Molecular Breeding

, Volume 19, Issue 1, pp 69–85 | Cite as

Field trial detects incomplete barstar attenuation of vegetative cytotoxicity in Populus trees containing a poplar LEAFY promoter::barnase sterility transgene

  • Hao Wei
  • Richard Meilan
  • Amy M. Brunner
  • Jeffrey S. Skinner
  • Caiping Ma
  • Harish T. Gandhi
  • Steven H. Strauss
Article

Abstract

We tested the efficacy of an attenuation system developed to preclude the deleterious effects of floral promoter::cytotoxin genes on vegetative growth of transgenic sterile plants. We tested the promoter (2.6 kb 5′ region) of the poplar LEAFY gene PTLF driving barstar, combined on the same T-DNA with barstar driven by either the CaMV 35S basal promoter +5 to −72 fragment (35SBP), 35SBP fused to the TMV omega element (35SBP omega), or the NOS promoter. The unattenuated pPTLF::barnase construct failed to give rise to any transgenic events, suggesting substantial non-reproductive expression from this promoter. The barstar-attenuated constructs enabled transformation, but the rate was reduced by nearly one-third. Four events (7% of attenuated events) had highly abnormal morphology, and were identified during the early phases of propagation; these events had significantly higher barnase:barstar expression ratios based on quantitative RT-PCR. A greenhouse study showed that phenotypically normal attenuated plants grew at the same rate as wild-type and barnase-lacking transgenic plants. A statistically significant positive linear association was found between relative growth rate (RGR) and barstar:barnase ratio in the attenuated events, and graphical analysis suggested a threshold for barstar attenuation of barnase, above which additional levels of barstar did not provide further attenuation. Surprisingly, the appearance and growth rate of the nearly all of the attenuated events were substantially reduced after one or two growing seasons in the field, and the extent of growth reduction was associated with barstar:barnase expression ratio. These results demonstrate the importance of field testing during early phases of research to identify pleiotropic effects of transgenic sterility genes in trees.

Keywords

Ablation Biosafety Containment Biotechnology Forestry Gene flow Genetic engineering Trees Sterility Genetic modification Barnase Barstar LEAFY 

References

  1. Allen GC, Hall G Jr, Michalowski G, Newman W, Spiker S, Weissinger AK, Thompson WF (1996) High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco. Plant Cell 8:899–913PubMedCrossRefGoogle Scholar
  2. Allen GC, Spiker S, Thompson WF (2000) Use of matrix attachment regions (MARs) to minimize transgene silencing. Plant Mol Biol 43:361–376PubMedCrossRefGoogle Scholar
  3. Beals TP, Goldberg RB (1997) A novel cell ablation strategy blocks tobacco anther dehiscence. Plant Cell 9:1527–1545PubMedCrossRefGoogle Scholar
  4. Bhalero R, Nilsson O, Sandberg G (2003) Out of the woods: forest biotechnology enters the genomic era. Curr Opin Biotechnol 14:206–213CrossRefGoogle Scholar
  5. Bi YM, Rothstein SJ, Wildeman AG (2001) A novel strategy for regulated expression of a cytotoxic gene. Gene 279:175–179PubMedCrossRefGoogle Scholar
  6. Block M, Debrouwer D (1993) Engineered fertility control in transgenic Brassica napus L.: histochemical analysis of anther development. Planta 189:218–225CrossRefGoogle Scholar
  7. Block M, Debrouwer D, Moens T (1997) The development of a nuclear male sterility system in wheat. Expression of the barnase gene under the control of tapetum specific promoters. Theor Appl Genet 95:125–131CrossRefGoogle Scholar
  8. Boerjan W (2005) Biotechnology and the domestication of forest trees. Curr Opin Biotechnol 16:159–166PubMedCrossRefGoogle Scholar
  9. Brunner AM, Rottmann WH, Sheppard LA, Krutovskii K, DiFazio SP, Leonardi S, Strauss SH (2000) Structure and expression of duplicate AGAMOUS orthologues in poplar. Plant Mol Biol 44:619–634PubMedCrossRefGoogle Scholar
  10. Brunner A, Li J, DiFazio SP, Shevchenko O, Montgomery B, Mohamed R, Wei H, Ma C, Elias A, Van Wormer K, Strauss SH (2006) Genetic containment of forest plantations. Tree Genet Genomes (in press)Google Scholar
  11. Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BioMed Central Plant Biology 4:14. DOI:10.1186/1471-2229-4-14Google Scholar
  12. Burgess DG, Ralston EJ, Hanson WG, Heckert M, Ho M, Jenq T, Palys JM, Tang K, Gutterson N (2002) A novel, two-component system for cell lethality and its use in engineering nuclear male-sterility in plants. Plant J 31:113–125PubMedCrossRefGoogle Scholar
  13. Filichkin SA, Meilan R, Busov VB, Ma C, Brunner AM, Strauss SH (2006) Alcohol-inducible gene expression in transgenic Populus. Plant Cell Rep 25:660–667PubMedCrossRefGoogle Scholar
  14. Goldman MH, Goldberg RB, Mariani C (1994) Female sterile tobacco plants are produced by stigma-specific cell ablation. EMBO J 13:2976–2984PubMedGoogle Scholar
  15. Han KH, Ma C, Strauss SH (1997) Matrix attachment regions (MARs) enhance transformation frequency and transgene expression in poplar. Transgenic Res 6:415–420CrossRefGoogle Scholar
  16. Hartley RW (1988) Barnase and barstar: expression of its cloned inhibitor permits expression of a cloned ribonuclease. J Mol Biol 202:913–915PubMedCrossRefGoogle Scholar
  17. Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaus PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832PubMedCrossRefGoogle Scholar
  18. Höfig KP, Möller R, Donaldson L, Putterill J, Walter C (2006) Towards male-sterility in Pinus radiata – a stilbene synthase approach to genetically engineer nuclear male sterility. Plant Biotechnol J 4:333–343PubMedCrossRefGoogle Scholar
  19. Holsters M, de Waele D, Depicker A, Messens E, Montagu MV, Schell J (1978) Transfection and transformations of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187PubMedCrossRefGoogle Scholar
  20. James R, DiFazio S, Brunner A, Strauss SH (1998) Environmental effects of genetically engineered woody biomass crops. Biomass Bioenerg 14:403–414CrossRefGoogle Scholar
  21. Kyozuka J, Harcourt R, Peacock WJ, Dennis ES (1997) Eucalyptus has functional equivalents of the Arabidopsis AP1 gene. Plant Mol Biol 35:573–584PubMedCrossRefGoogle Scholar
  22. Lemmetyinen J, Sopanen T (2004a) Modification of flowering in forest trees. In: Kumar S, Fladung M (eds) Molecular genetics and breeding of forest trees. Haworth Press Inc., NY, pp 263–292Google Scholar
  23. Lemmetyinen J, Sopanen T (2004b) Prevention of the flowering of a tree, silver birch. Mol Breeding 13:243–249CrossRefGoogle Scholar
  24. Leuchtenberger S, Perz A, Gatz C, Bartsch JW (2001) Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells. Nucleic Acids Res 29:1–6CrossRefGoogle Scholar
  25. Mariani C, Beuckeleer MD, Truettner J, Leemans J, Goldberg RB (1990) Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature 347:737–741CrossRefGoogle Scholar
  26. Mariani C, Gossele V, Beuckeleer MD, Block MD, Goldberg RB, Greef WD, Leemans J (1992) A chimaeric ribonuclease-inhibitor gene restores fertility to male sterile plants. Nature 357:384–387CrossRefGoogle Scholar
  27. Meilan R, Ellis D, Pilate G, Brunner AM, Skinner J (2004) Accomplishments and challenges in genetic engineering of forest trees. In: Strauss SH, Bradshaw HD (eds) The bioengineered forest: challenges to science and society. Resources for the Future, Washington, DC, pp 36–51Google Scholar
  28. Merkle SA, Dean JFD (2000) Forest tree biotechnology. Curr Opin Biotechnol 11:298–302PubMedCrossRefGoogle Scholar
  29. Nilsson O, Wu E, Wolfe DS, Weigel D (1998) Genetic ablation of flowers in transgenic Arabidopsis. Plant J 15:799–804PubMedCrossRefGoogle Scholar
  30. Paddon CJ, Hartley RW (1986) Cloning, sequencing and transcription of an inactivated copy of Bacillus amyloliquefaciens extracellular ribonuclease (barnase). Gene 40:231–239CrossRefGoogle Scholar
  31. Paddon CJ, Hartley RW (1987) Expression of Bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivating mutation. Gene 53:11–19PubMedCrossRefGoogle Scholar
  32. Peña L, Séguin A (2001) Recent advances in genetic transformation of trees. Trends Biotechnol 19:500–506PubMedCrossRefGoogle Scholar
  33. Rottmann WH, Meilan R, Sheppard LA, Brunner AM, Skinner JS, Ma C, Cheng S, Jouanin L, Pilate G, Strauss SH (2000) Diverse effects of over expression of LEAFY and PTLF, a poplar (Populus) homolog of LEAFY/FLOICAULA, in transgenic poplar and Arabidopsis. Plant J 22:235–245PubMedCrossRefGoogle Scholar
  34. Skinner JS, Meilan R, Brunner AM, Strauss SH (2000) Options for genetic engineering of floral sterility in forest trees. In: Jain SM, Minocha SC (eds) Molecular biology of woody plants. Kluwer Academic Pubishers, Dordrecht, The Netherlands, pp 135–153Google Scholar
  35. Skinner JS, Meilan R, Ma C, Strauss SH (2003) The Populus PTD promoter imparts floral-predominant expression and enables high levels of floral–organ ablation in Populus, Nicotiana, and Arabidopsis. Mol Breed 12:119–132CrossRefGoogle Scholar
  36. Southerton SG, Strauss SH, Olive MR, Harcourt RL, Decroocq V, Zhu X, Llewellyn DJ, Peacock WJ, Dennis ES (1998a) Eucalyptus has a functional equivalent of the Arabidopsis floral meristem identity gene LEAFY. Plant Mol Biol 37:897–910CrossRefGoogle Scholar
  37. Southerton SG, Marshall H, Mouradov A, Teasdale RD (1998b) Eucalypt MADS-box genes expressed in developing flowers. Plant Physiol 118:365–372CrossRefGoogle Scholar
  38. Strauss SH, Rottmann WH, Brunner AM, Sheppard LA (1995) Genetic engineering of reproductive sterility in forest trees. Mol Breed 1:5–26CrossRefGoogle Scholar
  39. Strauss SH, DiFazio SP, Meilan R (2001) Genetically modified poplars in context. Forest Chron 77:271–279Google Scholar
  40. Valenzuela S, Strauss SH (2005) Lost in the woods. Nature Biotechnol 23:532–533CrossRefGoogle Scholar
  41. Velten J, Schell J (1985) Selection-expression plasmid vectors for use in genetic transformation of higher plants. Nucleic Acids Res 13:6981–6998PubMedGoogle Scholar
  42. Walter C, Fenning T (2004) Deployment of genetically-engineered trees in plantation forestry – An issue of concern? In: Plantation forestry biotechnology for the 21st century. Research Signpost, Kerala, India, pp 423–446Google Scholar
  43. Wei H, Meilan R, Brunner AM, Skinner JS, Ma C, Strauss SH (2006) Transgenic sterility in Populus: expression properties of the poplar PTLF, Agrobacterium NOS, and two minimal 35S promoters in vegetative tissues. Tree Physiol 26:401–410Google Scholar
  44. Weigel D et al (19 authors) (2000) Activation tagging in Arabidopsis. Plant Physiol 122:1003–1014Google Scholar
  45. Yoo WY, Bomblies K, Yoo SK, Yang JW, Choi MS, Lee JS, Weigel D, Ahn JH (2005) The 35S promoter used in a selectable marker gene of a plant transformation vector affect the expression of the transgene. Planta 221:523–530PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Hao Wei
    • 1
    • 5
  • Richard Meilan
    • 2
  • Amy M. Brunner
    • 3
  • Jeffrey S. Skinner
    • 4
  • Caiping Ma
    • 1
  • Harish T. Gandhi
    • 1
  • Steven H. Strauss
    • 1
  1. 1.Department of Forest ScienceOregon State UniversityCorvallisUSA
  2. 2.Department of Forestry and Natural ResourcesPurdue UniversityWest LafayetteUSA
  3. 3.Department of ForestryVirginia Polytechnic Institute and State UniversityBlacksburgUSA
  4. 4.Department of HorticultureOregon State UniversityCorvallisUSA
  5. 5.Linus Pauling InstituteOregon State UniversityCorvallisUSA

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