Transformation of barley scutellum protoplasts: regeneration of fertile transgenic plants

 A system for barley transformation via polyethyleneglycol-mediated DNA uptake into protoplasts isolated directly from scutella and the regeneration of transgenic plants is reported. Scutellum protoplasts (cv. Clipper, an Australian malting cultivar) were co-transformed with plasmids Act 1-DGUS, containing the marker uidA gene, and pCaIneo, which contains the selectable marker neomycin phosphotransferase gene. Protoplast-derived calluses were selected on medium containing the antibiotic G418 (25 and 15 mg.l^–1) and macroscopic antibiotic resistant colonies were recovered. Fertile plants were regenerated from a callus line and molecular analysis confirmed transgene integration. Received: 11 October 1999 / Revision received: 11 February 2000 / Accepted: 11 February 2000     

Transformation and regeneration of transgenic aspen plants via shoot formation from stem explants

An Agrobacterium -mediated transformation procedure for aspen ( Populus tremula L.), involving the direct regeneration of shoot-buds from stem explants, is described. Disarmed Agrobacterium tumefaciens strain EHA101 harboring the binary plasmid pKIW1105 (which carries the uidA and nptII genes, coding for β-glucuronidase [GUS] and neomycin phosphotransferase II, respectively) was used for the transformation of stem explants. An incubation period of 48 to 72 h was found to be most effective in terms of transient GUS expression on the cut surface of the stem explants. Adventitious shoots regenerated after 2–3 weeks of culture in a woody plant medium (WPM) supplemented with TDZ (1-phenyl-3-[1,2,3-thiadiazol-5-yl]-urea, Thidiazuron) and carbenicillin. Three different kanamycin-based selection schemes were evaluated for optimization of transformation efficiency: (1) Kanamycin was added only to the rooting medium (5 to 6 weeks post-inoculation), or (2) to the regeneration medium 10–14 days after inoculation, or (3) after 2 days of co-cultivation. The third selection scheme was found to be optimal for adventitious shoots with regard to both the time required and the transformation efficiency, the latter being much higher than with the other schemes. Leaf samples from kanamycin-resistant shoots and plantlets were tested for GUS expression, and subjected to polymerase chain reaction (PCR) analysis of uidA and nptII genes. A Southern blot of the corresponding PCR-amplified fragments confirmed their authenticity and Southern blots of total plant DNA confirmed integration of the nptII gene into the plant genome.     

Transformation of maize embryogenic calli mediated by ultrasonication and regeneration of fertile transgenic plants

An insecticidal protein gene isolated fromBacillus thuringiensis was transferred into maize by using ultrasonication. The fertile transgenic plants and their progeny were obtained. The Southern hybridization results indicated that the foreign gene had integrated into the maize genome. It has been found that the acoustic intensity and the duration of treatment are the important parameters influencing transformation efficiency by ultrasonication. The maximum relative transformation frequency of 34.1 % was achieved after 30 min of sonication at 0. 5 W/cm² acoustic intensity. With appropriate parameters the ultrasonication can make a number of micropores formed on the cell surface and minimize the treatment damage to the foreign DNA molecules, thus facilitating the DNA molecules to enter the cells. Project supported by “863” State High Technology Development Program.     

Transformation of maize (Zea mays L.) protoplasts and regeneration of haploid transgenic plants

Transgenic haploid maize (Zea mays L.) plants were obtained from protoplasts isolated from microspore-derived cell suspension cultures. Protoplasts were electroporated in the presence of plasmid DNA containing the gus A and npt II genes encoding ß-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II), respectively. Transformed calli were selected and continuously maintained on kanamycin containing medium. Stable transformation was confirmed by enzyme assays and DNA. analysis. Stably transformed tissue was transferred to regeneration medium and several plants were obtained. Most plants showed NPT II activity, and some also showed GUS activity. Chromosome examinations performed on representative plants showed that they were haploid. As expected, these plants were infertile.     

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.     

Transformation of grape (Vitis vinifera L.) zygotic-derived somatic embryos and regeneration of transgenic plants

Summary Transgenic grape plants were regenerated from somatic embryos derived from immature zygotic embryos of seedless grape (Vitis vinifera L.) selections. Somatic embryos were bombarded twice with 1 m gold particles using the Biolistic PDS-1000/He device (Bio-Rad Laboratories) and then exposed to Agrobacterium tumefaciens strain C58/Z707 containing the binary plasmid pGA482GG or pCGN7314. Following cocultivation, secondary embryos were allowed to proliferate on Emershad/Ramming proliferation (ERP) medium for 6 weeks before selection on ERP medium containing 20–40 g/ml kanamycin (kan). Transgenic embryos were identified after 3–5 months under selection and allowed to germinate and develop into rooted plants on Woody Plant Medium containing 1 M 6-benzylaminopurine (BAP), 1.5% sucrose, 0.3% activated charcoal and 0.75% agar. Integration of the foreign genes into these grapevines was verified by growth in the presence of kan, positive GUS and PCR assays, and Southern analysis.     

发根农杆菌LBA9402Bin19转化红豆草及再生转基因植株

Hypocotyl segments of Onobrychis viciaefolia were transformed by Agrobacterium rhizogenes LBA9402 which harboured pBin19 and pRi1855. Seedling age and preculture time of hypocotyl segments influenced the transformation frequency. Paper electrophoresis revealed that 70% of single hairy root cultures could synthesize agropine. Calli were induced from hairy root segments on MS medium containing 0-9.05 mumol/L 2,4-D and 0-2.22 mumol/L 6-BA at first, then they were transferred onto MS0 medium without kanamycin for regeneration. Constitution and concentration of phytohormones in callus induction media affected subsequent regeneration of calluses on MS0 medium remarkably. Regeneration frequency and shoot number per callus declined when 2,4-D concentration in callus induction media increased from 4.52 to 9.05 mumol/L, while they ascended when 6-BA in callus induction media increased from 0 to 2.22 mumol/L. On MS medium supplemented with 4.52 mumol/L 2,4-D and 2.22 mumol/L 6-BA, only 14.2% hairy root segments could produce calluses, but the regeneration frequency reached 58.1% and the shoot number per callus was 37.2. In 32 analysed plants regenerated from 8 kanamycin resistant hairy root lines, 25 were nptII positive and showed different copy numbers.     

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 transformation of sweet orange and regeneration of transgenic plants

Summary Transgenic sweet orange (Citrus sinensis L. Osbeck) plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer. An hypervirulent A. tumefaciens strain harboring a binary vector that contains the chimeric neomycin phosphotransferase II (NPT II) and ß-glucuronidase (GUS) genes was cocultivated with stem segments from in vivo grown seedlings. Shoots regenerated under kanamycin selection were harvested from the stem segments within 12 weeks. Shoot basal portions were assayed for GUS activity and the remaining portions were shoot tip grafted in vitro for production of plants. Integration of the GUS gene was confirmed by Southern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for Citrus.Abbreviations BA benzyladenine - CaMV cauliflowermosaic virus - GUS ß-glucuronidase - LB Luria Broth - MS Murashige and Skoog - NAA naphthalenacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PEG polyethylene glycol - RM rooting medium - SRM shoot regeneration medium     

Electroporation of immature maize zygotic embryos and regeneration of transgenic plants

Using the pulse-discharging electroporation system HPES-3, we have transferred the neomycin phosphotransferase II (nptII) gene and -glucuronidase (gus) gene into mechanically-woulded immature zygotic embryo cells of an elite local maize cultivar Huanong Supersweet No. 42 and have produced transgenic maize plants. DNA hybridization and NPTII dot assay showed that the foreign genes were integrated into the genomes and expressed stably in the cells of the transgenic calluses and plants.     

Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants

Summary A method for Agrobacterium-mediated transformation of Citrus and organogenic regeneration of transgenic plants is reported. Internodal stem segments were co-cultured with Agrobacterium harboring binary vectors that contained the genes for the scorable marker ß-glucuronidase (GUS) and the selectable marker NPT-II. A low but significant percentage ( 5%) of the shoots regenerated in the presence of 100 g/ml kanamycin were GUS⁺. Polymerase chain reaction (PCR) analysis confirmed that GUS⁺ shoots contained T-DNA. Two plants established in soil were shown to be transgenic by Southern analysis.     

Biolistic transformation of chincherinchee (Ornithogalum) and regeneration of transgenic plants

Ornithogalum (chincherinchee) is a genus in the Hyacinthaceae. It is popular as a cut flower or pot plant. However, susceptibility to disease, especially ornithogalum mosaic virus, prevents commercial exploitation of micropropagated hybrids. Provided that it is possible to transform Ornithogalum , this problem might be alleviated by the transfer of genes that code for resistance to the virus. The purpose of this study was to develop a transformation protocol using the pat gene as a selectable marker. Callus, induced on leaf segments of an Ornithogalum thyrsoides × O. dubium hybrid cultured in vitro, was bombarded with a particle gun 4–6 weeks after initiation. We first used β -glucuronidase transient expression to optimise the bombardment parameters and then for stable transformation used both a conventional microprojectile-mediated method as well as a modification that entailed complexing single-stranded p35SAC DNA, containing the pat gene, with histone H1 prior to bombardment. Transgenic plants were regenerated from the bombarded tissues and cultured on a medium containing 15 μ M phosphinothricin as selective agent. Rooted plants were tested for the presence of the pat gene by polymerase chain reaction. Integration of the gene into the genomic DNA was verified by Southern blotting. Northern blots, enzyme-linked immunosorbent assay, and leaf paint assays with the herbicide Ignite® (glufosinate ammonium) confirmed expression of phosphinothricin acetyl transferase, the enzyme that detoxifies the herbicide.     

Stable transformation and regeneration of transgenic plants of Pinus radiata D. Don

A biolistic particle delivery system was used to genetically transform embryogenic tissue of Pinus radiata. The introduced DNA contained a uidA reporter gene under the control of either the tandem CaMV 35S or the artificial Emu promoter, and the npt II selectable marker controlled by the CaMV 35S promoter. The average number of stable, geneticin-resistant lines recovered was 0.5 per 200 mg fresh weight bombarded tissue. Expression of the uidA reporter gene was detected histochemically and fluorimetrically in transformed embryogenic tissue and in derived mature somatic embryos and regenerated plants. The integration of uidA and npt II genes into the Pinus radiata genome was demonstrated using PCR amplification of the inserts and Southern hybridisation analysis. The expression of both genes in transformed tissue was confirmed by Northern hybridisation analysis. More than 150 transgenic Pinus radiata plants were produced from 20 independent transformation experiments with four different embryogenic clones. Received: 9 May 1997 / Revision received: 18 September 1997 / Accepted: 18 October 1997     

Agrobacterium tumefaciens-mediated transformation of Artemisia annua L. and regeneration of transgenic plants

Summary A transformation system was developed for Artemisia annua L. plants. Leaf explants from in vitro grown plants developed callus and shoots on medium with 0.05 mg/L naphthaleneacetic acid and 0.5 mg/L N⁶-benzyladenine after transformation with the C58C1 Rif^R (pGV2260) (pTJK136) Agrobacterium tumefaciens strain. A concentration of 20 mg/L kanamycin was added in order to select transformed tissue. Kanamycin resistant shoots were rooted on naphthaleneacetic acid 0.1 mg/L. Polymerase chain reactions and DNA sequencing of the amplification products revealed that 75% of the regenerants contained the foreign genes. 94% of the transgenic plants showed a -glucuronidase-positive response.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - GM germination medium - GMVIT germination medium with vitamins - GUS -glucuronidase - Kin kinetin (N⁶-furfurylaminopurine) - NAA -naphthaleneacetic acid - NPT II neomycin phosphotransferase II - PCR Polymerase Chain Reaction - T-DNA transfer-DNA - X-glucuronide 5-bromo-4-chloro-3-indolyl -D-glucuronide     

Effective selection and regeneration of transgenic rice plants with mannose as selective agent

A new method for the selection of transgenic rice plants without the use of antibiotics or herbicides has been developed. The phosphomannose isomerase (PMI) gene from Escherichia coli has been cloned and consitutively expressed in japonica rice variety TP 309. The PMI gene was transferred to immature rice embryos by Agrobacterium-mediated transformation, which allowed the selection of transgenic plants with mannose as selective agent. The integration and expression of the transgene was confirmed by Southern and northern blot analysis and the activity of PMI indirectly proved with the chlorophenol red assay. The results of genetic analysis showed that the transgenes were segregated in a Mendelian fashion in the T₁ generation. The establishment of this selection system in rice provides an efficient way for producing transgenic plants without using antibiotics or herbicides with a transformation frequency of up to 41%.     

Agrobacterium-mediated transformation of quaking aspen (Populus tremuloides) and regeneration of transgenic plants

Summary Agrobacterium-mediated gene transformation of Populus tremuloides Michx was accomplished by co-cultivation of leaf disks excised from greenhouse plants with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase (NPT II) and ß-glucuronidase (GUS) genes. Shoot regeneration in the presence of kanamycin was achieved when thidiazuron (TDZ) was used as a plant growth regulator. Transformation was verified by amplification of NPT II and GUS gene fragments from genomic DNA of transgenic plants with polymerase chain reaction (PCR) and integration of these genes into nuclear genome of transgenic plants was confirmed by genomic Southern hybridization analysis. Histochemical assay revealed the expression of GUS gene in leaf, stem and root tissues of transgenic plants, further confirming the integration and expression of T-DNA in these plants. This protocol allows effective transformation and regeneration of quaking aspen using greenhouse-grown materials as an explant source. Whole plant regeneration from cuttings of fieldgrown mature quaking aspen and hybrid poplar (P. alba x P. grandidentata) was also readily achieved by using this protocol, which represents a potential system for producing transgenic quaking aspen and hybrid poplar of valuable genotypes.Abbreviations AMV RNA4 Alfalfa mosaic virus RNA4 - BA 6-benzyladenine - CaMV cauliflower mosaic virus - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraacetic acid - FAA formalin-acetic acid-alcohol - GUS ß-glucuronidase - NAA 1-naphthylacetic acid - NPT II neomycin phosphotransferase II - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - TE Tris-Cl/EDTA - TDZ N-phenyl-N-1,2,3-thiadiazol-5-yl-urea (thidiazuron) - WPM woody plant medium (Lloyd and McCown 1980) - X-GLUC 5-bromo-4-chloro-3-indolyl-ß-glucuronic acid     

Influence of light intensity and selection scheme on regeneration time of transgenic flax plants

This study aimed at establishing a protocol to increase the number of regenerated shoots and to limit the recovery of “escapes” during the regeneration of transgenic flax plants (cv Barbara). Here, we describe how light, adapted media and selection scheme could stimulate the transformation process, the organogenic potentiality of calli (by a factor of 3.2) and accelerate the transgenic shoot regeneration (by a factor of about 2). On comparison of the transformation rate observed while using low light (LL) and high light (HL) a considerable enhancement from 0.12 to 5.7% was evident. The promotive effect of light might also had a direct beneficial effect on transgenic plant production time leading to a reduction of more than 4 months in the time need to obtain transgenic seeds. All data indicate that HL plays a role on growth and on protein, rubisco and pigment contents by stimulating the gene implicated in photosynthetic and Calvin cycle processes.     

Transfer ofE. coli gutD gene into maize and regeneration of salt-tolerant transgenic plants

GutD gene, encoding a key enzyme (glucitol-6-phosphate dehydrogenase) of sugar alcohol metabolic pathway inE. coli, was transferred into maize. Results of Southern and Western blotting analysis certified that this gene had integrated and been expressed in transgenic maize plants and their progeny. The synthesis and accumulation of sorbitol were detected in transgenic maize plants and a preliminary nutrient solution culture experiment showed thatgutD transgenic maize plants had an increased tolerance to salt stress compared with nontransgenic ones.     

High frequency transformation and regeneration of transgenic plants in the model legume Lotus japonicus

The molecular analysis of plant genes involved in nodulationhas been slowed by the inability to produce high numbers oftransgenic legume lines. The high efficiency gene transfer andplant regeneration systems of the model legume Lotus japonicusis described. A collection of wild-type A. rhizogenes strainswas tested for infectivity and the most virulent strains, 9402and AR10, were selected for further use. Growth conditions forplantlets, induction of hairy roots and nodulation of compositeplants were optimized for large-scale screening in Petri dishes.A cluster of 3–10 nodules was regularly formed on transgenichairy roots 7–12 d after inoculation with the effectiveRhizobium loti strain NZP2235. There were no apparent morphologicaldifferences between nodulation of hairy and wildtype roots.To test the applicability of the hairy root system for the trappingof symbiotic genes, transformation experiments with binary vectorspossessing a ß-glucuronidase (gus, uidA) or a luciferase(luc) reporter driven by a cauliflower mosaic virus (CaMV) 35Spromoter were performed. The frequency of cotransfer of a binaryT-DNA with a root-inducing (Ri) T-DNA was 70%. Positive expressionsuggests that gus and luc trap vectors can be used for genetagging in L. japonicus. To open the possibility of searchingfor mutant phenotypes, a regeneration system has been developedenabling the regeneration of large numbers of transgenic plantsfrom hairy root cultures in about 5–6 months. At the sametime, the A. tumefaciens hypocotyl transformation regenerationin L. japonicus has been improved. This new version providesfertile transgenic plants in about 4 months. Key words: Agrobacterium, luciferase, nodulation, Rhizobium, symbiosis     

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%).