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ThesisItem Open Access DEVELOPMENT OF MOLECULAR DIAGNOSIS TECHNIQUE FOR X{mthonzoiuts axonopodis pv. punicae(MPKV,Rahuri, 2015) PATIL NILAM PRAKASHRAOPomegranate (Punica granatum L.) is an important fruit crop oflndia and other subtropical countries. Bacterial blight (Oily spot) of pomegranate has recently emerged as a very serious disease and resulted in decline pomegranate orchards, particularly in pomegranate belt of peninsular Indi~ . The oily spot caused by Xanthomonas axonopodis pv. punicae is becoming a major threat to pomegranate production in Maharashtra as well as other pomegranate growing states of India. It causes yield loss to the extent of 80-90 %. Considering importance of this disease, present study entitled "Development of molecular diagnosis technique for the Xanthomonas axonopodis pv. punicae" was undertaken. A survey was conducted using various isolates of X axonopodis pv. punicae from Nashik, Ahmednagar, and Solapur districts along with related Xanthomonas species were used for PCR based molecular diagnosis shtdies.ThesisItem Open Access BIOCHEMICAL INVESTIGATIONS RELATED TO INHERITANCE OF DOWNY MILDEW RESISTANCE IN PEARL MILLET f.i.:tennzsetum glaucum (L.))(MPKV,Rahuri, 2013) SUSHEEL KUMAR T; R.M.NaikThe present investigations were undertaken to study the inheritance of metabolites and enzymes involved in disease resistance by analyzing parents and crosses after post inoculation by Sclerospora graminicola. The total phenols were less in non inoculated seedlings of both resistant and susceptible parents, though they were significantly more in resistant genotypes. In seedlings of both resistant and susceptible genotypes and their crosses (RxS, SxR and RxR) there was significant increase in total phenol content after inoculation. The level of 0. D. phenols after inoculation of seedlings with pathogen however decreased in all the resistant parents, susceptible parents and the crosses (SxR, RxS and RxR).There was no defmate trend in the level of reduction of O.D. phenols in resistant or susceptible parents, although the susceptible parent 70428 recorded comparatively more reduction in the level of O.D. phenols. It is however interesting to note that the peroxidase activity was much higher in the susceptible genotype 7042S even in the absence of the pathogen against the resistant genotype. The crosses involving downy mildew susceptible genotype (7042S) either as male or female parent also exhibited much higher peroxidase activity than the resistant genotypes both in the absence and presence of the pathogen inoculum. The level of induction as evident from activity differences between control and inoculated seedlings however was much less in susceptible parent and the crosses involving 7042S as a male parent. A significant variation was observed in the polyphenol oxidase activity of the resistant and susceptible parents. The polyphenol oxidase activity was much higher in the downy mildew susceptible genotypes 7042S both in the absence of the pathogen and when the seedlings were dipped in pathogen suspension for 72 hrs. The crosses involving susceptible genotype (7042S) as a male parent exhibited comparatively lower level induction of polyphenol oxidase with higher downy mildew field incidance. A reduction in CAT activity after inoculation was also observed in RxR cross. As crosses involving resistant genotype as a n;:tale parent (SxR) recorded minimum reduction of CAT upon pathogen inoculation. Superoxide dismutase (SOD) activity was analyzed in resistant, susceptible genotypes of pearl millet and their crosses after inoculation with S. graminicola. In the resistant genotypes increases in enzyme activity over that of uninoculated control seedlings was recorded, while in susceptible genotype a decrease in SOD activity was noticed. It thus appears that superoxide dismutase induction on inoculation with the downy mildew pathogen can be correlated with resistance and nuclear gene appears to control the downy mildew resistance, as crosses involving resistant genotype as a male parent (SxR) also recorded induction of SOD upon pathogen inoculation. Phenylalanine Ammonia Lyase (PAL) activity was studied in resistant and susceptible genotype of pearl millet and theirThesisItem Open Access LEVEL OF OSMOLYTES ACCUMULATION, ANTIOXIDATIVE ENZYMES AND OTHER BIOCHEMICAL CONSTITUENTS IN LEAVES OF HORSEGRAM (MACROTYLO.MA UNIFLORUMLAM.) CULTIVARS DURING STRESS(MPKV,Rahuri, 2012) Naik Prat.ima Ezak; D.P. KachareThe present research work entitled "Level of osmolytes accumulation, antioxidative enzymes and other biochemical constituents in leaves of horsegram (Macrotyloma unijlorum Lam.) cultivars during stress" was carried out with a view to evaluate the effect of moisture stress by withholding irrigation water on five cultivar of horsegram. The levels of water stress was evaluated in terms of RWC. Five cultivars of horsegram viz. SHG-40, SHG-42, SHG-64, SHG-66 and SHG-67 were subjected to moisture stress and levels of accumulation of proline, glycine betaine, superoxide dismutase, peroxidase, soluble proteins, free amino acids and polyphenols were estimated. Moreover, isozyme pattern of catalase and extracted soluble proteins from leaf material were resolved on a polyacrylamide gel in order to observe the difference if any in banding pattern under stress condition.It revealed that levels of proline and glycine betaine increased in cultivars of horsegram when exposed to moisture stress. Comparatively highest fold (29.58) increase in free proline was observed in cultivar SHG-40, whereas highest fold (8.49) increase in glycine betaine was observed in cultivar SHG-42. Activities of antioxidative enzymes viz., superoxide dismutase (SOD) and peroxidase (POD) increased under stress condition in all horsegram cultivars. The highest fold (1.68) increase in superoxide dismutase was observed in cultivar SHG-66, where as the highest fold (1.92) increase in peroxidase was observed in SHG-66. An isozyme pattern of catalase resolved on polyacrylamide gel which exhibited dark single band with molecular weight 66kDa and showed no difference in band intensity. Soluble proteins also increased under stress condition in all horsegram cultivars in response to imposed moisture stress, but highest fold increase (1.47) was observed in cultivar SHG-40. Similarly free amino acids were also observed to be increased in stress condition. Highest fold (1.79) increase was observed in cultivar SHG- 40. Polyphenols were estimated which showed similar elevated level in stress condition in all the five cultivars of horsegram. The highest fold (2.25) increase was observed in cultivar SHG-40. Relative leaf water content was reduced in stressed plants. It ranged from 97-98% in normal plants and it was reduced to 74-76% in stressed plants. Similarly SDS-PAGE analysis of soluble proteins from the leaves of five cultivars demonstrated four bands of approximate molecularThesisItem Open Access "INFLUENCE OF STAGE OF HARVESTING ON CHANGES IN JUICE SUGARS AND ETHANOL YIELD OF SWEET SORGHUM GENOTYPES AND GRAIN COMPOSITION STUDIES DURING'WINTER SEASON"(MPKV,Rahuri, 2012) Borade Ranajitsinh Manasing; U.S.DalviThe present investigations were undertaken to study the sugar and juice quality of promising sweet sorghum genotypes. Fourteen selected sweet sorghum genotypes were grown in rabi season of 20 11 on the research farm of All India Co-ordinated Sorghum Improvement Project, M.P.K.V. Rahuri. The juice from stalks of each genotypes were extracted at different stages of harvesting i.e. 10, 20, 30, and 45, days after 50 % flowering and analyzed for Obrix, reducing sugar, nonreducing sugar, total sugar, juice yield and juice extraction %, The calculated ethanol yield was computed only at last stage of harvesting. The matured and dry grains of above fourteen genotypes of sweet sorghum were analyzed for moisture, total sugar, ash, starch, total protein, polyphenol, and crude fiber. The sweet sorghum genotypes analyzed for juice quality showed significant differences in respect of 0 brix, reducing sugar, non-reducing sugar, total sugar of the juice at all the stages of harvesting. The obrix, reducing sugar, non-reducing sugar, total sugar increased from flowering to physiological maturity stage of cane. The mean °brix value of all these genotypes ranged from 14.33 to 17.00 with a mean of 15.92 per cent at physiological maturity. The reducing sugar content was also increased from flowering to maturity. The genotypes RSSV- 220 and RSSV-120 had the highest (2.07 %) reducing sugar content. The non-reducing sugar ranged from 8.81-10.84 per cent with the mean 9.66 % at 45 days after 50 % flowering. The genotype RSSV-275 had the lowest (10.68 %) total sugar in juice and the genotype RSSV -104 showed the highest total sugar content (12.60 %). The green cane yield in all the genotypes ranged from 18.94-29.43 tjha .The genotype RSSV-167 showed highest juice yield (6610 L/ha), while the lowest juice extraction per cent was recorded in RSSV-99, (13.59 %). All the sweet sorghum genotypes significantly differed in respect of calculated ethanol yield (L/ha). The genotype RSSV-167 recorded the highest ethanol yield of 432.7 L/haThesisItem Open Access "Studies on nitrate and ammonia assimilating enzymes . in the leaves of three aestivum wheat genotypes"(MPKV,Rahuri, 2011) MANE PRAVIN SARJERAO; S. V. MunjalThe present investigation aims at evaluating the nitrate and ammonia assimilating enzymic potentials in the leaves of three aestivum wheat genotypes. In addition, the photosynthetic rate of these genotypes was determined by using IRGA method. Further, correlation between NR with GS and photosynthetic rate in leaves was determined in the genotype HD-2189. The nitrate reductase enzyme activity from the leaves of 10 day old wheat seedlings was standardized by an in vivo method. Diurnal variability in NR activity of wheat leaves was noticed with a single peak of activity at 2 p.m. All the standardization experiments were performed with 10-day-old seedlings of the genotype, NIAW-301. The wheat plants were grown in pots filled with medium black calcareous soil under natural day-light The standardized infiltration medium (5.0 ml) for in vivo NR activity in plus KN03 assays in wheat leaves was: sodium phosphate buffer (pH 7.5) 100 mM; KN03, 100 mM; n-propanol, 1% (vfv) and Triton-X-100, 0.1% (v jv). The asssays that did not include nitrate in the infiltration medium demonstrate the actual amount of NOj'· reduced. The in vivo NR activity was linear with time over a 2-hr incubation period The enzyme was extracted with 0.1 M phosphate buffer (pH 7.5) containing 1mM cysteine and 0.5 mM EDTA and assayed in test tubes containing 100 1-1M sodium phosphate buffer (pH 7.5), 20 1-1M KN03, 0.75 mM NADH in a total volume of 2 ml and a crude enzyme extract 0.2 ml. The in vitro reaction rate was linear with time over a 30-min incubation period. The in vitro NR activity exceeded the in vivo NR activity, probably because the former was assayed under optimized conditions of substrate and reductant availability. The NiR activity of these genotypes was also determined by an in vivo method. The activities of ammonia assimilating enzymes such as GS, GOGAT and GDH was determined by the standardized in vitro methods. The NR potentials from aestivum wheat genotypes demonstrated the highest NR activity in the leaves of NIAW-34, whereas the minimum was in NIAW-301. In the studies on assessment of in vivo NR leaf potential in wheat genotypes, the observed NR activities were higher in the assays where exogenous nitrate ( 100 mM) was added than those performed without KN03. The NiR activity in these cultivars was comparatively high than that of NR. The genotype condition during the months from November, 2010 to February, 2011.ThesisItem Open Access ASSESSMENT OF PROTEIN, LYSINE AND STARCH CONTENTS IN GRAINS AND BIOCHEMICAL MARKERS ASSOCIATED WITH DROUGHT TOLERANCE IN SORGHUM GENOTYPES(MPKV,Rahuri, 2010) PATIL SUNIL DILIPRAO; S. V. DAMAMEThe present investigation was undertaken to evaluate the ~ levels of crude protein, lysine and starch from the sorghum grains of seven each of drought tolerant and susceptible genotypes. In addition, the levels of proline, glycine betaine, the activity of PSCS and soluble proteins were determined from the leaves of these genotypes by imposing stress created by polyethylene glycol-6000. Among the drought tolerant genotypes under study, the highest crude protein content of 9.63% was observed in the grains of RSV -962, whereas among the drought susceptible ones, the highest of 9.24% was observed in CSV-216. Amongst the drought tolerant genotypes, the highest lysine content of 2.19 g 16 g-1 N was observed in the grains of RSV-458 and among the susceptible ones, the highest of 2.17 g 16g-1 N was observed in SPV-504. As regards the starch content, a drought tolerant genotype SPV-1546 recorded the highest of 77.40 per cent in the grains, whereas among the drought susceptible ones, RSLG-1119 recorded the highest of 76.50 per cent. The seedlings of 2-weeks-old sorghum genotypes were evaluated for the accumulation of free proline, glycine betaine and P5CS activity under control and PEG induced stress of -0.5 MPa. On imposition of stress the mean proline accumulation increased by 7 -folds in drought tolerant genotypes as compared to a 3-folds increase in drought susceptible ones. The highest proline content of 544.4 pmol g-I fr.wt. was recorded in a drought tolerant genotype, RSLG-262. The activity of the rate-limiting enzyme, P5CS in leaf materials of drought tolerant genotypes demonstrated correspondingly higher activity than the susceptible ones under PEG induced stress. The mean fold increase of 2.44 in P5CS activity of leaf materials was observed in drought tolerant genotypes, whereas there was 1.44-fold in drought susceptible ones. The maximum fold increase of 3.42 in P5CS activity was observed in a drought tolerant genotype, Sel-3. As regards the accumulation of glycine betaine, the mean fold increase of 3.71 was observed in drought tolerant genotypes as compared to 1.57 -fold in leaf materials of a drought susceptible ones.ThesisItem Open Access ASSESSMENT OF BIOCHEMICAL AND MOLECULAR MARKERS IN DIFFERENTIATING SUGARCANE GENOTYPES FOR SALT TOLERANCE(MPKV,Rahuri, 2010) MAHAJAN SUCHETA TAPIRAM; R.M.NaikSugarcane (Saccharum officinarum L.) crop shows high sensitivity to salinity at various growth stages. T.he present investigation entitled, Assessment of biochemical and molecular markers in differentiating sugarcane genotypes for salt tolerance" was undertaken with a view to analyse the effect of salt stress on different sugarcane genotypes. The sets of sugarcane were planted in normal soils. Then after one !TIOnth, the seedlings were transplanted to bigger earthen pots in normal, aline and sadie soils. The leaves from 75 days old plants were excised or the estimation of various biochemical parameters viz., proline, glycine etaine,soluble proteins, in vivo NR activity and the activity of PSCS. f urther,the leaf soluble proteins were extracted from sugarcane leaves grown on normal, saline and sadie soils and resolved on SDS-PAGE. The ifferences in isozyme forms of peroxidase and esterase were also bserved. The genomic DNA' isolated and purified from the sugarcane enotypes was amplified using different random primers.The leaf free proline content of all the sugarcane varieties increased_, the mean proline accumulation fFom 99.97 to 114.43 J..1 mole g-1 fr.wt. in saline soil and from 99.97 to 122.76 J..1 mole g-1 fr.wt. m sorlic soil. Comparison of the leaf prelme €ontents wblen gFOwn on normal, saline and sodic soils revealerl ~ferenees m feld increase in proline. Sugarcane genotypes CoM-0265,Co:-740 and Co-62175 showed an increase in proline content of 1.26, 1.25 and 1.22 .. £old in sa1ine soil and 1.34, 1.49and 1.28 -fold in sodic S0ilS, respectively. Amongst the promising clones, CoM -08011 appeared to 1De sa1t tolera.I'lt showing an increase in leaf proline content of 1.1 ~and 1.25 fold under salline and sodic conditions. It thus appears that fold increase in pF®line level is a sensitive parameter while evaluating salt toleFance and pFoline accumulatoin is an adaptive parameter unde:r stress c0naition. The early maturing and high sugar genetypes viz., CoC-671 and Co-940 12 appeared to be salt susceptible. ·A significant increase in mean g~cine ,betaine content was recorded with 4.34-fold increase in saline soils and 16.63 fold in sodic soils. A maximum fold iiacrease in glyci~e betaine content of 10.44 and 51.06 fold was reco~ded in CoM-0265, followed by Co-7 40 with a fold increase of 8.19 and 27.28 in saline and sodic soils~ respectively . The early maturing aE.d nigfu suga:r centaining sugarcane genotypes,.,CoC-671 and Co-94012 showed the minimum fold increase of 2.45 and 1.68, respectively when grown on saline soils and 7.42 and 6.77 when grown on sodic soils.The soluble protein content also increased under stress aE.dWlehighest fold increase ef 2.52 and 4.18 was observed in CoM-0265 in saline and sodic soils,respeetively. 1'he increase in free proline content is not due te proteolysis but rather due to increased de-novo synthesis. The in vivo leaf nitrate reductase activity was drastically reduced when grown on saline and sodie soils. A :m.inimum reduction in the in vivo NR activity from 0.62 in normal soH to 0.54 J..1 mole N02~ fromed g-1 fr.wt.hr-1 in saline soil was recorded in CoM-0265, followed by a reduction of 14.46 % in Co-740. It is interesting to note that early aturing and high sugar genotypes, CoC-671 and Co-94012 showed igher in vivo NR activity of 1.63 and 1.61 j..1 mole N02- formed g -~ r.wt.hr-1 , respectively when grown on normal soils. However , the eduction in the in vivo NR activity was significant to the extent of 32.90 Yo in CoC-671 and 43.52% in Co-94012 in saline soils and 62.64% and 4.66% in sodic soils.ThesisItem Open Access "Investigations on nitrate metabolism during light I dark transitions in spinach ( Spinacia o leracea L.) leaves"(MPKV,Rahuri, 2009) POWALE RUPALI NIVAS; S.V.MunjalThe present studies involving "Investigations on nitrate metabolism during light/dark transitions in spinach (Spinacia oleraceae L.)" were undertaken with a view to understand the dynamics of nitrate metabolism and regulation of key nitrate assimilatory enzyme, nitrate reductase (NR) during light/ dark transitions.The enzyme NR was characterized by in vivo and in vitro methods from light-grown spinach plants with the optimum concentrations of substrate and reductant. When the light-grown plants were transferred to dark, the in vivo and in vitro NR activities were drastically reduced after 2 h..-s of exposure. The drastic reduction of in vitro NR activity, despite the saturating levels of both limitation under light as the plants were periodically irrigated with 15 mM KN03 soon after emergence. The in vitro NR activity in light progressively increased with an increase in NADH concentration and the maximum in vitro NR activity was observed at NADH concentration of 200 11M. However, when the light-grown plants were transferred to dark for 2 hrs, there was an increase in nitrate content in leaves on account of diminished NR activity. With increasing NADH concentration from 50 to 250 11M in in vitro NR assays of dark-grown plants, there was no significant increase in NR activity even at a saturating concentration of nitrate. The decline in NR activity in dark is not due to the availability of nitrate but the limitation of enzyme per se besides the involvement of complex regulatory mechanism. With fixed and optimum NADH concentration, there was a gradual increase in N02 production with incremental additions of substrate, N03 in in vitro assays of lightgrown spinach plants and the maximum NO; production was observed at 40 mM concentration of KN03. As nitrate is also reduced to nitrite when the enzyme was extracted froin the dark-groWn plants and the NR activity determined with varying NADH concentrations, there was no significant change in the activity in dark as compared to the activity in light-grown plants. It thus confirms that No-;- is reduced to NO-; even in the dark, although at a much slower rate than in the light. The disappearance of nitrite under aerobic condition was substrate N03 and NADH ruled out the possibility of substrateThesisItem Open Access ANTIOXIDANT POTENTIAL OF POMEGRANATE PEEL AQUEOUS EXTRACTS AND SYNTHESIS OF SILVER NANOPARTICLES(MPKV, Rahuri, 2023) Dhakne Aishwarya Sudhakar; Bhalerao, B.M.Nanotechnology has marked a significant revolution in various areas of science and involved in the production and application of nanoparticles that can be used in different fields. Silver nanoparticles among other nanoparticles, have received substantial attention because of their unique properties. This study focuses on the green synthesis of silver nanoparticles using pomegranate peel extracts. Green synthesized silver nanoparticles have great advantages, including energy efficiency, low toxicity, high yields, cost-effectiveness, eco-friendliness, and ready availability. The Antioxidant potential of pomegranate peel aqueous extracts and synthesis of silver nanoparticles was carried out in Department of Biochemistry, Mahatma Phule Krishi Vidyapeeth, Rahuri. In the present study total five different solvents in order to polarity were used namely water, ethanol 60%, methanol 60%, ethyl acetate and petroleum ether respectively. In the extraction process solvent selection was a very important step because the solubility of required compounds was depend upon the polarity of the solvent. Method of extraction is widely affect the yield of the extracts. In this study four different extraction methods were used viz ultrasonic assisted extraction (UAE), soxhlet extraction (SE), microwave assisted extraction (MAE), and maceration extraction (ME) for comparison. The earlier scientists reported that pomegranate peel has high antioxidant activity than its flower, leaf and seed. The pomegranate peel extracts act as a natural inhibitors of plant pathogens like fungi and bacteria. In the present study Bhagwa, Superbhagwa and Ganesh pomegranate cultivar peels as agro waste material were used for comparative antioxidant study.
