Salinity Responsiveness in Finger Millet Analysis

Salinity Responsiveness in find Millet AnalysisIntroductionSalinity re enters a strong limitation for pastoral production worldwide, especially in arid and semi-arid and restricts efficient utilization of accessible land resources. It is estimated that about 7% of world agricultural land that nearly one half of the natural argona of irrigated land could be adversely affected by salinization (Kosova 2013). Most of the cereal functions are sensitive to common t commensurate salt and have limited amount of genetic variation for common salt border in their germplasm. Hence genetic improvement of crops for their perimeter against common salt leave behind be helpful in achieving targeted food production to meet the demands of growth population.Conventional plant breeding approaches have resulted in limited winner in developing salt bighearted crop varieties payable to multigenic personality of salt valuation account mechanisms and presence of low genetic variation in ma jor crops. Another problem associated with conventional breeding is that if the gene is present in a wild relative of the crop, there is difficulty in transferring it to the domesticated cultivar, due to reproductive barriers and linkage drag.Recently, substantial progress in elucidation of salt border mechanisms, especially salt ion signaling and merchant marine, has been achieved due to utilization of modern genetic approaches and high-throughput modes of functional genomics. Genetic engineer has been show to be successful in developing salt tolerant crop plants (Zhang et al. 2001 Su and Wu 2004 Zhang et al. 2001). Genetic engineering strategies targeting various metabolic parcel of lands that is to say, accumulation of os moleytes, antioxidant enzymes and up ruler of genes deald in show solvents handle ion transporters, ion channels, transcriptional factors and various signaling pathway components have resulted in production of genetically modified crop plants exhibiti ng ameliorate level of table salt gross profit margin (Turan et al. 2012).Identifying overbold genes, analyzing their expression patterns in response to salt examine and deter bitation of their potential functions in salt stress adaptation will provide the basis for effective genetic engineering strategies to enhance tolerance against salt stress (Cushman and Bohnert 2000). Responses against coarseness stress involve many molecular processes such as ion homeostasis (membrane proteins involved in bean transport), osmotic adjustment and water regime regulation (osmolytes) and scavenging of toxic compounds (Munns and examiner 2008). During recent years, considerable attention has been given towards elucidating the molecular basis of salt tolerance in crop plants. Several important pathways involved in common salt tolerance have been identified in model plants like Arabidopsis and sieve (Zhu 2003 Walia et al. 2005 Cotsaftis et al. 2011).It is hypothesized that exploitation of halophytes or distantly related crops or wild progenitors of cereal food crops exhibiting master copy levels of salinity tolerance whitethorn lead to identification of novel metabolic pathways/mechanisms/genes involved in modulating salinity stress tolerance in crop plants. Several research groups are working on chthonicstanding mechanisms of salinity tolerance in genus Pennisetum glaucum (Mishra et al. 2007), Avecinnia marina (Mehta et al. 2005), Porteresia coarctata (Garg et al. 2014) with a view to identify novel genes for genetic engineering of salinity tolerance in crop plants. But much more conjunctive efforts are needed to identify and exploit diverse crop species exhibiting superior level of salinity tolerance which will help in identifying novel genes associated with salinity tolerance.Finger millet (Eleusine coracanaL.) is an important minor cereal crop astray grown in Africa and Asia, known for its high degree of tolerance against drought, salinity and blast disease (S hailaja and Thirumeni 2007 Agarwal et al. 2011). Investigating the mechanisms and pathways involved in salt-tolerance of dactyl millet could allay better to a lower placestanding of the molecular basis of salt tolerance and then enable the effective use of genetic and genomic approaches to improve salt tolerance in major cultivated crops. Although a wide range of probative physiologic mechanisms and genetic adaptations to salinity stress has been observed, the on a lower floorlying mechanisms of salt-tolerance in plants are still poorly understood. The best possible approach to explore tolerance mechanisms is to compare the components involved in stress response in tolerant as compared to sensitive plants. The other alternative to overcome this limitation would be to pick up some selected conserved genes which may be employ to perform limited transcriptome analysis among the diverse genotypes.With this background, we planned to understand the physiological and molecular bas is of salinity reactiveness in feel millet in comparison to the major cereal food crop, strain. Comparative physiological studies were conducted with a view to prove the high quality of finger millet genotypes over strain in terms of salinity tolerance. Two separate finger millet genotypes were used for physiological studies and expression analysis of already identified salinity responsive genes was done. This is the start-off study conducted to compare molecular basis of salinity tolerance in finger millet with rice.Material and MethodGenetic Materials UsedSeeds of two secernate genotypes of rice (Oryza sativa) FL478 (tolerant), White Ponni (Susceptible) and finger millet (Eleusine coracona) Trichy 1 (tolerant), CO12 (Susceptible) in terms of salinity tolerance were evaluated for their responses against salinity stress under nursery conditions. Nucleus seeds of rice genotypes were obtained from Paddy Breeding institutionalise, Tamil Nadu Agricultural University, Coimbatore , India and finger millet genotypes were obtained from Millet Breeding Station of Tamil Nadu Agricultural University, Coimbatore, India.Effect of salinity stress during germination contrastive genotypes of rice FL478 (tolerant), White Ponni (Susceptible) and finger millet Trichy 1 (tolerant), CO12 (Susceptible) genotypes were assessed for their qualification to germinate under salinity stress. Twenty seeds of both rice and finger millet genotypes were allowed for germination under different concentrations of NaCl solutions (0 mM, 50 mM, degree centigrade mM, 200 mM NaCl solution) in petri-dishes with seemly replications. Germination fate was calculated establish on the number of seeds successfully germinated and vitality forefinger was calculated based on the shoot distance and root length on 10th twenty-four hour period of germination.Effect of salinity stress during vegetative stageImposition of salinity stress severalize genotypes of rice and finger millet genotypes (thre e seedlings per pot) were grown in perforated pots of 15 cm diameter and 20 cm height (having 35 mm holes on the side walls and bottom) filled with 2 kg of field smut mixed with required amount of fertilizer 1.25 g of (NH4)2SO4, 0.08 g Muriate of potassium hydroxide (KCl), and 0.08 g single superphosphate (SSP). Three pots were placed inside a swelled tray containing irrigation water and grown up to 20 twenty-four hourss under greenhouse conditions. Plants were grown during JuneAugust when air temperature ranged from 26 to 34 C during the day and from 20 to 27 C during the night and relative humidity ranged from 60 to 80 %. Salinity stress was imposed on 21st day when plant has reached to 5 leaf stage by adding desired concentrations of NaCl viz. 150 mM and 300 mM along with suitable engage pots irrigated with normal water. Progression of salinity stress was monitored by periodically criterion the electrical conductivity (EC) of soil (from pot) and water (collected from tray) s amples collected from both conceal and salinity stressed trays.Physiological and biochemical responses of contrasting rice and finger millet genotypes under salinity stressContrasting genotypes of rice viz., FL478 (tolerant) and White Ponni ( subject) and finger millet viz., CO 12 (susceptible) and Trichy 1 (tolerant) were evaluated for their physiological and biochemical responses viz., osmotic tolerance ability, salt accumulation pattern and sugar accumulation pattern during salinity stress.Measurement of Osmotic tolerance abilityFor assessing the osmotic tolerance ability of contrasting rice and finger millet genotypes, freshly emerged leaf (5-6cm) was marked and summation in leaf length was measured at every 24hrs time interval during the initial 6 years of salinity stress along with stamp down plants. Terminal leaf elongation rate per day (24 h) was calculated based on the observations recorded.Salt accumulation patternSalt (Na+ and K+) uptake, transport and accumulation p attern of contrasting rice and finger millet genotypes was assessed by find out the (Na+ and K+) subject fields in shoots and top 3 leaves collected under normal and salinity stress conditions. Tissue samples collected at 21 DAS ( years after stress) were washed with de-ionized water, dry in a igneous air oven (70 C) and then ground into fine powder. Ground samples were digested with common chord erosive mixture (sulfuric acid, perchloric acid and nitric acid in the ratio 921 v/v). Na+ and K+) concentrations in the triple acid digested extract were estimated development Flame Photometer (Elico, CL378).Determination of total soluble sugar content hit soluble sugar (TSS) content in the top three leaves of defy and salinity stressed plants (21 days after stress) of contrasting rice and finger millet genotypes was determined utilize anthrone reagent method (Yemm and Willis 1954). Fresh leaf sample (100 mg) was ground in liquid normality and pigments were removed using acetone extraction. TSSs were extracted in 80 % ethanol and were estimated by the anthrone reagent method using glucose as the standard.Other physiological responses of contrasting finger millet genotypes to salinity stressGas exchange parameters were recorded in the trey leaf (from top) of control and salinity stressed plants of rice and finger millet genotypes amidst 1000 hours and 1200 noon at 11 DAS (days after stress) using LI-COR 6400-XT photosynthesis system (LI-COR Biosciences, Nebraska, USA). The instrument was set with the following conditions photo-synthetically active radiation 1,500 mol of photon m2s1 ambient levels of CO2 and temperature leaf area 3 cm2 and flow rate of 500 mol s1.RNA isolation, northerly blotting and hybridizationExpression analysis of already reported salinity responsive candidate genes in response to salinity stress in the leaves of contrasting rice and finger millet genotypes were studied by northern blotting. Top 3 leaves of both rice and finger millet genotypes were collected and frozen immediately in liquid nitrogen from both control and stressed plant (300mM NaCl) when susceptible rice variety viz. White Ponni has shown salinity symptoms i.e., 11 days after salinity stress. Total RNA was isolated from stressed and control leaf samples using One Step RNA Reagent (Biobasic Inc., Canada) as per shapers protocol. The integrity of RNA was assessed by formaldehyde agarose gel electrophoresis. Total RNA was quantified using Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). 20ug of RNA mixed with RNA core dye (11) was denatured at 75C for 10mins and separated on denaturing agarose gel as describe by Streit et al. (2008). The gel was stained with ethidium cliche and photographed. Gel was processed and RNAs were transferred to positively charged nylon membrane (Pal Corporation) using 20XSSC buffer. after capillary transfer to the membrane, RNAs were fixed by exposing the membrane to UV brand linker ( Hoeffer, Piscataway). DNA fragment of candidate genes to be used as canvass were isolated from rice cloned in pTZ57R TA cloning vector and confirm by sequencing. Double-stranded probes were radioactively labelled with (-32P) dCTP using DecaLabel DNA Labeling kit (Fermentas) and probes were purified using Sephadex G-50 spin column (GE Healthcare). Radiolabelled probes were denatured on boiling water bath manner snap cooled on ice and used for hybridization as described by Streit et al. (2008). RNA blots were pre-hybridized in ULTRAhyb at 45C for 48 h. The blots were hybridized with 32P-labelled denatured probes at 45Cfor 20 h in the same but fresh buffer. The blots were initially washed at room temperature with 2XSSC and 0.1% SDS followed by twice wash with 1XSSC and 0.1%SDS at 45C for 20 min each.The blots were initially washed at room temperature with 2XSSC and 0.1% SDS for 30 min and then washed with different stringencies for different probes to decrease background. Hybridized membrane were dried on blotting paper and exposed to Kodak XAE-5 film with cassette having Kodak intensifying screen for 16 d. The resulting radiograms were scanned in an LKB 2201 densitometric scanner.ResultsEffect of salinity stress on rice and finger millet genotypes during germination stageScreening of contrasting genotypes of both rice and finger millet against salinity stress at germination stage revealed the superiority of finger millet over rice in terms of salinity tolerance at germination stage. At lower concentration of salinity stress (i.e 50mM NaCl) the susceptible genotypes of both finger millet (CO12) and rice (White ponni) has shown better germination shareage and vigor index as compared to tolerant genotypes. Tolerant rice genotype FL478 was found to possess better germination portionage (352.9%) and vigor index (128.210.6) in comparison to susceptible White Ponni where germination percent and vigor index was found to be 16.71.7% and 70.97.1 respectively. Both fin ger millet genotypes i.e. CO12 and Trichy1 has shown almost interchangeable germination percent and vigor index at 100mM of NaCl stress. Both rice genotypes (viz. FL476 and White Ponni) did not show any germination beyond 100 mM NaCl stress (Table 1) whereas both susceptible (CO12) and tolerant (Trichy 1) finger millet genotypes were able to germinate even at 300 mM NaCl stress (Table 1). At 300 mM NaCl stress Trichy 1 has shown better germination percent (40.01.6) and vigor index (32.01.3) as compared to CO 12 germination percent (24.40.9) and vigor index 24.40.9.