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Selectable antibiotic resistance marker gene-free transgenic rice harbouring the garlic leaf lectin gene exhibits resistance to sap-sucking planthoppers

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Abstract

Rice, the major food crop of world is severely affected by homopteran sucking pests. We introduced coding sequence of Allium sativum leaf agglutinin, ASAL, in rice cultivar IR64 to develop sustainable resistance against sap-sucking planthoppers as well as eliminated the selectable antibiotic-resistant marker gene hygromycin phosphotransferase (hpt) exploiting cre/lox site-specific recombination system. An expression vector was constructed containing the coding sequence of ASAL, a potent controlling agent against green leafhoppers (GLH, Nephotettix virescens) and brown planthopper (BPH, Nilaparvata lugens). The selectable marker (hpt) gene cassette was cloned within two lox sites of the same vector. Alongside, another vector was developed with chimeric cre recombinase gene cassette. Reciprocal crosses were performed between three single-copy T0 plants with ASAL- lox-hpt-lox T-DNA and three single-copy T0 plants with cre-bar T-DNA. Marker gene excisions were detected in T1 hybrids through hygromycin sensitivity assay. Molecular analysis of T1 plants exhibited 27.4% recombination efficiency. T2 progenies of L03C04(1) hybrid parent showed 25% cre negative ASAL-expressing plants. Northern blot, western blot and ELISA showed significant level of ASAL expression in five marker-free T2 progeny plants. In planta bioassay of GLH and BPH performed on these T2 progenies exhibited radical reduction in survivability and fecundity compared with the untransformed control plants.

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Abbreviations

ANOVA:

Analysis of variance

ASAL:

Allium sativum agglutinin from leaf

BPH:

Brown planthopper

ELISA:

Enzyme-linked immunosorbent assay

GLH:

Green leafhopper

PCR:

Polymerase chain reaction

References

  • Bai X, Wang Q, Chu C (2008) Excision of a selective marker in transgenic rice using a novel Cre/loxP system controlled by a floral specific promoter. Transgenic Res 17:1035–1043. doi:10.1007/s11248-008-9182-7

    Article  CAS  PubMed  Google Scholar 

  • Bandyopadhyay S, Roy A, Das S (2001) Binding of garlic (Allium sativum) leaf lectin to the gut receptors of homopteran pests is correlated to its insecticidal activity. Plant Sci 161:1025–1033

    Article  CAS  Google Scholar 

  • Bar M, Lesham B, Gilboa N, Gidoni D (1996) Visual characterization of recombination at FRT-gusA loci in transgenic tobacco mediated by constitutive expression of the native FLP recombinase. Theor Appl Genet 43:407–413. doi:10.1007/BF00223183

    Article  Google Scholar 

  • Chakraborti D, Sarkar A, Mondal HA, Schuermann D, Hohn B, Sarmah BK, Das S (2008) Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect. Plant Cell Rep 27:1623–1633. doi:10.1007/s00299-008-0585-y

    Article  CAS  PubMed  Google Scholar 

  • Chakraborti D, Sarkar A, Mondal HA, Das S (2009) Tissue specific expression of potent insecticidal, Allium sativum leaf agglutinin (ASAL) in important pulse crop, chickpea (Cicer arietinum L.) to resist the phloem feeding Aphis craccivora. Transgenic Res. doi:10.1007/s11248-009-9242-7

  • Cotsaftis O, Sallaud C, Breitler JC, Meynard D, Greco R, Pereira A, Guiderdoni E (2002) Transposon-mediated generation of T-DNA and marker free rice plants expressing a Bt endotoxin gene. Mol Breed 10:165–180. doi:10.1023/A:1020380305904

    Article  CAS  Google Scholar 

  • Dahal G, Shrestha RB, Thapa RB (1997) Species composition and relative abundance of rice green leafhoppers (Nephotettix spp.) in different altitudinal regions of Nepal. Int J Pest Manag 43:49–58

    Article  Google Scholar 

  • Dale EC, Ow DW (1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA 88:10558–10562

    Article  CAS  PubMed  Google Scholar 

  • Daley M, Knauf V, Summerfelt KR, Turner JC (1998) Co-transformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants. Plant Cell Rep 17:489–496

    Article  CAS  Google Scholar 

  • Djukanovic V, Lenderts B, Bidney D, Lyznik LA (2008) A Cre:FLP fusion protein recombines FRT or loxP sites in transgenic maize plants. Plant Biotechnol J 6:770–781. doi:10.1111/j.1467-7652.2008.00357.x

    Article  CAS  PubMed  Google Scholar 

  • Dutta I, Saha P, Majumder P, Sarkar A, Chakraborti D, Banerjee S, Das S (2005a) The efficacy of a novel insecticidal protein, Allium sativum leaf lectin (ASAL), against homopteran insects monitored in transgenic tobacco. Plant Biotechnol J 3:601–611. doi:10.1111/j.1467-7652.2005.00151.x

    Article  CAS  PubMed  Google Scholar 

  • Dutta I, Majumder P, Saha P, Ray K, Das S (2005b) Constitutive and phloem specific expression of Allium sativum leaf agglutinin (ASAL) to engineer aphid (Lipaphis erysimi) resistance in transgenic Indian mustard (Brassica juncea). Plant Sci 169:996–1007. doi:10.1016/j.plantsci.2005.05.016

    Article  CAS  Google Scholar 

  • Fitches E, Gatehouse AMR, Gatehouse JA (1997) Effect of snowdrop lectin (GNA) delivered via artificial diet and transgenic plants on the development of tomato moth (Lacanobia oleracea) larvae in laboratory and glasshouse trials. J Insect Physiol 43:727–739. doi:10.1016/S0022-1910(97)00042-5

    Article  CAS  PubMed  Google Scholar 

  • Foissac X, Loc NT, Christou P, Gatehouse AMR, Gatehouse JA (2000) Resistance to green leafhopper (Nephotettix virescens) and brown planthopper (Nilaparvata lugens) in transgenic rice expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA). J Insect Physiol 46:573–583. doi:10.1016/S0022-1910(99)00143-2

    Article  CAS  PubMed  Google Scholar 

  • Gatehouse AMR, Gatehouse JA (1998) Identifying proteins with insecticidal activity: use of encoding genes to produce insect resistant transgenic crops. Pest Sci 52:165–175. doi:10.1002/(SICI)1096-9063(199802)52:2

    Article  CAS  Google Scholar 

  • Gilbertson L (2003) Cre–lox recombination: creative tools for plant biotechnology. Trends Biotechnol 21:550–555. doi:10.1016/j.tibtech.2003.09.011

    Article  CAS  PubMed  Google Scholar 

  • Hare P, Chua NM (2002) Excision of selectable marker genes from transgenic plants. Nat Biotechnol 20:575–580. doi:10.1038/nbt0602-575

    Article  CAS  PubMed  Google Scholar 

  • Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282. doi:10.1046/j.1365-313X.1994.6020271.x

    Article  CAS  PubMed  Google Scholar 

  • Hilder VA, Powell KS, Gatehouse AMR, Gatehouse J, Gatehouse LN, Shi Y, Hamilton W, Merryweather A, Newell CA, Timans JC (1995) Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids. Transgenic Res 4:18–25. doi:10.1007/BF01976497

    Article  CAS  Google Scholar 

  • Hoa TTC, Bong BB, Huq E, Hodge TK (2002) Cre/lox site-specific recombination controls the excision of a transgene from the rice genome. Theor Appl Genet 104:518–525. doi:10.1046/j.1365-313X.1994.6020271.x

    Article  CAS  PubMed  Google Scholar 

  • Hoff T, Schnorr KM, Mundy J (2001) A recombinase-mediated transcriptional induction system in transgenic plants. Plant Mol Biol 45:41–49. doi:10.1023/A:1006402308365

    Article  CAS  PubMed  Google Scholar 

  • Loc NT, Tinjuangjun P, Gatehouse AMR, Christou P, Gatehouse JA (2002) Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance. Mol Breed 9:231–244. doi:10.1023/A:1020333210563

    Article  CAS  Google Scholar 

  • Lu HJ, Zhou XR, Gong ZX, Upadhyaya NM (2001) Generation of selectable marker-free transgenic rice using double right-border (DRB) binary vectors. Aust J Plant Physiol 28:241–248. doi:10.1071/PP00129

    CAS  Google Scholar 

  • MacKenzie D (2000) International comparison of regulatory frameworks for food products of biotechnology. Canadian Biotechnology Advisory Committee, Ottawa, Canada, p 62

    Google Scholar 

  • Moore SK, Srivastava V (2006) Efficient deletion of transgenic DNA from complex integration locus of rice mediated by Cre/lox recombination system. Crop Sci 46:700–705. doi:10.2135/cropsci2005.08-0289

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Nap J, Metz PLJ, Escaler M, Conner AJ (2003) The release of genetically modified crops into the environment. Plant J 33:1–18

    Article  PubMed  Google Scholar 

  • Odell JT, Hoopes JL, Vermerris W (1994) Seed-specific gene activation mediated by the Cre/lox site-specific recombination system. Plant Physiol 106:447–458

    Article  CAS  PubMed  Google Scholar 

  • Ow DW (2001) The right chemistry for marker gene removal? Nat Biotechnol 19:115–116. doi:10.1038/84362

    Article  CAS  PubMed  Google Scholar 

  • Powell KS, Gatehouse AMR, Hilder VA, Gatehouse AJ (1995a) Antifeedant effects of plant lectins and an enzyme on the adult stage of the rice brown planthopper, Nilaparvata lugens. Entomol Exp Appl 75:51–59. doi:10.1007/BF02382779

    Article  CAS  Google Scholar 

  • Powell KS, Gatehouse AMR, Hilder VA, Van Damme EJM, Peumans WJ, Boonjawat I, Horsham K, Gatehouse AJ (1995b) Different antimetabolic effects of related lectin towards nymphal stages of Nilaparvata lugens. Entomol Exp Appl 75:61–65. doi:10.1007/BF02382780

    Article  CAS  Google Scholar 

  • Ramesh S, Nagadhara D, Reddy VD, Rao KV (2004) Production of transgenic indica rice resistant to yellow stem borer and sap-sucking insects, using super-binary vectors of Agrobacterium tumefaciens. Plant Sci 166:1077–1085. doi:10.1016/j.plantsci.2003.12.028

    Article  CAS  Google Scholar 

  • Rao KV, Rathore KS, Hodges TK, Fu X, Stoger E, Sudhakar S, Williams P, Christou P, Bharathi M, Bown DP, Powell KS, Spence J, Gatehouse A, Gatehouse JA (1998) Expression of snowdrop lectin (GNA) in transgenic plants confers resistance to rice brown plant hopper. Plant J 15:469–477. doi:10.1046/j.1365-313X.1998.00226.x

    Article  CAS  PubMed  Google Scholar 

  • Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dynamics. Proc Natl Acad Sci USA 81:8014–8018

    Article  CAS  PubMed  Google Scholar 

  • Saha P, Majumder P, Dutta I, Ray T, Roy SC, Das S (2006a) Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sapsucking insect pests. Planta 223:1329–1343. doi:10.1007/s00425-005-0182-z

    Article  CAS  PubMed  Google Scholar 

  • Saha P, Dasgupta I, Das S (2006b) A novel approach for developing resistance in rice against phloem limited viruses by antagonizing the phloem feeding hemipteran vectors. Plant Mol Biol 62:735–752. doi:10.1007/s11103-006-9054-6

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Smeets K, Van Damme EJM, Verhaert P, Barre A, Rouge P, Leuven FV, Peumans WJ (1997) Isolation, characterization and molecular cloning of the mannose-binding lectins from leaves and roots of garlic (Allium sativum L.). Plant Mol Biol 33:223–234. doi:10.1023/A:1005717020021

    Article  CAS  PubMed  Google Scholar 

  • Sreekala C, Wu L, Gu K, Wang D, Tian D, Yin Z (2005) Excision of a selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre/loxP system. Plant Cell Rep 24:86–94. doi:10.1007/s00299-004-0909-5

    Article  CAS  PubMed  Google Scholar 

  • Sripriya R, Raghupathy V, Veluthambi K (2008) Generation of selectable marker free sheath blight resistant transgenic rice plants by efficient co-transformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain. Plant Cell Rep 27:1635–1644. doi:10.1007/s00299-008-0586-x

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Chen B, Hu Y, Li J, Lin Z (2005) Inducible excision of selectable marker gene from transgenic plants by the Cre/lox site-specific recombination system. Transgenic Res 14:605–614. doi:10.1007/s11248-005-0884-9

    Article  CAS  PubMed  Google Scholar 

  • Yoder JI, Goldsbrough AP (1994) Transformation systems for generating marker-free transgenic plants. Nat Biotechnol 12:263–267. doi:10.1038/nbt0394-263

    Article  CAS  Google Scholar 

  • Zhang W, Subbarao S, Addae P, Shen A, Armstrong C, Peschke V, Gilbertson L (2003) Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants. Theor Appl Genet 107:1157–1168. doi:10.1007/s00122-003-1368-z

    Article  CAS  PubMed  Google Scholar 

  • Zuo J, Niu QW, Moller SG, Chua NH (2001) Chemical-regulated, site-specific DNA excision in transgenic plants. Nat Biotechnol 19:157–161. doi:10.1038/84428

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors are grateful to Department of Biotechnology, Government of India for financial contributions and Bose Institute, Kolkata, India for providing infrastructure to carry out the work. The authors are also thankful to Regional Rice Research Station, Chinsurah, West Bengal, India, for providing seed stock of IR64 rice cultivar.

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Author notes

  1. Dipankar Chakraborti

    Present address: PG Department of Biotechnology, St Xavier’s College, 30, Park Street, Kolkata, 700016, India

Authors and Affiliations

  1. Plant Molecular and Cellular Genetics, Bose Institute, Centenary Campus, P1/12 CIT Scheme VII M, Kankurgachi, Kolkata, 700054, India

    Subhadipa Sengupta, Dipankar Chakraborti, Hossain A. Mondal & Sampa Das

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  1. Subhadipa Sengupta
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  2. Dipankar Chakraborti
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  3. Hossain A. Mondal
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  4. Sampa Das
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Correspondence to Sampa Das.

Additional information

Communicated by L. Jouanin.

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ESM_2 PCR analysis of T

3 progenies homozygous forASALgene Nineteen T3 progenies of L03C04(1) 01 parent showed presence of 0.362 kb amplicons for the presence of ASAL coding gene using gene-specific primer pairs F1/R1. Lane ‘-’ represents DNA from untransformed rice plant as negative control. Lane M, DNA molecular weight marker (Generuler TM, MBI Fermentas,uk) Supplementary material 2 (TIFF 842 kb)

ESM_1 Hygromycin sensitivity assay of T

1 hybrid lines (a) germinating T1 seeds in medium without hygromycin. (b)Untransformed control seeds in germination media with hygromycin 50 mg l−1, (c) T1 seeds of L03C04 hybrid (d) T1 seeds of L04C06 hybrid and (e) T1 seeds of L07C09 hybrid in germination medium supplemented with 50 mg l−1 hygromycin Supplementary material 1 (TIFF 2999 kb)

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Sengupta, S., Chakraborti, D., Mondal, H.A. et al. Selectable antibiotic resistance marker gene-free transgenic rice harbouring the garlic leaf lectin gene exhibits resistance to sap-sucking planthoppers. Plant Cell Rep 29, 261–271 (2010). https://doi.org/10.1007/s00299-010-0819-7

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