Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 g/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 g/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.NRCC No. 31491During the editorial process, a report has appeared on transformation of strawberry (James et al. 1990 Plant Sci 69:79–94).     

Genetic transformation of cocoa leaf cells using Agrobacterium tumefaciens

Leaf strips from cocoa tree (Theobroma cacao L.) clones ICS-16 and SIC-5 were cocultivated with the supervirulent Agrobacterium tumefaciens strain A281-Kan. A281-Kan contains a wild-type Ti plasmid and an additional plasmid, pGPTV-Kan, which confers kanamycin resistance to transformed plant cells after integration and expression of the neomycin phosphotransferase II (nptII) gene. Transformed cells were selected on callusing medium containing 100 g ml^-1 kanamycin. NptII assays confirmed that kanamycin-resistant cultures of ICS-16 and SIC-5 expressed the nptII gene, whereas control cultures did not. Genomic Southern blot analyses demonstrated single T-DNA insertions into ICS-16 and SIC-5. T-DNA/cocoa DNA border regions from transformed cultures were cloned and sequenced, revealing that in both transformed cell lines, the right T-DNA border was at the 5 end of the 25 bp right border repeat. Cocoa DNA probes from the T-DNA/cocoa DNA insertion sites were used in Southern blot analyses and showed that T-DNA from pGPTV-Kan had inserted into a unique region in ICS-16 and into a repetitive region in SIC-5. This study establishes that foreign genes can be inserted and expressed in cocoa using A. tumefaciens-mediated gene transfer.     

Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants

Transgenic muskmelon (Cucumis melo L.) plants were produced efficiently by inoculating cotyledon explants with Agrobacterium tumefaciens strain LBA4404 bearing a Ti plasmid with the NPT II gene for kanaymcin resistance. After co-cultivation for three days, expiants were transferred to melon regeneration medium with kanamycin to select for transformed tissue. Shoot regeneration occurred within 3–5 weeks; excised shoots were rooted on medium containing kanamycin before transferring to soil. Morphologically normal plants were produced in three months. Southern blot analysis confirmed that ca. 85% of the regenerated plants contained the NPT gene. Dot blot analysis and leaf callus assay of progeny of transgenic plants verified transmission of the introduced gene(s) to the next generation. Factors affecting transformation efficiency are discussed.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - IAA indole 3 acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NPT II neomycin phosphotransferase II     

Genetic transformation of Ginkgo biloba by Agrobacterium tumefaciens

A reproducible protocol has been established for the transformation of Ginkgo biloba by Agrobacterium tumefaciens . Embryos were co-cultivated with Agrobacterium tumefaciens GV3101 (pGV2260) carrying the binary vector pTHW136, which contained the gus reporter gene and the nptII selectable gene, encoding the enzymes β -glucuronidase (GUS) and neomycin phophotransferase II, respectively. Transient GUS activity has been used to screen the effects of different factors on the transfer of DNA into embryos (age of embryos, infection method, composition of co-cultivation medium). Then, experimental conditions have been defined to obtain transgenic kanamycin-resistant G. biloba calluses expressing GUS activity. The highest rate of transformation (45%) was reached using 1.5-month-old embryos co-cultivated on a medium lacking mineral elements. The integration of gus and nptII genes in calluses was confirmed by polymerase chain reaction analysis and Southern blot analysis.     

Genetic transformation of Robinia pseudoacacia by Agrobacterium tumefaciens

Transgenic Robinia pseudoacacia plants were obtained by Agrobacterium tumefaciens mediated gene transfer. Agrobacterium strain LBA4404 harbouring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes was co-cultivated with hypocotyl segments of in vitro raised seedlings of Robinia. Parameters important for high efficiency regeneration and transformation rates included type of explant, pre-conditioning of explants and appropriate length of co-cultivation period with Agrobacterium. A transformation frequency 16.67% was obtained by 48 hr of pre-conditioning followed by 48 hr of co-cultivation. Transformed tissue was selected by the ability to grow on kanamycin containing medium. Successful regeneration was followed after histochemical GUS assay for the detection of transgenic tissue. This transformation procedure has the potential to expand the range of genetic variation in Robinia.     

Genetic transformation of Populus nigra by Agrobacterium tumefaciens

Two clones of Populus nigra L. were tested in vivo and in vitro for their susceptibility to three different Agrobacterium tumefaciens wild-type strains evaluating number and size of resulting calluses. Strain C58 proved to be the most virulent.Various parameters affecting Agrobacterium-mediated transformation of P. nigra clones were further analyzed using ß-glucuronidase gene transient expression. The clone Jean Pourtet proved to be more susceptible than the clone San Giorgio. A. tumefaciens strain A281 pKIWI105 proved to be the most virulent. The optimal procedure involved dipping of leaf discs into a bacterial suspension (7×10⁸ cells/ml) for 20 min, followed by a 48 h co-cultivation period on semi-solid regeneration medium.Leaf explants were co-cultivated with two disarmed A. tumefaciens strains. Plantlets of San Giorgio were regenerated, tested for ß-glucuronidase activity and rooted on selective medium containing kanamycin. Polymerase chain reaction analysis and Southern blot hybridization confirmed the integration of the neomycin phosphotransferase II gene into the poplar genome.Abbreviations BAP 6-benzyl-aminopurine - CaMV Cauliflower Mosaic Virus - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS and gus ß-glucuronidase - hpt hygromycin phosphotransferase - IBA indole-3-butyric acid - KIN kinetin - LB Luria Bertani - MS Murashige and Skoog - NAA ßnaphthaleneacetic acid - NOS Nopaline synthase - NPTII and nptII neomycin phosphotransferase II - PCR Polymerase chain reaction - PVC poly-vinyl-cloride - SDS sodium dodecyl sulfate - SSC sodium cloride-sodium citrate - Tris tris(hydroxymethyl)amino-methane - WPM Woody Plant Medium     

Genotype-independent transformation of lettuce using Agrobacterium tumefaciens

The genetic manipulation of lettuce (Lactuca sativa) necessitatesa reliable and efficient, genotype-independent method of transformation.Thirteen lettuce cultivars have been assessed for their amenabilityto Agrobacterrum-mediated gene transfer linked to their tissueculture responsiveness, including callus induction and shootregeneration. A reliable protocol has been developed for theroutine production of transgenic plants for all 13 cultivarsinvestigated. Key words: Agrobacterium-mediated transformation, Lactuca sativa genotypes, lettuce     

Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens

Summary Transformation and regeneration procedures for obtaining transgenic Brassica rapa ssp. oleifera plants are described. Regeneration frequencies were increasedby using silver nitrate and by adjusting the duration of exposure to 2,4-D. For transformation, Agrobacterium tumefaciens strain EHA101 containing a binary plasmid with the neomycin phosphotransferase gene (NPT II) and the b-glucuronidase gene (GUS) was cocultivated with hypocotyl explants from the oilseed B. rapa cvs. Tobin and Emma. Transformed plants were obtained within three months of cocultivation. Transformation frequencies for the cultivars Tobin and Emma were 1–9%. Evidence for transformation was shown by NPT II dot blot assay, the GUS fluorometric assay, Southern analysis, and segregation of the kanamycin-resistance trait in the progeny. The transformation and regeneration procedure described here has been used routinely to transform two cultivars of B. rapa and 18 cultivars of B. napus.     

Transformation and regeneration of Lobelia erinus using Agrobacterium tumefaciens

A transformation/regeneration system was developed for the common garden Lobelia (Lobelia erinus). Using an Agrobacterium-based protocol, over 40 transformants have been generated with four different binary vectors. The explant source was hypocotyl-root sections from axenically grown seedlings. Stable transformation was demonstrated by Southern hybridization analysis, β-glucuronidase staining, and transmission of the T-DNA to progeny. This extends the ever-widening range of transformable plant species to the Campanulaceae and will allow molecular studies of development and physiology in this easily cultured and popular garden plant. Received: 16 September 1997 / Revision received: 3 June 1998 / Accepted: 14 July 1998     

Genetic transformation and regeneration of transgenic plants from protocorm-like bodies of vanilla (Vanilla planifolia Andrews) using Agrobacterium tumefaciens

An efficient transformation protocol was developed for vanilla (Vanilla planifolia) using protocorm-like bodies (PLBs) derived from shoot tips as explants. Of the ten media tested, Murashige and Skoog (MS) medium containing 0.45 μM thidiazuron (TDZ) produced maximum PLBs per shoot tip. Genetic fidelity of PLB-derived plantlets was confirmed by random amplified polymorphic DNA (RAPD) using 23 random primers. PLBs were co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pBI121 containing the β-glucuronidase (gusA) and neomycin phosphotransferase II (npt II) genes for 3 days in MS medium supplemented with acetosyringone and transferred to selective regeneration medium containing 4.43 μM benzyladenine (BA), 2.68 μM naphthalene acetic acid (NAA) supplemented with 50 mg l^?1kanamycin and 250 mg l^?1 cefotaxime. After 15 days of culture, the surviving explants were transferred to the same regeneration medium but with a higher concentration of kanamycin (75 mg l^?1). Finally, explants surviving after 30 days were subjected to more stringent selection in the regeneration medium supplemented with 100 mg l^?1 kanamycin. Strong β glucuronidase activity was detected in the transformed plantlets by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and Southern hybridization, while expression of transgene was confirmed by northern hybridization. This protocol allows effective and high frequency transformation of vanilla.     

Genetic transformation of Populus deltoides and P.x euramericana clones using Agrobacterium tumefaciens

The susceptibility of different Populus euramericana (Neva, PE68-022 x P. nigra, 71-060 x P. nigra) and P. deltoides (PE68-022 x P. deltoides) clones to wild-type Agrobacterium tumefaciens strains (A281 and 82.139) was evaluated in an inoculation experiment, and differences in the frequency of tumor formation (0-48) were found. Co-cultivation experiments demonstrated high transformation ability of oncogenic binary A. tumefaciens strains as compared to disarmed strains. Using oncogenic binary strains, transgenic calluses were obtained from all tested clones. The presence of acetosyringone did not influence the transformation frequency of the disarmed strains. Co-inoculation experiments were performed using leaf discs and a bacterial suspension containing both wild-type and disarmed strains. No positive effects on transformation efficiency were noticed in these conditions either. The transformation of tumors and kanamycin resistant calluses was confirmed by DNA analysis.     

Genetic transformation of tobacco NT1 cells with Agrobacterium tumefaciens

This protocol is used to produce stably transformed tobacco (Nicotiana tabacum) NT1 cell lines, using Agrobacterium tumefaciens-mediated DNA delivery of a binary vector containing a gene encoding hepatitis B surface antigen and a gene encoding the kanamycin selection marker. The NT1 cultures, at the appropriate stage of growth, are inoculated with A. tumefaciens containing the binary vector. A 3-day cocultivation period follows, after which the cultures are rinsed and placed on solid selective medium. Transformed colonies ('calli') appear in approximately 4 weeks; they are subcultured until adequate material is obtained for analysis of antigen production. 'Elite' lines are selected based on antigen expression and growth characteristics. The time required for the procedure from preparation of the plant cell materials to callus development is approximately 5 weeks. Growth of selected calli to sufficient quantities for antigen screening may require 4-6 weeks beyond the initial selection. Creation of the plasmid constructs, transformation of the A. tumefaciens line, and ELISA and Bradford assays to assess protein production require additional time.     

Genetic transformation using Agrobacterium rhizogenes

UDP-glucose pyrophosphorylase (EC 2.7.7.9) has been highly purified from the plant fraction of soybean ( Glycine max L. Merr. cv Williams) nodules. The purified enzyme gave a single polypeptide band following sodium docecyl sulphate polyacryla-mide gel electrophoresis, but was resolved into three bands of activity in non-denaturing gels. The enzyme appeared to be a monomer of molecular weight between 30 and 40 kDa. UDP-glucose pyrophosphorylase had optimum activity at pH 8.5 and displayed typical hyperbolic kinetics. The enzyme had a requirement for divalent metal ions, and was highly specific for the substrates pyrophosphate and UDP-glucose in the pyrophosphorolysis direction, and glucose-1-phosphate and UTP in the direction of UDP-glucose synthesis. The K_m values were 0.19 m M and 0.07 m M for pyrophosphate and UDP-glucose, respectively, and 0.23 m M and 0.11 m M for glucose-1-phosphate and UTP. The maximum velocity in the pyrophosphorolysis direction was almost double that for the reverse reaction. UDP-glucose pyrophosphorylase did not appear to be subject to a high degree of fine control, and activity in vivo may be regulated mainly by the availability of the substrates.     

Chloroplast transformation by Agrobacterium tumefaciens

A chimeric gene consisting of the promoter region of the nopaline synthase gene (Pnos) fused to the coding sequence of the chloramphenicol acetyltransferase gene (cat gene) of Tn9 was introduced by co-cultivation in tobacco protoplasts followed by selection with 10 μg/ml chloramphenicol. The chloramphenicol-resistant plants derived from these selected calli were unable to transmit the Cm^R phenotype through pollen. A typically maternal inheritance pattern was observed. Southern blot analysis showed that the chimeric Pnos-cat gene was present in the chloroplasts of these resistant plants. Furthermore, the chloramphenicol acetyltransferase activity was shown to be associated with the chloroplast fraction. These observations are the first proof that the Agrobacterium Ti-plasmid vectors can be used to introduce genes in chloroplasts.     

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.     

Genetic transformation of Medicago species by Agrobacterium tumefaciens and electroporation of protoplasts

Shoot and leaf segments of a non-regenerable Medicago sativa L. genotype were cocultivated with the shooty mutant of Agrobacterium tumefaciens carrying the pGV 2206 plasmid. Transformed callus lines were selected and regenerated on the hormone free B5 medium. Southern blot analysis demonstrated integration of T-DNA in to the genome of the regenerated plants.Transgenic plants resistant to kanamycin were obtained by electroporation of Medicago borealis protoplasts with the pGA 472 plasmid DNA.Abbreviations 2.4 D 2.4 dichlorophenoxyacetic acid - BAP 6-benzyladenine - T-DNA transferred DNA into plants from Ti-plasmid of A. tumefaciens