Carrot (Daucus carota) hypocotyl transformation usingAgrobacterium tumefaciens

Summary Daucus carota hypocotyl sections were transformed withAgrobacterium tumefaciens LBA4404 containing CaMV 35S promoter, -glucuronidase coding sequence and the nopaline synthase (Nos) poly adenylation sequences in Bin 19. Sliced sterile seedling hypocotyl segments were preincubated for 2 days, co-cultivated withAgrobacterium for an additional 2 days, and then transferred to medium containing 100ug/ml of kanamycin and 400ug/ml carbenicillin. In 6 weeks kanamycin resistant calli were obtained in 5.8% of the explants from one variety. Calli were subcultured on solid medium, and in 4 weeks introduced into suspension culture. NPTII and Southern blot analysis confirmed that three selected lines were transformed with 1–3 copies of the GUSII construction. GUS activity in transformants was 5 to 250 fold over background.Abbreviations NPT II neomycin phosphotransferase II - Nos nopaline synthase - GUS -glucuronidase - CaMV cauliflower mosaic virus - 2,4-D 2,4 dichlorophenoxyacetic acid - PMSF phenylmethylsulfonyl fluoride     

Transgenic Brassica napus plants obtained by cocultivation of protoplasts with Agrobacterium tumefaciens

Hypocotyl protoplasts of German winter oilseed, rape (Brassica napus) lines of double-low quality were transformed using Agrobacterium tumefaciens harbouring pGV 38501103 neo (dimer) containing chimaeric kanamycin resistance reporter genes. Transformed protoplasts were regenerated to fertile and phenotypically normal plants. Transformation was confirmed by kanamycin resistance, nopaline production, neomycinphosphotransferase II activity, and Southern blot hybridization. Seed progeny from self-pollinated transformants expressed the introduced kanamycin resistance as a Mendelian trait.Abbreviations BAP 6-benzylaminopurine - Cf Claforan^R - 2.4D 2,4-dichlorophenoxy acetic acid - Km kanamycin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - NPT II neomycinphosphotransferase - npt II neomycinphosphotransferase II gene - NOS nopaline synthase - nos nopaline synthase gene - ocs octopine synthase gene - IAA indole-3-acetic acid     

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     

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     

High efficiency Agrobacterium-mediated gene transfer to flax

Summary Using an Agrobacterium tumefaciens binary vector (pAL4404, pBI131), we have demonstrated the transfer of the -glucuronidase gene into the flax (Linum usitatissimum L.) cultivar Glenelg after selection for kanamycin resistance. The transformed lines were obtained by inoculation and subsequent regeneration of hypocotyl segments. The callus that formed on the cut surfaces of the hypocotyl segments was isolated three weeks after infection and was subsequently subcultured to yield shoots. This procedure generated a large number of transgenic shoots over a relatively short period of time. The transformation efficiencies obtained were the highest reported so far for this plant species.Abbreviations 2,4-D, 2,4 dichlorophenoxyacetic acid - GUS glucuronidase - MS Murasbige and Skoog (1962) medium - MU 4-methyl-umbelliferone - MUG 4-methylumbelliferyl-glucuronide - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

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 tumefaciens mediated gene transfer in peanut (Arachis hypogaea L.)

Transgenic peanut plants were produced using Agrobacterium mediated gene transfer. Primary leaf explants of peanut were co-cultivated with Agrobacterium tumefaciens LBA 4404 harbouring the binary plasmid pBI 121 (conferring -glucuronidase activity and resistance to kanamycin) and cultured on regeneration medium supplemented with kanamycin to select putatively transformed shoots. They were rooted and plants were transferred to soil. Stable integration and expression of the transgenes were confirmed by NPT II assay, Southern blot hybridization and GUS assay.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - SDS Lauryl sulfate     

Transformation of peas (Pisum sativum L.) using immature cotyledons

A reliable Agrobacterium tumefaciens-mediated transformation method has been developed for peas (Pisum sativum) using immature cotyledons as the explant source. Transgenic plants were recovered from the four cultivars tested: Bolero, Trounce, Bohatyr and Huka. The method takes approximately 7 months from explant to seed-bearing primary regenerant. The binary vector used carried genes for kanamycin and phosphinothricin resistance. Transformed pea plants were selected on 10 mg/l phosphinothricin. The nptII and bar genes were shown to be stably inherited through the first sexual generation of transformed plants. Expression of the phosphinothricin-resistance gene in the transformed plants was demonstrated using the Buster (=Basta) leaf-paint test and the phosphinothricin acetyl transferase enzyme assay.Abbreviations BA 6-benzylaminopurine     

Transformation of Liquidambar styraciflua using Agrobacterium tumefaciens

Summary We describe the molecular transformation of Liquidambar styraciflua using Agrobacterium tumefaciens. A binary TI-plasmid vector containing a chimeric neomycin phosphotransferagene which confers resistance to kanamycin and either a chimeric Bacillus thuringiensis toxin gene, a chimeric E. coli -glucuronida(GUS), or a chimeric tobacco anionic peroxidase gene was introduced into sweetgum by co-cultivation with Agrobacterium tumefaciens. Sweetgum shoots regenerated in the presence of kanamycin were confirmed to be transformed by genomic DNA blots or the presence of GUS activity. The optimization of the transformation protocol and the incorporation of molecular transformation into a rapid germplasm improvement protocol are discussed.     

Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro)

Cotyledon explants of muskmelon (Cucumis melo L., cv. Amarillo Oro) seedlings were co-cultivated with disarmed Agrobacterium tumefaciens strain LBA4404 that contained the binary vector plasmid pBI121.1. The T-DNA region of this binary vector contains the Nopaline synthase/neomycin phosphotransferase II (NPTII) chimeric gene for kanamycin resistance and the Cauliflower Mosaic Virus 35S/-glucuronidase (GUS) chimeric gene. After infection, the cotyledon pieces were placed in induction medium containing 100 mg/l kanamycin. Putative transformed shoots were obtained, followed by the development of morphologically normal plantlets. The transgenic nature of regenerants was demonstrated by polymerase chain reaction, Southern blot analysis, plant growth on medium selective for the transgene (NPTII) and expression of the co-transformed GUS gene. Factors affecting the transformation procedure are discussed.Abbreviations CaMV Cauliflower Mosaic Virus - Cf Cefotaxime - GUS -glucuronidase - Km Kanamycin - MS Murashige and Skoog - NOS nopaline synthase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

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%).     

Transformation of potato using mannopine and cucumopine strains of Agrobacterium rhizogenes

Mannopine and cucumopine strains of Agrobacterium rhizogenes were used for genetic transformation in two cultivars of potato (Solanum tuberosum L.). An overnight pretreatment of stem fragments with NAA prior to bacterial infection was necessary to induce root formation, otherwise very few roots were produced. Whatever the potato cultivar used, rhizogenesis induced by NAA pretreament depended on the bacterial strain. In fact, when explants from both potato cultivars were pretreated with 26.5 M NAA, on average 84.4% and 71.9% produced roots after inoculation with the strains 2659 and 2659 GUS respectively. On the contrary, few rhizogenic responses (2.0–17.0%) or no response at all (0.0%) were obtained with the strains 15834 and 8196 GUS whatever NAA concentration used. Tests for confirming stable transformation of plant explants by examining both -glucuronidase activity and the presence of opines showed that 85% of the selected roots were cotransformed. Most of the transformed roots were highly branched and grew rapidly, compared to non-transformed roots with no branching and poor growth. Transgenic plants were readily regenerated with a frequency reaching 80% of total explants tested for both potato cultivars.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - df degree of freedom - F F distribution - GA₃ gibberellic acid - MS Murashige and Skoog basal medium - NAA -naphthaleneacetic acid - P probability - T-DNA transferred DNA     

Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting gene transfer efficiency during early transformation steps

The factors influencing transfer of an intron — containing -glucuronidase gene to apple leaf explants were studied during early steps of an Agrobacterium tumefaciens-mediated transformation procedure. The gene transfer process was evaluated by counting the number of -glucuronidase expressing leaf zones immediately after cocultivation, as well as by counting the number of -glucuronidase expressing calli developing on the explants after 6 weeks of postcultivation in the presence of 50 mg/l kanamycin. Of three different tested disarmed A. tumefaciens strains, EHA101(pEHA101) was the most effective for apple transformation. Cocultivation of leaf explants with A. tumefaciens on a medium with a high cytokinin level was more conducive to gene transfer than cocultivation on media with high auxin concentrations. Precultivation of leaf explants, prior to cocultivation, slightly increased the number of -glucuronidase expressing zones measured immediately after cocultivation, but it drastically decreased the number of transformed calli appearing on the explants 6 weeks after infection. Other factors examined were: Agrobacterium cell density during infection, bacterial growth phase, nature of the carbon source, explant age, and explant genotype.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - CaMV35S 35S RNA of cauliflower mosaic virus - EDTA ethylenediaminetetraacetate - FeNaEDTA ethylenediaminetetraacetate ferric-sodium salt - GusA -glucuronidase - gusA ß-glucuronidase gene of Escherichia coli - gusA-intron ß-glucuronidase gene containing an intron in the coding region - IBA indole butyric acid - 2iP N6-2-isopentenyl adenine - NAA naphthaleneacetic acid - nptII neomycinphosphotransferase II gene - X-Gluc 5-bromo-4-chloro-3-indolyl ß-D-glucuronide     

Agrobacterium tumefaciens-mediated transformation of Vigna mungo (L.) Hepper

Transformed Vigna mungo (blackgram) calli were obtained by cocultivating segments of primary leaves with Agrobacterium tumefaciens vir helper strains harbouring the binary vector pGA472 having kanamycin resistance gene as plant transformation marker. Transformed calli were selected on Murashige and Skoog medium supplemented with 50 mg/l kanamycin and 500 mg/l carbenicillin. Transformed calli were found to be resistant to kanamycin up to 900 mg/l concentration. Expression of kanamycin resistance gene in transformed calli was demonstrated by neomycin phosphotransferase assay. Stable integration of transferred DNA into V. mungo genome was confirmed by Southern blot analysis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP 6-(,-dimethylallylamino)purine - Kn kinetin - nptII neomycin phosphotransferaseII - MS Murashige and Skoog (1962) medium     

Shoot regeneration in stem expiants and its amenability to Agrobacterium tumefaciens mediated gene transfer in Brassica carinata

Immature stem segments of seven different genotypes of Brassica carinata produced shoots with variable frequencies when cultured in MS medium with BAP and picloram at 0.2 mg/l each. Line 171, which produced shoots with 100% efficiency from both cut ends of the expiant, was selected for testing the amenability of this regeneration protocol for genetic transformation. A non-oncogenic Agrobacterium tumefaciens containing plasmid PCV 730, a binary vector carrying resistance genes for kanamycin and hygromycin, was used. A cocultivation period of 4 d with a bacterial concentration of approximately 2.5×10 cells/ml, followed by a recovery period of 2 d, produced transformed shoots that could be selected and rooted in the presence of kanamycin at 15 mg/l. Transformation was confirmed by neomycin phospho-transferase assay and Southern blot analysis. Seed analysis of transformed plants indicated that kanamycin resistance was inherited in the progeny.Abbreviations BAP 6-Benzylaminopurine - Kn Kinetin - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - ATP Adenosine triphosphate - MS Murashige and Skoog (1962) medium     

Improved protoplast culture and regeneration of shoots in pea (Pisum sativum L.)

An improved system involving a modification of the bead culture system was developed for culturing pea protoplasts. Using this method, sustained divisions and callus growth could be obtained in all 10 cultivars tested. In the best responding cultivar division frequency could be raised from 17% in liquid culture to 80% in the bead system. Shoot regeneration with a reproducible frequency of about 1% could be obtained from protoplast-derived calli in two of the tested cultivars.Abbreviations ABA abscisic acid - BA N⁶-benzyladenine - NAA -naphthylacetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid     

A modified method for routine Agrobacterium-mediated transformation of in vitro grown potato microtubers

Summary In vitro-grown potato (Solanum tuberosum L.) microtubers were used as an explant source in the production of transgenic plants by Agrobacterium-mediated gene transfer. In this study we tested four diverse potato cultivars, Lemhi Russet, Russet Burbank, Wauseon, and Yankee Chipper on various levels of zeatin riboside and 3-indoleacetyl-DL-aspartic acid for their ability to regenerate transgenic plants after infection with Agrobacterium tumefaciens. Culturing microtuber blocks from the medullary area separately from cortex and epidermal tissue containing the eyes resulted in fewer transgenic plants, with transgenic shoots arising only from the tissue with the eyes. Lemhi and Russet Burbank microtuber discs were also transformed with a chimeric gene, CLaSP, designed to increase resistance to blackspot bruise in the tuber. This method resulted in transformed plants in every experiment, with an efficiency that appeared to be genotype dependent.Abbreviations GUS -glucuronida (uidA) - IAA-AA 3-indoleacetyl-DL-aspartic acid - LB Luria-Bertani - LSP larval serum storage protein - nos nopaline synthase - npt II neomycin phosphotransferase - MS Murashige and Skoog - PHA phytohemaglutinin - ZR zeatin riboside     

Accumulation of essential oils by Agrobacterium tumefaciens-transformed shoot cultures of Pimpinella anisum

Axenic transformed shoot cultures of Pimpinella anisum (anise) were established following inoculation of plant stems with the nopaline strain T37 of Agrobacterium tumefaciens. The stable incorporation of T-DNA in the transformed tissues was demonstrated by polymerase chain reaction. Total essential oil accumulated by transformed shoot cultures grown under continuous light was found to be 18% lower (per unit fresh weight of tissue) than that produced by untransformed shoot cultures incubated under similar conditions, but more than 89% lower than the yield of oil from the intact plant. The relative amounts of the principal components of the essential oil of the transformed shoot cultures, namely geraniol, -bisabolene, trans-pseudoisoeugenol-2-methylbutyrate and transanethole, were similar to those present in the parent plant, but significantly different from those of the untransformed shoot cultures.Abbreviations T-DNA transfer-DNA - MS Murashige and Skoog medium - 2,4-D 2,4-dichlorophenoxyacetic acid - TY tryptone and yeast extract medium - t_D doubling time - GC-MS gas chromatography coupled with mass spectrometry - FID flame ionisation detector - PCR polymerase chain reaction - TE Tris-HCl, EDTA buffer - TBE Tris, borate, EDTA buffer     

Agrobacterium-mediated transformation of Arabis gunnisoniana

An efficient method for adventitious shoot regeneration for Arabis drummondii and a transformation protocol for A. gunnisoniana from hypocotyl explants are described. Hypocotyl explants from 7-day-old aseptically grown seedlings were cultured on MS medium containing plant growth regulators (6-benzylaminopurine, 1-phelyl-3- (1,2,3-thiadiazol-5-yl) urea, -naphthaleneacetic acid and 2,4-dichlorophenoxy-acetic acid). After 4 weeks in culture, high frequency of adventitious shoot regeneration was observed. Regenerated shoots were rooted on half-strength MS basal medium supplemented 1% (w/v) sucrose, with or without NAA. This protocol was then used to produce transformed Arabis gunnisoniana plants. A. gunnisoniana hypocotyl explants were co-cultivated with Agrobacterium tumefaciens strain GV3101 harbouring pBJ40. Transgenic shoots were selected on MS 21 medium supplemented with 50 mg l kanamycin. PCR analysis verified the presence of the nptII gene in the plant DNA isolated from kanamycin resistant shoots.     

Agrobacterium tumefaciens-mediated transformation of severalRubus genotypes and recovery of transformed plants

Experiments in shoot regeneration and virulentAgrobacterium tumefaciens-mediated transformation were used to develop a protocol forRubus transformation. This protocol was then used to produce transformedRubus plants fromin vitro internodes inoculated with anAgrobacterium tumefaciens encoding neomycin phosphotransferase on its disarmed T-DNA. Two transformed plants were selected from 800 inoculations on a medium containing 10 µg ml^–1 kanamycin. Results indicated that this level of kanamycin successfully selected against non-transformed cells but did not reduce the number of transformed, kanamycin-resistant, shoots formed. Enzyme assays and Southern blot analysis verified the presence of the -glucuronidase gene in the plant genome.