(1995) Agrobacterium Tumefaciens Transformation of Monocotyledons

CROP SCIENCE

Volume 35 March-April 1995 Number 2

REVIEW AND INTERPRETATION

Agrobacterium tumefaciens Transformation of Monocotyledons

Roberta H. Smith* and Elizabeth E. Hood

ABSTRACT their host ranges overlap, they are not identical. Most Agrobacterium tumefaciens (Smith and Townsend, 1907) has been of the wild-type A. tumefaciens strains that have been an extremely useful vector to transfer foreign genes into dicotyledonous isolated from tumors maybe classified as octopine, nopa- plants. Monocotyledonousplants, particularly the cereals, have been line, succinamopine, or L,L-succinamopine types, ac- consideredoutside the host range for A. tumefaciens, which has necessi- cording to the opine synthesis encoded by their T-DNAs. tated the developmentof other transformation systems such as naked The opines are tumor-specific products from plants in- DNAdelivery to protoplasts and, most recently, microprojectile bom- fected by A. tumefaciens and are catabolized by the bardment delivery of DNAto cells and tissues. Both systems have A. tumefaciens strain producing the tumor. Genes that worked, but there are still many difficulties encountered in routine encode enzymesinvolved in the biosynthesis of the opines transformation of any monocotyledon. Recently, there has been re- newed interest in using the A. tutnefaciens system to transform econom- are transferred into the plant genome. Opines can be ically important grasses and other monocotyledons. This paper exam- utilized by the bacteria as N and C sources. Significantly, ines the literature and steps involved in transformation of opines are not produced by the A. tumefaciens strains monocotyledons by A. tu~nefaciens. The many recent advances in themselves because opine genes have eukaryotic regula- understanding the biology of the infection process (meristematic target tory sequences for expression; thus their expression by cell, vir gene inducing compounds, and wide host range strains of infected host tissue is an indicator of T-DNAtransfer. A. tumefaciens), and availability of more monocotyledongene promot- The virulence (vir) genes mediate the process of ers and improved selectable markersgreatly improvethe opportunities T-DNAtransfer. Approximately 25 vir genes are ar- of developing monocotyledon transformation systems with A. tumefaciens. ranged into seven operons (Stachel and Nester, 1986) and are located on the Ti plasmid. Vir gene transcription is/induced at low pH by low molecular weight phenolic AGROBACTERIUM TUMEFACIENS compoundsproduced by woundedplant cells; this effect BIOLOGY can be further increased by opines and monosaccharides WhenA. tumefaciens attaches to a plant cell at a wound (Bolton et al., 1986; Usami et al., 1988; Veluthambi site, it introduces a piece of its Ti plasrnid, the T-DNA et al., 1989; Zambryski, 1992). Induction requires the (transferred DNA),into the plant nucleus (Nester et al., products of the virA and virG genes, members of the 1984; Hooykaas-Van Slogteren et al., 1984; Hohn et two component transcriptional regulatory system (Chen al., 1989; Zambryski, 1992). Tumorformation, or the et al., 1991). Vir gene action generates and processes a oncogenic response, following A. tumefaciens infection T-DNAcopy, and facilitates T-DNAmovement out of is a result of the transfer of the onc genes into the plant the bacterium and into the plant cell. Helper plasmids for chromosome. These genes code for auxin and cytokinin non-oncogenic plant transformation have been developed biosynthesis and result in cell proliferation giving rise that utilize the vir gene functions with T-DNAscontaining to the typical crown gall on the plant. Tumorcells grow genes of choice (Hoodet al., 1993). Someof these helper in vitro independent of plant growth regulators. plasmids are based on the supervirulent phenotype of Several A. tumefaciens strains have been identified strain A281(Sciaky et al., 1978; Hood et al., 1984, that harbor broad host range Ti plasmids. Each of these 1986), as well as, several octopine and nopaline strains strains infects a large variety of plants, and although (Hoodet al., as cited in 1993). In dicotyledonous plants, successful infection and sub- R.H. Smith, Dep. of Soil & Crop Sciences, Texas A&MUniversity, sequent crown gall formation depend on age and physio- College Station, TX 77843, and E.E. Hood, Utah State Univ., Dep. of logical state of the plant as well as the infection procedure Biology, Logan, UT 84322. Contribution from the Texas Agric. Exp. Stn, 31677. This paper was supported by funds from the Eugene Butler Professorship. Received 5 April 1994. *Corresponding author (rsmith@ Abbreviations: onc genes, oncogenic genes in the T-DNA;vir genes, ppserver.tamu.edu). virulence genes in the T-DNA;T-DNA, transferred DNA;PEG, polyethyl- eneglycol; GUS, 13-glucuronidasegene;NPTII, neomycinphosphotrans- Published in Crop Sci. 35:301-309 (1995). ferase gene; MSV,maize streak virus; LB, Luria broth. 301 302 CROPSCIENCE, VOL. 35, MARCH-APRIL1995 utilized, even in species sensitive to infection (Hernal- DNA,and the evidence for occurence of these steps in steens et al., 1984). Theinoculation of manyimportant monocotyledonousplants will be presented (Table 1). dicotyledonousspecies has not resulted in tumorformation (De Cleene and De Ley, 1976). The A. tumefaciens EVENTS OCCURRING IN strain will also makea differencein regardto successful A. TUMEFACIENS-HOST INFECTION infection of a plant, as different strains have different thoughoverlapping host ranges, the majorityof identified Activation of the vir Genes strains havingwide host ranges (Dommisseeet al., 1990; by Phenolic Compounds Thomashowet al., 1981; De Cleene and De Ley, 1976; The bacteriumis attracted to woundedplants presum- Komariet al., 1986). ably by followingsignal moleculesreleased by the plant cell to whichit then attaches (Hohnet al., 1989; Shaw, 1991as cited in Zambryski,1992; Stachel et al., 1985). TRANSFORMATION OF Woundedtobacco (Nicotiana sp.) cells exude phenolic MONOCOTYLEDONS compoundssuch as acetosyringone and a-hydroxy- Current successful transformation systems for mono- acetosyringonethat activate vir genesthat are responsible cotyledons include protoplast uptake of foreign DNA for the transfer of T-DNAfrom A. tumefaciens to the and biolistics (particle gun). In general, these systems woundedhost cell (Stachel et al., 1985). Bolton et al. have a very low efficiency of transformation(Potrykus, (1986) utilized sevenphenolic compounds(catechol, gal- 1990; Wilminkand Dons, 1993). A major problem with lic acid, pyrogallic acid, p-hydroxybenzoicacid, protoca- the former methodis regeneration of transgenic proto- techuic acid, [~-resorcylic acid, and vanillin) to induce plasts into fertile plants. Protoplasts frommost monocot- vir-gene activity. Thesesignal moleculesappear to be yledonswill not regenerate, and the ones that do, do so very important in allowing A. tumefaciens to recognize at low frequencies (Potrykus, 1990). Additionally, the suitable hosts, and they activate the vir loci on the Ti plasmid. The vir loci mediate T-DNAprocessing and biolistic methodis often used to bombardcells with morphogenicpotential, somatic embryos, zygotic em- delivery steps (Binns, 1990). Monocotyledons,particularly the grasses, may not bryos, and shoot tips, and again subsequentnormal plant produce these compounds,or if they do, then not at regeneration can be a problem with many important sufficient levels to serve as signal molecules.Usami et monocotyledons. al. (1987) showedseven monocotyledonseedlings lacked Althoughmonocotyledons as a group have been con- vir-inducing compoundsand suggested that this might sidered outside the host range ofA. tumefaciens,informa- blockA,tumefaciens infection ofmonocotyledons.Later, tion on tumor formation, opine production, hormone Usami et al. (1988) showedthat after homogenizing autonomousgrowth of tumors, and presence of T-DNA tissue of wheat(Triticurn aestivumL.) and oats (Arena is accumulating and indicates that monocotyledonous sativa L.), vir inducingcompounds could be identified. plants can be infected by A. tumefaciens. Transformation In the recent reports of successful T-DNAexpression of plants with A. tumefacienshas advantagesover proto- in monocotyledons, these inducing compoundswere plast uptakeof DNAor biolistics. This methodgenerally addedto the A. tumefaciens suspensionto activate the results in higher rates of transformation (0.1-5% as vir genes prior to inoculation of the monocottissue. comparedwith 0.01-1%)and moreefficient and predict- The compoundsincluded acetosyringone and nopaline able patterns for integration of the foreign DNA(Chan in maize(Gould et al., 1991), and potato woundexudate et al., 1993; Wilminkand Dons, 1993; Binns, 1990). for yam(Schafer et al., 1987) and rice (Chanet al., Becauseisolated shoot apices of any monocotyledonous 1993). Manyinvestigators believe that inoculation of species or cultivar will rapidly regeneratefertile, normal monocotyledonswith A. tumefaciens treated with induc- plants on a very simple mediumin 3 to 5 wk, the shoot ing compoundswill significantly increase the numberof apex is an excellent choice as a transformationtarget. transformation events in monocotyledonsresulting from Therefore, the developmentof a transformation system A. tumefaciens treatment (Schafer et al., 1987; Gould for monocotyledonsusing Agrobacterium-mediatedgene et al., 1991; Chanet al., 1993). However,Dommisse transfer and the meristematicshoot tip could be a very et al. (1990) presented evidence showingthat although efficient technology(Gould et al., 1991). Alternatively, added acetosyringone resulted in tumor induction 1 wk recent reports of rapid formationand regeneration from earlier in inoculatedonion, the final frequencyof tumors somatic embryos(Hess and Carman,1993; Chanet al., wasnot affected. Ritchie et al. (1993)did not find that 1993) from wheat and rice also offers a viable system octopine and acetosyringoneenhanced the transient expression of the [~-glucuronidasegene (GUS)in maize(Zea for cereal transformationin combinationwith A. tumefaciens as a vector system. maysL.) seedlingtissue cocultivatedwith A. tumefaciens; In general, monocotyledonshave not been suitable however,acetosyringone was routinely used in all their experiments. hosts for Agrobacterium-mediatedcrown gall formation. However,this doesnot rule out the possibility that A. tumefacienshas the ability to transfer T-DNAinto a mono- A. TUMEFACIENS ATTACHMENT TO CELLS cotyledon. This paper examinesthe sequence of steps Attachmentof the bacteriumto the host plant cell wall recognized in Agrobacterium-mediatedtransfer of T- is an initial step in the processof infection(Lippincott and SMITH & HOOD: AGROBACTERIUMTRANSFORMATION OF MONOCOTS 303

Table 1. Evidence for transformation of monocotyledonous species. Family/species Evidence~" Reference Liliaceae Allium cepa L. tumorous growth, opines Dommisseeet al., 1990 Aloe ferox Mill. tumor formation In De Clcene and De Ley, 1976 Aloe densiflorus (Kunth.) Jessop tumor formation In De Cleene and De Ley, 1976 Asparagusoffwinulis L. kant, ~-gluc, T-DNApresence Delbriel et al., 1993 hormoneautotrophic growth Hernalsteens et al., 1984 T-DNApresence, opines Bytebier et al., 1987 auxin and cytokinin expression Prinsen et al., 1990 Asparagustetragonus Bresler tumorous growth, nopaline Suseelanet al., 1987 Asparagussprengeri Regel tumorous growth, nopaline Suseelanet al., 1987 Chlorophytumcapense ~.) Voss small swellings, nonpaline Hooykaas-vanSlogteren et al., 1984 Chlorophytum comosum(Thumb.) Jacques opines Fenget al., 1988 Hippeastrumrutilum$ (Ker-Gawl.) Herb. opines Fenget al., 1988 Hemerocalliscitrina~ Baroni. opines Jianping et al., 1988 Lycorus radiata~ (L’H~r.) Herb. opines Jianping et al., 1988 Agavaceae Aga~e salmiana Otto tumor formation In De Cleene and De Ley, 1976 Cordyline terminalis~ Kunth tumorous growth, nopaline Suseelan et al., 1987 Cordyline rubro~ Hugel tumorous growth, nopaline Suseelan et al., 1987

Narcissus canaliculatus Guss small swellings, nopaline Hooykaas-vanSlogteren et al., 1984 Iridaceae Gladiolussp. tumors, opines Graves and Goldman, 1987 Oioscoreaceae Dioscoreasp. hormone independent growth Feng et al., 1986 Dioscoreabulbifera~ L. hormone independent growth tumors, T-DNApresence Schafer et al., 1987 Commelinaceae Commelina communi~ L. Ann. opines Deng and Shao, 1988 Graminae (Poaceae) A~ena sat~a L. agroinfection Donsonet al., 1988 Hordeum~ulgare L. opines Denget al., 1988 Oryza sa6~a L. ~-gluc, T-DNApresence Chanet al., 1992, 1993 hormone independent growth Ralneri et al., 1990 T-DNApresence, kanR, ~-gluc Liu et al., 1992 transient ~-gluc histochemistry Liu et al., 1992 TrR~cumaes~vum L. kant, nopaline synthase activity Mooneyet al., 1991 T-DNApresence opines Denget al., 1988 agroinfection Dale et al., 1989 Woolstonet al., 1988 Zea mays L. p-gluc Ritchie et al., 1993 kant, I~-gluc, T-DNApresence Gould et al., 1991 opines Graves and Goldman, 1986 Agroinfection of MSV Grimsley et al., 1987, 1988 I~-gluc histochemistry Shen et al., 1993 Araceae Calla sp. tumor formation In De Cleene and De Ley, 1976 Philodendron sp. tumor formation In De Clcene and De Ley, 1976 Anthurium andraeanum~Linden. tumor formation, opines Kuehnle and Sugii, 1991 kant, kanomycinresistance; [~-gluc, I~-glucuronidase. Family designation f~omMabberley (1989).

Lippincott, 1969; Lippincott et al., 1977). Attachment et al., 1988), and in Asparagus o~cinalis L. (Draper et can be affected by plant or tissue age, cell type, cell al., 1983). The numberof bacterial cells attaching was cycle stage, and other physiological parameters (Graves shownto vary using different A. tumefaciens strains with et al., 1988). Most monocotyledonsdo not form tumors A66 and T37 appearing to show greater affinity, and as a result of A. tumefaciens inoculation, and it was cells in the vascular tissue had the most bacteria attached assumedthat the bacterium could not attach to monocoty- (Graves et al., 1988). ledonous cells because monocotyledoncells lacked sites The reports of monocotyledontransformation by A. tu- for A. tumefaciens attachment (Lippincott and Lippincott, mefaciens support the idea that meristematic cells in 1978). Lippincott and Lippincott (1978) did not directly monocotyledonsmay be a critical target for successful measure bacterial attachment to monocotyledon cell bacterial attachment (Grimsley et al., 1988; Gould et walls. Morerecently, Douglaset al. (1985) showedattach- al., 1991; Chanet al., 1993; Hernalsteens et al., 1984; ment to bamboocell walls, and the kinetics of attachment Raineri et al., 1990; Chanet al., 1992; Delbreil et al., to monocotyledonand dicotyledon cell walls was similar 1993). Because the basic anatomy of monocotyledons (20%of added bacteria were attached after approximately and dicotyledons is different, and the location of meriste- 2 h). Bacterial attachment has also been demonstrated matic cell types is somewhatdifferent, past attempts to in Zea mays, Gladiolus sp., Triticum aestivum (Graves transform monocotyledons(stem and leaf tissue inocula- 304 CROP SCIENCE, VOL. 35, MARCH-APRIL1995 tions) probablydid not specifically target meristematic inducing genes, and subsequenttumor formation as com- cells or cells that were competentto dedifferentiate. pared with dicotyledons. Some monocotyledons, Graveset al. (1988) remindus in their discussion that including the agronomicallyimportant grasses, lack the monocotyledonouscells in general appear to lose the tumor-formingresponse to the phytohormonegenes on ability to dedifferentiateat a veryearly stage in develop- the T-DNA,so DNAtransfer detection, relying on this ment and maylose the ability to respond to bacterial responsealone, has probablyresulted in an underestima- infections. The process of differentiation mayinvolve tion of the host range for this bacterium(Graves et al., changesin the cell wall that reduceor inhibit A. tumefa- 1988). ciens attachment.It can also be speculatedthat meriste- The growth of monocotyledonoustumors following matic cells maysecrete compoundsthat induce the vir A. tumefaciens inoculation, on hormone-free medium genes. has been shownfor Asparaguso~cinalis (Hernalsteens et al., 1984), Gladiolus (Graves and Goldman,1987), Tumor Formation Dioscoreasp. (Prinsen et al., 1990), Dioscoreabulbifera L. (Schafer et al., 1987), and Oryzasativa L. (Raineri Perhaps the earliest report of tumor formation on et al., 1990). Tumorformation has also been reported monocotyledonous species was by De Cleene and De for Anthuriumandraeanum Lind. (Kuehnle and Sugii, Ley(1976). Theydid an extensivesurvey of the literature 1991) frominternode, leaf and petiole explants. in addition to their owninoculations to determinethe Early attempts to culture callus from monocotyledon host range for A. tumefaciens. Theseauthors used only stem and leaf sections, met with minimalsuccess. These one strain of A. tumefaciens,B6, for their inoculations explants simply wouldnot respond to auxins and cytoki- and noted that most literature reporting plant infection nins in the culture mediumto produce the woundor by A. tumefaciens was carried out with strains Hop, callus responseas did dicotyledonleaf and stem sections. ChrlIb, B23, and B6. However,because not all the Thus, the commonlyheld belief in the 1960s and early strains ofA. tumefacienshave the samehost range, these 1970s was that monocotyledonswere not amenable to reports are a minimumestimate of monocotyledontumor cell culture. However,in the 1980sit wasfound that if formation. Sixty percent of gymnospermsand dicotyle- the monocotyledonexplant contained meristematic or donous angiosperms examined were susceptible and undifferentiated cells, callus could readily be initiated formedcrown galls upon inoculation. Theyalso estab- (Bhaskaranand Smith,1990). It wasnot surprising, then, lished that two monocotyledonousfamilies, Liliales and that even ifA. tumefaciensdid infect monocotyledonsand Arales, had species that weresusceptible to A. tumefa- transfer genes for auxin and cytokinin biosynthesis, a ciens infection. Theinoculations included 15 of the 53 major wound(callus proliferation) or tumor wouldnot monocotyledonousfamilies, and five out of 79 inocula- result from the presence of auxin and cytokinin as was tions produced crown galls. De Cleene and De Ley commonlyobserved on dicotyledons. (1976) certainly established by conventionalprocedures that monocotyledons,although not broadly susceptible T-DNATransfer: Opine Biosynthesis, to tumorformation, are susceptible. With moreA. tume- Agroinfection, GUSActivity faciens strains available and morecurrent knowledgeof factors important for gene transfer and expression such Early claims for T-DNAtransfer to monocotyledons as vir genes, vir gene activation, monocotgene promot- by A. tumefaciens wasthe documentationof opine pro- ers, and signaling molecules,it is conceivablethat many duction from woundsite tissue. Opine production from moremonocotyledons and dicotyledons will fall within tumors or from inoculation sites has been detected in the host range of A. tumefaciens. manymonocotyledons including Allium cepa L. (Dom- A report in Nature (Hooykaas-VanSlogteren et al., misse et al., 1990), Asparaguso~cinalis (Bytebier 1984)showing the expressionof a foreign genein aspara- et al., 1987; Hernalsteens et al., 1984), Chlorophytum gus after A. tumefaciensinfection, initiated discussion capense(L.) Voss and Narcissus canaliculatus Gusscv. on whetherA. tumefaciensis a suitable vector for mono- Paperwhite(Hooykaas-Van Slogteren et al., 1984), Zea cotyledon transformation. The manifestation of tumor mays (Graves and Goldman,1986), Gladiolus (Graves formationfollowing A. turnefaciensinfection is the phe- and Goldman, 1987), Asparagus tetragonus Bresler, notypic responseof host plants, whereasnon-host plants Asparagusspringeri Regel, Cordyline terminalis (L.) do not form tumors. In a strict definition of the host- Kunth. and Cordyline rubra (Suseelan et al., 1987), parasite interaction, the tumorprovides substrates for Hemerocalliscitrina Baroni and Lycorus radiata Herb. A. tumefaciens to growon the plant; therefore, plants (Jianping et al., 1988), Hippeastrumrutilum Herb. and that do not produce tumors do not provide a preferred Chlorophytumcomusum Wood (Prinsen et al., 1990), An- habitat for A. tumefaciens (Grimsley, 1990). Many thurium andraeanum (Kuehnle and Sugii, 1991), monocotyledonousplants inoculated with A. tumefaciens Hordeumvulgare L. and Triticum aestivum (Prinsen et do not form a tumor or crown gall; therefore, it was al., 1990), and Commelinacommunis L. (Prinsen et assumedthat monocotyledonswere not capable of receiv- al., 1990). Additionally, the presence of T-DNAfrom ing DNAfrom A. tumefaciens. Prinsen et al. (1990) A. tumefaciens has been shownin Asparagusofficinalis have shownthat in asparagustumor tissue an active onc (Byetebieret al., 1987;Delbreil et al., 1993), Dioscorea gene 1 seemedto be lethal, confirmingthat monocotyle- bulbifera (Schaferet al., 1987), Zea mays(Gould et al., dons mayreact very differently to the onc, or tumor- 1991), Triticum aestivum (Mooneyet al., 1991), and SMITH & HOOD: AGROBACTERIUMTRANSFORMATION OF MONOCOTS 305

Oryzasativa (Chanet al., 1992, 1993). Prinsen et al. presence of opines. Nopaline and agrocinopine were (1990) reported expression of the onc genes for auxin present in this tissue demonstratingstable T-DNAtrans- and cytokinins in transgenic plants and molecular evi- fer and expression. Transient expression of the opine dence for the presence of the onc genes in Asparagus compoundswas ruled out by the demonstration of con- o.fficinalis followingtransformation with wild type A. tu- stant levels of nopaline and agrocinopineproduction in mefaciens. establishedcallus cultures fromthe inoculatedplant tis- The source of the opines must be the plant tissue sues (Hernalsteenset al., 1984). Opinelevels werecom- becauseA. tumefaciens-mediated production of opines is parable to those foundin dicotyledoncrown gall tissue. not possible since these genesare regulatedby eukaryotic Thetissues also grew without plant growthregulators sequences whichA. tumefaciens cannot read. Addition- indicating genes for hormone-independentgrowth from ally, opine production by the bacterium has not been the Ti plasmid were being expressed. Untransformed detected. Christou et al. (1986)presented evidence show- asparagus does not grow without exogenoushormones ing opines can be detected in normal callus and plant in the medium.It is significant that the apical or youngest tissue as a result of arginine metabolismin the absence region of the stem wasmost susceptible, supportingthe of A. tumefaciens infection. Therefore, these workers idea that careful choice of monocotyledonplant material cautionedthat opineproduction following bacterial treat- is critical. mentmay not be sufficient evidenceto support the claim Hooykaas-VanSlogteren et al. (1984)described A. tu- of A. tumefaciens T-DNAtransfer and integration. To mefaciensinfection of Chlorophytumcapense (Liliaceae) showthat plant tissues can synthesize opine compounds and Narcissus cv. Paperwhite(Amaryllidaceae) with the without A. tumefaciensinfection, hypocotyl, leaf, and developmentof small swellings at the woundsites. At callus tissue from several dicotyledons[cotton (Gossyp- 21 d after inoculation, tissue wasanalyzed for nopaline, ium hirsutum L.), soybean [Glycine max (L.) Merr.], whichwas present in infected cells and not in uninfected and tobaccoI were incubated for 3 to 5 d on a medium control tissue. It wassignificant that not all strains of containing 100 mMarginine, and in somecases ¢t-keto- A. tumefacienstested producedthis result. LBA1010and glutaric acid was added (Christou et al., 1986). Low LBA1023incited swellings that producedoctopine, and levels of opines werethen detected in the samples.Sev- LBA2318and LBA2347were responsible for nopaline eral areas of concernappear in interpreting these data production in infected monocotyledontissue. Twoaviru- to refute opine production as proof of T-DNAtransfer lent strains, LBA288and LBA1516,did not form swell- particularly for monocotyledons. ings or incite opine production.Therefore, vir genesare 1. Dicotyledonousand not monocotyledonoustissue important for monocotyledoninfection. was used. Therefore, one could question whether In a report on maizetransformation, 4-d-old seedlings similarly treated monocotyledonoustissue would were woundedand inoculated with A. tumefaciens also showarginine metabolism. (Graves and Goldman,1986). Meristematic tissue from 2. Comparablefeeding experiments on monocotyle- the base of the scutellar nodethrough the mesocotylarea donoustissue (Hernalsteenset al., 1984)incubated was the site of inoculation. After 2 wk, the tissue was overnight in 10 mMarginine and 10 mM~t-keto- analyzed for lysopine (forming octopine) or nopaline glutaric acid detectedopine in the transgenictissue dehydrogenaseactivity. Octopineor nopaline waspresent and not in control tissue. The study by Christou in transformedplant tissue but not in control tissue. et al. (1986) used 10 times the concentration Transformation frequencies were approximately 60%. precursors in the arginine metabolicpathway, and Transformationof Gladiolus cells was shownby ex- an incubation period three to five times longer, pression of octopine and nopaline synthesizing enzyme perhaps stimulating the production of abnormal activities (Graves and Goldman,1987). Only virulent metabolic products. A. tumefaciens strains could induce tumor formation. 3. Backgroundarginine metabolismproducing opines Additionally, tumorcalli grew on hormone-freemedium in the monocotyledonoustissue would have been in the presenceof antibiotics that kill the bacterium. seen in the controls but wasnot observed(Hernal- Convincingevidence that documentsT-DNA transfer steens et al., 1984). has been reported by Grimsleyet al. (1986). Strains A. tumefaciens containing maize streak virus (MSV) If control, uninoculated, monocottissue, i.e., unex- genomesin the T-DNAwere used to inoculate maize posed to T-DNAfrom A. tumefaciens, is negative for seedling tissue ( Grimsleyet al., 1986,1987). The plants opinesand treated tissue is positive, then the monocotyle- displayed the symptomsof the virus indicating T-DNA dontissue is at least demonstratingtransient expression had been transferred though not verifying T-DNAinte- of the T-DNAand thus establishes the T-DNAtransfer gration. Theterm agroinfectionwas defined as the intro- event. Dommisseet al. (1’990) demonstratedlevels ductionof plant infectious agentsinto plants via A. tume- opines in onion tumorsto be at least 100 times higher faciens (Grimsley et al., 1986; Hohnet al., 1989). than backgroundlevels. Surface inoculation of MSVis not naturally infective to Asparagusogfficinalis (Liliaceae) apical stem sections maizebut requires an insect vector. However,seedlings were infected by wild-type A. tumefaciens C58 and in agroinfected developed viral disease symptoms2 wk 30 d produced large, tumorousoutgrowths on the stem after plant inoculation. No symptomswere observed on (Hernalsteenset al., 1984). Thetumor tissue waspropa- seedlings from control inoculations with naked MSV gated on hormone-free mediumand analyzed for the DNA,or by A. tumefaciens containing a mutation in 306 CROPSCIENCE, VOL. 35, MARCH-APRIL1995 the virA locus, or a T-DNAlacking borders. Southern formed by A. tumefaciens. The A. tumefaciens strains analysis of digested DNAfrom infected plants using were harboring wild-type nopaline and octopine tumor- MSVDNA as a probe verified the identity of the virus. inducingplasmi. "ds. Selectionof transgenictissue on anti- These bands did not result from endogenousA. tumefa- bacterial, antibiotic-containing medium,followed by ciens because (i) the pEAP37binary vector was much DNAblot hybridization was used to confirm transforma- larger than the fragmentsdetected, (ii) the probe only tion and integration of the T-DNAinto the asparagus detected restriction fragments from MSV,and (iii) the genome. They concluded that T-DNAwas transferred leaves used for DNAisolation weredistant fromthe site and integrated into a monocotgenome with A. tumefa- of bacterial inoculation. Agroinfectiondemonstrates that ciens as a vector, and T-DNAintegration in Asparagus A. tumefaciens can transfer DNAinto cells of grass was comparableto that in dicotyledons. species (Potrykus, 1990; Grimsley, 1990). Other reports Small tumorswere producedin vitro on bulbil tissue in which agroinfection wasused to transfer T-DNAinto of yam, Dioscorea bulbifera, after inoculation with wheat (Dale et al., 1989; Woolstonet al., 1988) and A. tumefaciens (Schafer et al., 1987), and the growth oats (Donsonet al., 1988) confirmedthat A. tumefaciens of this tumor tissue was hormone-independent.Tumors delivered T-DNAinto cereals and grasses. did not result if the bacteriumwas not preincubatedwith Grimsley et al. (1988) demonstratedthat the shoot woundexudates from potato. The presence of nopaline apical meristematicregion of maizeseedlings is the tissue was confirmedin the tumors; whereas, woundedbulbil susceptible to A. tumefaciens infection. Non-meri- tissue, bulbil tissue treated withoutthe potato exudate, stematic tissues generally did not express the T-DNA or tissue treated with an avirulent A. tumefaciensstrain froman A. tumefaciensinfection, indicating a uniqueness did not produce nopaline. A Southern blot demonstrated of these meristematiccalls for bacterial attachmentand/ the integration of T-DNAinto the monocotyledonnu- or T-DNAtransfer. clear DNA. AlthoughMooney et al. (1991) were not able to regen- Raineri et al. (1990) have demonstrated T-DNAex- erate wheatplants fromtransgenic callus, they did dem- pression and integration into genomicDNA of rice fol- onstrate the presence of T-DNAin the callus by Southern lowingA. tumefacienstreatment. A supervirulent strain, hybridizations.In an attemptto rule out bacterial contam- A281that contains pTiBo542,was used to inoculate ination of the callus, callus tissue pieces werechecked embryosof rice (Oryza sativa). Southern blots demon- by streaking onto an LBmedium plate; however,a better strated the integration of the T-DNA.A secondinocula- control wouldhave been a Southernblot utilizing a non tion with an octopine strain producedrooty callus in T-DNAor chromosomalprobe from A. tumefaciens. No whichoctopine wasdetected. Octopinewas not detected bacterial growthwas obs.erved throughoutthe study. in uninoculatedtissue. Recently A. tumefaciens was shownto transfer T-DNA Agrobacterium-mediated transformation of maize containing the GUSgene into maize seedling shoot tips shoot tips from seedlings and expression of the GUS (Shen et al., 1993). The GUSgene had an intron and NPTII genes has been reported (Gouldet al., 1991). prevent GUSactivity from A. tumefaciens. Whenvir Foreign DNAwas integrated into the host genomicDNA, mutants of A. tumefaciens were used, no transfer or and progenyshowed segregation of the foreign genes. expressionof the foreign gene wasdetected. Thesework- Foreign gene expression was also demonstratedin the ers also reported that transformationwas affected by the progeny. maize genotype used with A188being successful. The Transformationof rice seedling tissue with A. tumefa- strain ofA. tumefaciensused wasalso critical demonstra- ciens was shownby Chanet al. (1992). Southern blot ting the importanceof vir gene type. Similar experiments analysis showedintegration of T-DNA,and lack of hy- werealso recently reported by Ritchie et al. (1993). bridization with vir probes indicated A. tumefacienswas Experimentsby Li et al. (1992) also showedtransient not present. Additionally, enzymeactivity of neomycin GUSexpression in several rice cultivars. Theyobserved phosphotransferase II was demonstrated. This report a slight enhancementof GUSactivity if an auxin was stresses the importanceof the use of meristematictissue, present in the culture mediumduring cocultivation of and the use of phenolic compoundsto induce vir genes. the explantswith the bacteria. Indica cultivars had higher Transformation did not occur without addition of the frequencies of GUSexpression as comparedto japonica potato woundexudate. cultivars. Additionally, Li et al. (1992) observedthat vir genes of an agropine-type Ti-plasmid were more In a very recent report, immaturerice embryos(10- 12 d after pollination) weretransformed with A. tumefa- effective in GUSexpression that nopaline and octopine- type plasmids. Liu et al. (1992)also reported that addi- ciens A281containing the GUSand NPTII genes (Chan tional copies of the virG genes in A. tumefaciens en- et al., 1993). A maizeAdhlpromoter was used to drive hanced the frequency of transient GUSexpression in expression of the GUSgene, and phenolic compounds rice explants. from a potato suspension culture appeared to improve transformation efficiency. Transformationand integration of T-DNAinto genomic DNAwas confirmed by T-DNAIntegration into Host Cell Genomic DNA Southernblot analysis of the progeny,and sexual inheri- Bytebier et al. (1987) demonstratedT-DNA integration tance of the genesin a 3:1 ratio wasconfirmed. Transfor- into genomicDNA of Asparagusoj~cinalis identical to marionfrequencies of 6.8 %were obtained. Contamina- T-DNAsegments found in dicotyledonous plants trans- tion by A. tumefaciensin the transgenicplants wasruled SMITH & HOOD: AGROBACTERIUM TRANSFORMATION OF MONOCOTS 307 out by stripping the Southern blot and rehybridizing with them will be useful (Hood et al., 1993), as well as C58 a probe for virB DNA. (Grimsley et al., 1986, 1987, 1988; Ritchie et al., 1993; Delbreil et al. (1993) reexamined Asparagus officinalis Shenet al., 1993). transformation by A. tumefaciens carrying the genes for GUS and NFT II. Kanamycin-resistant embryogenic lines CONCLUSION were obtained from three genotypes of asparagus. An X-Gluc (5-bromo-4-chloro-3-indolyl glucuronide) assay Fundamental scientific knowledge is critically absent for expression of the GUS gene confirmed 3-glu- in regard to the biology of monocotyledon regeneration curonidase activity in the cultures, and a Southern geno- from protoplasts. In addition, when regeneration occurs, mic blot of the cultures and a regenerated plant supported fertility and albino problems are common. The general T-DNA integration. overall difficulty of putting the most elite lines of important monocotyledons into cell culture is also a major problem. The pursuit of transformation with protoplast KEY FACTORS INVOLVED IN and DNA uptake by polyetheleneglycol or electropora- A. TUMEFACIENS-MEDUXED tion will not be routine, efficient, or generally applicable TRANSFORMATION OF methods for gene transfer (Potrykus, 1990). Therefore, MONOCOTYLEDONS until this knowledge is established, transformation by Use of Meristematic Monocotyledonous Tissue protoplasts is not going to be a routine, inexpensive, for Cocultivation with A. tumefaciens rapid, or easily-transferred technology. Biolistics is also plagued by cell culture problems The success of transformation using embryonic or because generally the best targets are somatic embryos meristematic tissues could be attributed to the following: or embryogenic callus that can be produced in large (i) v/r-inducing substances are produced by embryonic quantities, thus making good target tissue. Shoot tips tissues, (ii) low production of bacteriotoxic substances, are also used and are an excellent target. However, the (iii) favorable endogenous hormone levels, (iv) availabil- transformation of shoot tips is very time consuming ity of receptors for attachment of Agrobacterium (Chen because about 1000 shoots need to be prepared to obtain et al., 1993), and (v) these cells are actively dividing one potentially transformed shoot. If transgenic plants and host DNA synthesis is occuring. DNA synthesis are produced, they are likely to be chimeric, a condition may be required for T-DNA integration (Gheysen et al., that increases screening problems. 1987). This may be significant because most differenti- Much progress has been made in the last 5 yr to ated monocotyledon cells fail to divide and thus cannot understand better steps involved in Agrobacterium- produce tumors. Many researchers believe that meriste- mediated gene transfer, and the role of the plant in this matic target cells are essential (Gould et al., 1991; Hohn process. With further understanding of these parameters, et al., 1989; Grimsley et al., 1988). These factors may the host range of A. tumefaciens may perhaps be extended all contribute to cellular competence to receive foreign routinely to include most dicotyledons and monocotyle- DNA. dons. A foundation of basic knowledge is now available and very specific parameters are lending themselves to vir Gene Activation experimental testing. Many monocotyledons may not produce the phenolic factors to activate the expression of the vir genes on the ACKNOWLEDGMENTS Ti plasmid (Usami et al., 1987). Therefore, addition of I would like to thank Mr. Eugene Butler for his support of these compounds to the cocultivation mixture is probably my research projects on the transformation of monocotyledons beneficial. Monocotyledons may produce these com- (RHS). pounds from meristematic, undifferentiated cells. Monocotyledon Gene Promoters such as Actin, Ubiquione, and a-Amylase May Be Much More Effective Than Is the 35S Promoter Tumors may not form in monocotyledons because of the lack of transcription of the one genes or because of a different endogenous hormone physiology as compared with cotyledons. The use of monocotyledon promoters has been shown to enhance significantly gene expression by monocotyledon cells.

Wide Host Range A. tumefaciens Strains Success in monocotyledon transformation most certainly will depend on using the correct, compatible A. tumefaciens strains. In this regard, wide host range bacterial strains and new helper plasmids derived from 308 CROP SCIENCE, VOL. 35, MARCH-APRIL 1995 WATERWORTH & DONNELLY: FOREIGN TURFGRASS GERMPLASM AND FEDERAL QUARANTINE 309