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. tumefa- ciens. 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 tumorforma- tion (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