Skip to main content
SpringerLink
Log in

Direct somatic embryogenesis using leaf explants and short term storage of synseeds in Spathoglottis plicata Blume

  • Original Article
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

Spathoglottis plicata Blume is a vulnerable orchid species in various parts of the world, and the conventional propagation provides limited success to its cultivation and conservation. Therefore, present study deals with the direct induction of somatic embryos (SEs) from the leaf explants of S. plicata. Murashige and Skoog’s (MS) medium fortified with various types and concentrations of plant growth regulators were used to induce somatic embryogenesis and plantlet production. The highest percentage of somatic embryo formation (93.7 ± 0.56%) was achieved on MS medium supplemented with 1.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), whereas, maximum proliferation and increase in fresh weight (FW) of SEs (149.5 ± 0.24 mg/ 50 mg initial FW) was achieved on MS medium fortified with 2.0 mg L−1 6-benzylaminopurine (BAP) within 5 weeks of incubation. The light microscopic observation revealed that SEs emerged directly from the leaf surface. The viable synthetic seeds (SS) from SEs were prepared by encapsulating with gel matrix of 3% (w/v) sodium alginate and 100 mM calcium chloride. The SS were successfully stored for 60 days at − 4 °C with 97.4% germination frequency and shoot proliferation using MS medium with 2.0 mg L−1 BAP and 0.25 mg L−1 indole-3-acetic acid. The synergistic rooting frequency was observed on half-strength MS medium with 1.0 mg L−1 indole-3-butyric acid. The rooted shoots were acclimatized in a greenhouse with a 90% survival rate using soilrite® and vermicompost. This is an efficient short-term storage and regeneration protocol for S. plicata, which could help in reducing pressure on its wild population and could also be extended to the cryopreservation of this orchid species.

Key message

The somatic embryos were induced directly from the leaf explants of Spathoglottis plicata and an efficient short-term storage protocol was developed by encapsulating these somatic embryos. The light microscopic analysis confirmed the formation of somatic embryos. The protocol could be convenient for the commercial cultivation and conservation of S. plicata.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Adhikari S, Bandyopadhyay TK, Ghosha P (2014) Assessment of genetic stability of Cucumis sativus L. regenerated from encapsulated shoot tips. Sci Hortic 170:115–122

    Article  CAS  Google Scholar 

  • Aktar S, Nasiruddin KM, Huq H (2007) In vitro root formation in Dendrobium orchid plantlets with IBA. J Agric Rural Dev 5:48–51

    Google Scholar 

  • Arditti J (1992) Fundamentals of orchid biology. Wiley, New York, p 691

    Google Scholar 

  • Aswathi, Shibu S, Gopinath A, Mohan A (2017) In vitro propagation of Spathoglottis plicata Blume via asymbiotic seed germination. Int J Adv Res 5:431–438

    Google Scholar 

  • Bhattacharyya P, Kumar V, Van Staden J (2018) 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 Org Cult 133:299–310. https://doi.org/10.1007/s11240-018-1382-0

    Article  CAS  Google Scholar 

  • Cardoso JC, Zanello CA, Chen JT (2020) An overview of orchid protocorm-like bodies: mass propagation, biotechnology, molecular aspects, and breeding. Int J Mol Sci 21:985. https://doi.org/10.3390/ijms21030985

    Article  CAS  PubMed Central  Google Scholar 

  • Chung H, Chen J, Chang W (2005) Cytokinins induce direct somatic embryogenesis of Dendrobium chiengmai pink and subsequent plant regeneration. In Vitro Cell Dev Biol-Plant 41:765–769. https://doi.org/10.1079/IVP2005702

    Article  CAS  Google Scholar 

  • Cueva-Agila A, Medina J, Concia L et al (2016) Effects of plant growth regulator, auxin polar transport inhibitors on somatic embryogenesis and CmSERK gene expression in Cattleya maxima (Lindl.). In: Mujib A (ed) Somatic embryogenesis in ornamentals and its applications. Springer, India, pp 255–267. https://doi.org/10.1007/978-81-322-2683-3_16

    Chapter  Google Scholar 

  • Dagar HS, Dagar JC (2003) Plants used in ethnomedicine by the Nicobarese of Islands in Bay of Bengal, India. In: Singh V, Jain AP (eds) Ethnobotany and medicinal Plants of India and Nepal. Scientific Publishers (India), Jodhpur, pp 773–784

    Google Scholar 

  • Deb CR, Pongener A (2011) Asymbiotic seed germination and in vitro seedling development of Cymbidium aloifolium (L.) Sw.: a multipurpose orchid. J Plant Biochem Biotechnol 20:90–95. https://doi.org/10.1007/s13562-010-0031-4

    Article  Google Scholar 

  • Decruse WS, Gangaprasad A, Seeni S, Menon SV (2003) A protocol for shoot multiplication from foliar meristem of Vanda spathulata (L.) Spreng. Indian J Exp Biol 41:924–927

    PubMed  Google Scholar 

  • Dewir YH, El-Mahrouk ME, Murthy HN, Paek KY (2015) Micropropagation of Cattleya: improved in vitro rooting and acclimatization. Hort Environ Biotechnol 56:89–93

    Article  Google 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 354–365

    Google Scholar 

  • Environment Protection and Biodiversity Conservation (EPBC) Act (1999) Spathoglottis plicata conservation advice, pp 1-3

  • Feng JH, Chen JT (2014) A novel in vitro protocol for inducing direct somatic embryogenesis in Phalaenopsis aphrodite without taking explants. Sci World J. https://doi.org/10.1155/2014/263642

    Article  Google Scholar 

  • Gaintait S, Kundu S, Ali NC, Sahu NC (2015) Synthetic seed production of medicinal plants: a review on influence of explants, encapsulation agent and matrix. Acta Physiol Plant 37:98. https://doi.org/10.1007/s11738-015-1847-2

    Article  CAS  Google Scholar 

  • Govaerts R (2012) World checklist of orchidaceae. http://apps.kew.org/wcsp. Accessed on 11 July 2020

  • Haque MS, Ghosh B (2017) Regeneration of cytologically stable plants through dedifferentiation, redifferentiation and artificial seeds in Spathoglottis plicata Blume (Orchidaceae). Hortic Plant J 3:199–208

    Article  Google Scholar 

  • Horstman A, Bemer M, Boutilier K (2017) A transcriptional view on somatic embryogenesis. Regeneration 4:201–216. https://doi.org/10.1002/reg2.91

    Article  PubMed  PubMed Central  Google Scholar 

  • Hossain MM, Dey R (2013) Multiple regeneration pathways in Spathoglottis plicata Blume—a study in vitro. S Afr J Bot 85:56–62

    Article  CAS  Google Scholar 

  • Johansen DA (1940) Plant microtechnique, 1st edn. McGraw Hill Book Co, New York, pp 182–197

    Google Scholar 

  • Kaewubon P, Meesawat U (2016) Histological examination of callogenesis in bisected protocorm culture of pigeon orchid (Dendrobium crumenatum Swartz). Walailak J Schi Tech 13:745–756

    Google Scholar 

  • Khor E, Ng WF, Loh CS (1998) Two-coat systems for encapsulation of Spathoglottis plicata (Orchidaceae) seeds and protocorms. Biotechnol Bioeng 59:635–639

    Article  CAS  Google Scholar 

  • Kulus D (2019) Application of synthetic seeds in propagation, storage, and preservation of Asteraceae plant species. In: Faisal M, Alatar A (eds) Synthetic Seeds. Springer, Cham. https://doi.org/10.1007/978-3-030-24631-0_6

    Chapter  Google Scholar 

  • Kulus D, Tymoszuk A (2020) Induction of callogenesis, organogenesis, and embryogenesis in non-meristematic explants of bleeding heart and evaluation of chemical diversity of key metabolites from callus. Int J Mol Sci 21:5826. https://doi.org/10.3390/ijms21165826

    Article  CAS  PubMed Central  Google Scholar 

  • Mahendran G, Bai VN (2012) Direct somatic embryogenesis and plant regeneration from seed derived protocorms of Cymbidium bicolor Lindl. Sci Hortic 135:40–44

    Article  CAS  Google Scholar 

  • Mahendran G, Bai VN (2016) Direct somatic embryogenesis of Malaxis densiflora (A. Rich.) Kuntze. J Genet Eng Biotechnol 14:77–81

    Article  CAS  Google Scholar 

  • Mahendran G, Muniappan V, Ashwini M, Muthukumar T, Narmatha Bai V (2013) Asymbiotic seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal fungus on seedling development. Acta Physiol Plant 35(3):829–840

    Article  Google Scholar 

  • Manokari M, Latha R, Priyadharshini S, Jogam P, Shekhawat MS (2020) Short-term cold storage of encapsulated somatic embryos and retrieval of plantlets in grey orchid (Vanda tessellata (Roxb.) Hook. ex G.Don). Plant Cell Tiss Org Cult. https://doi.org/10.1007/s11240-020-01899-y

    Article  Google Scholar 

  • Martinez MT, San José MC, Vieitez AM, Cernadas MJ, Ballester A, Corredoira E (2017) Propagation of mature Quercus ilex L. (holm oak) trees by somatic embryogenesis. Plant Cell Tiss Org Cult 131:321–333. https://doi.org/10.1007/s11240-017-1286-4

    Article  CAS  Google Scholar 

  • Mayer JLS, Stancato GC, AppezzatoDa-Gloria B (2010) Direct regeneration of protocorm-like bodies (PLB) from leaf apices of Oncidium flexuosum Sims (Orchidaceae). Plant Cell Tiss Org Cult 103:411–416

    Article  CAS  Google Scholar 

  • Micheli M, Standardi A (2016) From somatic embryo to synthetic seed in Citrus spp. through the encapsulation technology. In: Germana MA, Lambardi M (eds) In vitro embryogenesis in higher plants. Springer, New York, pp 515–522

    Chapter  Google Scholar 

  • Mohanraj R, Ananthan R, Bai VN (2009) Production and storage of synthetic seeds in Coelogyne breviscapa Lindl. Asian J Biotechnol 1:124–128

    Article  CAS  Google Scholar 

  • Mollik AH, Hossan S, Islam T, Jahan R, Rahmatullah M (2009) Medicinal plants used against rheumatoid arthritis by traditional medicinal practitioners of Bangladesh. Planta Med. https://doi.org/10.1055/s-0029-1234537

    Article  Google Scholar 

  • Moradi S, Daylami SD, Arab M, Vahdati K (2017) Direct somatic embryogenesis in Epipactis veratrifolia, a temperate terrestrial orchid. J Hortic Sci Biotechnol 92:88–97

    Article  CAS  Google Scholar 

  • Mose W, Indrianto A, Purwantoro A, Semiarti E (2017) The influence of thidiazuron on direct somatic embryo formation from various types of explant in Phalaenopsis amabilis (L.) Blume Orchid. HAYATI J Biosci 24:201–205

    Article  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:495–497

    Article  Google Scholar 

  • Nawy T, Lukowitz W, Bayer M (2008) Talk global, act local-patterning the Arabidopsis embryo. Curr Opin Plant Biol 11:28–33. https://doi.org/10.1016/j.pbi.2007.10.007

    Article  CAS  PubMed  Google Scholar 

  • Nongdam P, Tikendra L (2014) Establishment of an efficient in vitro regeneration protocol for rapid and mass propagation of Dendrobium chrysotoxum Lindl. using seed culture. Sci World J. https://doi.org/10.1155/2014/740150

    Article  Google Scholar 

  • Novak O, Pencik A, Ljung K (2014) Identification and profiling of auxin and auxin metabolites. In: Zazímalová E, Petrásek J, Benková E (eds) Auxin and its role in plant development. Springer, Vienna, pp 39–60. https://doi.org/10.1007/978-3-7091-1526-8_3

    Chapter  Google Scholar 

  • Otero JT, Flanagan NS (2006) Orchid diversity: beyond deception. Trends Ecol Evol 21:64–65

    Article  Google Scholar 

  • Parthibhan S, Rao MV, Teixeira da Silva JA, Kumar ST (2018) Somatic embryogenesis from stem thin cell layers of Dendrobium aqueum. Biol Plant 62:439–450

    Article  CAS  Google Scholar 

  • Pradhan S, Tiruwa B, Subedee BR, Pant B (2014) In vitro germination and propagation of a threatened medicinal orchid, Cymbidium aloifolium (L.) Sw. through artificial seed. Asian Pac J Trop Biomed 4:971–976

    Article  CAS  Google Scholar 

  • Recart W, Ackerman JD, Cuevas AA (2013) There goes the neighbourhood: apparent competition between invasive and native orchids mediated by a specialist florivorous weevil. Biol Invasions 15(2):283–293. https://doi.org/10.1007/s10530-012-0283-0

    Article  Google Scholar 

  • Rittirat S, Thammasiri K, Te-chato S (2012) Effect of media and sucrose concentrations with or without activated charcoal on the plantlet growth of Phalaenopsis cornucervi (Breda) Blume & Rchb. F. J Agr Tech 8:2077–2087

    CAS  Google Scholar 

  • Riva SS, Islam A, Hoque ME (2016) In vitro regeneration and rapid multiplication of Dendrobium bensoniae, an indigenous ornamental orchid. Agriculturalists 14:24–31

    Article  Google Scholar 

  • Saiprasad GVS, Polisetty R (2003) Propagation of three orchid genera using encapsulated protocorm-like bodies. In Vitro Cell Dev Biol Plant 39:42–48. https://doi.org/10.1079/IVP2002360

    Article  Google Scholar 

  • Sebastinraj J, Muhirkuzhali S (2014) Asymbiotic seed germination and micropropagation of Spathoglottis plicata Blume. I J Adv Pharm Biol Chem 3:495–501

    CAS  Google Scholar 

  • Shen HJ, Chen JT, Chung HH, Chang WC (2018) Plant regeneration via direct somatic embryogenesis from leaf explants of Tolumnia Louise Elmore ‘Elsa.’ Bot Stud. https://doi.org/10.1186/s40529-018-0220-3

    Article  PubMed  PubMed Central  Google Scholar 

  • Sherif AN, Benjamin FJH, Kumar ST, 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 Cult 132:303–316

    Article  CAS  Google Scholar 

  • Sinha P, Hakim LM, Alam FM (2009) In vitro mass clonal propagation of Spathoglottis plicata Blume. Plant Tissue Cult Biotechnol 19:151–160

    Article  Google Scholar 

  • Soonthornkalump S, Yamamoto S, Nakkanong K, Meesawat U (2019) The investigation of condition for cryopreservation of Snow-White Venus’s Slipper orchid protocorm [Paphiopedilum niveum (Rchb.f.) Stein] using V cryo-plate method. Songklanakarin J Plant Sci 6:10–18

    Google Scholar 

  • Stella RY, Priya MT, Begam MFK, Manimekalai V (2015) In vitro seed germination, somatic embryogenesis and protocorm based micropropagation of a terrestrial ornamental orchid—Spathoglottis plicata Blume. Eur J Biotechnol Biosci 3:20–23

    Google Scholar 

  • Stevens PF (2012) Angiosperm phylogeny. http://www.mobot.org/MOBOT/research/APweb. Accessed on 9 June 2020

  • Su YH, Zhang XS (2009) Auxin gradients trigger de novo formation of stem cells during somatic embryogenesis. Plant Signal Beh 4:547–576

    Google Scholar 

  • Teixeira da Silva JA, Winarto B (2016) Somatic embryogenesis in two orchid genera (Cymbidium, Dendrobium). In: Germana M, Lambardi M (eds) In vitro embryogenesis in higher plants. Methods in molecular biology. Humana Press, New York. https://doi.org/10.1007/978-1-4939-3061-6_18

    Chapter  Google Scholar 

  • Teixeira da Silva JA, Hossain MM, Sharma M, Dobranszki J, Cardoso JC, Songjun Z (2017) Acclimatization of in vitro derived Dendrobium. Hortic Plant J 3:110–124

    Article  Google Scholar 

  • Teng W, Nicholson L, Teng M (1997) Micropropagation of Spathoglottis plicata. Plant Cell Rep 16:831–835. https://doi.org/10.1007/s002990050329

    Article  CAS  PubMed  Google Scholar 

  • Teng W, Nicholson L, Yu YY (1997) Clonal propagation of Spathoglottis plicata from young plants. Acta Hortic 447:193–198. https://doi.org/10.17660/ActaHortic.1997.447.37

    Article  CAS  Google Scholar 

  • Teoh ES (2016) Medicinal orchids of Asia. Springer, Singapore, pp 644–646

    Book  Google Scholar 

  • Vondrakova Z, Eliasova K, Fischerova L, Vágner M (2011) The role of auxins in somatic embryogenesis of Abies alba. Open Life Sci 6:587–596. https://doi.org/10.2478/s11535-011-0035-7

    Article  CAS  Google Scholar 

  • Wagner WI, Herbst DR, Sohmer SH (1999) Manual of the Flowering Plants of Hawaii. Revised edition. University of Hawaii Press, Honolulu

    Google Scholar 

  • Wu G-Y, Wei X-L, Wang X, Wei Y (2020) Induction of somatic embryogenesis in different explants from Ormosia henryi Prain. Plant Cell Tiss Org Cult. https://doi.org/10.1007/s11240-020-01822-5

    Article  Google Scholar 

  • Yang X, Zhang X (2010) Regulation of somatic embryogenesis in higher plants. Crit Rev Plant Sci 29:36–57

    Article  CAS  Google Scholar 

  • Zavattieri MA, Frederico AM, Lima M et al (2010) Induction of somatic embryogenesis as an example of stress-related plant reactions. Electron J Biotechnol 13:1–9. https://doi.org/10.2225/vol13-issue1-fulltext-4

    Article  CAS  Google Scholar 

  • Zhao P, Wang W, Sun M (2011) Characterization and expression pattern analysis of DcNAC gene in somatic embryos of Dendrobium candidum Wall ex Lindl. Plant Cell Tiss Org Cult. https://doi.org/10.1007/s11240-011-9968-9

    Article  Google Scholar 

Download references

Acknowledgements

Authors MSS and PS are grateful to the National Medicinal Plants Board, Ministry of AYUSH, Government of India for providing financial support to their laboratory (grant number NMPB/IFD/GIA/NR/PL/2018-19/187).

Author information

Authors and Affiliations

  1. Biotechnology Unit, Kanchi Mamunivar Government Institute for Postgraduate Studies and Research, Puducherry, 605008, India

    M. Manokari, S. Priyadharshini & Mahipal S. Shekhawat

  2. Siddha Clinical Research Unit, Central Council for Research in Siddha, Palayamkottai, Tirunelveli, TN, 627002, India

    M. Manokari

Authors
  1. M. Manokari
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. Priyadharshini
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mahipal S. Shekhawat
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

MSS and MM: Conceptualization, investigation, methodology. PS and MM: Conducted experiments. MM and MSS: Writing the original draft. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to Mahipal S. Shekhawat.

Ethics declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Informed consent

Informed consent was obtained from all individual participants included in the study. Additional informed consent was obtained from all individual participants for whom identifying information is included in this article.

Research involving human and/or animal participants

This research did not involve experiments with human or animal participants.

Additional information

Communicated by So-Young Park.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Manokari, M., Priyadharshini, S. & Shekhawat, M.S. Direct somatic embryogenesis using leaf explants and short term storage of synseeds in Spathoglottis plicata Blume. Plant Cell Tiss Organ Cult 145, 321–331 (2021). https://doi.org/10.1007/s11240-021-02010-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-021-02010-9

Keywords

Search

Navigation