In vitro encapsulation based short term storage and assessment of genetic homogeneity in regenerated Ansellia africana (Leopard orchid) using gene targeted molecular markers
- 161 Downloads
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.
KeywordsAromatic cytokinin Gene targeted markers Phenolic elicitors Short term storage Topolins Zulu pharmacopoeia
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.
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.
- Ara H, Jaiswal U, Jaiswal VS (2000) Synthetic seed: prospects and limitations. Curr Sci 78:1438–1444Google Scholar
- Bhattacharyya P, Kumaria S, Job N, Tandon P (2016b) En-masse production of elite clones of Dendrobium crepidatum: a threatened, medicinal orchid used in Traditional Chinese Medicine (TCM). J Appl Res Med Aromat Plants 4:6–11Google Scholar
- Bogaert I, Van Cauter S, Werbrouck SPO, Doležal K (2004) New aromatic cytokinins can make the difference. In: V International symposium on in vitro culture and horticultural breeding 725, Giza, pp 265–270Google Scholar
- Bose B, Kumaria S, Choudhury H, Tandon P (2016) Assessment of genetic homogeneity and analysis of phytomedicinal potential in micropropagated plants of Nardostachys jatamansi, a critically endangered, medicinal plant of alpine Himalayas. Plant Cell Tissue Organ Cult 38:179–185Google Scholar
- Dohling S, Das MC, Kumaria S, Tandon P (2007) Conservation of splendid orchids of North-East India. Biodiversity and its significance. IK Int Publishers, New Delhi, pp 354–365Google Scholar
- Redenbaugh K, Slade D, Viss P, Fujii JA (1987) Encapsulation of somatic embryos in synthetic seed coats. Hortic Sci 22:803–809Google Scholar
- Rohlf JF (1998) NTSYSpc, Numerical Taxonomy and Multivariate Analysis System Version 2.0 User GuideGoogle Scholar
- Ross S, Grasso R (2010) In vitro propagation of “Guayabo del país”(Acca sellowiana (Berg.) Burret). Fruit Veg Cereal Sci Biotech 4:83–87Google Scholar
- Singh F (1991) Encapsulation of Spathoglottis plicata protocorms. Lindleyana 6:61–63Google Scholar
- Stover RH (1986) Somaclonal variation in Grande Naine and Saba bananas in the nursery and field. In: International workshop on banana and plantain breeding strategies. pp 136–139Google Scholar
- Szüčová L, Spíchal L, Doležal K, Zatloukal M, Greplová J, Galuszka P, Kryštof V, Voller J, Popa I, Massino FJ, Jørgensen JE, Strnad M (2009) Synthesis, characterization and biological activity of ring-substituted 6-benzylamino-9-tetrahydropyran-2-yl and 9-tetrahydrofuran-2-ylpurine derivatives. Bioorg Med Chem 17:1938–1947CrossRefPubMedGoogle Scholar
- Tao J, Yu L, Kong F, Zhao D (2011) Effect of plant growth regulators on in vitro propagation of Cymbidium faberi Rolfe. Afr J Biotechnol 10:15639–15646Google Scholar