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Transgenic Research

, Volume 17, Issue 5, pp 827–838 | Cite as

Plant development inhibitory genes in binary vector backbone improve quality event efficiency in soybean transformation

  • Xudong YeEmail author
  • Edward J. Williams
  • Junjiang Shen
  • James A. Esser
  • Amy M. Nichols
  • Michael W. Petersen
  • Larry A. Gilbertson
Original Paper

Abstract

Conventional Agrobacterium-mediated plant transformation often produces a significant frequency of transgenic events containing vector backbone sequence, which is generally undesirable for biotechnology applications. We tested methods to reduce the frequency of transgenic plants containing vector backbone by incorporating genes into the backbone that inhibit the development of transgenic plants. Four backbone frequency reduction genes, bacterial levansucrase (sacB), maize cytokinin oxidase (CKX), Phaseolus GA 2-oxidase (GA 2-ox), and bacterial phytoene synthase (crtB), each expressed by the enhanced CaMV 35S promoter, were placed individually in a binary vector backbone near the left border (LB) of binary vectors. In transformed soybean plants, the lowest frequency of backbone presence was observed when the constitutively expressed CKX gene was used, followed by crtB. Higher backbone frequencies were found among the plants transformed with the GA 2-oxidase and sacB vectors. In some events, transfer of short backbone fragments appeared to be caused by LB readthrough and termination within the backbone reduction gene. To determine the effect of the backbone genes on transformation frequency, the crtB and CKX vectors were then compared to a control vector in soybean transformation experiments. The results revealed that there was no significant transformation frequency difference between the crtB and control vectors, but the CKX vector showed a significant transformation frequency decrease. Molecular analysis revealed that the frequency of transgenic plants containing one or two copies of the transgene and free of backbone was significantly increased by both the CKX and crtB backbone reduction vectors, indicating that there may be a correlation between transgene copy number and backbone frequency.

Keywords

Soybean transformation T-DNA, Vector backbone Cytokinin oxidase crtB GA 2-oxidase sacB 

Notes

Acknowledgments

The authors thank the Monsanto Middleton Soybean Transformation Team for transgenic plant production, the Middleton Trait Development team for greenhouse care, Drs. D. Somers and Y. Wan for critical reading manuscript and stimulating discussion, C. Lawson for helpful suggestions, and C. Marquez for statistical analysis.

References

  1. Agrawal P, Kohli A, Twyman R, Christou P (2005) Transformation of plants with multiple cassettes generates simple transgene integration patterns and high expression levels. Mol Breed 16:247–260CrossRefGoogle Scholar
  2. Alterpeter A, Baisakh N, Bechy R, Bock B, Capell T, Christou P et al (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Transgenic Res 15:305–327Google Scholar
  3. Armstrong DJ (1994) In: Mok DWS, Mok MC (eds) Cytokinins: chemistry, activity and function. CRC, Boca Raton, pp 139–154Google Scholar
  4. Barker RF, Idler KB, Thompson DV, Kemp JD (1983) Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol 2:335–350CrossRefGoogle Scholar
  5. Barry G, Kishore G, Padgette S, Taylor M, Kolacz K, Weldon M, Re D, Eichholtz D, Fincher K, Hallas L (1992) In: Singh BK, Flores HE, Shannon JC (eds) Biosynthesis and molecular regulation of amino acids in plants. American Society of Plant Physiologists, pp 139–145Google Scholar
  6. Burkhardt PK, Beyer P, Wünn J, Klöti A, Armstrong GA, Schledz M, von Lintig J, Potrykus I (1997) Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis. Plant J 11:1071–1078PubMedCrossRefGoogle Scholar
  7. Caimi PG, McCole LM, Kerr PS (1996) Fructan accumulation and sucrose metabolism in transgenic maize endosperm expressing a Bacillus amyloliquefaciens sacB gene. Plant Physiol 110:355–363PubMedGoogle Scholar
  8. Cairns AJ (2003) Fructan biosynthesis in transgenic plants. J Exp Bot 54:549–567PubMedCrossRefGoogle Scholar
  9. Cross MA, Warne SR, Thomas CM (1986) Analysis of the vegetative replication origin of broad-host-range plasmid RK2 by transposon mutagenesis. Plasmid 15:132–146PubMedCrossRefGoogle Scholar
  10. De Buck S, Wilde CD, Van Montague M, Depicker A (2000) T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation. Mol Breed 6:459–468CrossRefGoogle Scholar
  11. Dellaporta SL, Wood J, Hicks JB, (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21CrossRefGoogle Scholar
  12. Depicker A, Stachel S, Dhaese P, Zambryski P, Goodman HM (1982) Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet 1:561–573PubMedGoogle Scholar
  13. Dube T, Kovalchuk I, Hohn B, Thomson JA (2004) Agrobacterium tumefaciens-mediated transformation of plants by the pTF-FC2 plasmid is sufficient and strictly dependent on the MobA protein. Plant Mol Biol 55:531–539PubMedCrossRefGoogle Scholar
  14. Fray RG, Wallace A, Fraser PD, Valero D, Hedden P, Bramley PM, Grierson D (1995) Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway. Plant J 8:693–701CrossRefGoogle Scholar
  15. Fu XD, Duc LT, Fontana S, Bong BB, Tinjuangjun P, Sudhakar D, Twyman RM, Christou P, Kohli A (2000) Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns. Transgenic Res 9:11–19PubMedCrossRefGoogle Scholar
  16. Gamborg OL, Miller RA, Ojima K (1968) Plant cell culture I. Nutrient requirement of suspension cultures of soybean root cells. Exp Cell Res 50:151–158PubMedCrossRefGoogle Scholar
  17. Hanson B, Engler D, Moy Y, Newman B, Ralston E, Gutterson N (1999) A simple method to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences. Plant J 19:727–734PubMedCrossRefGoogle Scholar
  18. Horsch RB, Klee HJ (1986) Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: Role of T-DNA borders in the transfer process. PNAS 12:4428–4432CrossRefGoogle Scholar
  19. Huang S, Raman AS, Ream JE, Fujiwara H, Cerny RE, Brown SM (1998) Overexpression of 20-oxidase confers a gibberellin-overproduction phenotype in Arabidopsis. Plant Physiol 118:773–781PubMedCrossRefGoogle Scholar
  20. Huang S, Cerny RE, Qi Y, Bhat D, Aydt CM, Hanson DD, Malloy KP, Ness LA (2003) Transgenic studies on the involvement of cytokinin and gibberellin in male development. Plant Physiol 131:1270–1282PubMedCrossRefGoogle Scholar
  21. Huang S, Gilbertson L, Adams T, Malloy K, Reisenbigler E, Birr D, Snyder M, Zhang Q, Luethy M (2004) Generation of marker-free transgenic maize by regular two-border Agrobacterium transformation vectors. Transgenic Res 13:451–461PubMedCrossRefGoogle Scholar
  22. Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236:1299–1302PubMedCrossRefGoogle Scholar
  23. Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396CrossRefGoogle Scholar
  24. Kononov ME, Bassuner B, Gelvin SB (1997) Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J 11:945–957PubMedCrossRefGoogle Scholar
  25. Kuraya Y, Ohta S, Fukuda M, Hiei Y, Murai N, Hamada K, Ueki T, Imaseki H, Komari T (2004) Suppression of transfer of non-T-DNA ‘vector backbone’ sequences by multiple left border repeats in vectors for transformation of higher plants mediated by Agrobacterium tumefaciens. Mol Breed 14:309–320CrossRefGoogle Scholar
  26. Lloyd G, McCown B (1980) Commercially feasible micropropagation of mountain laural (Kalmia latifolia) by use of shoot tip culture. Proc Int Plant Prop Soc 30:421–427Google Scholar
  27. Martinell B, Julson LS, Emler CA, Huang Y, McCabe DE, Williams EJ (2002) Soybean Agrobacterium transformation method. US Patent # US6384301Google Scholar
  28. Morris RO, Bilyeu KD, Laskey JG, Cheikh NN (1999) Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem Biophys Res Commun 255:328–333PubMedCrossRefGoogle Scholar
  29. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  30. Olhoft PM, Flagel LE, Somers DA (2004) T-DNA locus structure in a large population of soybean plants transformed using the Agrobacterium-mediated cotyledonary-node method. Plant Biotechnol J 2:289–300PubMedCrossRefGoogle Scholar
  31. Pielberg G, Day AE, Plastow GS, Andersson L. (2003) A sensitive method for detecting variation in copy numbers of duplicated genes. Genome Res 13:2171–2177PubMedCrossRefGoogle Scholar
  32. Podevin N, De Buck S, De Wilde C, Depicker A. (2006) Insights into recognition of the T-DNA border repeats as termination sites for T-strand synthesis by Agrobacterium tumefaciens. Transgenic Res 15:557–571PubMedCrossRefGoogle Scholar
  33. Ramanathan V, Veluthambi K (1995) Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA. Plant Mol Biol 28:1149–1154PubMedCrossRefGoogle Scholar
  34. Röber M, Geider K, Müller-Röber B, Willmitzer L (1996) Synthesis of fructans in tubers of transgenic starch-deficient potato plant does not result in an increased allocation of carbohydrates. Planta 199:528–536PubMedCrossRefGoogle Scholar
  35. Rommens CM, Humara JM, Ye J, Richael C, Zhang L, Perry R, Swords K (2004) Crop improvement through modification of the plant’s own genome. Plant Physiol 135:421–431PubMedCrossRefGoogle Scholar
  36. Sakamoto T, Morinaka Y, Ishiyama K, Kobayashi M, Itoh H, Kayano T, Iwahori S, Matsuoka M and Tanaka H (2003) Genetic manipulation of gibberellin metabolism in transgenic rice. Nat Biotechnol 21:909–913PubMedCrossRefGoogle Scholar
  37. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbour Laboratory Press, New YorkGoogle Scholar
  38. Sanger M, Daubert S, Goodman RM (1990) Characteristics of a strong promoter from figwort mosaic virus: comparison with the analogous 35S promoter from cauliflower mosaic virus and the regulated mannopine synthase promoter. Plant Mol Biol 14:433–443PubMedCrossRefGoogle Scholar
  39. Schardl CL, Byrd AD, Benzion G, Altschuler MA, Hildebrand DF, Hunt AG (1987) Design and construction of a versatile system for the expression of foreign genes in plants. Gene 61:1–11PubMedCrossRefGoogle Scholar
  40. Shewmaker CK, Sheehy JA, Daley M, Colburn S, Ke DY (1999) Seed-specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects. Plant J 20:401–412PubMedCrossRefGoogle Scholar
  41. Steinmetz M, Le Coq D, Aymerich S, Gonzy-Treboul G, Gay P (1985) The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites. Mol Gen Genet 200:220–228PubMedCrossRefGoogle Scholar
  42. Thomas SG, Phillips AL, Hedden P (1999) Molecular cloning and functional expression of gibberellin 2-oxidases, multifunctional enzymes involved in gibberellin deactivation. PNAS 96:4698–4703PubMedCrossRefGoogle Scholar
  43. Vancanneyt G, Schmidt R, O’Connor-Sanchez A, Willmitzer L, Rocha-Sosa M (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Genet Genomics 220:245–250Google Scholar
  44. van der Graaff E, den Dulk-Ras A, Hooykaas PJJ (1996) Deviating T-DNA transfer from Agrobacterium tumefaciens to plants. Plant Mol Biol 31:677–681PubMedCrossRefGoogle Scholar
  45. van Haaren MJ, Sedee NJ, Schilperoort RA, Hooykaas PJJ (1987) Overdrive is a T-region transfer enhancer which stimulates T-strand production in Agrobacterium tumefaciens. Nucleic Acids Res 15:8983–8997PubMedCrossRefGoogle Scholar
  46. van Haaren MJJ, Sedee NJA, Krul MJT, Schilperoort RA, Hooykaas PJJ (1988) Function of heterologous and pseudo border repeats in T-region transfer via the octopine virulence system of Agrobacterium tumefaciens. Plant Mol Biol 11:773–781CrossRefGoogle Scholar
  47. Wenck A, Czakó M, Kanevski I, Márton L (1997) Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation. Plant Mol Biol 34:913–922PubMedCrossRefGoogle Scholar
  48. Werner T, Motyka V, Strand M, Schmülling T (2001) Regulation of plant growth by cytokinin. PNAS 98:10487–10492PubMedCrossRefGoogle Scholar
  49. Werner T, Motyka V, Laucou V, Smets R, van Onckelen H, Schmülling T (2003) Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15:2532–2550PubMedCrossRefGoogle Scholar
  50. Yang S, Yu H, Xu Y, Goh CJ (2003) Investigation of cytokinin-deficient phenotypes in Arabidopsis by ectopic expression of orchid DSCKX1. FEBS Lett 555:291–296PubMedCrossRefGoogle Scholar
  51. Ye X, Wu X, Zhao H, Frehner M, Nösberger N, Potrykus I, Spangenberg G (2001) Altered fructan accumulation in transgenic Lolium multiflorum plants expressing a Bacillus subtilis sacB gene. Plant Cell Rep 20:205–212CrossRefGoogle Scholar
  52. Zupan J, Muth TR, Draper O, Zambryski P (2000) The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J 23:11–28PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Xudong Ye
    • 1
    Email author
  • Edward J. Williams
    • 1
  • Junjiang Shen
    • 1
  • James A. Esser
    • 1
  • Amy M. Nichols
    • 1
  • Michael W. Petersen
    • 1
  • Larry A. Gilbertson
    • 2
  1. 1.Agracetus CampusMonsanto CompanyMiddletonUSA
  2. 2.Monsanto CompanySt. LouisUSA

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