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Development of Flow Cytometric Protocol for Nuclear DNA Content Estimation and Determination of Chromosome Number in Pongamia pinnata L., a Valuable Biodiesel Plant

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Abstract

The potentiality of Pongamia pinnata L. as a sustainable source of feedstock for the biodiesel industry is dependent on an extensive knowledge of the genome structure of the plant. 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 β-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 P. pinnata leaves from different altitudes (37–117 m 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 1,300 Mb using Z. mays as standard. Coefficient of variation in flow cytometric analysis was within the limit of 5 % indicating that the results were reliable. Somatic chromosome numbers were counted from root–tip cells and was found to be 2n = 22 corresponding to the diploid level (x = 11). A decreasing trend in the nuclear DNA content was observed for the species of different altitudes.

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Acknowledgments

AMR and SB thank the Ministry of Human Resources Development (MHRD), Government of India for fellowship. Thanks to the Forest Department officials of Sila Forest, North Guwahati for kind supply of study material. LR acknowledges Department of Science and Technology (Science and Engineering Research Council) and Department of Biotechnology, Government of India for funding the research.

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Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, India, 781 039

    Aadi Moolam Ramesh, Supriyo Basak, Rimjhim Roy Choudhury & Latha Rangan

Authors
  1. Aadi Moolam Ramesh
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  2. Supriyo Basak
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  3. Rimjhim Roy Choudhury
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  4. Latha Rangan
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Corresponding author

Correspondence to Latha Rangan.

Additional information

Aadi Moolam Ramesh and Supriyo Basak equally contributed to this study.

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Supplementary Fig. 1

Step-by-step gating regime for P. pinnata using Z. mays as an internal standard (JPEG 476 kb)

Supplementary Fig. 2

Appearance of G2 peak of P. pinnata in internal standardization: a P. sativum with P. pinnata; b Z. mays with P. pinnata [1, 2 corresponds to G0/G1 and G2 peak of P. pinnata and 3 correspond to G0/G1 peak of standards used] (JPEG 320 kb)

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Ramesh, A.M., Basak, S., Choudhury, R.R. et al. Development of Flow Cytometric Protocol for Nuclear DNA Content Estimation and Determination of Chromosome Number in Pongamia pinnata L., a Valuable Biodiesel Plant. Appl Biochem Biotechnol 172, 533–548 (2014). https://doi.org/10.1007/s12010-013-0553-z

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  • DOI: https://doi.org/10.1007/s12010-013-0553-z

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