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The Allahabad Farmer Vol. LXVIII, January - 2013 No. 2Molecular Characterization of Moringa oleifera withRAPD MarkersAradhana Irene Charan , Nishant Kumar Srivastava , Sanjay Singh • , Amit Alexander Charan and Abhishek Sharan ABSTRACTA total of 7 RAPD primers were used to investigate the genetic diversity betweenand within cultivated and non-cultivated provenances of M. oleifera taken fromdifferent regions of Jharkhand, West Bengal and Bihar. Genetic identity anddiversity (Nei's genetic identity and Shannon information index) was measuredby POPGENE software. Cluster analysis was also done using POPGENE softwarewhich produced two clusters. Clustering pattern and genetic identity and geneticdiversity revealed a trend of genetic separation which could be attributed togenetic changes taking place in the individuals as they adapt to the naturalenvironment. Based on the results, selection of elite germplasm and conservationof M. oleifera genetic resources could be achieved.Keywords: Genetic Identity, Genetic Diversity, Clustering Pattern, POPGENE.INTRODUCTIONAn understanding of their genetic diversity is essential as they are the source ofgenetic material that is used to improve elite varieties (Makkar and Becker, 1997).M. oleifera belongs to the family Moringaceae. The family consists of a single genuswith about fourteen species, which follows the distribution pathway from Rajasthan (India)through South West Africa (Verdcourt, 2000). The family consists of trees and shrubsand inhabits the dry habitats of Old World Tropics (Olson, 2000). M. oleifera iscommonly known as drumstick tree. This name is also used for the golden shower tree.India is the largest producer of M. oleifera with an annual production of 1.1 to 1.3million tones of tender fruits from an area 380 km2 among the states. Andhra Pradeshleads in both area and production (156.65 km 2 ). It contains 38 % protein with all aminoStudent, • , Assistant Professor•Institute of Forest Productivity, Ranchi, Jharkhand., Jacob School of Biotechnology and Bioengineering, SHIATS.106
Aradhana Irene Charan, Nishant Kumar Srivastava, Sanjay Singh, Amit Alexander Charanand Abhishek Sharanacids, making it a richer source of proteins compared to other vegetables. The drumsticktree in fact has the highest protein ratio of any plant on earth, with calcium content of theleaves being 297 mg per 100 g of leaves. 100 g of freshly cooked drumstick leavesprovide for 75 % of iron and 50 % of proteins required by a growing child.MATERIALS AND METHODFresh leaves of M. oleifera were collected from Germplasm Bank established atNational Bureau of Plant Genetic Resources (NBPGR), Ranchi, which contains accessionof drumstick plants which were collected from different locations of Jharkhand. Extractionof DNA in M. oleifera was done by (Doyle and Doyle, 1990), with some modificationin the procedure, as M. oleifera contains some phenolic compounds which causestickiness in the sample and make it difficult to isolate the DNA . So, in context withabove an additional step is added, which uses wash buffer containing 0.1% PVP and4% - Mercaptoethanol to wash out the phenolic compounds (Hedrick, 1992).The genomic DNA was then subjected to spectrophotometric quantification at 260nm and 280 nm. 14 eppendorf tubes were taken each having 1 ml volume consisting of980 µl TE buffer and 20 µl DNA sample and another 1 ml eppendorf tube which is usedas blank consisting of 1000 µl TE buffer. Agarose gel electrophoresis was also done tocheck the presence of DNA in the sample. RAPD analysis was done with total sevenprimers which were already available in the laboratory and they were selected on thebasis of their genetic diversity and identity (Table 1). PCR was performed with followingsettings- total 18 µl in which 1.5 µl dNTPs, 0.5 µl Taq Polymerase, 1.0 µl Taq Buffer, 1.5µl Primer, 0.5 µl MgCl 2, 11 µl PCR grade water and 2 µl of DNA sample. Total 45 cycleswere performed. Amplification products were analyzed by electrophoresis using 1.5%Agarose gel in 1X TAE running buffer. The bands were visualized under the GelDocumentation System. Bands were scored, and analyzed with software POPGENEVersion 1.32 (32-bit).RESULTS AND DISCUSSIONIn preliminary screening, seven primers were chosen for the evaluation of geneticdiversity among the M. oleifera populations. Specific DNA band patterns were observedwith all the seven primers used. A total of twenty five bands were scored and all werepolymorphic (Arnold and Emms, 1998). On average each primer produced 3.2 RAPDbands, the largest number was obtained from RPI 8 and RPI 15 and lowest number wasobserved from RPI 5 after running on Agarose gel. UPGMA cluster was prepared usingNei's Genetic distance method UPGMA (Nei, 1973) which is modified from NEIGHBOR107
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Verma Rekha and Parvez RaziaINTRODU
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Page 81 and 82: Neetesh Gupta, S. Saravanan and V.
Page 87 and 88: Payal Sao, J.P.Collis, S. Saravanan
Page 89 and 90: Payal Sao, J.P.Collis, S. Saravanan
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Page 97 and 98: Pradeep Kumar, S.S. Sengarinvolves
Page 99 and 100: Pradeep Kumar, S.S. SengarREFERENCE
Page 101 and 102: Avantika Pandey, Natasha Nageswaran
Page 103 and 104: Avantika Pandey, Natasha Nageswaran
Page 105: Avantika Pandey, Natasha Nageswaran
Page 109 and 110: Aradhana Irene Charan, Nishant Kuma
Page 111 and 112: Aradhana Irene Charan, Nishant Kuma
Page 117 and 118: Apala Gupta, Prema Devi and V.P. Sa
Page 125 and 126: Sudarshan Subedi, Neena Gupta, Vari
Page 133: Sudarshan Subedi, Neena Gupta, Vari
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