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ThesisItem Open Access Identification of gene for ADP-Glucose pyrophosphorylase from developing grains of wheat(CCSHAU, 2006) Saha, Abhigyan; Sikka, Virendera K.A step towards isolation of gene(s) has been undertaken in the proposed study. In order to achieve the target of AGPase cDNA gene library preparation, first of all total RNA was isolated from developing grains (14 DAA), of wheat varieties C306 and WH711. Modified RNA extraction protocol was employed, which gave fairly high quality RNA. Good quality mRNA was fractionated from total RNA using poly AT tract magnetic beads mRNA isolation system (Promega). Complementary DNA (cDNA) library was generated from the mRNA of C306 and WH711 using Lambda zap cDNA kit (Stratagene). Plaque lifts were hybridized with rice AGPase gene probe to identify clones containing wheat AGPase gene. However, exact clone could not be isolated. AGPase enzyme assay was carried out in wheat varieties C306, WH711, WH147M, SG70, MLU-2, at 14DAA and 28DAA. The enzyme activity showed an association with rate of starch accumulation and grain size and yield.ThesisItem Open Access Isolation of drought related MYB transcription factor gene from brassica carinata(CCSHAU, 2006) Supriya; Yadav, Neelam R.The present study was conducted to isolate the drought related MYB transcription factor gene from Brassica carinata using RT-PCR approach. Primers were designed using AtMyb2 gene sequence of Arabidopsis thaliana, a well established drought induced Myb gene. No similar sequence was found in Brassica species when gene sequence was used in BLASTN search using NCBI website www.ncbi.nlm.nih.gov. The primers were based on conserved DNA binding domain of Myb gene. Brassica carinata seeds were grown on MS medium for 14 days, seedlings were uprooted and then exposed to air for 1hr/2hrs.dehydration treatment. Total RNA was isolated using Trizol Reagent method. Total RNA yield varied from 2250-2262.5 μg/g fresh wt. and two distinct ribosomal bands (28S and 18S) were observed when total RNA was analysed on formaldehyde and agarose gels. Isolation of mRNA was done using Genelute mRNA miniprep kit and its concentration ranged from 148.8-211.7μg/g fresh wt. i. e. one-tenth of the total RNA. RT-PCR was carried out using Qiagen one step RT-PCR kit with Q-solution and total RNA as well as mRNA as template. All the three sets of primers specific to Myb gene showed amplification. Set III (GhMYB) amplified a product of around 200 bp. A common band of approx. 500 bp was observed with Set I and Set II with mRNA as template. The cDNA was eluted from gel and further amplified. The amplified fragment was transformed using pPCR ScriptTM Amp SK(+) Cloning Vector (Stratagene) and pDrive cloning Vector (Qiagen) in XL-Blue strain of E. coli using blue-white selection. Transformation frequency in the range of 10.00 to 21.05 per cent was observed. The transformed clones were characterized by carrying out plasmid DNA isolation, PCR amplification of plasmid DNA and restriction enzyme digestion of plasmid DNA. The isolated plasmid DNA from transformed (white) colonies showed higher vector size when analyzed on agarose gel suggesting that DNA fragment was inserted into vector. Further, the plasmid DNA restriction pattern confirmed the cloning event.ThesisItem Open Access RAPD analysis of male and female date palm (Phoenix dactylifera L.) plants(CCSHAU, 2006) Puja Singh; Chowdhury, Vijay KumarDate palm (Phoenix dactylifera L.) is a dioecious, monocotyledoneous woody perennial belonging to the family Palmae (Arecaecae) and grown in the arid regions of the world for its nutritious fruit. This study was thus undertaken to assess polymorphism among the male and female date palm genotypes and to identify sex specific RAPD markers in date palm. 31 RAPD primers were used to assess molecular polymorphism in 30 date palm genotypes (10 male and 20 female). A total of 246 amplified products were observed of which 32 were monomorphic and 214 polymorphic. Average polymorphism across the 30 genotypes was 87.19±2.92. A significant co-relation (0.957, p<0.01) was observed between the total number of bands and the number of polymorphic bands amplified by 31 RAPD primers. The number of amplified DNA bands varied between 4 and 19 with an average of 7.94±0.63 bands per primer. Primers were identified which produced unique alleles capable of differentiating between the males of unknown variety and the females of Shamran and/or Khadrawy variety. Analysis of this polymorphism profile generated using NTSYS-PC software, grouped the 30 genotypes into 2 major clusters. Three sub clusters consisted of the males, the females of Shamran variety and the females of Khadrawi variety. M2, K7, K9 and K10 were outgrouped showing them to be genetically more diverse. M10 was placed in the Shamran cluster and was almost 83 similar to S1. Genetic similarity based on ‘Simqual’ sub programme ranged from 0.50 to 0.83 indicating moderate genetic variability among genotypes. The average similarity of males with all the twenty females was found to be 0.6. Thus RAPD markers detected a high level of polymorphism and the fingerprint data generated in this study can be used for the identification of sex-linked markers in date palm.ThesisItem Open Access Assessment of diversity between male and female plants of date palm (Phoenix dactylifera L.) using ISSR markers(CCSHAU, 2006) Pandey, Neelam; Kharb, PushpaDate Palm is an important crop. It is considered as a fresh fruit. It ranks five in the production list of tropical and sub-tropical fruits after citrus, mangoes, bananas and pineapples. As a dried fruit, dates easily top the list over raisins, figs and prunes. It is a dioecious plant. This study was conducted to prepare DNA fingerprint database of 30 genotypes of Date Palm to assess the diversity between male and female plants so that sex of the plant could be determined at the juvenile stage. Out of total 44 ISSR primers used in this investigation, 34 primers amplified while 24 primers showed polymorphism. Using these 24 primers 193 alleles were obtained, out of which 139 bands were polymorphic and 54 bands were monomorphic. Analysis of this polymorphism profile generated using NTSYS-PC package, the 30 date palm genotypes were grouped separately into two clusters. Group I consisted of all male plants and females of Khadrawi. Group II consisted of all females of Shamran variety. The similarity matrix further showed the relatedness among 30 genotypes. A high polymorphism value of 71.08+1.49 among all the genotypes was detected. Also a moderately high polymorphism was observed among the males (71.24+2.98%), Shamran variety females (70.76+2.94%) and Khadrawi variety females (75.61+3.04%). The polymorphic study of Date Palm has revealed that one primer (KJ-12) can be a putative sex linked marker and needs further verification by using more male plants and female plants of Date Palm.ThesisItem Open Access Identification of gene for ADP-Glucose pyrophosphorylase from developing grains of wheat(CCSHAU, 2006) Saha, Abhigyan; Sikka, Virendera K.A step towards isolation of gene(s) has been undertaken in the proposed study. In order to achieve the target of AGPase cDNA gene library preparation, first of all total RNA was isolated from developing grains (14 DAA), of wheat varieties C306 and WH711. Modified RNA extraction protocol was employed, which gave fairly high quality RNA. Good quality mRNA was fractionated from total RNA using poly AT tract magnetic beads mRNA isolation system (Promega). Complementary DNA (cDNA) library was generated from the mRNA of C306 and WH711 using Lambda zap cDNA kit (Stratagene). Plaque lifts were hybridized with rice AGPase gene probe to identify clones containing wheat AGPase gene. However, exact clone could not be isolated. AGPase enzyme assay was carried out in wheat varieties C306, WH711, WH147M, SG70, MLU-2, at 14DAA and 28DAA. The enzyme activity showed an association with rate of starch accumulation and grain size and yield.ThesisItem Open Access Molecular cloning and expression of α-amylase gene of bacillus sp. to E. coli(CCSHAU, 2006) Kaushik, Naveen Kumar; Chaudhary, KamlaAn alpha-amylase producing Bacillus sp. Ns5, was isolated from soil. Growth and amylase production by Bacillus sp. Ns5 was studied under different cultivation conditions. Maximum growth was observed at 30°C. Among defined carbohydrates tested starch and glucose, starch supported good growth and amylase production, with the highest productivity recorded in the presence of starch 1%. The enzyme starts releasing reducing sugar after 2 min of incubation with substrate. The enzyme characterized for different parameters including temperature, pH, Ca++ requirement and its Km. Enzyme was found active under range of temperature 30-70°C with temperature optima at 45° C. pH optima of enzyme found to 5.5 with active under acidic range of pH having Km 0.67 mg/ml. Presence of Ca++ increase activity 2 fold. partial Sau3A fragment of Bacillus Sp. Ns5 DNA cloned in an Escherichia coli YEp FLAG-1 yeast E. coli shuttle vector system was shown to direct the synthesis of a alpha-amylase whose activity could be detected in situ on petri plates using the iodine staining method. A 2kb fragment containing plasmid Kn3, the active gene was shown to express in the periplasmic space and then to excreted out to medium. The restriction map of the plasmid was determined for three restriction enzymes. The enzyme production in E. coli transformant K3 was found seven fold less than parent bacterial strain. The optimum temperature and pH for enzyme activity were found to be 45°C and pH 5.5, respectively. Furthermore enzyme was found to be affected by Ca++ Which increase its activity two fold.ThesisItem Open Access Agrobacterium-mediated transformation of indica rice variety IR-72 with amaranthus seed albumin (AmA1) gene(CCSHAU, 2006) Priyadarshini; Jain, R.K.Agrobacterium-mediated transformation experiments were conducted to transfer the Amaranthus seed albumin (AmA1) gene, driven by CaMV35S promoter in indica rice variety IR-72; Pusa Basmati 1 was used as a control check variety. Calli were initiated from surface sterilized, dehusked mature seeds and immature embryos of two indica rice varieties on MS medium supplemented with 2,4-D (2.5 mg/l), proline (560 mg/l), casein hydrolysate (300 mg/l), maltose (30 mg/l) and gelrite (3 g/l). 15-18 day-old calli were co-cultivated with Agrobacterium strain EHA105 (pSB8) and then transferred on to selection medium containing kanamycin/ paromomycin (50 mg/l) and cefotaxime (250 mg/l) for 2-3 cycles of selection of 15 days each. The frequency of calli showing sustained proliferation (putatively transformed calli) in the selection medium varied between 5.3 to 43.2% depending upon the genotype and explant source. The frequency of putatively transformed calli was higher in case of Pusa Basmati 1 (11.9 and 43.2%) compared to IR-72 (9.0 and 5.3%). These putatively transformed calli were transferred on to MS based shoot regeneration medium. Two shoots each of IR-72 and Pusa Basmati were obtained, but these shoots turned albino later on. More experiments are required to understand the basis of albino plant regeneration and to find out the ways to recover green, fertile indica rice plants containing AmA1 gene.