Transgenic antibiotic resistance may be differentially silenced in germinating pollen grains

Transgenic tobacco (Nicotiana tabacum L.) plants, carrying the neomycin phosphotransferase (NPT II) gene from E. coli, are resistant to kanamycin when grown from seeds on kanamycin containing medium. Tissue and cell cultures derived from those transformants also express resistance and regenerate complete plantlets in the presence of the antibiotic. This unspecific response to the selective condition has led to the belief that the foreign gene is continuously active or uniformly inducible in all cells of the transgenic plant. However, our experiments show that this view is not true for pollen grains during in vitro germination. Pollen grains isolated from kanamycin resistant tobacco plants carry and transmit the foreign gene but do not express resistance when germinating in vitro. This data presents evidence for differential silencing of a foreign gene in a mature gamete. On the other hand, immature pollen grains (microspores) appear to express resistance. The point of the downregulation of the neomycin transferase gene during pollen maturation is discussed.Abbreviations kan kanamycin sulfate - NPT II neomycin phosphotransferase II - sr streptomycin sulfate     

Transformation of Brassica napus canola cultivars with Arabidopsis thaliana acetohydroxyacid synthase genes and analysis of herbicide resistance

Summary A survey of selected crop species and weeds was conducted to evaluate the inhibition of the enzyme acetohydroxyacid synthase (AHAS) and seedling growth in vitro by the sulfonylurea herbicides chlorsulfuron, DPX A7881, DPX L5300, DPX M6316 and the imidazolinone herbicides AC243,997, AC263,499, AC252,214. Particular attention was given to the Brassica species including canola cultivars and cruciferous weeds such as B. kaber (wild mustard) and Thlaspi arvense (stinkweed). Transgenic lines of B. napus cultivars Westar and Profit, which express the Arabidopsis thaliana wild-type AHAS gene or the mutant gene csr1-1 at levels similar to the resident AHAS genes, were generated and compared. The mutant gene was essential for resistance to the sulfonylurea chlorsulfuron but not to DPX A7881, which appeared to be tolerated by certain Brassica species. Cross-resistance to the imidazolinones did not occur. The level of resistance to chlorsulfuron in transgenic canola greatly exceeded the levels that were toxic to the Brassica species or cruciferous weeds. Direct selection of transgenic lines with chlorsulfuron sprayed at field levels under greenhouse conditions was achieved.     

In vitro study of transgenic tobacco expressing Arabidopsis wild type and mutant acetohydroxyacid synthase genes

Summary Genes coding for the enzyme acetohydroxyacid synthase, often referred to as acetolactate synthase (AHAS, ALS; EC 4.1.3.18), from wild type Arabidopsis thaliana and a sulfonylurea-resistant mutant line GH50 (csrl-1; Haughn et al. 1988) were introduced in Nicotiana tabacum. Both genes were expressed at high levels with the 35S promoter. The csrl-1 gene conferred high levels of resistance to chlorsulfuron whereas the wild type gene did not. As selectable markers, chimaeric AHAS genes yielded transgenic plants on chlorsulfuron but at much lower efficiencies than with a chimaeric neomycin phosphotransferase gene on kanamycin (Sanders et al. 1987). Shoot differentiation from leaf discs was delayed on chlorsulfuron by 4–6 weeks. This study indicated a role for mutant AHAS genes in the genetic manipulation of herbicide resistance in transgenic plants but as selectable markers for plant cells undergoing differentiation no advantage over other genes was perceived.     

Agrobacterium-mediated transformation of pepino and regeneration of transgenic plants

Regeneration of pepino (Solanum muricatum Ait.) shoots was achieved both by organogenesis and by embryogenesis. Shoots derived via organogenesis were easily rooted and most regenerated plants appeared phenotypically normal. Transgenic plants were obtained using the binary vector pKIWI110 in the avirulent Agrobacterium tumefaciens strain LBA4404. Optimization of transformation protocols was rapidly achieved by monitoring early expression of the GUS (-D-glucuronidase) reporter gene carried on pKIWI110. Transgenic plants expressed GUS and selectable marker genes for kanamycin resistance and chlorsulfuron resistance. PCR (polymerase chain reaction) and Southern analysis provided molecular evidence for transformation.     

Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.)

Summary Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding.     

Somatic hybrids between Solanum brevidens and Solanum tuberosum: Expression of a late blight resistance gene and potato leaf roll resistance

Hexaploid somatic hybrids resulting from mesophyll protoplast fusions between Solanum brevidens Phil., PI 218228, and Solanum tuberosum L., PI 203900 were tested for late blight resistance using two races of Phytophthora infestans Monte., de Bary. The S. tuberosum parent was a late blight differential possessing the R4 gene which confers resistance to race 0. The S. brevidens parent is resistant to potato leaf roll virus. Inoculations with both compatible (race 1.3.4.5) and incompatible (race 0) races of P. infestans clearly demonstrated the expression of the late blight resistance gene in all of the hybrid progeny tested. Most of the hybrids tested were also resistant to potato leaf roll virus (PLRV), indicating that the S. brevidens genes for PLRV resistance were present and expressed.     

Improved efficiency of the walnut somatic embryo gene transfer system

Summary AnAgrobacterium-mediated gene transfer system which relies on repetitive embryogenesis to regenerate transgenic walnut plants has been made more efficient by using a more virulent strain ofAgrobacterium and vectors containing genes for both kanamycin resistance and beta-glucuronidase (GUS) activity to facilitate early screening and selection. Two plasmids (pCGN7001 and pCGN7314) introduced individually into the disarmedAgrobacterium host strain EHA101 were used as inoculum. Embryos maintained on medium containing 100 mg/l kanamycin after co-cultivation produced more transformed secondary embryos than embryos maintained on kanamycin-free medium. Of the 186 GUS-positive secondary embryo lines identified, 70% were regenerated from 3 out of 16 primary embryos inoculated with EHA101/pCGN7314 and grown on kanamycin- containing medium, 28% from 4 out of 17 primary embryos inoculated with EHA101/ pCGN7001 and grown on kanamycin medium, and 2% from one out of 13 primary embryos inoculated with EHA101/pCGN7001 but not exposed to kanamycin. Because kanamycin inhibits but does not completely block new embryo formation in controls, identification of transformants formerly required repetitive selection on kanamycin for several months. Introduction of the GUS marker gene allowed positive identification of transformant secondary embryos as early as 5–6 weeks after inoculation. DNA analysis of a representative subset of lines (n=13) derived from secondary embryos confirmed transformation and provided evidence for multiple insertion events in single inoculated primary embryos.     

The characterization of herbicide tolerant plants in Brassica napus L. after in vitro selection of microspores and protoplasts

Brassica napus L.(cv Topas) plants tolerant to chlorsulfuron (CS) were isolated after selection experiments utilizing microspores and haploid protoplasts. The first microspore-derived plant (M-37,) was CS tolerant, haploid and sterile. Normal plant morphology and fertility was restored after colchicine doubling. A CS tolerant plant was also selected from protoplasts (P-26) isolated from microspore-derived embryo tissue and grown on medium containing CS. P-26 was aneuploid, CS tolerant and had very low fertility. The two selected lines produced selfed progeny which were tolerant to from 10–100 times the CS levels of the corresponding Topas plants. Microspores and protoplasts derived from the selfed plants were also CS tolerant. The segregation pattern for CS tolerance from reciprocally crossed progeny of M-37 and Topas was consistent with a semi-dominant nuclear mode of inheritance. Biochemical analysis of the two mutants indicated that the microspore-derived mutant and F1 crosses contained an altered acetohydroxyacid synthase (AHAS) enzyme, while the AHAS activity of the protoplast mutant was similar to Topas. Selfed seed from the M-37 plants have provided tolerance to CS in both greenhouse and field tests. S1 plants from a second microspore selected mutant (M-42) have tolerated 30 g/ha of CS in greenhouse tests. The two single-celled selection systems are discussed and the microspore selection system highlighted as a new method for in vitro selection.     

Characterization of transgenic sulfonylurea-resistant flax (Linum usitatissimum)

Summary Fourteen transgenic flax (Linum usitatissimum) lines, carrying a mutant Arabidopsis acetolactate synthase (ALS) gene selected for resistance to chlorsulfuron, were characterized for resistance to two sulfonylurea herbicides. Progeny of 10 of the 14 lines segregated in a ratio of 3 resistant to 1 susceptible, indicating a single insertion. Progeny of 1 line segregated in a 151 ratio, indicating two insertions of the ALS gene at independent loci. Progeny from 3 lines did not segregate in a Mendelian fashion and were likely the products of chimeric shoots. Resistance to chlorsulfuron was stably inherited in all lines. At the enzyme level, the transgenic lines were 2.5 to more than 60 times more resistant to chlorsulfuron than the parental lines. The transgenic lines were 25–260 times more resistant to chlorsulfuron than the parental lines in root growth experiments and demonstrated resistance when grown in soil treated with 20 g ha^-1 chlorsulfuron. The lines demonstrated less resistance to metsulfuron methyl; in root growth experiments, the transgenic lines were only 1.6–4.8 times more resistant to metsulfuron methyl than the parental lines. Resistance was demonstrated in the field at half (2.25 g ha^-1) and full (4.5 g ha^-1) rates of metsulfuron methyl.     

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector

Summary Kanamycin resistant plants of Solarium melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NPTII) gene as the selectable marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.Abbreviations CAT chloramphenicol acetyltransferase - MS Murashige and Skoog - NPTII neomycin phosphotransferase - NOS nopaline synthase - ZEA zeatin     

In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase

Selection genes are routinely used in plant genetic transformation protocols to ensure the survival of transformed cells by limiting the regeneration of non-transgenic cells. In order to find alternatives to the use of antibiotics as selection agents, we followed a targeted approach utilizing a plant gene, encoding a mutant form of the enzyme acetolactate synthase, to convey resistance to herbicides. The sensitivity of sugarcane callus (Saccharum spp. hybrids, cv. NCo310) to a number of herbicides from the sulfonylurea and imidazolinone classes was tested. Callus growth was most affected by sulfonylurea herbicides, particularly 3.6 μg/l chlorsulfuron. Herbicide-resistant transgenic sugarcane plants containing mutant forms of a tobacco acetolactate synthase (als) gene were obtained following biolistic transformation. Post-bombardment, putative transgenic callus was selectively proliferated on MS medium containing 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/l sucrose, 0.5 g/l casein, and 3.6 μg/l chlorsulfuron. Plant regeneration and rooting was done on MS medium lacking 2,4-D under similar selection conditions. Thirty vigorously growing putative transgenic plants were successfully ex vitro-acclimatized and established under glasshouse conditions. Glasshouse spraying of putative transgenic plants with 100 mg/l chlorsulfuron dramatically decreased the amount of non-transgenic plants that had escaped the in vitro selection regime. PCR analysis showed that six surviving plants were als-positive and that five of these expressed the mutant als gene. This report is the first to describe a selection system for sugarcane transformation that uses a selectable marker gene of plant origin targeted by a sulfonylurea herbicide.     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

Glyphosate tolerant flax plants from Agrobacterium mediated gene transfer

Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector containing a chimeric NPT-II gene and a glyphosate resistance plant-derived 5-enolpyruvylshikimate-3-phosphate synthase gene was used to transform flax hypocotyl tissues. Transformed shoots could be regenerated from the inoculated tissue and were proven to be transgenic by the combination of leaf callus assays, nopaline assays and progeny tests. Co-segregation was observed in the progeny for kanamycin and glyphosate resistance.     

Celery transformation by Agrobacterium tumefaciens: cytological and genetic analysis of transgenic plants

Transgenic celery plants were obtained following co-cultivation of petiole explants with Agrobacterlum tumefaciens containing pMON200, a cointegrate vector carrying genes for kanamycin resistance and nopaline synthase. Transformants were selected by ability of callus to grow in the presence of 50mg/l kanamycin. Transformation was confirmed either by the presence of nopaline or by Southern blots. Cytological analysis of 14 transformed plants revealed chromosomal aberrations, both in structure and number. Only 20% of the regenerated plants had the normal karyotype. Kanamycin resistance behaved as a monogenic, dominant trait, segregating in a 3:1 ratio in three families derived from plants with normal karyotypes.Abbreviations KB Kilobases - 2-4D 2,4-diphenoxyacetic acid     

Regeneration of transgenic Picea glauca, P. Mariana, and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens

Summary Transgenic plants of three Picea species were produced after coculture of embryogenic tissue with the disarmed strain of Agrobacterium tumefaciens C58/pMP90/pBIV10 and selection on medium containing kanamycin. In addition to the nptII selectable gene (conferring resistance to kanamycin), the vector carried the uidA (β-glucuronidase) marker gene. Transformation frequencies were dependent on the species, genotype, and post-cocultivation procedure. Of the three species tested, P. mariana was transformed at the highest frequency, followed by P. glauca and P. abies. The transgenic state of the embryogenic tissue was initially, confirmed by histochemical β-glucuronidase (GUS) assay followed by Southern hybridization. One to over five copies of T-DNA were detected in various transgenic lines analyzed. Transgenic plants were regenerated for all species using modified protocols for maturation and germination of somatic embryos.     

Novel insect resistance in Brassica napus developed by transformation of chitinase and scorpion toxin genes

Transgenic plants with introduced pest-resistant gene offer an efficient alternative insect control. The novel insect-resistant gene combination, chitinase(chi) and BmkIT(Bmk), containing an insect-specific chitinase gene and a scorpion insect toxin gene was introduced into Brassica napus cultivar via Agrobacterium-mediated transformation. Fifty-seven regenerated plantlets with kanamycin-resistance were obtained. Transgenic plants were verified by Southern blot analysis. Enzyme-linked immunosorbent assay (ELISA) and bioassay of artificial inoculation with diamondback moth (Plutella maculipenis) (DBM) larvae indicated that some of the transgenic plants were high-level expression for both chitinase and scorpion toxin proteins and performed high resistance against the tested pest infestation. The genetic analysis of T₁ progeny confirmed that the inheritance of introduced genes followed the Mendelian rules.     

Agrobacterium tumefaciens-mediated transformation of embryogenic tissues of tea (Camellia sinensis (L.) O. Kuntze)

Embryogenic tissues of tea were cocultivated withAgrobacterium tumefaciens LBA4404. The plasmid pBi121, which contains the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the β-glucuronidase (uidA) reporter gene, was used as binary vector. The highest transformation frequency (12 transformants/g fresh weight [FW] of treated embryogenic tissue) was obtained with 5-day-old tissues grown in liquid medium and cocultivated withAgrobacterium for 2 d in the same medium but containing 50 μM acetosyringone. There was improvement in the recovery of kanamycin-resistant tissues when tissues were first grown for 10 d on a medium containing 350 mg/L Timentin to prevent bacterial overgrowth, before application of the selection pressure. Resistant tissues obtained after 6 wk on kanamycin-selection medium showed stableuidA expression. Polymerase chain reaction demonstrated the presence of the transgenes, while Southern hybridization confirmed their integration into the genome. Transgenic plants were regenerated from transformed tissues within 4 mo after coculture.     

Insect-resistant transgenic Pinus radiata

Transgenic radiata pine (Pinus radiata D. Don) plants containing a Bacillus thuringiensis (Bt) toxin gene, crylAc, were produced by means of biolistic transformation of embryogenic tissue. Using the selectable marker gene nptII and corresponding geneticin selection, 20 independent transgenic lines from five genotypes were established. Over 200 plants regenerated from ten transgenic lines were successfully transferred to soil. The integration and expression of the introduced genes in transgenic tissue and/or plants were confirmed by PCR, Southern hybridisation and neomycin phosphotransferase II (NPTII) and Bt ELISA assays. Bioassays with larvae of the painted apple moth, Teia anartoides, demonstrated that transgenic plants displayed variable levels of resistance to insect damage, with one transgenic line being highly resistant to feeding damage.     

Transgenic cereal (tritordeum) plants obtained at high efficiency by microprojectile bombardment of inflorescence tissue

Transgenic cereal plants expressing the β-glucuronidase (uidA) and neomycin phosphotransferase (neo) genes were obtained via microprojectile bombardment of immature inflorescence tissue of tritordeum (the fertile Hordeum x Triticum amphiploid, H^chH^chAABB). A total of 17 independent transgenic plants were recovered from 32 bombardments (on average four inflorescences per shot). Of the bombardment and culture parameters tested, explant preculture had the most influence on stable transformation frequency. The uidA and neo genes were supplied on two separate plasmids (co-transformation) and 88% of the transgenic plants recovered expressed both genes. Southern analysis confirmed the results of histochemical GUS and NPT II assays. Transgenic plants were grown to maturity and flowered and set seed. Pollen from four T₀ GUS⁺ plants analysed showed GUS activity and T₁ seedlings derived from one of the transgenic plants showed a segregation of 2.75:1 (GUS⁺:GUS⁻) for uidA gene activity.     

Transgenic tobacco plants containing <Emphasis Type="Italic">Bt</Emphasis> and <Emphasis Type="Italic">GNA</Emphasis> genes

A new plant expression vector (pBSbtCry1Ac-GNA) containing two insect resistant genes, a synthetic chimeric gene SbtCry1Ac encoding the insecticidal protein CrylAc and a gene GNA encoding snowdrop lectin (Galanthus nivalis agglutinin) was constructed. Transgenic tobacco plants containing these two genes were obtained through Agrobacterium-mediated transformation of tobacco leaf discs. Results from PCR detection and genomic DNA Southern blot analysis indicated that both SbtCrylAc gene and GNA gene were integrated into the genome of these plants. Results of Western blot analysis indicated that these two proteins were expressed in the analyzed plants. Bioassays of Myzus persicae and Helicoverpa assulta on detached leaves of transformed tobacco plants were carried out. The average aphid inhibition rate of these plants tested at 12 d post-infestation was 71.9 %. The average H. assulta mortality of these plants tested at 6 d post-infestation was up to 89.8 %. The kanamycin resistance of the T₁ progeny of these transgenic plants was analyzed and a typical 3:1 segregation was observed.