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ThesisItem Open Access MOLECULAR SCREENING OF RED BANANA MUTANT FOR BBTV (Banana Bunchy Top Virus) AND DWARFNESS THROUGH MOLECULAR MARKERS(Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli, 2024-01-29) KOTA REVANTH REDDY; Sawardekar S. V.; Deshpande R. S.; Parulekar Y. R.This research project aimed to assess the molecular characteristics of red banana mutants concerning BBTV tolerance and dwarfness using various molecular markers. A comprehensive examination of 3,200 in vitro-raised red banana plant samples involved gamma irradiation at varying doses, identifying an LD50 dose of 20 Gy for optimal survival and growth.ThesisItem Open Access STUDIES ON MICROPROPAGATION TECHNIQUES IN BANANA Cv. NENDRAN AND NANJANGUD RASABALE(Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli, 2024-01-31) MEHTRE ANKITA RAJENDRA; Sawardekar S. V.; Deshpande R. S.; Malshe K. V.The goal of the current study, is to create methods for surface sterilization, optimizing growth hormones for in vitro organogenesis of banana Cv. Nendran and Nanjangud Rasabale. Three replications and a fully randomized design were used for the investigation. The study was conducted in Plant Biotechnology Centre, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli, Dist- Ratnagiri (M. S.) during the academic years 2022-2023.ThesisItem Open Access CALLUS INDUCTION AND INVESTIGATION OF DOMINANT ENZYMES PRESENT IN CALLUS MASS OF ALPHONSO MANGO (Mangifera indica L.)(Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli, 2024-01-31) TANDALE ANURAG SANDEEP; Bedse T. J.; Sawardekar S. V.; Deshpande R. S.A laboratory experiment was conducted during year 2022-2023 at Plant Biotechnology centre, College of Agriculture, Dapoli, Dr. B.S. Konkan Krishi Vidyapeeth, Dapoli to CALLUS INDUCTION AND INVESTIGATION OF important ENZYMES PRESENT IN CALLUS MASS OF ALPHONSO MANGO (Mangifera indica L.). The experiment was laid out in completely randomized design comprising of 10 treatments with three replications.ThesisItem Open Access SCREENING OF RICE GENOTYPES FOR BIOTIC AND ABIOTIC STRESS USING SPECIFIC MARKERS(Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli., 2021-06) PATIL, SHUBHAM DADASAHEB; DESHPANDE, R. S.; Sawardekar, S. V.; Mane, A. V.Present study, aimed to identify tolerant rice varieties for biotic and abiotic stresses and to establish rice genotype profiles through SSR markers. Amongst the 33 rice varieties evaluated 2 varieties were tolerant to Blast, 4 genotypes were tolerant to BB, 1 genotype was tolerant to BPH, 3 varieties were tolerant to Gall midge, 3 genotypes were salt tolerant and 2 varieties were tolerant to drought. All 19 primers amplified and showed 100% polymorphism. Total 100 alleles were observed with average of 5.26 alleles per primer. PIC values ranged from 0.27 to 0.894 with an average PIC value of 0.65 per primer. The genetic distance ranged from 0.022 to 0.92 and disclosed wide variability. All 33 rice genotypes divided into two clusters, I cluster containing 19 genotypes and II cluster containing 14 genotypes. This concludes that the molecular screenings of rice varieties using SSR markers provided sufficient knowledge on traits of biotic and abiotic tolerance at molecular level.ThesisItem Open Access DIVERSITY ANALYSIS OF PROMISING FINGER MILLET (Eleusine coracana L.) MUTANT LINES THROUGH ISSR MARKER (Accession No. T06472)(Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli , Dist : Ratnagiri, 2018-05) BORADE, SUPRIYA MOHAN; Sawardekar, S. V.The molecular marker technology has a great potential for assessing genetic variability and relationship among the selected mutant lines. In the present study, promising mutants of finger millet along with 3 check varieties showing distinct morphological differences were screened using 18 ISSR markers. The DNA was extracted from the green leaf samples collected from 15 days old seedlings of finger millet from 10 mutant lines along with 3 check varieties by rapid DNA extraction method. The most suitable combination of extraction buffer was found to be 200 mM Tris-HCl having pH 8.0, 25 mM EDTA, 250 mM NaCl which showed clear and specific banding pattern when subjected to PCR. Initially the PCR master mix was standardized by changing the quantity of each component and the optimum concentration of each component in master mix was used for further ISSR analysis. In which 10 mM (1μl) dNTPs concentration and Taq polymerase 3 U/μl (0.5 μl) gave better amplification. The annealing temperature ranging from 45.40C to 54.80C for 1 minute yielded good results. The finger millet DNA showed better amplification with 18 ISSR primers studied. A total of 760 bands were amplified and out of which 747 were polymorphic which showed 98.83 % polymorphism. The primer UBC-834 showed 79.03 minimum per cent polymorphism while the average bands per primer were 42.22. The ISSR profile generated by each of the primer was analyzed using standard DNA ladder (1353-310bp and 1800-200bp) and compared with their respective banding pattern. The average size of amplified fragment ranged from 388.8 bp to 1627.78bp. The primer UBC-814 and UBC-891(0.82) recorded minimum PIC value (0.25), whereas primer UBC-881 gave maximum PIC value 0.91 and average polymorphic information content is 0.50 among the all 10 mutants. It indicates that ISSR markers have a great potential to show the polymorphism among the finger millet mutants. The data of 10 mutants along with 3 check varieties of finger millet were used to generate pair-wise matrix based on the Jaccard‟s co-efficient. The genetic distance was calculated on the basis of pooled data and the dendrogram was constructed. The similarity coefficient ranged from 0.112 (between the mutant lines M-3 and M- 21) to 0.49 (between mutant lines M-1 and Dapoli-2) indicating the similarities and distinctness of these mutants, respectively. Cluster analysis was carried out based on UPGMA analysis and it divided 10 mutants along with 3 check varieties into two main clusters and each having two sub-clusters. The first sub-clusters of first major cluster comprised of 6 mutants and the second sub-clusters also comprised of 6 mutants. The first sub-clusters of second major cluster had 8 mutants. While the second sub-clusters of second major cluster consisted of 6mutant lines.ThesisItem Open Access STANDARDIZATION OF IN VITRO REGENERATION TECHNIQUE IN RED BANANA (Musa acuminata) (Accession No. T06471)(Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli , Dist : Ratnagiri, 2018-05) Pandurang, Keskar Kiran; S. V., SawardekarThe present investigation entitled, ―Standardization of in vitro regeneration technique in red banana (Musa acuminata)‖ was aimed to develop surface sterilization techniques and optimization of growth hormones for in vitro regeneration of red banana and fidelity testing of tissue cultured red banana plantlets through molecular markers. The study was undertaken in completely randomized design with 3 replications. The surface sterilization treatment of 5ml/L Tween20 for 10min, (5ml Dettol + 45ml savlon)/L for 30min ,1% Carbendazim for 30 min, 70% Ethanol for 1 min, 5% NaOCl for 10 min, 250 mgL-1 cefotaxime (I) for 20 min, 250 mgL-1 cefotaxime (II) for 40 min was found to be best combination to achieve highest percentage of contamination free healthy cultures (67.80%) and gave best response for minimum days to shoot initiation i.e. 22 days. Media combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgLMedia combination MS + 4.5 mgL-1 BAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgLBAP + 0.175 mgL-1 IAA + 20 mgLIAA + 20 mgLIAA + 20 mgLIAA + 20 mgLIAA + 20 mgLIAA + 20 mgLIAA + 20 mgLIAA + 20 mgLIAA + 20 mgLIAA + 20 mgLIAA + 20 mgLIAA + 20 mgL-1 ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment (ascorbic acid showed highest establishment ( 78%) of explants. The media combination MS + 4.5 mgL-1 BAP + 0.175 mgL-1 IAA + 20 mgL-1 ascorbic acid was found effective for maximum number of shoots initiation (96.67%) and maximum rate of multiple shooting (560.29). Same media combination with addition of 1gL-1 charcoal was also found effective for highest percentage of root regeneration (93.33) and maximum number of roots per shoot with an average of 15.34 roots per shooted plant. The days required for shoot initiation ranged between 21-30 days and for rooting ranged between 14 to 20 days. The rooted plants transferred in potting mixture of sand, soil and FYM present in equal proportion (1:1:1) recorded 90 per cent survivability. The best month for inoculation of explants for culture was July for highest proliferation. In fidelity testing of red banana, out of 20 ISSR primers, 11 primers resulted in clear and scorable amplification products. Average per cent polymorphism was 0. Range of size of product within bulked DNA was 200-2000bp. Average number of alleles produced per marker was 4.81 The monomorphic banding patterns between the selected plantlets from single mother plant indicates the uniformity of tissue culture banana plantlets. Key word : red banana, surface sterilization, growth hormones,fidelityThesisItem Open Access GENETIC DIVERSITY ANALYSIS IN COWPEA (Vigna unguiculata (L.) Walp.) BY USING RAPD MARKERS(DBSKKV., Dapoli, 2012-05) Mr. Patil, Dagadu Magan; Sawardekar, S. V.GENETIC DIVERSITY ANALYSIS IN COWPEA (Vigna unguiculata (L.) Walp.) BY USING RAPD MARKERS D. M. Patil 2012 Dr. S. V. Sawardekar Research Guide ABSTRACT Assessment of genetic variability within Vigna unguiculata (L.) Walp. is fundamental for the conservation of genetic resources and its utilization in hybridization programme. The objective of this study was to estimate the genetic diversity among 30 genotypes of cowpea through molecular characterization by using RAPD markers. RAPD profiles for all 30 genotypes were generated with 20 random decamer primers. Out of 20 primers screened 17 primers gave scorable DNA fragments and each of the 17 random primers revealed polymorphism. The primers generated 1238 DNA fragments in the average range of 381.94 bp to 1131.71 bp, of which 908 were polymorphic. The level of polymorphism generated by the primers was high (71.20%). The primers OPA-04, OPA-05, OPC-02, OPC-05 and OPC-08 produced distinct RAPD patterns (100% polymorphism) for all the 30 genotypes. These primers can be used for identification of the genotypes studied. The average discrimination power among the 17 primers was 62 per cent. The overall range of the similarity among 30 genotypes was found to be very wide, ranging from 0.321 to 0.800 which indicates there was high variability among the cowpea cultivars under study. The cluster analysis based on RAPD data showed that genotypes formed two main groups. The genotype PCP-97223 occupied a unique position and was most diverse from rest of the genotypes. The study indicated that RAPD markers are suitable for the assessment of genetic diversity among group of genotypes and identification of diverse sources in crop germplasm collection. This study could identify the diverse genotype like DCP-11 and Pusa Phalguni for their use in hybridization programme of cowpea.ThesisItem Open Access FIDELITY TESTING IN TISSUE CULTURE DEVELOPED BANANA PLANTLETS OF Safed velchi AND DETECTION OF BBTV THROUGH ELISA (Accession No. T06031)(DBSKKV., Dapoli, 2016-05) Mr. KADAM, SAURABH; Gokhale, N. B.PLANT BIOTECHNOLOGY CENTRE COLLEGE OF AGRICULTURE, DAPOLI DR. BALASAHEB SAWANT KONKAN KRISHI VIDYAPEETH, DAPOLI Name of Student : Mr. Kadam Saurabh Subhash Registration No. : 0018 Name and Designation : Dr. N. B. Gokhale of Research Guide Incharge Plant Biotechnology Centre College of Agriculture, Dapoli. Dist.- Ratnagiri Title of Thesis : Fidelity testing in tissue culture developed banana plantlets of Safed velchi and detection of BBTV through ELISA Academic Year : 2014-2016 ABSTRACT The present study was carried out with an objectives to test fidelity of tissue cultured banana plantlets through molecular markers, to standardize protocol for viral detection and to test tissue cultured banana plantlets for Banana Bunchy Top Virus (BBTV) through ELISA technique. The DNA was extracted from tissue cultured tender leaf samples of banana plantlets. A total of 200 plant samples were selected for extraction of genomic DNA from 3 mother plants (mother plant no. 15, 16 and 21). The standardization of buffer constituents for DNA isolation was carried out. Modifications in extraction procedure and in PCR parameters like PCR master mixture and thermo profile showed clear and specific banding pattern. ISSR primer UBC-891 showed 0.12 per cent average polymorphism in third lot of mother plant no. 15. All other primers produced monomorphic banding pattern in remaining lots of mother plant no. 15, 16 and 21. The results revealed that, the monomorphic banding patterns between the selected plantlets and respective mother plant indicate the uniformity of tissue culture banana plantlets. It also indicates that, primer UBC-891 will be helpful in future to identify variation in tissue cultured plantlets of Safed velchi. For detection of BBTV infection, 200 plant samples were used from mother plant no. 15, 16 and 21. It was observed that, only positive control 141 and BBTV infected sample of banana plants showed positive results. While, all the remaining sample test wells showed the negative results. It is also observed that, ISSR-PCR analysis and ELISA Assay is an effective technique for testing fidelity/uniformity and for the detection of BBTV infection of tissue cultured plants, respectively. These two tests have been made mandatory to all the tissue culture laboratories so that genetically pure clones and virus free plantlets will be detected.
