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Evaluation of a morphological marker selection and excision system to generate marker-free transgenic cassava plants

  • Genetic Transformation and Hybridization
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Abstract

The efficacy of the ipt-type Multi-Auto-Transformation (MAT) vector system to transform the extensively grown cassava cultivar “KU50” was evaluated. This system utilizes the isopentenyltransferase (ipt) gene as morphological marker for visual selection of transgenic lines. The extreme shooty phenotype (ESP) of transgenic lines is lost due to the removal of ipt gene mediated by the yeast Rint/RS system. As a result, phenotypically normal shoots, considered marker-free transgenic plants, could be obtained. When transforming KU50 cassava cultivar with two different ipt-type MAT vectors, transformation frequency at 19–21% was observed. Among the total number of ESP explants, 32–38% regained normal extended shoot phenotype and 88–96% of which were confirmed to represent the marker-free transgenic plants. This is the first demonstration of the efficacy of Rint/RS system in promoting excision of ipt marker gene in cassava specie, with the consequent rapid production of marker-free transgenic plants. The high efficiency of this system should facilitate pyramiding a number of transgenes by repeated transformation without having to undergo through laborious, expensive and time-consuming processes of sexual crossing and seed production. The generation of marker-free, thus environmentally safe, genetically modified cassava clones should also ease the public concerns regarding the use of transgenic cassava in both food and nonfood industries.

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References

  • Araki H, Jearnpipatkula A, Tatsumi H, Sakurai T, Ushino K, Muta T, Oshima Y (1987) Molecular and functional organization of yeast plasmid pSR1. J Mol Biol 182:191–203

    Article  Google Scholar 

  • Ballester A, Cervera M, Pena L (2007) Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by sitespecific recombination. Plant Cell Rep 26:39–45

    Article  PubMed  CAS  Google Scholar 

  • Ballester A, Cervera M, Pena L (2008) Evaluation of selection strategies alternative to nptII in genetic transformation of citrus. Plant Cell Rep 27:1005–1015

    Article  PubMed  CAS  Google Scholar 

  • Chabuad M, de Carvalho-Niebel F, Barker DG (2003) Efficient transformation of Medicago truncata cv. Jemalong using the hypervirulent Agrobacterium tumefaciens strain AGL1. Plant Cell Rep 22:46–51

    Article  Google Scholar 

  • Cui M, Takayanagi K, Kamada H, Nishimura S, Handa T (2001) Efficient shoot regeneration from hairy roots of Antirrhinum majus L. transformed by the rol-type MAT vector system. Plant Cell Rep 20:55–59

    Article  CAS  Google Scholar 

  • Darbani B, Elimanifar A, Stewart CN, Camargo WN (2007) Methods to produce marker-free transgenic plants. Biotechnol J 2:83–90

    Article  PubMed  CAS  Google Scholar 

  • Ebinuma H, Komamine A (2001) MAT (Multi-Auto-Transformation) vector system. The oncogenes of Agrobacterium as positive markers for regeneration and selection of marker-free transgenic plants. In Vitro Cell Dev Biol Plant 37:103–113

    Article  CAS  Google Scholar 

  • Ebinuma H, Sugita K, Matsunaga E, Yamakado M (1997) Selection of marker-free transgenic plants using the isopenthenyl transferase gene. Proc Natl Acad Sci USA 94:2117–2121

    Article  PubMed  CAS  Google Scholar 

  • Endo S, Kasahara T, Sugita K, Matsunaga E (2001) The isopentenyl transferase gene is effective as a selectable marker gene for plant transformation in tobacco (Nicotiana tabacum cv. Petite Havana SRI). Plant Cell Rep 20:60–66

    Article  CAS  Google Scholar 

  • Endo S, Sugita K, Sakai M, Tanaka H, Ebinuma H (2002) Single-step transformation for generating marker-free transgenic rice using the ipt-type MAT vector system. Plant J 30:115–122

    Article  PubMed  CAS  Google Scholar 

  • Gleave AP, Mitra DS, Mudge SR, morris BAM (1999) Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene. Plant Mol Biol 40:223–235

    Article  PubMed  CAS  Google Scholar 

  • Goldstein DA, Tinland B, Gilbertson LA, Staub JM, Bannon GA, Goodman RE, Mc Coy RL, Silvanovich A (2005) Human safety and genetically modified plants: a review of antibiotic resistance markers and future transformation selection technologies. J Appl Microbiol 99:7–23

    Article  PubMed  CAS  Google Scholar 

  • Gonzalez AE, Schopke C, Taylor NJ, Beachy RN, Fauquet CM (1998) Regeneration of transgenic cassava plants (Manihot esculenta Crantz) through Agrobacterium-mediated transformation of embryogenic suspension cultures. Plant Cell Rep 17:827–831

    Article  CAS  Google Scholar 

  • Hohn B, Levy A, Putcha H (2001) Elimination of selection markers from transgenic plants. Curr Opin Biotechnol 12:139–143

    Article  PubMed  CAS  Google Scholar 

  • Khan RS, Chin DP, Nakamura I, Mii M (2006) Production of marker-free transgenic Nierembergia caerulea using MAT vector system. Plant Cell Rep 25:914–919

    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

    Article  PubMed  CAS  Google Scholar 

  • Li Y, Hagen G, Guifoyle J (1992) Altered morphology in transgenic tobacco plants that overproduce cytokinins in specific tissues and organs. Dev Biol 153:386–395

    Article  PubMed  CAS  Google Scholar 

  • Li H-Q, Huang YW, Liang CY, Guo JY, Liu HX, Potrykus I, Puonti-Kaerlas J (1998) Regeneration of cassava plants via shoot organogenesis. Plant Cell Rep 17:410–414

    Article  CAS  Google Scholar 

  • Matsunaga E, Sugita K, Ebinuma H (2002) Asexual production of selectable-marker free transgenic woody plants, vegetatively propagated species. Mol Breed 10:95–106

    Article  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Google Scholar 

  • Siritunga D, Sayre RT (2003) Generation of cyanogens-free transgenic cassava. Planta 217:367–373

    Article  PubMed  CAS  Google Scholar 

  • Soni R, Murray JAH (1994) Isolation of intact DNA and RNA from plant tissues. Anal Biochem 218:474–476

    Article  PubMed  CAS  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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Szabados L, Hoyos R, Roca W (1987) In vitro somatic embryo genesis and plant regeneration of cassava. Plant Cell Rep 6:248–251

    Article  CAS  Google Scholar 

  • Taylor N, Chavarriaga P, Raemakers K, Siritunga D, Zhang P (2004) Development and application of transgenic technologies in cassava. Plant Mol Biol 56:671–688

    Article  PubMed  CAS  Google Scholar 

  • Vetten N, Wolters AM, Raemkers K, Meer I, Stege R, Heeres E, Heeres P, Visser R (2003) A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop. Nat Biotechnol 21:4339–4442

    Google Scholar 

  • Zelasco S, Ressegotti V, Confalonieri M, Carbonera D, Calligari P, Bonadei M, Bisoffi S, Yamada K, Balestrazzi A (2007) Evaluation of MAT-vector system in white poplar (Populus alba L.) and production of ipt marker-free transgenic plants by ‘single-step transformation’. Plant Cell Tissue Organ Cult 91:61–72

    Article  CAS  Google Scholar 

  • Zhang P, Vanderschuren H, Futterer J, Gruissem W (2005) Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genes. Plant Biotechnol J 3:385–397

    Article  PubMed  Google Scholar 

  • Zubko E, Scutt C, Meyer P (2000) Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nat Biotechnol 18:442–445

    Article  PubMed  CAS  Google Scholar 

  • Zuo J, Niu QW, Ikeda Y, Chua NH (2002) Marker-free transformation: increasing transformation frequency by the use of regeneration-promoting genes. Curr Opin Biotechnol 13:173–180

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by a grant from National Center for Genetic Engineering and Biotechnology (BIOTEC) Project code BT-B-06-PG-14-460. LS would like to thank for a scholarship from Thailand Graduate Institute of Science and Technology (TGIST) No. TG-B-11-22-14-831M. We would like to thank Dr. Opas Boonseng from Rayong Agronomical Research Center, Thailand, for kindly providing cassava plants. We appreciated Onuma Tangsomsuk, Yaowalak Maengpang and Dalad Teainthong for their excellent plant tissue culture assistance.

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Authors and Affiliations

  1. Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok, 10400, Thailand

    Laddawan Saelim & Jarunya Narangajavana

  2. Scientific Equipment Center, KURDI, Kasetsart University, Bangkok, Thailand

    Salak Phansiri

  3. National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani, Thailand

    Malinee Suksangpanomrung & Supatcharee Netrphan

  4. Center for Cassava Molecular Biotechnology (CASS-MOL-BIOTECH), Faculty of Science, Mahidol University, Bangkok, Thailand

    Jarunya Narangajavana

Authors
  1. Laddawan Saelim
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  2. Salak Phansiri
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  3. Malinee Suksangpanomrung
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  4. Supatcharee Netrphan
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  5. Jarunya Narangajavana
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Correspondence to Jarunya Narangajavana.

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Communicated by H. Ebinuma.

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Saelim, L., Phansiri, S., Suksangpanomrung, M. et al. Evaluation of a morphological marker selection and excision system to generate marker-free transgenic cassava plants. Plant Cell Rep 28, 445–455 (2009). https://doi.org/10.1007/s00299-008-0658-y

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  • DOI: https://doi.org/10.1007/s00299-008-0658-y

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