Molecular Analysis of Genetic Variability and Relationship among Pearl Millet (Pennisetum glaucum) Genotypes by Using SSR Markers

Pearl millet has a place with sort Pennisetum, and family Poaceae. Pearl millet is the most broadly developed grain crop in Asia and Africa representing close to half of the worldwide millet creation. In India, pearl millet is the fourth most broadly developed food crop after rice, wheat, and maize. We used molecular techniques to investigate the genetic diversity and relatedness of six genotypes viz:ICMV155, Dhanshakti, PC-612, Sampada, HHB-67, and HHB-197.the six genotypes was collected from National agriculture research project Aurangabad. The present study was conducted at the Department of Plant Biotechnology at K. K. Wagh College of Agricultural Biotechnology, Nashik. DNA was isolated by fixing a sample in alcohol without using liquid nitrogen; six genotypes were analyzed through SSR primers to determine the extent of molecular characterization. PCR amplification using 10 SSR primers generated a total of 111 no. of bands were scored corresponding an average of 11 bands per primer with 77 bands showing polymorphism (67%) and 34 bands showing monomorphism (30%). The PIC value ranged from 0.35 to 0.68 with an average of 0.4. Jaccard’s coefficient based on SSR analysis 0.38 to 0.90. The dendrogram wasconstructed using the UPGMA method. It has two main clusters Cluster-1 consisting of C1- HHB 67, V6-ICMV-155, C2- HHB 197 and C4- Sampada. Cluster-2 comprised C3-Dhanshakti and V5-PC 612 as an out group. C2- HHB 197 and C4- Sampada genotypes have the highest similarity coefficient 0.76. Among all the genotypes, C1- HHB 67 and V5- PC 612 was found most diverse as it separated from all other genotypes at a very low similarity coefficient of 0.6. The identified markers can prove useful for identification of diverse germplasm and future DNA fingerprinting studies

Analysis of Genetic Diversity in Maize (Zea mays L.) Variety Using SSR Markers

The study was carried out to analyze the genetic diversity of five maize varieties at K. K. Wagh collage of agricultural Biotechnology, Nashik during May to October 2022 Genomic DNA was isolated from maize varieties by using CTAB method. The PCR amplification obtains with set of five SSR primers. The dendrogram was constructed based UPGMA method from the Jaccard’s similarity coefficient and the varieties subjected to cluster analysis. Among the five SSR primer used only three found polymorphic with total 13 alleles. The PIC value 0.358 to 0.868 of Phi-085, ssr-08 and ssr-06 shows polymorphism 50%, 80% and 83.34% respectively. The cluster analysis indicates the varieties were divided into 3 clades and one Simplicifolicus. Interpreting the results P. madhu is divers among the all varieties. The cluster analysis indicates the P. madhu variety shows the diversity. The genetic diversity of maize will help the breeder to select the parents for different breeding programs

An Efficient Protocol for In vitro Regeneration and Acclimatization of Banana (Musa spp.) cv. Grand Naine

Aim: The study was undertaken with a view to standardize a protocol for in vitro regeneration of Banana cv. Grand naine using shoot tip of sucker as an explant Methodology: In present study, explants sterilized with different sterilizing agents such as Tween-20 (1%), Bavistin (0.5-1%), Streptomycin sulphate (250 mg/L), Ascorbic acid (150 mg/L) + Citric acid (100 mg/L), HgCl2 (0.1%) and 70% ethanol.Sterilized explants  were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth hormones for shoot initiation BAP alone (1.5, 2.0, 4.0, 6.0 mg/L) and  BAP (3.0 mg/L) in combination with  IAA and IBA (2.0 mg/L), elongation BAP (3.0 mg/L) and NAA (1.0, 1.5, 2.0 mg/L) and rooting IAA (1.0, 1.5 mg/L) and IBA (1.0, 1.5 mg/L). Primary and secondaryhardening was done in potting mixture containing autoclaved black soil: vermicompost: cocopeat (1:1:1) and garden black soil, cocopeat and red soil (1:1:1) respectively. Results: In present investigation 1% Bavistin (fungicide) showed maximum respond to prevent fungal contamination. Highest shoot initiation (100%) was observed on a MS medium fortified with BAP (1.5 mg/L). Maximum shoot length (10.7 cm) was recorded on a MS medium supplemented with BAP (3.0 mg/L) + NAA (2.0 mg/L) + Activated charcoal. Maximum root initiation was observed on half strength MS medium supplemented with IAA (1.5 mg/L). In vitro regenerated plantlets hardened on the mixture of autoclaved black soil: vermicompost: cocopeat (1:1:1). After 14 weeks In vitro plantlets transferred in green house for acclimatization where, 80% survival rate was recorded. Conclusion: Regeneration protocol was successfully standardized. Therefore, itcan be used for large scale propagation of healthy, disease and virus free planting material and In vitro propagation helps to meet higher demand of healthy planting material within shorter period

Improvement of Safflower (Carthamus tinctorius L.) for Salinity Tolerance under in vitro Condition

Aim: The primary aim of the present study was to screen the salt tolerant calli and optimization of   in-vitro regeneration protocol from selected screen calli. Methodology: The cotyledonary leaf explants was sterilized by using 1% Bavistin, 0.1% Mercuric chloride and 70% ethanol followed by washing with distilled water. Sterilized explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentration of NaCl (i.e. 0, 50mM, 100mM, 200mM, 300 mM and 400 mM) to check the salinity tolerance ability of PKV pink genotype. In vitro screening of callus was studied by morphological characters like colour and texture of callus, callus growth percentage, relative water contained, cell survivility and proline content from saline stress and unstress calli to confirm the saline tolerance pressure to regenerate the PKV Pink safflower genotype. Results: MS medium supplemented with 150 mM NaCl showed 50% survival of calli, whereas no growth was obtained in high concentration of NaCl. Moreover, biochemical assay like proline estimation was done for its confirmation from different NaCl stress and unstress calli. The proline accumulation found to be highest callus grown on MS media supplemented with 150 mM NaCl as compared to control. Also studied the morphological observation i.e. colour and texture of calli, callus growth percentage, relative water content and cell viability under different NaCl stress to select the saline tolerance pressure to regenerate the tolerance line in PKV Pink. The saline tolerance shoots failed to produce in vitro rooting on the standardized rooting medium. So, the different approach like higher auxin shock and grafting experiment were attempted to overcome the rooting problem. Higher auxin shock experiment failed but grafting approach found satisfactory to overcome the rooting in saline tolerance shoot and for development of saline tolerance line in PKV Pink safflower genotype. Conclusion: The development of abiotic stress tolerance plants found the better understanding of physiological and biochemical changes in plants under in vitro stress conditions.&nbsp

Detection and Comparison of RAPD & SSR Primers in Genetic Diversity of Ocimum sanctum

Aims: To Analysis of genetic variability in Tulsi (Ocimum sanctum) genotypes by using RAPD & SSR Markers. Place and Duration of Study: Department of Plant Biotechnology at K.K.Wagh College of Agricultural Biotechnology, Nashik. Methodology: Ocimum tenuiflorum Linn., commonly known as Tulsi, is an aromatic plant with significant traditional and medicinal value. To assess the genetic diversity and relatedness of six Tulsi genotypes (Krishna, Ram, Lavangi, Pandharpuri, Daisil, and Kapoori), molecular techniques were employed. The genotypes were collected from Nagarjuna Medicinal and Aromatic Plant Park at Dr. P.D.K.V. Akola. DNA isolation was performed using alcohol fixation without liquid nitrogen, and the genotypes were analyzed using RAPD and SSR primers for molecular characterization. Results: Genetic diversity analysis of six Tulsi genotypes (Krishna, Ram, Lavangi, Pandharpuri, Daisil, and Kapoori) was performed using RAPD and SSR markers. Five RAPD primers produced 15 bands, with 11 bands showing polymorphism (73.3%) and 4 bands showing monomorphism (26.7%). The PIC value ranged from 0.28 to 0.49 (average: 0.40). Four SSR primers generated 9 bands, with 8 bands showing polymorphism and 1 band showing monomorphism. The PIC value ranged from 0.24 to 0.57 (average: 0.39). The Jaccard coefficient revealed moderate to high similarity in RAPD (0.40 to 0.73) and SSR (0.44 to 0.88) analyses. The UPGMA dendrogram separated the genotypes into two main clusters. Cluster 1 included Krishna, Lavangi, Ram, Pandharpuri, and Daisil Tulsi, while cluster 2 consisted of Kapoori Tulsi. The SSR dendrogram also formed two clusters, with Krishna, Lavangi, Ram, Daisil, and Kapoori genotypes in cluster 1, and Pandharpuri Tulsi showing dissimilarity and forming cluster 2