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Analysis of nuclear DNA content, genetic stability, Bacoside A quantity and antioxidant potential of long term in vitro grown germplasm lines of Bacopa monnieri (L.)

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Abstract

The true-to-type nature of shoot cultures of Bacopa monnieri maintained in vitro for 5 years has been confirmed by means of flow cytometry (FCM), inter simple sequence repeat (ISSR), directed amplification of mini satellite region DNA (DAMD) analysis and chemical profiling. The nuclear DNA content of B. monnieri has been estimated as 1.86 pg/2C based on the FCM analysis of nuclei from young leaves. It has also detected no significant variation in the nuclear DNA contents between long term maintained germplasm lines and field grown plants. ISSR and DAMD analysis produced about 93 amplification products and all of them were found to be monomorphic among the field grown and seven germplasm lines. Even though morphological growth parameters varied slightly between the lines, the bioactive compound production capacity has been retained. The mean amount of Bacoside A accumulated by the germplasm lines has been quantified as 25.02 mg g−1 dry weight (DW) using high performance liquid chromatography analysis which is comparable with the quantity of Bacoside A accumulated by the field grown plants (26 mg g−1 DW). The total phenolics (folin–ciocalteau method; expressed in milligram Gallic acid equivalents per gram dry weight of the sample) and the flavonoids content (aluminium chloride method; expressed in milligram Quercetin equivalents per gram dry weight of the sample) of the germplasm lines (53.50–56.62 mg GAE/g DW; 273.51–277.16 mg QRE/g DW) were found to be slightly higher than the field grown plants (46.66 mg/g GAE; 256.65 mg/g QRE) respectively. Further, the antioxidant potential of the methanolic whole plant extracts determined by means of (1,1-diphenyl-2-picrylhydrazyl) DPPH assay revealed that the germplasm lines showed little higher antioxidant potential with IC50 values ranging from 39.51 to 43.71 µg g−1 DW when compared to the IC50 value of field grown (45.68 µg g−1 DW). Hence the protocol devised using axillary bud culturing can be utilized effectively for the long term maintenance of aseptic clones with high genetic and chemical fidelity. The approach tackles the increasing market demand through the continuous availability of high quality plant materials all round the year besides preserving the lines under monitored in vitro conditions.

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Acknowledgments

The first author greatly acknowledges UGC for the financial support in the form of Basic Science Research (BSR) Fellowship. The authors sincerely acknowledge the Computational and Bioinformatics Facility provided by the Alagappa University Bioinformatics Infrastructure Facility (funded by the Department of Biotechnology, Government of India: Grant No.BT/BI/25/001/2006) for performing statistical analysis of the work. We are greatly thankful to Dr. G. S. Selvam, Professor and Head, Department of Biochemistry for providing the FCM instrumentation facility at UGC-Networking Resource Centre in Biological Sciences (NRCBS) Madurai Kamaraj University, Madurai and Dr. A. Mahesh, Project Faculty for his great help in doing the experiment.

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  1. Department of Biotechnology, Science Campus, Alagappa University, Karaikudi, 630 004, Tamil Nadu, India

    Muthiah Joe Virgin Largia, Jayabalan Shilpha, Govindan Pothiraj & Manikandan Ramesh

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  1. Muthiah Joe Virgin Largia
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  2. Jayabalan Shilpha
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  3. Govindan Pothiraj
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  4. Manikandan Ramesh
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Correspondence to Manikandan Ramesh.

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Largia, M.J.V., Shilpha, J., Pothiraj, G. et al. Analysis of nuclear DNA content, genetic stability, Bacoside A quantity and antioxidant potential of long term in vitro grown germplasm lines of Bacopa monnieri (L.). Plant Cell Tiss Organ Cult 120, 399–406 (2015). https://doi.org/10.1007/s11240-014-0602-5

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