An alternative approach for gene transfer in trees using wild-typeAgrobacterium strains

Micropropagated shoots of three forest tree species, poplar (Populus tremula × P.alba), wild cherry (Prunus avium L.) and walnut (Juglans nigra × J. regia), were inoculated each with six different wild-typeAgrobacterium strains. Poplar and wild cherry developed tumors that grew hormone-independently, whereas on walnut, gall formation was weak. On poplar and wild cherry, tumors induced by nopaline strains developed spontaneously shoots that had a normal phenotype and did not carry oncogenic T-DNA. From these observations, we have established a co-inoculation method to transform plants, using poplar as an experimental model. The method is based on inoculation of stem internodes with anAgrobacterium suspension containing both an oncogenic strain that induces shoot differentiation and a disarmed strain that provides the suitable genes in a binary vector. We used the vector pBI121 carryingneo (kanamycin resistance) anduidA (-glucuronidase) genes to facilitate early selection and screening. Poplar plants derived from kanamycin-resistant shoots that did not carry oncogenic T-DNA, were shown to contain and to expressneo anduidA genes. These results suggest that wild-typeAgrobacterium strains that induce shoot formation directly from tumors can be used as a general tool for gene transfer, avoiding difficult regeneration procedures.This work is dedicated to the late Marie-France Michel who initiated the poplar biotechnology project at INRA.     

Factors affecting Agrobacterium tumefaciens-mediated transformation in several black poplar clones

Transient expression of the uidA reporter gene was used in preliminary experiments with two oncogenic and two disarmed Agrobacterium tumefaciens strains in order to test the efficiency of T-DNA transfer to N084 x Populus nigra and N107 x P. nigra clones. The oncogenic strain A281 pKIWI105 produced the highest average number of GUS spots per leaf disc. In order to optimize the production of transgenic plantlets from different P. nigra clones (San Giorgio, Jean Pourtet, N084 x P. nigra and N107 x P. nigra, respectively), two A. tumefaciens strains (GV2260 p35S GUS, A281pKIWI105) and bacterial concentrations (7×10⁸; 1.2×0⁹ bacteria ml^-1) were used. Following co-cultivation with A281 pKIWI105, the frequency of leaf discs producing kanamycin-resistant calli was not significantly different between the clones and bacteria concentrations used. Transformed shoots were regenerated from all clones, except for Jean Pourtet. Co-cultivation of leaf discs with GV2260 p35S GUS produced very few calli which died when transferred to selective regeneration medium. In addition, the effects of acetosyringone and leaf wounding were evaluated for the San Giorgio and Jean Pourtet clones, using the same strains. Factors which significantly affected the transformation efficiency of leaf explants were the P. nigra clone, the A. tumefaciens strain, and the presence of acetosyringone. Genetic transformation of calli and regenerated plantlets was confirmed by their ability to grow and root on Woody Plant Medium containing kanamycin, by histochemical -glucuronidase assays, and Southern blot hybridization analyses.Abbreviations BA benzyladenine - GUS -glucuronidase - IBA indolebutyric acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid - nptII neomycin phosphotransferase II gene - uidA -glucuronidase gene - WPM Woody Plant Medium     

Factors Affecting the Agrobacterium-Mediated Transient Transformation of the Wetland Monocot,Typha latifolia

An Agrobacterium-mediated transformation system, using transient transformation assays, was used to evaluate conditions influencing transformation for the wetland monocot Typha latifolia. These studies were aimed at the long-term objective of evaluating candidate genes for phytoremediation. The binary plasmid vector pCAMBIA1301/EHA105, containing the -glucuronidase coding sequence, was used in combination with factors known to affect transformation. These included callus age at the time of cocultivation with Agrobacterium tumefaciens, type and concentration of auxin for explant growth, light or dark culture environment, the presence or absence of acetosyringone (AS), explant type, explant wounding and the number of days used for cocultivation. The number of days needed for the first detection of transient expression of the -glucuronidase gene was also examined. Three days of Agrobacterium cocultivation of 50-day-old seedling-derived calluses, grown on 20.7 µM (5 mg l^–1) picloram supplemented medium, in the dark, resulted in higher levels of transient -glucuronidase expression than were seen in calluses cultured on 4.5 or 22.6 µM (1 or 5 mg l^–1) 2,4-dichlorphenoxyacetic acid containing media. The addition of 100 µM acetosyringone significantly enhanced transient -glucuronidase activity. Wounding of explants, by cutting into two or three pieces, 3 days before cocultivation, increased expression of -glucuronidase only in calluses cultured under light conditions. Transient -glucuronidase expression was observed as early as 24 h after cocultivation and increased as the days post cultivation increased. The developed transient system will be used for stable transformation of Typha species.     

Genetic transformation of Medicago truncatula using Agrobacterium with genetically modified Ri and disarmed Ti plasmids

Summary Fertile transgenic plants of the annual pasture legume Medicago truncatula were obtained by Agrobacterium-mediated transformation, utilising a disarmed Ti plasmid and a binary vector containing the kanamycin resistance gene under the control of the cauliflower mosaic virus 35S promoter. Factors contributing to the result included an improved plant regeneration protocol and the use of explants from a plant identified as possessing high regeneration capability from tissue culture. Genes present on the T-DNA of the Ri plasmid had a negative effect on somatic embryogenesis. Only tissue inoculated with Agrobacterium strains containing a disarmed Ti plasmid lacking the T-DNA region or a Ri plasmid with an inactivated rol A gene regenerated transgenic plants. Fertile transgenic plants were only obtained with disarmed A. tumefaciens, and the introduced NPT II gene was transmitted to R₁ progeny.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - NPT neomycin phosphotransferase     

Cell competence forAgrobacterium-mediated DNA transfer inPisum sativum L.

Distribution and properties of pea (Pisum sativum L.) cells, competent forAgrobacterium-mediated transformation were analysed byin situ histochemical detection of GUS (-glucuronidase) activity, 4 d after inoculation with engineeredAgrobacterium tumefaciens. The vector system consisted of the hypervirulent disarmed strain EHA101 and the binary plasmid pIBGUS, carrying an intron-containing, 35S-promotor drivengusA (oruidA) gene and two selectable marker genes. Cells competent for transformation were mainly restricted to the dedifferentiating cells neighbouring the vascular system of cotyledon and epicotyl explants. A standardized assay was developed, allowing determination and quantification of factors influencing number and distribution of competent cells. In etiolated seedlings, competence for transformation decreased with the distance of the epicotyl explant from the shoot apex and was specifically induced by the exogenous application of auxins. Transient expression ofgusA afterAgrobacterium-mediated DNA transfer was dramatically reduced upon application of cell-cycle and DNA replication inhibitors aphidicolin, colchicine and nalidixic acid. GUS expression after direct DNA transfer of double-stranded plasmid DNA (via PEG into protoplasts or via particle bombardment of epicotyl segments) was independent of cell-division/DNA replication.A GUS-positive mutant of EHA101 was constructed to allowin situ analysis of attaching bacteria within the plant tissue. Attachment and invasion was inhibited by well-developed cuticula but was restored after chloroform treatment of the tissue surface. Moreover, no correlation was found between distribution of attaching bacteria and the pattern of transformation-competent cells.     

Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting gene transfer efficiency during early transformation steps

The factors influencing transfer of an intron — containing -glucuronidase gene to apple leaf explants were studied during early steps of an Agrobacterium tumefaciens-mediated transformation procedure. The gene transfer process was evaluated by counting the number of -glucuronidase expressing leaf zones immediately after cocultivation, as well as by counting the number of -glucuronidase expressing calli developing on the explants after 6 weeks of postcultivation in the presence of 50 mg/l kanamycin. Of three different tested disarmed A. tumefaciens strains, EHA101(pEHA101) was the most effective for apple transformation. Cocultivation of leaf explants with A. tumefaciens on a medium with a high cytokinin level was more conducive to gene transfer than cocultivation on media with high auxin concentrations. Precultivation of leaf explants, prior to cocultivation, slightly increased the number of -glucuronidase expressing zones measured immediately after cocultivation, but it drastically decreased the number of transformed calli appearing on the explants 6 weeks after infection. Other factors examined were: Agrobacterium cell density during infection, bacterial growth phase, nature of the carbon source, explant age, and explant genotype.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - CaMV35S 35S RNA of cauliflower mosaic virus - EDTA ethylenediaminetetraacetate - FeNaEDTA ethylenediaminetetraacetate ferric-sodium salt - GusA -glucuronidase - gusA ß-glucuronidase gene of Escherichia coli - gusA-intron ß-glucuronidase gene containing an intron in the coding region - IBA indole butyric acid - 2iP N6-2-isopentenyl adenine - NAA naphthaleneacetic acid - nptII neomycinphosphotransferase II gene - X-Gluc 5-bromo-4-chloro-3-indolyl ß-D-glucuronide     

Transient Expression of β-Glucuronidase in Embryo Axes of Cotton by Agrobacterium and Particle Bombardment Methods

Transient expression of -glucuronidase (GUS) in zygotic embryo axes of two cotton (Gossypium hirsutum L.) cultivars NHH-44 and DCH-32 was induced by Agrobacterium mediated transformation or by particle bombardment. For Agrobacterium transformation, disarmed A. tumefaciens strain GV 2260/p35SGUSINT was used. In cv. NHH-44, the maximum frequency of transient expression (14.28 %) was achieved on spotting Agrobacterium paste on the apical regions of the split embryo axes. The method resulted in a transformed callus line, which showed strong GUS activity. Integration of NPTII gene was confirmed by Southern analysis. Transgene expression by particle bombardment was achieved with p35SGUSINT and pIBGUS plasmids independently. The maximum frequency of GUS expression in 29.16 % explants was observed in cultivar NHH-44 with gold microcarriers (1.1 µm) when bombarded once with rupture disc of 7586 kPa at target cell distance of 6 cm. A transformed callus line was obtained when explants were bombarded with p35SGUSINT and cultured on Murashige and Skoog's medium supplemented with B₅ vitamins, 0.1 mg dm^–3 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, 0.01 mg dm^–3 -naphthaleneacetic acid, 3 % glucose + 50 mg dm^–3 kanamycin. High GUS activity was observed in callus tissue as well as in somatic embryo like structures achieved in liquid shake cultures.     

Factors affecting the rate of T-DNA transfer from Agrobacterium tumefaciens to Nicotiana glauca plant cells

Different factors involved in the early steps of the T-DNA transfer process were studied by using a -glucuronidase gene (gusA) as a reporter in Nicotiana glauca leaf disc transformation experiments. The levels of transient expression of the gusA gene in leaf discs infected with several strains or vir mutants correlated well with their virulence phenotype, except for virC mutants. The rate of T-DNA transfer was shown to be stimulated in the case of non-oncogenic strains by the co-transfer of small amounts of oncogenic genes. It was found that the location of the T-DNA in the Agrobacterium genome affected the T-DNA transfer rate especially in virC mutants. The virC mutants transferred the gusA-containing T-DNA located on a binary vector more efficiently than the oncogenic T-DNA of the Ti plasmid. Although wild-type strains induced high levels of gusA expression early after infection, the gusA expression appeared to be lost late after infection in the infected leaf discs. In contrast, in leaf discs infected by virC mutants the level of gusA expression increased steadily in time. A model explaining these results is presented.     

The use of in vitro-grown microtuber discs inAgrobacterium-mediated transformation of Russet Burbank and Lemhi Russet potatoes

We have usedin vitro-grown microtuber discs in the transformation of Russet Burbank and Lemhi Russet potato (Solanum tuberosum L.) cultivars byAgrobacterium-mediated gene transfer. Transformed plants were selected by their resistance to kanamycin and identified by -glucuronidase activity. Northern blot analysis confirmed the presence of the corresponding messenger RNA. The ability to transform these two cultivars promises significant improvements to agronomically important varieties.Abbreviations MS Murashige and Skoog - LB Luria-Bertani - KS high kanamycin selection medium - NAA naphthaleneacetic acid - BA benzyladenine - GUS -glucuronidase - EDTA ethylenediaminetetraacetic acid - MUG 4-methylumbelliferyl glucuronide     

Efficient transformation of potato (Solanum tuberosum L.) using a binary vector in Agrobacterium rhizogenes

Summary We transformed three potato (Solanum tuberosum L.) genotypes by using A. rhizogenes or a mixture of A. rhizogenes and A. tumefaciens. Inoculations of potato stem segments were performed with Agrobacterium rhizogenes AM8703 containing two independent plasmids: the wild-type Ri-plasmid, pRI1855, and the binary vector plasmid, pBI121. In mixed inoculation experiments, Agrobacterium rhizogenes LBA1334 (pRI1855) and Agrobacterium tumefaciens AM8706 containing the disarmed Ti-plasmid (pAL4404) and the binary vector plasmid (pBI121) were mixed in a 11 ratio. The T-DNA of the binary vector plasmid pBI121 contained two marker genes encoding neomycin phosphotransferase, which confers resistance to kanamycin, and -glucuronidase. Both transformation procedures gave rise to hairy roots on potato stem segments within 2 weeks. With both procedures it was possible to obtain transformed hairy roots, able to grow on kanamycin and possessing -glucuronidase activity, without selection pressure. The efficiency of the A. rhizogenes AM8703 transformation, however, was much higher than that of the mixed transformation. Up to 60% of the hairy roots resulting from the former transformation method were kanamycin resistant and possessed -glucuronidase activity. There was no correlation between the height of the kanamycin resistance and that of the -glucuronidase activity in a root clone. Hairy roots obtained from a diploid potato genotype turned out to be diploid in 80% of the cases. Transformed potato plants were recovered from Agrobacterium rhizogenes AM8703-induced hairy roots.     

Genetic transformation and regeneration of transgenic plants in grapevine (Vitis rupestris S.)

Isolated somatic embryos from petiole-derived callus cultures ofVitis rupestris Scheele have been employed in experiments on genetic transformation. Co-cultivation of somatic embryos during embryogenesis induction withAgrobacterium tumefaciens strain LBA4404, which contains the plasmid pBI121 carrying the neomycin phosphotranspherase and the-glucuronidase genes, produced transformed cellular lines capable of recurrent somatic embryogenesis. Precocious selection for high levels of kanamycin (100 mgl^-1) was an important part of our transformation protocol. Transformed lines still have strong-glucuronidase expression as well as stable insertion of the marker genes after 3 years of in-vitro culture, during which they have maintained their capacity to organize secondary embryos and to regenerate transgenic plants with an agreeable efficiency (13%).     

A simple procedure for the expression of genes in transgenic soybean callus tissue

Cotyledons from germinating seeds of the soybean cultivar Peking were inoculated with virulent Agrobacterium tumefaciens strain A281:pZA-7 which carries a wild type Ti plasmid pTiBo542 and a disarmed Ti plasmid (a binary vector)pZA-7 which contains the glucuronidase (uidA) and neomycin phosphotransferase (nptII) genes. Tumors were produced on all inoculated explants and 82% of these tumor lines were cotransformed by the nptII gene from the binary vector pZA-7 as shown by PCR analysis (18 of 22 lines tested). Eleven of these 18 lines were also resistant to kanamycin. Eleven lines expressed -glucuronidase activity (GUS), six of which were also kanamycin resistant. Since there is a high rate of coexpression of genes carried by the binary vector, this system provides a simple and rapid method for the expression of genes of interest in transformed soybean tissue which has been used successfully to test constructs designed for soybean transformation.     

Transgenic grasspea (Lathyrus sativus L.): Factors influencing Agrobacterium-mediated transformation and regeneration

A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD₆₀₀0.6 and diluted to a cell density of 10⁹ cells ml^–1 for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 g ml^–1). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l^–1 indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T₁ seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.     

Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro)

Cotyledon explants of muskmelon (Cucumis melo L., cv. Amarillo Oro) seedlings were co-cultivated with disarmed Agrobacterium tumefaciens strain LBA4404 that contained the binary vector plasmid pBI121.1. The T-DNA region of this binary vector contains the Nopaline synthase/neomycin phosphotransferase II (NPTII) chimeric gene for kanamycin resistance and the Cauliflower Mosaic Virus 35S/-glucuronidase (GUS) chimeric gene. After infection, the cotyledon pieces were placed in induction medium containing 100 mg/l kanamycin. Putative transformed shoots were obtained, followed by the development of morphologically normal plantlets. The transgenic nature of regenerants was demonstrated by polymerase chain reaction, Southern blot analysis, plant growth on medium selective for the transgene (NPTII) and expression of the co-transformed GUS gene. Factors affecting the transformation procedure are discussed.Abbreviations CaMV Cauliflower Mosaic Virus - Cf Cefotaxime - GUS -glucuronidase - Km Kanamycin - MS Murashige and Skoog - NOS nopaline synthase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

Treatment of Agrobacterium or leaf disks with 5-azacytidine increases transgene expression in tobacco

We have studied the effect of the demethylating agent azacytidine (azaC) on expression of a -glucuronidase (GUS) gene transferred to tobacco leaf disks by Agrobacterium-mediated transformation. In a system where no selection was performed, where shoot formation was partially repressed, and where Agrobacterium does not express the GUS gene, we were able to follow the early events of transient and stable expression. Two days after inoculation, 8% of the cells expressed GUS but this proportion rapidly decreased to near zero in the following week. Treatment of leaf disks with azaC just after transformation retarded this inactivation to some extent, while treatment of Agrobacterium prior to transformation increased the frequency of transient expression. Three weeks after inoculation the number of GUS-expressing cells increased 4- to 6-fold in the leaf disks treated with azaC and in the leaf disks transformed with azaC-treated bacteria, while the control remained low. These data suggest that DNA methylation is involved in transgene inactivation and that a large number of silent but potentially active transgenes become integrated.     

Transformation of barrel medic (Medicago truncatula Gaertn.) by (Agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with the MtENOD12

Summary Fertile and stable transgenic plants of the model legume Medicago truncatula Gaertn. were obtained through transformation of leaf tissue with the disarmed Agrobacterium tumefaciens strain LBA4404 and in vitro regeneration via somatic embryogenesis. An optimised transformation/regeneration protocol has been established for two genotypes of the cultivar Jemalong, including a previously described highly embryogenic line (Nolan et al. 1989, Plant Cell Rep. 8: 278–281). Using this protocol, transgenic plantlets were obtained within 4–10 months following cocultivation with Agrobacterium. We have introduced into M. truncatula a chimeric fusion between the early nodulin MtENOD12 promoter and the gus (-glucuronidase) reporter gene, and shown that symbiosis-specific gene expression can be elicited in the roots of such transgenic plants following the addition of purified Rhizobium nodulation factors.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediamine tetracetic acid - GUS gb-glucuronidase - IBA indolebutyric acid - MES 2-(N-morpholino) ethane sulfonic acid - OD optical density - X-Gluc 5-bromo-4-chloro-3-indolyl glucuronide     

High frequency <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and plant regeneration via direct shoot formation from leaf explants in <Emphasis Type="Italic">Beta vulgaris</Emphasis> and <Emphasis Type="Italic">Beta maritima</Emphasis>

We have developed a new procedure for Agrobacterium-mediated transformation of plants in the genus Beta using shoot-base as the material for Agrobacterium infection. The frequency of regeneration from shoot bases was analyzed in seven accessions of sugarbeet (Beta vulgaris) and two accessions of B. maritima to select materials suitable for obtaining transformed plants. The frequency of transformation of the chosen accessions using Agrobacterium strain LBA4404 and selection on 150-mg/l kanamycin was found to be higher than that in previously published methods. Genomic DNA analysis and -glucuronidase reporter assays showed that the transgene was inherited and expressed in subsequent generations. In our method, shoot bases are prepared by a simple procedure, and transformation does not involve the callus phase, thus minimizing the occurrence of somaclonal variations.     

Patterns of transformation intensity on flax hypocotyls inoculated with Agrobacterium tumefaciens

Summary Cellular transformation intensities on flax (Linum usitatissimum) hypocotyl explants using disarmed Agrobacterium tumefaciens were investigated through various preculture durations, cocultivation durations and removal of epidermis. The expression of an intron-containing -glucuronidase (GUS) gene driven by CaMV 35S promoter served as a reporter for determination of transformed tissues on hypocotyls. The binary plasmid p35SGUSINT in octopine-type Agrobacterium strain GV2260 was used as the vector system. A prolonged cocultivation duration (5–7 days) resulted in a much higher transformation staining intensity (frequency * tissue area) than 2- or 3-day-cocultivation duration on hypocotyls variously precultured prior to inoculation. A high staining intensity on the two cut ends was obtained from nonprecultured hypocotyls. A reduction in intensity on the upper cut end of hypocotyls was observed with preculture times greater than 6 days. Peeled hypocotyls with a post-peeling preculture of 2 or 3 days had a high proportion of superficial area covered by transformed tissues after a 7 day-cocultivation duration. These results will help to improve the efficiency of recovery of transgenic plants by increasing the proportion of transformation in the regenerable tissues.     

Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination

A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both -glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3–1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l^–1 hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.