In vitro encapsulation based short term storage and assessment of genetic homogeneity in regenerated Ansellia africana (Leopard orchid) using gene targeted molecular markers

  • Paromik Bhattacharyya
  • Vijay Kumar
  • Johannes Van Staden
original article


An efficient short term storage protocol was developed for Ansellia africana, a vulnerable medicinal orchid of Africa using encapsulated protocorm-like bodies (PLBs) induced from nodal segments of seedlings with highest response recorded on MS medium supplemented with 10 µM TDZ and 5 µM NAA. The gel matrix containing 3% sodium alginate and 100 mM calcium chloride was the best for the production of viable synthetic seeds. In the present study, the effects of meta-topolin (mT) and its derivatives i.e. meta-Topolin riboside (mTR) and meta-methoxy topolin 9-tetrahydropyran-2-yl (memTTHP) were studied on the viability of synthetic seeds, maintained at different temperatures (4, 8 and 25 °C) for varying duration (15, 30, 45, 60, 75 and 90 days). The highest response percentage (88.21%) of encapsulated PLBs was recorded in those cultivated on medium supplemented with 7.5 µM memTTHP. The alginate beads were successfully stored for 75 days at 8 °C with a recorded conversion frequency of 86.21%. Synergistic effect of auxin (IBA or IAA) and the phenolic elicitor phloroglucinol (PG) were tested on root induction and proliferation. The highest rooting frequency was achieved using 15 µM IBA and 30 µM phloroglucinol resulting in successful acclimatization of the plantlets. The clonal fidelity of the regenerated plantlets was also ascertained using inter-retrotransposon amplified polymorphism and start codon targeted markers which revealed a high degree of genetic homogenity amongst the in vitro raised plants. The study also documents the role of mT, mTR and memTTHP on the regeneration of artificial seed-derived plantlets in orchids. The regeneration protocol, would be helpful in reducing stress on fragmented natural habitats of A. africana and can also be extended to conserve other orchids which are encountering threats of extinction.


Aromatic cytokinin Gene targeted markers Phenolic elicitors Short term storage Topolins Zulu pharmacopoeia 





meta-topolin riboside


meta-methoxy topolin 9-tetrahydropyran-2-yl


Start codon targeted polymorphism



PB and VK thank the University of KwaZulu-Natal and the Claude Leon Foundation, South Africa for financial support in the form of postdoctoral fellowships. The authors are grateful to the Microscopy and Microanalysis Unit (MMU), UKZN, Pietermaritzburg for microscopic assistance.

Author contributions

PB and VK designed the experiment. PB performed, recorded and analyzed the data. PB and VK wrote and drafted the manuscript. JVS supervised the overall process, checked, edited and corrected the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

11240_2018_1382_MOESM1_ESM.doc (42 kb)
Supplementary material 1 (DOC 41 KB)


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Authors and Affiliations

  1. 1.Research Centre for Plant Growth and Development, School of Life SciencesUniversity of KwaZulu-Natal PietermaritzburgScottsvilleSouth Africa

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