IJSR - INTERNATIONAL JOURNAL OF SCIENTIFIC RESEARCH 27 Volume : 4 | Issue : 4 | April 2015 • ISSN No 2277 - 8179 Research Paper Botany Harpreet Kaur Department of Botany, Punjabi University, Patiala Geetika Sirhindi Department of Botany, Punjabi University, Patiala-147002, Punjab, India Renu Bhardwaj Department of Botanical & Environmental Sciences, GNDU, Amritsar Poonam Sharma Department of Botany, Punjabi University, Patiala 28-Homobrassinolide Modulation of Osmolytes in Brassica Juncea L. Under Salt Stress KEYWORDS : Brassica juncea, salt stress, 28-homobrassinolide, sugars, glycine betaine ABSTRACT e aim of present study was to explore the ameliorative impact of 28-homobrassinolide on morpho-physiological attributes and osmolytes of Brassica juncea L. exposed to oxidative stress caused by salt stress (180 mM NaCl). For this, experiments were carried out at the Plant Conservatory, Department of Botany, Punjabi University, Patiala. 28-homoBL (10-6, 10-9 and 10-12M) primed and unprimed seeds of B. Juncea L. were examined for total carbohydrates, total soluble sugars and accumulation of proline and glycine betaine. In conclusion 28-homoBL showed great potential in protecting the Brassica juncea L. plants from oxidative stress caused by (NaCl) salt stress but in very dose dependent manner 1. Introduction Salt stress is one of the most important environmental stresses that adversely affect plant growth and development thereby limit- ing plant productivity. It causes reduction in crop yield and altera- tion in plant metabolism including reduced water potential, ion imbalance and toxicity. Several hormones are implicated in modu- lating the plant responses to oxidative stress, including ethylene [1], abscisic acid [2], salicylic acid (SA) [3] and brassinosteroids (BRs) [4]. Brassinosteroids are a class of plant polyhydroxyster- oids that are ubiquitously distributed in the plant kingdom. ese compounds, when applied exogenously, improve plant quality and yield. ey have been further explored for stress-protective prop- erties in plants against a number of stresses like salt [5] and heat [6]. e studies conducted with brassinosteroid in biosynthetic mutants of pea (Pisum sativum) [7] and faba bean (Vicia faba) [8] provided compelling evidence for the vital role of this group of growth regulators for normal growth and development. However, it is unclear whether BRs are involved in the accumulation of os- molytes under oxidative stresses. Brassica juncea L. is an impor- tant oilseed crop known for its oil content, edible and medicinal uses. e present study was undertaken to observe the growth and accumulation of osmolytes under the influence of 28-homo- brassinolide (28-homoBL). 2. Materials and methods 2.1 Seed Treatments and Growth Conditions Seeds of B. juncea L. cultivar. (RLC-1) were procured from the Department of Plant Breeding, Punjab Agriculture University, Ludhiana, India. Seeds were surface sterilized with 0.01% HgCl 2 and rinsed 5-6 times with double distilled water. e sterilized seeds were soaked for 8h in different concentrations of 28-ho- moBL (Sigma-Aldrich. USA) (10-6, 10-9 and 10-12M). e treat- ed seeds were propagated in triplicate in cemented pots under natural field conditions. 3 kg soil was added to each pot and 1 litre solution of 180 mM NaCl was added to each pot at the time of sowing. Plants were sampled on the 30th day after sowing for measuring morphological and biochemical parameters. 2.2 Morphological parameters Shoot length of B. juncea L. plants was taken on 30th day after sowing. 2.3 Biochemical parameters 2.4 Total proline content Total proline content was determined by the method of Bates, [9]. Fresh plant material was homogenized in 3 % aqueous sul- phosalicylic acid and the homogenate was centrifuged to 10,000 rpm. Supernatant was taken for estimation of proline content. e reaction mixture consisting of 2 ml supernatant, 2 ml acid ninhydrin and 2 ml of glacial acetic acid was boiled at 100°C for 1 hr. After termination of the reaction in ice bath, the reaction mixture was extracted with 4 ml toluene and the absorbance was read at 520 nm. 2.5 Glycine betaine content Glycine betaine content was estimated according to the method of Grieve and Grattan, [10]. 0.5 g tissue was crushed and mechanically shaken in 20 ml deionized water for 24 h at 25 ° C. After filtration 0.5 ml extract was mixed with 1 ml of 2N H 2 SO 4 solution. Add 0.2 ml of potassium tri-iodide solution was added and reactants were gently stirred with a vortex mixture. Tubes were stored at 4 °C for 16 h and then 3 ml supernatant and 9 ml of 1,2 dichloroethane was pour in it. By passing continuous stream of air for 1-2 minutes two layers were separated, upper aqueous layer was discarded and op- tical density of organic layer was recorded at 365 nm. e concen- trations of glycinebetaine were estimated by using standard curve developed with different concentration of glycine betaine . 2.6 Total Soluble sugars Total soluble sugars content was estimated by following Loewus, [11]. Known weight of dried plant material was homogenised in 80% of ethanol then centrifuged at 3000x g for 15 minutes and the extract was collected for sugars estimation. For total sugars 0.05ml of extract was diluted to 2 ml by distilled water and add 3ml cold anthrone reagent and mixed thoroughly. en mixture was heated for 10 min in boiling water bath and cooled rapidly at room temperature. O.D. was recorded at 630 nm. Amount of total sugars was calculated and expressed as mg/g -1 DW tissue. 2.7 Total Carbohydrates Total carbohydrates content was determined according to Dubois, [12]. 0.05ml of extract was diluted to 2 ml by distilled water and 0.05 ml of phenol reagent was added to it and mixed thoroughly. en 5 ml of H 2 SO 4 was added rapidly. Blank was prepared by taking distilled water instead of extract. e sam- ples were allowed to stand at room temperature for 30 minutes and optical density was taken at 485 nm. Standard curve was prepared by using glucose (20-100 mg). e amount of carbohy- drates was calculated and expressed as mg/g -1 dry weight. e data obtained were presented as means ± SE. 3. Results and Discussion In the present study, B. juncea L. plants showed susceptibil- ity to salt stress as shoot length was 28% inhibited in 180 mM