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2013 - 20164
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Subject: Agricultural Biotechnology × End date: 2016 × Start date: 2010 ×
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Now showing 1 - 4 of 4
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    Allele mining for phosphorus starvation tolerance gene (pstoli) in wild species of rice
    (Punjab Agricultural University, Ludhiana, 2016) Thakur, Shiwali; Neelam Kumari
    In present study, 182 accessions of Oryza rufipogon having different countries of origin along with O. sativa cv. PR114, PR121, PR122, PB3 and Vandana (positive control) were screened with flanking, co-dominant and dominant (InDel specific) markers to assess the variability throughout 90 kb insertion region of Kasalath. Previously reported SSRs markers OsPupK-4, OsPupK-05, OsPupK-20, OsPupK-29, K-41, K-42, K-43, K-46, K-48, K-52, and K-59 were applied. Based on analysis, it is inferred that most of the O. rufipogon accessions under study had 90 kb insertion present, whereas O. sativa cv. PR114, PR121, PR122 didn't have amplification with K-46, a diagnostic marker for PSTOL 1. Further, the primers were designed for full length amplification of PSTOL 1 gene (1 kb) and sequencing of 69 representative accessions of O. rufipogon with Vandana were performed. Sequence alignment led to the detection of 74 single nucleotide polymorphism among O. rufipogon accessions when compared to Vandana/ Kasalath. Out of this, 21 turned out to be false positives on manual curation and ultimately fifty three true SNPs were observed in the transcribed region of PSTOL1 gene. Transitions were more common than transversions. There were 39 transitions and 14 transversions observed. The most common SNP was A/G SNP. Based on the nucleotide diversity, a total of 17 haplotypes were formed. Haplotype II forms a major group with 41 accessions of O. rufipogon including Vandana and Kasalath whereas other 16 Haplotype groups had O. rufipogon accessions ranging from 1 to 3. Further, protein sequences were also studied in order to detect if any functional variation is pressent. A total number of 28 conversions for amino acid at different positions with comparison to reference sequence were found. The most common SNP was Lysine/Arginine. Validation of O. rufipogon accessions carrying PSTOL1 gene was done in P-deficient soil along with checks and it was found that O.rufipogon accessions had higher tolerance against P starvation when compared to check Vandna under stress conditions. F1 plants were generated by cross-pollination between cultivars and O. rufipogon accessions in order to transfer the novel alleles of PSTOL1 gene to elite cultivars. Thus, this study found out that allelic variation for locus PSTOL1 is present in Wild germplasm.
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    ThesisItemOpen Access
    Characterisation of transformed sugarcane carrying anti-fungal β-1,3-glucanase gene
    (Punjab Agricultural University, Ludhiana, 2016) Nayyar, Shivani; Sandhu, Jagdeep Singh
    The present study entitled “Characterisation of transformed sugarcane carrying anti-fungal β-1,3-glucanase gene” was carried out to characterize in planta transformed sugarcane plants. The objective of this study was to induce red rot resistance caused by C. falcatum. The in planta transformed T0 putative transgenic plants (10) were propagated through plant to row progenies leading to formation of 295 axillary bud setts, out of which 127 setts sprouted representing the clonal generation 1 (CG1). The PCR analysis on T0 putative transgenic plants of CG1 (127 clones) revealed the amplification of 1764 bp β-1,3-glucanase transgene in six putative transgenic plants. The RT-PCR pointed towards the differential expression of transgene in all the six plants and the relative β-1,3-glucanase transgene expression by qRT-PCR confirmed that three plants had 3.197, 4.280 and 4.438 fold increase in transgene expression as compared to non-transformed plant. Bioassay for red rot on six qRT-PCR positive plants with Cf08 pathotype demonstrated that three plants showed resistance against red rot and with Cf09 pathotype demonstrated that two plants showed moderate resistance against red rot. The CG2 plant analysis through qRT-PCR revealed similar relative transgene expression in the clones suggesting absence of chimerism.
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    ThesisItemOpen Access
    Cloning and expression of delayed bitterness gene encoding limonoid glucosyltransferase from Kinnow mandarin (Citrus reticulata Blanco)
    (Punjab Agricultural University, Ludhiana, 2016) Sumedha; Mohanpuria, Prashant
    Kinnow mandarin (Citrus reticulata Blanco) is the major citrus fruit crop of north western India and it occupies maximum fruit area in Punjab. But citrus juice processing industry has been suffering from delayed bitterness problem which is mainly because of limonoid aglycones such as limonin content in citrus juices. In order to study citrus limonoid metabolism, limonoid glucosyltransferase (LGT) gene which encodes a natural debittering enzyme was isolated from the fruit tissues of Kinnow mandarin. After confirmation and characterization, this full length gene sequence (1533 bp) was submitted on NCBI. BLASTn and BLASTp analysis of this isolated LGT showed 98% nucleotide sequence identity and 94-99% identity with the amino acids of LGT from different Citrus spp. respectively. The transcript expression of LGT was evaluated in different tissues such as young leaf (YL), flavedo (F), albedo (A), sac covering (Sc) and seed (S) of Kinnow mandarin during early (90-120 days after flowering (DAF)), mid (150-210DAF) and late (240-270DAF) fruit developmental stages using semi-quantitative method. Its expression was highest in flavedo among other tissues. The LGT expression level increases with each fruit developmental stage but in F only, which signifies that delayed bitterness level which is inversely related to LGT transcript level, decreases till the late fruit developmental stage. Further, a prokaryotic overexpression construct was generated by cloning LGT gene into pET-28a vector. This pET28a-LGT construct plasmid was transformed into DH5alpha, and BL21 (DE3) Escherichia coli strain for its protein expression study in future.
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    ESTABLISHING EFFICIENT IN VITRO REGENERATION PROTOCOL AND GENETIC TRANSFORMATION OF PIGEONPEA (Cajanus cajan L. Millsp.) THROUGH BIOLISTIC GUN USING cry1Ac GENE
    (Punjab Agricultural University, Ludhiana, 2013) Atul Dev; Ajinder Kaur
    The present investigation entitled “Establishing efficient in vitro regeneration protocol and genetic transformation of pigeonpea (Cajanus cajan L. Millsp.) through biolistic gun using cry1Ac gene” was carried using two varieties of pigeonpea ICPL87 and ICPL 88039. Regeneration from callus and direct organogenesis, both approaches were used to establish a tissue culture baseline to carry out transformation. Different explants viz., leaves, cotyledons, epicotyls, embryonic axes, mature and immature embryos were used for callus induction. None of the calli regenerated into plants on any of the growth hormone combinations and concentrations used. Organogenesis approach worked well with the induction of multiple shoots from cotyledonary nodes. Both the varieties exhibited a high frequency of shoot buds (60-65%) on MS + 5mgL-1 BAP medium. Elongation of the shoot buds was carried out on MS + 30 mgL-1 adenine sulphate medium and an average of 5-6 shoots per explant was obtained. Rooting and hardening were done ex vitro with a success rate of 75-94%, using a rooting mixture ROOTEXTM. For genetic transformation, biolistic approach was used to introduce cry1Ac gene. Mature seeds after 12 days of culturing produced axillary meristems in cotyledonary nodes that were used as target tissue for bombardments. Bombarded explants were kept on shoot induction medium for further induction of shoot buds for 10-12 days that were later transferred to shoot elongation medium. In total, 8 transformation experiments were conducted and a total of 326 axillary meristem explants (169 of ICPL 87 and 157 of ICPL 88039) were bombarded, from which 416 shoots in ICPL 87 and 384 shoots in ICPL 88039 were regenerated. Transformation results were promising in both the varieties with transformation efficiencies of 1.18% in ICPL 87 and 3.18% in ICPL 88039