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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

Applied Biochemistry and Biotechnology volume 172, pages 533–548 (2014)Cite this article

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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References

  1. Jingura, R. M., Musademba, D., & Matengaifa, R. (2009). International Journal of Engineering, Science and Technology, 2(7), 115–122.

    Google Scholar 

  2. Kesari, V., Das, A., & Rangan, L. (2010). Biomass and Bioenergy, 34(1), 108–115.

    CAS  Article  Google Scholar 

  3. Kumar, A., & Sharma, S. (2008). An evaluation of multipurpose oil seed crop for industrial uses (Jatropha curcas L.): A review. Industrial Crops and Products, 28, 1–10.

    CAS  Article  Google Scholar 

  4. Kesari, V., Krishnamachari, A., & Rangan, L. (2008). Annals of Applied Biology, 152, 397–404.

    Article  Google Scholar 

  5. Bennett, M. D., & Leitch, I. J. (1995). Annals of Botany, 76, 113–176.

    CAS  Article  Google Scholar 

  6. Bennett, M. D., & Leitch, I. J. (2005). Annals of Botany, 95, 1–6.

    CAS  Article  Google Scholar 

  7. Bennetzen, J. L. (2007). Current Opinion in Plant Biology, 10, 176–181.

    CAS  Article  Google Scholar 

  8. Dolezel, J., Greilhuber, J., & Suda, J. (2007). Nature Protocols, 2, 2233–2244.

    CAS  Article  Google Scholar 

  9. Greilhuber, J., Temsch, E., & Loureiro, J. (2007). Nuclear DNA content measurement in Flow Cytometry with Plant Cells, In: J. Dolezel, J. Greilhuber, J. Suda (Eds.), Analysis of Genes, Chromosomes and Genomes, (pp. 67–101). Wiley-VCH.

  10. Price, H. J., Hodnett, G., & Johnston, J. S. (2000). Annals of Botany, 86(5), 929–934.

    CAS  Article  Google Scholar 

  11. Noirot, M., Barre, P., Duperray, C., Louarn, J., & Hamon, S. (2003). Annals of Botany, 92(2), 259–264.

    CAS  Article  Google Scholar 

  12. Loureiro, J. E., Rodriguez, J., Dolezel, C., & Santos. (2007). Annals of Botany, 100(4), 875–888.

    CAS  Article  Google Scholar 

  13. Kanellos, M (2009). http://www.greentechmedia.com/articles/read/boosting-biofuels-with-extra-chromosomes.

  14. Atchinson, E. (1951). American Journal of Botany, 538

  15. Sarbhoy, R. K. (1977). Cytologia, 42, 415.

    Article  Google Scholar 

  16. Bukhari, Y. M. (1997). Hereditas, 126, 45–51.

    CAS  Article  Google Scholar 

  17. Krishan, A. (1975). Journal of Cell Biology, 66, 188–193.

    CAS  Article  Google Scholar 

  18. Dolezel, J., Bartos, J., Voglmayr, H., & Greilhuber, J. (2003). Cytometry, 51, 127–128.

    CAS  Article  Google Scholar 

  19. Skornickova, J. L., Sida, O., Jarolimova, V., Sabu, M., Fer, T., Travnicek, P., & Suda, J. (2007). Annals of Botany, 100, 505–526.

    Article  Google Scholar 

  20. Ott, L., Longnecker, M., (1998). Boston-MA: PWS-KENT.

  21. Dolezel, J., & Bartos, J. (2005). Annals of Botany, 95, 99–110.

    CAS  Article  Google Scholar 

  22. Loureiro, J. E., Rodriguez, J., Dolezel, C., & Santos. (2006). Annals of Botany, 98(3), 515–527.

    CAS  Article  Google Scholar 

  23. Noirot, M., Barre, P., Louarn, J., Duperray, C., & Hamon, S. (2000). Annals of Botany, 86(2), 309–316.

    CAS  Article  Google Scholar 

  24. Endres, H. (1961). Leder, 12, 294–297.

    CAS  Google Scholar 

  25. O'Brien, I. E., Smith, D. R., Gardner, R. C., & Murray, B. G. (1996). Plant Science, 115, 91–99.

    Article  Google Scholar 

  26. Rayburn, A. L., Birdari, D. P., Bullock, D. G., Nelson, R. L., Gourmet, C., & Wentzel, J. B. (1997). Annals of Botany, 80, 321–325.

    CAS  Article  Google Scholar 

  27. Bharathan, G., Lambert, G., & Galbraith, D. W. (1994). American Journal of Botany, 81, 381–386.

    Article  Google Scholar 

  28. Baranyi, M., & Greilhuber, J. (1995). Plant Systematic and Evolution, 194, 231–239.

    Article  Google Scholar 

  29. Suda, J., & Leitch, I. J. (2010). Cytometry. Part A, 77A, 717–720.

    CAS  Article  Google Scholar 

  30. Geilhuber, J. (1998). Annals of Botany, 82(A), 27–35.

    Article  Google Scholar 

  31. Galbraith, D. W., Harkins, K. R., & Knapp, S. (1991). Plant Physiology, 96, 985–989.

    CAS  Article  Google Scholar 

  32. Bennett, M. D., Leitch, I. J., Price, H. J., & Johnston, J. S. (2003). Annals of Botany, 91, 1–11.

    Article  Google Scholar 

  33. Dolezel, J., Greilhuber, J., Lucretti, S., Meister, A., Lysak, M. A., Nardi, L., & Obermayer, R. (1998). Annals of Botany, 82(A), 17–26.

    CAS  Article  Google Scholar 

  34. Bennett, M. D., & Leitch, I. J. (2010). Annals of Botany, 1–124.

  35. Hartman, T. P. V., Jones, J., Blackhall, N. W., Power, J. W., Cocking, E. C., & Davey, M. R. (2000). Cytogenetics, molecular cytogenetics and genome size in Leucaena (Leguminosae, Mimosoideae), In: H. Guttenberger, Z. Borzan, S.E. Schlarbaum, T.P.V. Hartman (Eds.), Special Issue: Forrest Genetics (pp. 57–70).

  36. Bennett, M. D., Smith, J. B., & Heslop-Harrison, J. S. (1982). Proceedings of the Royal Society of London, B16, 179–199.

    Article  Google Scholar 

  37. Mukherjee, S., & Sharma, A. K. (1993). Cytobios, 75, 33–36.

    Google Scholar 

  38. Price, H. J., Dillon, S. L., Hadnett, G., Rooney, W. L., Ross, L., & Johnston, J. S. (2005). Annals of Botany, 95, 219–227.

    CAS  Article  Google Scholar 

  39. Coulaud, J., Brown, S. C., & Siljak-Yakovlev, S. (1995). Annals of Botany, 75, 95–100.

    CAS  Article  Google Scholar 

  40. Bennett, M. D., & Smith, J. B. (1976). Philosophical Transactions of the Royal Society of London Series B-Biological Sciences, 274, 227–274.

    CAS  Article  Google Scholar 

  41. Parida, A., Raina, S. N., & Narayan, R. K. J. (1990). Genetica, 82, 125–133.

    CAS  Article  Google Scholar 

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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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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.

Electronic supplementary material

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

Keyword

  • Flow cytometry
  • Internal standardization
  • Nuclear DNA content
  • PI intercalation
  • Pseudo-internal standardization
  • Pongamia pinnata