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Transgenic white clover. Studies with the auxin-responsive promoter, GH3, in root gravitropism and lateral root development

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Abstract

We report an improved method for white clover (Trifolium repens) transformation usingAgrobacterium tumefaciens. High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed. Transgenic plants were recovered routinely from over 50% of treated cotyledons. Thebar gene and phosphinothricin selection was shown to be a more effective selection system thannptII (kanamycin selection) oraadA (spectinomycin selection). White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (β-glucuronidase) to study the involvement of auxin in root development. Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied. The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered. Expression of the GH3:GUS fusion was not enhanced by other phytohormones. A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues. In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated. For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated. Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development. Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature. These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses.

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References

  • An, G., Watson, B.D., Gordon, M.P. and Nester, E.W. (1985) New cloning vehicles for transformation of higher plants.EMBO J. 4, 277–84.

    Google Scholar 

  • Chabaud, M., Passiatore, J.E., Cannon, F. and Buchanan-Wollaston, V. (1988) Parameters affecting the frequency of kanamycin resistant alfalfa obtained byAgrobacterium tumefaciens mediated transformation.Plant Cell Rep. 7, 512–6.

    Google Scholar 

  • Chee, P.P., Fober, K.A. and Slightom, J.L. (1989) Transformation of soybean (Glycine max) by infecting germinating seeds withAgrobacterium tumefaciens.Plant Physiol. 91, 1212–8.

    Google Scholar 

  • Damiani, F., Nenz, E., Paolocci, F. and Arcioni, S. (1993) Introduction of hygromycin resistance inLotus spp throughAgrobacterium rhizogenes transformation.Transgenic Res. 2, 330–5.

    Google Scholar 

  • Deak, M., Kiss, G.B., Koncz, C. and Dudits, D. (1986) Transformation ofMedicago byAgrobacterium mediated gene transfer.Plant Cell Rep. 5, 97–100.

    Google Scholar 

  • De Block, M., Botterman, J., Vandewiele, M., Dockx, J., Thoen, C., Gossele, V. and Rao Movva, N. (1987) Engineering herbicide resistance in plants by expression of a detoxifying enzyme.EMBO J. 6, 2513–8.

    Google Scholar 

  • Evans, M.L. (1991) Gravitropism: interaction of sensitivity modulation and effector redistribution.Plant Physiol. 95, 1–5.

    PubMed  Google Scholar 

  • Gamborg, O.J. and Eveleigh, D.E. (1968) Culture methods and glucanases in suspension cultures of wheat and barley.Can. J. Biochem. 46, 417–21.

    PubMed  Google Scholar 

  • Garfinkle, D.J. and Nester, E.W. (1980)Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism.J. Bacteriol. 144, 732–43.

    PubMed  Google Scholar 

  • Gee, M.A., Hagen, G. and Guilfoyle, T.J. (1991) Tissue-specific and organ-specific expression of soybean auxin-responsive transcripts GH3 and SAURs.Plant Cell 3, 419–30.

    PubMed  Google Scholar 

  • Golds, T.J., Lee, J.Y., Husnain, T., Ghose, T.K. and Davey, M.R. (1991)Agrobacterium rhizogenes mediated transformation of the forage legumesMedicago sativa andOnobrychis viciifolia.J. Exp. Botany 42, 1147–57.

    Google Scholar 

  • Guilfoyle, T.J., Hagen, G., Li, Y., Ulmasov, T., Liu, Z. and Gee, M. (1993) Auxin-regulated transcription.Aust. J. Plant Physiol. 20, 489–502.

    Google Scholar 

  • Hagen, G., Kleinschmidt, A. and Guilfoyle, T. (1984) Auxin-regulated gene expression in intact soybean hypocotyl and excised hypocotyl sections.Planta 162, 147–53.

    Google Scholar 

  • Hagen, G., Martin, G., Li, Y. and Guilfoyle, T.J. (1991) Auxin-induced expression of the soybean GH3 promoter in transgenic tobacco plants.Plant Molec. Biol. 17, 567–79.

    Google Scholar 

  • Harrison, M.A. and Pickard, B.G. (1989) Auxin asymmetry during gravitropism by tomato hypocotyls.Plant Physiology (Besthesda) 89, 652–7.

    Google Scholar 

  • Higgins, T.J.V. and Spencer, D. (1991) The expression of a chimeric cauliflower mosaic virus (CaMV-35S)-pea vicilin gene in tobacco.Plant Sci. 74, 89–98.

    Google Scholar 

  • Horton, R.F. (1993) Gravitropism in rice seedlings: the effects of anoxia andN-1-naphthylphthalamic acid.Plant Sci. 95, 41–7.

    Google Scholar 

  • Iino, M. (1991) Mediation of tropisms by lateral translocation of endogenous IAA in maize coleoptiles.Plant Cell Environment 14, 279–86.

    Google Scholar 

  • Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. TheEMBO J. 6, 3901–7.

    Google Scholar 

  • Jones, J.D.G., Shlumukov, L., Carland, F., English, J., Scofield, S.R., Bishop, G.J. and Harrison, K. (1992) Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants.Transgenic Res. 1, 285–97.

    PubMed  Google Scholar 

  • Jordan, M.C. and Hobbs, S.L.A. (1993) Evaluation of a cotyledonary node regeneration system forAgrobacterium-mediated transformation of pea (Pisum sativum L.).In Vitro Cell. Devel. Biology — Plant 29P, 77–82.

    Google Scholar 

  • Khan, M.R.I., Tabe, L.M., Heath, L.C., Spencer, D. and Higgins, T.J.V. (1994)Agrobacterium-mediated transformation of subterranean clover (Trifolium subterraneum L).Plant Physiol. 105, 81–8.

    PubMed  Google Scholar 

  • Khandjian E.W. (1986) UV crosslinking of RNA to nylon membrane enhances hybridization signals.Molec. Biol. Rep. 11, 107–15.

    Google Scholar 

  • Lazo, G.R., Stein, P.A. and Ludwig, R.A. (1991) A transformation-competentArabidopsis genomic library inAgrobacterium.Bio/Technology 9, 963–7.

    PubMed  Google Scholar 

  • Legué, V., Vilaine, F., Tepfer, M. and Perbal, G. (1994) Modification of the gravitropic response of seedling roots of rapeseed (Brassica napus) transformed byAgrobacterium rhizogenes A4.Physiologia Plantarum 91, 559–66.

    Google Scholar 

  • Li, Y., Hagen, G. and Guilfoyle, T.J. (1991) A auxin-responsive promoter is differentially induced by auxin gradients during tropisms.Plant Cell 3, 1167–75.

    PubMed  Google Scholar 

  • Liu, Z.B., Ulmasov, T., Shi, X.Y., Hagen, G. and Guilfoyle, T.J. (1994) Soybean GH3 promoter contains multiple auxin-inducible elements.Plant Cell 6, 645–57.

    PubMed  Google Scholar 

  • Manners, J.M. (1987) Transformation ofStylosanthes species usingAgrobacterium.Plant Cell Rep. 6, 204–7.

    Google Scholar 

  • Manners, J.M. (1988) Transgenic plants of the tropical pasture legumeStylosanthes humulis.Plant Science 55, 61–8.

    Google Scholar 

  • McClure, B.A. and Guilfoyle, T. (1989) Rapid redistribution of auxin-regulated RNAs during gravitropism.Science 243, 91–3.

    PubMed  Google Scholar 

  • Morris, P. and Robbins, M.P. (1992) Condensed tannin formation byAgrobacterium rhizogenes transformed root and shoot organ cultures ofLotus corniculatus.J. Exp. Botany 43, 221–31.

    Google Scholar 

  • Napier, R.M. and Venis, M.A. (1995) Auxin action and auxin binding.New Phytologist 129, 167–201.

    Google Scholar 

  • Ozias-Akins, P., Schnall, J.A., Anderson, W.F., Singsit, C., Clemente, T.E., Adang, M.J. and Weissinger, A.K. (1993) Regeneration of transgenic peanut plants from stably transformed embryogenic callus.Plant Sci. 93, 185–94.

    Google Scholar 

  • Parrott, W.A. (1991) Auxin-stimulated somatic embryogenesis from immature cotyledons of white clover.Plant Cell Rep. 10, 17–21.

    Google Scholar 

  • Petit, A., Stougaard, J., Kuhle, A., Marcker, K.A. and Tempe, J. (1987) Transformation and regeneration of the legumeLotus corniculatus: a system for molecular studies of symbiotic nitrogen fixation.Mol. Gen. Genet. 207, 245–50.

    Google Scholar 

  • Phillips, G.C. and Collins, G.B. (1984) Red clover and other forage legumes. In Sharp, W.R., Evans, D.A., Ammirato, P.V. and Yamada, Y., eds.,Handbook of Plant Cell Culture Vol 2. Crop Species, pp. 169–210. New York: Macmillan Publishing.

    Google Scholar 

  • Pietrzak, M., Shillito, R.D., Hohn, T. and Potrykus, I. (1986) Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector.Nucl. Acids Res. 14, 5857–68.

    PubMed  Google Scholar 

  • Rolfe, B.G., Gresshoff, P.M. and Shine, J. (1980) Rapid screening for symbiotic mutants ofRhizobium and white clover.Plant. Sci. Lett. 19, 326–33.

    Google Scholar 

  • Rogers, S.O. and Bendich, A.J. (1985) Extraction of DNA from milligram quantities of fresh herbarium and mummified plant tissue.Plant. Mol. Biol. 5, 69–76.

    Google Scholar 

  • Salisbury, F.B., Gillespie, L. and Rorabaugh, P. (1988) Gravitropism in higher plant shoots. V. Changing sensitivity to auxin.Plant Physiol. 88, 1186–94.

    PubMed  Google Scholar 

  • Sarria, R., Calderon, A., Thro, A.M., Torres, E., Mayer, J.E. and Roca, W.M. (1994)Agrobacterium-mediated transformation ofStylosanthes guianensis and production of transgenic plants.Plant Science 96, 119–27.

    Google Scholar 

  • Schnall, J.A. and Weissinger, A.K. (1993) Culturing peanut (Arachis hypogaea L.) zygotic embryos for transformation via microprojectile bombardment.Plant Cell Rep. 12, 316–19.

    Google Scholar 

  • Schroeder, H.E., Khan, M.R.I., Knibb, W.R., Spencer, D. and Higgins, T.J.V. (1991) Expression of a chicken ovalbumin gene in three lucerne cultivars.Australian J. Plant Physiol. 18, 495–505.

    Google Scholar 

  • Shi, L., Miller, I. and Moore, R. (1993) Immunocytochemical localization of indole-3-acetic acid in primary roots ofZea mays.Plant Cell Environ. 16, 967–73.

    Google Scholar 

  • Spencer, T.M., Gordon-Kamm, W.J., Daines, R.J., Start, W.G. and Lemaux, P.G. (1990) Bialophos selection of stable transformations from maize cell culture.Theor. Appl. Genet. 79, 625–31.

    Google Scholar 

  • Tabe, L.M., Wardley-Richardson, T., Ceriotti, A., Aryan, A., McNabb, W., Moore, A. and Higgins, T.J.V. (1995) A biotechnological approach to improving the nutritive value of alfalfa.J. Animal Sci. (in press).

  • Thomas, M.R., Rose, R.J. and Nolan, K.E. (1992) Genetic transformation ofMedicago truncatula usingAgrobacterium with genetically modified Ri and disarmed Ti plasmids.Plant Cell Rep. 11, 113–7.

    Google Scholar 

  • Vlachova, M., Metz, B.A., Schell, J. and de Bruijn, F.J. (1987) The tropical legumeSesbania rostrata: tissue culture, plant regeneration and infection withAgrobacterium tumefaciens andrhizogenes.Plant Sci. 50, 213–23.

    Google Scholar 

  • Voisey, C.R., White, D.W.R., Dudas, B., Appleby, R.D., Ealing, P.M. and Scott, A.G. (1994)Agrobacterium-mediated transformation of white clover using direct shoot organogenesis.Plant Cell Rep. 13, 309–14.

    Google Scholar 

  • White, D.W.R. and Voisey, C. (1994) Prolific direct plant regeneration from cotyledons of white clover.Plant Cell Rep. 13, 303–8.

    Google Scholar 

  • White, D.W.R. and Greenwood, D. (1987) Transformation of the forage legumeTrifolium repens L using binaryAgrobacterium vectors.Plant Molec. Biol. 8, 461–9.

    Google Scholar 

  • Wright, R.M., Hagen, G. and Guilfoyle, T.J. (1987) An auxin-induced polypeptide in dicotyledonous plants.Plant Mol. Biol. 9, 625–34.

    Google Scholar 

  • Wyatt, R.E., Ainley, W.M., Nagao, R.T., Conner, T.W. and Key, J.L. (1993) Expression of theArabidopsis AtAux2-11 auxin-responsive gene in transgenic plants.Plant Mol. Biol. 22, 731–49.

    PubMed  Google Scholar 

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Authors and Affiliations

  1. CSIRO Division of Plant Industry, GPO Box 1600, 2601, Canberra, ACT, Australia

    P. J. Larkin, J. M. Gibson & G. J. Tanner

  2. Plant-Microbe Interaction Group, Research School of Biological Sciences, Australian National University, PO Box 475, 2601, Canberra City, ACT, Australia

    U. Mathesius, J. J. Weinman, E. Gartner, E. Hall, B. G. Rolfe & M. A. Djordjevic

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  1. P. J. Larkin
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  2. J. M. Gibson
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  3. U. Mathesius
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  4. J. J. Weinman
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  5. E. Gartner
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  6. E. Hall
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  7. G. J. Tanner
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  8. B. G. Rolfe
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  9. M. A. Djordjevic
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Larkin, P.J., Gibson, J.M., Mathesius, U. et al. Transgenic white clover. Studies with the auxin-responsive promoter, GH3, in root gravitropism and lateral root development. Transgenic Research 5, 325–335 (1996). https://doi.org/10.1007/BF01968942

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  • DOI: https://doi.org/10.1007/BF01968942

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