Agrobacterium-mediated transformation and plant regeneration of buckwheat (Fagopyrum esculentum Moench.)

Genetic transformation of buckwheat (Fagopyrum esculentum Moench.) and regeneration of transgenic plants were obtained by using Agrobacterium tumefaciens strains as vectors. Buckwheat cotyledons were excised from imbibed seeds, co-cultivated with A. tumefaciens and subjected to previously reported protocols for callus and shoot regeneration. The transformation with oncogenic strains was confirmed by opine and DNA analyses of tumour tissue extracts. Plants were regenerated on cotyledon fragments incubated with strain A281, harboring pGA472, which carries the neomycin phosphotransferase II gene for kanamycin resistance. The transformation of resistant shoot clones was confirmed by NPTII enzyme assay and DNA hybridization. A large number of transformed shoots were rooted and fertile plantlets were raised in the greenhouse. Transgenic plants comprised pin and thrum clones, which were allowed to cross-pollinate. In about 180 R₂ seeds tested for kanamycin resistance, the ratio of resistant to sensitive seedlings was roughly 3:1.Abbreviations BAP 6-benzylaminopurine - 2,4-D dichloro-phenoxyacetic acid - 2iP 6-(, ,-dimethylallyl-amino)-purine - IBA indole-3-butyric acid - IAA indole-3-acetic acid - Km kanamycin - NPTII neomycin phosphotransferase II     

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     

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     

Genetic transformation ofPelargonium X hortorum

Summary TransgenicPelargonium X hortorum have been producedvia Agrobacterium tumefaciens-mediated transformation. The regeneration protocol used provided a regeneration frequency approximately to 95 percent. Clumps of regenerants, from cotyledons and hypocotyls ofPelargonium X hortorum seedlings, were inoculated with the disarmed strain EHA101 ofAgrobacterium tumefaciens. This strain contains a binary vector carrying neomycin phosphotransferase II, hygromycin B phosphotransferase and ß-glucuronidase genes. Selection on the regeneration medium supplemented with hygromycin allowed production of transgenic plants in up to 20% of the inoculated explants. The insertion of foreign DNA was demonstrated by Southern and polymerase chain reaction analysis: these experiments indicated that the inserted T-DNA is not full length for most of the plants. All RO transgenic plants exhibited a normal phenotype and are fertile.Abbreviations GUS ß-glucuronidase coding sequence - PCR polymerase chain reaction - CaMV cauliflower mosaic virus - NPTII neomycin phosphotransferase coding sequence - NOS nopaline synthase gene promoter and terminator - HPH hygromycin B phosphotransferase coding sequence - SDS sodium dodecyl sulphate - EDTA (ethylenedinitro trilo)tetra-acetic acid disodium salt     

Transformation of haploid Datura innoxia protoplasts and analysis of the plasmid integration pattern in regenerated transgenic plants

We developed a highly efficient transformation protocol for the PEG-mediated direct transfer of plasmid DNA into protoplasts of haploid Datura innoxia. Vectors harbouring a neomycin phosphotransferase II gene or a hygromycin B phosphotransferase gene under the control of different promoters were used in the transformation experiments. Various amounts of plasmid DNA were applied without any carrier DNA to show the direct influence of the plasmid DNA concentration on the transformation efficiency. Approximately 95% of the selected calli were regenerated to plants; 20% of them remained haploid. Total DNA of different transgenic plants was analysed with regard to the integration pattern of the plasmid DNA. Plants carrying only one or two copies of the vector DNA were observed as well as individuals with multi-copy integration (up to ten or more copies).Abbreviations ATF/RTF absolute/relative transformation frequency - BAP 6-benzylaminopurine - CaMV cauliflower mosaic virus - CTAB N-cetyl-N,N,N-trimethyl-ammonium bromide - HPT hygromycin B phosphotransferase gene - PEG polyethyleneglycol - MES 2-(N-morpholino) ethanesulfonic acid - NPT II neomycin phosphotransferase II gene     

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     

Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs,plant ecotypes and Agrobacterium strains

Summary The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the ß-glucuronidase (GUS) reporter gene showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80–90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.Abbreviations AIM Agrobacterium infection medium - CIM callus-inducing medium - CTAB cetyltrimethylammonium bromide - 2,4-D 2,4-dichlorophenoxy-acetic acid - GUS ß-glucuronidase - hph hygromycin phosphotransferase - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip N -(2-isopentenyl) adenine - NPTII neomycin phosphotransferase II - RIM root-inducing medium - 35S cauliflower mosaic virus 35S promoter - SIM shoot-inducing medium     

Shuttle vectors conferring hygromycin B resistance toE. coli and to mammalian cells

Shuttle vectors expressing resistance to hygromycin B in bothE. coli and in mammalian cells were constructed. A combination of the simian virus 40 early promoter upstream of the native bacterial promoter of theneo gene from transposon Tn5 was found to express hygromycin B resistance better in both types of host cells than a combination of the Tn5 promoter followed by the promoter of the Herpes simplex virus thymidine kinase gene. Hygromycin phosphotransferase fusion proteins with extensions at the carboxyterminus were also tested and found to be marginally less effective as selection markers in eukaryotic cells but virtually inactive inE. coli.Abbreviations HM hygromycin - hpt hygromycin phosphotransferase gene - neo neomycin (geneticin) phosphotransferase gene - DHFR dihydrofolate reductase     

The Pseudomonas savastanoi tryptophan-2-mono-oxygenase is biologically active in Nicotiana tabacum

It has been proposed that the eukaryotic T-DNA-encoded indole-3-acetic acid (IAA) biosynthesis genes of Agrobacterium tumefaciens and their prokaryotic counterpart in Pseudomonas savastanoi originated from common ancestor genes. This paper provides additional evidence for the functional similarity between the gene products. We have demonstrated that a chimeric gene consisting of the coding sequence of the P. savastanoi tryptophan-2-mono-oxygenase (iaaM gene) and a plant promoter encodes an active enzyme in Nicotiana tabacum. Transformants obtained with this chimeric gene grew as a callus on hormone-free media. No stably transformed plantlets could be isolated. The callus tissues contained extremely high levels of indole-3-acetamide and slightly elevated levels of IAA. Either indole-3-acetamide by itself has a low auxin activity or, alternatively, it is converted aspecifically and at low rates into IAA. The P. savastanoi tryptophan-2-mono-oxygenase activity in plants is also able to detoxify the amino-acid analogue 5-methyltryptophan. This property can be used for positive selection of transformed calli.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IAM indole-3-acetamide - NAA naphthalene-1-acetic acid - NPT-II neomycin phosphotransferase II - T-DNA transferred DNA     

Single-copy T-DNA insertions in Arabidopsis are the predominant form of integration in root-derived transgenics,whereas multiple insertions are found in leaf discs

Different patterns of T-DNA integration in Arabidopsis were obtained that depended on whether a root or a leaf-disc transformation method was used. An examination of 82 individual transgenic Arabidopsis plants, derived from 15 independent Agrobacterium-mediated transformations in which different cointegrate and binary constructs were used, indicated that the transformation method had a significant influence on the type and copy number of T-DNA integration events. Southern hybridizations showed that most of the transgenic plants produced by a leaf-disc method contained multiple T-DNA insertions (89%), the majority of which were organized as right-border inverted repeat structures (58%). In contrast, a root transformation method mostly resulted in single T-DNA insertions (64%), with fewer right-border inverted repeats (38%). The transformation vectors, including cointegrate and binary types, and the plant selectable markers, hygromycin phosphotransferase and dihydrofolate reductase, did not appear to influence the T-DNA integration patterns.     

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     

New plant binary vectors with selectable markers located proximal to the left T-DNA border

Five new binary vectors have been constructed which have the following features: (1) different plant selectable markers including neomycin phosphotransferase (nptII), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), phosphinothricin acetyl transferase (bar), and bleomycin resistance (ble); (2) selectable markers are located near the T-DNA left border and; (3) selectable marker and -glucuronidase (uidA) reporter genes are divergently organized for efficient expression, and can easily be removed or replaced as needed.     

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     

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     

Agrobacterium-mediated transformation of Bangladeshi Indica rices

Morphologically normal, fertile transgenic plants were obtained by co-culturing embryogenic calli of the Bangladeshi Indica rice cultivars BR26 and Binni with Agrobacterium tumefaciens strain LBA4404 carrying the super binary vector pTOK233. Acetosyringone (100 microM) in the medium during co-culture (25-28 degrees C) and selection on hygromycin B (50 mg l(-1)) were essential for efficient transformation. Stable integration and expression of beta-glucuronidase, neomycin phosphotransferase and hygromycin phosphotransferase genes in regenerated plants were confirmed by histochemical and fluorometric assays, ELISA and Southern analysis. Two to 3 copies of T-DNA were integrated into regenerated plants; transgene expression did not correlate with gene copy number. Mendelian segregation of transgenes occurred in T1 seed progeny.     

Agrobacterium-mediated transformation of five wild Solanum species using in vitro microtubers

Summary A genetic transformation method, using in vitro microtubers and Agrobacterium-mediated transformation has been developed for five wild Solanum species: S. verrucosum, S. hjertingii, S. papita, S. stoloniferum, S. demissum, which range in ploidy from diploid to hexaploid. A disarmed A. tumefaciens strain, C58 harbouring the co-integrate vector pGV3580::pKU2 with the genes of neomycin phosphotransferase (NPTII) and hygromycin phosphotransferase (HPTII) as selectable markers, was used. Selection of putative transformants was based on their ability to grow and produce roots on a medium containing 150 mg/l kanamycin. The transgenic nature of the putative transformants was confirmed by Polymerase Chain Reaction analysis and by NPTII dotblot assay to show the expression of the NPTII gene. Additionally, the transmission of transgenes, NPTII and HPTII in selfed-sexual progeny of some transgenic plants was also determined.Abbreviations MS Murashige and Skoog - NPTII neomycin phosphotransferase - HPTII hygromycin phosphotransferase - PCR polymerase chain reaction - GA₃ gibberellic acid - CCC chlorocholine chloride - BAP benzylaminopurine - NAA naphthalene acetic acid - ZR zeatin riboside - IAA indole acetic acid - LB Luria Broth     

Efficient genetic transformation of red raspberry,Rubus ideaus L.

We have developed an efficient transformation system for red raspberry (Rubus ideaus L.) using Agrobacterium mediated gene transfer. Using this system we have successfully introduced a gene that encodes an enzyme, S-adenosylmethionine hydrolase (SAMase), in raspberry cultivars Meeker (MK), Chilliwack (CH) and Canby (CY). Leaf and petiole expiants were inoculated with disarmed Agrobacterium tumefaciens strain EHA 105 carrying either of two binary vectors, pAG1452 or pAG1552, encoding gene sequences for SAMase under the control of the wound and fruit specific tomato E4 promoter. Primary shoot regenerants on selection medium were chimeral containing both transformed and non-transformed cells. Non-chimeral transgenic clones were developed by iterative culture of petiole, node and leaf explants, on selection medium, from successive generations of shoots derived from the primary regenerants. Percent recovery of transformants was higher with the selection marker gene hygromycin phosphotransferase (hpt), than with neomycin phosphotransferase (nptII). Transformation frequencies of 49.6%, 0.9% and 8.1% were obtained in cultivars Meeker, Chilliwack and Canby respectively from petiole expiants using hygromycin selection. Genomic integration of transgenes was confirmed by Southern hybridization. Transgenic plants from a total of 218 independent transformation events (161 MK, 4 CH, 53 CY) have been successfully established in soil.Abbreviations ACCO amincocyclopropane-1-carboxylic acid oxidase - AS acetosyringone - BA 6-benzylaminopurine - CH cultivar Chilliwack - CY cultivar Canby - cv cultivar - hpt hygromycin phosphotransferase - IBA indolebutyric acid - MK cultivar Meeker - npt II neomycin phosphotransferase - SAMase S-adenosylmethionine hydrolase - TDZ Thidiazuron (N-phenyl-N'-l,2,3-thidiazol-5-ylurea)     

Agrobacterium-mediated transformation of white mustard (Sinapis alba L.) and regeneration of transgenic plants

Summary A procedure for the regeneration of fertile transgenic white mustard (Sinapis alba L.) is presented. The protocol is based on infection of stem explants of 7–9 day old plants with an Agrobacterium tumefaciens strain harboring a disarmed binary vector with chimeric genes encoding neomycin phosphotransferase and -glucuronidase. Shoots are regenerated from callus-forming explants within 3–4 weeks. Under selection, 10% of the explants with transgenic embryonic callus develop into fertile transgenic plants. Rooting shoots transferred to soil yield seeds within 14–16 weeks following transformation. Integration and expression of the T-DNA encoded marker genes was confirmed by histochemical glucuronidase assays and Southern-DNA hybridization using primary transformants and S1-progeny. The analysis showed stable integration and Mendelian inheritance of trans-genes in transformed Sinapis lines.Abbreviations BAP 6-benzylaminopurine - CaMV cauliflower mosaic virus - GUS -glucuronidase - IBA indole-3-butyric acid - IM infection medium - NAA 1-naphthalene acetic acid - neo gene encoding NPTII - NPTII neomycin phosphotransferase - RIM root-inducing medium - SEM shoot-elongation medium - SIM shoot-inducing medium - t-nos polyadenylation site of the nopaline synthase gene - uidA gene encoding GUS - WM wash medium - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronide     

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     

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