Nopaline Strains Research Articles

Development of a real-time PCR assay using locked nucleic acid probe for the detection of octopine, nopaline, and vitopine strains of tumorigenic Allorhizobium vitis in grapevines

Crown gall disease caused by Allorhizobium vitis is one of the most destructive diseases in grapevine cultivation from an economical perspective. The bacterial pathogen exists systemically in grapevines and spreads through cuttings used for propagation material obtained from grapevines, living vines, and mother blocks, which poses a high risk for certified grapevine nursery material cultivation, nursery industry, and growers. Therefore, in this study, real-time PCR-based methods were developed for effectively and accurately determining the presence and the density of the bacterial pathogen in grapevine nursery material and the data used for indexing programs. Octopine-, nopaline-, and vitopine-catabolizing A. vitis strain-specific primer and locked nucleic acid (LNA) probe sets were determined depending on the ocs, nos, vis, vitopine iaaM, and vitopine virD2 genes. Primer-probe sets were then used to amplify the octopine synthase gene (62 bp) from octopine strains, the nopaline synthase gene (78 bp) from nopaline strains, and the vitopine synthase gene (60 bp) from vitopine strains, as well as the vitopine iaaM gene (60 bp) and the vitopine virD2 gene (66 bp) of A. vitis. The detection of A. vitis strains from bacterial suspension or extracted plant sap could be achieved within a short time (i.e., 20–25 min) with real-time PCR-based assays using the designed primer and probe sets. For the real-time PCR-based methods, the bacterial cell sensitivity threshold was defined as sensitivity threshold for one bacterial cell and at the DNA level, which was 1.4–1.9 fg. In real-time PCR-based methods, the corresponding PCR efficiencies for octopine and nopaline strains were 97.12% and 97.87%, respectively and for vitopine strains were 94.39%, 96.76%, and 99.01%. In this study, a new real-time PCR-based method was developed that uses the LNA probe, and different opine strains of A. vitis were detected and quantified from vegetative materials of infected grapevine.

Constitutive expression of the tzs gene from Agrobacterium tumefaciens virG mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation.

KEY MESSAGE : virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from the tzs gene in the virG mutant strains is responsible for the improvement. Strains of Agrobacterium tumefaciens were tested for their ability to improve cotton meristem transformation frequency. Two disarmed A. tumefaciens nopaline strains with either a virGN54D constitutively active mutation or virGI77V hypersensitive induction mutation significantly increased the transformation frequency in a cotton meristem transformation system. The virG mutant strains resulted in greener explants after three days of co-culture in the presence of light, which could be attributed to a cytokinin effect of the mutants. A tzs knockout strain of virGI77V mutant showed more elongated, less green explants and decreased cotton transformation frequency, as compared to a wild type parental strain, suggesting that expression of the tzs gene is required for transformation frequency improvement in cotton meristem transformation. In vitro cytokinin levels in culture media were tenfold higher in the virGN54D strain, and approximately 30-fold higher in the virGI77V strain, in the absence of acetosyringone induction, compared to the wild type strain. The cytokinin level in the virGN54D strain is further increased upon acetosyringone induction, while the cytokinin level in the virGI77V mutant is decreased by induction, suggesting that different tzs gene expression regulation mechanisms are present in the two virG mutant strains. Based on these data, we suggest that the increased cytokinin levels play a major role in increasing Agrobacterium attachment and stimulating localized division of the attached plant cells.

Nopaline-type Ti plasmid of Agrobacterium encodes a VirF-like functional F-box protein.

During Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF.

Membrane and Core Periplasmic Agrobacterium tumefaciens Virulence Type IV Secretion System Components Localize to Multiple Sites around the Bacterial Perimeter during Lateral Attachment to Plant Cells

ABSTRACTType IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain of Agrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in the A. tumefaciens octopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression following vir induction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles. vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiple vir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates.

Metabolic changes in Agrobacterium tumefaciens-infected Brassica rapa

Agrobacterium has the ability to transfer its genetic material, T-DNA, into the plant genome. The unique interaction between the bacterium and its host plant has been well studied at the transcriptome, but not at the metabolic level. For a better understanding of this interaction it is necessary to investigate the metabolic changes of the host plant upon infection with Agrobacterium tumefaciens. This study investigated the metabolic response of Brassica rapa to infection with disarmed and tumor-inducing strains of A. tumefaciens using (1)H nuclear magnetic resonance spectroscopy combined with multivariate data analysis. The partial least square-discriminant analysis (PLS-DA) of two varieties of B. rapa showed that there was a clear differentiation in the metabolite profiles of B. rapa leaves infected with the disarmed strain LBA4404 and with tumor-inducing octopine and nopaline strains, particularly in the flavonoid, phenylpropanoid, sugar and free amino/organic acid contents. However, individual PLS-DA of each type of infection suggests that, in general, some flavonoids and phenylpropanoids were suppressed as a consequence of these infections. The results obtained in this study indicate that the disarmed strain LBA4404 and tumor-inducing strains have different effects on the metabolite profile of B. rapa.

Detection of Agrobacterium vitis by PCR using novel virD2 gene-specific primers that discriminate two subgroups

Tumour tissue samples were collected from vines grown in various regions of Italy and other parts of Europe and extracted for detection of Agrobacterium vitis. Fifty strains were isolated on agar plates and screened by PCR with consensus primers from the virD2 gene. They were confirmed as A. vitis with a species-specific monoclonal antibody. The isolates were further analyzed by PCR for their opine synthase genes and ordered into octopine, nopaline and vitopine strains. Primers designed on the octopine synthase gene did not detect octopine strains of Agrobacterium tumefaciens. For quantitative PCR, virD2 fragments were sequenced: two classes of virD2 genes were found and two primer sets designed, which detected octopine and nopaline strains or only vitopine strains. For simultaneous identification of all opine-type strains, multiplex real-time PCR with either primer pair and SYBR Green was performed: the combined sets of primers gave signals with DNA from any A. vitis strain. Specificity of the new primers for real-time PCR was evaluated using several unidentified bacterial isolates from grapevines and other plant species. An elevated level of non-specific background was observed when the combined primer sets were used in multiplex PCR assays. The real-time PCR protocol was also used to detect A. vitis cells directly from grapevine tumours; avoiding direct isolation procedures a sensitivity in the range of one to ten cells per assay was found. Inhibition of the PCR reaction by plant material was overcome by treating tumour extracts with a DNA purification kit as a step for the isolation of nucleic acids.

Topology of the VirB4 C Terminus in the Agrobacterium tumefaciens VirB/D4 Type IV Secretion System

Gram-negative type IV secretion systems (T4SSs) transfer proteins and DNA to eukaryotic and/or prokaryotic recipients resulting in pathogenesis or conjugative DNA transfer. VirB4, one of the most conserved proteins in these systems, has both energetic and structural roles in substrate translocation. We previously predicted a structural model for the large C-terminal domain (residues 425-789) of VirB4 of Agrobacterium tumefaciens. Here we have defined a homology-based structural model for Agrobacterium VirB11. Both VirB4 and VirB11 models predict hexameric oligomers. Yeast two-hybrid interactions define peptides in the C terminus of VirB4 and the N terminus of VirB11 that interact with each other. These interactions were mapped onto the homology models to predict direct interactions between the hexameric interfaces of VirB4 and VirB11 such that the VirB4 C terminus stacks above VirB11 in the periplasm. In support of this, fractionation and Western blotting show that the VirB4 C terminus is localized to the membrane and periplasm rather than the cytoplasm of cells. Additional high resolution yeast two-hybrid results demonstrate interactions between the C terminus of VirB4 and the periplasmic portions of VirB1, VirB8, and VirB10. Genetic studies reveal dominant negative interactions and thus function of the VirB4 C terminus in vivo. The above data are integrated with the existing body of literature to propose a structural, periplasmic role for the C-terminal half of the Agrobacterium VirB4 protein.

Characterization of Agrobacterium tumefaciens strains isolated from grapevine

Grapevine crown gall is predominantly induced by Agrobacterium vitis , but rarely, Agrobacterium tumefaciens may also cause this disease. We have collected 18 grapevine isolates of A. tumefaciens from distinct geographical regions and various grapevine varieties. Characterization of these strains showed that 7 of the 18 strains carried A. vitis -type octopine/cucumopine Ti plasmids, utilized tartrate, were avirulent on pea and did not respond to the virC-specific PCR primers like A. vitis . Eleven of the 18 strains carried A. tumefaciens -type Ti plasmids, did not grow on tartrate, were virulent on pea and reacted positively to virC-specific PCR primers. Two of these strains induced octopine/agropine-, and two strains induced agropine producing tumors on grapevine and/or Kalanchoe tubiflora plants. The remaining 7 strains showed the characteristic properties of A. tumefaciens nopaline strains and were sensitive to agrocin 84.

Efficient vir gene induction in Agrobacterium tumefaciens requires virA, virG, and vir box from the same Ti plasmid.

The vir genes of octopine, nopaline, and L,L-succinamopine Ti plasmids exhibit structural and functional similarities. However, we observed differences in the interactions between octopine and nopaline vir components. The induction of an octopine virE(A6)::lacZ fusion (pSM358cd) was 2.3-fold higher in an octopine strain (A348) than in a nopaline strain (C58). Supplementation of the octopine virG(A6) in a nopaline strain with pSM358 did not completely restore virE(A6) induction. However, addition of the octopine virA(A6) to the above strain increased virE(A6) induction to a level almost comparable to that in octopine strains. In a reciprocal analysis, the induction of a nopaline virE(C58)::cat fusion (pUCD1553) was two- to threefold higher in nopaline (C58 and T37) strains than in octopine (A348 and Ach5) and L,L-succinamopine (A281) strains. Supplementation of nopaline virA(C58) and virG(C58) in an octopine strain (A348) harboring pUCD1553 increased induction levels of virE(C58)::cat fusion to a level comparable to that in a nopaline strain (C58). Our results suggest that octopine and L,L-succinamopine VirG proteins induce the octopine virE(A6) more efficiently than they do the nopaline virE(C58). Conversely, the nopaline VirG protein induces the nopaline virE(C58) more efficiently than it does the octopine virE(A6). The ability of Bo542 virG to bring about supervirulence in tobacco is observed for an octopine vir helper (LBA4404) but not for a nopaline vir helper (PMP90). Our analyses reveal that quantitative differences exist in the interactions between VirG and vir boxes of different Ti plasmids. Efficient vir gene induction in octopine and nopaline strains requires virA, virG, and vir boxes from the respective Ti plasmids.

A second T-region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid.

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadoriopines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 co-inherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.

Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium tumefaciens.

Three VirB proteins (VirB1*, VirB2, and VirB5) have been implicated as putative components of the T pilus from Agrobacterium tumefaciens, which likely mediates binding to plant cells followed by transfer of genetic material. Recently, VirB2 was indeed shown to be its major component (E.-M. Lai and C. I. Kado, J. Bacteriol. 180:2711-2717, 1998). Here, the influence of other Vir proteins on the stability and cellular localization of VirB1*, VirB2, and VirB5 was analyzed. Solubility of VirB1* and membrane association of VirB2 proved to be inherent features of these proteins, independent of virulence gene induction. In contrast, cellular levels of VirB5 were strongly reduced in the absence of other Vir proteins, indicating its stabilization by protein-protein interactions. The assembly and composition of the T pilus were analyzed in nopaline strain C58(pTiC58), its flagellum-free derivative NT1REB(pJK270), and octopine strain A348(pTiA6) following optimized virulence gene induction on solid agar medium. In all strains VirB2 was the major pilus component and VirB5 cofractionated during several purification steps, such as ultracentrifugation, gel filtration, and sucrose gradient centrifugation. VirB5 may therefore be directly involved in pilus assembly, possibly as minor component. In contrast, secreted VirB1* showed no association with the T pilus. In-frame deletions in genes virB1, virB2, virB5, and virB6 blocked the formation of virulence gene-dependent extracellular high-molecular-weight structures. Thus, an intact VirB machinery as well as VirB2 and VirB5 are required for T-pilus formation.

Withanolide synthesis in cultures of Withania somnifera transformed with Agrobacterium tumefaciens

Transformed organ cultures of Withania somnifera were established following infection with wild type nopaline and octopine strains of Agrobacterium tumefaciens. The oncogenic strains had different levels of virulence on two genotypes studied, the main difference was found in the nature of the galls formed and in their subsequent morphological competence. Ten percent of the galls obtained following infection with nopaline strain N2/73 spontaneously developed shooty teratomas of altered phenotype. The shooty teratomas grew in unsupplemented basal medium and were able to synthesize both the major withanolides of the parent plants. Withanolide synthesis in shooty teratomas was much higher (0.07–0.1% withaferin A and 0.085–0.025% withanolide D) than in non-transformed shoot cultures.

The presence and characterization of a virF gene on Agrobacterium vitis Ti plasmids.

Octopine and nopaline strains of Agrobacterium tumefaciens differ in their ability to induce tumors on Nicotiana glauca. The presence of a virF locus on the octopine Ti plasmid makes N. glauca a host plant for these strains, indicating that the VirF protein is a host-range determinant. Here we show the presence of a virF locus not only on the Agrobacterium vitis octopine/cucumopine plasmids pTiAg57 and pTiTm4, but also on the nopaline Ti plasmids pTiAT1, pTiAT66a, and pTiAT66b. On the octopine Ti plasmids from A. tumefaciens the virF gene is located between the virE locus and the left border of the T-region. In contrast, the virF gene on Ti plasmids of A. vitis is located at the very left end of the vir-region near the virA locus. The virF gene of pTiAg57 has been sequenced and codes for a protein of 202 amino acids with a molecular mass of 22,280 Da. Comparison showed that the virF gene from A. vitis strain Ag57 is almost identical to that from A. tumefaciens octopine strains. The transcription of the pTiAg57 virF is inducible by the plant phenolic compound acetosyringone through the presence of a vir-box consensus sequence in its promoter region. The VirF protein from pTiAg57 can complement octopine A. tumefaciens strains deleted for virF as shown by tumor formation on N. glauca.

Genetic transformation of Artemisia annua by Agrobacterium tumefaciens and artemisinin synthesis in transformed cultures

Transformed organ cultures of Artemisia annua were established following infection with two wild type nopaline strains of Agrobacterium tumefaciens. Parameters like explant type, strain type, age of the plant source for explants, affected the tumorigenesis frequency significantly. Crown galls were formed both on in vivo and in vitro plants: 2–3% of the in vitro galls regenerated spontaneously to produce shooty teratoma of altered phenotype. Artemisinin contents were measured in all transformed as well as non-transformed clones. While shooty teratomas synthesized 0.063 ± 0.002 g/100 g DW artemisinin, non-transformed shoots were found to synthesize only 0.0179 ± 0.002 g/100 g DW of the compound.

Agrobacterium-mediated transformation of commercial mints

Commercial peppermint (P) (Mentha × piperita L. ev. Black Mitcham), native spearmint (NS) (M. spicata L.) and Scotch spearmint (SS) (M. × gracillis Sole cv Baker) petioles and orange mint (OM) (M. citrata Ehrh.) leaf disks were cocultivated with a number of Agrobacterium tumefaciens strains. P, SS and OM initiated tumor-like callus tissue on growth regulator-free MS medium after cocultivation with strain A281, a hypervirulent agropine strain containing Ti plasmid pTiBo542. Callus did not initiate from explants cocultivated with strain C58, a virulent nopaline strain; with A 136, a plasmidless strain, or from uninoculated controls. A281-derived callus was maintained on growth regulator-free medium in the absence of antibiotics for up to two years with no bacterial outgrowth. No shoots regenerated from any of the tumors on regeneration medium. Five of seven OM callus lines assayed gave a positive signal for agropine. DNA extracted from OM tumor tissue hybridized to a DNA probe specific to the T-DNA region of pTi plasmid. Genomic Southern analysis of DNA from tumors of P and SS indicated that one to a few copies of the T-DNA integrated into the mint chromosomes. PCR amplification of genomic DNA with primers specific for one of the T-DNA encoded genes yielded fragments that, when analyzed by restriction enzyme mapping and on Southern blots, corresponded to the cytokinin biosynthesis gene ipt. These results demonstrate transformation of three species of mint and the potential for using A. tumefaciens to transfer economically important genes into commercial mint cultivars.

Spontaneous Transfer of the Ti Plasmid ofAgrobacterium tumefaciensand the Nopaline Catabolism Plasmid ofA. radiobacterStrain K84 in Crown Gall Tissue

Spontaneous transfer of the Ti plasmid from Agrobacterium tumefaciens to strain K84 of A. radiobacter was observed and studied for the first time in an experiment on biological control of crown gall. This transfer was detected in a tumor from a K84-treated plant grown in soil inoculated with a nopaline strain of A. tumefaciens biovar 1 sensitive to agrocin 84. The transconjugant strain was virulent and produced agrocin 84. Southern blot hybridization analysis with several probes (T-DNA and right adjacent regions and vir genes) showed important changes at the Ti plasmid, suggesting that recombination between Ti plasmid and pAtK84b in K84 could have happened, resulting in a new Ti plasmid. Transfer of both plasmids of strain K84, pAtK84b and pAgK84, responsible for nopaline catabolism and agrocin 84 production, respectively, to A. tumefaciens also was detected in isolates from the same tumor. Southern blot hybridization of plasmids from one of these avirulent isolates with a nopaline plasmid-specific probe of strain K84 indicated there was a replacement of Ti plasmid by pAtK84b in A. tumefaciens, explaining its avirulence. These results show that plasmid exchanges can occur spontaneously between A. tumefaciens and A. radiobacter. This kind of transfer generates genetic diversity in Agrobacterium and may influence the biocontrol efficiency of A. radiobacter.

Sequence and mutational analysis of a tartrate utilization operon from Agrobacterium vitis

The grapevine is the natural host of the tumorigenic bacterium Agrobacterium vitis. Most of the A. vitis isolates can use tartrate, an unusually abundant compound in grapevine. The nopaline strain, AB4, contains a 170-kb conjugative plasmid (pTrAB4) encoding tartrate utilization. A 5.65-kb pTrAB4 region which enables non-tartrate-utilizing Agrobacterium tumefaciens to grow on tartrate was sequenced and mutagenized with the transcriptional fusion transposon Tn5-uidA1. This DNA fragment contains four intact open reading frames (ORFs) (ttuABCD) required for tartrate-dependent growth. The mutant phenotypes of each ORF, their homologies to published sequences, and their induction patterns allowed us to propose a model for tartrate utilization in A. vitis. ttuA encodes a LysR-like transcriptional activator and is transcribed in the absence of tartrate. ttuB codes for a protein with homology to transporter proteins and is required for entry of tartrate into bacteria. ttuC codes for a tartrate dehydrogenase, while ttuD lacks homology to known sequences; the growth properties of ttuD mutants suggest that TtuD catalyzes the second step in tartrate degradation. A fifth incomplete ORF (ttuE) encodes a pyruvate kinase which is induced by tartrate and required for optimal growth. Although the ttuABCD fragment allows growth of A. tumefaciens on tartrate, it does not provide full tartrate utilization in the original A. vitis background.

An Agrobacterium virulence factor encoded by a Ti plasmid gene or a chromosomal gene is required for T-DNA transfer into plants.

Mutagenesis of the vir region on the Ti plasmid of Agrobacterium tumefaciens revealed a new locus, virJ, that is induced by the plant-wound signal molecule, acetosyringone (AS). virJ lies between virA and virB, and is transcribed in the same direction. The amino acid sequence of virJ is similar to a region of a previously characterized chromosomal gene, acvB, required for virulence. virJ can complement the avirulent phenotype of an acvB mutant, indicating that virJ and acvB encode the same factor required for tumorigenesis. Southern analysis revealed that virJ is present on the Ti plasmid of an octopine but not a nopaline strain whereas acvB is present on the chromosomes of both octopine and nopaline strains. While virJ is regulated by AS under the control of the virA/virG two-component regulatory system, acvB is not induced by AS. VirJ possesses a putative signal peptide and was found predominantly in the periplasmic fraction. The strain lacking both acvB and virJ had an impaired ability to transfer T-DNA into plant cells, suggesting that the factor encoded by virJ or acvB is required for T-DNA transfer from A. tumefaciens to plant cells. acvB is the first chromosomal gene implicated in T-DNA transfer, but whether it functions specifically for this process is not clear. We hypothesize that virJ evolved from acvB, presumably for a more specialized role in tumorigenesis.

Accumulation of essential oils by Agrobacterium tumefaciens-transformed shoot cultures of Pimpinella anisum

Axenic transformed shoot cultures of Pimpinella anisum (anise) were established following inoculation of plant stems with the nopaline strain T37 of Agrobacterium tumefaciens. The stable incorporation of T-DNA in the transformed tissues was demonstrated by polymerase chain reaction. Total essential oil accumulated by transformed shoot cultures grown under continuous light was found to be 18% lower (per unit fresh weight of tissue) than that produced by untransformed shoot cultures incubated under similar conditions, but more than 89% lower than the yield of oil from the intact plant. The relative amounts of the principal components of the essential oil of the transformed shoot cultures, namely geraniol, β-bisabolene, trans-pseudoisoeugenol-2-methylbutyrate and transanethole, were similar to those present in the parent plant, but significantly different from those of the untransformed shoot cultures.

175 GENETIC TRANSFORMATION OF COFFEE (COFFEA ARABICA L.) BY AGROBACTERIUM SPP.

Efficient genetic transformation could enhance coffee breeding, which is limited by its long generation time and narrow genetic base. Three explant types of three coffee cultivars were inoculated with 14 strains of Agrobacterium spp. Callus and hairy roots were produced with 13 of the 14 strains tested. With A. tumefaciens, nopaline strains were more effective than octopine strains. Cucumopine and mannopine strains of A. rhizogenes were both effective in inducing hairy roots and callus. PCR amplification of a 0.72 Kb fragment of T-DNA encoding a portion of the ipt gene was achieved with DNA from A. tumefaciens strain A208 and with putatively transformed tissue inoculated with A208. No amplification was observed with virB in putatively transformed tissue which indicates it was not contaminated with Agrobacterium. We conclude that coffee can be genetically transformed by some Agrobacterium strains.