Agrobacterium-mediated transformation of white clover using direct shoot organogenesis

Summary White clover (Trifolium repens L.) plants from the cultivars Grasslands Huia and Grasslands Tahora have been transformed using Agrobacterium-mediated T-DNA transfer. Transgenic plants regenerated directly from cells of the cotyledonary axil. To transform white clover, shoot tips from 3 day old seedlings were co-cultivated with A. tumefaciens strain LBA4404 carrying the plasmid vector pPE64. This vector contains the neomycin phosphotransferase II gene (nptII) and -glucuronidase reporter gene (gus) both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 42 days after transfer onto selective media. Integration of the nptII and gus genes into the white clover genome was confirmed using Southern blotting, and histochemical analysis indicated that the gus gene was expressed in a variety of tissues. In reciprocal crosses between a primary transformant and a non-transformed plant the introduced gus gene segregated as a single dominant Mendelian trait.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MS Murashige and Skoog - GUS -glucuronidase - X-GLUc 5-bromo-4-chloro-3-indolyl--D-glucuronide - MUG methylumbelliferyl--D-glucuronide - CaMV Cauliflower Mosaic Virus - NPTII neomycin phosphotransferase II - OCS octopine synthase - 4-MU 4-methyl umbelliferone     

Evidence for non-proteinaceous inhibitor(s) of β-glucuronidase in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>L.) leaf and root tissues

The GUS gene of E. coli, encoding -glucuronidase, has been widely used as a reporter gene in plant transformation. However, -glucuronidase activity in transgenic wheat leaf or root tissue is rarely observed or reported. To address this question, we investigated three wheat lines transformed with the GUS reporter gene. We found all three lines expressed GUS mRNA as well as -glucuronidase protein in their leaf and root tissues as detected by RNA gel blot, ELISA, and immunoblot analyses. However, -glucuronidase enzyme activity was only detected in pollen grains from the transgenic plants. Fluorometric and histochemical assays performed in the presence of wheat tissue extracts indicated that wheat leaf and root tissues contain inhibitor(s) of -glucuronidase activity, but pollen grains contain much lower concentrations. Further characterizations indicated that the inhibitor(s) is of low molecular weight (<10 kDa) and is non-proteinaceous.     

Truncated presequences of mitochondrial F1-ATPase β subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco

The mitochondrial F₁-ATPase subunit (ATPase-) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH₂-terminal extension. By sequencing the mature N. tabacum ATPase-, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3 deletions in the sequence coding for the 90 NH₂-terminal residues of ATPase-. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and -glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase- remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.     

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

Transformation of haploid, microspore-derived cell suspension protoplasts of rice (Oryza sativa L.)

We compared the transient activity of three cereal gene-derived promoter-gus fusions and the efficiency of selection mediated by three different selectable genes in a polyethylene glycol transformation system with haploid cell suspension protoplasts of rice. The maize ubiquitin promoter was found to be the most active in transformed protoplasts, and selection on ammonium glufosinate mediated by the bar gene was the most efficient for producing resistant calluses. Cotransformation of protoplasts with two separate plasmids carrying the gus and the bar genes, at either a 21 or 11 ratio, led to 0.8 × 10^–5 and 1.6 × 10^–5 resistant callus recovery frequencies and 59.7 and 37.9 cotransformation efficiencies respectively. No escapes were detected in dot blot analyses of 100 resistant calluses with a probe consisting of the bar coding region. Cotransformation efficiency, based on resistance to basta and -glucuronidase staining of the leaf tissue of 115 regenerated plants, was 47%. Resistance tests and Southern analysis of seed progenies of three diploid transgenic plants demonstrated homozygous integration of multiple copies of the transgene at one locus at least in the first plant, heterozygous integration at one locus in the second plant and heterozygous integration at two loci in the third plant.Abbreviations PEG polyethylene glycol - T₀ regenerated transgenic plant - GUS -glucuronidase - CaMV cauliflower mosaic virus - ARE anaerobic responsive element - OCS octopine synthase - T₁ first generation progeny of transgenic plants     

Spurious localizations of diX-indigo microcrystals generated by the histochemical GUS assay

For several models expressing theuidA orgus reporter gene with or without a presequence for mitochondrial targeting, we have demonstrated that the compartmentation of -glucuronidase (E.C. 3.2.1.31) activity was not in agreement within situ localization of the diX-indigo microcrystals generated by the cytoenzymological GUS assay. These crystals were generally associated with the various cytomembranes and lipid inclusions. Experiments with purified -glucuronidase or withgus-expressing bacteria incubated with 5-bromo-4-chloro-3-indolyl--d-glucuronide and maize oil-phosphate buffer emulsion indicated that the intermediate products resulting from the GUS assay actively diffused and crystallized preferentially in association with lipids, sometimes far from the site of enzyme activity. This phenomenon could not be suppressed by the addition of potassium ferricyanide in the incubation medium. These findings are discussed with regard to previously reported biochemical and histochemical data on animal tissues, and focus on the necessity for caution in studies of tissue-specific gene expression using the GUS assay, particularly for lipid-rich plant models.     

Introduction of foreign genes into potato cultivars Bintje and Désirée using an Agrobacterium tumefaciens binary vector

Tuber discs of Solanum tuberosum cv Bintje and Désirée were cocultivated with an Agrobacterium tumefaciens binary vector, carrying both the neomycine phosphotransferase and the E. coli -glucuronidase gene fused to resp. the nopaline synthase and Cauliflower mosaic virus 35S promotor.Inoculated tuber discs produce transgenic shoots in selective media containing kanamycin. The transgenic plants are phenotypically normal and contain the euploid number of chromosomes. Both the neomycin phosphotransferase as well as the -glucuronidase gene are expressed conferring resp. kanamycin resistance and -glucuronidase activity to the plants.Abbreviations GUS -glucuronidase - NPT neomycin phosphotransferase - CaMV Cauliflower Mosaic Virus - BAP 6-benzylaminopurine - GA₃ gibberellic acid - NAA naphthalineacetic acid - LB Luria Broth - MU methylumbelliferone     

Regeneration and Agrobacterium-mediated transformation of Forsythia×intermedia "Spring Glory"

Internode explants ofin vitro plants ofForsythia x intermedia Spring Glory were transformed with thegus andnpt II genes after inoculation with theA. tumefaciens strain EHA 101 harbouring the plasmid pFAJ3000. Shoot organogenesis took place from callused edges of explants. The first transformed buds were detected 4 to 6 weeks after transfer on regeneration medium, containing 25 mg/l kanamycin as selective agent. An average of 1% of explants regenerated transgenic shoots.-glucuronidase assays and culture on kanamycin-containing medium provided the first indication of integration and expression of introduced genes in transformants. Southern blot and polymerase chain reaction amplification analyses gave molecular confirmation of genetic transformation. Transgenic plants were acclimatized in the greenhouse. Enzymatic assays on several organs of mature plants still showed -glucuronidase activity, thus confirming stable integration of T-DNA in the plant genome.Abbreviations BAP 6-benzyl-aminopurine - CaMV Cauliflower Mosaic Virus - GUS andgus -glucuronidase - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog - NOS nopaline synthase - NPT II andnpt II neomycin phosphotransferase II - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - SSC sodium chloride-sodium citrate - X-Gluc 5-bromo-4-cbloro-3-indolyl glucuronide     

Agrobacterium rhizogenes-mediated transformation of Rubia peregrina L.: in vitro accumulation of anthraquinones

An Agrobacterium rhizogenes-mediated transformation system for Rubia peregrina L. has been established by co-cultivation of callus cultures or by direct infection of explants with A. rhizogenes LBA 9402 harbouring the binary vector pMON 9703 containing gus and npt-II genes as markers. The putative transformed roots were selected on medium containing kanamycin (25 mg l^-1). Antibiotic resistant root clones were subjected to histochemical analysis for the localisation of -glucuronidase activity. Polymerase chain reaction was used to confirm the presence of gus, npt-II and T _L border sequences in the transformed root clones. Spontaneous regeneration of shoots was observed from 30 day-old transgenic roots. Total anthraquinone and alizarin contents of transgenic root cultures were measured by spectrophotometry and by high performance liquid chromatography. The accumulation of total anthraquinones in transformed roots was found to be approximately 2-fold higher than that found in one year-old field grown roots (2.12±0.12 and 1.23±0.12 mg g^-1 dry weight, respectively). Alizarin was found to be the major anthraquinone in transformed root cultures and was found to be approximately 3-fold higher than in field grown roots.Abbreviations BA 6-benzyladenine - B5 Gamborg B5 medium - gus -glucuronidase gene - GUS -glucuronidase - HPLC high performance liquid chromatography - MS Murashige and Skoog medium - NAA -naphthalene acetic acid - npt-II neomycin phosphotransferase II gene - OD₆₀₀ optical density at 600 nm - PCR polymerase chain reaction - T _L left border sequence of T-DNA - vir D1 virulence D1 gene - YMB yeast mannitol broth     

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     

Calluses initiated from thin mature embryo fragments are suitable targets for wheat transformation as assessed by long-term GUS expression studies

Microprojectile- or Agrobacterium-mediated DNA delivery into calluses initiated from immature embryos has proven to be effective in transforming wheat. Yet, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process. To circumvent these limitations, we propose an alternative technique applying the particle bombardment technology to calluses derived from fragmented mature embryos rather than immature tissues. The phosphinothricin acetyl transferase (bar) and -glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimise the performance of the proposed technique. Primary requirement for genetic transformation method development, the regeneration capacity of bombarded calluses was established. A preculture duration of 6 days was identified as optimal for DNA uptake and -glucuronidase (GUS) expression. The highest activity was recorded when calluses were selected. Long-term GUS expression studies (1–7weeks subsequent to bombardment), showed differentiated behaviours for tissues obtained from mature versus immature embryos. Notably, mature embryos exhibited the greatest number of cells stably expressing the reporter gene, thus providing an excellent source material for developing a stable transformation procedure.     

Transformation of white spruce (Picea glauca) somatic embryos by microprojectile bombardment

Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of 605 embryos (2.2%) remained GUS positive after two months in culture. Three of those thirteen (23%) embryo-derived tissues consistently showed GUS activity for eight months in culture. These putatively transfomed embryogenic tissues were subjected to Southern blot analysis and the results suggested integration of the gus::nptll gene expression cassette in the white spruce genome.Abbreviations ABA (±)abscisic acid - BA benzyladenine - bp base pair - 2,4-D 2,4-dichlorophenoxyacetic acid - kb kilobase - gus E. coli gene uid A for -glucuronidase - nptll neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronic acid     

High frequency transformation ofKalanchoe laciniata

Leaf explants ofKalanchoe laciniata were cocultivated for different days (2, 4, 6 and 8 days) with disarmedAgrobacterium tumefaciens strains A208SE, GV3111SE and EHA101 carring a binary vector pROA93. The vector contains a cauliflower mosaic virus 35S promotor which drives the coding sequence of neomycin phosphotransferase II (NPT-II) in one direction and -glucuronidase (GUS) in the opposite direction. Prolonged cocultivation (6 days) resulted in a marked increase of GUS gene transient expression, in terms of, the number of explants with transformed cells (up to 100%) and the percent area of transformed tissue ( 50%). Explants cocultivated for 6–7 days showed a dramatic increase in the frequency of stable transformation and 75–80% of the inoculated explants produced transgenic plants. Cocultivation with the nopaline strain A208SE for 7 days gave as high as 10 transgenic plants per explant.Abbreviations GUS -glucuronidase - NPT neomycin phosphotransferase - MS Murashige and Skoog (1962) - BA 6-benzylaminopurine - IAA indol-3-acetic acid - KT kinetin     

5′-Regulatory region of Agrobacterium tumefaciens T-DNA gene 6b directs organ-specific,wound-inducible and auxin-inducible expression in transgenic tobacco

The regulatory activity of a 826 bp DNA fragment located upstream of the pTiBo542 TL-DNA gene 6b coding region was analysed in transgenic tobacco, using -glucuronidase (gus) as a reporter gene. The region was shown to drive organ-specific, wound- and auxin-inducible expression of the reporter, the effect being dependent on the type and concentration of auxin.     

Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens

Fertile transgenic plants of peanut (Arachis hypogaea L. cv. New Mexico Valencia A) were produced using an Agrobacterium-mediated transformation system. Leaf section explants were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBI121 containing the genes for -glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). Approximately 10% of the shoots regenerated on selection medium were GUS-positive. Five independent transformation events resulted in the production of 52 fertile transgenic peanut plants. On average, 240 d were required between seed germination for explant preparation and the production of mature t₁ seed by T₀ plants. Molecular analysis of transgenic plants confirmed the stable integration of the transgenes into the peanut genome. GUS expression segregated in a 31 Mendelian ratio in most T₁ generation plants.Abbreviations GUS -glucuronidase - NPTII neomycin-phosphotransferase II - MS medium, Murashige and Skoog medium (1962) - BA N₆, enzyladenine - NAA 1-naphthaleneacetic acid     

Transient expression of uidA constructs in in vitro onion ( Allium cepa L.) cultures following particle bombardment and Agrobacterium‐mediated DNA delivery

Particle bombardment and Agrobacterium-mediated DNA delivery into immature embryos and microbulbs were used to investigate the expression of the uidA gene in in vitro onion cultures. Both methods were successful in delivering DNA and subsequent uidA expression was observed. Optimal transient -glucuronidase activity was observed in immature embryos that had been pre-cultured for three days and bombarded at a distance of 3 cm from the stopping plate, under 25 in Hg vacuum, using 900–1300 psi rupture discs. The CaMV35S-uidA gene construct gave five fold higher transient -glucuronidase activity than the uidA gene construct regulated by any of four other promoters initially chosen for high experession in monocotyledonous tissues.Abbreviations GUS -glucuronidase - IE immature embryo - MUG methylumbelliferyl -D-glucuronide     

TheCaMV 35S promoter is highly active on floral organs and pollen of transgenic strawberry plants

We have evaluated the expression of the reporter -glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter in flowers and pollen from 14 independent transgenic strawberry lines. Of the 14 lines evaluated, 13 (92.8%) showed GUS activity—as estimated by the histochemical GUS assay—in some floral organs, with expression being most common in the flower stem, sepals, petals, ovary and stigma. Ten of these thirteen transgenic lines (77%) showed GUS activity in pollen, although the percentages of positive pollen per flower varied greatly among the different lines. A study of the GUS expression during pollen maturation showed that the (CaMV 35S) promoter showed low expression in pollen from flower buds before anthesis but was activated in mature pollen following anther dehiscence. The percentages of pollen grains that showed GUS activity ranged from 2.1% to 46.3%. These percentages were similar or even higher when mature pollen was stored dry at room temperature for 2 weeks. After 5 weeks of storage, the percentages of GUS-positive pollen decreased in two of the six lines analysed but remained at similar values in the other four lines. GUS activity was also measured in protein extracts of mature pollen by means of the fluorometric GUS assay, with the values obtained ranging from 3.8 mol MU mg protein^–1 h^–1 to 0.26 mol MU mg protein^–1 h^–1. Contrary to the generally held view that the CaMV 35S promoter is virtually silent in pollen, we conclude that it is highly expressed in transgenic strawberry pollen.Abbreviations CaMV 35S Cauliflower mosaic virus promoter - GUS -Glucuronidase (EC 3.2.1.31) - MU 4-Methyl umbelliferone - nos Nopaline synthase promoter - nptII Neomycin phosphotransferase - X-Gluc 5-Bromo-4-chloro-3-indolyl--d-glucuronic acid     

Analysis of an ABA-responsive rice gene promoter in transgenic tobacco

We have analyzed in transgenic tobacco the expression of a chimeric gene containing 5 sequences of the rice rab-16B gene fused to the -glucuronidase (GUS) reporter gene. This construct, a translational fusion (–482 to +184) including 14 amino acids of the RAB-16B protein, is expressed only in zygotic and pollen-derived embryos. In zygotic embryos, GUS activity begins to accumulate 10 days after flowering (daf), and increases until seed maturation at 25 daf. Immunological measurements of endogenous abscisic acid (ABA) accumulation in these seeds showed a close parallel between hormone levels and GUS activity. However, GUS activity could not be reproducibly induced by treatment of immature embryos with ABA (10 M). Neither GUS activity nor GUS mRNA could be detected in leaves of transgenic tobacco even after ABA treatment. In contrast, GUS activity could be induced to high levels in pollen-derived embryos by treatment with ABA. Our results show that 482 bp of 5 sequences of the rice rab-16B promoter can confer in transgenic tobacco developmentally regulated expression in embryos but not ABA-responsive expression in vegetative tissues.     

Agrobacterium-mediated transient transformation of annatto (Bixa orellana) hypocotyls with the GUS reporter gene

Hypocotyls from annatto seedlings, were inoculated with Agrobacterium tumefaciens harboring a binary vector, pBI.121 or pCAMBIA2301, containing the -glucuronidase (gus) gene. Histochemical GUS assay of infected hypocotyls from two annatto varieties showed transient gus gene expression between 3 and 12 days after inoculation.These authors contributed equally to this work.)     

The rice metallothionein gene promoter does not direct foreign gene expression in seed endosperm

We generated transgenic tobacco and rice plants harboring a chimeric gene consisting of the 5-upstream sequence of the rice metallothionein gene (ricMT) fused to the -glucuronidase (GUS) gene. The activity and tissue-specific expression of the ricMT promoter were demonstrated in these transgenic plants. In the transgenic rice plants, despite substantial levels of GUS activity in the shoot and root, almost no GUS signal was detected in the endosperm. Thus, the ricMT promoter could be useful in avoiding accumulation of undesired proteins in the seed endosperm.