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The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants

  • Genetic Transformation and Hybridization
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Abstract

A selection system based on the phosphomannose-isomerase gene (pmi) as a selectable marker and mannose as the selective agent was evaluated for the transformation of apple (Malus domestica Borkh.). Mannose is an unusable carbon source for many plant species. After uptake, mannose is phosphorylated by endogenous hexokinases to mannose-6-phosphate. The accumulation of mannose-6-phosphate leads to a block in glycolysis by inhibition of phosphoglucose-isomerase, resulting in severe growth inhibition. The phosphomannose-isomerase is encoded by the manA gene from Escherichia coli and catalyzes the conversion of mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis. Transformed cells expressing the manA gene can therefore utilize mannose as a carbon and survive on media containing mannose. The manA gene along with a β-glucuronidase (GUS) gene was transferred into apple cv. ‘Holsteiner Cox’ via Agrobacterium tumefaciens-mediated transformation. Leaf explants were selected on medium supplemented with different concentrations and combinations of mannose and sorbitol to establish an optimized mannose selection protocol. Transgenic lines were regenerated after an initial selection pressure of 1–2 g l−1 mannose in combination with 30 g l−1 sorbitol followed by a stepwise increase in the mannose concentration up to 10 g l−1 and simultaneous decrease in the sorbitol concentration. Integration of transgenes in the apple genome of selected plants was confirmed by PCR and southern blot analysis. GUS histochemical and chlorophenol red (CPR) assays confirmed activity of both transgenes in regenerated plants. The pmi/mannose selection system is shown to be highly efficient for producing transgenic apple plants without using antibiotics or herbicides.

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Abbreviations

BAP:

6-Benzylaminopurine

CaMV:

Cauliflower mosaic virus

CMPS:

Cestrium yellow leaf curling virus promoter shorter version

GUS:

β-Glucuronidase

IBA:

Indole-3-butyric acid

Man:

Mannose

MS:

Murashige and Skoog

pmi :

Phosphomannose-isomerase

Sor:

Sorbitol

Suc:

Sucrose

TDZ:

Thidiazuron

YEP:

Yeast extract broth

References

  • Aswath CR, Mo SY, Kim DH, Park SW (2006) Agrobacterium and biolistic transformation of onion using non-antibiotic selection marker phosphomannose isomerase. Plant Cell Rep 25:92–99

    Article  PubMed  CAS  Google Scholar 

  • Belfanti E, Silfverberg-Dilworth E, Tartarini S, Patocchi A, Barbieri M, Zhu J, Vinatzer BA, Gianfranceschi L, Gessler C, Sansavini S (2004) The HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety. Proc Natl Acad Sci USA 101:886–890

    Article  PubMed  CAS  Google Scholar 

  • Bolar JP, Brown SK, Norelli JL, Aldwinckle HS (1999) Factors affecting the transformation of ‘Marshall McIntosh’ apple by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 55:31–38

    Article  Google Scholar 

  • Bolar JP, Norelli JL, Wong KW, Hayes K, Harman GE, Aldwinckle HS (2000) Expression of endochitinase from Trichoderma harzianum in transgenic apple increases resistance to apple scab and reduces vigor. Phytopathology 90:72–77

    CAS  Google Scholar 

  • Bolar JP, Norelli JL, Wong KW, Hayes K, Harman GE, Brown SK, Aldwinckle HS (2001) Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (T. harzianum) against the pathogenic fungus (Venturia inaequalis) in transgenic apple plants. Transgenic Res 10:533–543

    Article  PubMed  CAS  Google Scholar 

  • Boscariol RL, Almeida WAB, Derbyshire MTVC, Mourão Filho FAA, Mendes BMJ (2003) The use of the pmi/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck). Plant Cell Rep 22:122–128

    Article  PubMed  CAS  Google Scholar 

  • Cheng L, Zhou R, Reidel EJ, Sharkey TD, Dandekar AM (2005) Antisense inhibition of sorbitol synthesis leads to up-regulation of starch synthesis without altering CO2 assimilation in apple leaves. Planta 220:767–776

    Article  PubMed  CAS  Google Scholar 

  • De Bondt A, Eggermont K, Penninckx I, Goderis I, Broekaert WF (1996) Agrobacterium-mediated transformation of apple (Malus × domestica Borkh.): An assessment of factors affecting regeneration of transgenic plants. Plant Cell Rep 15:549–554

    Article  CAS  Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Faize M, Malnoy M, Dupuis F, Chevalier M, Parisi L, Chevreau E (2003) Chitinases of Trichoderma atroviride induce scab resistance and some metabolic changes in two cultivars of apple. Phytopathology 93:1496–1504

    CAS  Google Scholar 

  • Flachowsky H, Birk T, Hanke V (2004) Preliminary results to establish an alternative selection system for apple transformation. Acta Hortic 663:425–430

    Google Scholar 

  • Ghorbel R, La-Malfa S, López MM, Petit A, Navarro L, Peña L (2001) Additional copies of virG from pTiBo542 provide a super-transformation ability to Agrobacterium tumefaciens in citrus. Physiol Mol Plant Pathol 58:103–110

    Article  CAS  Google Scholar 

  • Goldsworthy A, Street HE (1965) The carbohydrate nutrition of tomato roots VIII. The mechanism of the inhibition by d-mannose of the respiration of excised roots. Ann Bot 29:45–58

    CAS  Google Scholar 

  • He Z, Fu Y, Si H, Hu G, Zhang S, Yu Y, Sun Z (2004) Phosphomannose-isomerase (pmi) gene as a selectable marker for rice transformation via Agrobacterium. Plant Sci 166:17–22

    Article  CAS  Google Scholar 

  • Holefors A, Xue ZT, Welander M (1998) Transformation of apple rootstock M26 with the rolA gene and its influence on growth. Plant Sci 136:69–78

    Article  CAS  Google Scholar 

  • Holefors A, Xue ZT, Zhu LH, Welander M (2000) The Arabidopsis phytochrome B gene influences growth of the apple rootstock M26. Plant Cell Rep 19:1049–1056

    Article  CAS  Google Scholar 

  • Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium vectors for plant transformation. Transgenic Res 2:208–218

    Article  CAS  Google Scholar 

  • Hsu JC (1996) Multiple comparisons—Theory and methods. Chapman & Hall, London, p 277

    Google Scholar 

  • Jefferson RA (1989) The GUS reporter system. Nature 342:837–838

    Article  PubMed  CAS  Google Scholar 

  • Joersbo M, Donaldson I, Kreiberg J, Petersen SG, Brunstedt J, Okkels FT (1998) Analysis of mannose selection used for transformation of sugar beet. Mol Breed 4:111–117

    Article  CAS  Google Scholar 

  • Joersbo M, Okkels T (1996) A novel principle for selection of transgenic plant cells: Positive selection. Plant Cell Rep 16:219–221

    Article  CAS  Google Scholar 

  • Ko K, Norelli JL, Reynoird JP, Aldwinckle HS, Brown S (2002) T4 lysozyme and attacin genes enhance resistance of transgenic ‘Galaxy’ apple against Erwinia amylovora. J Am Soc Hortic Sci 127:515–519

    CAS  Google Scholar 

  • Ko K, Norelli JL, Reynoird JP, Boresjza-Wysocka E, Brown SK, Aldwinckle HS (2000) Effect of untranslated leader sequence of AMV RNA 4 and signal peptide of pathogenesis-related protein 1b on attacin gene expression, and resistance to fire blight in transgenic apple. Biotechnol Lett 22:373–381

    Article  CAS  Google Scholar 

  • Kramer C, DiMaio J, Carswell G, Shillito R (1993) Selection of transformed protoplast-derived Zea mays colonies with phosphinotricin and a novel assay using the pH indicator chlorophenol red. Planta 190:454–458

    Article  CAS  Google Scholar 

  • Krens FA, Pelgrom KTB, Schaart JG, den Nijs APM, Rouwendal GJA (2004a) Clean vector technology for marker free transgenic fruit crops. Acta Hortic 663:431–435

    CAS  Google Scholar 

  • Krens FA, Pelgrom KTB, Schaart JG, den Nijs APM, Rouwendal GJA (2004b) Clean vector technology for marker free transgenic ornamentals. Acta Hortic 651:101–105

    CAS  Google Scholar 

  • Liu Q, Ingersoll J, Owens L, Salih S, Meng R, Hammerschlag F (2001) Response of transgenic Royal Gala apple (Malus × domestica Borkh.) shoots carrying a modified cecropin MB39 gene, to Erwinia amylovora. Plant Cell Rep 20:306–312

    Article  Google Scholar 

  • Lucca P, Ye X, Potrykus I (2001) Effective selection and regeneration of transgenic rice plants with mannose as selective agent. Mol Breed 7:43–49

    Article  CAS  Google Scholar 

  • Maximova SN, Dandekar AM, Guiltinan MJ (1998) Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: High transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate-limiting. Plant Mol Biol 37:549–559

    Article  PubMed  CAS  Google Scholar 

  • Miles J, Guest J (1984) Nucleotide sequence and transcriptional start point of the phosphomannose-isomerase gene (manA) of Escherichia coli. Gene 32:41–48

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Negrotto D, Jolley M, Beer S, Wenck AR, Hansen G (2000) The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Rep 19:798–803

    Article  CAS  Google Scholar 

  • Puite KJ, Shaart JG (1996) Genetic modification of the commercial apple cultivars Gala, Golden Delicious and Elstar via an Agrobacterium tumefaciens-mediated transformation. Plant Sci 119:125–133

    Article  CAS  Google Scholar 

  • Pego JV, Weisbeek PJ, Smeeken SCM (1999) Mannose inhibits Arabidopsis germination via a hexokinase-mediated step. Plant Physiol 119:1017–1023

    Google Scholar 

  • Radchuk VV, Korkhovoy VI (2005) The rolB gene promotes rooting in vitro and increases fresh root weight in vivo of transformed apple scion cultivar ‘Florina’. Plant Cell Tissue Organ Cult 81:203–212

    Article  CAS  Google Scholar 

  • Ramesh SA, Kaiser BN, Franks T, Collins G, Sedgley M (2006) Improved methods in Agrobacterium-mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols. Plant Cell Rep DOI 10.1007/s00299-006-0139-0

  • Sedira M, Holefors A, Welander M (2001) Protocol for transformation of the apple rootstock Jork 9 with the rolB gene and its influence on rooting. Plant Cell Rep 20:517–524

    Article  CAS  Google Scholar 

  • Szankowski I, Briviba K, Fleschhut J, Schönherr J, Jacobsen HJ, Kiesecker H (2003) Transformation of apple (Malus domestica Borkh.) with the stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa). Plant Cell Rep 22:141–149

    Article  PubMed  CAS  Google Scholar 

  • Wang AS, Evans RA, Altendorf PR, Hanten JA, Doyle MC, Rosichan JL (2000) A mannose selection system for production of fertile transgenic maize plants from protoplasts. Plant Cell Rep 19:654–660

    Article  CAS  Google Scholar 

  • Welander M, Pawlicki N, Holefors A, Wilson F (1998) Genetic transformation of the apple rootstock M26 with the RolB gene and its influence on rooting. J Plant Physiol 153:371–380

    CAS  Google Scholar 

  • Wong KW, Harman GE, Norelli JL, Gustafson HL, Aldwinckle HS (1999) Chitinase-transgenic lines of ‘Royal Gala’ apple showing enhanced resistance to apple scab. Acta Hortic 494:595–599

    Google Scholar 

  • Wright M, Dawson J, Dunder E, Suttie J, Reed J, Kramer C, Chang Y, Novitzky R, Wang H, Artim-Moore L (2001) Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker. Plant Cell Rep 20:429–436

    Article  CAS  Google Scholar 

  • Zhu LH, Li XY, Ahlman A, Xue ZT, Welander M (2004) The use of mannose as a selection agent in transformation of the apple rootstock M26 via Agrobacterium tumefaciens. Acta Hortic 663:503–505

    CAS  Google Scholar 

  • Zhu YJ, Agbayani R, McCafferty H, Albert HH, Moore PH (2005) Effective selection of transgenic papaya plants with the PMI/Man selection system. Plant Cell Rep 24:426–432

    Article  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank Syngenta, Switzerland, for providing the plasmid pNOV2819 and Dr. Michael Wallbraun, Centrum Grüne Gentechnik, Germany, for introducing the GUS gene into pNOV2819. The work was supported by CAPES, Brazil, by a scholarship to Juliana Degenhardt and Stiftung Gisela.

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Authors and Affiliations

  1. Institute of Biological Production Systems, Fruit Science Section, University of Hannover, Herrenhaeuser Str. 2, 30419, Hannover, Germany

    Juliana Degenhardt, Annika Poppe, Jurith Montag & Iris Szankowski

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  1. Juliana Degenhardt
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  2. Annika Poppe
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  3. Jurith Montag
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  4. Iris Szankowski
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Correspondence to Iris Szankowski.

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Communicated by H. Lörz

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Degenhardt, J., Poppe, A., Montag, J. et al. The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants. Plant Cell Rep 25, 1149–1156 (2006). https://doi.org/10.1007/s00299-006-0179-5

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  • DOI: https://doi.org/10.1007/s00299-006-0179-5

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