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Agrobacterium -mediated transformation of cotton (Gossypium hirsutum) using a heterologous bean chitinase gene

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Abstract

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l−1 kanamycin and 200 mg l−1 cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and βglucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T1 plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. ‘Western’ immunoblot analysis of leaves isolated from T0 transformants and progeny plants (T1) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae.Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.

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Abbreviations

B5 vitamins:

Gamborg et al. (1968) vitamins

CaMV:

cauliflower mosaic virus

chi :

chitinase

2,4-D:

2,4-dichlorophenoxyacetic acid

GUS:

β-glucuronidase

MS:

murashige and shoog (1962)

NAGA:

N-acetyl glucosamine

nptII :

neomycin phosphotransferase gene

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

References

  • G An (1987) ArticleTitleBinary TI vectors for plant transformation and promoter analysis Methods Enzymol 153 292–293

    Google Scholar 

  • T Boller A Gehri F Mauch U Vogeli (1983) ArticleTitleChitinase in bean leaves: induction by ethylene, purification, properties and possible function Planta 157 22–37 Occurrence Handle10.1007/BF00394536

    Article  Google Scholar 

  • T Boller F Mauch (1988) Colorimetric assay for chitinase WA Wood ST Kellog (Eds) Methods in Enzymology, Pectin and Chitin. Academic Press San Diego 430–435

    Google Scholar 

  • VK Borole DB Dhumale JP Rajput (2000) ArticleTitleEmbryo culture studies in interspecific crosses between arboretum and hirsutum cotton Indian J. Genet. 60 105–214

    Google Scholar 

  • MM Bradford (1976) ArticleTitleA rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding Anal. Biochem. 72 248–254 Occurrence Handle942051

    PubMed  Google Scholar 

  • K Broglie I Chet M Holliday C Robert P Biddle S Knolton CJ Mauvais R Broglie (1991) ArticleTitleTransgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani Science. 245 1194–1197

    Google Scholar 

  • WX Chen GF Xiao Z Zhu (2002) ArticleTitleObtaining high pest-resistant transgenic upland cotton cultivars carrying cryIAc3 gene driven by chimeric OM promoter Acta Bot. Sin. 44 963–970

    Google Scholar 

  • DB Collinge KM Kragh JD Mikkelsen KK Nielsen K RasmussenUand Vad (1993) ArticleTitlePlant Chitinases Plant J. 3 31–40 Occurrence Handle10.1046/j.1365-313X.1993.t01-1-00999.x Occurrence Handle8401605

    Article  PubMed  Google Scholar 

  • PA Donaldson DH Simmonds (2000) ArticleTitleSusceptibility to Agrobacterium tumefaciens and cotyledonary node transformation in short-season soybean Plant Cell Rep. 19 478–484 Occurrence Handle10.1007/s002990050759

    Article  Google Scholar 

  • C Emani JM Garcia E Lopata-Finch MJ Pozo P Uribe DJ Kim G Sunilkumar DR Cook CM Kenerley KS Rathore (2003) ArticleTitleEnhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens Plant. Biotech. J. 1 321–336 Occurrence Handle10.1046/j.1467-7652.2003.00029.x

    Article  Google Scholar 

  • E Firoozabady DL DeBoer D Merlo E Halk L Amerson KE Rashka EE Murray (1987) ArticleTitleTransformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants Plant Mol. Biol. 10 105–116 Occurrence Handle10.1007/BF00016148

    Article  Google Scholar 

  • E Firoozabady DW Galbraith (1983) ArticleTitleThe effects of cell wall and cell cycle on crown gall tumorigenesis HortScience. 18 441–449

    Google Scholar 

  • E Firoozabady DW Galbraith (1984) ArticleTitlePresence of a plant cell wall is not required for transformation of Nicotiana by Agrobacterium tumefaciens. Plant Cell Tiss Org. Cult. 3 175–188 Occurrence Handle10.1007/BF00033739

    Article  Google Scholar 

  • OL Gamborg RA Miller K Ojima (1968) ArticleTitleNutrient requirements of suspension cultures of soyabean root cells Exp. Cell Res. 50 151–158 Occurrence Handle10.1016/0014-4827(68)90403-5 Occurrence Handle5650857

    Article  PubMed  Google Scholar 

  • LS Graham MB Sticklen (1994) ArticleTitlePlant Chitinases Can. J. Bot. 72 1057–1083

    Google Scholar 

  • RA Jefferson TA Kavanagh MW Bevan (1987) ArticleTitleGus fusion: glucuronidase as a sensitive and versatile gene fusion marker in higher plants EMBO J. 6 3901–3907 Occurrence Handle3327686

    PubMed  Google Scholar 

  • ME John G Keller (1996) ArticleTitleMetabolic pathway for engineering cotton: biosynthesis of polyhydroxybutyrate in fiber cells Proc. Natl. Acad. Sci. 93 12768–12773 Occurrence Handle10.1073/pnas.93.23.12768 Occurrence Handle11038522

    Article  PubMed  Google Scholar 

  • K Kishimoto Y Nishizawa Y Tabei T Hibi A Nakajima K Akutsu (2002) ArticleTitleDetailed analysis of rice chitinase gene expression in transgenic cucumber plants showing different levels of disease resistance to grey mold (Botrytis cinerea) Plant Sci. 162 655–662 Occurrence Handle10.1016/S0168-9452(01)00602-1

    Article  Google Scholar 

  • S Kumar D Pental (1998) ArticleTitleRegeration of Indian cotton variety MCU-5 through somatic embryogenesis Curr. Sci. 74 538–540

    Google Scholar 

  • UK Laemmli (1970) ArticleTitleCleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature. 227 680–685 Occurrence Handle5432063

    PubMed  Google Scholar 

  • H Li L Jinhua JK Hemphill JT Wang J Gould (2001) ArticleTitleA rapid and high yielding DNA miniprep for cotton (Gossypium spp). Plant Mol Biol Rep 19 183a–183e

    Google Scholar 

  • W Lin CS Anuratha K Datta I Potrykus S Muthukrishnan SK Datta (1995) ArticleTitleGenetic engineering of rice for resistance to sheath blight Biotechnology 13 686–691 Occurrence Handle10.1038/nbt0795-686

    Article  Google Scholar 

  • F Mauch B Mauch-Mahdi T Boller (1988) ArticleTitleAntifungal hydrolases in pea tissue, II: Inhibition of fungal growth by combination of chitinase and β-1,3-glucanase Plant Physiol 88 936–942

    Google Scholar 

  • P Meyer (1985) ArticleTitleUnderstanding and controlling transgene expression Trends Biotechnol. 13 332–337 Occurrence Handle10.1016/S0167-7799(00)88977-5

    Article  Google Scholar 

  • M Mohammadi AL Karr (2002) ArticleTitleβ1,3-Glucanase and chitinase activities in soybean root nodules J. Plant Physiol. 159 245–256

    Google Scholar 

  • T Murashige F Skoog (1962) ArticleTitleA revised medium for rapid growth and bioassays with tobacco tissue cultures Plant Physiol. 15 472–497

    Google Scholar 

  • J-M Neuhaus (1999) Plant chitinases (PR-3, PR-4, PR-8, PR-11) SK Datta S Muthukrishnan (Eds) Pathogenesis-related Proteins in Plants CRC Press Boca Raton Florida 77–105

    Google Scholar 

  • Y Nishizawa Z Nishio K Nakazono M Soma E Nakajima M Ugaki T Hibi (1999) ArticleTitleEnhanced resistance to blast (Magnaporthe grisea) in transgenic japonica rice by constitutive expression of rice chitinase Theor. Appl. Genet. 99 383–390 Occurrence Handle10.1007/s001220051248

    Article  Google Scholar 

  • HT Raharjo HO Hernandez YY Zhang ZK Punja (1990) ArticleTitleTransformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens. Plant Cell Rep. 15 591–596 Occurrence Handle10.1007/s002990050080

    Article  Google Scholar 

  • H Rashid S Yokoi K Toriyama K Hinata (1996) ArticleTitleTransgenic plant production mediated by Agrobacterium indica rice Plant Cell Rep. 15 727–730 Occurrence Handle10.1007/s002990050108

    Article  Google Scholar 

  • VK Rohini K Sankara Rao (2000) ArticleTitleTransformation of peanut (Arachis hypogaea L.) with tobacco chitinase gene: variable response of transformants to leaf spot disease Plant Sci. 160 889–898 Occurrence Handle10.1016/S0168-9452(00)00462-3

    Article  Google Scholar 

  • J Sambrook DW Russell (2001) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press. Cold Spring Harbor NY

    Google Scholar 

  • SQ San XS Ye J Kuc (1992) ArticleTitleInduction of chitinases in tobacco plants systemically protected against blue mold by Peronospora tabacina or tobacco mosaic virus Phytopathology 82 119–123

    Google Scholar 

  • H Schickler I Chet (1997) ArticleTitleHeterologous chitinase gene expression to improve plant defence against phytopathogenic fungi J. Indust. Microbiol. Biotech. 19 196–201 Occurrence Handle10.1038/sj.jim.2900447

    Article  Google Scholar 

  • A Schlumbaum F Mauch K Vögeli T Boller (1986) ArticleTitlePlant chitinases are potent inhibitors of fungal growth Nature. 324 365–367 Occurrence Handle10.1038/324365a0

    Article  Google Scholar 

  • MB Sela-Buurlage AS Ponstein SA Vloemans LS Melchers PJM Elzen Particlevan den BJC Cornelissen (1993) ArticleTitleOnly specific tobacco (Nicotiana tabacum) chitinases and β-1,3-glucanases exhibit antifungal activity Plant Physiol. 101 857–863 Occurrence Handle12231736

    PubMed  Google Scholar 

  • Y Shneyour A Zelcer S Izhar JS Backman (1984) ArticleTitleA simple feeder layer technique for the plating of cells and protoplasts at low density Plant Sci. Lett. 33 293–302 Occurrence Handle10.1016/0304-4211(84)90020-8

    Article  Google Scholar 

  • T Terakawa N Takaya H Horiuchi M Koike (1997) ArticleTitleA fungal chitinase gene from Rhizopus oligosporus confers antifungal activity to transgenic tobacco Plant Cell Rep. 16 439–443

    Google Scholar 

  • P Umbeck G Johnson K Barton W Swain (1987) ArticleTitleGenetically transformed cotton (Gossypium hirsutum L.) plants Bio/technology. 5 263–266 Occurrence Handle10.1038/nbt0387-263

    Article  Google Scholar 

  • TA Wilkins K Rajasekaran M Anderson (2000) ArticleTitleCotton biotechnology Crit. Rev. Plant Sci. 19 511–550 Occurrence Handle10.1016/S0735-2689(01)80007-1

    Article  Google Scholar 

  • T Yamamoto H Iketani H Ieki Y Nishizawa K Notsuka T Hibi N HayashiTand Matsuta (2000) ArticleTitleTransgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens Plant Cell Rep. 19 639–646 Occurrence Handle10.1007/s002999900174

    Article  Google Scholar 

  • D Yun RA Bressan P Hasegawa (1997) ArticleTitlePlant antifungal proteins Hort. Rev. 14 39–87

    Google Scholar 

  • CS Zapata H Park KM EI-Zik RH Smith (1998) ArticleTitleTransformation of a Texas cotton cultivar by using Agrobacterium and the shoot apex Theor. Appl. Genet. 98 252–256 Occurrence Handle10.1007/s001220051065

    Article  Google Scholar 

  • BH Zhang R Feng F Liu Q Wang (2001) ArticleTitleHigh frequency somatic embryogenesis and plant regeneration of an elite Chinese cotton variety Bot. Bull. Acad. Sin. 42 9–16

    Google Scholar 

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Authors and Affiliations

  1. Agricultural Biotechnology, Research Institute of Iran (ABRII), P.O. Box v, Mâhdasht Road, Karaj, Iran

    Masoud Tohidfar & Behzad Ghareyazie

  2. Department of Plant Pathology Entomology College of Agriculture, University of Tehran, Karaj, 31587-11167, Iran

    Mojtaba Mohammadi

Authors
  1. Masoud Tohidfar
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  2. Mojtaba Mohammadi
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  3. Behzad Ghareyazie
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Correspondence to Masoud Tohidfar.

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Tohidfar, M., Mohammadi, M. & Ghareyazie, B. Agrobacterium -mediated transformation of cotton (Gossypium hirsutum) using a heterologous bean chitinase gene. Plant Cell Tiss Organ Cult 83, 83–96 (2005). https://doi.org/10.1007/s11240-004-6155-2

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  • DOI: https://doi.org/10.1007/s11240-004-6155-2

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