Skip to main content
SpringerLink
Log in

Dendrobium protoplast co-culture promotes phytochemical assemblage in vitro

  • Original Article
  • Published:
Protoplasma Aims and scope Submit manuscript

Abstract

The present study is intended to analyze the occurrence of potent, low produce, naturally occurring stilbenes in protoplasts of wild species and hybrids of Dendrobium. The wild species selected for the study was Dendrobium ovatum, endemic to Western Ghats of India. Protoplasts were isolated from leaves and tepal tissues of all the species and were cultured purely to generate homofusants and cross-cultured to raise heterofusants. Phytochemical composition of protoplast culture with atypical and pure microcolonies was performed using mass spectrometry. Enzyme cocktail of 4% pectinase together with 2% cellulase displayed the highest competence for protoplast isolations. Maximum protoplast density of 30.11 × 104/ml was obtained from D. ovatum leaves in 2 h. Subcellular features such as the presence of partially formed cell wall, the position of the nucleus, chloroplast density, colony existence, and integrity of the plasma membrane were analyzed. Among the pure and cross-cultured protoplasts, the number of heterofusants and homofusants formed were enumerated. The spectral feature extraction of the mass spectrometry indicated the presence of five phenolic marker compounds, viz., tristin, confusarin, gigantol, moscatilin, and resveratrol, some of them in pure and others in assorted protoplast cultures raised from Dendrobium leaves and tepals. The study demonstrated that protoplast fusion technique enabled phytochemical assemblage in vitro as stilbenes tend to get restricted either in a tissue or species specific manner. This is the first report showing the presence of resveratrol, moscatilin, tristin, gigantol, and confusarin in wild and hybrid species from cultured Dendrobium protoplasts in vitro.

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
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

6-BAP:

6-Benzylaminopurine

FACS:

Fluorescence-activated cell sorter

G :

Centrifugation speed in gravitational force

Rpm:

Revolutions per minute

References

  • Belarmino MM, Abe T, Sasahara T (1996) Asymmetric protoplast fusion between sweet potato and its relatives, and plant regeneration. Plant Cell Tissue Organ Cult 46:195–202. doi:10.1007/BF02307095

    Article  CAS  Google Scholar 

  • Chen CC, Wu LG, Ko FN, Teng CM (1994) Antiplatelet aggregation principles of Dendrobium loddigesii. J Nat Prod 57:1271–1274. doi:10.1021/np50111a014

    Article  CAS  PubMed  Google Scholar 

  • Chen TH, Pan LS, Guh JH, Liao CH, Huang DY, Chen CC, Teng CM (2008) Moscatilin induces apoptosis in human colorectal cancer cells: a crucial role of c-Jun NH2-terminal protein kinase activation caused by tubulin depolymerization and DNA damage. Clin Cancer Res 14:4250–4258. doi:10.1158/1078-0432.CCR-07-4578

    Article  CAS  PubMed  Google Scholar 

  • Committee of Chinese Pharmacopoeia (2005) Pharmacopoeia of People’s Republic of China, vol 1. Chemical Industry Press, Beijing, p. 62

    Google Scholar 

  • Fakhrai H, Haq H, Evans PK (1988) Enucleation of protoplasts derived from suspension cultures of winged bean from a crown gall cell line of Parthenocissus tricuspidata. Biol Plantarum 30:401. doi:10.1007/BF02890507

    Article  Google Scholar 

  • Fan CQ, Wang W, Wang YP, Qin GW, Zhao WM (2001) Chemical constituents from Dendrobium densiflorum. Phytochemistry 57:1255–1258. doi:10.1016/s0031-9422(01)00168-6

    Article  CAS  PubMed  Google Scholar 

  • Gong YQ, Fan Y, Wu DZ, Yang H, Hu ZB, Wang ZT (2004) In vivo and in vitro evaluation of erianin, a novel anti-angiogenic agent. Eur J Cancer 40:554–565. doi:10.1016/j.ejca.2004.01.041

    Article  Google Scholar 

  • Hellwig S, Drossard J, Twyman RM, Fischer R (2004) Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 22:1415–1422. doi:10.1038/nbt1027

    Article  CAS  PubMed  Google Scholar 

  • Jarl CI, Rietveld EM, de Haas JM (1999) Transfer of fungal tolerance through interspecific somatic hybridization between Solanum melongena and S. torvum. Plant Cell Rep 18:791–796. doi:10.1007/s.002990050663

    Article  CAS  Google Scholar 

  • Jiang F, Zhu J, Liu HL (2013) Protoplasts: a useful research system for plant cell biology, especially dedifferentiation. Protoplasma 250:1231–1238. doi:10.1007/s00709-013-0513-z

    Article  CAS  PubMed  Google Scholar 

  • Khentry Y, Paradornuvat A, Tantiwiwat S, Phansiri S, Thaveechai N (2006) Protoplast isolation and culture of Dendrobium Sonia “Bom 17”. Kasetsart J (Nat Sci) 40:361–369

    Google Scholar 

  • Kirimura K, Sato T, Nakanishi N, Terada M (1997) Breeding of starch-utilizing and itaconic-acid-producing koji molds by interspecific protoplast fusion between Aspergillus terreus and Aspergillus usamii. Appl Microbiol Biotechnol 47:127–131. doi:10.1007/s002530050900

    Article  CAS  Google Scholar 

  • Kiyohara H, Watanabe T, Imai J, Takizawa N, Hatta T, Nagao K, Yamamoto A (1990) Intergeneric hybridization between Monascus anka and Aspergillus oryzae by protoplast fusion. Appl Microbiol Biotechnol 33:671–676. doi:10.1007/BF00604935

    Article  CAS  Google Scholar 

  • Kunasakdakul K, Smitamana P (2003) Dendrobium Pratum Red Protoplast. Thai J Agric Sci 36:1–8

    Google Scholar 

  • Lee YH, Park JD, Baek NI, Kim SI, Ahn BZ (1995) In vitro and in vivo antitumoral phenanthrenes from the aerial parts of Dendrobium nobile. Planta Med 61:178–180. doi:10.1055/s2006958043

    Article  CAS  PubMed  Google Scholar 

  • Lienard D, Sourrouille C, Gomord V, Faye L (2007) Pharming and transgenic plants. Biotechnol Annu Rev 13:115–147

    Article  CAS  PubMed  Google Scholar 

  • Lin TH, Chang SJ, Chen CC, Wang JP, Tsao LT (2001) Two phenanthraquinones from Dendrobium moniliform. J Nat Prod 64:1084–1086. doi:10.1021/np010016i

    Article  CAS  PubMed  Google Scholar 

  • Liu F, Ryschka U, Marthe F, Klocke E, Schumann G, Zhao H (2007) Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis. Protoplasma 231:89–97. doi:10.1007/s00709-006-0228-5

    Article  PubMed  Google Scholar 

  • Majumdar PL, Sen RC (1987) Moscatilin, a bibenzyl derivative from the orchid Dendrobium moscatum. Phytochemistry 26:2121–2124. doi:10.1016/s0031-9422(00)81777-x

    Article  Google Scholar 

  • Majumdar PL, Guha S, Sen S (1999) Bibenzyl derivatives from the orchid Dendrobium amoenum. Phytochemistry 52:1365–1369. doi:10.1016/S0031-9422(99)00370-2

    Article  Google Scholar 

  • Masumura T, Morita S, Miki Y, Kurita A, Morita S, Shirono H, Koga J, Tanaka K (2006) Production of biologically active human interferon-a in transgenic rice. Plant Biotechnol 23:91–97

    Article  CAS  Google Scholar 

  • Melchers G, Mohri Y, Watanabe K, Wakabayashi S, Harada K (1992) One-step generation of cytoplasmic male sterility by fusion of mitochondrial-inactivated tomato protoplasts with nuclear-inactivated Solanum protoplasts. Proc Natl Acad Sci 89:6832–6836

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Miyazawa M, Shimamura H, Nakamura S, Kameoka H (1997) Antimutagenic activity of gigantol from, Dendrobium nobile. J Agric Food Chem 45:2849–2853. doi:10.1021/jf9603902

    Article  CAS  Google Scholar 

  • Miyazawa M, Shimamura H, Nakamura S, Kameoka H (1999) Moscatilin from Dendrobium nobile, a naturally occurring bibenzyl compound with potential antimutagenic activity. J Agric Food Chem 47:2163–2167. doi:10.1021/jf970930a

    Article  CAS  PubMed  Google Scholar 

  • Moloney MM (2000) In seed technology and its biological basis; Black, M., Bewley, J.D., Eds., CRC Press, Boca Raton, Florida. 226–253

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x

    Article  CAS  Google Scholar 

  • Pati PK, Sharma M, Ahuja PS (2008) Rose protoplast isolation and culture and heterokaryons selection by immobilization in extra thin alginate film. Protoplasma 232:165–171. doi:10.1007/s00709-008-0297-8

    Article  Google Scholar 

  • Pertoft H (2000) Fractionation of cells and subcellular particles with Percoll. J Biochem Biophys Methods 44:1–30. doi:10.1016/S0165-022x(00)00066-x

    Article  CAS  PubMed  Google Scholar 

  • Schroder J, Schroder G (1990) Stilbene and chalcone synthases: related enzymes with key functions in plant-specific pathways. Z Naturforsch 45c:1–8

    Google Scholar 

  • Shetty V, Thomas A, Pujari I, Babu VS (2015) Asymbiotic hypergeneration of Protocorm like bodies- an efficient and simple micropropagation strategy to conserve the therapeutic ornamental—Dendrobium ovatum. Int J Recent Sci Res 6:8009–8015

    Google Scholar 

  • Shirono H, Morita S, Miki Y, Kurita A, Morita S, Koga J, Tanaka K, Masumura T (2006) Highly efficient production of human interferon-a transgenic culture rice cells. Plant Biotechnol 23:283–289

    Article  CAS  Google Scholar 

  • Song JI, Kang YJ, Yong HY, Kim YC, Moon A (2012) “Denbinobin”, a phenanthrene from Dendrobium nobile, inhibits invasion and induces apoptosis in SNU-484 human gastric cancer cells. Oncol Rep 27:813–818. doi:10.3892/or.2011.1551

    CAS  PubMed  Google Scholar 

  • Sunagawa M, Miura K (1992) Interspecific heterokaryon formation between Auricularia auricula-judue and A. polytricha by electrical protoplast fusion using two metabolic inhibitors. Trans Mycol Soc Japan 33:375–383

    CAS  Google Scholar 

  • Veilleux RE, Compton ME (1996) Isolation, culture and fusion of tobacco and potato protoplasts. In: Trigiano RN, Gray DJ (eds) Plant tissue culture concepts and laboratory exercises. CRC Press, Boca Raton, pp. 215–226

    Google Scholar 

  • Yang H, Chou GX, Wang ZT, Guo YW, Hu ZB, Xu LS (2004) Two new compounds from Dendrobium chrysotoxum. Helv Chim Acta 87:394–399. doi:10.1002/hlca.200490037

    Article  CAS  Google Scholar 

  • Yang L, Qing LH, Bligh SWA, Bashall A, Zhang CF, Zhang M, Wang ZT, Xu LS (2006) A new phenanthrene with a spirolactone from Dendrobium chrysanthum and its anti-inflammatory activities. Bioorg Med Chem 14:3496–3501. doi:10.1016/j.bmc.2006.01.004

    Article  CAS  PubMed  Google Scholar 

  • Zhang GN, Zhong LY, Bligh SWA, Guo YL, Zhang CF, Zhang M, Wang ZT, Xu LS (2005) New bi-bicyclic and bi-tricyclic from Dendrobium thyrsiflorum. Phytochemistry 66:1113–1120. doi:10.1016/j.phytochem.2005.04.001

    Article  CAS  PubMed  Google Scholar 

  • Zhao J, Chang ST (1995) Intraspecific hybridization between Coprinus cinereus and Schizophyllum commune by PEG-induced protoplast fusion and electro-fusion. World J Microbiol Biotechnol 11:585–590. doi:10.1007/BF00286378

    Article  CAS  PubMed  Google Scholar 

  • Zhao J, Chang ST (1996) Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune by PEG-induced protoplast fusion. World J Microbiol Biotechnol 12:573–578. doi:10.1007/BF00327717

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Science and Engineering Research Board (SERB) of Department of Science and Technology (DST), Government of India, Grant No. SR/FT/LS-174/2009 for funding the research project. We thank Manipal University for providing the necessary facilities for implementing the research program. We would also like to thank the editor and the anonymous reviewers for their constructive comments regarding the improvement of the manuscript.

Author information

Authors and Affiliations

  1. Department of Plant Sciences, School of Life Sciences, Planetarium Complex, Manipal University, Manipal Udupi, Karnataka, 576 104, India

    Abitha Thomas, Ipsita Pujari, Vasudeep Shetty & Vidhu Sankar Babu

  2. Department of Ageing Research, School of Life Sciences, Planetarium Complex, Manipal University, Manipal Udupi, Karnataka, 576 104, India

    Manjunath B. Joshi

  3. Department of Biotechnology, School of Life Sciences, Planetarium Complex, Manipal University, Manipal Udupi, Karnataka, 576 104, India

    Padmalatha S. Rai

  4. Department of Cell and Molecular Biology, School of Life Sciences, Planetarium Complex, Manipal University, Manipal Udupi, Karnataka, 576 104, India

    Kapaettu Satyamoorthy

Authors
  1. Abitha Thomas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ipsita Pujari
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vasudeep Shetty
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Manjunath B. Joshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Padmalatha S. Rai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kapaettu Satyamoorthy
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Vidhu Sankar Babu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vidhu Sankar Babu.

Ethics declarations

Declaration

All authors declare that the content of the manuscript is original and it has not been submitted elsewhere for publication. All authors agree on the content of the manuscript. Laws on nature protection were not violated during this study.

Conflicts of interest

The authors declare that they have no conflict of interest.

Additional information

Handling Editor: Burkhard Becker

Abitha Thomas and Ipsita Pujari are contributed as first author

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Thomas, A., Pujari, I., Shetty, V. et al. Dendrobium protoplast co-culture promotes phytochemical assemblage in vitro. Protoplasma 254, 1517–1528 (2017). https://doi.org/10.1007/s00709-016-1043-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00709-016-1043-2

Keywords

Search

Navigation