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Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011SECTION I: AGRICULTURAL BIOTECHNOLOGYISOPEROXIDASE PROFILES IN SOYBEAN IN VITRO CULTURES UNDEROSMOTIC STRESS8GEORGINA KOSTURKOVA 1 , GEORGI ANGELOV 2 , MARGARITA DIMITROVA 1 ,KRASIMIRA TASHEVA 11 Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria; Phone: + 3592 974 6228; Fax: + 359 2 978 5512 Institute for Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria; Phone:+359 2 871 8259; Fax: + 359 2 871 9032Correspondence to: Georgina Kosturkova; E-mail: gkosturkova@yahoo.comAbstract. Soybean is commonly used grain legume with multiple product applications. However, itsyield is reduced by environmental stress with drought being one of them. Due to the complex nature ofdrought tolerance, the problem has been studied from different aspects. Biotechnological methodscomplement classical ones and make possible modeling of stress in vitro. Biochemical analysis of stressrelated proteins contribute to better evaluation of the tolerant genotypes. The aim of the present work isto compare isozyme profiles of peroxidase in organogeneic soybean cultures cultivated in normal andstress conditions. Adventitious shoots and callus cultures from cv Rosa were induced on modifiedFraytag medium. In vitro response of cotyledonary explant was recorded, shoot and callus developmentin stress conditions was characterized. Clusters of shoots were transferred to control medium and mediacontaining polyethylene glycol (PEG) of concentrations of 4 %, 6%, and 8%. Totally five peroxidaseisoforms were identified. Some of them cannot be detected in the control, thus induced under stressconditions. Induction is weaker at PEG concentration of 4% and more pronounced at 6% and 8% PEG.Enzyme profiles in shoots subjected to stress for periods of 1, 3, 6 and 23 days were identified.Differential response was recorded depending on PEG concentration and time treatment.Keywords: peroxidase, osmotic stress, soybean, Glycine max, in vitro cultures1. INTRODUCTIONGlycine max is considered a world wide strategic crop and commonly used grainlegume with multiple product applications. Soybean has a leading position among thelegume species as a source of high quality protein and oil and beneficiary valuablesubstances as isoflavones, phenolic compounds and saponins. (Sakthivelu et al., 2008a)especially recognized by vegetarian and healthy food diets. However, abiotic environmentalstresses including drought, high temperatures and salinity reduce yield potentialsubstantially (Boyer, 1982). Due to the great importance of the problem and the complexnature of the tolerance efforts and studies for improvement of plant performance are ofdifferent aspects. Classical breeding methods have been complemented with newtechniques of plant biotechnology and molecular biology (Bajji et al, 2000, Diab et al.,2008) giving possibility for simulating the desired stress in vitro, providing selection on celllevel and manipulating genes for resistance.
Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011Plant defensive mechanisms are also in the focus of environmental stress research. Anumber of stress factors provoke a burst of active oxygen species in plants (Foyer et al.,1994). These free radicals are chemically highly active and cause injuries in plant cellstructure. Several enzymes, including peroxidase, are the main defensive mechanismagainst oxidative stress in plants as they eliminate H 2 O 2 and free radicals. Peroxidases (PO)are a large family of enzymes which oxidize different substrates in the presence of H 2 O 2 ,thus regulating its concentration in plant cell. It has been demonstrated that these enzymesare involved in several biochemical and physiological processes including different stressresponses (Medina et al., 1999). Increase of peroxidases activity is a general response ofplants to stress factors (Fang & Kao, 2000). Effect of water stress (flooding and drought) onantioxidant enzymes have been studied in rice varieties (Mali and Mehta, 1977) andAnthemis (Gorjizad et al., 2010)In Bulgaria soybean breeding have been directed to improvement of yield, biotic andabiotic stress tolerance (Goranova and Todorova, 2005). Recently tissue cultures ofsoybean have been developed and they were used for in vitro modeling of abiotic stress andscreening for genotypes with higher tolerance (Kosturkova 2005, Kosturkova et al., 2006,2008, Nedev et al., 2007, Sakthivelu et al., 2008b)The aim of the present work is to compare the development and the isoenzyme profilesof peroxidase in soybean calli and shoots cultivated in vitro under normal and stressconditions in attempt to analyze plant response to the drought stress factor.2. MATERIALS AND METHODSPreparation of in vitro cultures.Seeds of soybean cv Rosa were provided by Dr. R. Todorova (patent holder, Todorovaand Goranova, 2010) from the Soybean Experimental Station – Pavlikeni. After rinsed withwater seeds were surface sterilized by immersion in 70 % ethanol for 1 min, followed by 30% v/v commercial bleach for 20 min and rinsed three times in sterile distilled water. Seedswere plated under aseptic conditions on basal medium of Murashige and Skoog (1962) forgermination. After 10-14 days cotyledonary nodes were excised from the seedlings beforethe formation of the first leaf and were plated on modified Fraytag’s medium (MSF)containing 4 mg/l benzylaminopurine (BAP) and 0.1 mg/l 3-indolebutyric acid (IBA)(Nedev et al., 2007) - normal control conditions. To simulate drought conditionspolyethylene glycol (PEG) with molecular weight 6000 was added into the media beforeautoclaving to have final concentrations of 4 %, 6 % and 8 % w/v. For peroxidase profilingexpants responding in vitro on MSF medium were transferred to media containing PEG.After induction of organogenesis and callogenesis tissues from the green shoots and fromthe calli were biochemical analyzed on the 1 st , 3 rd , 6 th , 23 rd , and 30 th day of cultivation instress (cultured on media containing PEG) and normal control conditions (cultured onmedia lacking PEG).Cultivation conditions.In vitro cultures were maintained in phytotrone rooms at temperature of 25±1°C,photoperiod of 16/8 h and illumination of 40 µmolm –2 s –1 .Biochemical analysis.9
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