34 European Journal of Biotechnology and Bioscience Online ISSN: 2321-9122, Impact Factor: RJIF 5.44 www.biosciencejournals.com Volume 4; Issue 3; March 2016; Page No. 34-40 Primary level of defense reaction vary upon challenged Arbuscular mycorrhizal fungal inoculation among the differently responsive rice varieties 1 Ramkrishna Saha, 2 Bidisha Mondal 1 Assistant Director of Agriculture, Department of Agriculture, Government of West Bengal, India. 2 Scientist, Directorate of Research, Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India. Abstract Rice having a varying level of responsiveness to Arbuscular Mycorrhizal Fungi (AMF) mostly High Yielding and hybrid varieties which have a high PO 4 demand with comparatively less root growth rate showed more responsiveness than the varieties having less PO4 demand with comparatively higher root growth. As Arbuscular Mycorrhiza inoculation trigger a primary level of defense mechanism the quantitative & qualitative changes in the whole root peroxidase studies at the early stage of AMF colonization showed a rise in the peroxidase activity in negatively or non- responsive rice varieties whereas declined the activity in highly responsive rice varieties. The zymogram of rice roots by Peroxidase polyacrylamide gel electrophoresis showed five distinct bands. Increased activity of peroxidase in negatively responsive variety may due to transitional defense reaction of the plant showing a level of incompatibility where as in responsive variety the activity may suppress. Densiometric estimation also supported the above results. Keywords: Arbuscular Mycorrhiza, Rice, Densiometric, Peroxidase, Zymogram 1. Introduction In response to microbial attack, plants elaborate an array of inducible defense reactions many of which involve the transcriptional activation of the corresponding defense genes [1] . These include the genes that encode enzymes involved in the phenylpropanoid pathway – synthesis of lignin and phytoalexins, PR-proteins, Cell wall hydrolases and Reinforcement of plant cell walls, such as hydroxyproline rich glycoproteins (HRGP). During initial stages of AM colonization, only weak and transient increases in gene expression for cell wall hydrolases, HRGP, and those involved in phenylpropanoid pathway are observed [2] . Production of reactive O2 intermediates through an oxidative burst is the hallmark of plant’s defense responses, especially through the phenylpropanoid pathway. Peroxidases participate in a variety of defense mechanism [3] in which H 2 O 2 is often supplied by the oxidative burst. The cell wall appears to be the site of defense related peroxidase polymerization reactions, such as lignification, suberization and cross linking of structural cell wall proteins [4, 5] . This peroxidase activity is utilized for studying the difference between AM responsive and non-responsive varieties of rice. Both quantitative and qualitative expression showed the same result. 2. Materials and Methods Four selected rice varieties 2 non-responsive varieties – Black Gora (BG) and ARC- 12737 (ARC) and 2 responsive varieties – Jhingasal (Jhing), and MTU-7029 (MTU) [6] were grown in small volume sterile soil with or without AM inoculation for 10 and 25 days. Experimental plants were grown in pots over cemented racks in a partially covered net house under ambient light, temperature and humidity. For the purpose of biochemical analysis, small volume plastic cups were kept covered under transparent fine polythene nets. Irrigation was provided with fresh demineralized water. For the purpose of this experiment rice seedlings were grown in low volume sterilized soil in 250 ml thermocol cups keeping 3 seedlings per cup for 10 days and 25 days. Sterilized soil was inoculated with pure root based AMF inoculum at the rate of 5 g per kg soil to raise the infective inoculum density to approx. 3.5 X 10 5 propagules per g soil. Control plants also inoculated with sterilized root inoculum at the same rate. Seedlings were raised from surface sterilized seeds in inoculated soil and control cups and whole plant harvests were made between 10 and 25 days of seedling growth. Roots taken from the harvested plants was processed and analyzed for peroxidase by colorimetry [7] at 10 and 25 days and polyacrylamide gel electrophoresis [8] at 10 days, keeping adequate number of both plant and replicates. 1g of plant root stored overnight at –20ºC, was extracted with 1 ml of phosphate buffer (pH 7) by grinding in a pre-cooled glass mortar and pestle at 0-4ºC. The homogenate was centrifuged at 18000 g at 5ºC for 15 minutes and the supernatant was used as enzyme source immediately. 0.2 ml enzyme extract and 0.1 ml freshly prepared O-dianosidine solution was added to 3.5 ml phosphate buffer in a dry cuvette. The temperature of the assay mixture was immediately brought to 28-30ºC and the cuvette was placed in spectrophotometer at 430 nm. 0.2 ml 0.2 Molar H2O2 was then added immediately. The initial absorbance at 0 time immediately after addition of H 2 O 2 was recorded and followed further by recording at every 30 sec interval up to 3 minutes. A blank with reaction mixture minus enzyme extract was run parallel. Enzyme activity was expressed as rate of increase in absorbance per unit time per mg protein. Soluble root protein was estimated by Lowry’s colorimetric method. Whole root crude protein extract (1 g root in 1 ml phosphate buffer) (pH 6.8) was centrifuged at 10000 rpm for 15 minutes. The extract was diluted 20 times with buffer and