Gene amplification in glyphosate tolerant tobacco cells

Plant Science, 72 (1990) 53--62

53

Elsevier Scientific Publishers Ireland Ltd.

Gene amplification in glyphosate tolerant tobacco cells* Peter B. Goldsbrough", Evelyn M. Hatch a, Bin Huang a,**, William G. Kosinski a, William E. Dyer a,t, Klaus M. H e r r m a n n b and Stephen C. Weller a aDepartment of Horticulture and ~Department of Biochemistry, Purdue University, West Lafayette IN 47907(U.S.A.) (Received March 19th, 1990; revision received June 28th, 1990; accepted June 29th, 1990) A series of tobacco (Nicotiana tabacum L.) cell lines have been selected for growth in the presence of normally lethal concentrations of glyphosate, up to 20 raM. Activity of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), which is inhibited by glyphosate, is elevated in tolerant cells. However, EPSPS isolated from tolerant cells is still inhibited by the herbicide. Increased activity of EPSPS is the result of increased levels of the enzyme in tolerant cells, as determined by immunoblotting, rather than a change in enzyme activity. RNA blots with a petunia EPSPS cDNA demonstrate a correlation between levels of tolerance and steady state level of EPSPS mRNA. Increased expression of EPSPS mRNA results from amplification of at least two genes encoding this enzyme in the tobacco genome. The degree of gene amplification also increases with level of glyphosate tolerance. Selected cells grown in the absence of glyphosate have an elevated EPSPS mRNA abundance and maintain some gene amplification, indicating that selection has resulted in a stable genetic modification.

Key words: tobacco; glyphosate; 5-enolpyruvylshikimate 3-phosphate synthase; gene amplification

Introduction

Glyphosate (N-[phosphonomethyl]glycine) is a highly effective broad spectrum herbicide. The primary mechanism of action of glyphosate is inhibition of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), the penultimate enzyme of the common pre-chorismate portion of the shikimic acid pathway [1]. Disruption of this pathway prevents synthesis of aromatic amino acids, their secondary metabolites and other products derived from chorismate. Glyphosate is a competitive inhibitor of EPSPS with respect to phosphoenolpyruvate [2] and has been shown to block import *This research was supported in part by a grant from the Indiana Corporation for Science and Technology. This is Journal Paper No. 12,026 from the Purdue University Agricultural Experiment Station. **Present address: Plant Research Centre, Agriculture Canada, Ottawa, Ontario, Canada KIA OC6. tpresent address: Department of Plant and Soil Science, Montana State University, Bozeman, MT 59717. Correspondence to: Dr. Peter B. Goldsbrough, Department of Horticulture, Purdue University, West Lafayette, IN 47907, U.S.A.

of the EPSPS preprotein into chloroplasts [3], the subcellular location of the shikimic acid pathway [4]. Glyphosate resistant mutants of both Escherichia coli [5] and Salmonella typhimurium [6] have been characterized and result from mutations at the aroA locus encoding EPSPS proteins that are not inhibited by glyphosate. Similarly, plants resistant to other herbicides have arisen [7], or been selected [8,9] that are the result of amino acid substitutions in the primary target proteins of these herbicides. However, there has been no report to date of a naturally occurring or selected plant EPSPS that is insensitive to glyphosate. A number of plant cell culture lines have nevertheless been selected for their ability to grow in the presence of normally lethal concentrations of glyphosate [10--14]. These tolerant cell lines contain elevated levels of EPSPS activity, although the enzymes are still sensitive to glyphosate inhibition. In petunia [15] and carrot [16] the molecular basis for overproduction of EPSPS is amplification of genes encoding EPSPS, whereas in Corydalis sempervirens transcriptional or posttranscrip-

0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

54 tional alterations have led to the increased level of EPSPS, in the absence of changes in EPSPS gene copy number [17]. In this paper, we report a detailed analysis of EPSPS gene expression in a series of tobacco (Nicotiana tabacurn L.) cell lines selected for increased tolerance to glyphosate. In these cell lines, amplification o f at least two genes encoding EPSPS is correlated with increased levels o f EPSPS enzyme activity, protein and mRNA and tolerance to glyphosate. Methods

Plant cell cultures Tobacco (cv. Wisconsin 38) cell cultures were initiated and maintained as described [10] and unselected cells are referred to as W38. The isolation of the original glyphosate tolerant cell line has been described [10]. These cells (designated Indiana 7 in Ref. 10) have been maintained for 3 years in the absence of glyphosate and are designated I7/0. I7/0 were gradually exposed to increasing concentrations o f glyphosate and three cell lines have been selected that are tolerant of 5, 10 and 20 mM glyphosate; these cell lines are referred to as I7/5, I7/10 and I7/20, respectively, and have been grown in the presence of the appropriate glyphosate concentration for at least 1 year. Growth of cells and determination of IDs0 for glyphosate were performed as described [10]. For enzyme assays and nucleic acid isolation, cells were harvested in the middle of the logarithmic phase of growth. EPSPS assay Cells were assayed for EPSPS activity as described [10]. One unit of EPSPS is the amount of protein that catalyzes the disappearance of 1 /amol of phosphoenolpyruvate per min. Immunoblotting Fifty micrograms of protein from cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose as described [18]. EPSPS proteins were detected using rabbit antiserum prepared against the petunia enzyme [14]

and visualized by reaction with goat anti-rabbit immunoglobulin-horseradish peroxidase conjugate secondary antibodies [ 18].

Nucleic acid analysis Total RNA was isolated from cells as described [19]. DNA was prepared as described [20] with further purification by CsCI buoyant density gradient centrifugation in the presence of ethidium bromide [21]. For blot analysis, RNA was denatured in 7.5°7o (v/v) formaldehyde containing 5007o (v/v) formamide and separated by electrophoresis in 1.5070 agarose gels containing 7.5070 (v/v) formaldehyde. The gels were stained with ethidium bromide (1/ag/ml) for 5 min, destained overnight in water, photographed and the RNA transferred to a nitrocellulose filter [22]. After transfer, the filters were baked at 80°C, boiled in 20 mM T r i s - HCI (pH 7.5), for 5 min and prehybridized at 42°C for 4 h in a solution containing 5 × SSPE (1 x SSPE containing 0.15 M NaC1, 10 mM NaH2PO 4 and 1 mM ethylenediamine tetraacetic acid (EDTA), pH 7.4), 5007o (v/v) formamide, 0.1 070 (w/v) each of bovine serum albumin, ficoll, polyvinylpyrrolidone (PVP) and SDS and 100/ag/ ml denatured salmon sperm DNA. Hybridization was carried out for 16 h in the same solution containing 5 x 105 cpm/ml of 32p-labelled DNA. Filters were washed in 3 × SSC (1 x SSC contains 0.15 M NaCI, 15 mM Na-citrate, pH 7.0) containing 0.1°70 (w/v) SDS at 42°C, dried and exposed to Kodak XAR5 film at - 70°C with an intensifying screen. For DNA blots, DNA was digested with EcoRI (BRL, Gaithersburg, MD), separated on 0.7070 agarose gels and transferred to nitrocellulose [23]. Filters were prehybridized at 68 °C for 4 h in a solution containing 6 x SSC, 0.1 07o (w/v) each of bovine serum albumin, ficoll, PVP and SDS and 100/ag/ml denatured salmon sperm DNA. Hybridization was carried out for 16 h in the same solution containing 5 X 105 cpm/ml of 32p-labelled DNA. Filters were washed in 3 X SSC containing 0.1 07o (w/v) SDS at 68 °C and subjected to autoradiography as described above. The following probes were used for hybridization: pMON9543, a cDNA for petunia EPSPS [15]; pBD14, a cDNA for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) from

55

potato [24]; pOC, a cDNA for tobacco osmotin [25] and p112, a plasmid containing Arabidopsis genomic DNA encoding ubiquitin [26]. DNA inserts were separated from plasmid vector sequences and labelled with 32P-dCTP to specific activities of approximately 1 × 10s cpm//Jg using a nick translation kit (Amersham, Arlington Heights, IL) essentially as described [21]. Results

Characterization o f glyphosate tolerant cells The original cell line selected for glyphosate tolerance (I7/0) is stably tolerant and unaltered in glyphosate uptake [10]. I7/0 cells exhibited increased activity of two enzymes in the shikimic acid pathway, DAHPS and EPSPS, with no alteration in the sensitivity of EPSPS to glyphosate [10]. I7/0 cells have been used in a stepwise selection scheme to isolate three additional cell lines (17/5, I7/10, I7/20) that grow in the presence of 5, 10 and 20 mM glyphosate, respectively. These cells exhibit a longer lag phase than W38 cells, similar to that observed for I7/0 cells [10], but grow to approximately the same maximum fresh weight as W38 cells. Growth of cells in media containing increasing glyphosate concentrations was measured and the IDs0 for glyphosate for each cell line is given in Table I. The IDso for 17/0 cells is similar to that previously reported [10] and for the other cell lines the IDs0 reflects the concentration of glyphosate at which they have been selected for growth.

Table 1.

EPSPS in tolerant cells The levels of EPSPS enzyme activity were measured in extracts from cells grown in media containing the appropriate concentrations of glyphosate (Table I). Cell lines that were more tolerant to glyphosate yielded higher EPSPS specific activity and I7/20 cells had more than 20 times the EPSPS activity of W38 cells. EPSPS activity from all cell lines was inhibited by more than 93% when assayed in the presence of 1 mM glyphosate. Therefore, even though tolerant cell lines contain elevated levels of EPSPS activity over that found in W38 cells, the enzyme isolated from these cells remains sensitive to glyphosate inhibition. To determine if the increased EPSPS activity in tolerant cell lines resulted from overproduction of this enzyme, proteins extracted from the unselected and glyphosate tolerant cells were analyzed by immunoblotting using a polyclonal antiserum to petunia EPSPS (Fig. 1). All cell lines contained a protein of M 46 000 that cross-reacted with this antibody. The size of this protein is similar to that reported for EPSPS from petunia [14] and C. sempervirens [12], indicating that this immunoreactive protein from tobacco is indeed EPSPS. The level of EPSPS protein is higher in I7/0 cells than in W38. In the absence of glyphosate there is a constitutive increase in EPSPS abundance in I7/ 0 cells. The highest level of EPSPS was observed in the most tolerant cells, I7/20. Other experiments have shown that I7/10 cells contain a level of EPSPS protein intermediate between that observed in I7/5 and I7/20 cells (data not shown).

Glyphosate tolerance (growth) and EPSPS activity of selected tobacco ceils.

Cell type

W38 17/0 17/5

17/10 I7/20 "E.U., enzyme units. b Mean ± S.E. (n = 2). Mean ± S.E. (n = 3).

IDa0 glyphosate (mM)

0.48 ± 0.02 b 1.95 ± 0.05 5.65 ± 2.15

12.60± 2,20 2 2 . 2 0 ± 0,60

EPSPS spec. act. No additions (E.U.'/mg prot.)

+ 1 mM Glyphosate (% of no additions)

0.012 ± O.OOV 0.057 ± 0.004 0.167 ± 0.004

5.6 ± 0.0b 4.4 ± 1.6 6.4 ± 1.3

0.175 ± 0.032 0.250 ± 0.012

2.8 ± 0.1 3.1 ± 0.6

56

1

2

3

4

~ ii!ii~!9!i!i!~!ii~!i¸ ~~ Fig. 1. Immunoblot analysisof EPSPS from glyphosatetolerant cells. Fifty microgramsof protein extracted from cells of W38 (1), 17/0 (2), 17/5 (3) and I7/20 (4) were subjected to SDS-PAGE and transferred to a nitrocellulose filter. The blot was incubated with antiserum raised against petunia EPSPS. The migration of protein molecular weight standards is shown on the right.

The elevated EPSPS activity in tolerant cells results from an increase in protein abundance rather than an alteration in activity of the enzyme. The antibody reacts with more than a single band in this region o f the blot. This may indicate that tobacco contains isozymes o f EPSPS that vary slightly in size. Alternatively, since glyphosate inhibits import of the EPSPS precursor into chloroplasts [3], the larger proteins may represent precursor molecules that still contain part of the transit peptide.

Messenger R N A levels in tolerant cells The elevated levels of EPSPS protein in 17 cell lines led us to examine the abundance of the m R N A encoding this protein. R N A blot analysis showed that, in all cell lines, a m R N A o f approximately 1.9 kb hybridized to the petunia EPSPS

cDNA probe (Fig. 2). The size of this R N A is similar to that reported for EPSPS m R N A in petunia [15] and C. sempervirens [17]. There is a clear relationship between the amount of EPSPS m R N A and the level of glyphosate tolerance in I7 cells. Cells with a higher IDs0 for glyphosate contain elevated steady state levels of EPSPS m R N A . In addition, I7/0 cells that are grown in the absence of glyphosate contain more EPSPS m R N A than W38 cells grown under the same conditions. As a control for these R N A blots, the levels of m R N A s for osmotin and ubiquitin were determined (Fig. 2). Some variation was detected between cell lines in the steady state levels o f these mRNAs. The m R N A s for osmotin and ubiquitin, although present at lower levels in W38 than I7 cells, could be readily detected in R N A prepared

57

1

2

3

4

i ¸

5 ,

EPSPS

OSMOTZN

UBIOUITIN

Fig. 2. mRNA levels in glyphosate tolerant cells. Ten micrograms of iotal RNA from cells of W38 (1), I7/0 (2), I7/5 (3), I7/10 (4) and I7/20 (5) were separated by agarose gel electrophoresis (direction of migration was from top to bottom) and transferred to nitrocellulose filters. The figure shows autoradiographs of filters after hybridization. Filters were hybridized with the 32P-labelledDNA probes as indicated on the left. The approximate sizes of these mRNAs are: EPSPS, 1.9 kb; ubiquitin, 1.5 kb; osmotin, 1.1 kb. EPSPS mRNA could be detected in W38 cells after prolonged exposure of the blots.

f r o m all cell lines. H o w e v e r , E P S P S m R N A c o u l d o n l y be d e t e c t e d in R N A i s o l a t e d f r o m W 3 8 cells after p r o l o n g e d e x p o s u r e o f the blots. T h e r e is no correlation between abundance of ubiquitin and o s m o t i n m R N A s a n d g l y p h o s a t e tolerance. T h e increase in E P S P S m R N A o b s e r v e d in I7 cells is t h e r e f o r e a specific c h a n g e in gene expression.

Genome changes in tolerant cells T o d e t e r m i n e the basis f o r increased levels o f E P S P S m R N A in I7 cells, g e n o m i c D N A i s o l a t e d f r o m these cell lines was a n a l y z e d b y S o u t h e r n blotting. T h e p e t u n i a E P S P S c D N A h y b r i d i z e d to six EcoRI f r a g m e n t s in D N A i s o l a t e d f r o m all the t o b a c c o cell lines (Fig. 3). H y b r i d i z a t i o n was

58

I

2

3

4

5

6

7

8

9

10 23 kb

9.4 6,6

4.3

2.3 2.0

0.5

Fig. 3.

EPSPSgenecopynumber in glyphosatetolerant cells. Five microgramsof DNA from tobaccoleaves (5), W38 cells (6), I7/0 (7), I7/5 (8), 17/10 (9) and I7/20 (10) were digested with EcoRI separated on a 0.7% agarosegel and transferred to a nitrocellulose filter. The filter was hybridizedwith the petunia EPSPS cDNAfrom pMON9543. An autoradiograph of the filter after hybridization is presented. Lane 4 contains the same genome equivalents of EcoRI digested petunia leaf DNA. Lanes 1, 2 and 3 contain 10, 3 and 1 gene copy reconstructions, respectively, of pMON9543 digested with EcoRI. The positions of DNA size markers are shown on the right.

greater to two fragments of 11 kb and 14 kb in DNA from I7 cells, indicating that these DNA sequences have been amplified in glyphosate tolerant cells. As observed with the R N A analysis, the degree of gene amplification increases with the level of glyphosate tolerance. The elevated levels of EPSPS protein and m R N A in the glyphosate tolerant cells are therefore at least partly the result of amplification of D N A sequences that encode this enzyme. Changes in gene transcription and m R N A stability may also contribute to the elevated steady state levels of EPSPS mRNA. Four

other E c o R I fragments in the tobacco genome also hybridize to the petunia EPSPS cDNA but do not show any evidence o f amplification in I7 cells. The nature of the EPSPS gene amplification in I7 cells was further examined by hybridizing D N A from I7/20 cells with three fragments o f the petunia EPSPS cDNA, representing the 5', central and 3' regions of the gene transcription unit. The patterns of hybridization produced by these three probes were identical (Fig. 4), indicating that each of the six E c o R I fragments could potentially encode a complete EPSPS gene. The two E c o R I

59

I

2

Discussion

3 23kb

9.4 6.6

4.3 Fig. 4. Analysisof amplified genes in tobacco cells. Aliquots of 5/~gof DNA from I7/20 cells were digested with EcoRI separated by agarose gel electrophoresis and transferred to nitrocellulose filters. The filters were hybridized with 3 fragments of the petunia EPSPS cDNA in pMON9543 (see Ref. 22) as follows: EcoRI-BamHI (5') l, BamHI-HindIII (central) 2 and HindIII-EcoRI (3') 3. Autoradiographs of these filters after hybridization are shown here. The positions of DNA size markers are shown on the right.

fragments that are amplified in glyphosate tolerant cells therefore represent at least two separate EPSPS genes. This is supported by the observation that they are amplified independently. In I7/0 cells, only the 14 kb EcoRI fragment is amplified and increased copy number of the 11 kb EcoRI fragment is apparent only in cells selected for tolerance to higher glyphosate concentrations. A blot of D N A f r o m the five cell lines was also hybridized with a potato D A H P S c D N A (Fig. 5). A small number o f EcoRI fragments in D N A f r o m each cell line hybridized to this probe, but there was no difference in intensity of hybridization between the cell lines. Therefore the amplification of genes encoding EPSPS in glyphosate tolerant cells is specific and genes encoding other enzymes of the shikimate pathway are not amplified in response to glyphosate selection.

A variety of metabolic inhibitors have been used to select resistant or tolerant mutants in plant and animal cells. In m a n y cases tolerance results from over-expression of the target enzyme as a consequence of gene amplification [27]. As a specific competitive inhibitor o f EPSPS, glyphosate is well suited to this analytical approach. Cell lines of several plant species have developed resistance to glyphosate as a result o f increased EPSPS activity [10--14]. In both petunia [15] and carrot [16] cells, elevated enzyme activity is accompanied by amplification of genes encoding this enzyme. This report characterizes a family of glyphosate tolerant tobacco cell lines and shows a clear relationship between EPSPS enzyme activity, protein, m R N A , gene copy number and tolerance to the herbicide. Cells of C. sempervirens selected for glyphosate tolerance show similar increases in EPSPS enzyme activity and m R N A without any gene amplification, suggesting an alteration in either gene transcription or post-transcriptional processes [17]. It is unclear why none of the selections for glyphosate tolerance in plants has resulted from a mutation giving rise to an EPSPS that is insensitive to the herbicide, as found in several bacterial species [5,6]. The frequency of point mutations in plant EPSPS genes may be rare compared with amplification, or a single mutation may be insufficient to give herbicide insensitivity. In this context, it is of interest to compare the genetic basis for resistance to two other herbicides, phosphinothricin and sulfonylureas. Phosphinothricin is an inhibitor of glutamine synthetase. Alfalfa cell lines selected for resistance to this herbicide contained amplified genes for glutamine synthetase [28]. In contrast, resistance to sulfonylureas in both tobacco [9] and Arabidopsis [8] resulted f r o m point mutations in genes encoding acetolactate synthase, the target enzyme for these herbicides. Both methods of obtaining herbicide resistance, overproducing or altering the target, have been observed. In addition, genetic modifications to either over-express EPSPS [15] or produce a herbicide insensitive enzyme [29,30] have been successfully applied to produce transgenic plants that are more tolerant to glyphosate.

1

2

3

4

5

9.4kb 6.6 4.3

2.3 2.0

0.6 Fig. 5. DAHPS gene copy number in glyphosate tolerant ceils. Five micrograms of DNA from cells of W38 (1), 17/0 (2), 17/5 (3), 17/10 (4) and 17/20 (5) were digested with EcoRI, separated by agarose gel electrophoresis and transferred to nitrocellulose. The filter was hybridized with the potato DAHPS cDNA from pBDI4. An autoradiograph of the filter after hybridization is shown here. The positions of DNA size markers are shown on the left.

W h y different strategies o f g e n o m e modification are observed in response to selection for resistance to particular herbicides is still unclear. The original t o b a c c o cell line selected for glyphosate tolerance (17/0) has been g r o w n in the absence o f the herbicide for 3 years. Tolerance and E P S P S gene amplification have been maintained in the absence o f selection pressure. Therefore the amplified E P S P S genes in 17/0 ceils are stable and m a y be integrated in a c h r o m o s o m e . The stability o f genes that are amplified to a higher degree in ceils selected for increased glyphosate tolerance has not yet been examined. The E P S P S genes present on the EcoRI fragments o f 14 and 11 kb must be functional as amplification

o f pseudogenes would confer no selective advantage. The other four EcoRI fragments in the tobacco genome that hybridize to the petunia E P S P S c D N A are not amplified in glyphosate tolerant cell lines. These four fragments m a y represent E P S P S pseudogenes or contain functional genes located in regions o f the t o b a c c o g e n o m e that are s o m e h o w less susceptible to gene amplification. Tolerance o f I7 cells to glyphosate clearly involves over-production o f E P S P S to o v e r c o m e the block in the shikimate pathway. However, we cannot rule out the possibility o f other genetic or epigenetic modifications contributing to glyphosate tolerance in these cells. Differences in protein

61

synthesis between unselected and glyphosate tolerant cells have been described [12]. I7/0 cells also contained elevated levels of DAHPS activity [10]. Further experiments are required to determine if changes in expression of genes other than EPSPS contribute to glyphosate tolerance.

10

ll

12

Acknowledgments We thank Nancy Petretic for her excellent assistance with growth and maintenance of cell cultures, Deb Altman and Colleen Thomas for preparation of the manuscript and Drs. Avtar Handa and Randy Woodson for critical reading of the manuscript. We also thank Dr. Dilip Shah (Monsanto) for providing the petunia EPSPS cDNA and Dr. Nikolaus Amrhein for supplying antiserum against petunia EPSPS.

13

14

15

References 1

2

3

4

5

6

7

8

9

H. Steinrucken and N. Amrhein, The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid 3phosphate synthase. Biochem. Biophys. Res. Commun., 94 (1980) 1207--1212. M.R. Boocock and J.R. Coggins, Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate. FEBS Lett., 154 (1983) 127--133. G. della-Cioppa and G.M. Kishore, Import of a precursor protein into chloroplasts is inhibited by the herbicide glyphosate. EMBO J., 7 (1988) 1299--1305. P.M. Mousdale and J.R. Coggins, Subcellular localization of the common shikimate pathway enzymes in Pisum sativum L. Planta, 163 (1985) 241--249. G.M. Kishore, L. Brundage, K. Kolk, S.R. Padgette, D. Rochester, K. Huynh and G. della-Cioppa, Isolation, purification and characterization of a glyphosate tolerant mutant E. coli EPSP synthase. Fed. Proc., 45 (1986) 1506. L. Comai, L.C. Sen and D.M. Stalker, A n altered aroA gene product confers resistance to the herbicide glyphosate. Science, 221 (1983) 370---371. J. Hirschberg and L. Mclntosh, Molecular basis of herbicide resistance in Amaranthus hybridus. Science, 222 (1983) 1346--1349. G.W. Haughn, J. Smith, B. Mazur and C. Somerville, Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonylurea herbicides. Mol. Gen. Genet., 211 (1988) 266--271. K.Y. Lee, J. Townsend, J. Tepperman, M. Biack, C.F. Chui, B. Mazur, P. Dunsmuir and J. Bedbrook, The molecular basis of sulfonylurea herbicide resistance in tobacco. EMBO J., 7 (1988) 1241--1248.

16

17

18

19

20

21

22

23

W.E. Dyer, S.C. Weller, R.A. Bressan and K.M. Herrmann, Glyphosate tolerance in tobacco (Nicotiana tabacum L.). Plant Physiol., 88 (1988) 661--666. E.D. Nafzinger, J.M. Widholm, H.C. Steinrucken and J.L. Killmer, Selection and characterization of a carrot cell line tolerant to glyphosate. Plant Physiol., 76 (1984) 571--574. C.C. Smart, D. Johanning, G. Muller and N. Amrhein, Selective overproduction of 5-enol-pyruvylshikimic acid 3-phosphate synthase in a plant cell culture which tolerates high doses of the herbicide glyphosate. J. Biol. Chem., 260 (1985) 16338--16346. C.M. Smith, D. Pratt and G.A. Thompson, Increased 5enolpyruvylshikimic acid 3-phosphate synthase activity in a glyphosate-tolerant variant strain of tomato cells. Plant Cell Rep., 5 (1986) 298--301. H.C. Steinrucken, A. Schulz, N. Amrhein, C.A. Porter and R.T. Fraley, Overproduction of 5-enolpyruvylshikimate 3-phosphate synthase in a glyphosate-tolerant Petunia hybrida cell line. Arch. Biochem. Biophys., 244 0986) 169--178. D.M. Shah, R.B. Horsch, H.J. Klee, G.M. Kishore, J.A. Winter, N.E. Turner, C.M. Hironaka, P.R. Sanders, C.S. Gasser, S.A. Aykent, N.R. Siegel, S.G. Rogers and R.T. Fraley, Engineering herbicide tolerance in transgenic plants. Science., 233 (1986) 478--481. R.M. Hauptmann, G. della-Cioppa, A.G. Smith, G.M. Kishore and J.M. Widholm, Expression of glyphosate resistance in carrot somatic hybrid cells through the transfer of an amplified 5-enolpyruvylshikimic acid 3-phosphate synthase gene. Mol. Gen. Genet., 211 (1988) 357-363. H. Hollander-Czytko, D. Johanning, H.E. Meyer and N. Amrhein, Molecular basis for the overproduction of 5enolpyruvylshikimate-3-phosphate synthase in a glyphosate-tolerant cell suspension culture of Corydalis sempervirens. Plant Mol. Biol., l 1 0988) 215--220. J.E.B.P. Pinto, W.E. Dyer, S.C. Weller and K.M. Herrmann, Glyphosate induces 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in potato (Solanum tuberosum L.) cells grown in suspension culture. Plant Physiol., 87 (1988) 891--893. P.B. Goldsbrough, S.B. Gelvin and B.A. Larkins, Expression of maize zein genes in transformed sunflower cells. Mol. Gen. Genet., 202 (1986) 374--381. S.L. Dellaporta, J. Wood and J.B. Hicks, A plant DNA minipreparation: version II. Plant Mol. Biol. Rep., 1/4 (1983) 19--21. T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York, 1982. P.S. Thomas, Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 5201--5205. E.M. Southern, Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98 (1975) 503--517.

62 24

25

26

27

W.E. Dyer, L.M. Weaver, J. Zhao, D.N. Kuhn, S.C. Weller and K.M. Herrmann, A cDNA encoding 3-deoxyD-arabino-heptulosonate 7-phosphate synthase from Solanum tuberosum L. J. Biol. Chem., 265 (1990) 1608-1614. N.K. Singh, D.E. Nelson, D.N. Kuhn, P.M. Hasegawa, R.A. Bressan, Molecular cloning of osmotin and regulation of its expression by ABA and adaptation to low water potential. Plant Physiol., 90 0989) 1096--1101. T.J. Burke, J. Callis and R.D. Vierstra, Characterization of a polyubiquitin gene from Arabidopsis thaliana. Mol. Gen. Genet., 213 (1988) 435--443. G.R. Stark and G.M. Wahl, Gene amplification. Annu. Rev. Biochem., 53 (1984) 447--491.

28

29

30

G. Donn, E. Tischer, J.A. Smith and H.M. Goodman, Herbicide-resistant alfalfa cells: an example of gene amplification in plants. J. Mol. Appl. Genet., 2 (1984) 621--635. L. Comai, D. Facciotti, W.R. Hiatt, G. Thompson, R.E. Rose and D.M. Stalker, Expression in plants of a mutant aroA gene from Salmonella typhimurium confers tolerance to glyphosate. Nature, 317 (1985) 741--744. G. della-Cioppa, S.C. Bauer, M.L. Taylor, D.E. Rochester, B.K. Klein, D.M. Shah, R.T. Fraley and G.M. Kishore, Targeting a herbicide-resistant enzyme from Escherichia coli to chloroplasts of higher plants. Bio/ technology, 5 (1987) 579--584.