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.

In vitro seed germination of economically important edible bamboo Dendrocalamus membranaceus Munro.

An in vitro propagation protocol using mature seeds of D. membranaceus was successfully established. Scarcity of seeds in bamboos because of their long flowering periods and irregular seed set resulting in low viability and germination potential, motivated us to undertake the present study. The effects of sterilants, light conditions, exogenous application of plant growth regulators and temperature in overcoming germination barriers in ageing seeds of bamboo were studied. It was found that HgCl2 (0.1%) along with bleach (15%) was more effective in raising aseptic cultures. Dark conditions, high temperatures around 30 degrees C and soaking of seeds in GA3 solution (50 ppm) overnight stimulated high percent of seed germination with corresponding increase in shoot length (2.7 +/- 0.7 mm) and number of sprouts (2.1 +/- 0.7) per explants during culture initiation. 6-benzylaminopurine acted synergistically with kinetin to give optimum germination rate of 70 +/- 13.9% as compared to 63.13% when used individually. For prolonged maintenance of cultures, 2% sucrose was found to be suitable for promoting photomixotrophic micropropagation. Following this procedure, about 65% survival of plantlets could be achieved during hardening. Biochemically seeds consume starchy endosperm for emergence of radicle which is taken as a sign of germination as also evident from the present study. Loss of viability and vigour after a year was confirmed by Tetrazolium chloride test. Micropropagation protocol developed here will ensure regeneration of large number of plants in a relatively short time. Conclusively, in vitro propagation protocol developed in D. membranaceus using mature seeds as an explants is reported for the first time.

Identification and elimination of bacterial contamination during in vitro propagation of Guadua angustifolia Kunth.

BACKGROUND: Guadua angustifolia Kunth is a very important bamboo species with significant utility in pharmaceutical, paper, charcoal, and construction industries. Microbial contamination is a major problem encountered during establishment of in vitro cultures of Guadua. OBJECTIVE: This study has been designed to analyze the identity of contaminating bacteria and to develop the strategy to eliminate them during micropropagation of Guadua. MATERIALS AND METHODS: We isolated and consequently analyzed partial sequence analysis of the 16S rRNA gene to identify two contaminating bacteria as (1) Pantoea agglomerans and (2) Pantoea ananatis. In addition, we also- performed antibiotic sensitivity testing on these bacterial isolates. RESULTS: We identified kanamycin and streptomycin sulfate as potentially useful antibiotics in eliminating the contaminating bacteria. We grew shoots on multiplication medium containing BAP (2 mg/l) and adenine sulfate (10 mg/l) supplemented with kanamycin (10 µg/ml) for 10 days and transferred them to fresh medium without antibiotics and found that bacterial growth was inhibited. Moreover, we observed intensive formation of high-quality shoots. Streptomycin sulfate also inhibited bacterial growth but at higher concentration. We also demonstrated that shoots grown in streptomycin sulfate tended to be shorter and had yellow leaves. CONCLUSION: Thus, we have developed a novel strategy to identify and inhibit intriguing microbial contaminations of (1) Pantoea agglomerans and (2) Pantoea ananatis during establishment of in vitro cultures of Guadua. This would improve in vitro establishment of an important bamboo, Guadua angustifolia Kunth for large scale propagation.