Optimizing the production of transformed pea (Pisum sativum L.) callus using disarmed Agrobacterium tumefaciens strains

For optimization of the transformation procedure with Pisum sativum L. stern explant callus was used to test the effect of disarmed Agrobacterium tumefaciens strains, cocultivation procedures (preconditioning of explants; use of Nicotiana tabacum L. nurse cultures), duration of cocultivation (2, 3 or 4 days), and agents for selection (kanamycin or hygromycin). The succinamopine strain EHA101(pBI1042) produced the highest percentage of transformed calli (77%) when used in conjunction with tobacco nurse culture during four days of cocultivation. Using this strain, kanamycin (76%) and hygromycin (77%) were equally effective selective agents, but for strain LBA4404(pBI1042) percentage of transformed calli was higher for hygromycin (63%) than for kanamycin (17%). The procedures and strains shown to be optimal for transformation of pea callus will now be complemented by a pea regeneration system.     

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector

Summary Kanamycin resistant plants of Solarium melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NPTII) gene as the selectable marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.Abbreviations CAT chloramphenicol acetyltransferase - MS Murashige and Skoog - NPTII neomycin phosphotransferase - NOS nopaline synthase - ZEA zeatin     

Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector

The disamed Ti-binary vector pBIN 6 in Agrobacterium tumefaciens has been used in leaf disc transfomations to produce transgenic apple (Malus pumila Mill.) plants with a nomal phenotype except for a somewhat reduced capacity to root. The presence of the genes for nopaline synthase and neomycin phosphotrans ferase (conferring kanamycin resistance), inserted into the host genome by the vector, was confirmed by Southern blot analysis, the detection of nopaline synthase activity and rooting in the presence of the antibiotic.The nopaline synthase gene continued to be expressed in glasshouse-grown plants several months after removal from in vitro growth conditions.     

Transformation of Medicago arborea L. with an Agrobacterium rhizogenes binary vector carrying the hygromycin resistance gene

Plants of Medicago arborea have been infected with Agrobacterium rhizogenes strain LBA9402 harbouring the plasmids Ri 1855 and AGS125 carrying a gene conferring resistance to the antibiotic hygromycin. About 7056 of the hairy roots showed callus formation on hygromycin-supplemented medium. Regeneration took place on antibiotic free medium only. Plantlets suitable for transfer to soil were obtained after the manual removal of most of the leaves. Plant morphology showed the usual alterations induced by the Ri plasmid; moreover, two years after soil-transfer, transformants have not flowered. Molecular analysis indicates the presence of T-DNA from both pAGS 125 and p1855. The expression of the hygromycin phosphotransferase gene allowed callus and protoplasts of transformed plants to grow on media supplemented with the antibiotic. This trait will be utilized as a marker in protoplast fusion between Medicago arborea and Medicago sativa (alfalfa).Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - kin kinetin - GA3 Gibberellic acid - IAA Indole-3-acetic acid - HPT hygromycin phosphotransferase - NOS nopaline synthase - MS Murashige and Skoog (1962) - B5 Gamborg et al. (1968) - B5hy B5 supplemented with 20 mg 1-1 of hygromycin - YMB yeast mannitol broth     

Parameters affecting the frequency of kanamycin resistant alfalfa obtained by Agrobacterium tumefaciens mediated transformation

Kanamycin resistant plants of Medicago varia A2 were obtained by an optimized procedure for high frequency transformation using Agrobacterium tumefaciens infection of leaf and petiole tissue. Parameters which affected the frequency were explant type, the Agrobacterium strain used and the time allowed for cocultivation. Under optimum conditions, i.e., using the Agrobacterium strain A281 and a 4 day cocultivation period, the frequency of transformed leaflets obtained was greater than 70%.     

Transgenic antibiotic resistance may be differentially silenced in germinating pollen grains

Transgenic tobacco (Nicotiana tabacum L.) plants, carrying the neomycin phosphotransferase (NPT II) gene from E. coli, are resistant to kanamycin when grown from seeds on kanamycin containing medium. Tissue and cell cultures derived from those transformants also express resistance and regenerate complete plantlets in the presence of the antibiotic. This unspecific response to the selective condition has led to the belief that the foreign gene is continuously active or uniformly inducible in all cells of the transgenic plant. However, our experiments show that this view is not true for pollen grains during in vitro germination. Pollen grains isolated from kanamycin resistant tobacco plants carry and transmit the foreign gene but do not express resistance when germinating in vitro. This data presents evidence for differential silencing of a foreign gene in a mature gamete. On the other hand, immature pollen grains (microspores) appear to express resistance. The point of the downregulation of the neomycin transferase gene during pollen maturation is discussed.Abbreviations kan kanamycin sulfate - NPT II neomycin phosphotransferase II - sr streptomycin sulfate     

A chimaeric hygromycin resistance gene as a selectable marker in plant cells

Summary We show here that plant cells are sensitive to the antibiotic hygromycin-B⁴. We also show that a chimaeric gene consisting of the nopaline synthase (nos) gene regulatory elements and the E. coli derived hygromycin phosphotransferase (hpt) gene, when transferred to plants' cells, confers resistance to hygromycin B. The chimaeric nos-hpt gene enables efficient selection of DNA transfer to plant cells when used in conjunction with Ti plasmid-derived binary vectors in cocultivation experiments.     

Toxicity of antibiotics on zygotic embryos of white spruce (Picea glauca) cultured in vitro

The toxicity of kanamycin, hygromycin B, geneticin, methotrexate and cefotaxime on zygotic embryos ofPicea glauca was studied. Embryos placed on bud induction medium produced approximately 20 adventitious buds per embryo under control conditions. Addition of antibiotics reduced the number of bud-forming embryos. Using the percentage of embryos with buds as an indication of antibiotic toxicity, two-day-old explants were found to be more sensitive than nine-day-old. Kanamycin toxicity was enhanced by cefotaxime and this effect increased with increases in concentration of either antibiotic. Although no morphological difference was observed after 21 days, embryos growing on medium containing 20 g ml^–1 kanamycin showed a decrease of 73% in dry weight and 23% in protein content per embryo when compared to control embryos. Similarly, a decrease of 38% in dry weight and 40% in protein content per embryo was found in embryos on medium containing 300 g ml^–1 cefotaxime.Abbreviations BA 6 benzyl-aminopurine - EDTA ethylenediamine-tetraacetic acid - PVP 10 polyvinylpyrrolidone (MW 10,000) - Tris-HCl Tris [hydroxymethyl] amino hydrochloride NRCC No. 30262     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated genetic transformation in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> [L.] O. Kuntze)

We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7 d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L giberellic acid (GA₃). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern hybridization to confirm gene integration.     

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     

Effect of Some Antibiotics on the In Vitro Morphogenetic Response from Callus Cultures of Coryphantha Elephantidens

The effect of five antibiotics: carbenicillin, chloramphenicol, cefotaxime, kanamycin and hygromycin on the organogenesis from callus cultures of Coryphantha elphantidens (Lem.) Lem. have been studied. Carbenicillin and cefotaxime stimulated shoot regeneration from callus. All antibiotics under study suppressed rooting of in vitro formed shoots. After five sequential subcultures on kanamycin supplemented medium, antibiotic resistant callus was obtained. To study the impact of kanamycin on resistant callus, total protein content was also studied. Selected callus showed a remarkable increase in callus mass. Antibiotic resistant plants have been selected by screening callus pieces on kanamycin supplemented media. Total protein content increased with subsequent subcultures in kanamycin resistant callus. The kanamycin selected shoots withstood the stability test after 2 months on antibiotic free medium. Plants were raised from the callus, which formed roots in 20 mg dm^–3 kanamycin, which was under study.     

Evaluation of selectable markers for obtaining stable transformants in the gramineae

Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 × 10⁻⁵ to 2 × 10⁻⁶) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.     

Stable integration and expression of β-glucuronidase and NPT II genes in mango somatic embryos

Summary Somatic proembryos of mango (Mangifera indica L. cv. Hindi) were co-cultivated withAgrobacterium tumefaciens strain A208 harboring pTiT37-Se::pMON 9749 (9749 ASE). Transformed somatic proembryos capable of growing on selection medium containing 200 μg/ml kanamycin produced the characteristic indigo blue precipitate in the presence of 5-bromo-4-chloro-3-glucuronic acid. These proembryos were chimeral consisting of transformed (blue) and nontransformed (yellow/white) cells. A stepwise selection strategy was found necessary to eliminate chimeras. a) Initial screening at 200 μg/ml kanamycin to enable growth of transformed cells, b) further screening at 400 μg/ml kanamycin to reduce chimeras, and c) recovery of pure transformed clones of proembryos in liquid selection medium with 100 μg/ml kanamycin. The integration of the NPT II and GUS genes into mango genome was confirmed by Southern hybridization.     

In vitro culture of ovules ofTriticum aestivum at early stages of embryogenesis

Because most of the intergenericGramineae embryos abort before they can be isolated and cultured, our object was to grow ovules at an early stage of embryogenesis. Ovules were at size 1 to 7 mm. The youngest stages represented ovules containing several-celled proembryos; the oldest stage consisted of embryos at the level of differentiation of the organs. 1357 ovules were jointly inoculated in 12 different media of which only 3 appeared to be most suitable for the growth and differentiation of globular proembryos. From several-celled proembryos (ovules at the size of 1.5 – 2.5 mm) only compact calluses developed. The capability of proembryos to differentiate and to form fully developed embryos and consequently plants started when the ovules were inoculated at the size of 2.5 – 3.0 mm. Since the medium plays an important role in the process of differentiation of proembryos, the application of similar culture conditions is suggested for the in vitro culture of haploid or hybrid proembryos obtained among wide crosses inGramineae.List of abbreviations COC Coconut milk - BAP 6-benzylaminopurine - YE Yeast extract - CH Casein hydrolysate - KT Kinetin - ZT Zeatin     

Genetic transformation of NERICA,interspecific hybrid rice between <Emphasis Type="Italic">Oryza glaberrima</Emphasis> and <Emphasis Type="Italic">O. sativa</Emphasis>, mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin. The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and segregation of the transgenes were demonstrated by molecular and genetic analyses in the T₀ and T₁ generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the frequency of transformation was lower and sterility was more frequent.     

Patterns of transformation intensity on flax hypocotyls inoculated with Agrobacterium tumefaciens

Summary Cellular transformation intensities on flax (Linum usitatissimum) hypocotyl explants using disarmed Agrobacterium tumefaciens were investigated through various preculture durations, cocultivation durations and removal of epidermis. The expression of an intron-containing -glucuronidase (GUS) gene driven by CaMV 35S promoter served as a reporter for determination of transformed tissues on hypocotyls. The binary plasmid p35SGUSINT in octopine-type Agrobacterium strain GV2260 was used as the vector system. A prolonged cocultivation duration (5–7 days) resulted in a much higher transformation staining intensity (frequency * tissue area) than 2- or 3-day-cocultivation duration on hypocotyls variously precultured prior to inoculation. A high staining intensity on the two cut ends was obtained from nonprecultured hypocotyls. A reduction in intensity on the upper cut end of hypocotyls was observed with preculture times greater than 6 days. Peeled hypocotyls with a post-peeling preculture of 2 or 3 days had a high proportion of superficial area covered by transformed tissues after a 7 day-cocultivation duration. These results will help to improve the efficiency of recovery of transgenic plants by increasing the proportion of transformation in the regenerable tissues.     

Agrobacterium mediated transfer of chlorsulfuron resistance to commercial flax cultivars

Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector containing a chimeric NPT-ll gene and a mutant acetolactate synthase gene (conferring resistance to the herbicide chlorsulfuron) from Arabidopsis was used to transform flax (Linum usitatissimum) hypocotyl tissue. Transgenic regenerants were recovered from the inoculated tissue and were tested for expression of the foreign genes by leaf callus assays on kanamycin and on chlorsulfuron. Transgenic plants were grown to maturity; selfed progeny were similarly tested to determine segregation pattern for the novel genes, and some were grown in chlorsulfuron containing soil. Lines from two major commercial cultivars express chlorsulfuron resistance in greenhouse tests.     

Transformation of the coryneform bacterium cellulomonas flavigena with plasmid DNA via electroporation

Summary Using electroporation we have transformed Cellulomonas flavigena with a shuttle vector (pJA85) derived from the E. coli plasmid pUC8 and the Brevibacterium lactofermentum plasmid pULRS8. Upon transformation this plasmid was found to be stable, not to undergo detectable deletion, and to express antibiotic resistance markers originating in Brevibacterium.     

影响农杆菌介导的麻疯树基因转化因素的研究(简报)

麻疯树(Jatropha curcas)为大戟科麻疯树属植物,是一种多年生木本油料植物。原产美洲,广泛分布于热带亚热带地区。因其种子含油量高达 60％,可作生物燃料之用,麻疯树是目前正在被开发利用的重要能源植物之一。除此之外,麻疯树的种子含有多种活性成分,有着重要的农药和医药价值;其生长能耐干旱贫瘠,可用于荒山造林。因此,麻疯树是一种具有多种用途、重要经济价值和学术研究价值的植物。目前对其研究多停留在植物组织培养、植物化学、毒理学、种植业方面等。     

Transformation of Medicago by Agrobacterium mediated gene transfer

Shoot segments of Medicago varia genotype A2 were co-cultivated with Agrobacterium tumefaciens strain bo42 carrying pGA471, a plasmid coding for the kanamycin resistant determinant as transferable positive selection marker in plant cells (An et al., 1985). Resistant plants were regenerated at high frequency from green calli developed on inoculated stem cuttings under kanamycin selection. DNA-DNA hybridization analysis showed the presence of the structural gene of the kanamycin resistant determinant in total DNA isolated from several independent transformants. All data presented clearly demonstrate the transfer, stable maintenance and functional expression of the kanamycin resistance marker in Medicago varia cells which retain their morphogenic property.Abbreviations Km kanamycin - KmR kanamycin resistant - Cb carbenicillin - 2,4-D 2,4 dichlorophenoxyacetic acid - BA 6-benzyladenine - T-DNA transferred DNA into plants