* On leave of absence from the Department of General Genetics, University of Oslo, Norway. Angelo Spena, Reidunn B. Aalen,* and Sabine C.

Transcription

1 The Plant Cell, Vol. 1, , December 1989 Q 1989 American Society of Plant Physiologists Cell-Autonomous Behavior of the rolc Gene of Agrobacterium rhizogenes during Leaf Development: A Visual Assay for Transposon Excision in Transgenic Plants Angelo Spena, Reidunn B. Aalen,* and Sabine C. Schulze Max-Planck-lnstitut für Züchtungsforschung, Carl-von-Linne-Weg 1 O, D-5000 Koln 30, Federal Republic of Germany We describe a genetic switch based on the Ac transposable element of maize and the rolc gene of Agrobacterium rhizogenes, a dominant gene, which has pleiotropic effects on plant growth and morphology. Moreover, rolc gene expression under the control of the 35s cauliflower mosaic virus promoter decreases chlorophyll content in transgenic tobacco plants. Chlorophyll is a visible cell-autonomous marker, and it is shown here that the reduction in chlorophyll content caused by the rolc gene product allows us to monitor, in palisade or spongy mesophyll cells, Ac excision events resulting in rolc gene expression as pale-green sectors and spots. Our results indicate that the folc gene product behaves in a cell-autonomous manner during leaf development, at least as far as chlorophyll accumulation is concerned. In addition, the rolc gene can be useful to evaluate visually if and when a transposable element is active. Most important, we propose the use of a transposable element as a tool to activate expression of morphogenetic genes in a clonal population of cells. This could be particularly useful when studying genes affecting growth and development whose constitutive expression can severely impair regeneration of transgenic plants. INTRODUCTION The biological effects established in transgenic plants by the expression of the CaMVC chimeric gene, constructed by positioning the rolc gene from Agrobacterium rhizogenes TL-DNA under the control of the 35s RNA promoter of cauliflower mosaic virus, include reduced internodal length, pale-green and lanceolate leaves, reduced flower size, male sterility, reduced apical dominance, and altered root geotropism (Schmülling, Schell, and Spena, 1988). In addition, the rolc gene product is able to induce root formation and to stimulate root growth in transformed tobacco roots (Spena et al., 1987; Schmülling et al., 1988). Although the rolc pleiotropic effects of expression are reminiscent of alterations in the activity of plant growth regulators, rolc-induced roots, obtained via leaf disc inoculation, are always clonally transformed (Schmülling et al., 1988). This suggests that root induction is restricted to transformed cells, and not neighboring, untransformed cells. To test whether the rolc gene product behaves in a cell-autonomous manner in other plant organs, a transposable element based system has been constructed so that To whom correspondence should be addressed. * On leave of absence from the Department of General Genetics, University of Oslo, Norway. rolc expression is specifically initiated in a clonal population of cells. Transposable elements can produce genetic mosaics composed of two genotypically distinct types of tissue. In the simplest case, one type of tissue contains a transposable element at a given locus, the other does not. In plants, most mutable alleles studied are in genes whose products affect easily screenable features such as flower, seed, or leaf pigmentation (Doring and Starlinger, 1986; Nevers, Shepherd, and Saedler, 1986). Our genetic switch is based on the transposable element Ac of Zea mays (McClintock, 1951), which is able to transpose in heterologous plant systems (Baker et al., 1986; Van Sluys, Tempe, and Fedoroff, 1987; Knapp et al., 1988; Yoder et al., 1988) and the rolc gene from Agrobacterium rhizogenes which, when expressed under the control of the 35s promoter from cauliflower mosaic virus, is able to decrease chlorophyll content (Schmülling, 1988). Chlorophyll is a visible cell-autonomous marker; consequently, the reduction in chlorophyll pigmentation caused by rolc gene expression allows us to monitor Ac excision events resulting in rolc gene expression in palisade and spongy mesophyll cells. Severa1 phenotypic assays for transposon excision have already been described (Baker et al., 1987; Van Sluys et al., 1987; Coupland et

2 11 58 The Plant Cell Bg n E n B K I I I I BL I 1Kb I Ac Transposablc elamenl p35s RolC coding region Figure 1. Schematic Drawing of the AcCaMVC Construction. The Ac transposable element was inserted between the 35s RNA cauliflower mosaic virus promoter and the rolc coding region. The entire construction is located between the EcoRl and Hindlll sites of the binary vector ppcvoo2. The small arrow indicates the direction of Ac transcription. Abbreviations: p35s, cauliflower mosaic virus 35s promoter; BL and BR, left and right border sequences of vector T-DNAs; pg5, truncated promoter of TL-DNA gene 5; pnos, promoter of nopaline synthase gene; paocs, polyadenylation sequence of octopine synthase gene; NPT-II, neomycin phosphotransferase gene of transposon Tn5; E, EcoRI; H, Hindlll; B, BamHI; K, Kpnl. al., 1988; Yoder et al., 1988; Jones et al., 1989), but none of them allows one to monitor excision events merely by visual inspection during the whole course of plant development in the field. RESULTS An Ac element (Behrens et al., 1984) was inserted in the untranslated leader region of the CaMVC gene in such a way that the chimeric gene is interrupted and its expression prevented. Figure 1 shows a schematic drawing of the AcCaMVC construction, which was delivered to SR1 tobacco plant cells using a Tiderived binary vector (Koncz and Schell, 1986). Transgenic kanamycin-resistant tobacco plants were regenerated via leaf disc transformation I I (Horsch et al., 1984). Thirteen out of 14 independent transformants had leaves variegated with pale-green sectors and/or spots, a feature indicative of the cell-autonomous behavior of the rolc gene product with respect to its effect on chlorophyll pigmentation. Figure 2A shows pale-green sectors ranging in size from relatively large to small ones observed in the progeny obtained by back-crossing a variegated AcCaMVC transgenic plant clone AcCaMVC7) to SR1 wild-type tobacco. We have never observed a completely pale-green leaf in variegated plants. Variegated plants, obtained either through tissue culture techniques or by back-crossing of transformants, were not affected either in their fertility or in their apical dominance. This observation suggests that clonal expression of the rolc gene is not sufficient to affect these traits. In the back-crossed progeny of variegated transgenic plants, we have also observed normal plants and dwarf pale-green plants with a phenotype resembling that of CaMVC transgenic plants (see the progeny analysis and Table 1). The dwarf pale-green plants most likely represent germinal excision events. Indeed, germinal excisions are expected to give rise to tobacco plants showing phenotypical alterations reminiscent of, although slightly less pronounced than, CaMVC transgenic plants. The reduction in the expressivity of the CaMVC-specific traits (i.e., less pronounced reduction in apical dominance and incomplete male sterility) is most likely due to the fact that Ac excision events will leave 87 bp of extra DNA containing 56 bp from the waxy gene of Z. mays plus DNA linkers used to build AcCaMVC. Consequently, linker sequences are present as inverted repeats in the untranslated leader sequence of the CaMVC gene; their pairing might decrease rolc mrna translational efficiency (Pelletier and Sonenberg, 1985). Figures 2A and 26 show that leaf pale sectors have a smooth appearance. Interestingly, the smooth a p pearance is restricted to those parts of the leaf that show a decrease in chlorophyll pigmentation (i.e., pale-green sectors). Among plants regenerated from variegated leaves, we have observed plants showing either the variegated phenotype or displaying alterations typical of CaMVC transgenic plants, although expressed at a lower level. Figure 2. Extra-Apical Chimeras in Subepidermal Tissues of Leaves from AcCaMVC Transgenic Tobacco Plants Are Easily Visualized as Pale-Green Sectors and Spots. (A) Early and late excision events observed in leaves of the progenies obtained by back-crossing plant clone AcCaMVC7. This clone contains at least four active copies of the AcCaMVC construction located on four unlinked loci (see Table 1). The presence of sectors with different intensities of pale green might represent restriction of individual sectors to one layer of the mesophyll or different levels of ro/c gene expression. This could be due to positional effect and/or to the activation of one or more copies of the CaMVC gene. Moreover, it is interesting to note that the leaf surface has a smooth appearance only in pale sectors. See, for example, the picture at the bottom right. (8) Large pale-green sector observed on a leaf from plant clone AcCaMVC8. Note the smooth appearance of the leaf surface within the area corresponding to the pale-green sector. The darkgreen spot within the pale-green sector may be due either to cell displacement (Poethig, 1987) or to inactivation of the CaMVC gene.

3

4 1160 The Plant Cell Table 1. Progeny Analysis of AcCaMVC Transgenic Plants Phenotypes x 2 Ratio Crosses WT" M a Tested Value AcCaMVd x SR1 SR1 x AcCaMVCI AcCaMVC2 x SR SR1 x AcCaMVC AcCaMVC4 x SR1 SR1 x AcCaMVC4 AcCaMVC6 x SR1 SR1 x AcCaMVCG AcCaMVC7 x SR1 SR1 x AcCaMVC7 AcCaMVCI x SR1 SR1 x AcCaMVCI AcCaMVC2 x SR1 SR1 x AcCaMVC (77 + 4) (121 +3) km" ( ) 152( ) 175( ) 116( ) 274 ( ) 254 ( ) km' :3 1:3 1:7 1:7 1: " 0.00" 2.23 b d b 0.13" 2.06" 1.99" 2.42 b 0.02 b 0.11" 0.30 b Transcripts hybridizing to ro/c-specific probes are detectable in polya + RNA extracted from variegated leaves of all transgenic plants tested. Figure 3 shows an RNA gel blot analysis of polya + RNA extracted from several independent plant clones. Although the level of accumulation of RNA varies among different samples, in all cases ro/c transcripts from AcCaMVC transgenic plants show a slower mobility than ro/c transcripts extracted from CaMVC transgenic plants. (Compare lane 1 with lanes 2, 3, 4, 5, 6, and 7 of Figure 3.) This is to be expected, as Ac excision events produce a CaMVC gene with approximately an additional 87 bp in its untranslated leader sequence, and, consequently, ro/c transcripts with slightly slower mobility under denaturing conditions. Of course, imprecise excision events may take place; however, differences in a few bases cannot be resolved on RNA gel blots. The extra 87 bp contain 56 bp from the waxy gene of Z. mays plus DNA linkers used to build AcCaMVC. When polya + RNA from transgenic leaf tissue was hybridized to either a waxy-specific or an ro/c-specific probe, the same transcripts of approximately 820 bases were detected (data not shown). No transcripts are observed in plant clone AcCaMVCI 0 that did not show leaf variegation (data not shown). Thus, the RNA gel blot analysis indicates a correlation between the phenotypic assay (i.e., pale-green sectors) and ro/c-waxy-specific transcripts. The variation in transcript level between clones could represent a difference in the extent of variegation and/or a position effect on the level of ro/c expression following excision, and/or be due to the activation of several copies of the gene. DNA gel blot analysis of DNA extracted from leaves of AcCaMVC transgenic plants shows that ro/c gene expression was due to reconstruction of a functional CaMVC ^ O> CD F- lo v? O O O O O O AcCaMVC4 x SR SR1xAcCaMVC :3 1:3 0.70" 0.22" AcCaMVC6 x SR1 SR1 x AcCaMVC6 NT" NT :7 0.00" Resistance to kanamycin sulfate (50 mg/l) was tested in vitro on MS medium (Murashige and Skoog, 1962). The seeds analyzed for each cross represent the progeny obtained from a single capsule. Pollen from more than one flower has been used. "WT, wild type; M, number of variegated and dwarf pale-green plants. Numbers in parentheses are the numbers of plants with variegated and dwarf pale-green phenotypes, respectively. b Not significant (P = 0.05). 0 Significant at P = a Significant at P = e NT, not tested Figure 3. RNA Gel Blot Analysis of PolyA* RNA Extracted from Leaves Transgenic for the AcCaMVC Construction. Lane 2, clone 11; lane 3, clone 9; lane 4, clone 8; lane 5, clone 7; lane 6, clone 6; lane 7, clone 4. Lane 1 contains 0.5 ^g of polya* RNA from CaMVC transgenic tissue. In all other cases, 10 ng were loaded per each lane and separated on a 1.7% agaroseformaldehyde gel. The RNA gel blot was hybridized as previously described to a probe spanning ro/c coding and terminator regions. Molecular weight was calculated using a standard marker (Bethesda Research Laboratories).

5 rolc Extra-Apical Chimeras 1161 **> Kb Figure 4. DMA Gel Blot Analysis of DMA Extracted from Leaves of Independent AcCaMVC Transformants. Lane 1, DNA radiolabeled marker (Bethesda Research Laboratories); lanes 2, 4, 6, and 8 contain Hindlll digests of genomic DNA from plants transgenic for CaMVC, ppcv002, AcCaMVC-4, and AcCaMVC-6, respectively. Lanes 3, 5, 7, and 9 contain Hindlll/ EcoRI double digests of the same DNA in the same order. The 2.8-kb Hindlll bands are composed of the right end of the transposable element Ac plus the ro/c coding and terminator regions (see Figure 1), and they represent /Jc-containing donor sites. Empty donor sites are detected as bands of higher and variable molecular weight in Hindlll digests (lanes 6 and 8). This is because one Hindlll site is located in the plant DNA to the left of the T- DNA integration site. In EcoRI/Hindlll double digests, empty donor sites appear as bands of 1.6 kb. The probe was identical to that used in the RNA gel blot analysis. gene by Ac excision. Figure 4 shows a genomic DNA gel blot displaying Hindlll and Hindlll-EcoRI digests of DNA extracted from leaves of independent AcCaMVC transgenic plants hybridized to a probe spanning the whole rolc coding and termination regions. In the Hindlll digests, the 2.8-kb band represents the /Ac-interrupted CaMVC gene (see Figure 1), whereas excision events produce bands of higher molecular weight. The multiple bands most likely represent independent excision events because genetic crosses show that several plant clones contain T-DNA inserts at several unlinked loci (see progeny analysis). In particular, plant clone AcCaMVC4, which shows two active Ac copies on a DNA gel blot (Figure 4, lane 6), has T-DNA inserts at two unlinked loci. Plant clone AcCaMVCS, which shows five active Ac copies on a DNA gel blot (Figure 4, lane 8), has T-DNA inserts at three unlinked loci. When the DNA samples are also cut with EcoRI, the 2.8-kb band remains unaffected, the bands of higher molecular weight disappear, and a new band of approximately 1.6 kb is generated. This band represents the reconstructed CaMVC gene and, due to waxy and linker sequences left after Ac excision, it has a slightly higher molecular weight as compared with the band representing the original chimeric CaMVC gene in DNA extracted from CaMVC transgenic plants (Figure 4, lane 3). However, no empty donor site bands could be detected in plant clone AcCaMVC 10, the only AcCaMVC transgenic plant that did not show leaf variegation (data not shown). Plants transgenic for the AcCaMVC construction were back-crossed to normal SR1 plants, and their progeny were analyzed for somatic and germinal excision events either directly in the soil (Table 1) or after kanamycin selection in vitro. Table 1 shows that the back-crossed progenies of variegated AcCaMVC transgenic plants are composed of normal, variegated, and dwarf pale-green plants. The genetic data confirm the presence of multiple unlinked copies in some AcCaMVC transgenic plants (e.g., AcCaMVC4, -6, and -7), as expected from the DNA gel blot data. To test co-segregation of the construct presence with variegated and dwarf pale-green phenotype, backcrossed progenies of AcCaMVC transgenic plants were first selected in vitro for kanamycin resistance, and then further grown in soil. Out of 101 kanamycin-resistant AcCaMVC7 plants (cross AcCaMVC7 x SR1), 92 had variegated leaves, whereas nine were pale-green dwarfs. Similarly, out of 71 AcCaMVC4 kanamycin-resistant plants (cross AcCaMVC4 x SR1), 44 were variegated, 25 dwarf pale-green, and one looked normal. Out of 47 AcCaMVC6 kanamycin-resistant plants (cross AcCaMVCG x SR1), 36 were variegated and 11 were dwarf pale-green; out of 38 AcCaMVCI kanamycin-resistant plants (cross AcCaMVCI x SR1), 35 were variegated and three were dwarf palegreen. The presence of normal-looking plants can be most easily explained by Ac inactivation. In this respect, Table 1 also shows that the back-crossed progeny of plant clone AcCaMVC2 (one active copy as estimated by DNA gel blotting and by analysis of kanamycin-resistant plants), which showed sectors only in the oldest leaves, is composed of normal plants only. This indicates that the Ac transposable element in this plant clone was inactivated during development.

6 11 62 The Plant Cell DISCUSSION Expression of the rolc gene of A. rhizogenes T-DNA establishes pleiotropic alterations in transgenic plants indicative of a modification in the activity of plant growth regulators (Oono et al., 1987; Schmülling et al., 1988). However, perturbations in the biological activities of phytohormones could be achieved by different and diverse biochemical processes affecting either hormonal biosynthetic or catabolic pathways or by altering the ratio between free and conjugated hormones or by interfering with hormone transport. To exclude that rolc biological effects are due to the rolc-mediated synthesis of a transported growth factor, we have constructed a genetic switch based on the Ac element of maize (McClintock, 1951) in such a way that rolc gene expression is dependent on Ac excision event and, consequently, specifically initiated in a clonal population of cells. Because plants transgenic for the wildtype rolc gene &e., under the control of a promoter conferring gene expression mainly to leaf vascular tissue; Schmülling, Schell, and Spena, 1989) do not show palegreen leaves, but plants transgenic for the CaMVC chimeric gene do, we conclude that rolc gene expression in mesophyll cells is required to implement the pale-green phenotype (Schmülling et al., 1988). Consequently, excision events taking place during leaf development generate palegreen sectors and spots. lrradiation of axillary buds in tobacco lines heterozygous for two unlinked, epistatic chlorophyll mutations, a7 and a2, generates extra-apical chimeras visualized as sectors or spots depending upon at which stage of leaf development the irradiation is performed (Poethig and Sussex, 1985). Therefore, by comparison, we can determine the stage of leaf development at which excision events took place. Large sectors represent early events, small sectors and spots represent late excision events. The sharp borders of these sectors indicate that the rolc gene product behaves in a cell-autonomous manner not only in root induction on leaf discs (Schmülling et al., 1988), but also in leaf tissue, with respect to chlorophyll pigmentation. Moreover, the smooth appearance of pale-green leaf sectors suggests that the cell-autonomous behavior of the rolc gene product is not restricted simply to chlorophyll pigmentation during leaf development. However, the effects of rolc expression on plant morphology and leaf shape are more difficult to assess, as no whole-leaf sectors were observed. Our results show that rolc biological effect on leaf pigmentation, and very likely also on leaf morphology, is expressed only in transformed cells. We interpret this finding as evidence that rolc biological effect is not due to the 'ynthesis Of a growh factor that transported in leaf tissue. In this respect, it is worthwhile to mention that X-raYinduced chimeras have been used to study the cell auton- omy (Harberd and Freeling, 1989) or the non-cell-autonomous behavior of the products of dominant morphological genes (Hake and Freeling, 1986; Poethig 1988). In consideration of the crucial role of indoleacetic acid in the control of apical dominance (Philips, 1975), it is conceivable that rolc biological effects observed in transgenic plants (Oono et al., 1987; Schmülling et al., 1988), could be caused by reducing auxin biological activity in a cellautonomous way. The fact that such an effect could be achieved by different mechanisms altering auxin activity either directly (e.g., auxin inactivation, inhibition of auxin transport, alteration of the auxin receptor system, inhibition of hormonal synthesis) or indirectly through a cytokininlike effect would be challenging to test experimentally. The rolc extra-apical chimeras will be a useful material for the analysis of the hormonal constitution in pale-green sectors and neighboring dark-green leaf tissue. In summary, we have used the transposable element Ac as a stringent onloff switch to control expression of a gene affecting plant morphogenesis and growth. This approach could be useful to study any gene of interest affecting plant growth and development, especially in those cases in which their (0ver)expression severely impairs plant regeneration and growth. One possibility to circumvent this problem is to use promoters that trigger expression only under certain environmental or developmental conditions. Unfortunately, many inducible promoters (e.g., heat-inducible promoters) have a low basal leve1 of expression. An alternative, which we have presented here, is to deliver the gene as a construction in which the promoter is separated from the gene by a transposable element. Then gene expression can take place only after transposon excision, and will, therefore, allow us to regenerate plants even when the constitutive expression of the gene would be lethal for plant cells. Moreover, the AcCaMVC construction can be useful to evaluate, by visual inspection, if and when a transposable element is active. Because the CaMVC gene establishes similar developmental alterations in potato (M. Fladung and F. Salamini, unpublished data), the AcCaMVC genetic switch is a versatile tool that probably could be used in all plants in which a Ri phenotype is reported (for review see Birot et al., 1987). Finally, the CaMVC gene per se represents an attractive trap for those searching for transposable elements that have yet to be identified. METHoDS Construction of plasmids Standard techniques were used for the construction of recombinant plasmids (Maniatis, Fritsch, and Sambrook, 1982). The Ac transposable element was obtained as a Bglll/Kpnl fragment of 4591 bp from plasmid puac kindly provided by Detlev Becker and

7 rolc Extra-Apical Chimeras Peter Starlinger. The Ac element was inserted between the 35s RNA cauliflower mosaic virus promoter and the rolc coding region by ligation of BamHl cohesive ends at the 3' of the 35s promoter (EcoRl/BamHI fragment of 544 bp) to Bglll linker sequences present at the 5' of the Ac element. Kpnl cohesive,ends attached to the 3' of the Ac transposable element were ligated to Kpnl sticky ends located 9 bp upstream of the ATG initiation codon of the rolc coding region. The entire construction is located between the EcoRl and Hindlll sites of the binary vector ppcvoo2 (Koncz and Schell, 1986). The AcCaMVC construction was transferred to Escherichia coli strain 37-1 and then mobilized to Agrobacterium fumefaciens strain GV3101 as described (Koncz and Schell, 1986). Plant Tissue Culture and Transformation Transgenic Nicotiana fabacum Petit Havana SR1 (Nagy and Maliga, 1976) were raised by following a modified leaf disc transformation procedure (Horsch et al., 1984). RNA Extraction and Analysis RNA was extracted from transgenic leaves as described (Logemann, Schell, and Willmitzer, 1987). PolyA+ RNA was purified by oligo(dt) chromatography, separated by agarose-formaldehyde gel electrophoresis, transferred to Hybond-N membranes (Amersham), and hybridized to probes radioactively labeled by random priming (Boehringer Mannheim) as previously described (Schmülling et al., 1988). DNA Extraction and Analysis DNA was extracted from transgenic leaves as described (Dellaporta, Wood, and Hicks, 1983). Restriction analysis, agarose gel electrophoresis, transfer to Hybond-N membranes, and hybridization to radioactively labeled probes were done by standard techniques (Maniatis et al., 1982). ACKNOWLEDGMENTS We thank Peter Starlinger and Detlev Becker for plasmid puac, Zusanne Schwarz-Sommer for advice in the genomic DNA analysis, and Rick Walden for critical reading of the manuscript. We are indebted to Jeff Schell and Francesco Salamini for their encouragement. Received July 12, 1989; revised October 17, REFERENCES Baker, B., Schell, J., Lorz, H., and Fedoroff, N. (1986). Transposition of the maize controlling element "Activator" in tobacco. Proc. Natl. Acad. Sci. USA 83, Baker, B., Coupland, G., Fedoroff, N., Starlinger, P., and Schell, J. (1 987). Phenotypic assay for excision of the maize controlling element Ac in tobacco. EMBO J. 6, Behrens, U., Fedoroff, N., Laird, A., Miiller-Neumann, M., Starlinger, P., and Yoder, J. (1984). Cloning of the Zea mays controlling element Ac from the wx-m7 allele. MOI. Gen. Genet. 194, Birot, A.M., Boucher, D., Casse-Delbart, F., Durand-Tardif, M., Jouanin, L., Fautot, V., Robaglia, C., Tepfer, D., Tepfer, M., Tourneur, J., and Vilaine, F. (1987). Studies and uses of the Ri plasmids of Agrobacterium rhizogenes. Plant. Physiol. Biochem. 25, Coupland, G., Baker, B., Schell, J., and Starlinger, P. (1988). Characterization of the maize transposable element Ac by interna1 deletions. EM60 J. 7, Dellaporta, S.L., Wood, J., and Hicks, J.B. (1983). A plant DNA minipreparation: Version 11. Plant MOI. Biol. Rep. 1, Doring, H.-P., and Starlinger, P. (1986). Molecular genetics of transposable eleinents in plants. Annu. Rev. Genet. 20, Hake, S., and Freeling, M. (1986). Analysis of genetic mosaics shows that the extra epidermal cell divisions in Knotted mutant maize plants are induced by adjacent mesophyll cells. Nature 320, Harberd, N.P., and Freeling, M. (1989). Genetics of dominant gibberellin-insensitive dwarfism in maize. Genetics 121, Horsch, R., Fraley, R., Rogers, S., Sanders, P., Lloyd, A., and Hoffmann, W. (1984). lnheritance of functional foreign genes in plants. Science 223, Jones, J.D.G., Carland, F.M., Maliga, P., and Dooner, H.K. (1989). Visual detection of transposition of the maize element Activator (Ac) in tobacco seedlings. Science 244, Knapp, S., Coupland, G., Uhrig, H., Starlinger, P., and Salamini, F. (1988). Transposition of the maize transposable element Ac in Solanum tuberosum. MOI. Gen. Genet. 213, Koncz, C., and Schell, J. (1986). The promoter of TL-DNA gene 5 controls the tlssue-specific expression of chimaeric genes carried by a nove1 tye of Agrobacterium binary vector. MOI. Gen. Genet. 204, Logemann, J., Schell, J., and Willmitzer, L. (1 987). lmproved method for the isolation of RNA from plant tissues. Anal. Biochem. 163, Maniatis, T., Fritsch, E.F., and Sambrook, J. (1 982). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory). McClintock, B. (1 951). Mutable loci in maize. Carnegie Inst. Wash. Yearbook 50, Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant 15, Nagy, J.I., and Maliga, P. (1 976). Streptomycin-resistant plants from callus culture of haploid tobacco. Z. Pflanzenphysiol. 78, Nevers, P., Shepherd, N.S., and Saedler, H. (1986). Plant transposable elements. Adv. Bot. Res. 12,

8 11 64 The Plant Cell Oono, Y., Handa, T., Kanaya, K., and Uchimiya, H. (1987). The TL-DNA gene of Ri plasmid responsible for dwarfness of tobacco plants. Jpn. J. Genet. 62, Pelletier, J., and Sonenberg, N. (1985). lnsertion mutagenesis to increase secondary structure within the 5 noncoding region of a eukaryotic mrna reduces translational efficiency. Cell 40, Philips, I.D.J. (1975). Apical dorninance. Annu. Rev. Plant Physiol. 26, Poethig, R.S. (1987). Clonal analysis of cell lineage patterns in plant development. Amer. J. Bot. 74, Poethig, S. (1988). A non-cell-autonomous mutation regulating juvenility in maize. Nature 336, Poethig, R.S., and Sussex, I.M. (1985). The developmental morphology and growth dynamics of the tobacco leaf. Planta 165, Schmülling, T. (1988). Studien zum EinfluR der rola, 6, and C Gene der TL-DNA von Agrobacterium rhizogenes auf die Pflan- zenentwicklung. Ph.D. thesis, Universitat zu Koln. Schmiilling, T., Schell, J., and Spena, A. (1988). Single genes from Agrobacterium rhizogenes influence plant development. EMBO J. 7, Schmülling, T., Schell, J., and Spena, A. (1989). Promoters of the rola, 0, and C genes of Agrobacterium rhizogenes are differentially regulated in transgenic plants. Plant Cell 1, Spena, A., Schmiilling, T., Koncz, C., and Schell, J. (1987). lndependent and synergistic activity of rola, 6, and C loci in stimulating abnormal growth in plants. EMBO J. 6, Van Sluys, M.A., Tempe, J., and Fedoroff, N. (1987). Studies on the introduction and mobility of the maize Activator elernent in Arabidopsis thaliana and Daucus carota. EMBO J. 6, Yoder, J.I., Palys, J., Alpert, K., and Lassner, M. (1988). Ac transposition in transgenic tomato plants. MOI. Gen. Genet. 213,

9 Cell-autonomous behavior of the rolc gene of Agrobacterium rhizogenes during leaf development: a visual assay for transposon excision in transgenic plants. A Spena, R B Aalen and S C Schulze Plant Cell 1989;1; DOI /tpc This information is current as of March 4, 2014 Permissions etocs CiteTrack Alerts Subscription Information Sign up for etocs at: Sign up for CiteTrack Alerts at: Subscription Information for The Plant Cell and Plant Physiology is available at: American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY