full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 1064-1072 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1066Materials <strong>and</strong> MethodsPlant material <strong>and</strong> activation tagging byAgrobacterium infection: Nicotiana tabacumvar. SR1 was used for all studies detailed here.Leaf discs from 3 week old magenta boxgrownplants were transformed withAgrobacterium tumefaciens 3850 harbouringthe activation tagging T-DNA constructpPCVICEn4HPT. Agrobacterium wascultured in Luria-Bertani (LB) mediumcontaining 100 mg l -1 Ampicillin. Bacterialculture was initiated by inoculating singlecolony <strong>of</strong> A. tumefaciens into 25 ml flaskcontaining 5 ml <strong>of</strong> liquid media <strong>and</strong> kept at200 rpm on a rotary shaker at 28ºC for oneday. Subsequently, 2 ml <strong>of</strong> overnight grownbacterial culture was transferred to 500 ml flaskcontaining 200 ml <strong>of</strong> liquid media. When theoptical density (OD) <strong>of</strong> the culture reachedaround 0.6 at 660 nm, 100 µM acetosyringonewas added. Bacterial culture was spun downin a centrifuge at 2500 g, when culture reachedaround 1.0 OD. The pellet was gentlyresuspended in 2 ml MS medium <strong>and</strong> used asbacterial stock for infection. A finalconcentration <strong>of</strong> 100 µM acetosyringone wasadded to the bacterial culture <strong>and</strong> leaf explantswere infected for approximately 20 min. Afterinfection, explants were blot-dried onto sterilefilter paper <strong>and</strong> transferred onto platescontaining solid MS media. The infectedexplants were co-cultivated for 2 days in theculture room set at 25 ºC <strong>and</strong> then transferredonto shoot induction media. Ethanol (200mM)or 4-MT was added directly to culture media.Regenerated shoots were excised <strong>and</strong>transferred to rooting medium (MS mediumwithout hormones) containing either ethanolor 4-MT along with hygromycin <strong>and</strong>cefotaxime. The resistant mutants weretransferred to compost substrate <strong>and</strong> grown ina greenhouse.Antioxidant assays: 200 mg <strong>of</strong> fresh leaft<strong>issue</strong> was ground to a fine powder underliquid nitrogen. The powder was homogenizedin 2 mL <strong>of</strong> 2% (w/v) metaphosphoric acid <strong>and</strong>2 mM ethylenediaminetetraacetic acid(EDTA), <strong>and</strong> transferred to eppendorf tubes.The supernatant was collected aftercentrifugation at 14,000xg for 10 min at 4 o C.The supernatants were then split into two tubes<strong>of</strong> 900 uL each <strong>and</strong> neutralized with 600 uL<strong>of</strong> 10% sodium citrate (7). Total AsA wasanalysed with 500 uL <strong>of</strong> neutralized extractby measuring the change inspectrophotometric absorbance before <strong>and</strong>after adding ascorbate oxidase enzyme (8).Similarly, a spectrophotometric assay wasused for measuring total GSH (7).ROS measurement: About 300 mg <strong>of</strong> leaft<strong>issue</strong> was ground in liquid nitrogen <strong>and</strong>homogenized with 1 mL <strong>of</strong> TRIS(hydroxymethylaminomethane) buffer (pH7.2). The supernatant was collected aftercentrifugation at 14,000xg for 10 min at 4 o C.800 uL <strong>of</strong> this extract was mixed with 1 mM2’,7’-dichlorodihydr<strong>of</strong>luorescein diacetate(H 2DCFDA) <strong>and</strong> incubated in darkness for 10min at room temp (7,9). Fluorescence wasmeasured using a Victor Fluorescence platereader (Perkin Elmer). Total protein wasquantified using Bradford dye (Bio-Rad).Gas chromatography-mass spectrometry (GC-MS) analysis: Tobacco alkaloid content wasdetermined using an Agilent 6890 GC/Agilent5973 mass-selective detector (MSD) with anAgilent 7683 Autosampler. The GC was fittedwith a 30m x 0.2 mm X 0.25 µm HP-5MSglass capillary column <strong>and</strong> a helium flow rate<strong>of</strong> 1.0 ml/ min. Extracts from survivorsselected from mutant populations at aconcentration <strong>of</strong> 100 mg dried material/ mlSurvival selection <strong>of</strong> N. tabaccum activation tagged mutants