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Transformation of white spruce and other conifer species by Agrobacterium tumefaciens

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Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0–80% depending upon the
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  Plant Ccll Reports 1989) :16-20 ABSTRACTStudj es of the ability of Agrobacterium totransform whjte spruce (Picea glauca), Engelmann qnrrre o (P pnopimann i ) qi ika qnrrre a /P qii.hon--i -\ruruLs \1. lljjSjlll?-ll_l.]ar. JrLNo ouruLU \r. rrLLrrLrrrf>, and Douglas-fir (Pseudotsuga menziesii) showedfrequencies of ga11 formation from 0-80% depending rrnnn Fha qtrai. of Aprohacterirrm, and the COnifef species. Thirty six A. tumefaciens strains and oneA. rhizogenee strain were tested on 6 month o1dwhite spruce seedlings. Nine A. tumefaciens sLrainslnduced ga11 formation on more than 50% of theinoculated trees and at greater than 10% of theinoculated sites. One strain, 82/74 gave rise toga11s at 287" of the lnoculated sites on white spruceand induced the highesL overal 1 frequency of galIformation on all the conlfer species tested.Relative frequency oI gall formation was consisLentamong species, alEhough Lhe overall frequency wasmrrch h oher on Dorrpl s-f i r - Of Lhe wellcharacterized strains for which disarmed derlvativesare available only A2B1 (carrying the supervirulenL tllmnr indrre ino nlnc-'i/l nTiR^(/'?) oqrrp efFinipnt..---*..o /loDlulu, Pt LDUJ+L,/ 6Av E SILfLLEIIL transformation. Stable integration of T-DNA encoded L^^ L^^- ^onfirmed hv the exnrpssion of onine BglrqJ lldD UgEll Lvrr rrrrrLu u) LrrL L^pa Lro rvrr vr vl- synthesis and hormone autonomous growth. Thetransfer and Iong-Lerm sLabLe expression oIkanamvcin resisfance and fireilv Luciferasg 36livitvrrsi no h inarv ve.-t nr svsf ems was af So ach-Leved. INTRODUCTION The oenefic 6-^i-aar-i-^ ^r conifers offers anopportunity for the introduction of novel traitsinto important forestry species more rapidLy than bytraditional breeding. GeneEic engineering has beensuccessful for a number of agricultural species,howpver- thp anolication of these methodS oforestry, in particular conifers, has been limitedby the lack of transformation methods andregeneration systems. Recent advances such as thedevelopment of embryogenesis in Norway spruce(Hakman and von Arnold 1985) and the regeneration ofnrof on asf s f rnrn ^-hr.'^^a--i ^,,1 tures of PinuS Laeda r vuvprqu Y vFvrrle vui (Gupta and Durzan 1987) and Picea glauca (Attree etal. 1987) are expanding the methods available forthe regeneration of conifers. Methods ofintroducing DNA which are compatible with theseregeneration systems are sti11 required for theproduction of Lransgenic conifers. Agrobacterium-mediated transformatlon has been the most successfuland broadly applicable method for the introductionof foreign DNA nlo plants and has been successfullyapplied to woody plants (Flllatti et a1. 1987,McGranahan t al. 19BB). Until recently however, ihe host renop of Aorohacterinm "^^ I L^"^L + L _. ry was rnougnt to Delargely confined to dicotyledonous angiosperrns. DeCleene and De L"y ( \976) reported symploms on somegymnosperrn pecies following woundlng withAgrobacterium tumefaciens strain 86 (LMG187). MorerecenF v. t he t ransformaLj n of sevcral n nes  vevr' +J t (SederofF eL al. 1986, Stomp eL al. l9BB) andDouglas-fir (Dandekar et a1. 7987) has beenconfirmed through the analysis of biochemical andmolecular markers.The objecLive of Ehis research was to idenrifyAgrobacterium strains which could transformcommer( aIIv imnortant conifer Snecies in BrifishColumbia. As a first step, we reporL Lransformat'ion ^f pi I -"-- f r^rh t p <nrrre o) P ^-^61fr-hh ju, ]_j 5 tsrouLo \wrrLLs Dpr uLE,l t r , cttHefllliLllltl- (Enge1 mann spruce) , P. sitchensis (Sltka spruce) ,and Pseudotsuga menziesii (Douglas-fir) by ^^-^L^^F^*:"- Bv qcreen ino a laroe nrrmher nltsrwuogLEr f ulll , __ o_ Aprohacferirrm rrains we were ablc to ident [vstrains which transform these conifers at highfrequencies. Although we know of no reportscorrelating in vivo and in vitro infectionfrenrtencv. the identification of strains which  urrs r infecL at a high frequency in vivo should be goodcandidates for high frequency in vitro infectlon.Furthermore, marker genes were introduced intoconifer seedlings through binary vectors and stableexnress on of I rrcj erase and kanamvc n res i stancewas demonstrated.MATERIALS AND METHODSBacterial strains and plasrnidsAgrobacterium strains used are listed 1n Table1. Strains were obtained from E. Nester, Universityof Washington, Seattle, WA; C. Kado, University of'^rir^*-r^ ^^--isr CA; L. Moore,Oregon State drILUl ltad, udv I University, Corvallis, 0R; and J. De Ley,Laboratorium voor Microbiologie en MicrobieleGenetica, Cent, Belgium. Cultures were gro\{n for 2days on agar plates containing PDA (Potato DextroseApar) medium orior to inoculation. Carrot slices o" / were inoculated as positive controls (Ryder et al.1 qRq Ilari rrari ves of A2B1 and \12/73 carryingbinary vectors were generated by transformation (Anet a1. i9BB) with purified plasrnid DNA (Maniatis eta1. I9B2). The two binary vectors used were: pEND4K(K1ee et al. 1985) with neomycin phosphotransferase(nptII) linked to the nopaline synthetase (nos)promoter and 3' sequences and, pLUX2 which carries achimeric firefly luciferase gene from pDO432 (0w etal. 1986) and is a derivative of pEND4K acking thenptTI gene. Strains R1601 and A2BI/pTVK291 carrythe plasmld pTVK291, which contains the virulence1 Present uddress; Department of Horticulture, University of Wisconsin-Madison, 1575 Linden Drive, Madison. WI 53706, USAO./lprint r(.quests o: D. Ellis (at his prcscnt address) PlantCell Reports @ Springer-Verlag 989 Tiansformation of white spruce and other conifer speciesby Agrobucterium tamefuciens David Ellis r, Dane Roberts, Ben Sutton, Wayne Lazaroff, David Webb, and Barry Flinn Forest Biotechnology Centre. BC Research Corporation, 3650 Wesbrook Mall, Vancouver B.C., Canada, V6T 2L2, CanadaReceived September 19. l988iRevised version received November 28, 1988 - Communicated by F. Constabel  region from the supervirulent tumor-inducing plasmid,pTiBo542 (Komari et a1. 1986).Growth and inoculation of seedlinFive month o1d dormant Picea glauca (whitespruce), P. engelmanii (nngeTilEnn spmce), p.sitchensis (Sitka spruce) and Pseudotsuga menziesiiO;D.rg1. -fir ) seedlings were pTaEEa-Zt-h'oC und .20 h/day photoperiod for one month prior toinoculation with Agrobacterium to a11ow the buds toflush. Seedlings were watered twice weekly andfertilized with a 20-20-20 soluble plant food (ShurGro, Greenleaf Products, Burnaby, B.C.), at 1/10 therecommended rate every other watering.For the initial screening, white spruceseedlings were j-noculated with thirty sixAgrobacterium tumefaciens strains and one A.rhizogenes strain by piercing the stem with a 26gauge hypodermic needle dipped 1n Agrobacterium or byrnaking a tangential slice into the stem such that aflap of tissue rernained attached. Agrobacterium wasthen placed on the cut surface of the stern and theflap pressed against the stem. For inoculation,seedlings were divided into new growth (stem tissueforrned during the period of flushing) and o1d growth(stem tissue formed prior to bud set). Each ageclass was inoculated four times with the piercingmethod and once with a slice for a total of 10inoculation sites per seedling. Twenty trees wereinoculated per Agrobacterium strain. In subsequentTable 1. Results from the screening of Agrobacteriumstrains for infectivity to white spruce.PRODUCEDGALL_LIKE OPINESTRAIN GENOTYPE SYMPTOMS DETECTEDT3l wild type +Bo543 wild type + agropineC5B wild type rSI/73 wild type +/-Id7/73 wild type + rr^ l-^ W'2/73 wild type + agropineK75/73 wild type +A2B1 A136 (pTiBo542) + agropineBo542 wild type + agropine1D135 wild type +A34B A136(pTiA6) + agropine'+R1601 RA4 (pTVK291 ) +/- ^^ l- t C3/74 wild type + nopalineK6/13 wild type + nopalineC2/74 wild type +4I wi-1d type r a.^ l-F I7O/75 wild type + nopaline116/75 wild type +82/74 wild type + nopaline40 wild type +142/73 wild type + nopalineCG56 wild type +/-LMG301I1 wild type +/-LMG3O1I2 wild type +/- nopalineLMG1B7 wild type + agropineLMG137 witd type +/-A2BI/ A136( pT1Bo542/ + agropinepTVK291 pTVK291)762 wild typeA6 wild tvoe37 73 wild typeSII/73 wild typeRR5 wild typelDl wild type1D156 wild type1D1109 wild typeLMG136 wild typeLMG340 wild type* Note A34B is an octopine producing strain. t7 experiments wlth selected strains, the slicing wassubstituted with an additional piercing because noincreased frequency of infection occurred with theslice method. A total of 40 trees were inoculatedwith each strain in these subsequent experiments.Following wounding, seedlings were covered with aplastic sandwich bag for one week to minimizedessicatin of the wound sites. Decapitation of someseedlings occurred during inoculation and when thishappened the cut surface was also inoculated.Control tissue was wounded identical to inoculatedseedlings with a needle dipped in Agrobacterium freemedlum.Ga11 formation was visually assessed at weeklyintervals and galls were harvested for opine analysis5 months after inoculation. In addition to gallswhich were morphologically identifled as A.tumefaciens-induced ga11s, ga1l-1ike symptoms such asabnormal swelli-ngs were also recorded. Wound sitesfrom control trees and lnoculated sites whlch did notform gal1-like synptoms, were also harvested andassayed for opine synthesis. The frequency of ga11formation by each strain was determined at the end ofthe experiment by counting the number of ga11s formedand dlviding this by the number of i-noculation sites(lO sites per tree).Opine analvsisTissue excised for opine analysis was harvestedand approximately 2 U1 of B0% ethanol per mg freshweight of tissue was added to the sample. The tissuewas homogenized, centrifuged at 16,000 x g for 10 minand the supernatant used for analysis by high voltagepaper electrophoresis (Otten and Schilperoort 1978).Eight U1 of sample supernatant was spotted onto 3 mm\{hatman chronatography paper and octoplne, nopaline(Sigma Chemical Company) and agroplne (P. Guyon)standards were run with each analysis. The paper wasplaced in a BioRad DNA sub ce11 unit containing BO0ml formic acid: acetic acid: water (5:15:80) runningbuffer, and electrophoresed at 300 V for 1.5 h. Toensure ldentity, oplne standards, and opines fromselected samples, were also electrophoresed at adifferent pH utillzing a running buIfer of pyridine:acetic acid: water (50:0.25:94.75).For visualization of agropine, the driedelectropherogram was dipped in acetone containingO.2% silver nitrate for 1 min, rinsed briefly inwater and transferred I:o I% NaOH n methanol for 2-3min (Parsons et al. 1986). The reaction was stoppedwith Kodak photographlc fixer, and under theseconditions agropine appeared as a distinctive brownspot which comigrated with the standard.Octopine and nopaline were deLected by sprayingthe dried paper with an BO% ethanol solutioncontalning O.022 phenanthrenequinone and 22 NaOH.Octoplne and nopaline were identified by theappearance of a yellow fluorescent spot underultraviolet light. Non-transformed tissue containedcompounds that co-mlgrated with nopaline standardsbut were a light blue color, and could easily bedistinguished from nopaline.In vitro culture of sa1l tissueed in a IO% chlorox(5.257 sodium hydrochorite), 0.1% Tween 20 solutionfor 10 min, rinsed 3x in sterile distilled water andthen placed on a hormone free basal medium (Rei11yand \,{asher, I977), contaj-ning 250 ug/ml cefotaxime ta and 500 Uglm1 carbenicillin. After 2 months inculture, tissue was homogenized, plated on bacterialmedium and determined to be bacteria free by theabsence of bacterial growth. Resistance to kanamycinwas tested in galls induced with \12/73 containing theplasmid pEND4K by placing the gall tissue on mediumcontaining 50 pg/ml kanamycin. Ga11s formed by thewild type strain without the binary vector were alsocultured as non-kanamycin resistant control tissue.  l8 Luciferase assaDouglas-fir and carrot galls jnduced by strainscontalning the binary plasmld pLUX2, as well ascontrol gal1s induced by the same strains without theplasmid, were cultured as above, on a hormone freemedium containing cefotaxime (250 pg/ni_) andcarbenicillin (500 Ug/m1). Approxlmately 200 mg oftlssue was placed in 500 m1 assa" h"ffar /]nn -M sodium itrate prl .o, "ioi- a7^Z;i.r;";;;;")ii"l',"' containing I mM D-luciferin and the luminescence wasrecorded on a scintillation counter (BeckrnanInstruments, Model 1801) in the single photon monitormode. RESULTS Gall forrnation and o rne roduction White spruce0f the 37 Aprobacterirrm strains tested for their ^1-ili+ r +^ :-g^l-- durLLL) LU rrrrect white spruce twenty one of thestrains induced galls with a characteristic greenglobular appearance and in some cases, these gaverlse to secondary gal1s (Fig. 1). An additional sixstrains, including the A. rhizogenes strain R1601,induced swe111ngs at the wound site and were rated+/-. Both ga11s and swellings were tested for their nn i np .nnt ont The nroqenep nf ai t har -^r^n-i -^parrs LUtlLstlL eLLIlef agfopang Of nopaline was confirmed in galls resul ting from I 3strains including one strain (LMG3O1I2) rated as +/-(Table 1). Opines were not detected in controltissues or frorn wound sltes which did not Droducega11s or swelllngs.Of the 13 strains that produced detectableopines fron gaIis, nine projuccd gall s o,r morc thiln507 of the 20 seedllngs inoculated in the initialwhite spruce experimenl (TabIe 2). The two strains(K6/73 and B2/74) which produced ga1ls on greaterLhan 902 of the seedllngs inoculated also produced the h'i phest f,renrrcncv nf on I I f ormal i on on a ner 6uff wound basis (Figure 2).The Frequency of ga1 formation depended on bolhthe strain and the age of the tissue (Fig. 2).Overall strains B2/74, C3/74 and K6/73 lnfectedwhite spruce at the highest frequencies. 0f the wellcharacterized A. tumefaciens strains for whichdisarmed de.i,r.tiuesl.e auailable for use in geneticengineering, A2Bl was a relative_Ly efficienL strain.A2B1 has the C5B chromosomal background and carriesthe supervirulent Ti plasmid, pTiBo542. It wastherefore of interest to compare the efficiency ofA2B1 with 8o542 and with A2B1/pTVK291., wlni.ch carriesaditional copies of the vir region from 8o542.Although results from the screening experlnent (Fig.2) suggested some interesting differences, subsequentexperiments showed that there were no reproducibledifferences in the efficiency of these strains 1ninducing galls (data not shown). The other strainstested in these subsequent experiments (Bo543, \12/73,K6/13 and 82/14), gave similar relative frequenciesof infection as in the first experinent with B2/74being the rnost efficient strain.Other conifer speciesThe Agrobacterium strains found to lnfect whitespruce at a frequency of greater than 10% were usedto inoculate Douglas-fir, Sitka spruce and Engelmannspruce. In addition, vectors carrying eiEher fireflyluciferase (pLUX2) or kanamycin resistance (pEND4K)constructs were introduced into A2B1 and \12/73 toprovide additional markers which could be tested inplanta. Relative frequencies of infectlon weresimilar for those other conifer sDecies as was foundwith white spruce (Ffg. 3). A11 the strains testedon Douglas-fir, Sitka spruce and Engelmann spruceinduced galls, yet frequencies of ga11 formationvaried between strains. Opines were detected inga11s induced by all strains on Douglas-fir andEngelmann spruce with the exceptlon of strain 40.Figure 1. Typical ga11 symptoms on A, white spruceano D, uougLas-Iar.Table 2.infectedgalls.STRAINS7. 65 65 t5 4t 21 66 61 95 53 94 32 29 61 AGROBACTERIUM TRAINS Figure 2. Gal1 formation induced by Agrobacteriumstrains on white spruce. Total height of barsrepresents the percentage of wound sites which formedga11s. The portion of the bars representing o1dgrowth and new growth reflect the fraction of ga11sappearing on each age class. The detection of opinesin the tissue is represented by 'A' , for agropine and'N' for nopaline. 0pines were not detected 1n ga1lswhich are not correspondingly 1abe11ed. Ga1l-1ikesynptoms which did not appear as typical ga11s are rllt -LaDeI-LeO +/ - . Percent of total white spruce seedlingswith Agrobacterium strains which formed a FlFl z aE-(n z Fbs c\ O'\ Ln = F-c.) cO N -\(-n -$ cO l-- \t CO O @-$ F- F-.,1 \t @ - Z r- F\ -\ r-- F_ COul CO u') :f CO> -\ -\ O --\ (J C)a g N o co c.lF (. \o N N = =Fa3 r n @ n nn r) - N D @- -.+ n +.- 6 n.+ O n @ -Nrrn { n F- F..N N@.+ n.rto € N N r+ r rr * r 6 + Or) * H'* +FEe ?: RDU EEb s = s;; y = A gg== N )=Y - GALL FORMATION N ITHITE SPRUCE  t9 Opines were not detected in galls formed onSltka spruce. It was noted that in Sitka spruce,arginine, (which is clearly visible i-n electro-pherograms used for analysis of opine) was present,but in much lower concentrations than in otherspecies (Fig. 4). This may account for the lack ofopine accumulalion in gall tissue, since arginine isa precursor of opines. It would be interesting totest this hypothesis by isolatlng ga11 tissue andadding arginine to the medium.Ga11 formation on Engelmann spruce and Sitkaspruce was strongly dependent on the age of thetissue with less than 10% of the ga11s formed on o1dgrowth. In contrast, Douglas-fir formed ga1lsequally on new and old growth. It is alsointeresting to note that in spruce, galls were greenand nodular, whereas Douglas-fir ga1ls were lightbrown and frlable (Fig. 1).Expression of foreign genes in gall tissueDouglas-fir gall tissue was used Lo confirm theintegration and expression of introduced genes frombinary vectors. Bacteria free ga11 tissue induced by\12/73 carrying binary vectors (pEND4K and pLUX2) wastested for hormone autonomous growth, the expressionof kanamycin resistance and the expresslon ofluciferase actlvity. Kanamycin resistance wasdemonstrated by the ability of ga11 tissue inducedby \12/73/pEND4K to grow on hormone free rnedlumcontainlng 50 pg/ml kanamycln. Control gal1s inducedby strains lacking pEND4K were unable to grow on thisleve1 of kanamycin. Luciferase activity wasexpressed in in vitro culLured gal1 Lissue derivedfrcm \12/73/p ,UX2 and assessed in in vitrc culturedgall Eissue of both Douglas-fj. und .r..ot (Tab-Le3). Luminescence was recorded at various times afterthe addltion of the substrate D-luciferin andincreased with tlme in both carrot and Douglas-firtissue transformed with pLUX2. Lurninescence of thetransformed tissue was at least 100 fold higher thantissue transformed with the wild type strain l,l2/13(Table 3).DISCUSSIONF-nl^"i-^ ^a11 forrnation and biochemicalanalysis, we showed that many A. Lumefaciens strainsare able to transform white spruce, Engelmann spruceand Douglas-fir. Agrobacterlum infection ofDouglas-fir has been previously conflrmed (Dandekaret al. I9B7 yet this is the first reportdemonstrating infectivity by a large number ofdlfferent strains. Although opines were not detectedin ga11s formed on Sitka spruce, the fact that galJ-sformed on seedlings inoculated with A. tumefaciensand not in the controls suggested tfit lnfe.tion ofSitka spruce did occur. The lack of opineaccumulation may be related to the 1ow levels ofarginine detected, or possibly due to the lack ofexpression of genes involved in opine biosynthesis.Transformation frequency varied and was stronglydependent upon the Agrobacterium strain and the ageof the tlssue. 0f the well characterized strains,C5B, T37, A2BI, for which disarmed (non-oncogenic)derivatlves are available, only A2B1 produced a highfrequency of gal1s with detectable opines on whitespruce. Other wild type strains in particular, B2/74and C3/74 gave higher frequencies of ga11 formationin the conifers tested.One A. rhizogenes strain, R1601, which containscopies of the virulence region (pTVK291) and Tiplasmid pTlBo542 in RA4 was tested. Since the wildtype 8o542 formed gal1s on white spruce, it wasexpected that R1601 would be capable of transformingwhite spruce based on a si-mj-lar comparison withinfection of poplar by these strains (Pythoud et al.1987). This would result in either a rooty or gallphenotype depending on the response of white spruce 100 90BO706050403020l00 AGROBACTERIUM TRAINS Figure 3. Gall formation induced by Agrobacterium onEngelmann spruce, SiLka spruce and Douglas-fir. Barslabelled as in Fisure 1. {t-n 1 2 3 4 12 3 4 't 2 3 4 5Figure 4. Analysis of opines in ga11 tissue. A,Agropine from white spruce: Lane 1, Agroplnestandard1' Iane 2, ga11 induced by strain A2BI; Iane3, control tissue; Iane 4, gal1 tissue induced byBo543. B, nopaline in Douglas-fir: Lane 1, controltissue; lanes 2 and 3, gall induced by strains II0/75and 82/74 respectively; lane 4; octopine and nopalinestandards. C, nopaline in Sitka spruce and Engelmannspruce: Lanes 1 and 2 Silka spruce gall from 82/74and control tissue respectively; lane 3 argininestandard, lane 5, Engelmann spruce ga11 from stralnC2/74. ( tA', agropine; 'Ar', arginine; tN',nopaline). 24,, 20IBl6t4t210Bo4 , 0 24 22201Bt6t41210,B.640ct) Fl z F:<(t)F a z F14be d3R6;Ilo n{oN:<:zNN EBS S:\\*=Ed== =6;->ueESge=o (JoJ{Nr@Y@r\ t eli t DOUGLAS-FIRENGELMANN SPRUCE SPRUCE  GALLTISSUE STRAINELAPSED TIME LUMINESCENCE(min) (cpmxlOs ) 20 Table 3. Luciferase activity in ga11 tissuefollowing infection wiLh \t2/73 containing pLUX2.Douglas-fir gal1s in vitro. The latter resultsuggests that kanamycin resistance may serve as auseful selectable marker for transformation invitro. Strains carrying pLUX2 also gave rise toga11s which express luclferase activity. Theseresults indicate that promoters which are commonlyused in binary vectors, the nopaline synthetaseprornoter (linked to nptIT) and the Cauliflower MosaicVirus 35S promoter (which drive the luciferase gene)function effectively in conifer tissue.The identification of Agrobacterium strainswhich transform conifers efficiently is an importantstep in the development of genetic engineering ofthese specles. I,Ve ave demonstrated that thechimerlc genes encodlng useful markers can beintroduced and expressed uslng these strains. We arecurrently combinlng transformation and in vitroregeneration protoco-Ls to develop systems for tn.genetic engineering of conifer species.ACKNOWLEDGEMENTSThe authors express their appreclation to EugeneNester for his advlce and comments throughout thiswork. \,{e also thank L. Moore, C. Kado and C. De Leyfor strains and P. Guyon and Y. Dessaux for agropinestandards.REFERENCESAn G, Ebert P, Mitra A, Ha S (1988) In: Gelvin andSchilperoort (eds), Plant Molecular BiologyManual. Martinus Nijhoff Pub., Dordrecht, TheNetherlands, A3:1-12Attree S, B:kkaoui F, Dunstan D, Fowke L (1987)Plant Cell Reports 6:480-483Bolton G, NesterE, Gordon M (1986) Science232:983-985Dandekar A, Gupta P, Durzan D, Knauf V (1987)Bio/Technology 5:587-590De Cleene M, De Ley J (1976) Bot. Rev. 42:389-466Fillatti J, Sellmer J, McCownB, Haissig B, Comai L(1987 ) Mo1. Gen. Genet. 2062192-199Gupta P, Durzan D (1987) Bio/Technology 5:7IO-7I2Hakman , von Arnold S (1985) J. Plant Physiol.I2I: I49-I58Hakman , Fowke L (1987) Can. J. Bot. 65:656-659KLee H, Yanofsky M, Nester E (1985) Bio/Technology3:637-642Komari T, Halperln W, Nester E (1986) J. Bacteriol.766:BB-94Maniatis T, Fritsch E, Sarnbrook (1982) MolecularCloning: A Laboratory Manual. Cold Spring HarborLaboratoryMcGranahan G, Leslie C, Uratsu S, Martin L, DandekarA (i9BB) Bio/Technology 6:BO0-804Otten L, Schilperoort R (1978) Biochim. Biophys.Acta. 527:497-5000w D, Wood K, Deluca M, deWet J, Helinskl D, Howe11S (1986) Science 234:856-859Parsons T, Sinkar V, Stettler R, Nester E, Gordon M(1986) Bio/Technology 4:533-536Pythoud F, Sinkar V, Nester E, Gordon M (1987).Bio/Technology 5 : 1323-1327Reilly K, Washer J (1977) N.Z. J. For. Sc1.7 2):199-206Ryder M, Tate M, Kerr A (1985) Plant Physiol.77:215-227Sederoff R, Stomp A-M, Chilton W, Moore L (1986)Bio/Technology 4 :647 649Stomp A-M, Loopstra C, Sederoff R, Chilton S,Fillatti J, Dupper G, Tedeschi P, Kinlaw C (1988)In: Hanover and Keathley (eds), The geneticmanipulation of woody plants. Plenum Press, N.Y.pp. 23I-241 CARROT W2/73/pLUX2 O 280.767.22II.02CARROT \]2/733.7664.3014.57216.0300.0860.0630.0600.06959.06366.59235.6840.2290.126 0.r2r DOUGLAS W2/73/pLUX2-FIRDOUGLAS VI2/73-FIR to A. rhizogenes transformation. Slnce opines nor atypical phenotype were noted it was not possible todetermine whether transfornation occurred by thisstrain.In addltion to a strong influence of differentstrains on gall formaLion, Lhe Lhree spruces showedgreater gall formaLjon on young Lissue, with only asmall- percenLage of the galis Forming orr old 5rc,wLir.This hipher occrtrrcn.c nF oall formation on the newgrowth has also been observed in poplar (W. Chellack,personal commun ication) and oLher conifers where highfrequency of gall formation was associated with a 1owdegree of stern woodiness (Stomp et al. 19BB). Thiscould be the result of new growth being in an activegrowing phase as the frequency of ga1 l formationdecreased in white spruce if the seedlings were notinoculated within one month of bud break. Inaddltion, no ga11s formed on the cut surface ofdecapitated seedlings.The differential production of phenoJicsfollowing wounding could also play a role in thelnfection process as phenolic compounds have beenfound to induce virulence of Agrobacterium spp.(Bolt-en eL a1. I986). Several phenolic compounds nwhlte spruce increased in concentration followingwounding of the old growLh, while only one increasedin the new growth. The leve1 of phenolic compoundsin the o1d growth remained at an elevated level forover 24 hours whereas the 1evel in the young tissuewas only elevated for three hours (D. Roberts,unpublished). To investigate if these phenolics hada possible role in the infection process, it would beinteresting to determine lf wound-induced phenolicproduction in Douglas-fir followed a slmilar tissuespecific pattern. If specific phenollcs affectedinfection, we would expect similar phenolic patternsin both young and old Douglas-fir tissue because newand old growth are equally susceptible Lo infectionby A. tumefaciens.The wild typ,e T-DNA of A. tumefaciens carriesgenes for opine synthesis, as well as, auxin andcytokinin biosynthesis. The expression of thesegenes in planta depends upon the transfer andintegration of the T-DNA. We have, therefore,presented evidence showing the transfer andintegration of T-DNA in a number of strains bydetecting the presence of opines. Furthermore, wehave confirmed that at least the nopali-ne synthetaseand agropine synthetase promoters are functional inconifer species. In addition, hormone autonomousgrowth and kanamycin resistance were exhibited by 0.270. 681.1011.28r .574 529.651 08s.6814.01
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