A
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`
`Wolfgang R. Streber1•2* and Lothar Willmitzer2
`, Schering AG Pflanzcnschutzfor,dnmg, Miillerstrasse 178, D-1000 Berlin 6::i, FRG. 2 Institut for Genbiologische Forschung Berlin
`GmbH, lhncs-trasse 63, D-1000 Berlin 33, FRG. "'Corresponding author.
`
`We have used a gene from the soil bacteri-
`trophus
`JMP134 to intro-
`um Alcaligenes eu
`degra
`duce a herbicide
`ding mechanism
`into plants. The gene, tfdA, which encodes
`a 2,4-dichlorophenoxyacetate monooxy­
`ge
`nase (DP AM) catalyzing the first step in
`the
`bacterial 2,4-D degradative pathway,
`has recently been cloned and sequenced.
`The coding sequence, fused with
`lant
`oduc
`· nto
`expression sequences was intr
`1
`plants
`via Agrobacterium-mediated gene
`transfe
`r. The chimeric gene was ex•
`pressed in tob_acco (Nicotiana. tabacum) un�
`der the control of either a constitutive or a
`ansgenic
`to-
`light-inducible promoter. Tr
`evels
`of 2,4-
`bacco lines tolerated elevated I
`D in tissue culture, and regenerated plants
`showed resistance when sprayed with the
`herbicide.
`
`benoxyacetic acids, such as 2,4-D and MCPA (4•
`chloro-2-mer.hylphenoxyaceti<: add) sho\'I' an aux­
`in-like homlone action when applied to plants. It
`is postulated they mimic the function of Lhe
`intrinsic auxin, indole-3-acctic acid, on an internal auxin
`receptor of the plant. The fact that higher concentrations
`of the iiynthetk auxins are toxic to many dicotyledonous,
`plants,
`has been widely
`but not to monocotyledonous,
`·
`)i
`pplicanon
`as her!. cides with high selectiv-
`used for their a
`broadleaf we
`ity to control
`ed:o; in cereal and grass crops.
`Considerable effort has been spent to create herbidde
`resistant crop plant.5. Although various auempts to isolate
`such mutants or variams by conver1tional breeding and
`tissue culture methods have met so far with limited
`s-U<:cess, the methods of genetic engineering opened up
`the way to transfer effective resistance mechanisms lo
`gl
`plants'. Resistance to at1·a.tine2 , yphosate', and sulfonyl­
`chie
`urea herbiddes·1 have been a
`ved by the introduction
`ncoding
`of foreign genes e
`modified insensitive target
`to phosp
`hinmridn5 and bromoxynil"
`proteins. Resistimce
`were based on the expression of detoxifying enzymes
`originally isolated from microorganisms.
`Attention has also been given to the possibility of
`generating dico1yledonous plant� resistant to 2,4-n. In
`addition to it! agricultural value, the study of 2,4-D
`resistant plants ma)' improve our knowledge ab!)ut the site
`· of action and the molecular mechaniuns by which auxins
`exert their effects. 2,4-O-resista.nt mutant., have been
`·
`tage11es1s of Arabidopsis tlial
`iatltl7
`• Similar
`isolated after mu
`es*, these nnnants showed
`obacco eel
`I !in
`to 2,4-0 resistant t
`a severely altered phenotype. Genetic and biod1emical
`studies suggested that resistance was due to a mutation
`
`affecting a function involved in the action and not in the
`metabolism of 2,4-D.
`A potentially useful approach to the production of 2,4-
`D 1·esistant plants that show a normal phenotype would
`gen
`therefore be to i11Lroduce a
`e into plant cells known to
`f 2,4-D
`into a less toxic com-
`encode the metabolism o
`pound. A gene fulfilling the�c needs has recently been
`cloned and sequenced11
`• The tfdA gene from the soil
`bacterium Alcaligtnes ttutn1Jhtts JMP 134 encodes the first
`enzyme involYed in a 2,4-D dcgradative parbway: A single
`polypeptide
`that is ,1ble to remove Lhe acetate side chain
`from 2,4-D
`in a monooxygenase-Jike reaction.
`[l was not known if the bacterial degradative monooxy­
`genase would be enzymatically active in plams, 01· if it.s
`broad substrate specificity wouJd interfere with the pla.ot
`metabolism. Here we report the expression of tjdA, en­
`coding a 2,4-dichlorophenoxyacecate monooxygenase
`(DPAM) in genetically engineered tobacco plam�. We
`show that transgenic plants that ex.prets DPAM are resist­
`ant to the hel'bidde 2,4-D, and we de�crib.: the phenotrpic
`respon.st: of the transgenic pl.ants to differently substituted
`phenoxy herbicides in tissue culture.
`
`RESULTS
`Contruction of plasmids for constitutive and light•
`ind1.u::ed DPAM expression in plants. To create a plant/
`bacterial hybrid gene for DPAM the procaryotic tramila­
`tion initiation codon GTG of tfdA was replaced by an ATG
`codon. This was achieved by a synthetic double stranded
`oligonudeotide designed to fit a 5' promzding eml gener­
`ated by F'okf 8 bp downstream from the 1ranslation
`initiation codon. The oJigonudeotide contained a s<�­
`quence reconstituting the original open reading frame for
`DPAM, the consensus sequence for plant translational
`start sites upstream from the ATC start c:odon 1-0 and a 5'
`protruding end cornpatible with BamHI cloning sites.
`·n,e oligonucleotide was linked with the coding sequence
`f tjdA
`o
`truncated at its N-tenninaJ end by a partial digest
`with Fok! (Fig. l
`). I11e correct ligation of the oligcmucleo-
`ified
`by sequence analysi!!I.
`tide was ver
`The new sequence was fused at its B' er1d to a 200 bp
`fragment contai11ing the polyadenyfatlon signal of the
`octopine synthase gene (()CS-poly A) 11 and at its 5' end to
`a 540 bp fragmem of the cauliflower mosaic: virns (CaMV)
`35S promoter 1
`2.1s. The resulting plasmid, designated
`MCJC
`1007 (Fig. I), was bm1ed on tbe intermediate vector
`p
`MPKI
`nteg
`rates whh clis-
`p
`10 which is able to form coi
`tu11111fad
`.irmed Ti-plasmids of
`,t.
`em
`such
`as
`pGV
`3850kan'◄• In a similar construction the constitutive
`CaM
`V 35S promc>ter was replaced by Lhe light-inducible
`promoter of gene ST-1.S l '� to form a plasmid designated
`pMlJCl 005 (data not shown).
`Toxicity of the DPAM reaction product. Gas chroma­
`tography and mass spectrometry of£. coli pUJCI00J
`expressing a DPAM-fusion gene showed that 2,4dichloro­
`phenol (2.4-DCP) wall the react.ion product thus confirm­
`ing previous indirect resu!ts9•
`In order to evaluate the possible toxicity of 2,4-DCP lo
`
`B10/TECHNOLcx;Y VOL 7 AUGUST 1989
`
`811
`
`Bayer EX1045
`
`

`

`II- I Construction of plasmid
`integration and expte,ssion of the D
`first doning step the procaryotk
`cleOlide.
`GTG of gene l}iJJt was replaced by a.
`ite the o 1gonudcotide
`To avoid dimcrisation al the Ba
`wn linked in a two itcp ligation fint to the Baml-ll end of
`linearised plJC\8 and lhen 1.0 the DPAM C<>ding 5equ('.nce. In
`
`two following cloning steps, the BamHI-Sphl fragment cort(cid:173)
`taining the hybrid conMruct was im1erted into I.he plam
`exprcision vector plasmid pA8, bet1~ccn the CaMV !!:is
`promoter and the OCS S' end. As pAS contains a ,econd Sphl
`er, pA5 was used Lo form ar,
`site OUl5ide
`on pllC18.
`int.enncdi.atc
`
`pUC18
`
`j BamHI
`
`Sphl
`
`5'-G ATCCAACA A TGAG CG TCG T~:r
`
`3'-G TTG TT AC TCGC AGC AGCGT-5"
`
`~ B ■ mHI
`
`pTJSX535
`
`Fok!
`GATCCAIICAATCIAGCGTC GT
`GT TOTT AC TCOCAG CAGCGT
`
`/»i,.1
`
`✓ !iphl
`
`C:
`
`pA6
`
`Llgue
`
`/41
`
`partial
`
`/
`
`TAO
`
`,&p Ip ::lphl
`
`1-tl,,,cUH
`
`j BamHI
`
`Hindlll
`
`.,,,,.$.
`
`orl pBA322
`
`. '
`1 "•"'
`c !!el -- Ir
`
`Hindlll
`
`looFII
`
`Kpnl
`
`pMCJC1007
`
`Sm/Sp
`
`or! pBR322
`
`812
`
`B10/IECHNOLCGY VOi- 7 AUGUST 1989
`
`Bayer EX1045
`
`

`

`plant cells, callus cultures of tobacco were placed onto MS(cid:173)
`plat.es containing I mgtl NAA, 0.1 mgfl BAP and 2,4-
`in concentrations ranging from l to l O mgfl. No
`toxic effects could be seen with 10 rrtg/1 2,4-DCP, whereas
`more than 2 mg/I 2,4-D severely reduced
`wrned calli brown. Perkins e.t al. 16 reported
`inhibition of lObacco suspension cultures by l O mg,1 2.4-
`pCP, but a generally much lower toxicity of 2,4-DCP on
`the development of leaves and roots when compared to
`2,4-D. The3e results were encouraging with re11pect to the
`idea that transformation of tobacco with the DPAM gene
`could result in resistance to 2,4-0.
`Tnmsformation of tobacco with DP AM genes and
`transcript 1malyai11. Both constructs containing the hybrid
`DPAM genes in combination either with the constitutive
`(pMC]Cl007) or with the light inducible promoter
`(pMLJClOOS) were used to transform tobacco (Nicotiana
`u1bacum W38). Gene transfer was mediated by Agrobact1ri-
`1wi st.rain C58C I canying the disarmed Ti-plasmid
`pGV3850kanH that is able to form cointegrates with
`pBR322 derived vectors. Shoots were regenerated from
`infected leaf discs by selection on kanamycin, screened for
`nopaline production and later analyzed by Northern blot
`hybridi1.ation for I.he synthesis of DPAM-RNA. Twenty
`out of 25 transformed plants S)•nthesized a speci6cally
`hybridizing transcript of the expected size of L3 kb (data
`not shown).
`Expression of the DPAM gene in tissue culture of
`tobacco. 2,4-D is known to promote callus growth and lo
`suppress shoot ~generation from explantecl tobacco leaf
`discs in tissue culture when applied in concentrations
`higher than O. I mgil. To test transgenic: plants for their
`ability lo tolerale elevated levels of 2,4-D, we chose a
`"shoot regeneration" medium containing LO mg/l BAP
`and 2,4-D at concentrations of 0.1, 0.2, 0.4, LO, 2.0, and
`4.0 mg/l. Leaf discs from tobacco plants where the DPAM
`gene was transcribed from the constitLttive 35S promoter
`were able to develop normal shoots on a medium contain(cid:173)
`ing LO mg/I 2,4-D (Fig. 2A). Depending on the individual
`transformed plant tested, minimal shoot inhibition is
`observed at concentrations of 2,0 or 4.0 mg/I 2,4-D,
`whereas leaf discs from untransformed control plants
`(tobacco W38) did not show any differentiation on 0.2 rng/
`l 2,4-D, indicaLing that
`ression of the DPAM
`could result in up to 20-
`higher tolerance again
`D.
`u
`A significanlly different reaction was shown by
`where the DPAM gene is under the co1mol of the
`t-
`inducible promoter of the ST-LSI gene. As this promoter
`1.s only active in photosynthetically active tissues, a11d shoot
`differentiation from ex planted tobacco leaf discs proceeds
`1•ia a (photosynthetically not aclive) callus stage, as expect(cid:173)
`ed in the shoot suppression assay, the transgenic DPAM-
`rlants showed 1he same sensit' ·
`· st 2,4-D as the
`nontransformed tobacco W38 (
`).
`R.e1ponse to differently substituted phenoxyaCfltic a.c(cid:173)
`ids. In Alcalig,n.n 11utn,phu., JMP1S4 from which the
`DPAM gene was
`' · Uy isolated, DPAM is able to
`degrade 2,4-D, MCP
`CPA and unsubstituted phen(cid:173)
`oxyacetk acid. To invest.igate how transgenic tobacco
`expressing DPAM responds to the auxin activiLy of differ•
`emly substituted phenox.yacetic adds, the suhslances listed
`tn Figure 3 were ·
`· MS-a
`together with
`LO mg/l BAP.
`co WS7-7 and
`ransgenic t
`control W38 were placed on the media and shoots arising
`were counted after 18 days. Figure 3 shows, that trans-
`plants expressed a similar tolerance lO 4-CPA as Lo
`, a slightly reduced 1:olerance to MCPA and no
`tolerance against tri-:rnbstituted phenoxyacetic acids and
`all the phenoxypropionic acids we tested.
`
`I
`
`B R- t Tulcrance <>f transgenic tobacco against 2,4•D in
`
`tissue culture. Exp!ant.s fr<>m Nico#a1111. tab.ieu111 W38 contrcll
`pla'nts (left) and
`·
`incubated on
`medium containing
`oots develop
`from leaf disc:i which c0tut1tutively cx.preu DPAM l;A,right}.
`'
`· explanu with a light-inducible DPAM gen (B,
`the !ame sensitivity ag-.tinal 2,4-D as the nonmms(cid:173)
`(lcft}.
`
`Transgenic plants are resistant to the herbicidal activi(cid:173)
`ty of 2,4•0. Replicates of transgenic plant8 obtained by
`clonal propagation were tl'anllferrcd w soil and grown in
`the gre~nhouse. No difference i.n growlh behaviour and
`final hei.ght could be seen between untransformed and
`transformed tobacco. After two weeks c)f growth in soil
`control tobacco plants and transgenic tobac<:o plants were
`sprayed with dose!> equivalent to I ltiba, 3 kgtha and 10
`kg/ha 2,4-D-Na. Plants were furthc
`n for 2-3 week.s
`in the greenhouse and then ch
`for resistance.
`Whereas 1 kg/ha totally h1hibited growth ()f nont.rans(cid:173)
`formed tobacco planes, plants of the transgenic line WS7-7
`which expressed the DPAM gene under the comrol of the
`constitutive 35S promoter were resistant to up to l O kg/ha
`2,4-D-Na (Fig. 4 A).
`Plants of the transgenic line WS4-7 lhat expressed the
`DPAM gene under a light-inducible promoter active
`in green tissue and thus did not show tolerance in. a tissue
`culture assay, were also resistant to the herbicide when
`sprayed with 1 kg/ha 2,4-D-Na (Fig. 4 B). No difference in
`growth behaviour and final height was observed between
`transgenic plants treated with the herbicide and non(cid:173)
`treated plants (Fig. 4 C).
`
`Bayer EX1045
`
`

`

`Inheritance of 2,4•D resistance. Seeds from selfed
`WS7-7 plants were germinat~d on sterile MS medium with
`k.anamydn. Two hundred and fifty eight seedr
`resistam to kanamycin and 83 were sensitive i
`active locus. Leaf seg-
`Mendelian inheritance of a sin
`Fl plan1.s were placed
`ments from 16 two weeks old
`on 2,4-D medium to check for 2,4-D resistance, another
`20 plants were sprayed with S kg/ha 2,4-D-Na .. lit all cases
`inheriumce of 2,4-D resistance was observed. Southern
`blots showed that the pare11tal line WS7-7 contained at
`least three copies of the DPAM gene, which could also be
`found in 14 out of 15 analysed transgenic Fl plants (data
`ests that the three T-DNA copies in
`not shown). This
`d.
`line WS7-7 were
`DPAM as a &electable marker. The use of the constitu-
`1.ively expressed DPAM gene as a selectable marker for
`A.grobacterfam mediated leaf disc transformation was ex(cid:173)
`amined, In a standard transformation procedure, kana(cid:173)
`mycin was replaced by 2,4-D at a concentration of 0.5
`mg/l, which was previously !hown to be appropriate to
`distinguish between untransformed and completely trans•
`formed tissue, but no shoot development was observed
`under these condit.ions.
`In order to test if DPAM expression could be used for
`selection of germinating seeds, root formation from seeds
`was measured iu response to different. concentnttions of
`2,4-D. Inhibition curves {Fig. 5) demonstrate that no
`significant difference in sensitivity is seen between un(cid:173)
`u·ansformed seedlings and seedlings containing the 35S(cid:173)
`DP AM gene.
`
`DISCUSSION
`The tfdA gene from Alcaligmes rotmphu.s JMP134 en(cid:173)
`codes the en:z.yme 2,4-dichlorophenoxyacetate monooxy(cid:173)
`genase {DP AM) that degrades the herbicide, 2,4-D. The
`degradation product, 2,4-dichlorophenol, has been shown
`to be non-wxic to plants at least up to concentrations of 10
`mgn. We bave fused the DPAM coding sequence to bolh a
`constitutive and a light-inducible promoter and trans•
`£erred the constructs in tobacco using an Agrobactl!rium
`nts transformed
`vector system. Transgenic tobacco
`sistant to high
`with the chimeric DPAM-genes were
`doses of 2,4-D. In addition, leaf cells from transgenic
`tobacco that constitutively expreu D.PAM are able to
`redifferentiatc into shoots in the pre$ence of concentra(cid:173)
`tions of 2,4-D that normally suppress shoot formati()n.
`Unlike 1.he NPT gene" or the PAT genes, the DPAM
`gene COLLld not be used a~ a selectable marker in leaf disc
`transformation. One reason may be that in a chimeric
`tissue transformed cells cannot develop into shoots be(cid:173)
`cause they are overgrown or otherwise inhibited by un(cid:173)
`transformed callLts that is rnpidly developing in the pre~(cid:173)
`ence of 2,4-D. It remains to be examined whether trans(cid:173)
`formation of protoplasts instead of leaf disc., might
`overcome this problem.
`It is also not possible to select transgenic DPAM plants
`during germination on 2,4-D. The laclt. of resistance of
`roots against 2,4-D may have several reasons, e.g. a lower
`expression of the S5S promoter in rools, a higher sensitiv(cid:173)
`ity of roots against 2,4-D or 2,4-DCP compared to shoots
`or an accumulation of 2,4-0 or 2,4-DCP by transport.
`The analysis of Lhe DP AM-specific: mRNA demonslrat(cid:173)
`cd a substantial variation in ex.pression levels of around 20
`to 80-fold between independent transformants. Ne,·erthe•
`less, the highest expression level ofthe DPAM gene in line
`WS7-7 corresponded to only lhree T-DNA copies in the
`genome (data not slmwn). A similar observation has been
`made by DeBklCk et aP who reported three copi.es of the
`expressing plant. He suggested
`PAT gene for the hi
`that the observed 30-fol differences in the expression of
`
`4-CP
`
`2,4-DP 2,4,5-TP
`
`4-CPA MCPA 2,4,5-T
`2.4-D
`RIIII I Crmi tolerance of transgenic tobacco against other
`phcnoxy herbicides. Suh,tances li:11ed below were incorponll(cid:173)
`in the 1ex1. The graph
`ed imo 2MS agal" as, de11cribed
`demona.trates the respon11e of concrol t()hacco (W38, black.
`c<ilumn:s and tramgenic tobacco (WS7-7, hatched columns)
`fferent synthctk auxins. The hl'.ight of the ,;olumns
`·
`m 1cates the upper limit thal allowed cells from explamed
`leaf dhic, to differentiate into normal shoots. At hi her
`as observed. 2,4-
`co11ccntrati011 only callu~
`: 4--chlQrO
`acetic
`noxyacctlc
`hlom-2
`4-CP: 2-(4.-chlorophen.
`2,4,5-trichlorophenoxyacetic a
`oxy)propionic acid, 2,4-DP: 2-(2.4·dkhlornphenc1xy)prn(cid:173)
`pion1c acid, 2,4,5-TP: 2-(2,4,5-trichlornphenoxy)propionic
`acid.
`
`wbacco plants. A. J:,.
`MUa 4 Effect of 2.4-D on
`ft) and a dona! rcpb-
`Nicotia1ta labacv,11 W38 contr
`cate of the 'IN57-7 line that expressei t e DPAM gene, uuder
`the control of the CaM V 353 promoter (right) were sprayed
`4-D-Na. B. A Nicoliana tabac11111 W38 control
`1d a clonal replica1.e of the WS-4-7 line that
`exprcs~es the DPAM gene under' the control of the light-
`I kg/ha
`moter (right) were$
`inducible ST-LS
`rayed
`e w
`tes of the WS4-
`2,4-D-Na. C.
`). The
`either with water (ldt) or with I kg/ha 2,4*D-Na (
`picture, ,~ere taken three weeks af~r spraying.
`
`Bayer EX1045
`
`

`

`pAT-RNA maybe due to effects of the T-DNA position in
`rhe chromosome.
`Expression ofDPAM-RNA in transformants where the
`DPAM gene is under the control of the light-inducible
`promoter varied in the same range, but we never observed
`similarly high expressing variants compared to the consti(cid:173)
`tutively expressing plants. The highest observed expres(cid:173)
`sion corresponded to a resistance·against sprayed doses of
`1 kg/ha 2,4-D-Na. No qualitative difference could be seen
`between resistance phenotypes generated by the constitu(cid:173)
`tive or the light-inducible DP AM gene.
`transgenic plants expressing DP AM were
`When
`sprayed with 2,4-D in the greenhouse, they were fully
`resistant to amounts of 2,4-D commercially applied in the
`field. For example, line WS4-7 tolerates 1 kg/ha, line WS7-
`7 tolerates IO kg/ha 2,4-D-Na. Depending on the crop and
`weed species, 2,4-D is effectively applied in amounts
`ranging between 0.3 kg/ha and 3 kg/ha. This result is
`encouraging with respect to the application of the DP AM
`gene to create 2,4-D resistance in other commercially
`interesting crop plants. As 2,4-D is already a selective
`herbicide in monocotyledonous crop cultures, dicotyle(cid:173)
`donous crop plants will be the preferred targets for
`genetic engineering. Additional investigation of the fate
`of the DPAM reaction product, 2,4-dichlorophenol, in the
`TJlant are required. It may be worthwhile to think about
`1troducing further genes of the bacterial 2,4-D degrada(cid:173)
`tive pathway into plants. In any case, toxicology and
`persistance of 2,4-D and its degradation products have to
`be newly evaluated.
`Other attempts to obtain 2,4-D resistant plants have
`been made7 •18 • The introduction of the detoxifying en(cid:173)
`zyme DP AM by genetic engineering is up to now the only
`approach that results in fully resistant and phenotypically
`normal plants. This strategy promises to have broad
`application, especially with herbicides for which detoxify(cid:173)
`ing pathways can be found in nature or created by
`genetically modifying existing pathways. Future develop(cid:173)
`ment of new herbicidal compounds should favour sub(cid:173)
`stances that are easily degraded by microorganisms, thus
`providing environmentally safe, non-persisting herbicides
`together with the opportunity to introduce these selective
`resistances into crop plants by genetic engineering.
`
`EXPERIMENTAL PROTOCOL
`Strains. E. coli TB I is a derivative of strain JMlOl 19 with the
`genotype; F'(tmD.86, proAB, lad, lacZllM15) '1(lac, pro), supE, thi,
`11'tA., Srl::TulO(Tc') (B;;i_rt Barrel, pers. communication).£. coli
`GJ23~" w11s u.sed as_ a helper strain for triparem;;il matin~.
`',gro/:latte,im11 Utm,facitns strain C58Cl (pGV.3830kan) 1i contams
`a disarmed cointegrate-type Ti-plasmid.
`Plasmids. pUCl8 wa1 described by Norrander21 . pA5 (Fig. 1)
`contains 200 bp of the octopine i-.ynthue 3 '-end 11 inserted into
`pUC18 (A. von Schacwcn., in preparation). pA8 (Fig. 1) is a plant
`expression vector constructed by insertion of the CaMV 35S
`promoter 12 and the OCS-3' endll into pMPKl 10 (A. von
`Schaewen, in preparation). The multiple cloning site of pA8 was
`derived from pUC18. pTJSX535 contains the coding sequence of
`tfdA from Alcaligents eutmphus JMP134 on a 1.5 kh Xbal-Sall
`fragment 9.
`Media. Synthetic media for plant transformation and shoot
`regeneration from transformed and u11tra11sformed tissue l'l'ere
`based on MS medium~~ comaining 20
`ucrose. Basic chemicals
`and 2,4-D were from Men:k,D,m1ist:,ia1:, FRG. All other syrtthtLic
`hormones were from Sigma, Si. Louis, MO, USA.
`:lcc)ticics w,~r,e symhesizerl
`Synthe
`nucleo~d.es. Ol
`ipment and purified hy
`Biosystems 380
`by an A
`prep;native polyacrylamide gel electrophoresis as described iu
`the Applied Biosystems User Bulletin issue No. 13, 1984 (Ap(cid:173)
`plied Biosystems, Foster City, California).
`Plasmid contructions. Pairs of complementary oligonucleo(cid:173)
`tides, each consisting of 20 bases, were phosphorylated by poly(cid:173)
`nucleotide kinase, annealed by shifting temperature from 60°C to
`37°C, and then ligated with pUC18 that had been digested with
`
`16
`
`14
`
`12
`
`10
`
`8
`
`6
`
`4
`
`2
`
`0
`
`E
`§.
`.c
`CJ
`C
`~
`0
`E
`
`0
`
`WS7
`W38
`
`2,4-D (mg/I]
`
`RMI S Effect. of 2,'1-D on rooting of seeds. Transgenic
`tobacco seeds comah1ing the 55S-DPAM gene (WS7) and
`untransformed .'leeds (W38) were genllinated on ~teril,! MS
`ning 2,4-0 at concentrations of 0, -4, 10, 20,
`d 100 µ.g/1. After 14 day& of incub,nion in the light, root
`length wat measured. Vertical ban indicate tlw standard
`
`error. -BamHI and SphJ. The linear product was se arated from au
`
`rcsis using
`excess ()f oligonucleotides by agaro,e
`DEAE membrane NA45 (S<;hlcicher &
`X.535 plas•
`mid DNA waa digellted with Xbal and Sphl and 5eparated by
`ontaining 1he
`agarnse gel dtctrophoJ"esis. The 1.2 k
`by electrodu-
`lie ucnce wa.s i:wl:ated lf1·0
`DPAM <:<1din
`tion, putia
`ted by Foltl, and repurili
`A fragmenu of around l .1 kb
`electro
`were ligated with the linearised pUCl8 that contained the
`leotide. The resulting plasmid pUIClO0J 1~.ts cloned in
`BI. DNA nf pUJCIO0J was digested with B,m1HI and
`Sphl lo ex.cise the insert, imd with PvuI to 11void religation of the
`vector, and then ligated witb pA5 that had been dige5ted with
`BamHI and SphI. pA5 was used as an. intermediate vector as pA8
`contained a second Sphl site outside th
`er. The result•
`ing plasmid pUJAO was cloned in E.
`TBl. DNA of both
`with Barn HI and Sphl, mixed
`pUJ A01002 and pA8 was
`pMCJA1006 wasdoned in E.
`and ligated. The result'
`coli TBl.
`Tram£onnation of tobacco pliu1ts with cbhneric genes,
`pMCJC1007 and p.ML.JC10O5 were mobilized from E. c!!u TBl
`mto the Agritboclenum strain C58C1 (pGV!ISS0kan) by a triparen-
`E. coli GJ2!1 ;;is a helper strain. AgrobactcriM
`.
`ybridfaat.ion were shown 1.0 contain the DPAM
`gene were mbscquent!y used for leaf disc infection of Nicotiom1-
`tab.2C1u11 W38. Tra1nfonnation and n:gcneration of tobacco plants
`followed established procedures".
`Cloning, Construction of recombinant DNA molecules, trans(cid:173)
`formation into E. coli, and analysis of recombinant clone5 were
`performed according to established prncedurc!fl,
`DNA sequencing. DNA wa, sequenced by the dldeoxy mcth-
`lasrnid DNA as a ternplate•0 •
`d25 u:sin
`lbl1ri1ii1D~ tion e.1tperiment1, RNA isolation from leaves and
`Northern hybridization ex.perirncnts were performed as de(cid:173)
`from
`fragment
`scribed by Jones 11• A BamHI-Hind[lI
`pU]ClOOl, can~·ing the DPAM gt:ne, was radioactivel>• labek:d
`an<l used H a probe l() detect l)PAM RNA. Plant DNA was
`isolated eisentially as described by Dellaporta~ 1• For Southern
`hybridization experimentst8 plant DNA wa cut wilh HindlII
`and probed with a cloned fragment of the nopaline synthase
`gene.
`Hcrbkide application. Each pl.mt was spl'lt)'ed with 4 ml of a
`l % aqueous solution of 2:,4-D, Na-~alt. With an e:nimated ground
`surface of 400 cm2 per plant, this roughly corresponds to 10 kg/
`ha. l.ower quantities were achie1•ed b)· appropriate dilution of the
`spray solution.
`
`Acknowledgments
`We are grateful to C. Recknagel for excellent performance of
`plant transformation, to R. Breitfeld for propagation of plants, to
`U. Kutschka for analysis of plants, to A. v. Schaewen for kind
`gifts of plasmids, and to H.-J. Scheuermann for gas chromatogra(cid:173)
`phy and mass spectrometry.
`
`Received 18January 1989; accepted 30 March 1989.
`
`B10/IECHNOLOGY VOL 7 AUGUST 1989
`
`815
`
`Bayer EX1045
`
`

`

`1 !
`
`./
`.!
`
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`BIO'TECHf\lOLCGY VOL 7 AUGUSf 1989
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`
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