Abstract
Aenhenrya rotundifolia is a critically endangered terrestrial jewel orchid. It is monotypic and endemic to evergreen forests of southern western ghats of India. In the present study, identification of this plant species is validated with DNA barcoding using matK and rbcL chloroplast markers. Further, germ-free juvenile axillary bud explants were cultured on Mitra medium supplemented with different kinds of cytokinins like 6-benzyladenine, 6-furfurylaminopurine, N6-(Δ2-isopentyl) adenine, thidiazuron, zeatin and meta-topolin as well as auxins such as α-naphthaleneacetic acid, indole-3-acetic acid and indole-3-butyric acid at different concentrations and combinations for successful proliferation and establishment in vitro. After 12 weeks of culture, axillary bud explants produced an average of 30.12 ± 0.71 shoots per explant, 3.87 ± 0.06 cm shoot length, 1671 ± 2.82 mg fresh mass of proliferated shoots with a proliferation frequency of 100% on Mitra medium supplemented with 6.20 µM meta-topolin and 2.25 µM thidiazuron. No root formation was observed in in vitro proliferated microshoots. However, tiny hair like projections were observed in some elongated shoots on Mitra medium pertaining to 5.37 µM NAA. The tiny hair like structure bearing plantlets were hardened and acclimatized with 100% survival rate in the polytunnel chamber. After 8–10 months of establishment ex vitro, flowering was observed. Additionally, the genetic fidelity of in vitro derived plants was tested with ISSR and SCoT marker profiling. The test results revealed that the plants derived from the protocol has 99% genetic similarity to that of the donor mother plant. This study can be applied in forensic interventions of this species, describes the maintenance of germplasm in vitro and establishment of new viable population in its original habitats by restoring existing sites of this critically endangered jewel orchid.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abubakar MB, Salleh MF, Shamsir Omar MS, Wagiran A (2017) Review: DNA barcoding and chromatography fingerprints for the authentication of botanicals in herbal medicinal products. Evid Based Complement Altern Med. https://doi.org/10.1155/2017/1352948
Amoo SO, Finnie JF, Van Staden J (2011) The role of meta-topolin in alleviating micropropagation problems. Plant Growth Regul 63:197–206. https://doi.org/10.1007/s10725-010-9504-7
Aremu AO, Bairu MW, Szucova L, Finnie JF, Van Staden J (2012) The role of meta-topolins on the photosynthetic pigment profiles and foliar structures of micropropagated “Williams” bananas. J Plant Physiol 169:1530–1541. https://doi.org/10.1016/j.jplph.2012.06.006
Bairu MW, Strike WA, Dolezal K, Van Staden J (2007) Optimizing the micropropagation protocol for the endangered Aloe polyphylla: can meta-topolin and its derivatives serve as replacement for benzyladenine and zeatin? Plant Cell Tiss Org 90:15–23. https://doi.org/10.1007/s11240-007-9233-4
Balachandran KRS, Mohanasundaram S, Ramalingam S (2015) DNA barcoding: a genomic-based tool for authentication of phytomedicinals and its products. Bot Targets Ther 5:77–84. https://doi.org/10.2147/BTAT.S61121
Balakrishnan NP (1966) Nomenclature notes on some flowering plants. J Bombay Nat Hist Soc 63:327–331
Barbi G, Wann SB, Saikia SP (2020) DNA barcodes for delineating Clerodendrum species of North East Inida. Sci Rep 10:13490. https://doi.org/10.1038/s41598-020-70405-3
Bhattacharjee A, Chowdhery HJ (2012) Jewel orchids of India: an overview. International seminar on “multidisciplinary approaches in angiosperm systematics”, pp 554–556
Bhattacharyya P, Kumaria S, Diengdoh R, Tandon P (2014) Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered medicinal orchid. Meta Gene 2:489–504. https://doi.org/10.1016/j.mgene.2014.06.003
Bhattacharyya P, Kumaria S, Tandon P (2016) High frequency regeneration protocol for Dendrobium nobile: a model tissue culture approach propagation of medicinally important orchid species. S Afr J Bot 104:232–243. https://doi.org/10.1016/j.sajb.2015.11.013
Bhattacharyya P, Kumar V, Van Staden J (2017) Assessment of genetic stability amongst micropropagated Ansellia africana, a vulnerable medicinal orchid species of Africa using SCoT markers. S Afr J Bot 108:294–302. https://doi.org/10.1016/j.sajb.2016.11
Blatter E (1928) List of orchids with some new species from the high wavy mountains (Madura District). J Bombay Nat Hist Soc 32:518–523
Chittora M (2018) Assessment of genetic fidelity of long term micropropagated shoot cultures of Achras sapota L. var. ‘Cricket Ball’ as assessed by RAPD and ISSR markers. Indian J Biotechnol 17:492–495
Christenhusz MJ, Byng JW (2016) The number of known plant species in the world and its annual increase. Phytotaxa 261:201–217. https://doi.org/10.11646/phytotaxa.261.3.1
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. https://doi.org/10.1007/s11105-008-0060-5
De Boer HJ, Ghorbani A, Manzanilla V, Raclariu AC, Kreziou A, Ounjai S, Osathanunkul M, Gravendeel B (2017) DNA metabarcoding of orchid-derived products reveals widespread illegal orchid trade. Pro R Soc B Biol Sci. https://doi.org/10.1098/rspb.2017.1182
Dohling S, Kumaria S, Tandon P (2012) Multiple shoot induction from axillary bud cultures of the medicinal orchid. Dendrobium longicornu. AoB Plants. https://doi.org/10.1093/aobpla/pls032
Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Heled J, Kearse M, Moir R, Stones-Haves S, Sturrock S, Thierer T, Wilson A (2010) Geneious v5.1. http://www.geneious.com. Accessed May 2018
Fay MF (2018) Orchid conservation: how can we meet the challenges in the twenty-first century? Bot Stud 59:16–21. https://doi.org/10.1186/s405229-018-0232-2
Gashi B, Abdullai K, Sota V, Kongjika E (2015) Micropropagation and in vitro conservation of the rare and threatened plants Ramonda serbica and Ramonda nathaliae. Physiol Mol Biol Plants 21:123–136. https://doi.org/10.1007/s12298-014-0261-3
Ghorbani A, Gravendeel B, Selliah S, Zarre S, De Boer HJ (2016) DNA barcoding of tuberous Orchidoideae: a resource for identification of orchids used in Salep. Mol Ecol Resour 17:342–352. https://doi.org/10.1111/1755-0998.12615
Ghoush S, Ganga M, Ratna Priyanka R, Manimaran P (2017) Endangered ornamental plant species in India and strategy for their conservation a review. Chem Sci Rev Lett 6:1457–1464
Gopalan R (1994) Aenhenrya: a new genus of Orchidaceae from Southern India. J Bombay Nat Hist Soc 90:270–273
Goyal AK, Pradhan S, Basistha BC, Sen A (2015) Micropropagation and assessment of genetic fidelity of Dendrocalamus strictus (Roxb.) nees using RAPD and ISSR markers. 3 Biotech 5:473–482. https://doi.org/10.1007/s13205-014-0244-7
Grover A, Sharma PC (2016) Development and use of molecular markers: past and present. CRC Rev Biotechnol 36:290–302. https://doi.org/10.3109/07388551.2014.959891
Hinsley A, de Boer HJ, Fay MF, Gale SW, Gardiner LM, Gunasekara RS, Kumar P, Masters S, Metusala D, Veldman S, Wong S, Phelps J (2018) A review of the trade in orchids and its implications for conservation. Bot J Linn Soc 186:435–455. https://doi.org/10.1093/botlinnean/box083
Hussain A, Qarshi IA, Nazir H, Ullah I (2012) Plant tissue culture: current status and opportunities. In: Leva A (ed) Recent advances in plant in vitro culture, InTech Open Access Publisher, pp 1–28. https://doi.org/10.5772/50568
Jalal JS, Jayanthi J (2012) Endemic orchids of Peninsular India: a review. JoTT 4:3415–3425. https://doi.org/10.11609/JoTT.o3091.3415-25
Jiang W, Zhao M, Fu C (2011) Studies on in vitro regeneration competence of pseudobulb cultures in ChangnieniaamoenaChein. Chin Sci Bull 56:2580–2585. https://doi.org/10.1007/s11434-011-4596-7
Kumar P, Gupta VK, Misra AK, Modi DR, Pandey BK (2009) Potential molecular markers in plant biotechnology. Plant Omic J 2:141–162
Laiou A, Mandolini LA, Piredda R, Bellarosa R, Simeone MC (2013) DNA barcoding as a complementary tool for conservation and valorisation of forest resources. ZooKeys 365:197–213. https://doi.org/10.3897/zookeys.365.5670
Li FW, Kuo LY, Rothfels CJ, Ebihara A, Chiou WL, Windham MD, Pryer KM (2011) rbcL and matK earn two thumbs up as the core DNA barcode for ferns. PLoS ONE 6:e26597. https://doi.org/10.1371/journal.pone.0026597
Malik S, Priya A, Babbar SB (2019) Employing barcoding markers to authenticated selected endangered medicinal plants traded in Indian markets. Physiol Mol Biol Plant 25:327–337. https://doi.org/10.1007/s12298-018-0610-8
Mitra GC, Prasad RN, Choudhury RA (1976) Inorganic salts and differentiation of protocorms in seed callus of orchid correlative changes in its free amino acid content. Indian J Exp Biol 4:350–351
Nayar MP, Sastry ARK (2000) Red data book of Indian plants, vol 1–3. Botanical Survey of India, Calcutta
Ngezahayo F, Liu B (2014) Axillary bud proliferation approach for plant biodiversity conservation and restoration. Int J Biodiver. https://doi.org/10.1155/2014/727025
Pant B (2013) Medicinal orchids and their uses: tissue culture a potential alternative for conservation. Afr J Biotechnol 7:448–467. https://doi.org/10.5897/AJPS2013.1031
Parveen I, Singh HK, Malik S, Raghuvanshi S, Babbar SB (2017) Evaluating five different loci (rbcL, rpoB, rpoCl, matK and ITS) for DNA barcoding of Indian orchids. Genome 60:665–671. https://doi.org/10.1139/gen-2016-0215
Phillips GC, Garda M (2019) Plant tissue culture media and practices: an overview. Vitro Cell Dev Biol Plant 55:242–257. https://doi.org/10.1007/s11627-019-09983-5
Raj K, Mahadani P, Kishore R, Loyanganba Meitei A, Sigh DR (2016). DNA barcoding of Indian Orchids. Technical bulletin no. 48, ICAR-National Research Centre for Orchids, Sikkim, India. https://doi.org/10.13140/RG.2.2.15969.61289
Ramos-Castella A, Iglesias-Andreu LG, Bello-Bello J, Lee-Espinosa H (2014) Improved propagation of vanilla (Vanilla planifolia Jacks. Ex Andrews) using a temporary immersion system. Vitro Cell Dev Biol Plant 50:576–581. https://doi.org/10.1007/s11627-014-9602-8
Rohela GK, Jogam P, Bylla P, Reuben C (2019) Indirect regeneration and assessment of genetic fidelity of acclimated plantlets by SCoT, ISSR, and RAPD markers in Rauwolfia tetraphylla L.: an endangered medicinal plant. Biomed Res Int. https://doi.org/10.1155/2019/3698742
Ronquist F, Teslenko M, Van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542. https://doi.org/10.1093/sysbio/sys029
Sarwat M (2012) ISSR: a reliable and cost-effective technique for detection of DNA polymorphism. Plant DNA fingerprinting and barcoding: methods and protocols. Methods Mol Biol 862:103–121. https://doi.org/10.1007/978-1-61779-609-8_9
Sathish Kumar C, Rasmussen FN (1997) The reappearance of Odontochilus rotundifolius blatter and its transfer to Aenhenrya Gopalan (Orchidaceae). Missouri Bot Gard 7:81–84. https://doi.org/10.2307/3392079
Seth S, Panigrahi J (2019) In vitro organogenesis of Abutilon indicum (L.) Sweet from leaf derived callus and assessment of genetic fidelity using ISSR markers. J Hortic Sci Biotechnol 94:70–79. https://doi.org/10.1080/14620316.2018.1447314
Seth S, Rath SC, Rout GR, Panigrahi J (2016) Somatic embryogenesis in Abutilon indicum (L.) sweet and assessment of genetic homogeneity using SCoT markers. Plant Biosyst 151:704–714. https://doi.org/10.1080/11263504.2016.1211193
Sherif NA, Senthil Kumar T, Rao MV (2016) In vitro regeneration by callus culture of Anoectochilus elatus Lindley, an endangered terrestrial jewel orchid. In Vitro Cell Dev Biol Plant 52:72–80. https://doi.org/10.1007/s11627-015-9741-6
Sherif NA, Senthil Kumar T, Rao MV (2017) In vitro propagation and genetic stability assessment of an endangered terrestrial jewel orchid Anoectochilus elatus Lindl. Indian J Exp Biol 55:853–863
Sherif NA, Franklin Benjamin JH, Senthil Kumar T, Rao MV (2018) Somatic embryogenesis, acclimatization and genetic homogeneity assessment of regenerated plantlets of Anoectochilus elatus Lindl., an endangered terrestrial jewel orchid. Plant Cell Tiss Org 132:303–316. https://doi.org/10.1007/s11240-017-1330-4
Souza LM, Silva MMA, Herculano L, Ulisses C, Camara TR (2019) Meta-topolin: an alternative for the prevention of oxidative stress in sugarcane micropropagation. Hoehnea 46:e1072018. https://doi.org/10.1590/2236-8906-107/2018
Tamura K, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA 5: molecular evolutionary genetic analysis using maximum likelihood, evolutionary distance and maximum parsimony methods. Mol Biol Evol 28:2731–2739. https://doi.org/10.1093/molbev/msr121
Thakur J, Dwivedi MD, Sourabh P, Uniyal PL, Pandey AK (2016) Genetic homogeneity revealed using SCoT, ISSR and RAPD markers in micropropagated Pittosporum eriocarpum Royle: an endemic and endangered medicinal plant. PLoS ONE. https://doi.org/10.1371/journal.pone.0159050
Tikendra L, Koijam AS, Nongdam P (2019) Molecular markers based genetic fidelity assessment of micropropagated Dendrobium chrysotoxum Lindl. Meta Gene 20:100562. https://doi.org/10.1016/j.mgene.2019.100562
Udomdee W, Wen PJ, Chin SW, Chen FC (2012) Shoot multiplication of Paphiopedilum orchid through in vitro cutting methods. Afr J Biotechnol 11:14077–14082. https://doi.org/10.5897/AJB12.2047
Vasudevan R, Van Staden J (2011) Cytokinin and explant types influence in vitro plant regeneration of Leopard orchid (Ansellia africana Lindl.). Plant Cell Tiss Org 107:123–129. https://doi.org/10.1007/s11240-011-9964-0
Vu THT, Le TL, Nguyen TK, Tran DD, Tran HD (2017) Review on molecular markers for identification of orchids. Vietnam J Sci Technol Eng 59:62–75. https://doi.org/10.31276/VJSTE.59(2).62
Wattoo JI, Saleem MZ, Shahzad MS, Arif A, Hameed A, Saleem MA (2016) DNA barcoding amplification and sequence analysis of rbcL and matK genome regions in three divergent plant species. Adv Life Sci 4:03–07
Zhang JC, Xie WG, Wang YR, Zhao XH (2015) Potential of start Codon targeted (SCoT) markers to estimate genetic diversity and relationships among Chinese Elymus sibiricus accessions. Molecules 20:5987–6001. https://doi.org/10.3390/molecules20045987
Zhang L, Chen F, Zhang GQ, Zhang YQ, Niu S, Xiong JS, Lin Z, Cheng ZM, Liu ZJ (2016) Origin and mechanism of crassulacean acid metabolism in orchids as implied by comparative transcriptomics and genomics of the carbon fixation pathway. Plant J 86:175–185. https://doi.org/10.1111/tpj.13159
Acknowledgements
The authors are grateful to Mr. R. W. Stewart and Mrs. Tanya Balcar (Late), Vattakanal Conservation Trust, Pambarpuram, Kodaikanal, Dindugal District, Tamil Nadu, India for providing A. rotundifolia germplasm for in vitro studies. The Corresponding author MVR is grateful to UGC, Government of India for providing Emeritus Fellowship (2016–2018). One of the authors TSK acknowledges the DST-SERB, Govt. of India for providing financial assistance through Project (Sanction Number CRG/2019/000367). The author NAS is thankful to DST, Government of India for providing the FIST scheme to the institution.
Author information
Authors and Affiliations
Contributions
All authors are equally contributed to bring out the manuscript in successive manner. Dr. NAS designed and performed the experiments, analysed the data and wrote the manuscript. Dr. TSK and Dr. MVR supervised the work.
Corresponding author
Ethics declarations
Conflict of interest
There is no conflict of interest with the authors.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Sherif, N.A., Senthil Kumar, T. & Rao, M.V. DNA barcoding and genetic fidelity assessment of micropropagated Aenhenrya rotundifolia (Blatt.) C.S. Kumar and F.N. Rasm.: a critically endangered jewel orchid. Physiol Mol Biol Plants 26, 2391–2405 (2020). https://doi.org/10.1007/s12298-020-00917-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12298-020-00917-9