrose with 6micromol/L Kn, 200mmol/L sucrose with 10micromol/L GA3 and 200mmol/L sucrose with 2micromol/L ABA significantly improved somatic embryogenesis in T. indica whereas glucose alone or in combination with sucrose had an inhibitory role. The embryos obtained developed normally and were easily converted into plants.

Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: Changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes.

We examined the effects of genetic transformation by Agrobacterium rhizogenes on the production of tylophorine, a phenanthroindolizidine alkaloid, in the Indian medicinal plant, Tylophora indica. Transformed roots induced by the bacterium grew in axenic culture and produced shoots or embryogenic calli in the absence of hormone treatments. However, hormonal treatment was required to regenerate shoots in root explants of wild type control plants. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes, which include, short internodes, small and wrinkled leaves, more branches and numerous plagiotropic roots. Plants regenerated from transformed roots showed increased biomass accumulation (350-510% in the roots and 200-320% in the whole plants) and augmented tylophorine content (20-60%) in the shoots, resulting in a 160-280% increase in tylophorine production in different clones grown in vitro.

Genetic transformation of Tylophora indica with Agrobacterium rhizogenes A4: growth and tylophorine productivity in different transformed root clones.

We have developed an efficient transformation system for Tylophora indica, an important medicinal plant in India, using Agrobacterium rhizogenes strains LBA9402 and A4 to infect excised leaf and stem explants and intact shoots at different sites. The induction of callus and transformed roots was dependent on the bacterial strain, explant type and inoculation site used. Transformed roots were induced only in explants infected with A. rhizogenes strain A4, while an optimal transformation frequency of up to 60% was obtained with intact shoots inoculated at the nodes. The presence of the left-hand transferred DNA (T(L)-DNA) in the genome of T. indica roots induced by A. rhizogenes was confirmed by PCR amplification of the rooting locus genes of A. rhizogenes. Root growth and the production of tylophorine, the major alkaloid of the plant, varied substantially among the nine root clones studied. Both parameters increased over time in liquid cultures, with maximum biomass and tylophorine accumulation occurring within 4-6 weeks of growth in fresh medium. Interestingly, in liquid culture, the culture medium also accumulated tylophorine up to concentrations of 9.78+/-0.21 mg l(-1).