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ThesisItem Open Access MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF ENDOPHYTIC BACTERIA ISOLATED FROM SUGARCANE IN RESPONSE TO SALINITY STRESS(MPKV,Rahuri, 2019) More Janardhan Dattatraya; Dr. A. S. JadhavLike plants, bacteria that are in association with sugarcane, exhibit variation in salinity tolerance and its characteristics which promote plant growth, such as nitrogen fixation, production of phytohormones, solubilization of inorganic phosphate, among others may be affected. This salt stress situation in sugarcane can be mitigated by using endophytic bacterial strains tolerant to salt stress. These bacteria are known as halotolerant, not require salt for metabolism but tolerate salts at different concentrations. With this view, the present investigation entitled "Molecular and biochemical characterization of endophytic bacteria isolated from sugarcane in response to salinity stress" was unde1taken to isolate endophytic bacteria and characterized with different biochemical tests and molecular analysis. These endophytic bacteria can be recommended as bio inoculants for crop like sugarcane which can help to sustain crop in saline habitat and provide a step forward towards sustainable agriculture. In the present investigation, a total three sugarcane plant pmts viz., leaf sheath, stem and root cortex were collected from different salinity location viz. , Ahmednagar, Sangli and Kolhapur district of Maharashtra. The pH and ECe of the three salinity locations of Ahmednagar, Sangli and Kolhapur were measured and it was 8.23, 8.38 and 8.24 and 5.56, 5.63 and 6.00 respectively. A total 9 endophytic bacterial isolates each three of Gluconacetobacter diazotrophicus, Herbaspirillum spp. and Azospirillum spp. were isolated from these plant pa1ts. Gluconacetobacter diazotrophicus, Herbaspirillwn spp. and Azospirillum spp. isolates showed positive test for catalase and oxidase however, negative test for starch hydrolysis and gelatin hydrolysis. Isolates of Herbaspirillum spp. and Azospirillum spp. showed negative test for H2S production and isolates of Gluconacetobacter diazotrophicus showed positive test. In case of casein hydrolysis test, Isolate 2 and 3 of Gluconacetobacter diazotrophicus and isolate 7 of Azospirillum spp. showed positive test and all other remaining isolates showed negative test. Gluconacetobacter diazotrophicus, Herbaspirillum spp. and Azospirillum spp. isolates were screened for salinity tolerance by inoculating on nutrient agar medium with different concentrations of NaCl (2.5, 5.0, 7.5, 10.0 and 12.5%) and observed that Isolate 6 of Herbaspirillum spp. and Isolate- 7, 8, 9 of Azospirillum spp. have tolerance limit upto 12.5% NaCI. However, Isolate 2 of Gluconacetobacter diazotrophicus tolerated upto 10.0% NaCl concentration. These nine endophytic bacterial isolates were tested for IAA production and phosphorus solubilization and it was observed that Isolates 7, 8, 9 of Azospirillum spp., isolates I and 2 of Gluconacetobacter diazotrophicus and isolate 5 of Herbaspirillum spp. showed positive test for IAA production and in case of phosphorus solubilization, isolates 7, 8, 9 of Azospirillum spp. and isolates 1, 2 of Gluconacetobacter diazotrophicus showed positive result, however, isolates 4, 5, 6 of Herbaspirillum spp. and isolate 3 of Gluconacetobacter diazotrophicus showed negative results.ThesisItem Open Access ISOLATION AND CHARACTERIZATION OF ANTIMICROBIAL GENE FROM ONION(MPKV,Rahuri, 2019) PATIL PRIYANKA DNYANOBA; Dr. A. S. JadhavThe thesis embodies the results of investigations on the molecular mechanisms controlling antimicrobial activity of Ace-AMP1 (Allium cepa-antimicrobial peptides) in the cultivated onion (Allium cepa L.) genotypes. The main objective of the investigation was to fmd out the gene sequences which are involved in the molecular mechanism of antimicrobial activity. The present investigation entitled " Isolation and characterization of antimicrobial gene from onion" was carried out with the objective to characterize candidate gene imparting antimicrobial activity in onion. Onion (2n=l6) is well known traditional neutraceutical and medicinal plant that is cultivated and used around the world. It possess several biological properties like antibacterial, antiviral, antifungal and antioxidant activities. The antimicrobial activity of four onion genotypes were investigated against Bacillus subtilis and Pseudomonas fluorescens by disc diffusion method by using onion juice extract. Highest zone of inhibition was shown by genotype N 2-4-1 ( 17 mrn), followed by BRBO 1001 (13 mrn) for Bacillus subtilis and BRBO 1004 (15 mm), followed by BRBO 1001(14 mrn) for Pseudomonas jluorescens. The genotype Sel 19 showed lowest zone of inhibition for Bacillus subtilis and Pseudomonas fluorescens respectively.ThesisItem Open Access CANDIDATE GENE ANALYSIS FOR SALINITY STRESS IN CHICKPEA (Cicer arietinum L.)(MPKV,Rahuri, 2019) INAMDAR SHAHEEN BADSHAH; Dr. A. S. JadbavSalinity is complex abiotic stress which affects almost all stages of crop growth leading to reduction in yield. The main causes of salinity are excess of irrigation and poor drainage facilities. The area under salt affected soils is increasing at alraming rate. As the soil reclamation is difficult and expensive procedure, to overcome yield reduction caused by salinity , breeding for salt tolerant cultivars with bioteclmological approaches is better option than conventional breeding. In the present investigation, 48 genotypes of desi chickpea comprising released varieties and breeding lines were used for performing candidate gene (CG) analysis. Twenty two candidate genes responsible for salinity stress tolerance were analysed and their sequence were retieved from NCB! website for designing candidate gene specific primers by using Primer-BLAST tool. More than 1 primer was designed and used for studying the individual gene. The coding sequences (CDS) range for different CGs varied from 300 bp (DSTP) to 3432 bp (SOS 1) were used for designing primers. The primer covering one or more than one exon was selected for analysis. Total of 45 candidate gene specific markers were designed by using Primer-BLAST tool from NCB!. In addition 3 CG specific markers available in literature reported for salinity tolerance in sugarcane were also used for analysis after performing homology search with chickpea genome. Out of total 48 CG specific markers, only 24 markers amplified the DNA of which, 4 markers showed length polymorphism, 17 showed presence/ absence polymorphism and 3 showed non-specific amplification. The three CG based markers from sugarcane failed toThesisItem Open Access "GENE EXPRESSION PROFILING IN SUGARCANE UNDER SALINITY STRESS CONDITION "(MPKV,Rahuri, 2019) SHELKE PRASAD DILIP; Dr. A. A. KaleThe present investigation entitled "Gene expression profiling in sugarcane under salinity stress condition" was carried out with the objective to study the differential transcript expression in sugarcane under salinity stress. The two genotypes of sugarcane, salinity stress tolerant CoM 0265 (Phule 265) and salinity stress susceptible CoC 671 (Vasant I) were exposed against salt stress viz., 0, 4.6, 7.8, 9.8 and 13.4 dS m- 1 induced by Sodium Chloride (NaCI) solution for the period of 14 hrs and 38 hrs. Present study was conducted using I 0 gene specific primers, which has predicted functions in detoxification of sodium, sodium transport, proline biosynthesis, antioxidant and various stress signaling protein under salt stress conditions. In the present investigation, expression of various genes under salt stress conditions were upregulated or highly expressed in salt tolerant genotype CoM 0265 (Phule 265) at 14 hrs and 38 hrs after salt stress application. However, downregulation or poor expression of gene was observed in salt susceptible genotype CoC 671 (Vas ant l) under various salt stress levels and period. NHX, SOSJ, HKTJ gene encoding membrane transporter protein, involve in extrusion ofNa+ from plant cell. NHX gene was upregulated in salt tolerant genotype CoM 0265 with increase in salinity stress levels. The increase in NHX gene expression in salt tolerant genotype CoM 0265 indicates NHX gene may involve in the sequestration ofNa+ in vacuoles and thus detoxify the effect ofNa+_ NHX gene expression was decreased in salt susceptible genotype CoC 671 with increase in salt stress level, it may resulted in inadequate Na+ sequestration in vacuoles causing a damage to the cell. Cation exchanger (CAXJ) gene was upregulated in salt tolerant genotype CoM 0265 and gene expression was comparatively higher at 38 hrs after salt stress application than that of 14 hrs. The upregulation of CAXJ gene may regulated calcium (Ca2+) level in cytosol under salt stressThesisItem Open Access "EFFECT OF POLYAMINE ON GENE EXPRESSION TO ALLEVIATE DROUGHT TOLERANCE IN SUGARCANE"(MPKV,Rahuri, 2019) MULE KRUSHNA DNYANDEV; Dr. A. A. KaleAs water resources on planet becoming limited, the development of more efficient cultivars for water usage is extremely important. It has become a challenge to better understand sugarcane response to water deficit. The present investigation entitled " Effect of polyamine on gene expression to alleviate drought tolerance in sugarcane" was carried out with the objective, to study effect of putrescine on gene regulation under induced drought condition in sugarcane. The two genotypes of sugarcane, a drought tolerant Co 740 and a drought susceptible Co 7219 were evaluated against water stress of 0, -0.1, -0.4 and -0.6 MPa, PEG induced stress with putrescine application of 0, I , 10 and 100 )!M cone. at 24 hours. Present study was conducted using nine gene specific primers, which are reported perfonning functions of detoxification, water and sugar transp01ts, proline biosynthesis and root elongation in water deficit stress conditions. The genes fJ-a ctin, P5CS, APX, Aquaporin, SUTJ, Mn-SOD and LEA were found expressed in both sugarcane genotypes, however, IGS gene was expressed only in drought tolerant genotype after 24 hours of PEG induced stress conditions. The highest fold expression of JGS gene was found in Co-740 is 1.49 fold with 100 )!M of putrescine cone. at -0.6 MPa PEG induced stress. The gene fJ-a ctin was used as an internal control or reference gene. Among the nine drought stress responsive genes used in this investigation, Aquaporin gene involved in water transportation chmmels was found to be decreased at each PEG induced stress levels viz., 0, -0.1, -0.4 and -0.6 MPa in both tolerant and susceptible sugarcane genotypes. The dehydration responsive element binding (DREB) protein gene was expressed in Co 740 genotype after 24 hrs of drought stress. Application of putrescine resulted in synthesis of transcript of DREB in susceptible Co 7219 genotype at -0.1, -0.4 and -0.6 MPa induced water stress. The highest fold expression of DREB gene was found in Co 740 genotype, it was 1.44 fold with 100 )!M cone. of putrescine at -0.4 MPa stress. Expression level of LEA gene was found to be decreased at -0.1 MPa induced stress with 10 )!M application of putrescine compared to 1 )!M application of putrescine in Co 740 a drought tolerant genotype. Highest fold expression of LEA gene was found in Co 7219, 1.51 fold at -0.4 MPa stress with 100 )!M of putrescine cone., however, in Co 740, it was 1.37 fold at -0.6 MPa stress with 100 )!M of putrescine application. The highest fold expression of superoxide dismutase (Mn-SOD) gene was found in both Co-740 and Co 7219 genotype at -0.6 MPa PEG induced stress with 100 )!M putrescine cone., which was 1.57 and 1.59 folds, respectively. The sucrose transporter (SUTJ) gene expression was found highestThesisItem Open Access QTL ANALYSIS FOR BLAST RESISTANCE IN RICE (Oryza sativa L.)(MPKV,Rahuri, 2018) Dudbabhate Jayashri Goroba; Dr.P.I.KulwalThe present investigation entitled "QTL analysis for blast resistance in rice ( Oryza sativa L.)" was carried out with the objective to identify marker(s) linked to blast resistance in rice. Rice (Oryza sativa L.) is one of the staple foods in the world, belonging to the family Poaceae. One of the !imitating factors in the production of rice is the attack of fungal pathogen Magnaporthe oryzae, causes blast disease which is one of the most destructive diseases affecting rice production worldwide and can cause heavy economic losses in a susceptible cultivar. The mapping population used in this study was the derived from cross of two local genotypes EK 70 (highly susceptible to blast) and RDN-98-2-3-5-14 (resistant to blast). The F9 generation of the RIL mapping population was developed using modified single seed descent method. Two hundred and four RILs along with parents were used. Bulk segregant analysis method was employed to identify molecular markers associated with blast resistance in rice. Among the 32 SSR markers used, 11 were polymorphic among the parents, whereas five markers viz., RM8225, RM204, AP5659-5, RM247 and RM208 were polymorphic between susceptible and resistant parent and respective bulks indicating their possible association with blast resistance in rice. The phenotypic and genotypic data obtained were used for the identification of single marker analysis. The chi-square (X2) analysis showed that three markers RM8225, AP5659-5, RM247 showed expected segregation ratio 1:1 and RM204 gave skewing in favor of EK70 and RM206 in favor of RDN98-2-3-5-14. Two markers NMSMPi9-l and RM21 showed highly distorted segregation towards the resistant parent RDN98-2-3-5-14. Simple regression analysis with phenotypic data indicated that the marker RM8225,ThesisItem Open Access "MICROPROPAGATION STUDIES IN ASHWAGANDHA (Withania somnifera)"(MPKV,Rahuri, 2010) Thokale Suwarna Jagannath; S.C.PatilThe investigation "Microprogagation studies m Ashwagandha (Withania somnifera)" was carried out at State Level Biotechnology Research Centre, M.P.K.V., Rahuri during 2009-10. The objectives of the research work were to· study the response of various genotypes on different media for callus induction response, induction of somatic embryogenesis and regeneration system in ashwagandha. For this experiment five different genotypes were selected. Auxiliary bud, nodes, internodes, leaves were used as a explants for establishment, callus induction and regeneration studies. MS medium was used as basal medium throughout the experiment were supplemented with various levels and combinations of2, 4-D, Kinetin, BAP, NAA and IAA for establishment callus ' induction, regeneration of shoots and ca.se,~n hydrolysate for induction of somatic embryogenesis, where as IBA was added for root induction. There were significant differences among genctypes, media and interaction for days required for establishment, days to callus induction, fresh and dry weight of callus, shoot length (em) 45 days after regeneration, number of shoots per explants, days required for rooting, number of roots and average length of roots. {- 7 I 3_9 Least period for the establishment for all genotypes responded on MS media supplemented with 2 mg/1 BAP and 0.4 mg/1 KIN. Callus induction required 9 to 13 days on MS media supplemented with various levels of 2, 4-D. However embryogenic callus was formed on MS medium containing 2, 4-D (0.5 mg/1) after 2 weeks of subculture. Regeneration was noticed within 3.66 to 12.33 days. Shoot length was observed 45 days after regeneration was 1.225 to 2 .617 em and produced 1.33 to 7 shoots per explants on the medium added with various levels and combinations of BAP, Kinetin, NAA and IAA. Regenerated shoots were rooted on MS media with two levels of IBA (1.0 and 1.5 mg/1). The root induction was noticed within 13.833 to 14.333 days with 13 to 38.167 number of roots and 3.250 to 4.567 em root length. Complete in vitro produced shootlets were transferred to soil and allowed to grow till maturity.ThesisItem Open Access Marker-Trait association study for protein content in Chickpea (Cicer arietinum L.).(MPKV, Rahuri, 2013) Jadhav Amol Asaram; Kulwal, P.L.ThesisItem Open Access Micropropagation studies in Sarpgandha (Rauvolfia serpentina L.)(MPKV, Rahuri, 2010) Koskewar Sandeep Uttamrao; Pawar, S.V.
