Abstract
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.
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Abbreviations
- mT:
-
meta-topolin
- mTR:
-
meta-topolin riboside
- memTTHP:
-
meta-methoxy topolin 9-tetrahydropyran-2-yl
- SCoT:
-
Start codon targeted polymorphism
References
Adhikari S, Bandyopadhyay TK, Ghosh P (2014) Assessment of genetic stability of Cucumis sativus L. regenerated from encapsulated shoot tips. Sci Hortic 170:115–122
Amoo SO, Aremu AO, Moyo M, Szüčová L, Doležal K, Van Staden J (2014) Physiological effects of a novel aromatic cytokinin analogue in micropropagated Aloe arborescens and Harpagophytum procumbens. Plant Cell Tissue Organ Cult 116:17–26
Ara H, Jaiswal U, Jaiswal VS (2000) Synthetic seed: prospects and limitations. Curr Sci 78:1438–1444
Aremu AO, Bairu MW, Doležal K, Finnie J, Van Staden J (2012) Topolins: a panacea to plant tissue culture challenges? Plant Cell Tissue Organ Cult 108:1–16
Asghar S, Ahmad T, Ahmad I, Yaseen M (2011) In vitro propagation of orchid (Dendrobium nobile) var. Emma white. Afr J Biotechnol 10:3097–3103
Bhattacharyya P, Van Staden J (2016) Ansellia africana (Leopard orchid): a medicinal orchid species with untapped reserves of important biomolecules: a mini review. S Afr J Bot 106:181–185
Bhattacharyya P, Kumaria S, Tandon P (2016a) High frequency regeneration protocol for Dendrobium nobile: a model tissue culture approach for propagation of medicinally important orchid species. S Afr J Bot 104:232–243
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 4:6–11
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 265–270
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 38:179–185
Capuano G, Piccioni E, Standardi A (1998) Effect of different treatments on the conversion of M. 26 apple rootstock synthetic seeds obtained from encapsulated apical and axillary micropropagated buds. J Hortic Sci Biotechnol 73:289–297
Cassells AC, Curry RF (2001) Oxidative stress and physiological, epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineers. Plant Cell Tissue Organ Cult 64:145–157
Chang C, Chang W (2000) Effect of thidiazuron on bud development of Cymbidium sinense Willd in vitro. Plant Growth Regul 30:171–175
Cheruvathur MK, Abraham J, Mani B, Thomas TD (2010) Adventitious shoot induction from cultured internodal explants of Malaxis acuminata D. Don, a valuable terrestrial medicinal orchid. Plant Cell Tissue Organ Cult 101:163–170
Chinsamy M, Finnie JF, Van Staden J (2011) The ethnobotany of South African medicinal orchids. S Afr J Bot 77:2–9
Chinsamy M, Finnie JF, Van Staden J (2014) Anti-inflammatory, antioxidant, anti-cholinesterase activity and mutagenicity of South African medicinal orchids. S Afr J Bot 91:88–98
Collard BCY, Mackill DJ (2009) Start codon targeted (SCoT) polymorphism: a simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Mol Biol Rep 27:86–93
Danso KE, Ford-Lloyd BV (2003) Encapsulation of nodal cuttings and shoot tips for storage and exchange of cassava germplasm. Plant Cell Rep 21:718–725
de Klerk G-J, van der Krieken W, de Jong JC (1999) Review the formation of adventitious roots: new concepts, new possibilities. In Vitro Cell Dev Biol Plant 35:189–199
Debergh PC, Maene LJ (1981) A scheme for commercial propagation of ornamental plants by tissue culture. Sci Hortic 14:335–345
Devi SP, Kumaria S, Rao SR, Tandon P (2013) In vitro propagation and assessment of clonal fidelity of Nepenthes khasiana Hook. f.: a medicinal insectivorous plant of India. Acta Physiol Plant 35:1–8
Devi SP, Kumaria S, Rao SR, Tandon P (2014) Single primer amplification reaction (SPAR) methods reveal subsequent increase in genetic variations in micropropagated plants of Nepenthes khasiana Hook. f. maintained for three consecutive regenerations. Gene 538:23–29
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 354–365
Doležal K, Popa I, Krystof V, Spíchal L, Fojtíková M, Holub J, Lenobel R, Schmülling T, Strnad M (2006) Preparation and biological activity of 6-benzylaminopurine derivatives in plants and human cancer cells. Bioorg Med Chem 14:875–884
Doležal K, Popa I, Hauserová E, Spíchal L, Chakrabarty K, Novák O, Kryštof V, Voller J, Holub J, Strnad M (2007) Preparation, biological activity and endogenous occurrence of N6-benzyladenosines. Bioorg Med Chem 15:3737–3747
Feyissa T, Welander M, Negash L (2007) Genetic stability, ex vitro rooting and gene expression studies in Hagenia abyssinica. Biol Plant 51:15–21
Gantait S, Sinniah UR (2013) Storability, post-storage conversion and genetic stability assessment of alginate-encapsulated shoot tips of monopodial orchid hybrid Aranda Wan Chark Kuan “Blue” × Vanda coerulea Grifft. ex. Lindl. Plant Biotechnol Rep 7:257–266
Gantait S, Bustam S, Sinniah UR (2012) Alginate-encapsulation, short-term storage and plant regeneration from protocorm-like bodies of Aranda Wan Chark Kuan “Blue” × Vanda coerulea Grifft. ex. Lindl. (Orchidaceae). Plant Growth Regul 68:303–311
Gantait S, Kundu S, Ali N, Sahu NC (2015) Synthetic seed production of medicinal plants: a review on influence of explants, encapsulation agent and matrix. Acta Physiol Plant 37:1–12
Ghosh B, Sen S (1994) Plant regeneration from alginate encapsulated somatic embryos of Asparagus cooperi Baker. Plant Cell Rep 13:381–385
Gorji A, Poczai P, Polgar Z, Taller J (2011) Efficiency of arbitrarily amplified dominant markers (SCoT, ISSR and RAPD) for diagnostic fingerprinting in tetraploid potato. Am J Potato Res 88:226–237
Guo B, Abbasi BH, Zeb A, Xu LL, Wei YH (2011) Thidiazuron: a multi-dimensional plant growth regulator. Afr J Biotechnol 10:8984–9000
Gupta AK, Harish Rai MK, Phulwaria M, Agarwal T, Shekhawat NS (2014) In vitro propagation, encapsulation, and genetic fidelity analysis of Terminalia arjuna: a cardioprotective medicinal tree. Appl Biochem Biotechnol 173:1481–1494
Häggman HM, Ryynänen LA, Aronen TS, Krajnakova J (1998) Cryopreservation of embryogenic cultures of Scots pine. Plant Cell Tissue Organ Cult 54:45–53
Jones MPA, Yi Z, Murch SJ, Saxena PK (2007) Thidiazuron-induced regeneration of Echinacea purpurea L.: micropropagation in solid and liquid culture systems. Plant Cell Rep 26:13–19
Kalendar R, Schulman AH (2006) IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nat Protoc 1:2478–2484
Kilinç FM, Süzerer V, Çiftçi Y, Onay A, Yıldırım H, Uncuoğlu AA, Tilkat E,, Metin OK, Ibrahim K, Akdemir OF (2015) Clonal micropropagation of Pistacia lentiscus L. and assessment of genetic stability using IRAP markers. Plant Growth Regul 75:75–88
Kim HS, Lee K, Kang KA, Lee NH, Hyun JW, Kim HS (2012) Phloroglucinol exerts protective effects against oxidative stress–induced cell damage in SH-SY5Y cells. J Pharmacol Sci 119:186–192
Kumar V, Moyo M, Van Staden J (2016) Enhancing plant regeneration of Lachenalia viridiflora, a critically endangered ornamental geophyte with high floricultural potential. Sci Hortic 211:263–268
Larkin PJ, Scowcroft WR (1981) Somaclonal variation-a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214
Malabadi RB, Mulgund GS, Nataraja K (2004) Efficient regeneration of Vanda coerulea, an endangered orchid using thidiazuron. Plant Cell Tissue Organ Cult 76:289–293
Mallón R, Rodríguez-Oubiña J, González ML (2010) In vitro propagation of the endangered plant Centaurea ultreiae: assessment of genetic stability by cytological studies, flow cytometry and RAPD analysis. Plant Cell Tissue Organ Cult 101:31–39
Mok MC, Mok DWS (1985) The metabolism of [14C]-thidiazuron in callus tissues of Phaseolus lunatus. Physiol Plant 65:427–432
Mok MC, Mok DWS, Armstrong DJ, Shudo K, Isogai Y, Okamoto T (1982) Cytokinin activity of N-phenyl-N-1,2,3-thiadiazol-5-yl-urea (thidiazuron). Phytochem 21:1509–1511
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Palombi M, Damiano C (2002) Comparison between RAPD and SSR molecular markers in detecting genetic variation in kiwifruit (Actinidia deliciosa A. Chev). Plant Cell Rep 20:1061–1066
Papenfus HB, Naidoo D, Pošta M, Finnie JF, Van Staden J (2016) The effects of smoke derivatives on in vitro seed germination and development of the Leopard orchid Ansellia africana. Plant Biol 18:289–294
Peyvandi M, Noormohammadi Z, Banihashemi O, Farahani F, Majd A, Hosseini-Mazinani M, Sheidai M (2009) Molecular analysis of genetic stability in long-term micropropagated shoots of Olea europaea L.(cv. Dezful). Asian J Plant Sci 8:146–152
Phan CT, Hegedus P (1986) Possible metabolic basis for the developmental anomaly observed in in vitro culture, called “vitreous plants”. Plant Cell Tissue Organ Cult 6:83–94
Piao MJ, Ahn MJ, Kang KA, Kim KC, Zheng J, Yao CW, Cha JW, Hyun CL, Kang HK, Lee NH, Hyun JW (2014) Phloroglucinol inhibits ultraviolet B radiation-induced oxidative stress in the mouse skin. Int J Radiat Biol 90:928–935
Piao MJ, Ahn MJ, Kang KA, Kim KC, Cha JW, Lee NH, Hyun JW (2015) Phloroglucinol enhances the repair of UVB radiation-induced DNA damage via promotion of the nucleotide excision repair system in vitro and in vivo. DNA Repair 28:131–138
Poczai P, Varga I, Laos M, Cseh A, Bell N, Valkonen JPT, Hyvönen J (2013) Advances in plant gene-targeted and functional markers: a review. Plant Methods 9:6
Podlešáková K, Zalabák D, Čudejková M, Plíhal O, SzüčováL K, Doležal K, Spíchal L, Strnad M, Galuszka P (2012) Novel cytokinin derivatives do not show negative effects on root growth and proliferation in submicromolar range. PLoS ONE 7:e39293
Prevost A, Wilkinson MJ (1999) A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theor Appl Genet 98:107–112
Redenbaugh K, Slade D, Viss P, Fujii JA (1987) Encapsulation of somatic embryos in synthetic seed coats. Hortic Sci 22:803–809
Rohlf JF (1998) NTSYSpc, Numerical Taxonomy and Multivariate Analysis System Version 2.0 User Guide
Ross S, Grasso R (2010) In vitro propagation of “Guayabo del país”(Acca sellowiana (Berg.) Burret). Fruit Veg Cereal Sci Biotech 4:83–87
Roy J, Naha S, Majumdar M, Banerjee N (2007) Direct and callus-mediated protocorm-like body induction from shoot-tips of Dendrobium chrysotoxum Lindl.(Orchidaceae). Plant Cell Tissue Organ Cult 90:31–39
Roy AR, Sajeev S, Pattanayak A, Deka BC (2012) TDZ induced micropropagation in Cymbidium giganteum Wall. Ex Lindl. and assessment of genetic variation in the regenerated plants. Plant Growth Regul 68:435–445
Saiprasad GVS, Polisetty R (2003) Propagation of three orchid genera using encapsulated protocorm-like bodies. In Vitro Cell Dev Biol-Plant 39:42–48
Salvi ND, George L, Eapen S (2001) Plant regeneration from leaf base callus of turmeric and random amplified polymorphic DNA analysis of regenerated plants. Plant Cell Tissue Organ Cult 66:113–119
Singh F (1991) Encapsulation of Spathoglottis plicata protocorms. Lindleyana 6:61–63
Singh AK, Varshney R, Sharma M, Agarwal SS, Bansal KC (2006) Regeneration of plants from alginate-encapsulated shoot tips of Withania somnifera (L.) Dunal, a medicinally important plant species. J Plant Physiol 163:220–223
Smith JSC, Chin ECL, Shu H, Smith OS, Wall SJ, Senior ML, Mitchell SE, Kresovich S, Ziegle J (1997) An evaluation of the utility of SSR loci as molecular markers in maize (Zea mays L.): comparisons with data from RFLPs and pedigree. Theor Appl Genet 95:163–173
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 136–139
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 17:1938–1947
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 10:15639–15646
Vasudevan R, Van Staden J (2010) In vitro asymbiotic seed germination and seedling growth of Ansellia africana Lindl. Sci Hortic 123:496–504
Werbrouck SPO, Strnad M, Van Onckelen HA, Debergh PC (1996) Meta-topolin, an alternative to benzyladenine in tissue culture? Physiol Plant 98:291–297
Acknowledgements
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.
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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.
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Communicated by Sergio J. Ochatt.
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Bhattacharyya, P., Kumar, V. & Van Staden, J. In vitro encapsulation based short term storage and assessment of genetic homogeneity in regenerated Ansellia africana (Leopard orchid) using gene targeted molecular markers. Plant Cell Tiss Organ Cult 133, 299–310 (2018). https://doi.org/10.1007/s11240-018-1382-0
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DOI: https://doi.org/10.1007/s11240-018-1382-0