Skip to main content
SpringerLink
Log in

Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium-mediated co-transformation with the same selectable marker gene

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Rice chitinase (chi11) and tobacco osmotin (ap24) genes, which cause disruption of fungal cell wall and cell membrane, respectively, were stacked in transgenic rice to develop resistance against the sheath blight disease. The homozygous marker-free transgenic rice line CoT23 which harboured the rice chi11 transgene was sequentially re-transformed with a second transgene ap24 by co-transformation using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector pGV2260∷pSSJ1 and a multi-copy binary vector pBin19∆nptII-ap24 in the same cell. pGV2260∷pSSJ1 T-DNA carried the hygromycin phosphotransferase (hph) and β-glucuronidase (gus) genes. pBin19∆nptII-ap24 T-DNA harboured the tobacco osmotin (ap24) gene. Co-transformation of the gene of interest (ap24) with the selectable marker gene (SMG, hph) occurred in 12 out of 18 T0 plants (67%). Segregation of hph from ap24 was accomplished in the T1 generation in one (line 11) of the four analysed co-transformed plants. The presence of ap24 and chi11 transgenes and the absence of the hph gene in the SMG-eliminated T1 plants of the line 11 were confirmed by DNA blot analyses. The SMG-free transgenic plants of the line 11 harboured a single copy of the ap24 gene. Homozygous, SMG-free T2 plants of the transgenic line 11 harboured stacked transgenes, chi11 and ap24. Northern blot analysis of the SMG-free plants revealed constitutive expression of chi11 and ap24. The transgenic plants with stacked transgenes displayed high levels of resistance against Rhizoctonia solani. Thus, we demonstrate the development of transgene-stacked and marker-free transgenic rice by sequential Agrobacterium-mediated co-transformation with the same SMG.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Abad LR, D’Urzo MP, Liu D, Narasimhan ML, Reuveni M, Zhu JK, Niu X, Singh NK, Hasegawa PM, Bressan RA (1996) Antifungal activity of tobacco osmotin has specificity and involves plasma membrane permeabilization. Plant Sci 118:11–23. doi:10.1016/0168-9452(96)04420-2

    Article  CAS  Google Scholar 

  • Broglie K, Chet I, Holliday M, Cressman R, Biddle P, Knowlton S, Mauvais CJ, Broglie R (1991) Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science 254:1194–1197. doi:10.1126/science.254.5035.1194

    Article  PubMed  CAS  Google Scholar 

  • Chilton MD, Currier TC, Farrand SK, Bendich AJ, Gordon MP, Nester EW (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage not detected in crown gall tumours. Proc Natl Acad Sci USA 71:3672–3676

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • 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  PubMed  CAS  Google Scholar 

  • Dale PJ, Clarke B, Fontes EMG (2002) Potential for the environmental impact of transgenic crops. Nat Biotechnol 20:567–574. doi:10.1038/nbt0602-567

    Article  PubMed  CAS  Google Scholar 

  • Daley M, Knauf VC, 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. doi:10.1007/s002990050430

    Article  CAS  Google Scholar 

  • Darbani B, Eimanifar A, Stewart CN Jr, Camargo WN (2007) Methods to produce marker-free transgenic plants. Biotechnol J 2:83–90. doi:10.1002/biot.200600182

    Article  PubMed  CAS  Google Scholar 

  • Francois IEJA, Broekaert WF, Cammue BPA (2002) Different approaches for multi-transgene-stacking in plants. Plant Sci 163:281–295. doi:10.1016/S0168-9452(02)00130-9

    Article  CAS  Google Scholar 

  • Goldsbrough AP, Lastrella CN, Yoder JI (1993) Transposition mediated re-positioning and subsequent elimination of marker genes from transgenic tomato. Bio/Technology 11:1286–1292. doi:10.1038/nbt1193-1286

    CAS  Google Scholar 

  • Halpin C (2005) Gene stacking in transgenic plants—the challenge for 21st century plant biotechnology. Plant Biotechnol J 3:141–155. doi:10.1111/j.1467-7652.2004.00113.x

    Article  PubMed  CAS  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  PubMed  CAS  Google Scholar 

  • Hohn B, Levy AA, Puchta H (2001) Elimination of selection markers from transgenic plants. Curr Opin Biotechnol 12:139–143. doi:10.1016/S0958-1669(00)00188-9

    Article  PubMed  CAS  Google Scholar 

  • Jacob SS, Veluthambi K (2002) Generation of selection marker-free transgenic plants by cotransformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain. Plant Sci 163:801–806. doi:10.1016/SO168-9452(02)00215-7

    Article  CAS  Google Scholar 

  • Jacob SS, Veluthambi K (2003) A cointegrate Ti plasmid vector for Agrobacterium tumefaciens-mediated transformation of indica rice cv Pusa Basmati 1. J Plant Biochem Biotechnol 12:1–9

    CAS  Google Scholar 

  • Kilby NJ, Davies GJ, Snaith MR, Murray JAH (1995) FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis. Plant J 8:637–652. doi:10.1046/j.1365-313x.1995.08050637.x

    Article  PubMed  CAS  Google Scholar 

  • Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165–174. doi:10.1046/j.1365-313X.1996.10010165.x

    Article  PubMed  CAS  Google Scholar 

  • Lapierre C, Pollet B, Petit-Conil M, Toval G, Romero J, Pilate G, Leple J-C, Boerjan W, Ferret V, Nadai V, Jouanin L (1999) Structural alterations of lignins in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or caffeic acid O-methyl transferase activity have an opposite impact on the efficiency of industrial kraft pulping. Plant Physiol 119:153–163. doi:10.1104/pp.119.1.153

    Article  PubMed  CAS  Google Scholar 

  • Lin W, Anuratha CS, Datta K, Potrykus I, Muthukrishnan S, Datta SK (1995) Genetic engineering of rice for resistance to sheath blight. Bio/Technology 13:686–691. doi:10.1038/nbt0795-686

    Article  CAS  Google Scholar 

  • Lorito M, Woo SL, Ambrosio MD, Herman GE, Hayes CK, Kubicek CP, Scala F (1996) Synergistic interaction between cell wall degrading enzymes and membrane affecting compounds. Mol Plant-Microbe Interact 9:206–213. doi:10.1094/MPMI-9-0206

    Article  CAS  Google Scholar 

  • Matzke MA, Primig M, Trnovsky J, Matzke AJM (1989) Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J 8:643–649

    PubMed  CAS  Google Scholar 

  • McKnight TD, Lillis MT, Simpson RB (1987) Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant Mol Biol 8:439–445. doi:10.1007/BF00017989

    Article  CAS  Google Scholar 

  • Medberry SL, Dale E, Qin M, Ow DW (1995) Intra-chromosomal rearrangements generated by Cre-lox site specific recombination. Nucleic Acids Res 23:485–490. doi:10.1093/nar/23.3.485

    Article  PubMed  CAS  Google Scholar 

  • Melchers LS, Sela-Buurlage MB, Vloemans SA, Woloshuk CP, Van Roekel JSC, Pen J, Van den Elzen PJM, Cornelissen MJC (1993) Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1, 3-glucanase in transgenic plants. Plant Mol Biol 21:583–593. doi:10.1007/BF00014542

    Article  PubMed  CAS  Google Scholar 

  • Miki B, McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J Biotechnol 107:193–232. doi:10.1016/j.jbiotec.2003.10.011

    Article  PubMed  CAS  Google Scholar 

  • Miller M, Tagilani L, Wang N, Berka B, Bidney D, Zhao Z-Y (2002) High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system. Transgenic Res 11:381–396. doi:10.1023/A:1016390621482

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Pawlowski K, Kunze R, De Vries S, Bisseling T (1994) Isolation of total, poly (A) and polysomal RNA from plant tissues. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual. Kluwer, Dordrecht, pp D5/1–D5/13

    Google Scholar 

  • Qin M, Bayley C, Stockton T, Ow DW (1994) Cre recombinase-mediated site-specific recombination between plant chromosomes. Proc Natl Acad Sci USA 91:1706–1710

    Article  PubMed  CAS  Google Scholar 

  • Rogers SO, Bendich AJ (1988) Extraction of total cellular DNA from plants. In: Gelvin SB, Schilperoort RA, Verma DPS (eds) Plant molecular biology manual. Kluwer, Dordrecht, pp A6/1–A6/11

    Google Scholar 

  • Rosati C, Simoneau P, Treutter D, Poupard P, Cadot Y, Cadic A, Duron M (2003) Engineering flower color in forsythia by expression of two independently-transformed dihydroflavonol 4-reductase and anthocyanidin synthase genes of flavonoid pathway. Mol Breed 12:197–208. doi:10.1023/A:1026364618719

    Article  CAS  Google Scholar 

  • Sridevi G, Sabapathi N, Meena P, Nandakumar R, Samiyappan R, Muthukrishnan S, Veluthambi K (2003) Transgenic indica rice variety Pusa Basmati1 constitutively expressing a rice chitinase gene exhibits enhanced resistance to Rhizoctonia solani. J Plant Biochem Biotechnol 12:93–101

    CAS  Google Scholar 

  • Sridevi G, Parameswari C, Rajamuni P, Veluthambi K (2006) Identification of hemizygous and homozygous transgenic rice plants in T1 generation by DNA blot analysis. Plant Biotechnol 23:531–534

    Article  CAS  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  PubMed  CAS  Google Scholar 

  • Sriram S, Raghuchander T, Vidhyasekaran P, Muthukrishnan S, Samiyappan R (1997) Genetic relatedness with special reference to virulence among the isolates of Rhizoctonia solani causing sheath blight in rice. J Plant Dis Prot 104:260–271

    Google Scholar 

  • Sugita K, Matsunaga E, Ebinuma H (1999) Effective selection system for generating marker-free transgenic plants independent of sexual crossing. Plant Cell Rep 18:941–947. doi:10.1007/s002990050688

    Article  CAS  Google Scholar 

  • Sugita K, Matsunaga E, Kasahara T, Ebinuma H (2000) Transgene stacking in plants in the absence of sexual crossing. Mol Breed 6:529–536. doi:10.1023/A:1026505615615

    Article  CAS  Google Scholar 

  • Toriyama K, Hinata K (1985) Cell suspension and protoplast culture in rice. Plant Sci 41:179–183. doi:10.1016/0168-9452(85)90086-X

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge Dr Leo S. Melchers, MOGEN international NV, Netherlands, for the ap24 gene. We thank the Department of Biotechnology (DBT), Govt. of India for the research funding to K. Veluthambi and for the Post doctoral fellowship to M. V. Ramana Rao.

Author information

Authors and Affiliations

  1. Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, 625 021, Tamil Nadu, India

    Mangu Venkata Ramana Rao, Chidambaram Parameswari, Rajasekaran Sripriya & Karuppannan Veluthambi

Authors
  1. Mangu Venkata Ramana Rao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chidambaram Parameswari
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rajasekaran Sripriya
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karuppannan Veluthambi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karuppannan Veluthambi.

Additional information

Communicated by H. Ebinuma.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ramana Rao, M.V., Parameswari, C., Sripriya, R. et al. Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium-mediated co-transformation with the same selectable marker gene. Plant Cell Rep 30, 1241–1252 (2011). https://doi.org/10.1007/s00299-011-1033-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-011-1033-y

Keywords

Search

Navigation