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In vitro clonal propagation and evaluation of genetic fidelity using RAPD and ISSR marker in micropropagated plants of Cassia alata L.: a potential medicinal plant

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Abstract

Present study reports successful in vitro clonal propagation of a potential medicinal plant, Cassia alata using mature nodal explants. Murashige and Skoog (MS) medium supplemented with different concentrations (0.5, 2.5, 5.0, 7.5, 10.0 and 12.5 μM) of 6-benzyladenine (BA), kinetin and 2-isopentenyl adenine (2-iP) singly as well as in combination with different auxins, α-naphthalene acetic acid (NAA), Indole-3-butyric acid (IBA) or Indole-3-acetic acid (IAA) (0.1, 0.5 and 1.0 μM) were used. MS medium enriched with 7.5 μM BA and 0.5 μM NAA yielded the highest regeneration frequency (92 %) with maximum multiple shoots (12.3 ± 0.6) and shoot length (4.7 ± 0.1 cm) after 12 weeks of culture. Shoots were rooted best on full MS containing 0.5 μM IBA. Ex vitro rooting of in vitro derived microshoots was also achieved in 150 μM IBA treatment for 20 min followed by transfer to thermocol cups containing sterile Soilrite™. About 85 % plantlets survived acclimatization procedure to the field. The genetic fidelity of in vitro regenerated plants was analyzed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Of the 20 RAPD primers, 18 primers produced clear, reproducible and scorable bands while out of 13 ISSR primers screened, only ten generated well-defined and scorable bands in all the tested plants. A total of 69 and 71 bands were scored with an average of 3.8 and 7.1 bands per primer for RAPD and ISSR primers respectively. All banding profiles from micropropagated plants were monomorphic and similar to of the mother plant, thus confirming the true-to-type nature of the in vitro-raised clones.

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Acknowledgments

Md. Rafique Ahmed is thankful to the University Grants Commission (UGC), Govt. of India, New Delhi for the award of Senior Research Fellowship (MANF/UGC). Research support provided by the Department of Science and Technology (DST) in the form of DST-FIST –II (2011–2016) and UGC in the form of SAP-DRS-II (2016–2021) Programme is duly acknowledged.

Author contribution

MR Ahmed designed and carried out the experiments, data collection and wrote the manuscript. AA Alatar and M Faisal helped in data analysis and M Anis guided the whole experiment and made suggestions to the manuscript.

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Authors and Affiliations

  1. Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh, 202 002, India

    Md. Rafique Ahmed & Mohammad Anis

  2. Department of Botany & Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia

    Mohammad Anis, Abdulrahman A. Alatar & Mohammad Faisal

Authors
  1. Md. Rafique Ahmed
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  2. Mohammad Anis
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  3. Abdulrahman A. Alatar
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  4. Mohammad Faisal
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Correspondence to Mohammad Anis.

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Ahmed, M.R., Anis, M., Alatar, A.A. et al. In vitro clonal propagation and evaluation of genetic fidelity using RAPD and ISSR marker in micropropagated plants of Cassia alata L.: a potential medicinal plant. Agroforest Syst 91, 637–647 (2017). https://doi.org/10.1007/s10457-016-9992-1

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  • DOI: https://doi.org/10.1007/s10457-016-9992-1

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