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An Efficient and Reproducible Method for in vitro Clonal Multiplication of Rauvolfia tetraphylla L. and Evaluation of Genetic Stability using DNA-Based Markers

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Abstract

An efficient protocol is described for the rapid in vitro clonal propagation of an endangered medicinal plant, Rauvolfia tetraphylla L., through high frequency shoot induction from nodal explants collected from young shoots of a field grown plant. Effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) 2iP, or α-naphthalene acetic acid (NAA)], carbohydrates, different medium [Murashige and Skoog (MS), Woody Plant Medium (WPM), Gamborg medium (B5), Linsmier and Skoog medium (LS)], and various pH levels on in vitro morphogenesis were investigated. The highest frequency of shoot regeneration (90 %) and maximum number of shoot (35.4 ± 2.3) per explant were observed on WPM medium supplemented with 7.5 μM BA, 2.5 μM NAA, and 30 g/l sucrose at pH 5.8. Well-developed shoots, 4–5 cm in length, were successfully rooted ex vitro at 90 % by a 30-min pulse treatment with 150 μM IBA prior to their transfer in planting substrates. The survival rate of transplantation reached 90 % when transferred to field condition. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats markers. No variation was observed in DNA fingerprinting patterns among the micropropagated plants, which were similar to that of the donor plant illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. The work contributed to a better in vitro regeneration and clonal mass multiplication of R. tetraphylla and to develop a strategy for the germplasm conservation of this endangered medicinal plant.

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Acknowledgment

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for financial assistance through the research group project No. RGP-VPP-175.

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Authors and Affiliations

  1. Department of Botany & Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh, 11451, KSA

    Mohammad Faisal, Abdulrahman A. Alatar & Ahmad K. Hegazy

  2. Department of Botany, Aligarh Muslim University, Aligarh, 202 002, India

    Naseem Ahmad & Mohammad Anis

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  1. Mohammad Faisal
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  2. Abdulrahman A. Alatar
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  5. Ahmad K. Hegazy
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Correspondence to Mohammad Faisal.

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Faisal, M., Alatar, A.A., Ahmad, N. et al. An Efficient and Reproducible Method for in vitro Clonal Multiplication of Rauvolfia tetraphylla L. and Evaluation of Genetic Stability using DNA-Based Markers. Appl Biochem Biotechnol 168, 1739–1752 (2012). https://doi.org/10.1007/s12010-012-9893-3

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  • DOI: https://doi.org/10.1007/s12010-012-9893-3

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