Summary
Transgenic tobacco plants (Nicotiana tabacum L.) expressing the rolC gene of Agrobacterium rhizogenes under the transcriptional control of the 35S RNA promoter are male sterile. When these plants are genetically crossed with others containing the rolC gene linked in antisense orientation to the 35S RNA promoter, hybrid progeny display restoration of male fertility. Moreover, hybrid progeny are revertant for other features of the rolC phenotype, such as restoration of plant height, leaf pigment content and female fertility. The level of restoration of the characteristics of untransformed tobacco appeared to be independent of the steady-state level of antisense RNA. Addition of six transcriptional enhancer sequences upstream of the 35S transcriptional start region in the antisense construct led to a higher steady-state level of antisense RNA than that produced using a promoter linked to a single enhancer sequence. However no significant difference was observed in the level of attenuation of the rolC phenotype in the progeny of crosses with either one or six transcriptional enhancers linked to the antisense rolC gene. Antisense constructs comprising only 189 by of the rolC 5′ coding region appeared less efficient in attenuating the rolC phenotype than those including the whole rolC coding region as well as its 3′ untranslated region. Furthermore, results from experiments on light-controlled rolC gene expression indicate that microsporogenesis is sensitive to rolC gene action during the early stages of flower development.
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References
Ahokas H (1982) Cytoplasmic male sterility in barley: evidence for the involvement of cytokinins in fertility restoration. Proc Natl Acad Sci USA 79:7605–7608
Barnes WM (1990) Variable patterns of expression of luciferase in transgenic tobacco leaves. Proc Natl Acad Sci USA 87:9183–9187
Benfey PN, Ren L, Chua NH (1989) The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue specific expression pattern. EMBO J 8:2195–2202
Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification in Escherichia coli J Mol Biol 41:459–468
Cannon M, Platz J, O'Leary M, Sookdeo C, Cannon F (1990) Organ-specific modulation of gene expression in transgenic plants using antisense RNA. Plant Mol Biol 15:39–47
Cornelissen M, Vandewiele M (1989) Both RNA level and translation efficiency are reduced by antisense RNA in transgenic tobacco. Nucleic Acids Res 17:833–843
Delauney AJ, Tabaeizadeh Z, Verma DPS (1988) A stable bifunctional antisense transcript inhibiting gene expression in transgenic plants. Proc Natl Acad Sci USA 85:4300–4304
Estruch JJ, Chriqui D, Grossmann K, Schell J, Spena A (1991) The plant oncogene rolC is responsible for the release of cytokinins from glucoside-conjugates. EMBO J 10:2889–2896
Firoozabady E (1986) Rapid plant generation from Nicotiana mesophyll protoplasts. Plant Sci 46:127–131
Fladung M (1990) Transformation of diploid and tetraploid potato clones with the rolC gene of Agrobacterium rhizogenes and characterization of transgenic plants. Plant Breeding 104:295–304
Fritz C (1988) Molekulare Analyse der Struktur und Expression einer pflanzlichen Genfamilie. Die rbcS Gene in Solanum tuberosum. PhD thesis, Universität Köln
Fritz CC, Herger T, Wolter FP, Schell J, Schreier P (1991) Reduced steady-state levels of rbcS mRNA in plants kept in the dark are due to differential degradation. Proc Natl Acad Sci USA 88:4458–4462
Fritze K (1992) T-DNA Insertionsmutagenese mit Transkriptionsverstärkerelementen in Nicotiana tabacum und die genetische Analyse der Resistenz gegen Methylglyoxal-bisguanylhydrazon. PhD thesis, Universität Köln
Guilley H, Dubley RK, Jonard G, Balazs E, Richards KE (1982) Transcription of cauliflower mosaic virus DNA: Detection of promoter sequences, and characterization of transcripts. Cell 30:763–773
Holden M (1976) Chlorophylls. In: Goodwin TW (ed) Chemistry and biochemistry of plant pigments. Academic Press, London New York San Francisco, pp 2–37
Horsch RB, Fry JE, Hoffmann NL, Eichhotz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231
Kaul MLH (1988) Male sterility in higher plants. (Monographs on theoretical and applied genetics, vol 10) Springer-Verlag, Berlin
Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236:1299–1302
Khochbin S, Lawrence JJ (1989) An antisense RNA involved in p53 mRNA maturation in murine erythroleukemia cells induced to differentiate. EMBO J 8:4107–4114
Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2:1201–1224
Koncz C, Schell J (1986) The promoter of the TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396
Kraus K (1988) Die Wirkung von antisense-RNA auf die Genexpression von Pflanzenviren. PhD thesis, Universität Köln
Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem 163:16–20
Maliga P, Sz-Breznovitis A, Morton L (1973) Streptomycin-resistant plants from callus culture from haploid tobacco. Nature 244:29–30
Mariani C, De Beuckeleer M, Truettner J, Leemans J, Goldberg RB (1990) Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature 347:737–741
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Moffatt B, Somerville C (1988) Positive selection for male-sterile mutants of Arabidopsis lacking adenine phosphoribosyl transferase activity. Plant Physiol 86:1150–1154
Plegt L, Bino RJ (1989) β-glucuronidase activity during development of the male gametophyte from transgenic and non-transgenic plants. Mol Gen Genet 216:321–327
Riker AJ, Banfield WM, Wright WH, Keitt GW, Sagen HE (1930) Studies on infectious hairy root on nursery apple tree. J Agric Res 41:507–540
Rothstein SJ, Lagrimi LM (1989) Silencing gene expression in plants Oxf Surv Plant Mol Cell Biol 6:221–246
Rueda J, Vazquez AM (1985) Effect of auxins and cytokinins upon the start of meiosis in cultured anthers of rye (Secale cereale L.). Can J Genet Cytol 27:759–765
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sandler SJ, Stayton M, Townsend JA, Ralston ML, Bedbrook JR, Dunsmuir P (1988) Inhibition of gene expression in transformed plants by antisense RNA. Plant Mol Biol 11:301–310
Schmülling T (1988) Studien zum Einfluß der rolA, B und C Gene der TL-DNA von Agrobacterium rhizogenes auf die Pflanzenentwicklung. PhD thesis, Universität Köln
Schmülling T, Schell J, Spena A (1988) Single genes from Agrobacterium rhizogenes influence plant development. EMBO J 7:2621–2629
Schmülling T, Schell J (1993) Transgenic tobacco plants regenerated from leave disks can be periclinal chimeras. Plant Mol Biol, in press
Schmülling T, Fladung M, Grossmann K, Schell J (1993) Hormonal content and sensitivity of transgenic tobacco and potato plants expressing single rol genes of Agrobacterium rhizogenes T-DNA. Plant J, in press
Schuch W (1991) The manipulation of plant gene expression using antisense RNA. In: Dennis ES, Llewellyn DJ (eds) Plant gene research — molecular approaches to crop improvement. Springer-Verlag, New York
Sheehy RE, Kramer M, Hiatt WR (1988) Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc Natl Acad Sci USA 85:8805–8809
Simon R, Preifer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Biotechnology 1:784–791
Slightom JL, Durand-Tardif M, Jouanin L, Tepfer D (1986) Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine-type plasmid: identification of open reading frames. J Biol Chem 261:108–121
Smith CJS, Warson CF, Ray J, Bird CR, Morris PC, Schuch W, Grierson D (1988) Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334:724–726
Spena A, Schmülling T, Koncz C, Schell J (1987) Independent and synergistic activity of rolA, B, and C loci in stimulating abnormal growth in plants. EMBO J 6:3891–3899
Spena A, Aalen RB, Schulz S (1989) Cell-autonomous behaviour of the rolC gene of Agrobacterium rhizogenes during leaf development: A visual assay for transposon excision in transgenic plants. Plant Cell 1:1157–1164
Taylor LP, Jorgensen R (1992) Conditional male fertility in chatcone synthase-deficient petunia. J Hered 83:11–17
Van der Krol AR, Lenting PE, Veenstra J, Van der Meer IM, Koes RE, Gerats AGM, Mol JNM, Stuitje AR (1988a) An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature 333:866–869
Van der Krol AR, Mol JNM, Stuitje AR (1988b) Antisense genes in plants: an overview. Gene 72:45–50
Van der Krol AR, Mur LA, De Lange P, Mol JNM, Stuitje AR (1990) Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect. Plant Mol Biol 14:457–466
Van der Meer IM, Stam ME, van Tunen AJ, Mol JNM, Stuitje AR (1992) Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility. Plant Cell 4:253–262
Wing D, Koncz C, Schell J (1989) Conserved function in Nicotiana tabacum of a single Drosophila hsp70 promoter heat shock element when fused to a minimal T-DNA promoter. Mol Gen Genet 219:9–16
Wolters FP, Fritz CC, Willmitzer L, Schell J, Schreier P (1988) rbcS genes in Solanum tuberosum: Conservation of transit peptide and exon shuffling during evolution. Proc Natl Acad Sci USA 85:846–850
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Schmülling, T., Röhrig, H., Pilz, S. et al. Restoration of fertility by antisense RNA in genetically engineered male sterile tobacco plants. Molec. Gen. Genet. 237, 385–394 (1993). https://doi.org/10.1007/BF00279442
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DOI: https://doi.org/10.1007/BF00279442