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Establishment of dedifferentiated callus of haploid origin from unfertilized ovaries of tea (Camellia sinensis (L.) O. Kuntze) as a potential source of total phenolics and antioxidant activity

  • Molecular Farming/Metabolic Engineering/Secondary Metabolism
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An Erratum to this article was published on 24 April 2013

Abstract

This is the first report of induction of haploid callus with significant antioxidant activity from unpollinated ovary cultures of tea. Out of the five cultivars tested, TV18 gave the highest percentage of callus induction. Within 1 wk of induction, ovules swelled to almost double their original size, and white, friable callus emerged. A high cytokinin/auxin ratio, provided by 8.5 μM benzyl adenine and 4.5 μM 2,4-dichlorophenxyacetic acid, and high-temperature treatment (33°C) for 10 d in the dark promoted maximum callus induction. Callus was maintained on MS medium containing 22.2 μM benzyl adenine and 9.8 μM indolebutyric acid (callus line RM 1) in the light at 25°C. Well-developed tracheids were formed within 4 wk in callus subcultured on MS medium containing 1.8 μM thidiazuron and 5.0 μM 2,3,5-triiodobenzoic acid (line RM 2). Flow cytometric analysis revealed that most cells were haploid. Both RM 1 and RM 2 produced phenolic compounds with significant antioxidant capacity. Phenolic content showed a positive linear correlation with antioxidant activity. The total phenolic content of RM 1 was 3.47 ± 0.21 gallic acid equivalents (GAE) mg/g dry weight and that of RM 2 was 2.39 ± 0.12 GAE mg/g dry weight. Antioxidant activity was measured using IC50, a measure of inhibitory concentration; a lower IC50 value reflects greater antioxidant activity. The IC50 value of RM 1 was 2,530 μg/ml and that of RM 2 was 3,170 μg/ml. The results suggested that the phenolic compounds contributed significantly to the antioxidant capacity of the in vitro cell lines.

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References

  • Agarwal PK, Agarwal P, Custers JBM, Liu C, Bhojwani SS (2006) PCIB an antiauxin enhances microspore embryogenesis in microspore culture of Brassica juncea. Plant Cell Tissue Organ Cult 86:201–210

    Article  CAS  Google Scholar 

  • Alan AR, Mutschler MA, Brants A, Cobb E, Earle ED (2003) Production of gynogenic plants from hybrids of Allium cepa L. and A. roylei Stearn. Plant Sci 165:1201–1211

    Article  CAS  Google Scholar 

  • Astill C, Birch MR, Dacombe C, Humphrey PG, Martin PT (2001) Factors affecting the caffeine and polyphenol contents of black and green tea infusions. J Agric Food Chem 49:5340–5347

    Article  PubMed  CAS  Google Scholar 

  • Bhagyalakshmi N (1999) Factors influencing direct shoot regeneration from ovary explants of saffron. Plant Cell Tissue Organ Cult 58:205–211

    Article  CAS  Google Scholar 

  • Bhat JG, Murthy HN (2007) Factors affecting in-vitro gynogenic haploid production in niger (Guizotia abyssinica (L.f.) Cass.). Plant Growth Regul 52:241–248

    Article  CAS  Google Scholar 

  • Bhojwani SS, Razdan MK (1996) Plant tissue culture: theory and practice, a revised edition. Elsevier, Amsterdam

    Google Scholar 

  • Boskou G, Salta FN, Chrysostomou S, Mylona A, Chiou A, Andrikopoulos NK (2006) Antioxidant capacity and phenolic profile of table olives from the Greek market. Food Chem 94:558–564

    Article  CAS  Google Scholar 

  • Braca A, Tommasi ND, Bari LD, Pizza C, Politi M, Morelli I (2001) Antioxidant principles from Bauhinia tarapotensis. J Nat Prod 64:892–895

    Article  PubMed  CAS  Google Scholar 

  • Cai Y, Luo Q, Sun M, Corke H (2004) Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci 74:2157–2184

    Article  PubMed  CAS  Google Scholar 

  • Campion B, Azzimonti MT, Vicini E, Schiavi M, Falavigna A (1992) Advances in haploid plants induction in onion (Allium cepa L.) through in vitro gynogenesis. Plant Sci 86:97–104

    Article  Google Scholar 

  • Chan EWC, Lim YY, Chew YL (2007) Antioxidant activity of Camellia sinensis leaves and tea from a lowland plantation in Malaysia. Food Chem 102:1214–1222

    Article  CAS  Google Scholar 

  • Chaturvedi R, Razdan MK, Bhojwani SS (2003) Production of haploids of neem (Azadirachta indica A. Juss.) by anther culture. Plant Cell Rep 21:531–537

    PubMed  CAS  Google Scholar 

  • Chen JF, Cui L, Malik AA, Mbira KG (2011) In vitro haploid and dihaploid production via unfertilized ovule culture. Plant Cell Tissue Organ Cult 104:311–319

    Article  Google Scholar 

  • Dolezel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc 2:2233–2244

    Article  PubMed  CAS  Google Scholar 

  • Duangporn P, Siripong P (2009) Effect of auxin and cytokinin on phyllanthusol A production by callus cultures of Phyllanthus acidus Skeels. American-Eurasian J Agric Environ Sci 5:258–263

    CAS  Google Scholar 

  • Eden T (1958) The development of tea culture. In: Eden T (ed) Tea. Longman, London, pp 1–4

    Google Scholar 

  • Gemes-Juhasz A, Balogh P, Ferenczy A, Kristof Z (2002) Effect of optimal stage of female gametophyte and heat treatment on in vitro gynogenesis induction in cucumber (Cucumis sativus L.). Plant Cell Rep 21:105–111

    Article  CAS  Google Scholar 

  • Gugsa L, Sarial AK, Lorz H, Kumlehn J (2006) Gynogenic plant regeneration from unpollinated flower explants of Eragrostis tef (Zuccagni) Trotter. Plant Cell Rep 25:1287–1293

    Article  PubMed  CAS  Google Scholar 

  • Gulcin I (2006) Antioxidant and antiradical activities of L-carnitine. Life Sci 78:803–811

    Article  PubMed  Google Scholar 

  • Heiss ML (2008) The story of tea—a cultural history and drinking guide. Ten Speed Press, Berkeley

    Google Scholar 

  • Huetteman CA, Preece JE (1993) Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tissue Organ Cult 33:105–119

    Article  CAS  Google Scholar 

  • Kapil RN, Sethi SB (1963) Development of male and female gametophytes in Camellia sinensis (L.) O. Kuntze. Proc Natl Inst Sci India B 29:567–574

    Google Scholar 

  • Khalaf NA, Shakya AK, Al-Othman A, El-Agbar Z, Farah H (2008) Antioxidant activity of some common plants. Turk J Biol 32:51–55

    Google Scholar 

  • Kobayashi RS, Sinden SL, Bouwkamp JC (1993) Ovule cultures of sweet potato (Ipomoea batatas) and closely related species. Plant Cell Tissue Organ Cult 32:77–82

    Article  Google Scholar 

  • Loureiro J, Rodriguez E, Dolezel J, Santos C (2007) Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Ann Bot 100:875–888

    Article  PubMed  CAS  Google Scholar 

  • Mantell SH, Smith H (1984) Cultural factors that influence secondary metabolites accumulations in plant cell and tissue cultures. In: Mantell SH, Smith H (eds) Plant biotechnology. Society for experimental biology, seminar series 18. Cambridge University Press, Cambridge, p 75

    Google Scholar 

  • Matkowski A (2008) Plant in vitro culture for the production of antioxidants—a review. Biotechnol Adv 26:548–560

    Article  PubMed  CAS  Google Scholar 

  • Mondal TK, Bhattacharya A, Laxmikumaran M, Ahuja PS (2004) Recent advances in tea (Camellia sinensis) biotechnology. Plant Cell Tissue Organ Cult 76:195–254

    Article  CAS  Google Scholar 

  • Mondal TK, Bhattacharya A, Sood A, Ahuja PS (1998) Micropropagation of tea using thidiazuron. Plant Growth Regul 26:57–61

    Article  CAS  Google Scholar 

  • Mondal TK, Parathi R, Mohankumar P (2005) Micrografting—a technique to shorten the hardening time of micropropagated shoots of tea (Camellia sinensis (L.) O. Kuntze). Sri Lankan J Tea Sci 70:5–9

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with the tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Nakajima I, Kobayashi S, Nakamura Y (2000) Embryogenic callus induction and plant regeneration from unfertilized ovule of ‘Kyoho’ grape. J Jpn Soc Horticult Sci 69:186–188

    Article  CAS  Google Scholar 

  • Niedz RP, Smith SS, Dunbar KB (1989) Factors influencing shoot regeneration from cotyledonary explants of Cucumis melo. Plant Cell Tissue Organ Cult 18:313–319

    Article  Google Scholar 

  • Ouyang TW, Hu H, Chuang CC, Tseng CC (1973) Induction of pollen plants from anther of Triticum aestivum L. cultured in vitro. Sci Sin 16:79–95

    Google Scholar 

  • Pedroso MC, Pais MS (1993) Induction of microspore embryogenesis in Camellia japonica cv. Elegans. Plant Cell Tissue Organ Cult 37:129–136

    Article  Google Scholar 

  • Russell SD (1993) The egg cell: development and role in fertilization and early embryogenesis. Plant Cell 5:1349–1359

    PubMed  Google Scholar 

  • Saha K, Lajis NH, Israf DA, Hamzah AS, Khozirah S, Khamis S, Syahida A (2004) Evaluation of antioxidant and nitric oxide inhibitory activities of selected Malaysian medicinal plants. J Ethnopharmacol 92:263–267

    Article  PubMed  CAS  Google Scholar 

  • Saravanan M, Maria John KM, Raj Kumar R, Pius PK, Sasikumar R (2005) Genetic diversity of UPASI tea clones (Camellia sinensis (L.) O. Kuntze) on the basis of total catechins and their fractions. Phytochemistry 66:561–565

    Article  PubMed  CAS  Google Scholar 

  • Shalaby TA (2007) Factors affecting haploid induction through in vitro gynogenesis in summer squash (Cucurbita pepo L.). Sci Hortic 115:1–6

    Article  Google Scholar 

  • Shibasaki-Kitakawa N, Takeishi J, Yonemoto T (2003) Improvement of catechin productivity in suspension cultures of tea callus cells. Biotechnol Prog 19:655–658. doi:10.1021/bp025539a

    Article  PubMed  CAS  Google Scholar 

  • Singh SK, Syamal MM (2000) Anti-auxin enhance Rosa hybrid L. micropropagation. Biol Plant 43:279–281

    Article  CAS  Google Scholar 

  • Slinkard K, Singleton VL (1977) Total phenol analyses: automation and comparison with manual methods. Am J Enol Viticult 28:49–55

    CAS  Google Scholar 

  • Sopory S, Munshi M (1996) Anther culture. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants, vol. 1. Kluwer, Dordrecht, pp 145–176

    Chapter  Google Scholar 

  • Sugimura Y, Adachi T, Kotani E, Furusuwa T (1998) Efficient induction of shoot organogenesis from leaves of mulberry seedlings using 2,3,5-triiodobenzoic acid. Plant Biotechnol 16:123–127

    Google Scholar 

  • Thomas TD, Bhatnagar AK, Razdan MK, Bhojwani SS (1999) A reproducible protocol for the production of gynogenic haploids of mulberry, Morus alba L. Euphytica 110:169–173

    Article  Google Scholar 

  • Tsou CH (1997) Embryology of the Theaceae—anther and ovule development of Camellia, Franklinia, and Schima. Am J Bot 84:369–381

    Article  PubMed  CAS  Google Scholar 

  • Vieitez AM, Barciela J (1990) Somatic embryogenesis and plant regeneration from embryonic tissues of Camellia japonica L. Plant Cell Tissue Organ Cult 21:267–274

    Article  CAS  Google Scholar 

  • Wakizuka T, Nakajima T (1975) Development of proembryo in cultured ovules of Petunia hybrida Vilm. Jpn J Breed 25:161–167

    Google Scholar 

  • Watson L, Dallwitz MJ (1992 onwards) The families of flowering plants: descriptions, illustrations, identification, and information retrieval. Version: 4th March 2011. http://delta-intkey.com

  • Weisburger JH (1996) Tea antioxidants and health. In: Cadenas E, Packer L (eds) Handbook of antioxidants. Dekker, New York, pp 469–486

    Google Scholar 

  • Wight W (1962) Tea classification revised. Curr Sci 31:298–299

    Google Scholar 

  • Wu HK (1962) Embryogenesis in the tea plant. Bot Bull Acad Sinica 1:165–168

    Google Scholar 

  • Yang Z, Xu Y, Jie G, He P, Tu Y (2007) Study on the antioxidant activity of tea flowers (Camellia sinensis). Asia Pac J Clin Nutr 16:148–152

    PubMed  CAS  Google Scholar 

  • Yeoman MM (1987) Techniques, characteristics, properties and commercial potential of immobilized plant cells. In: Constabel F, Vasil IK (eds) Cell culture and somatic cell genetics, vol. 4. Cell culture in phytochemistry. Academic, San Diego, pp 197–215

    Chapter  Google Scholar 

  • Zheng W, Wang S (2001) Antioxidant activity and phenolic compounds in selected herbs. J Agric Food Chem 49:5165–5170

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We gratefully acknowledge the Department of Biotechnology (DBT), Government of India for providing financial assistance and Tocklai Experimental Station, Tea Research Association (TRA), Jorhat, Assam for providing access to the plant material.

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  1. Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India

    Hazarika Rashmi Rekha & Chaturvedi Rakhi

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  1. Hazarika Rashmi Rekha
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Correspondence to Chaturvedi Rakhi.

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Editor: Pamela Weathers

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Rekha, H.R., Rakhi, C. Establishment of dedifferentiated callus of haploid origin from unfertilized ovaries of tea (Camellia sinensis (L.) O. Kuntze) as a potential source of total phenolics and antioxidant activity. In Vitro Cell.Dev.Biol.-Plant 49, 60–69 (2013). https://doi.org/10.1007/s11627-013-9490-3

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