Browsing by Author "Ramesh, Aadi Moolam"

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    Cyto-genetic studies in elite genotype of Pongamia pinnata (L.), a versatile legume
    (2014) Ramesh, Aadi Moolam
    The leguminous tree, Pongamia pinnata (L.) Pierre is one of the major biofuel crop which produces oilseed suitable for biodiesel production. The potentiality of Pongamia, as a sustainable source of feedstock for the biodiesel industry is dependent on an extensive knowledge of the genome structure of the plant. In the present study, earlier characterized (based on vegetative and reproductive characters) candidate plus tree (CPT-NGPP46) of Pongamia germplasm was collected from Sila forest range, North Guwahati, Assam for further experimental study. cDNA library was constructed from early immature seeds (90-DAF) and full length genes that are involved in desaturation of fatty acid biosynthesis were fished out. Full length cDNA clone encoding for steroyl-ACP desaturase (SAD) gene was isolated from the cDNA library characterized based on the sequence similarities in NCBI database. The clone (PpSAD) contains an open reading frame of 1182 bp and shares similarity with SAD from other plants. Characteristic of the deduced protein were predicted and when analyzed using molecular modeling, its 3-Dimensional structure strongly resembled the crystal structure of Ricinus communis (RSAD). Southern blot and expression analysis using quantitative real time PCR indicated that PpSAD is a multiple copy gene having marked distinct expression during different stages of seed development. The PpSAD expression analysis, combined with existing research, suggests that SAD may be involved in the regulation of plant seed growth and development. Further two full length cDNA clones were also isolated from the library coding for fatty acid desaturase enzymes (FAD2-1 and FAD2-2). The deduced amino acid sequence revealed that both the sequences have 83 % homology among them, contain eight histidines essential for membrane-bound desaturases activity and motif in the C-terminal for endoplasmic reticulum retention which are conserved in other oil yielding plant species too. Semi-quantitative RT-PCR and northern blot analysis showed that PpFAD2-1 gene is restricted to the various seed developmental stages and PpFAD2-2 gene is constitutively expressed in both vegetative tissues and developing seeds. Southern hybridization revealed that PpFAD2 is a multi copy gene. Flow cytometry, with propidium iodide (PI) as the DNA stain, was used to estimate the nuclear DNA content of P. pinnata, with respect to Zea mays `CE-777 as standard. The internal and pseudo-internal standardization was followed on account of the inhibitory effect of secondary compounds on PI intercalation. The antioxidants (PVP-40 and -mercaptoethanol) were added to the nuclear isolation buffer for the reduction of inhibitory effect of P. pinnata cytosol. Nuclear DNA content estimation was done for Pongamia leaves from different altitudes (37-117m height from sea level) of Assam. Flow cytometry analysis indicated that the nuclear DNA content of P. pinnata is 2.66 pg with predicted 1C value of 1300 Mbp using Z. mays as standard. Coefficient of variation in flow cytometric analysis was within the limit of 5 % indicating that the results were reliable. The similar results were obtained with little variation in nuclear genome content when analyzed with different tissue types except flowers. The nuclear genome content was also estimated in individuals collected from various parts of Assam and India sh...