Effect of ageing on metabolite profiling of in vitro grown cultures of Gentiana kurroo
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Date
2019-07
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
Gentiana kurroo is a critically endangered medicinal plant found in mid hills of Himalayas (1500-5000 meters asl) and is rich in metabolites of pharmaceutical importance like Gentiopicrine, Gentiamarin, Amaroswerin, etc. and possess stomachic, hypoglycemic, hypotensive, antiinflammatory, anti-periodic, etc. activities. Therefore, study related to Gentiana kurroo was aimed for comparing micropropagation potential of source cultures of different ages; metabolite profiling of in vitro grown cultures viz a viz wild plant using GC-MS and spectroscopic techniques; Ca quantification using AAS; and detection of CBPs in leaves of wild plant and in vitro grown cultures. In vitro grown cultures were maintained in M2C-2 medium with 2 ppm extra calcium pantothenate. No significant difference was observed during micropropogation in terms of shoot height but total number of leaves increased from 75 ± 4.24 to 94.50 ± 13.43 in 5- and 15-month old source cultures, respectively. In order to compare the effect of extra Ca on in vitro growth, the morphological parameters during first sub culturing in M2C-2 and second & third sub culturings in M2C-1 (1 ppm Ca) media were recorded. The percent increase in number of leaves and shoot height at 45 DAI were 403.5% and 413.95% during first subculture; 152.38% and 185.92% during second subculture; and 164.81% and 172.4% during third subculture, respectively. The qualitative phytochemical analysis confirmed the higher phytochemical contents (total phenolic-, flavanoid- and antioxidant content) in WFR viz a viz in vitro grown cultures, both because the wild plants had been growing in natural habitat, and had also experienced temperature stress during transition from their wild habitat to the laboratory. Amongst the in vitro grown cultures (15mR-in and 3mR-in), 3mR-in the exhibited lower values of TPC, TFC and TAC due to less stressed and optimized in vitro growth conditions whereas the 15mR-in cultures had experienced culture medium nutrient depletion generated stress. GC-MS analysis indicated the presence of few major compounds such as Nitroisobutylglycerol (6.58%), Methyl palmitate (8.3%), Linoleoyl chloride (5.15%) and Z-9-Hexadecenal (12.8%) in WSR-; 2,3- Epoxyhexanol (40.79%), Oxirane, (3,3-dimethylbutyl)- (15.25%), Oleic acid, 3- (octadecyloxy)propyl ester (10.52%) and 1-Methyl-4-phenyl-1,4-dihydro-tetrazol-5-one (20.61%) in WFR; Trehalose (77.5%) and Alloinositol (6.08%) in the 15mR-in-; 2-Methoxy-4-vinylphenol (6.36%), Mome inositol (41.34%), Phthalic acid (6.47%) and gamma-sitosterol (5.15%) in the 3mRin methanolic extracts. At least, 13 common compounds were found in higher quantity in 3mR-in as compared to 15mR-in. Ca quantification through AAS was in the order of 864, 608, 552 and 160 ug/g of dry weight of propagule in root samples i.e. 3mR-in, 15mR-in, WFR and wCR-in, respectively and 5.24 and 2.42 ug/g of dry weight of green (G-WLF) vs. yellow (Y-WLF) leaf samples, respectively. Total soluble protein content in leaf samples (FL-in and WFL) by Bradford method was recorded to be 418.18±0.004 and 336.36±0.001 ug/g of fresh weight of leaf, respectively. Separation of soluble proteins of wild and in-vitro leaves on SDS-PAGE indicated more than 8 identical bands ranging from 14.3 to 97.4 kDa. Calcium specific staining by Stains-All revealed the presence of more than 5 CBPs which were identical in both the leaf samples.
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