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In Vitro Regeneration via Somatic Embryogenesis of Schizozygia Coffeoides Baill (Mpelepele)

Received: 20 March 2017     Accepted: 29 March 2017     Published: 19 April 2017
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Abstract

Schizozygia coffeoides (Mpelepele) is an endangered medicinal shrub with substantial antifungal and antibacterial properties. The sustainable utilization of this shrub is hindered by its limited regeneration potential since there exists no protocol for In vitro regeneration of not only this plant but the entire Apocynaceae family. The aim of this study was to develop an appropriate sterilization and regeneration protocol for In vitro regeneration of S. coffeoides using leaf discs, with the specific aims of determining the best NaOCL concentrations for explant sterilization as well as the appropriate plant growth hormone combinations for development of embryogenic calli, shoot development and rooting. Sterilization was optimized using NaOCl at the concentrations of 5.2mM, 7.8mM, 10.4mM and 13mM at exposure times of 10, 15, 20 and 25 minutes. The different concentrations of NaOCl had a significant effect on the survival of explants to sterilization (P=<0.0001), with the lower concentrations (5.2mM and 7.8mM) showing high levels of explant contamination and high concentration (13mM) leading to explant scotching. Similarly, the time explants were subjected to the sterilization substances was also found to significantly affect their ability to survive the sterilization process (P=0.0027). The best time and NaOCl concentration interaction for the sterilization of leaf explants was found out to be 10.4mM for 20 minutes. This time-NaOCl concentration interaction was found out to significantly produce a higher number of clean surviving explants than all other interactions (P=<0.0001). The best callus formation frequency of 68% was observed in MS media supplemented with 2.0mg/lBAP+0.8mg/lKin+0.4mg/l NAA+0.5mg/lTDZ, compared to a 39% callus formation frequency observed in media supplemented with 1.5mg/lBAP+0.8mg/lKin+0.4mg/lNAA+0.5mg/lTDZ and a 13% callus formation frequency observed in media supplemented with 1mg/lBAP+0.4mg/lKin+0.2mg/lNAA+0.05mg/lTDZ. Although somatic embryos formed in all media types, only media supplemented with 2.0mg/lBAP+0.8mg/lKin+0.4mg/lNAA+0.5mg/lTDZ formed most somatic embryos which survived to maturity and formed shoots, as most calli and embryos forming in other PGR supplementations died with subsequent subcultures. Roots only formed in shoots cultured in media supplemented with 1.0 BAP+0.5 IBA. The successful development of this regeneration protocol is expected to greatly contribute to mass production and conservation of this important shrub.

Published in American Journal of Plant Biology (Volume 2, Issue 2)
DOI 10.11648/j.ajpb.20170202.14
Page(s) 66-72
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2017. Published by Science Publishing Group

Keywords

Sterilization, Plant Growth Regulators, Callus Induction, Somatic Embryo

References
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[3] Magadula JJ, and Erasto P. 2009. Bioactive natural products derived from the East African flora. R Soc Chemestry 26: 1535–1554.
[4] Kariba RM, Houghton PJ, and Yenesew A. 2002. Antimicrobial activities of a new schizozygane indoline alkaloid from Schizozygia coffaeoides and the revised structure of isoschizogaline. J Nat Prod 65: 566–569.
[5] Maurice D, and Henri J. 1971. Contribution à l’inventaire des plantes médicinales de la côte Est de Madagascar. ORSTOm 8: 35–47.
[6] Murashige T, and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant.
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[8] Singh P, Singh A, Shukla AK, Singh L, Pande V, and Nailwal TK. 2009. Somatic embryogenesis and in vitro regeneration of an endangered medicinal plant sarpgandha (rauvolfia serpentina l.). Life Sci J 6: 57–62.
[9] Ndakidemi CF, Mneney E, and Ndakidemi PA. 2013. Development of Sanitation Protocol for Leaf Explants of B. huillensis for in Vitro Culture. Am J Plant Sci 4: 2425–2430.
[10] Çölgeçen H, Koca U, and Toker G. 2011. Influence of different sterilization methods on callus initiation and production of pigmented callus in Arnebia densiflora Ledeb. Tubitak 35: 513–520.
[11] Tiwari V, Singh BD, and Tiwari KN. 1998. Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]. Plant Cell Rep 17: 538–543.
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[14] Hatanaka T, Arakawa O, Yasuda T, Uchida N, and Yamaguchi T. 1991. Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora. Plant Cell Rep 10: 179–182.
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  • APA Style

    Bashiloni Naaty, Cecilia Mweu Mbithe, Aggery Bernard Nyende, Peter Njenga, Joshua Kiilu Muli. (2017). In Vitro Regeneration via Somatic Embryogenesis of Schizozygia Coffeoides Baill (Mpelepele). American Journal of Plant Biology, 2(2), 66-72. https://doi.org/10.11648/j.ajpb.20170202.14

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    Bashiloni Naaty; Cecilia Mweu Mbithe; Aggery Bernard Nyende; Peter Njenga; Joshua Kiilu Muli. In Vitro Regeneration via Somatic Embryogenesis of Schizozygia Coffeoides Baill (Mpelepele). Am. J. Plant Biol. 2017, 2(2), 66-72. doi: 10.11648/j.ajpb.20170202.14

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    AMA Style

    Bashiloni Naaty, Cecilia Mweu Mbithe, Aggery Bernard Nyende, Peter Njenga, Joshua Kiilu Muli. In Vitro Regeneration via Somatic Embryogenesis of Schizozygia Coffeoides Baill (Mpelepele). Am J Plant Biol. 2017;2(2):66-72. doi: 10.11648/j.ajpb.20170202.14

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  • @article{10.11648/j.ajpb.20170202.14,
      author = {Bashiloni Naaty and Cecilia Mweu Mbithe and Aggery Bernard Nyende and Peter Njenga and Joshua Kiilu Muli},
      title = {In Vitro Regeneration via Somatic Embryogenesis of Schizozygia Coffeoides Baill (Mpelepele)},
      journal = {American Journal of Plant Biology},
      volume = {2},
      number = {2},
      pages = {66-72},
      doi = {10.11648/j.ajpb.20170202.14},
      url = {https://doi.org/10.11648/j.ajpb.20170202.14},
      eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajpb.20170202.14},
      abstract = {Schizozygia coffeoides (Mpelepele) is an endangered medicinal shrub with substantial antifungal and antibacterial properties. The sustainable utilization of this shrub is hindered by its limited regeneration potential since there exists no protocol for In vitro regeneration of not only this plant but the entire Apocynaceae family. The aim of this study was to develop an appropriate sterilization and regeneration protocol for In vitro regeneration of S. coffeoides using leaf discs, with the specific aims of determining the best NaOCL concentrations for explant sterilization as well as the appropriate plant growth hormone combinations for development of embryogenic calli, shoot development and rooting. Sterilization was optimized using NaOCl at the concentrations of 5.2mM, 7.8mM, 10.4mM and 13mM at exposure times of 10, 15, 20 and 25 minutes. The different concentrations of NaOCl had a significant effect on the survival of explants to sterilization (P=<0.0001), with the lower concentrations (5.2mM and 7.8mM) showing high levels of explant contamination and high concentration (13mM) leading to explant scotching. Similarly, the time explants were subjected to the sterilization substances was also found to significantly affect their ability to survive the sterilization process (P=0.0027). The best time and NaOCl concentration interaction for the sterilization of leaf explants was found out to be 10.4mM for 20 minutes. This time-NaOCl concentration interaction was found out to significantly produce a higher number of clean surviving explants than all other interactions (P=<0.0001). The best callus formation frequency of 68% was observed in MS media supplemented with 2.0mg/lBAP+0.8mg/lKin+0.4mg/l NAA+0.5mg/lTDZ, compared to a 39% callus formation frequency observed in media supplemented with 1.5mg/lBAP+0.8mg/lKin+0.4mg/lNAA+0.5mg/lTDZ and a 13% callus formation frequency observed in media supplemented with 1mg/lBAP+0.4mg/lKin+0.2mg/lNAA+0.05mg/lTDZ. Although somatic embryos formed in all media types, only media supplemented with 2.0mg/lBAP+0.8mg/lKin+0.4mg/lNAA+0.5mg/lTDZ formed most somatic embryos which survived to maturity and formed shoots, as most calli and embryos forming in other PGR supplementations died with subsequent subcultures. Roots only formed in shoots cultured in media supplemented with 1.0 BAP+0.5 IBA. The successful development of this regeneration protocol is expected to greatly contribute to mass production and conservation of this important shrub.},
     year = {2017}
    }
    

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  • TY  - JOUR
    T1  - In Vitro Regeneration via Somatic Embryogenesis of Schizozygia Coffeoides Baill (Mpelepele)
    AU  - Bashiloni Naaty
    AU  - Cecilia Mweu Mbithe
    AU  - Aggery Bernard Nyende
    AU  - Peter Njenga
    AU  - Joshua Kiilu Muli
    Y1  - 2017/04/19
    PY  - 2017
    N1  - https://doi.org/10.11648/j.ajpb.20170202.14
    DO  - 10.11648/j.ajpb.20170202.14
    T2  - American Journal of Plant Biology
    JF  - American Journal of Plant Biology
    JO  - American Journal of Plant Biology
    SP  - 66
    EP  - 72
    PB  - Science Publishing Group
    SN  - 2578-8337
    UR  - https://doi.org/10.11648/j.ajpb.20170202.14
    AB  - Schizozygia coffeoides (Mpelepele) is an endangered medicinal shrub with substantial antifungal and antibacterial properties. The sustainable utilization of this shrub is hindered by its limited regeneration potential since there exists no protocol for In vitro regeneration of not only this plant but the entire Apocynaceae family. The aim of this study was to develop an appropriate sterilization and regeneration protocol for In vitro regeneration of S. coffeoides using leaf discs, with the specific aims of determining the best NaOCL concentrations for explant sterilization as well as the appropriate plant growth hormone combinations for development of embryogenic calli, shoot development and rooting. Sterilization was optimized using NaOCl at the concentrations of 5.2mM, 7.8mM, 10.4mM and 13mM at exposure times of 10, 15, 20 and 25 minutes. The different concentrations of NaOCl had a significant effect on the survival of explants to sterilization (P=<0.0001), with the lower concentrations (5.2mM and 7.8mM) showing high levels of explant contamination and high concentration (13mM) leading to explant scotching. Similarly, the time explants were subjected to the sterilization substances was also found to significantly affect their ability to survive the sterilization process (P=0.0027). The best time and NaOCl concentration interaction for the sterilization of leaf explants was found out to be 10.4mM for 20 minutes. This time-NaOCl concentration interaction was found out to significantly produce a higher number of clean surviving explants than all other interactions (P=<0.0001). The best callus formation frequency of 68% was observed in MS media supplemented with 2.0mg/lBAP+0.8mg/lKin+0.4mg/l NAA+0.5mg/lTDZ, compared to a 39% callus formation frequency observed in media supplemented with 1.5mg/lBAP+0.8mg/lKin+0.4mg/lNAA+0.5mg/lTDZ and a 13% callus formation frequency observed in media supplemented with 1mg/lBAP+0.4mg/lKin+0.2mg/lNAA+0.05mg/lTDZ. Although somatic embryos formed in all media types, only media supplemented with 2.0mg/lBAP+0.8mg/lKin+0.4mg/lNAA+0.5mg/lTDZ formed most somatic embryos which survived to maturity and formed shoots, as most calli and embryos forming in other PGR supplementations died with subsequent subcultures. Roots only formed in shoots cultured in media supplemented with 1.0 BAP+0.5 IBA. The successful development of this regeneration protocol is expected to greatly contribute to mass production and conservation of this important shrub.
    VL  - 2
    IS  - 2
    ER  - 

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Author Information
  • Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya

  • Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya

  • Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya

  • Department of Botany, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya

  • Department of Botany, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya

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