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ThesisItem Open Access Characterization of DREB1A putative transgenic sugarcane plants using molecular techniques and tolerance assay(Punjab Agricultural University, Ludhiana, 2017) Kaura, Varun; Malhotra, Pawan KumarTransgenic sugarcane plants of variety CoJ88 augmented with DREB1A transgene were screened physiologically to test the susceptibility and tolerance for abiotic stresses (drought, cold and salt). The PCR positive plants (Twenty-two sugarcane lines) grown in pots were subjected to drought stress at grand growth phase and evaluated physiologically (viz. Chlorophyll content, Relative water content (RWC) and Chlorophyll fluorescence) in TC1 stage (Tissue culturally generated clonal stage 1). The values for chlorophyll content exhibits a variation of (1.78-7.72) in transgenic lines as compared to control plants (1.07) whereas chlorophyll fluorescence shows a minimal effect of drought stress in transgenic lines (0.694-0.755) with respect to control plants (0.628). On the basis of aforementioned parameters six (96, 93, 90, 82, 59 and 108) best-performing lines were selected for sowing in next stage. The selected lines were subjected to drought and salt stress (50 mM, 75 mM, 100 mM, 200 mM and 300 mM). After the appearance of stress symptoms (chlorosis, wilting, leaf rolling and necrosis) the physiological and biochemical parameters (Proline and LPO content) were recorded. In TC2 stage (Tissue culturally generated clonal stage 2) transgenic plants demonstrate a nominal effect of salt (1.16-1.61) and drought (1.10-1.49) stress on chlorophyll content values as compared to control (1.03 for salt stress and 0.9 for drought stress) plants. The MDA content varies from (3.57-6.02) under salt and (4.72-7.08) drought stress in transgenic lines with respect to control plants (4.97 for salt stress and 5.93 for drought stress). Three transgenic lines (93, 82 and 96) reveal a higher increase in proline content (4417.5, 2816.8 and 3965.3) under salt stress. On the basis of above-mentioned parameters, three transgenic lines (93, 82 and 96) subjected to drought, salt, and cold stress were selected for molecular studies at RNA level through reverse transcription-PCR by using DREB1A gene specific primers. The quantitative gene expression study was done by using fifteen different stress responsive genes p5CS1, APX1, CAT, GST, HSP, LEA, NAC1, NAC5, NAC6, SOD, NHX1, NFY, NCED, ERA1, and SOS1. In contrast to proline estimation, p5CS1 gene reveals an up-regulation of 65.2 fold and 61.1 fold in transgenic plants subjected to salt and drought stress respectively. Some other genes that showed an up-regulation in transgenic plants subjected to salt and drought stress were CAT, NAC1, NAC5, NHX1 and SOS1.ThesisItem Restricted Integration of antifungal β-1,3-glucanase gene in japonica rice variety Kitaake(Punjab Agricultural University, Ludhiana, 2017) Sandhu, Dilpreet Kaur; Sandhu, Jagdeep SinghAgrobacterium mediated genetic transformation was carried out in japonica rice cultivar ‘Kitaake’ for integration of β-1,3-glucanase gene. The callus obtained from mature seeds on culture media supplemented with 3 mg/L 2,4-D, 0.25 mg/L BAP, 600 mg/L proline, 300 mg/L casein hydrolysate, 40 g/L maltose and 3 g/L clarigel was co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying H1 plasmid with pBI121 backbone harbouring β-1,3-glucanase gene. The Agrobacterium broth at O.D-0.25-0.3 was used for infection and the co-cultivation was performed for two days in the presence of 100 µM acetosyringone. The 38% regeneration was obtained after infection on media containing 4 mg/L BAP, 0.2 mg/L NAA, 30 g/L sucrose, 8 g/L agar, 2 g/L clarigel supplemented with 250 ppm cefotaxime. Out of 1215 calli subjected to infection, 368 calli regenerated and produced 620 plantlets after rooting. When transferred to glasshouse, the surviving 500 plants were analysed by PCR which revealed 16 PCR positive plants. Their T1 generation was grown and the 10 lines were found PCR positive. The reverse-transcriptase PCR analysis showed the expression of gene in 5 plants. RT-PCR positive plants were further analysed by qRT-PCR using actin as the reference gene. The results revealed the 1.5 fold increase in expression of β-1,3-glucanase gene in two lines.ThesisItem Restricted Characterisation of Carrot (Daucus carota L.) genotypes Based on biochemical and molecular markers(Punjab Agricultural University, Ludhiana, 2017) Sandhu, Harsimerdeep Kaur; Sarao, Navraj KaurThe characterization of 45 genotypes of carrot grown during 2015-16 season was done for morphological biochemical as well as molecular markers. Variability was observed for all morphological as well as biochemical traits however juice content, dry weight, carotene content, anthocyanin content and total soluble sugars were having wide variability among various genotypes. Genetic diversity and fingerprint profiles of these 45 Daucus genotypes were also determined using 55 microsatellite markers. Twenty five markers out of total amplified fifty one were polymorphic yielding a total of 85 alleles at 2921 loci, with an average of 1.55 alleles per marker. A unique DNA profile was generated by 11 markers yielding 19 unique alleles thus identified 18 genotypes. Genetic variability was assessed by diversity analysis using Darwin 6.0. All the genotypes were clustered into six major clusters irrespective of their geographic origin. The major differences among genotypes were identified by markers belonging to genome sequenced SSRs and Bac sequenced SSRs. Maximum number of alleles were amplified by Daucus carota L. specific marker DCM-2. Therefore, the biochemical traits as well as the SSR markers are effective tools to identify diversity or closeness among genotypes which may contribute to fasten the breeding programs and be fruitful for successful carrot crop improvement programs.ThesisItem Restricted Phosphorus use efficiency in wheat and identification of the associated genes(Punjab Agricultural University, Ludhiana, 2017) Hari Gowthem G; Chhuneja, ParveenAgriculturally, phosphorus (P) is the second most essential element for the growth and development of the crop; due to its key role in various biochemical reactions in the plant system such as phosphorylation, DNA replication, membrane formation, etc. The population boom demanding a leap in the quantum of production of food grains increases the need for the external application of the nutrients in the form of fertilizers. Phosphatic fertilizers being the costliest among all the chemical fertilizers makes it difficult for a marginal farmer to meet his farm demands. This situation could be managed by breeding crops which would use phosphorus efficiently. A panel of 42 wheat genotypes consisting of 10 bread wheat genotypes, 16 synthetic wheat and 16 Triticum dicoccoides accessions was assembled for assessing phosphorus use efficiency and underlying genes. Thirty of these genotypes were analysed in a pot experiment at two Phosphorus treatments. The panel was evaluated for various agro-morphological traits such as phosphorus concentration in straw and seeds, shoot dry matter, number of grains and yield and Phosphorus deficiency tolerance index (PDTI) of every trait was determined for the panel. The synthetic wheat PBW114-Ae.tauschii acc. pau14200 was found to be an efficient genotype at phosphorus starvation as its relative growth at phosphorus starvation was 68.5%, 66.2%, 92.1% and 151.1% more in case of shoot dry matter, number of tillers, yield and number of grains per plant, respectively. T. dicoccoides were observed to be better at Phosphorus uptake as compared to other genotypes. In rice OsPstol1(Phosphorus starvation tolerance), a gene for phosphorus use efficiency, has been cloned and characterized which is responsible for a three-fold increase in phosphorus-uptake and grain-yield in rice. A similarity search was conducted in the wheat genome sequence (IWGSC) for the OsPstol1 orthologous sequences which led to identification of six homologous sequences, two at chromosome 5AS, one each on 3B, 3AL, 3DL and 6DS. Genes were predicted and primers were designed for in vitro amplification and matching transcripts for these sequences were mined from wheat transcriptome database from a Phosphorus starvation study. Amplifications in the germplasm panel showed the presence of homologous sequences in most of the genotypes. Investigation was proceeded with the first hit contig of the BLAST result ‘5AS_IWGSC_1484262’ for allele mining which resulted in the identification of seven SNPs forming five haplotypes which further translated to four different polypeptide sequences. The synthetic wheat PBW114-Ae.tauschii acc. pau14200 could be a source for the genes responsible for enhanced phosphorus use efficiency in wheat and its relation with the wheat orthologs of OsPstol1 could be deduced by further investigating through expression analysis and the promoter sequence analysis of the ortholog.ThesisItem Open Access Androgenesis response studies in Guava (Psidium guajava L.)(Punjab Agricultural University, Ludhiana, 2017) Bhupinder Singh; Mittal, AmandeepDevelopment of haploids and doubled haploids through in vitro androgenesis greatly accelerates the process of achieving homozygosity in crop plants including fruit trees. In tree species including Citrus, Papaya, Neem, Mulberry, Custard apple and Apple, androgenesis have been attempted successfully to overcome self in-compatibility barriers so as to raise homozygous trees. In current study we attempted haploid production in three genotypes of guava viz. Allahabad safeda, Purple Guava and Punjab Pink. Anthers with microspores at mid to late uninucleate stage on N6 basal media containing 1% PVP showed maximum callus induction of 16.3±0.27% in Allahabad Safeda followed by 10±0.41% in Punjab Pink and 4.6±0.27% in Purple Guava. The stereomicroscopic evaluation of callus indicated the callus emergence from microspores. The callus induction frequency was enhanced from 1.7±0.27% to 27.7±1.18% by cold (4 ºC) and mannitol (8%) pre-treatment for 3-7 days to unopened stage IV flower buds. 77% calli survived on best regeneration media, nourished with liquid form in the mother test tube without mechanical disturbance for 1 month. One callus exhibited secondary structure on regeneration media. Isolated microspores showed increase in size on MS and N6 media containing 2,4-D (2.5 mg/L) and Kinetin (2.5 mg/L) and few anomalies on MS media fortified with BAP (0.2 mg/L) and Kinetin (0.2mg/L). 2/50 ovaries underwent callusing after 4 months of in vitro culturing on 1% PVP containing basal MS media and callus finally underwent necrosis after one week of sub culturing on MS media fortified with BAP (0.2 mg/L) and Kinetin (0.2 mg/L). Haploid nature of callus can further be evaluated by counting the chromosome number in the cells.ThesisItem Open Access Allele mining for heat stress related starch synthase gene in cultivated and wild species of wheat(Punjab Agricultural University, Ludhiana, 2017) Dhawan, Mehakdeep SinghThe present study was carried out to identify allelic variants in soluble starch synthase I (SSI) gene in 20 wheat genotypes including nine wild wheats, one durum wheat and ten hexaploid wheats. Candidate SSI gene located on wheat chromosome 7 (7A, 7B, 7D) was selected. After primer designing, SSI gene was amplified, cloned and sequenced in these 20 wheat genotypes though sequences of three wheat genotypes could not be obtained. After manual curation of raw data of these sequences, contigs were assembled and 9 exons and 8 introns were predicted like the reference SSI gene. There were 286 intronic SNPs and 45 exonic SNPs contributing to 221 transitions and 110 transversions. First exon has 18 SNPs while 3-6 SNPs were detected in rest of the exons. No exonic SNP was detected in wild genotype Triticum dicoccoides acc. pau14801 and four cultivated genotypes Giza, Arbon, C306 and PBW343. Out of 45 exonic SNPs, 12 contributed towards non-synonymous amino acid substitutions and none of these substitutions lies in active sites of SSI protein. Also important catalytic domain of SSI gene glycosyltransferase 5 (GT-5) lies outside predicted eight active sites of protein. GT-5 domain lies from exons 1- 6 and six SNPs corresponding to non-synonymous substitutions falls in this region. These six SNP based alleles from four wild progenitor species of Aegilops tauschii acc. pau14102, Aegilops tauschii acc. pau3747, Aegilops speltoides acc. pau15081 and Triticum dicoccoides acc. pau7107 and two cultivated genotypes Impala and C591 could be candidate SNPs for improved heat stress tolerance.