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Study of Agrobacterium-mediated co-transformation of tea for blister blight disease resistance

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Abstract

Blister blight is the most prevalent leaf disease of tea, an economical perennial crop. The disease is caused by a biotrophic fungus, Exobasidium vexans. The objective was set to study whether the combine over-expression of Solanum tuberosum class I chitinase gene and Vigna radiata defensin genes in transgenic tea would give more disease resistance compared to transgene over-expressed singly. Taking two constructs containing S. tuberosum class I chitinase (AF153195) and mung bean defensin (AY437639) gene respectively, three Agrobacterium-mediated transformation events were conducted with two individual constructs and combination of both the constructs. This is a first report about the preparation of transgenic tea by co-transformation for blister resistance. Transformation was successful in all three events. Comparative analysis showed a very interesting result. Minimum overall transformation efficiency was noticed in transformation of tea with pCAMBIA 1301-Chi (2.20%) followed by pBI121-Def (2.39%) and co-transformation with pCAMBIA 1301-Chi and pBI121-Def (3.41%). The fungal bioassay with E. vexans for the transformation event with pCAMBIA 1301-Chi gave the best disease resistance having minimum diameter (7.05 mm) of hypersensitivity zone followed by 15.05 mm in pBI121-Def and 18.14 mm in combination of pCAMBIA 1301-Chi and pBI121-Def.

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Abbreviations

DMSO:

Dimethyl sulfoxide

SEM:

Somatic embryogenesis media

SSE:

Secondary somatic embryo

MM:

Multiplication media

References

  • Ali Z, Schumacher HM, Heine-Dobbernack E, El-Banna A, Hafeez FY, Jacobsen HJ et al (2010) Dicistronic binary vector system: a versatile tool for gene expression studies in cell cultures and plants. J Biotechnol 145:9–16

    CAS  PubMed  Google Scholar 

  • Amian AA, Papenbrock J, Jacobsen HJ, Hassan F (2011) Enhancing transgenic pea (Pisum sativum L.) resistance against fungal diseases through stacking of two antifungal genes (chitinase and glucanase). GM Crops 2(2):104–109. https://doi.org/10.4161/gmcr.2.2.16125

    Article  PubMed  Google Scholar 

  • Anuradha TS, Divya K, Jami SK, Kirti PB (2008) Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens. Plant Cell Rep 27(11):1777–1786

    Google Scholar 

  • Candela M, Vitali B, Matteuzzi D, Brigidi P (2004) Evaluation of the rrn operon copy number in Bifidobacterium using real-time PCR. Lett Appl Microbiol 38:229–232

    CAS  PubMed  Google Scholar 

  • Carvalho AO, Gomes VM (2009) Plant defensins—prospects for the biological functions and biotechnological properties. Peptides 30(5):1007–1020

    CAS  Google Scholar 

  • Chen K-C, Lin C-Y, Kuan C-C, Sung H-Y, Chen C-S (2002) A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid. J Agric Food Chem 50:7258–7263

    CAS  PubMed  Google Scholar 

  • Chen K-C, Hsu M-P, Tan C-H, Sung H-Y, Kuo CG, Fan M-J, Chen H-M, Chen S, Chen C-S (2005) Cloning and characterization of a plant defensin, VaD1 from azuki bean. J Agric Food Chem 53:982–989

    CAS  PubMed  Google Scholar 

  • Chen SC, Liu AR, Wang FH, Ahammed GJ (2009) Combined overexpression of chitinase and defensin genes in transgenic tomato enhances resistance in Botrytis cinerea. Afr J Biotechnol 8:5182–5188

    CAS  Google Scholar 

  • Dafny-Yelin M, Tzfira T (2007) Delivery of multiple transgenes to plant cells. Plant Physiol 145:1118–1128

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dana MM, Pintor-Toro JA, Cubero B (2006) Transgenic tobacco plants overexpressing chitinases of fungal origin show enhanced resistance to biotic and abiotic stress agents. Plant Physiol 142:722–730

    PubMed Central  Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Farrokhi N, Whitelegge JP, Brusslan JA (2007) Plant peptides and peptidomics. Plant Biotechnol J 6(2):105–134

    PubMed  Google Scholar 

  • Francois IEJA, Broekaert WF, Cammue BPA (2002) Different approaches for multi-transgene-stacking in plants. Plant Sci 163:281–295

    CAS  Google Scholar 

  • Gohain B, Borchetia S, Bhuyan LP, Rahman A, Sakata K, Mizutani M, Shimizu B, Gurusubramaniam G, Ravindranath R, Hazarika M, Das S (2012) Understanding Darjeeling tea flavour on a molecular basis. Plant Mol Biol 78:577–597

    CAS  PubMed  Google Scholar 

  • Halpin C, Boerjan W (2003) Stacking transgenes in forest trees. Trends Plant Sci 8:363–365

    CAS  PubMed  Google Scholar 

  • Jach G, Gornhardt B, Mundy J, Logemann J, Pinsdorf E, Leah R, Schell J, Maas C (1995) Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J 8:97–109

    CAS  PubMed  Google Scholar 

  • Jia Z, Gou J, Sun Y, Yuan L, Tang Q, Yang X, Pei Y, Luo K (2010) Enhanced resistance to fungal pathogens in transgenic Populus tomentosa Carr. by overexpression of an nsLTP-like antimicrobial protein gene from motherwort (Leonurus japonicus). Tree Physiol 30:1599–1605

    CAS  PubMed  Google Scholar 

  • Jongedijk E, Tigelaar H, Van Roekel JSC, Bres-Vloemans SA, Dekker I, Van den Elzen PJM (1995) Synergistic activity of chitinases and beta-1,3-glucanases enhances fungal resistance in transgenic tomato plants. Euphytica 85:173–180

    CAS  Google Scholar 

  • Kapila J, De Rycke R, Van Montagu M, Angenon G (1996) An Agrobacterium mediated transient gene expression system for intact leaves. Plant Sci. 122(101–108):26

    Google Scholar 

  • Lee C, Kim J, Shin SG, Hwang S (2006) Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol 123:273–280

    CAS  PubMed  Google Scholar 

  • Lee C, Lee S, Shin SG, Hwang S (2008) Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with Escherichia coli. Appl Microbiol Biotechnol 78:371–376

    CAS  PubMed  Google Scholar 

  • Liu Y-J, Cheng C-S, Lai S-M, Hsu M-P, Chen C-S, Lyu P-C (2006) Solution structure of the plant defensin VrD1 from mung bean and its possible role in insecticidal activity against bruchids. Proteins 63:777–786

    CAS  PubMed  Google Scholar 

  • Mackintosh CA, Garvin DF, Radmer LE, Heinen SJ, Muehlbauer GJ (2006) A model wheat cultivar for transformation to improve resistance to Fusarium head blight. Plant Cell Rep 25:313–319

    CAS  PubMed  Google Scholar 

  • Mackintosh CA, Lewis J, Radmer LE (2007) Overexpression of defence response genes in transgenic wheat enhances resistance to Fusarium head blight. Plant Cell Rep 26:479–488

    CAS  PubMed  Google Scholar 

  • Mondal TK, Bhattacharya A, Ahuja PS, Chand PK (2001) Transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryos. Plant Cell Rep 20:712–720

    CAS  Google Scholar 

  • Mukhopadhyay M, Mondal TK, Chand PK (2016) Biotechnological advances in tea (Camellia sinensis [L.] O. Kuntze): a review. Plant Cell Rep 13(11):1–33

    Google Scholar 

  • Niki T, Mitsuhara I, Seo S et al (1998) Antagonistic effect of salicylic acid and jasmonic acid on the expression of pathogenesis-related (PR) protein genes in wounded mature tobacco leaves. Plant Cell Physiol 39:500–507

    CAS  Google Scholar 

  • Olli S, Kirti PB (2006) Cloning, characterization and antifungal activity of Defensin Tfgd1 from Trigonella foenum-graecum L. J Biochem Mol Biol 39:278–283

    CAS  PubMed  Google Scholar 

  • Ordish G (1952) Untaken harvest. Constable & Co., Ltd, London, p 171

    Google Scholar 

  • Osborn RW, De Samblanx GW, Thevissen K, Goderis I, Torrekens S, Van Leuven F, Attenborough S, Rees SB, Broekaert WF (1995) Isolation and characterization of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae. FEBS Lett 368:257–262

    CAS  PubMed  Google Scholar 

  • Portieles R, Ayra C, Gonzales E, Gallo A, Rodriguez R, Chacon O, Lopez Y, Rodriguez M, Castillo J, Pujol M, Enriques G, Borroto C, Trujiullo L, Thomma BP, Borras-Hidalgo O (2010) NmDef02, a novel antimicrobial gene isolated from Nicotiana megalosiphon confers high-level pathogen resistance under greenhouse and field conditions. Plant Biotechnol J 8:678–690

    CAS  PubMed  Google Scholar 

  • Premkumar R (1996) Forecasting blister blight disease of tea. Annu Rep UPASI Sci Dept 70:47–48

    Google Scholar 

  • Rajalakshmi N, Ramarethinam S (2000) The role of Exobasidium vexans Massee in flavonoid synthesis by Camellia assamica Shneider. Indian J Plant Prot 28:19–29

    Google Scholar 

  • Rohini VK, Rao S (2001) Transformation of peanut (Arachi hypogaea L.) with tobacco chitinase gene: variable response of transformants to leaf spot disease. Plant Sci 160:889–898

    CAS  PubMed  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Schaefer SG, Gasic K, Cammue B, Broekaert W, van Damme EJ, Peumans WJ, Korban SS (2005) Enhanced resistance to early blight in transgenic tomato lines expressing heterologous plant defense genes. Planta 222:858–866

    CAS  PubMed  Google Scholar 

  • Shin S, Mackintosh CA, Lewis J, Heinen SJ, Radmer L, Dill-Macky R, Baldridge GD, Zeyen RJ, Muehlbauer GJ (2008) Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum. J Exp Bot 59(9):2371–2378

    CAS  PubMed  PubMed Central  Google Scholar 

  • Simmons C (1994) The physiology and molecular biology of plant 1,3-β-D-glucanases and 1,3; 1,4-β-D-glucanases. Crit Rev Plant Sci 13:325–387

    CAS  Google Scholar 

  • Singh HR, Bhattacharyya N, Agarwala N, Bhagawati P, Deka M, Das S (2014) Exogenous gene transfer in Assam tea [Camellia assamica (Masters)] by Agrobacterium-mediated transformation using somatic embryo. Eur J Exp Biol 4(3):166–175

    CAS  Google Scholar 

  • Singh HR, Deka M, Das S (2015) Enhanced resistance to blister blight in transgenic tea (Camellia sinensis [L.] O. Kuntze) by overexpression of class I chitinase gene from potato (Solanum tuberosum). Funct Integr Genom 15(4):461–480

    CAS  Google Scholar 

  • Singh HR, Hazarika P, Agarwala N, Bhatacharjee N, Bhagawati P, Gohain B, Bandyopadhyay T, Bharalee R, Gupta S, Deka M, Das S (2018) Transgenic tea over-expressing Solanum tuberosum endo-1,3-beta-d-glucanase gene conferred resistance against blister blight disease. Plant Mol Biol Rep 36:107–122

    CAS  Google Scholar 

  • Sridevi G, Parameswari C, Sabapathi N, Raghupathy V, Karuppannan V (2008) Combined expression of chitinase and-1,3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance against Rhizoctonia solani. Plant Sci 175(3):283–290

    CAS  Google Scholar 

  • Tanaka J, Taniguchi F (2006) Estimation of the genome size of tea (Camellia sinensis), camellia (C. japonica), and their interspecific hybrids by flow cytometry. J Remote Sens Soc Jpn 101:1–7

    Google Scholar 

  • Thevissen K, Terras FR, Broekaert WF (1999) Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl Environ Microbiol 65:5451–5458

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tripathi RM, Bisht HS, Singh RP (2010) Effect of acetosyringone and callus age on transformation for scutellum-derived callus of rice. Int J Pharma Bio Sci 1(4):163–171

    Google Scholar 

  • Tuzun S, Rao MN, Vogeli U, Schardl CL, Kuc J (1989) Induced systemic resistance to blue mold: early induction and accumulation of b-1,3-glucanases, chitinases, and other pathogenesis-related proteins (b-proteins) in immunized tobacco. Phytopathology 79:979–983

    CAS  Google Scholar 

  • Vilalta A, Whitlow V, Martin T (2002) Real-time PCR determination of Escherichia coli genomic DNA contamination in plasmid preparations. Anal Biochem 301:151–153

    CAS  PubMed  Google Scholar 

  • Wally O, Jayaraj J, Punja Z (2009a) Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase, b-1,3-glucanase and peroxidise. Eur J Plant Pathol 123:331–342

    CAS  Google Scholar 

  • Wally O, Jayaraj J, Punja ZK (2009b) Broad-spectrum disease resistance to necrotrophic and biotrophic pathogens in transgenic carrots (Daucus carota L.) expressing an Arabidopsis NPR1 gene. Planta 231(1):131–141

    CAS  PubMed  Google Scholar 

  • Xu Y, Chang P-FL, Liu D et al (1994) Plant defense genes are synergistically induced by ethylene and methyl jasmonate. Plant Cell 6:1077–1085

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yang L, Hu C, Li N, Zhang J, Yan J, Deng Z (2011) Transformation of sweet orange [Citrus sinensis (L.) Osbeck] with pthA-nls for acquiring resistance to citrus canker disease. Plant Mol Biol 75:11–23

    CAS  PubMed  Google Scholar 

  • Yuan L, Wang L, Han Z et al (2012) Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants. J Exp Bot 63(7):2513–2524

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang XH, Guo DJ, Zhang LM (2000) The research on the expression of rabbit defensin (NP-1) gene in transgenic tomato. Acta Genitica Sin 27:953–958

    CAS  Google Scholar 

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Acknowledgements

This study was supported by generous funding from Department of Biotechnology, Govt. of India. HRS was Senior Research Fellow supported by Council of Scientific and Industrial Research, Govt. of India. The help from Dr Bornali Gohain during real time PCR work is highly acknowledged. The authors also acknowledge the support of the Director, Tocklai Tea Research Institute, Jorhat, India.

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Correspondence to H. Ranjit Singh.

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Singh, H.R., Hazarika, P., Deka, M. et al. Study of Agrobacterium-mediated co-transformation of tea for blister blight disease resistance. J. Plant Biochem. Biotechnol. 29, 24–35 (2020). https://doi.org/10.1007/s13562-019-00508-0

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