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2S albumin seed storage proteins undergo a complex series of posttranslational proteolytic cleavages. In order to determine if this process is correctly carried out in transgenic plants, the gene AT2S1 encoding an Arabidopsis thaliana 2S albumin isoform has been expressed in transgenic tobacco. Initial experiments using a reporter gene demonstrated that the AT2S1 promoter directs seed specific expression in both transgenic tobacco and Brassica napus plants. The entire AT2S1 gene was then transferred into tobacco plants, where it showed a tissue specific and developmentally regulated expression. Arabidopsis 2S albumin accumulates up to 0.1% of the total high-salt extractable seed protein. Protein sequencing demonstrated that the amino termini of the two Arabidopsis 2S albumin subunits were correctly processed, suggesting that the protease(s) necessary for posttranslational processing of 2S albumin precursors may display common specificities among different dicot plant species. Immunocytochemical studies showed that the Arabidopsis 2S albumin is localized in the protein body matrix of tobacco endosperm and embryo. Correct processing and targeting of the 2S albumin in transgenic plants suggests that modified versions could be expressed, allowing the study of 2S albumin processing and in particular the possible roles of the processed fragments in protein stability and/or targeting.

We present the results of two sets of experiments designed to express high methionine proteins in transgenic seeds in three different plant species. In the first approach, two chimeric genes were constructed in which parts of the Arabidopsis 2S albumin gene 1 (AT2S1) were fused at different positions to a Brazil nut 2S albumin cDNA clone. Brazil nut 2S albumin was found to accumulate stably in transgenic Arabidopsis, Brassica napus, and tobacco seeds. In the second approach, methionine-enriched AT2S1 genes were constructed by deleting sequences encoding a region of the protein which is not highly conserved among 2S albumins of different species and replacing them with methioninerich sequences. Introduction of the modified AT2S1 genes into three different plant species resulted in the accumulation of the methionine-enriched 2S albumins in all three species at levels reaching 1 to 2% of the total high salt-extractable seed protein.

dormancy in trees, as this process shares substantial analogies An ABSCISIC ACID-INSENSITIVE 3 (ABI3) homologous gene to seed dormancy (Saure, 1985). To test this hypothesis, was isolated from poplar (Populus trichocarpa). The deduced PtABI3 was isolated from the woody plant Populus protein of 736 amino acids showed sequence similarity to trichocarpa. other VP1/ABI3 proteins within four regions. PtABI3 is encoded An ABI3 homologous fragment from genomic DNA of a by a single gene in poplar and, as in Arabidopsis, highly P. trichocarpa◊P. deltoides hybrid was generated in a polyexpressed in developing seeds. merase chain reaction (PCR) using degenerated oligonucleotides (Fig. 1). This PCR fragment was used as a probe to

Comme la plupart des grandes avancees scientifiques, la transgenese vegetale est l'aboutissement d'une serie de travaux, provenant de differents groupes. Pourtant, indeniablement, ses premiers pas ont eu lieu dans un laboratoire belge ou operaient trois jeunes chercheurs, bientot des pionniers: Walter Fiers, Marc van Montagu et Jeff Schell. L'un d'eux raconte.

The vast majority of proteins in the nematode cuticle are collagens. Cuticular collagen-encoding genes (col) have been described for the animal parasites Ascaris suum and Haemonchus contortus and for the free-living Caenorhabditis elegans. The proteins encoded by all these genes seem to have the same basic structure, indicating that there is a conserved subfamily of cuticular col in these nematodes. In this paper, we describe the identification and characterization of a cDNA (Lemmi 5) which corresponds to a cuticular col of the plant-parasitic nematode Meloidogyne incognita. The derived protein structure is very similar to the basic structure of the C. elegans cuticular collagens. Using PCR technology, we have shown the presence of Lemmi 5-related sequences in the genome of Ditylenchus destructor. Our data strongly support the existence of a cuticular col subfamily which is highly conserved in the phylum Nemata.

We have isolated three Apetala2 (Ap2)-like genes from petunia and studied their expression patterns by in situ hybridization. PhAp2A has a high sequence similarity to the A function gene Ap2 from Arabidopsis and a similar expression pattern during flower development, suggesting that they are cognate orthologs. PhAp2B and PhAp2C encode for AP2-like proteins that belong to a different subgroup of the AP2 family of transcription factors and exhibit divergent, nearly complementary expression patterns during flower development compared with PhAp2A. In contrast, all three PhAp2 genes are strongly expressed in endosperm. The phenotype of the petunia A-type mutant blind cannot be attributed to mutations in the petunia Ap2 homologs identified in this study, and reverse genetics strategies applied to identify phap2a mutants indicate that PhAp2A might not be essential for normal perianth development in petunia. Nevertheless, we show that PhAp2A is capable of restoring the homeotic transformations observed in flowers and seed of the ap2-1 mutant of Arabidopsis. Although the interspecific complementation proves that PhAp2A encodes a genuine Ap2 ortholog from petunia, additional factors may be involved in the control of perianth identity in this species.

Activation of cell division in the root apical meristem after germination is essential for postembryonic root development. Arabidopsis plants homozygous for a mutation in the ROOT MERISTEMLESS1 (RML1) gene are unable to establish an active postembryonic meristem in the root apex. This mutation abolishes cell division in the root but not in the shoot. We report the molecular cloning of the RML1 gene, which encodes the first enzyme of glutathione (GSH) biosynthesis, gamma-glutamylcysteine synthetase, and which is allelic to CADMIUM SENSITIVE2. The phenotype of the rml1 mutant, which was also evident in the roots of wild-type Arabidopsis and tobacco treated with an inhibitor of GSH biosynthesis, could be relieved by applying GSH to rml1 seedlings. By using a synchronized tobacco cell suspension culture, we showed that the G(1)-to-S phase transition requires an adequate level of GSH. These observations suggest the existence of a GSH-dependent developmental pathway essential for initiation and maintenance of cell division during postembryonic root development.

Both compatible and incompatible interactions between Arabidopsis thaliana and Xanthomonas campestris have been identified and, for the first time, a strong hypersensitive response has been characterized. A highly reproducible mass spray inoculation protocol has been established and was used together with the more commonly used infiltration inoculation procedure to study the defense responses occurring in mature A. thaliana plants. A series of bacterial strains have been tested on A. thaliana ecotype Columbia (Col-O). X. c. pv. campestris was the most effective pathogen in these tests and was used for further detailed analysis. Several X. c. pv. campestris isolates were tested on A. thaliana Col-O, and one particular X. c. pv. campestris strain (147) was tested on 27 Arabidopsis ecotypes. Symptom development of compatible and incompatible interactions, including the hypersensitive response, was extensively characterized in A. thaliana Col-O. Lesion structure, bacterial distribution, accumulation of polyphenolic compounds, and the deposit of callose in inoculated leaves were documented by microscopic analysis. Activation of the defense-associated genes coding for phenylalanine ammonia lyase (PAL), beta-1, 3-glucanases, chitinases, and peroxidases was evaluated by Northern blot analysis.

Exudate from Sesbania rostrata seedlings contains signaling compounds that induce the common nodABC operon of the bacterial symbiont Azorhizobium caulinodans ORS571. An Azorhizobium strain harboring a nodA::lacZ reporter fusion was used to monitor the nod-inducing activity of crude exudate fractions that were separated by reversed-phase chromatography. The major inducer was shown, by spectroscopic analysis and by comparison with chemically synthesized compounds, to be 7,4'-dihydroxyflavanone (liquiritigenin). Newly synthesized analogues, 7,3'-dihydroxyflavanone and 7,2'-dihydroxyflavanone, have only poor inducing activity.

Tobacco shooty or rooty teratomas and hairy roots were induced by Agrobacterium tumefaciens (pGV 3845), A. tumefaciens (pGV 3304) and A. rhizogenes (pRi 8196), respectively. The tobacco alkaloids, nicotine, nornicotine and anatabine, were produced in hairy roots and in rooty teratomas but not in shooty teratomas. However, the shooty teratomas have the ability to accumulate the alkaloids and to biotransform nicotine to nornicotine. These were established by co-culture experiments incubating hairy roots and shooty teratomas in a same dish and by biotransformation experiments with shooty teratomas.

We have explored the possibility of producing large amounts of biologically active peptides as part of chimeric plant seed storage proteins, the 2S albumins. A portion of an Arabidopsis thaliana 2S albumin gene, encoding a region of the protein whose sequence is not highly conserved among different species, was replaced by sequences encoding the neuropeptide Leu-enkephalin, flanked by tryptic cleavage sites. Using Agrobacterium mediated transformation, the modified gene was transformed into Arabidopsis and oilseed rape and the 2S albumins isolated from the seeds of the regenerated transgenic plants. These were digested with trypsin and the enkephalin containing peptide isolated and treated to remove the extra lysine residue. Up to 200 nmol of peptide were recovered per gram of seed.

The T-DNA transfer process of Agrobacterium tumefaciens is activated by the induction of the expression of the Ti plasmid virulence (vir) loci by plant signal molecules such as acetosyringone. The vir gene products act in trans to mobilize the T-DNA element from the bacterial Ti plasmid. The T-DNA is bounded by 25-base pair direct repeat sequences, which are the only sequences on the element essential for transfer. Thus, specific reactions must occur at the border sites to generate a transferable T-DNA copy. The T-DNA border sequences were shown in this study to be specifically nicked after vir gene activation. Border nicks were detected on the bottom strand just after the third or fourth base (+/- one or two nucleotides) of the 25-base pair transferpromoting sequence. Naturally occurring and base-substituted derivatives of the 25-base pair sequences are effective substrates for acetosyringone-induced border cleavage, whereas derivatives carrying only the first 15 or last 19 base pairs of the 25-base pair sequence are not. Site-specific border cleavages occur within 12 hours after acetosyringone induction and probably represent an early step in the T-DNA transfer process.

We have investigated the regulatory functions of the 5'-flanking sequences of a chlorophyll a/b-binding protein gene from Pisum sativum, using the neomycin phosphotransferase (II) activity from Tn5 as an enzymatic reporter. We show that 0.4 kb of the upstream flanking sequences of this gene are sufficient for both organ-specific and light-regulated expression of our chimaeric constructs in transformed tobacco plants. In addition, we show that sequences farther upstream have a significant influence on the level of transcription of these constructions.

The tumour-inducing T-DNA genes 1, 2 and 4 of the octopine Ti-plasmid pTiAch5 were cloned and introduced into tobacco cells by cocultivation or leaf disk transformation using pTi derived vectors. When a selectable marker was needed, we used a aminoglycoside phosphotransferase II (nos-APH(3′)II) chimeric gene conferring kanamycin resistance to plant cells. The expression of gene 4 in transformed tissue cultures precluded the regeneration of normal transformed plants. Normal transformed plants were obtained with the construction carrying genes 1 or 2. We report in vivo complementation of genes 1 and 2 after crosses of transformed plants. Strategies are described for the use of genes 1 and 2 as selection or screening markers in plant cells or regenerated plants.

This paper describes a method for the transfer to plant cells of any cloned gene, regardless of its termini or internal restriction enzyme cleavage sites. A broad host-range intermediate vector, pGV1117, was constructed containing HindIII-23, a right-end T-region fragment of the nopaline plasmid pTiC58. Using in vivo protection by EcoRI methylase and EcoRI linker ligation, a fragment of rabbit chromosomal DNA, carrying the beta-globin gene, was inserted into plasmid pGV1117. Following transmission to Agrobacterium tumefaciens, insertion of the gene into the T-region of pTiC58 occurred via in vivo recombination. Infection of axenic tobacco seedlings resulted in the transfer to the plant genome of an intact beta-globin gene, as part of the T-DNA. Although the gene was stably maintained during tissue culture, beta-globin-specific transcripts were not detected in the transformed plant cells.

We have isolated a single-copy gene from the plant Arabidopsis thaliana, called dbp, which encodes a lysine-rich, DNA-binding protein. The Dbp protein has a molecular weight and a composition resembling histone H1. When the dbp gene was expressed in bacteria, the protein product bound DNA nonspecifically. The dbp gene is expressed constitutively in all parts of the plant but is induced five times above this basal level in apical zones. In vitro hormone-depletion experiments showed that the expression in the shoot apex could be induced by exogenous auxin. In situ hybridizations in the root apex indicated that the expression of dbp is enhanced in the region of cell division.

Foreign genes introduced into plant cells with Ti-plasmid vectors are not expressed. We have constructed an expression vector derived from the promoter sequence of nopaline synthase, and have inserted the coding sequences of the octopine synthase gene and a chloramphenicol acetyltransferase gene into this vector. These chimaeric genes are functionally expressed in plant cells after their transfer via a Ti-plasmid of Agrobacterium tumefaciens.

We have isolated a 1.5-kb plant DNA fragment (called insert 7) from Nicotiana plumbaginifolia DNA that contains a protoplast-specific enhancer-like sequence. The presence of this sequence on a plasmid carrying a chimeric nos-npt-II gene conferring kanamycin resistance to plant cells, produces an overexpression of the npt-II gene during at least eight days after protoplast transformation. This effect on the expression of the nos promoter was independent of the orientation and was observed both on circular and linearized plasmids. On the contrary, insert 7 had no influence when present on another plasmid (in trans) in cotransformation experiments. The overexpression of the nos-npt-II gene due to the presence of insert 7 on the transforming plasmid is correlated with a higher level of synthesis of the corresponding RNA. Insert 7 did not affect the level of expression of the nos-npt-II gene in stably transformed calli, or in regenerated plants. However, the overexpression was again detected in protoplasts prepared from leaves of stably transformed plants. This 1.5-kb plant DNA fragment contains highly repetitive DNA sequences, specific to N. plumbaginifolia. However, the enhancer-like activity is localized on a 600-bp unique sequence of insert 7. Insert 7 had no detectable effect on the transient expression of another gene, the nopaline synthase gene present at a longer distance on the same plasmid.

Co-cultivation of Agrobacterium tumefaciens with plant cells leads to the induction of circular copies of the T-DNA segment of the large tumour-inducing (Ti) plasmid in the bacterial cells. These circular molecules are presumably intermediates in DNA transfer from the A. tumefaciens genome to the plant cells. In support of this suggestion, the junction of the T-DNA circles occurs precisely in the 25-base pair terminal sequence involved in T-DNA transfer.

The title apigenin triglycoside was isolated (4 mg/6000 seeds) by reversephase column chromatography as the major u.v.-absorbing compound in the exudate of germinating seeds of Sesbania rostrata . The structure was assigned on the basis of u.v. spectra, f.a.b.-mass-spectral and 2D-n.m.r. data. The triglycoside was released continuously from the germinating seeds, but at a decreasing rate during the first two weeks.

The lambda ral function modulates the restriction and modification activities of the Escherichia coli K12 and B restriction enzymes (Zabeau et al., 1980). In order to further analyse this function, ral deficient mutants have been isolated, using a method which exploits the property of the strong mutagen N-methyl-N'-nitro-N-nitrosoguanidine (N.G.) to induce multiple closely linked mutations. Hence, mutagenized phages carrying mutations in one locus were frequently found to contain additional mutations in adjacent loci. This very efficient mutagenesis procedure enabled us to isolate 27 independent Ral deficient mutants. Seven mutants were found to affect the ral gene directly and were located between the genes N anc cIII. Detailed mapping of two of these mutants showed that the lambda ral gene is located at position 70.6-70.9% on the physical map. The isolation and characterization of these mutants further supports the conclusion that ral is a gene different from the N gene, and demonstrates that the ral gene product is responsible for both counteracting restriction and enhancing modification.

Antifungal rhizobacteria were obtained from maize, barley and chicory using direct or indirect isolation procedures. Effective isolates were tested for broad-spectrum activity against a set of phytopathogenic fungi. Isolates with broad-spectrum activity were identified as Pseudomonas fluorescens, P. cepacia, Serratia liquefaciens, S. plymuthica and Bacillus sp. Broad-spectrum compounds produced by P. cepacia and Erwinia herbicola were characterized as pyrrolnitrin and herbicolin-like compounds respectively.

We show here that Agrobacterium tumefaciens virulence (Vir) gene expression is activated specifically by the plant molecules acetosyringone (AS) and α-hydroxyacetosyringone (OH-AS). These molecules induce the entire vir regulon in Agrobacterium as well as the formation of T-DNA intermediate molecules. AS and OH-AS occur specifically in exudates of wounded and metabolically active plant cells and probably allow Agrobacterium to recognize susceptible cells in nature.

A series of monoclonal antibodies specific for distinct regions of H1 histone from the plant Nicotiana tabacum were obtained from fusion experiments with spleen cells of mice immunized with tobacco nuclear extracts. These monoclonal antibodies were characterized and the evolutionary conservation of the epitopes in higher plants and animals studied by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Whereas some epitopes appear restricted to the Solanaceae plant family, others are common to all higher eukaryotes tested and even detectable on nuclear proteins of yeast. ELISA experiments performed with isolated tobacco chromatin give some indications of the differential accessibility of the epitopes after interaction of H1 histone with the nucleosome.

A new class of binary vectors has been constructed, containing the origin of replication of the W-type plasmid Sa. These vectors are designed to express foreign genes in plants under control of the TR gene 2' promoter or the promoter of a light-inducible ribulose-1,5-bisphosphate carboxylase small subunit gene of Arabidopsis thaliana.

We present a detailed analysis of the function of the right and left T-DNA border regions of the nopaline Ti plasmid of Agrobacterium tumefaciens. An avirulent deletion of the right border of the nopaline Ti plasmid (pGV3852) was used as an acceptor for 14 different T-DNA border constructs. The functional activities of these constructs were assayed by their ability to restore virulence, i.e. transformation on inoculated plants. Tumorigenicities were measured in several independent experiments over a 2 year period and the statistical significance of their relative levels was evaluated. The data indicate: (i) the entire sequence of the 25 bp direct repeat of the T-DNA is required to provide an efficient substrate for mediating T-DNA transfer events; deletion derivatives of either the conserved or the vaiable domain of the repeat are defective in T-DNA transfer; (ii) while the 25 bp direct repeat alone can promote the T-DNA transfer, the flanking sequences of the repeats enhance (on the right) or attenuate (on the left) their activity; and (iii) tumorigenicity measurements vary depending on the plant assay system: potato discs are more sensitive than wounded tobacco leaves in detecting differences in T-DNA border activity.

A plasmid-encoded crystal protein gene (bt2) has been cloned from Bacillus thuringiensis berliner 1715. In Escherichia coli, it directs the synthesis of the 130-kDa protein (Bt2) which is toxic to larvae of Pieris brassicae and Manduca sexta. Comparison of the deduced amino acid sequence of this Bt2 protein with the B. thuringiensis kurstaki HD1 Dipel, B. thuringiensis kurstaki HD73 and B. thuringiensis sotto crystal protein sequences suggests that homologous recombination between the different genes has occurred during evolution. Treatment of the Bt2 protein with trypsin or chymotrypsin yields a 60-kDa protease-resistant and fully toxic polypeptide. The minimal portion of the Bt2 protein required for toxicity has been determined by analysing the polypeptides produced by deletion derivatives of the bt2 gene. It coincides with the 60-kDa protease-resistant Bt2 fragment and it starts between amino acids 29 and 35 at the N-terminus and terminates between positions 599 and 607 at the C-terminus.

A set of MS2 mutants were shown to have an additional silent mutation met --> ile at position 108 of the coat protein. As transitions are more frequent than transversions one would have expected an AUA codon in this position in the mutant RNAs. As the AUA codon is one of the best candidates for a modulation role in the control of translation in E. coli, the presence of this AUA in the gene for the protein made in major amounts upon viral infection would impose serious doubt on the theory of modulation. We have directly proven by minifinger-printing of mutant RNA and further analysis of the relevant spots that, in fact, the isoleucine residue at position 108 of the coat protein gene is specified by the non-rate-limiting AUU codon, in agreement with a modulation type of control of protein synthesis.

A plasmid was constructed that directs expression of the TL-DNA gene 4 protein in E. coli. The different steps of the construction were as follows: i) a region of gene 4 encoding the amino-terminal portion of the protein was fused in frame to DNA encoding an enzymatically active carboxy-terminal fragment of beta-galactosidase. The hybrid gene was poorly expressed from the upstream lambda PL promoter carried by the vector. ii) in order to generate an efficient procaryotic ribosome binding site, a DNA fragment carrying the lambda PR promoter with the nearby Shine-Dalgarno (SD) sequence of gene cro was placed in front of the gene 4-lacZ fusion. A recombinant plasmid, termed pGV793, that expressed efficiently a fused protein 4-beta-galactosidase was identified among the Lac+ clones. DNA sequencing analysis showed that pGV793 carried a hybrid ribosome binding site composed of the cro SD sequence, a five bp sequence and the ATG codon of gene 4. Plasmid pGV793 directed the synthesis of three polypeptides of molecular weight 132 Kd, 126 Kd and 122 Kd that carried beta-galactosidase antigenic determinants. The largest polypeptide had the expected size for the hybrid protein. The fusion proteins which accounted for about 0.5% of the total cellular proteins were purified by immunoadsorption using anti-beta-galactosidase antiserum. iii) the complete gene 4 coding sequence was reconstituted, with the lambda PR promoter in place. The resulting pGV822 plasmid expressed a polypeptide whose molecular weight 27 Kd corresponded to the expected size for the gene 4 product. The pI was about 7.

We present the first evidence, in Bacillus subtilis, for gene regulation through the classical mechanism of repressor-operator interaction. The EcoRI-F immunity region (immF) of lysogenic phage φ105 contains two promoters, PM and PR, in divergent orientations. PM initiates transcription of the φ105 repressor (cφ105) gene, whereas PR most probably signals the onset of the lytic pathway. Fusions between each of these promoters and the cat-86 gene were constructed, and in-vivo promoter activities were determined, in both the presence and absence of the functional cφ105 product, using S1 nuclease analysis and chloramphenicol acetyl transferase assays. The results showed that transcription from PM is stimulated, whereas PR activity is negatively controlled by the repressor. This differential regulation appears to be mediated by recognition of a 14 base-pair (bp) sequence, 5′ GACGGAAATACAAG 3′, three identical copies of which are present as direct repeats. Two copies, OR1 and OR2, are located closely together in the non-transcribed region between PM and PR, but do not overlap with the −35 and −10 regions of these promoters. The third copy, OR3, is located some 250 bp downstream from PR, within the coding region (ORF3) of the proximal gene of the PR transcription unit. When a 231 bp restriction fragment containing only OR3 was inserted between a strong constitutive promoter (P138) and the cat-86 gene, the in-vivo expression of chloramphenicol resistance was considerably reduced in the presence, but not in the absence, of φ105 repressor. This hybrid P138-OR-cat-86 construct was subsequently used to select in vivo for operator-constitutive (Oc) mutations. Of 25 Oc mutants analyzed, all showed base alterations or deletions affecting the 14 bp sequence. We show further that insertion of a chemically synthesized oligonucleotide, containing the 14 bp OR sequence, at a site more than 100 bp downstream from the constitutive P138 is sufficient for transcription to become negatively controlled by φ105 repressor. In comparison with previously identified Gram-negative bacterial and phage operators, the most unusual aspect of the φ105 OR sequence appears to be its complete lack of 2-fold rotational symmetry.

The small genome size of Arabidopsis thaliana allows the isolation of genes expressed in specific tissues and under controlled conditions by the differential screening of a genomic library, as has been shown previously for yeast and Drosophila. cDNA probes, based on poly(A)+ mRNA isolated from different Arabidopsis organs, were used in colony hybridizations with 1145 randomly chosen genomic clones, representing 27,000 kb of Arabidopsis DNA. Twenty percent of the clones containing low-copy-number sequences hybridized with one or more of the cDNA probes that were synthesized from mRNA isolated from leaves, stems, seed pods, inflorescences, callus tissue, and light-grown and dark-grown plants. Comparison of the colony hybridizations led to the identification of a large variety of clones which contain differentially expressed genes. The pattern of expression was confirmed by Northern analysis. The advantage of the described method is that it yields directly genomic sequences that contain specifically expressed or induced genes. In particular, it circumvents the construction and differential screening of cDNA libraries for every tissue or environmental parameter to be analyzed.

A variety of expression systems and selection regimes have been developed to transform plants such as tobacco, petunia, and tomato. We investigated several of these to determine whether the promoters and selectable markers used in dicotyledonous plants are suitable for selecting transformed rice callus. We compared transient expression driven by constitutive and regulated promoters in rice (Oryza sativa) protoplasts and found that the 2' promoter of the octopine T-DNA is approximately 3 to 4 times more efficient than the CAMV 35S promoter, 10 times more efficient than the nos promoter and the 1' promoter, and more than 100 times better than two other regulated plant promoters. Similar results were obtained in tobacco (Nicotiana tabacum) protoplasts with the exception that the nos promoter was expressed nearly 10 times better in rice. Further studies demonstrated that rice callus growth is sensitive to low concentrations of methotrexate, phosphinothricin, and bleomycin, and to moderate concentrations of G418 and hygromycin, but is only partially inhibited by relatively high concentrations of kanamycin. Finally, we tested the ability of stably introduced resistance genes to protect callus against some of the selective agents. Genes that inactivated phosphinothricin or G418 permitted transformed calli to grow almost unimpeded on toxic concentrations of these selective agents. However, a gene conferring resistance to methotrexate could not be used to select for activily growing transformants. Southern analysis of the transformed cell lines demonstrated that 50% of the transformants contained a single plasmid copy and that nearly all integrated copies showed rearrangements. These results on the use of selectable markers in rice should facilitate efforts to obtain transformants of this important grain.

Eighteen strains of Agrobacterium tumefaciens isolated from crown galls were tested for agrocin production. Of six agrocin-producing strains, one (D286) produced a broad-host-range agrocin active against strains carrying nopaline, octopine, and agropine type Ti plasmids. Sensitivity to agrocin D286 was found to map in the 11- to 18-megadalton region of the nopaline Ti plasmid pTiC58. The agrocin was partially purified, and its physical characteristics were consistent with its being a nucleotide, as is agrocin 84. Agrocin D286 was shown to inhibit DNA, RNA, and protein syntheses. Strain D286 spontaneously lost its pathogenicity, and its potential for use in the biological control of crown gall is discussed.

The fundamental goals of present day genetic engineering are in fact identical to those of traditional plant breeding: to engineer plants that are more valuable as crop plants, as biomass or as sources of important pharmaceuticals and enzymes. Central to many schemes designed to genetically modify plants is the availability of an efficient host gene vector system. Ideally the gene vector should provide an easy and reproducible system for the introduction of isolated genes—or groups of genes—into the genome of plant cells. The foreign genes should be appropriately expressed in the genetically modified (transformed) plant cells and stably transmitted, both somatically and sexually, to the offspring of the initially transformed cells. An ideal gene vector should also make it possible to introduce, maintain, and express any gene in plant cells independent of its origin, whether it is from plants, animals, bacteria, fungi or viruses. Finally one would wish the gene vector to have a very broad host range in order to be able to engineer the widest possible range of different plants. Agrobacteria, with their so–called Ti plasmids, provide us with a natural gene vector that can readily be modified to fulfill most of the requirements of an ideal gene vector for plants.