Transgenic Italian ryegrass (Lolium multiflorum) plants from microprojectile bombardment of embryogenic suspension cells

Transgenic forage-type Italian ryegrass (Lolium multiflorum Lam.) plants have been obtained by microprojectile bombardment of embryogenic suspension cells using a chimeric hygromycin phosphotransferase (hph) gene construct driven by riceActl 5 regulatory sequences. Parameters for the bombardment of embryogenic suspension cultures with the particle inflow gun were partially optimized using transient expression assays of a chimeric-glucuronidase (gusA) gene driven by the maizeUbi1 promoter. Stably transformed clones were recovered with a selection scheme using hygromycin in liquid medium followed by a plate selection. Plants were regenerated from 33% of the hygromycin-resistant calli. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis. Expression of the transgene in transformed adult Italian ryegrass plants was confirmed by northern analysis and a hygromycin phosphotransferase enzyme assay.Abbreviations 2,4-D 2,4 Dichlorophenoxyacetic acid - GUS Glucuronidase - Hm Hygromycin - HPH Hygromycin phosphotransferase - MS medium Murashige and Skoog medium - PCR Polymerase chain reaction - X-Gluc 5-Bromo-4-chloro--indolyl--D-glucuronic acid     

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants

Summary An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BA 6-ben-zyladenine - hpt hygromycin phosphotransferase gene - IAA indole acetic acid, kin, kinetin - NAA -naphtalene acetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Transgenic turf-type tall fescue (Festuca amndinacea Schreb.) plants regenerated from protoplasts

To improve turfgrasses using genetic engineering, we have developed a transformation system in turf-type tall fescue, one of the most important turfgrass species. Embryogenic cell cultures were established after callus induction from embryos of mature seed. The agarose-bead method with nurse cells was used to culture protoplasts and plants were regenerated from protoplasts of tall fescue cultured cells. To develop transgenic tall fescue plants, the hygromycin resistance gene and the -glucuronidase gene were introduced into the tall fescue protoplasts by electroporation. A high concentration (200 mg/l) of hygromycin was required to select transformed cells because of the high level of endogenous resistance to the antibiotic in tall fescue. Most of the transformed cells exhibited GUS activity and several plants were regenerated from these cells. The presence of introduced genes was confirmed by Southern blot hybridization of PCR amplified DNA from transgenic plants.Abbreviations Adh alcohol dehydrogenase - BAP benzylaminopurine - bp base pair(s) - GUS -glucuronidase - Kb kilobase(s) - MS Murashige and Skoog's medium - PCR polymerase chain reaction     

Transformation of cotton (Gossypium hirsutum L.) via particle bombardment

Embryogenic suspension cultures of cotton (Gossypium hirsutum L.) were subjected to particle bombardment, where high density particles carrying plasmid DNA were accelerated towards the embryogenic plant cells. The plasmid DNA coating the particles encoded hygromycin resistance. One to two weeks following bombardment, embryogenic cotton cells were placed in proliferation medium containing 100 g/ml hygromycin. Clumps of tissue which grew in the presence of hygromycin were subcultured at low density into fresh hygromycin-containing proliferation medium. Following sequential transfer of embryogenic tissue to development and then germination media, plants were recovered from transgenic embryogenic tissue. Southern hybridization confirmed the presence of the hygromycin resistance gene in embryogenic suspension culture tissue and regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Aph IV aminoglycoside phosphotransferase type IV Salaries and research support were provided by State and Federal funds appropriated to OSU/OARDC and USDA-ARS. Mention of trademark or proprietary products does not constitute a guarantee or warranty of the product by OSU/OARDC or USDA, and also does not imply approval to the exclusion of other products that may also be suitable. Journal Article No. 354-89     

Improved method for the transformation of Arabidopsis thaliana with chimeric dihydrofolate reductase constructs which confer methotrexate resistance

Summary A modified root explant transformation method has been developed that is effective in producing transgenic Arabidopsis thaliana plants which are methotrexate resistant due to the integration of T-DNA vectors containing a chimeric dihydrofolate reductase gene. Molecular analysis shows that transformed methotrexate resistant plants contain the expected T-DNA construct with the chimeric gene. This transformation method also works well with other plant selectable markers, including hygromycin phosphotransferase and neomycin phosphotransferase II.Abbreviations DHFR (dihydrofolate reductase) - HPT (hygromycin phosphotransferase) - NPTII (neomycin phosphotransferase) - CaMV (Cauliflower Mosaic Virus) - MES (2-[N-morpholino]ethanesulfonic acid) - BAP (N6-benzylaminopurine) - NAA (naphthaleneacetic acid) - 2,4-D (2,4 dichlorophenoxyacetic acid) - 2iP (6-(dimethylallylamino)-purine) - 2iPAde (6-(dimethylallylamino)-ade-nine) - IAA (indole-3-acetic acid) - IBA (indole-3-butyric acid)     

Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake

Summary Efficient regeneration (80%) and high frequency genetic transformation (10–33%) were achieved by culturing protoplasts isolated from hypocotyl tissues of six day old Brassica oleracea seedlings and by subjecting these protoplasts to PEG mediated direct plasmid uptake. Three different plasmid vectors carrying marker genes for resistance to methotrexate (dhfr), hygromycin (hpt) and phosphinotricin (bar) were constructed and used for transformation. Large number of normal, fertile transformants were obtained with vectors carrying hpt and bar genes. No transformants could be regenerated for resistance to methotrexate as it severely suppressed shoot differentiation.Abbreviations bar/PAT bialaphos resistance gene/phosphinotricin acetyltransferase - 2,4-D 2,4-di-chlorophenoxyacetic acid - dhfr/DHPR dihydrofolate reductase gene/enzyme - gus/GUS -glucuronidase gene/enzyme - hpt/HPT hygromycin phosphotransferase gene/enzyme - Kn kinetin - PEG polyethylene glycol - RH relative humidity     

Transgenic Plants of Colonial Bentgrass from Embryogenic Callus via Agrobacterium-mediated Transformation

Colonial bentgrass (Agrostis tenuis Sibth. Fl. Oxen.) is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable and repeatable approach in transforming the grass using Agrobacterium (strain LBA4404), in which -glucuronidase (gus) gene was used as a reporter and hygromycin phosphotransferase (hpt) gene as a selectable marker. This vector was effective in transforming 7-week-old calluses derived from mature seeds cultured on MS medium supplemented with 2,4-D. A two-step solid medium selection with increasing hygromycin concentration (from 50 to 70 mg l^–1) was used to obtain resistant calluses. Hundreds of transgenic plants have been produced from several independent transformed calluses. The presence of functional -glucuronidase (GUS) was detected in hygromycin-resistant calluses, young leaves and roots of transgenic plants. The transgenic plants collected from greenhouse showed strong resistance to 50 mg l^–1 hygromycin solution. Four putative transgenic plants and one control plant were randomly chosen and analyzed by Southern blot analysis. Bands corresponding to the hpt gene were clearly shown in transgenic plants.     

High-frequency transformation of<Emphasis Type="Italic"> Lobelia erinus</Emphasis> L. by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated gene transfer

A highly efficient transformation procedure was developed for Lobelia erinus. Leaf or cotyledon discs were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector plasmid pIG121Hm, which contains a -glucuronidase gene with an intron as a reporter gene and both the neomycin phosphotransferase II and hygromycin phosphotransferase genes as selectable markers. The hygromycin-resistant calli produced on the selection medium were transferred to MS medium supplemented with 0.5 mg/l benzyladenine and 0.2 mg/l indole-3-acetic acid for regeneration of adventitious shoots. Transgenic plants were obtained as a result of the high regeneration rate of the transformed calli, which was as high as 83%. In contrast, no transgenic plant was obtained by the procedure of direct shoot formation following inoculation with A. tumefaciens. Transgenic plants flowered 3–4 months after transformation. Integration of the transgenes was detected using PCR and Southern blot analysis, which revealed that one to several copies were integrated into the genomes of the host plants. The transformation frequency at the stage of whole plants was very high—45% per inoculated disc.Abbreviations BA: 6-Benzyladenine - 2,4-D: 2,4-Dichlorophenoxyacetic acid - GUS: -Glucuronidase - IAA: Indole-3-acetic acidCommunicated by G.C. Phillips     

Enrichment for Nicotiana heterokaryons after protoplast fusion and subsequent growth in agarose microdrops

Protoplasts isolated from Nicotiana tabacum L. leaves and Nicotiana suaveolens Lehm. cell suspensions have been fused with polyethylene glycol (PEG). Enrichment for heterokaryons was based on a Percoll flotation protocol which allowed a preparation with 50% heterokaryons to be obtained. The heterokaryons developed into calli whose hybrid nature was shown by polyacrylamide gel electrophoresis of esterase isoenzymes. Sensitivity of the mesophyll protoplasts to PEG and different buoyant densities of the heterokaryon and cell-suspension protoplasts contribute to the enrichment. The 50%-fusion figure following purification is an improvement on standard PEG procedures.Heterokaryons obtained were embedded in 20l drops of agarose and placed in a liquid nurse culture that allows optimum growth of the heterokaryons and maintains a physical boundary between the heterokaryons and the nurse culture. Once colonies develop, the agarose microdrop is removed from the nurse culture and placed on shoot-induction medium. Agarose microdrops containing the heterokaryons can be readily removed at any stage and processed for electron microscopy to follow the early stages of colony development.The procedures we have utilised provide a robust physical selection method that allows the total variation from a heterokaryon population to be expressed.Abbreviations BAP N⁶-benzylaminopurine - BM basal medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - PEG polyethylene glycol - PKM modified Kao (1977) medium for protoplast culture     

Expression of the hygromycin B phosphotransferase gene confers tolerance to the herbicide glyphosate

Escherichia coli cells and tobacco (cv. Xanthi) plants transformed with the hygromycin B phosphotransferase gene were able to grow in culture medium containing glyphosate at 2.0 mM. The growth of tobacco calli in media containing increasing glyphosate concentrations was measured. The ID₅₀ for glyphosate was 1.70±0.03 mM for hygromycin-B resistant plants, and 0.45±0.02 mM for control plants. Regenerated plants and progeny selected for resistance to hygromycin B were tested for glyphosate tolerance by spraying them with Faena herbicide (formulated glyphosate with surfactant) at a dose equal to 0.24 kg/ha. This was two times the dose required to kill 100 percent of the control plants. Phosphotransferase activity was measured in the extracts of the transformed leaves by the incorporation of ³²P from [^–32P]ATP and it was observed that hygromycin B phosphotransferase was able to recognize the molecule of glyphosate as substrate.Abbreviations (Hyg) Hygromycin - (Km) Kanamycin - (Glp) Glyphosate - (Sarc) Sarcosine - (AMPA) Aminomethylphosphonic acid     

Polyethylene glycol and abscisic acid improve maturation and regeneration of<Emphasis Type="Italic"> Panax ginseng</Emphasis> somatic embryos

Embryogenic culture was initiated from mature zygotic embryos of Panax ginseng. Multiple somatic embryos formed and proliferated on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2.26 M) and kinetin (0.046 M). Mature as well as immature somatic embryos grew into plantlets lacking roots on the same media. Histomorphological analysis of somatic embryos treated with abscisic acid (ABA) and polyethylene glycol (PEG 4000) showed a slight improvement in the root meristem organization of torpedo-stage embryos (embryos were more compact and their cells exhibited a lower degree of vacuolation). Shoot regeneration of non-treated somatic embryos was 31% while that for somatic embryos treated with PEG 4000 and ABA was 70%. Moreover, 75% of plants regenerated from PEG- and ABA-treated embryos formed roots while plants from non-treated embryos did not form roots.Abbreviations ABA (±)-Abscisic acid - BAP N ⁶-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA ₃ Gibberellic acid - Kin Kinetin - MS Murashige and Skoog medium - PEG 4000 Polyethylene glycol 4000 - PGR Plant growth regulators Communicated by H. van Onckelen     

Transgenic Russian wildrye (<Emphasis Type="Italic">Psathyrostachys juncea</Emphasis>) plants obtained by biolistic transformation of embryogenic suspension cells

Russian wildrye (Psathyrostachys juncea (Fisch.) Nevski) is a cool-season forage species well adapted to semi-arid climates. We are interested in developing biotechnological methods to improve this monocot forage species. Single genotype-derived embryogenic suspension cultures were established from the Russian wildrye cultivar Bozoisky-Select, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric -glucuronidase (gusA) gene was co-transformed with hph. Resistant calli were obtained from 29% of the bombarded dishes after selection with 200 mg/l hygromycin. Plants were regenerated from 45% of the hygromycin resistant calli. Thirty-six transgenic Russian wildrye plants were recovered after microprojectile bombardment of suspension cells and subsequent hygromycin selection. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. When a second gene (gusA) was co-transformed with hph, a reasonably high co-transformation frequency of 78% was observed. Transgenic expression of gusA was confirmed by GUS staining of shoot and leaf tissues. Fertile transgenic plants were obtained after two winters of vernalization under field conditions. This is the first report on the generation of transgenic plants in Russian wildrye.     

Regeneration of fertile transgenic indica (group 1) rice plants following microprojectile transformation of embryogenic suspension culture cells

Regenerable embryogenic suspensions of elite Indica (group 1) rice varieties IR24, IR64, IR72 and an advanced Indica rice breeding line IR57311-95-2-3 were established within 6–8 weeks from 3–4 week old calli derived from mature seeds. Transgenic rice plants were obtained by introducing a plasmid carrying genes encoding hygromycin phosphotransferase (hph, conferring resistance to hygromycin B) and ß-glucuronidase (uidA), both driven by the CaMV 35S promoter, via particle bombardment of embryogenic suspensions. The effect of osmotic conditioning on transformation was evaluated. Regenerated plants were resistant to hygromycin B and expressed the uidA (GUS) gene. The growth of mother plants (R₀) was normal and seeds were produced. Southern blot analysis of R₀ and R₁ plants showed that hygromycin resistant plants contained intact hph genes that were inherited in a Mendelian fashion. A protocol for a simple, efficient, repeatable, genotype- and environment-independent Indica rice transformation system is described.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - kb kilobase - GUS ß-glucuronidase - hph hygromycin B phosphotransferase     

Factors affecting PEG-mediated stable transformation of maize protoplasts

Factors influencing the frequency of stable transformation and co-transformation of maize protoplasts utilizing a polyethylene glycol (PEG) mediated DNA uptake procedure have been investigated. Protoplast plating conditions, pre-treatment buffer composition, PEG concentration, and DNA concentration were all found to be important. Carrier DNA was not beneficial when transforming with circular plasmid DNA. The effect of linearizing plasmid DNA was inconsistent across experiments, and may be dependent on the presence of carrier DNA. Functional co-transformation of an unlinked marker gene (hygromycin phosphotransferase) was increased by increasing the ratio of nonselected:selected DNA, and varied from 39% at a 11 ratio to 65% at a 1001 ratio. Under optimum conditions, up to 300 transformed calli were recovered per million input protoplasts. The protocol is simple, inexpensive, and effective, and is useful for studies in maize requiring large numbers of stably transformed or co-transformed cell lines.Abbreviations PEG polyethylene glycol - BMS Black Mexican Sweetcorn - MS salts of Murashige and Skoog (1962) culture medium - MaCa 0.2M mannitol, 80mM calcium chloride - MaMg 0.4M mannitol, 15mM magnesium chloride, 0.1% MES, pH 5.6 - CM conditioned medium - SE standard error of the mean     

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium     

Water loss and polyethylene glycol-mediated acclimatization of in vitro-grown seedlings of 5 cultivars of date palm (Phoenix dactylifera L.) plantlets

Plantlets derived from shoot-tips of seedlings from five cultivars of date palm, Phoenix dactylifera L., were subjected to polyethylene glycol in liquid medium. Comparisons of water loss of detached leaves among in vitro-grown, polyethylene glycol-treated and greenhouse-grown plants showed significant differences with treatment for all cultivars studied. For each treatment, significant differences were also found among cultivars. The common result was that the percent of moisture loss of non-treated in vitro-grown plantlets was almost twice that of greenhouse-grown plants. Polyethylene glycol-treated plantlets showed a water loss of approximately 27%, similar to that of greenhouse plants as compared to an average of 40% in control plants. This demonstrates the possibility of using polyethylene glycol as an osmoticum for in vitro acclimatization of plantlets prior to transfer to soil.Abbreviations EW epicuticular wax - MS Murashige and Skoog (1962) medium - NAA naphthalene acetic acid - PEG polyethylene glycol - PPFD photosynthetic photon flux density     

A simple method for isolation,liquid culture,transformation and regeneration of Arabidopsis thaliana protoplasts

Summary An efficient technique was developed for the isolation, culture, transformation and regeneration of protoplasts derived from auxin conditioned Arabidopsis root cultures. On an average 30% of root protoplasts underwent cell division in liquid culture and formed somatic embryolike structures which regenerated to plants without embedding in Ca²⁺-alginate. The protoplast protocol was applicable to different landraces of Arabidopsis thaliana (L.) Heynh., such as RLD, Columbia or C24. PEG-mediated DNA uptake into protoplasts using different uidA reporter gene constructs yielded transient gene expression in over 25% of treated cells indicating that root-derived protoplasts are suitable recipients for transformation.Abbreviations BA 6-benzylaminopurine - 2,4D 2,4dichlorophenoxyacetic acid - IAA indole-3-acetic acid - ISA indole-3buryric acid - IPAR 6-(,-dimethylallylamino)purine riboside - NAA naphthaleneacetic acid - uidA ß-glucuronidase gene - GUS ß-glucuronidase enzyme - CaMV Cauliflower Mosaic Virus - nos nopaline synthase - MES 2[N-morpholino]ethane-sulfonicacid - PEG polyethylene glycol - X-gluc 5bromo-4-chloro-3-indolyl glucuronide - MUG 4-methyl umbelliferyl glucuronide - MU 4-methylumbelliferone     

Somatic hybridization between Solanum ochranthum and Lycopersicon esculentum

Incorporation of genes from wild species has been a major contributor to tomato improvement in recent years. Solanum ochranthum, a woody vine-like tomato relative, is a potential source of resistance against tomato diseases and insect pests but is genetically isolated from tomato. Somatic hybridization methods were developed to facilitate the use of S. ochranthum for tomato germplasm improvement. Leaf mesophyll protoplasts of S. ochranthum and selected Lycopersicon esculentum genotypes were chemically fused with polyethylene glycol. The protoplasts were initially cultured in Shepard's CL, a Murashige and Skoog-based medium, containing 1 mg l^-1 NAA, 0.5 mg l^-1 N⁶-benzyladenine and 0.5 mg l^-1 2,4-dichlorophenony-acetic acid. Tetraploid and hexaploid hybrid regenerants and regenerants of an L. esculentum parent were recovered; S. ochranthum did not regenerate. Hybridity was established by morphological characters, peroxidase isozyme and RAPD markers.Abbreviations MS Murashige and Skoog (1962) medium - CL Shepard (1980) cell layer medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP N⁶-benzyladenine - MES 2-N-morpholinoethanesulfonic acid - PEG polyethylene glycol - RAPD randomly amplified polymorphic DNA - PPM potato propagation medium - TPM tomato propagation medium - OM modified Murashige and Skoog (1962) medium - OM + AC modified Murashige & Skoog (1962) medium + activated charcoal     

Transient gene expression in electroporated banana (Musa spp., cv. ‘Bluggoe’, ABB group) protoplasts isolated from regenerable embryogenetic cell suspensions

Summary Electroporation conditions were established for transient expression of introduced DNA in banana (Musa spp., cv. Bluggoe) protoplasts isolated from regenerable embryogenic cell suspensions. The following parameters were found to be highly influential: electroporation buffer, polyethylene glycol treatment and its duration before electroporation, use of a heat shock, and chimaeric gene constructs. The maximum frequency of DNA introduction as detected by an in situ assay for transient expression of the uidA gene, amounted to 1.8% of total protoplasts. Since plants have recently been regenerated from banana protoplasts at a high frequency, the present results may contribute to the production of transgenic banana.Abbreviations AMV alfalfa mosaic virus - CaMV cauliflower mosaic virus - 2,4-D 2,4-dichlorophenoxyacetic acid - EGTA ethylene glycol-O-O'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid - GUS glucuronidase - HEPES 4-(2-nydroxyethyl)piperazine-1-etnanesulfonic acid - MES 2-morpholinoethanesulfonic acid - MS Murashige-Skoog - NOS nopaline synthase - NFTII neomycin phosphotransferase - PEG polyethylene glycol - TGE transient GUS expression - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronic acid