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Production of sweet orange somaclones tolerant to citrus canker disease by in vitro mutagenesis with EMS

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Abstract

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a quarantine disease worldwide. This disease is hard to be completely controlled by chemical sprays, and the selection of citrus genotypes resistant to the pathogen becomes one of the most important ways to control the disease. The limited genotypes that have been found up to now tolerant to Xcc are not commercially cultivated. Sweet orange [Citrus sinensis (L.) Osbeck] is one of the most important citrus species widely cultivated in world and all the orange cultivars are susceptible to canker disease. Therefore, this study aims at the establishment of an efficient protocol for in vitro ethyl methane sulphonate (EMS) mutagenesis and the selection of ‘Bingtang’ sweet orange somaclones tolerant to citrus canker disease. Mutation was introduced by treating the cell suspension of embryogenic callus with 1.5 % of EMS for 1 h (the lethal concentration). A crude extract produced from the pathogen solution was used as the selection agent. Sweet orange leaves inoculated by Xcc-crude extract showed the symptoms similar to those inoculated by Xcc bacterial inoculum. The young shoots of susceptive genotypes cultured in 10 % of Xcc-crude extract solution become brown and died in 1 week, while the resistant citron C-05 grew normally under the same conditions. After two steps (cell suspension and plants) of selection by Xcc-crude extract solution, the survival plants were tested by in vitro and in vivo inoculation with Xcc. One somaclone, named DG-2, was identified to be resistant to the canker disease. The results suggested that the established protocol for somaclones variation by treating sweet orange callus with EMS and in vitro selection of the somaclones tolerant to canker disease by the pathogen crude extract was effective. The gained sweet orange somaclone tolerant to X. citri subsp. citri will go into further cultivar selection procedures.

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Abbreviations

Xcc :

Xanthomonas citri subsp. citri

EMS:

Ethyl methane sulphonate

MT:

Murashige and Tucker medium

LMT:

Liquid MT medium with 0.5 g L−1 malt extract and 1.5 g L−1 l-glutamine

LB:

Luria–Bertani medium

TTC:

Triphenyl tetrazolium chloride

Dpi:

Days post inoculation

References

  • Ali A, Naz S, Alam SS, Iqbal J (2007) In vitro induced mutation for screening of red rot (Colletotrichum falcatum) resistance in sugarcane (Saccharum officinarum). Pak J Bot 39:1979–1994

    Google Scholar 

  • Barbosa-Mendes JM, Mourao Filho FAA, Bergaminfilho A, Harakava R, Beer SV, Mendes BMJ (2009) Genetic transformation of Citrus sinensis cv. hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker. Sci Hortic 122:109–115

    Article  CAS  Google Scholar 

  • Brunings AM, Gabriel DW (2003) Xanthomonas citri: breaking the surface. Mol Plant Pathol 4:141–157

    Article  CAS  PubMed  Google Scholar 

  • Bulk RWVD (1991) Application of cell and tissue culture and in vitro selection for disease resistance breeding—a review. Euphytica 56:269–285

    Article  Google Scholar 

  • Chevreau E, Brisset MN, Paulin JP, James DJ (1998) Fire blight resistance and genetic trueness-to-type of four somaclonal variants from the apple cultivar greensleeves. Euphytica 104:199–205

    Article  Google Scholar 

  • Das AK (2003) Citrus canker—a review. J Appl Hortic 5(1):52–60

    Google Scholar 

  • Deng ZA, Zhang WC, Wan SY (1989) In vitro mutation breeding for salinity tolerance in citrus. Mutat breed newsl 33:12–14

    Google Scholar 

  • Deng ZN, Gentile A, Domina F, Nicolosi E, Tribulato E (1995) Selecting lemon protoplasts for insensitivity to Phoma tracheiphila toxin and regenerating tolerant plants. J Am Soc Hortic Sci 120(6):902–905

    Google Scholar 

  • Deng ZN, Xu L, Li DZ, Long GY, Liu LP, Fang F, Shu GP (2010) Screening citrus genotypes for resistance to canker disease (Xanthomonas axonopodis pv. citri). Plant Breed 129(3):341–345

    Article  CAS  Google Scholar 

  • Distefano G, La Malfa S, Vitale A, Lorito M, Deng ZN, Gentile A (2008) Defence-related gene expression in transgenic lemon plants producing an antimicrobial Trichoderma harzianum endochitinase during fungal infection. Transgenic Res 17:873–879

    Article  CAS  PubMed  Google Scholar 

  • Duncan DB (1955) Multiple range and multiple F tests. Biometrics 11:1–42

    Article  Google Scholar 

  • Ge HJ, Long GY, Dai SM, Li DZ, Li N, Deng ZN (2015) The influence of ‘Bingtang’ sweet orange or citron C-05 on the growth characteristics of Xanthomonas axonopodis pv. citri. Scientia Agricultura Sinca 48(7):1383–1391

    Google Scholar 

  • Gmitter FG, Grosser JW, Moore GA (1992) Citrus. In: Hammerschlag FA, Litz RE (eds) Biotechnology of perennial fruit crops. CAB International, Wallingford, pp 335–369

    Google Scholar 

  • Gottwald TR, Graham JH (1992) A device for precise and nondisruptive stomatal inoculation of leaf tissue with bacterial pathogens. Phytopathology 82:930–935

    Article  Google Scholar 

  • Graham JH, Gottwald TR, Riley TD, Achor D (1992) Penetration through leaf stomata and growth of strains of Xanthomonas campestris in citrus cultivars varying in susceptibility to bacterial diseases. Phytopathology 82:1319–1325

    Article  Google Scholar 

  • Grosser JW, Gmitter FG (1990) Protoplast fusion and citrus improvement. Plant Breed Rev 8:339–374

    Google Scholar 

  • Hammerschlag FA (2000) Resistant responses of peach somaclone 122-1 to Xanthomonas campestris pv. pruni and to Pseudomonas syringae pv. syringae. HortScience 35:141–143

    Google Scholar 

  • Hammerschlag FA, Ritchie D, Werner D, Hashmil G, Krusberg L, Meyer R, Huettel R (1995) In vitro selection of disease resistance in fruit trees. Acta Hortic 392:19–26

    Google Scholar 

  • Hao YJ, Deng XX (2002) Occurrence of chromosomal of chromosomal variations and plant regeneration from long-term-cultured citrus callus. In Vitro Cell Dev Biol Plant 38:472–476

    Article  Google Scholar 

  • Hao YJ, Wen XP, Deng XX (2004) Genetic and epigenetic evaluations of citrus calluses recovered from slow-growth culture. J Plant Physiol 161:479–484

    Article  CAS  PubMed  Google Scholar 

  • Jander G, Baerson SR, Hudak JA, Gonzalez KA, Gruys KJ, Last RL (2003) Ethyl methane sulfonate saturation mutagenesis in Arabidopsis to determine frequency of herbicide resistance. Plant Physiol 131:139–146

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Janick J, Moore JN (1996) Fruit Breeding Vol. I: Tree and Tropical Fruits. Wiley, New York, pp 565–606

    Google Scholar 

  • Kenganal M, Hanchinal RR, Nadaf HL (2008) Ethyl methane sulfonate (EMS) induced mutation and selection for salt tolerance in sugarcane in vitro. Indian J Plant Physiol 13:405–410

    CAS  Google Scholar 

  • Kilbey B, Hunter F (1983) Factors affecting mutational yield from EMS exposures of yeast (Saccharomyces cerevisia). Mutat Res 122:35–38

    Article  CAS  Google Scholar 

  • Kochba J, Spiegel-Roy P, Safran H (1972) Adventive plants from ovules and nucelli in citrus. Planta 106:237–245

    Article  CAS  PubMed  Google Scholar 

  • Koizumi M, Kuhara S (1982) Evaluation of citrus plants for resistance to bacterial canker disease in relation to the lesion extension. Bul Fruit Tree Res Station, D (Kuchinotsu) 55(3):73–92

    Google Scholar 

  • Kumar K, Gill MIS, Kaur H, Choudhary OP, Gosal SS (2010) In vitro mutagenesis and somaclonal variation assisted salt tolerance in ‘Rough Lemon’ (Citrus jambhiri Lush.). Eur J Hortic Sci 75(6):233–238

    Google Scholar 

  • Li MZ, Xu L, Sun ZL (2007) Isolation and characterization of a phytotoxin from Xanthomonas campestris pv. retroflexus. Chin J Chem Eng 15:639–642

    Article  CAS  Google Scholar 

  • Lin HC, Chu MK, Lin YC, Deng WL, Chang H, Hsu ST, Tzeng KC (2011) A single amino acid substitution in PthA of Xanthomonas axonopodis pv. citri altering canker formation on grapefruit leaves. Eur J Plant Pathol 130:143–154

    Article  CAS  Google Scholar 

  • Liu LP, Qu JW, Li N, Li DZ, Long GY, Deng ZN (2012) In silico characterization and molecular modeling of GntR family regulators in Xanthomonas axonopodis Implications for primary metabolism or virulence. Plant Omics J 5(5):494–502

    CAS  Google Scholar 

  • Luan YS, Zhang J, Gao XR, An LJ (2007) Mutation induced by ethyl methanesulfonate (EMS), in vitro screening for salt tolerance and plant regeneration of sweet potato (Ipomoea batatas L.). Plant Cell Tiss Org Cult 88(1):77–81

    Article  CAS  Google Scholar 

  • Luan ZQ, Cao HC, Yan BX (2008) Individual and combined phytotoxic effects of cadmium, lead and arsenic on soybean in phaeozem. Plant soil Environ 54(9):403–411

    CAS  Google Scholar 

  • Marta IF, Peña A, Graham JH (2010) Detached leaf inoculation of germplasm for rapid screening of resistance to citrus canker and citrus bacterial spot. Eur J Plant Pathol 127:571–578

    Article  Google Scholar 

  • Masuda T, Yoshioka T (1997) In vitro selection of a mutant resistant to Alternaria blotch disease in ‘Indo’ apple. Techn News Inst Rad Breed 56:1–2

    Google Scholar 

  • Murashige T, Tuker DPH (1969) Growth factor requirement of citrus tissue culture. Proc 1st Int Citrus Symp 3:1155–1169

    CAS  Google Scholar 

  • Omar MS, Novak FJ, Brunner H (1989) In vitro action of ethyl methane sulphonate on banana shoot tips. Sci Hortic 40:283–295

    Article  CAS  Google Scholar 

  • Predieri S (2001) Mutation induction and tissue culture in improving fruits. Plant Cell Tissue Organ Cult 64(2–3):185–210

    Article  CAS  Google Scholar 

  • Predieri S, Zimmerman RH (2001) Pear mutagenesis: in vitro treatment with gamma-rays and field selection for productivity and fruit traits. Euphytica 3:217–227

    Article  Google Scholar 

  • Rai MK, Kalia RK, Singh R, Gangola MP, Dhawan AK (2011) Developing stress tolerant plant through in vitro selection—an overview of the recent progress. Environ Exp Biol 71:89–98

    Article  Google Scholar 

  • Rao AV, Farooqui A, Jaya Sree T, Ramana RV, Sadanandam A (1993) EMS-induced streptomycin resistance in Solanum melongena. Theor Appl Genet 87:527–530

    Article  CAS  PubMed  Google Scholar 

  • Reddy MRS (1997) Sources of resistance to bacterial canker in citrus. J Mycol Plant Pathol 27(1):80–81

    Google Scholar 

  • Sahi ST, Ghazanfar MU, Afzal M, Rashed A, Habib A (2007) Incidence of citrus canker disease caused by Xanthomonas campestris pv. citri (Hasse) dows on Kinnow (Citrus reticulata) and its chemotherapy. Pak J Bot 39(4):1319–1327

    Google Scholar 

  • Sanada T, Amano E (1998) Induced mutation in fruit trees. somaclonal variation and induced mutations in crop improvement. Curr Plant Sci Biotech in Agr 32:401–409

    Article  CAS  Google Scholar 

  • Stall RE, Civerolo EL (1991) Research relating to the recent outbreak of citrus canker in Florida. Annu Rev Phytopathol 29:339–420

    Article  Google Scholar 

  • Starrantino A, Caponnetto P (1990) Effect of cytokinins on embryogenic callus formation from undeveloped ovules of orange. Acta Hortic 280:191–193

    Google Scholar 

  • Svabova L, Lebeda A (2005) In vitro selection for improved plant resistance to toxin-producing pathogens. J Phytopathol 153:52–64

    Article  CAS  Google Scholar 

  • Vardi A, Spiegel-Roy P (1982) Plant regeneration from citrus from citrus protoplast: variability in methodological requirements among cultivars and species. Theor Appl Genet 62:171–176

    Article  CAS  PubMed  Google Scholar 

  • Viloria Z, Drouillard DL, Graham JH, Grosser JW (2004) Screening triploid hybrids of ‘Lakeland’ limequat for resistance to citrus canker. Plant Dis 88(10):1056–1060

    Article  Google Scholar 

  • Viseur J (1990) Evaluation of fire blight resistance of somaclonal variants obtained from the pear cultivar ‘Durondeau’. Acta Hortic 273:275–284

    Google Scholar 

  • Yang L, Hu CH, Li N, Zhang JY, Yan JW, Deng ZN (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

    Article  CAS  PubMed  Google Scholar 

  • Zhang XD, Francis MI, Dawson WO, Graham JH, Orbović V, Triplett EW, Mou ZL (2010) Over-expression of the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker. Eur J Plant Pathol 128:91–100

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was financially supported by China Spark Program (Grant No. 2012GA770001), International S&T Cooperation Program of China (Grant No. 2011DFA32030), and China Agriculture Research System (Grant No. CARS-27-01A).

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  1. Ziniu Deng

    Present address: College of Horticulture and Landscapes, Hunan Agricultural University, Changsha, 410128, Hunan, People’s Republic of China

Authors and Affiliations

  1. Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, Hunan Agricultural University, Changsha, 410128, People’s Republic of China

    Hongjuan Ge, Hongyan Fu & Ziniu Deng

  2. College of Horticulture and Landscape, Hunan Agricultural University, Changsha, 410128, Hunan, People’s Republic of China

    Hongjuan Ge, Yi Li, Hongyan Fu, Guiyou Long, Li Luo, Ronghua Li & Ziniu Deng

  3. National Center for Citrus Improvement, Changsha, 410128, Hunan, People’s Republic of China

    Hongjuan Ge, Yi Li, Hongyan Fu, Guiyou Long, Li Luo, Ronghua Li & Ziniu Deng

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  1. Hongjuan Ge
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Correspondence to Ziniu Deng.

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Ge, H., Li, Y., Fu, H. et al. Production of sweet orange somaclones tolerant to citrus canker disease by in vitro mutagenesis with EMS. Plant Cell Tiss Organ Cult 123, 29–38 (2015). https://doi.org/10.1007/s11240-015-0810-7

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  • DOI: https://doi.org/10.1007/s11240-015-0810-7

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