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Nuclear DNA content of Pongamia pinnata L. and genome size stability of in vitro-regenerated plantlets

Protoplasma volume 251, pages 703–709 (2014)Cite this article

Abstract

Pongamia pinnata L. is a multipurpose versatile legume that is well known as a prospective feedstock biodiesel species. However, to date, there has been little genomic research aimed at the exploitation of the biotechnological potential of this species. Genetic characterization of any plant is a challenging task when there is no information about the genome size and organization of the species. Therefore, the genome size of P. pinnata was estimated by flow cytometry with respect to two standards (Zea mays and Pisum sativum), and compared with that of in vitro-raised plants (nodal segment, in vitro-rooted plantlets and acclimatized in vitro plants) to study the potential effect of somaclonal variation on genome size. This method can be used to support the establishment of true-to-type plants to encourage afforestation programs. Modified propidium iodide/hypotonic citrate buffer was used for isolation of the intact nuclei. The 2C DNA value of this species was estimated to be 2.51 ± 0.01 pg. Statistically, there was no significant difference in the DNA content of the in vitro-grown plants and mother plant at α = 0.05. As a result of the low genome size of P. pinnata, a species that has adapted itself to a wide range of edaphic and ecological condition, we can now proceed for its next generation sequencing and genomic diversity studies.

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Fig. 1

Abbreviations

AVM:

Acclimatized in vitro plants

CPT:

Candidate plus tree

CV:

Coefficient of variation

FCM:

Flow Cytometry

IRP:

In vitro-rooted plants

NGPP:

North Guwahati Pongamia pinnata

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Acknowledgments

RRC and LR thank Department of Science and Technology (DST), Government of India for Fellowship and support for funding the research. AMR and SB thank Ministry of Human Resources Development (MHRD), Government of India for Senior Research Fellowship (SRF). Thanks to Prof. Awtar Krishan, University of Miami, USA and Dr. H. Krishnamurthy, NCBS, India for their technical guidance and kind suggestions relating FACS analysis.

Conflicts of interest

The authors declare that they have no conflicts of interest.

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Affiliations

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

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

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

Correspondence to Latha Rangan.

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Electronic supplementary material

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Supplementary Fig. 1
figure 2

A step-by-step gating regime followed for P. pinnata to resolve our histograms obtained from FACSCalibur analyzer (BD Bioscience, California, USA). (a) Side scatter vs. Forward scatter profile (b) Selecting an interest zone on PI fluorescence width vs area profile to eliminate doublets and other clumps. (c) Selecting an interest zone on SS log vs PI fluorescence profile to eliminate debris and disintegrated nuclei. (d) Resolved histogram showing G0/G1 and G2/M peaks of Pongamia and reference standard peak. (e) Resolved histogram showing the debris removed via the gating regime (red) (JPEG 1217 kb)

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Supplementary Fig. 2
figure 3

Linear regression between the mean fluorescence intensity of test and standard nuclei (linear regression equation and R2 coefficient are also provided). The regression was plotted based on the histogram plot of the MFI during internal standardization. (JPEG 856 kb)

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Supplementary Fig. 3
figure 4

Test for presence of inhibitors. Histograms of relative fluorescence intensity of reference standard nuclei separately processed (a-b: P. pinnata; c: P. sativum d: Z. mays) and co processed (e: with P. sativum; f: with Z. mays. Peak 1 = P. pinnata G0/G1; Peak 2 = Reference standard G0/G1. (JPEG 964 kb)

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Choudhury, R.R., Basak, S., Ramesh, A.M. et al. Nuclear DNA content of Pongamia pinnata L. and genome size stability of in vitro-regenerated plantlets. Protoplasma 251, 703–709 (2014). https://doi.org/10.1007/s00709-013-0545-4

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  • DOI: https://doi.org/10.1007/s00709-013-0545-4

Keywords

  • Biodiesel
  • Flow cytometry
  • Genome size
  • In vitro culture
  • Pongamia pinnata